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Yang B, Wang SQ, Yang HQ. β-adrenergic regulation of Ca 2+ signaling in heart cells. BIOPHYSICS REPORTS 2024; 10:274-282. [PMID: 39539286 PMCID: PMC11554573 DOI: 10.52601/bpr.2024.240906] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2024] [Accepted: 04/15/2024] [Indexed: 11/16/2024] Open
Abstract
β-adrenergic receptors (βARs) play significant roles in regulating Ca2+ signaling in cardiac myocytes, thus holding a key function in modulating heart performance. βARs regulate the influx of extracellular Ca2+ and the release and uptake of Ca2+ from the sarcoplasmic reticulum (SR) by activating key components such as L-type calcium channels (LTCCs), ryanodine receptors (RyRs) and phospholamban (PLN), mediated by the phosphorylation actions by protein kinase A (PKA). In cardiac myocytes, the presence of β2AR provides a protective mechanism against potential overstimulation of β1AR, which may aid in the restoration of cardiac dysfunctions. Understanding the Ca2+ regulatory signaling pathways of βARs in cardiac myocytes and the differences among various βAR subtypes are crucial in cardiology and hold great potential for developing treatments for heart diseases.
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Affiliation(s)
- Bo Yang
- Cyrus Tang Medical Institute, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Soochow University, Suzhou, Jiangsu 215123, China
| | - Shi-Qiang Wang
- State Key Lab of Membrane Biology, College of Life Sciences, Peking University, Beijing 100871, China
| | - Hua-Qian Yang
- Cyrus Tang Medical Institute, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Soochow University, Suzhou, Jiangsu 215123, China
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Janicek R, Camors EM, Potenza DM, Fernandez-Tenorio M, Zhao Y, Dooge HC, Loaiza R, Alvarado FJ, Egger M, Valdivia HH, Niggli E. Dual ablation of the RyR2-Ser2808 and RyR2-Ser2814 sites increases propensity for pro-arrhythmic spontaneous Ca 2+ releases. J Physiol 2024; 602:5179-5201. [PMID: 39316734 PMCID: PMC11493507 DOI: 10.1113/jp286453] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2024] [Accepted: 08/21/2024] [Indexed: 09/26/2024] Open
Abstract
During exercise or stress, the sympathetic system stimulates cardiac contractility via β-adrenergic receptor (β-AR) activation, resulting in phosphorylation of the cardiac ryanodine receptor (RyR2). Three RyR2 phosphorylation sites have taken prominence in excitation-contraction coupling: S2808 and S2030 are described as protein kinase A specific and S2814 as a Ca2+/calmodulin kinase type-2-specific site. To examine the contribution of these phosphosites to Ca2+ signalling, we generated double knock-in (DKI) mice in which Ser2808 and Ser2814 phosphorylation sites have both been replaced by alanine (RyR2-S2808A/S2814A). These mice did not exhibit an overt phenotype. Heart morphology and haemodynamic parameters were not altered. However, they had a higher susceptibility to arrhythmias. We performed confocal Ca2+ imaging and electrophysiology experiments. Isoprenaline was used to stimulate β-ARs. Measurements of Ca2+ waves and latencies in myocytes revealed an increased propensity for spontaneous Ca2+ releases in DKI myocytes, both in control conditions and during β-AR stimulation. In DKI cells, waves were initiated from a lower threshold concentration of Ca2+ inside the sarcoplasmic reticulum, suggesting higher Ca2+ sensitivity of the RyRs. The refractoriness of Ca2+ spark triggering depends on the Ca2+ sensitivity of the RyR2. We found that RyR2-S2808A/S2814A channels were more Ca2+ sensitive in control conditions. Isoprenaline further shortened RyR refractoriness in DKI cardiomyocytes. Together, our results suggest that ablation of both the RyR2-Ser2808 and RyR2-S2814 sites increases the propensity for pro-arrhythmic spontaneous Ca2+ releases, as previously suggested for hyperphosphorylated RyRs. Given that the DKI cells present a full response to isoprenaline, the data suggest that phosphorylation of Ser2030 might be sufficient for β-AR-mediated sensitization of RyRs. KEY POINTS: Phosphorylation of cardiac sarcoplasmic reticulum Ca2+-release channels (ryanodine receptors, RyRs) is involved in the regulation of cardiac function. Ablation of both the RyR2-Ser2808 and RyR2-Ser2814 sites increases the propensity for pro-arrhythmic spontaneous Ca2+ releases, as previously suggested for hyperphosphorylated RyRs. The intra-sarcoplasmic reticulum Ca2+ threshold for spontaneous Ca2+ wave generation is lower in RyR2-double-knock-in cells. The RyR2 from double-knock-in cells exhibits increased Ca2+ sensitivity. Phosphorylation of Ser2808 and Ser2814 might be important for basal activity of the channel. Phosphorylation of Ser2030 might be sufficient for a β-adrenergic response.
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Affiliation(s)
| | - Emmanuel M Camors
- Department of Pediatrics, Division of Cardiology, University of Tennessee Health Science Center, Le Bonheur Children’s Hospital Research Center, Memphis, Tennessee 38103, USA
| | | | | | - Yanting Zhao
- Department of Medicine, Division of Cardiovascular Medicine, and Cardiovascular Research Center, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin 53705, USA
| | - Holly C. Dooge
- Department of Medicine, Division of Cardiovascular Medicine, and Cardiovascular Research Center, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin 53705, USA
| | - Randall Loaiza
- Department of Medicine, Division of Cardiovascular Medicine, and Cardiovascular Research Center, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin 53705, USA
| | - Francisco J Alvarado
- Department of Medicine, Division of Cardiovascular Medicine, and Cardiovascular Research Center, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin 53705, USA
| | - Marcel Egger
- Department of Physiology, University of Bern, Bern, Switzerland
| | - Hector H. Valdivia
- Department of Medicine, Division of Cardiovascular Medicine, and Cardiovascular Research Center, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin 53705, USA
| | - Ernst Niggli
- Department of Physiology, University of Bern, Bern, Switzerland
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Zheng J, Dooge HC, Valdivia HH, Alvarado FJ. Ablation of three major phospho-sites in RyR2 preserves the global adrenergic response but creates an arrhythmogenic substrate. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.07.08.602617. [PMID: 39026734 PMCID: PMC11257526 DOI: 10.1101/2024.07.08.602617] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/20/2024]
Abstract
Background Ryanodine receptor 2 (RyR2) is one of the first substrates undergoing phosphorylation upon catecholaminergic stimulation. Yet, the role of RyR2 phosphorylation in the adrenergic response remains debated. To date, three residues in RyR2 are known to undergo phosphorylation upon adrenergic stimulation. We generated a model of RyR2 phospho-ablation of all three canonical phospho-sites (RyR2-S2031A/S2808A/S2814A, triple phospho-mutant, TPM) to elucidate the role of phosphorylation at these residues in the adrenergic response. Methods Cardiac structure and function, cellular Ca 2+ dynamics and electrophysiology, and RyR2 channel activity both under basal conditions and under isoproterenol (Iso) stimulation were systematically evaluated. We used echocardiography and electrocardiography in anesthetized mice, single-cell Ca 2+ imaging and whole-cell patch clamp in isolated adult cardiomyocytes, and biochemical assays. Results Iso stimulation produced normal chronotropic and inotropic responses in TPM mice as well as an increase in the global Ca 2+ transients in isolated cardiomyocytes. Functional studies revealed fewer Ca 2+ sparks in permeabilized TPM myocytes, and reduced RyR2-mediated Ca 2+ leak in intact myocytes under Iso stimulation, suggesting that the canonical sites may regulate RyR2-mediated Ca 2+ leak. TPM mice also displayed increased propensity for arrhythmia. TPM myocytes were prone to develop early afterdepolarizations (EADs), which were abolished by chelating intracellular Ca 2+ with EGTA, indicating that EADs require SR Ca 2+ release. EADs were also blocked by a low concentration of tetrodotoxin, further suggesting reactivation of the sodium current ( I Na ) as the underlying cause. Conclusion Phosphorylation of the three canonical residues on RyR2 may not be essential for the global adrenergic responses. However, these sites play a vital role in maintaining electrical stability during catecholamine stimulation by fine-tuning RyR2-mediated Ca 2+ leak. These findings underscore the importance of RyR2 phosphorylation and a finite diastolic Ca 2+ leak in maintaining electrical stability during catecholamine stimulation.
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Takenaka M, Kodama M, Murayama T, Ishigami-Yuasa M, Mori S, Ishida R, Suzuki J, Kanemaru K, Sugihara M, Iino M, Miura A, Nishio H, Morimoto S, Kagechika H, Sakurai T, Kurebayashi N. Screening for Novel Type 2 Ryanodine Receptor Inhibitors by Endoplasmic Reticulum Ca 2+ Monitoring. Mol Pharmacol 2023; 104:275-286. [PMID: 37678938 DOI: 10.1124/molpharm.123.000720] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2023] [Revised: 08/21/2023] [Accepted: 08/29/2023] [Indexed: 09/09/2023] Open
Abstract
Type 2 ryanodine receptor (RyR2) is a Ca2+ release channel on the endoplasmic (ER)/sarcoplasmic reticulum that plays a central role in the excitation-contraction coupling in the heart. Hyperactivity of RyR2 has been linked to ventricular arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia and heart failure, where spontaneous Ca2+ release via hyperactivated RyR2 depolarizes diastolic membrane potential to induce triggered activity. In such cases, drugs that suppress RyR2 activity are expected to prevent the arrhythmias, but there is no clinically available RyR2 inhibitors at present. In this study, we searched for RyR2 inhibitors from a well-characterized compound library using a recently developed ER Ca2+-based assay, where the inhibition of RyR2 activity was detected by the increase in ER Ca2+ signals from R-CEPIA1er, a genetically encoded ER Ca2+ indicator, in RyR2-expressing HEK293 cells. By screening 1535 compounds in the library, we identified three compounds (chloroxylenol, methyl orsellinate, and riluzole) that greatly increased the ER Ca2+ signal. All of the three compounds suppressed spontaneous Ca2+ oscillations in RyR2-expressing HEK293 cells and correspondingly reduced the Ca2+-dependent [3H]ryanodine binding activity. In cardiomyocytes from RyR2-mutant mice, the three compounds effectively suppressed abnormal Ca2+ waves without substantial effects on the action-potential-induced Ca2+ transients. These results confirm that ER Ca2+-based screening is useful for identifying modulators of ER Ca2+ release channels and suggest that RyR2 inhibitors have potential to be developed as a new category of antiarrhythmic drugs. SIGNIFICANCE STATEMENT: We successfully identified three compounds having RyR2 inhibitory action from a well-characterized compound library using an endoplasmic reticulum Ca2+-based assay, and demonstrated that these compounds suppressed arrhythmogenic Ca2+ wave generation without substantially affecting physiological action-potential induced Ca2+ transients in cardiomyocytes. This study will facilitate the development of RyR2-specific inhibitors as a potential new class of drugs for life-threatening arrhythmias induced by hyperactivation of RyR2.
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Affiliation(s)
- Mai Takenaka
- Department of Cellular and Molecular Pharmacology (M.T., M.K., T.M., T.S., N.K.) and Department of Clinical Laboratory Medicine (M.S.), Juntendo University Graduate School of Medicine, Tokyo, Japan; Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan (M.I.-Y., Sh.M., R.I., H.K.); Department of Physiology, University of California San Francisco, San Francisco, California (J.S.); Department of Physiology, Nihon University School of Medicine, Tokyo, Japan (K.K., M.I.); Department of Legal Medicine, Hyogo Medical University, Nishinomiya, Japan (A.M., H.N.); and Department of Health Sciences at Fukuoka, International University of Health and Welfare, Fukuoka, Japan (Sa.M.)
| | - Masami Kodama
- Department of Cellular and Molecular Pharmacology (M.T., M.K., T.M., T.S., N.K.) and Department of Clinical Laboratory Medicine (M.S.), Juntendo University Graduate School of Medicine, Tokyo, Japan; Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan (M.I.-Y., Sh.M., R.I., H.K.); Department of Physiology, University of California San Francisco, San Francisco, California (J.S.); Department of Physiology, Nihon University School of Medicine, Tokyo, Japan (K.K., M.I.); Department of Legal Medicine, Hyogo Medical University, Nishinomiya, Japan (A.M., H.N.); and Department of Health Sciences at Fukuoka, International University of Health and Welfare, Fukuoka, Japan (Sa.M.)
| | - Takashi Murayama
- Department of Cellular and Molecular Pharmacology (M.T., M.K., T.M., T.S., N.K.) and Department of Clinical Laboratory Medicine (M.S.), Juntendo University Graduate School of Medicine, Tokyo, Japan; Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan (M.I.-Y., Sh.M., R.I., H.K.); Department of Physiology, University of California San Francisco, San Francisco, California (J.S.); Department of Physiology, Nihon University School of Medicine, Tokyo, Japan (K.K., M.I.); Department of Legal Medicine, Hyogo Medical University, Nishinomiya, Japan (A.M., H.N.); and Department of Health Sciences at Fukuoka, International University of Health and Welfare, Fukuoka, Japan (Sa.M.)
| | - Mari Ishigami-Yuasa
- Department of Cellular and Molecular Pharmacology (M.T., M.K., T.M., T.S., N.K.) and Department of Clinical Laboratory Medicine (M.S.), Juntendo University Graduate School of Medicine, Tokyo, Japan; Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan (M.I.-Y., Sh.M., R.I., H.K.); Department of Physiology, University of California San Francisco, San Francisco, California (J.S.); Department of Physiology, Nihon University School of Medicine, Tokyo, Japan (K.K., M.I.); Department of Legal Medicine, Hyogo Medical University, Nishinomiya, Japan (A.M., H.N.); and Department of Health Sciences at Fukuoka, International University of Health and Welfare, Fukuoka, Japan (Sa.M.)
| | - Shuichi Mori
- Department of Cellular and Molecular Pharmacology (M.T., M.K., T.M., T.S., N.K.) and Department of Clinical Laboratory Medicine (M.S.), Juntendo University Graduate School of Medicine, Tokyo, Japan; Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan (M.I.-Y., Sh.M., R.I., H.K.); Department of Physiology, University of California San Francisco, San Francisco, California (J.S.); Department of Physiology, Nihon University School of Medicine, Tokyo, Japan (K.K., M.I.); Department of Legal Medicine, Hyogo Medical University, Nishinomiya, Japan (A.M., H.N.); and Department of Health Sciences at Fukuoka, International University of Health and Welfare, Fukuoka, Japan (Sa.M.)
| | - Ryosuke Ishida
- Department of Cellular and Molecular Pharmacology (M.T., M.K., T.M., T.S., N.K.) and Department of Clinical Laboratory Medicine (M.S.), Juntendo University Graduate School of Medicine, Tokyo, Japan; Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan (M.I.-Y., Sh.M., R.I., H.K.); Department of Physiology, University of California San Francisco, San Francisco, California (J.S.); Department of Physiology, Nihon University School of Medicine, Tokyo, Japan (K.K., M.I.); Department of Legal Medicine, Hyogo Medical University, Nishinomiya, Japan (A.M., H.N.); and Department of Health Sciences at Fukuoka, International University of Health and Welfare, Fukuoka, Japan (Sa.M.)
| | - Junji Suzuki
- Department of Cellular and Molecular Pharmacology (M.T., M.K., T.M., T.S., N.K.) and Department of Clinical Laboratory Medicine (M.S.), Juntendo University Graduate School of Medicine, Tokyo, Japan; Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan (M.I.-Y., Sh.M., R.I., H.K.); Department of Physiology, University of California San Francisco, San Francisco, California (J.S.); Department of Physiology, Nihon University School of Medicine, Tokyo, Japan (K.K., M.I.); Department of Legal Medicine, Hyogo Medical University, Nishinomiya, Japan (A.M., H.N.); and Department of Health Sciences at Fukuoka, International University of Health and Welfare, Fukuoka, Japan (Sa.M.)
| | - Kazunori Kanemaru
- Department of Cellular and Molecular Pharmacology (M.T., M.K., T.M., T.S., N.K.) and Department of Clinical Laboratory Medicine (M.S.), Juntendo University Graduate School of Medicine, Tokyo, Japan; Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan (M.I.-Y., Sh.M., R.I., H.K.); Department of Physiology, University of California San Francisco, San Francisco, California (J.S.); Department of Physiology, Nihon University School of Medicine, Tokyo, Japan (K.K., M.I.); Department of Legal Medicine, Hyogo Medical University, Nishinomiya, Japan (A.M., H.N.); and Department of Health Sciences at Fukuoka, International University of Health and Welfare, Fukuoka, Japan (Sa.M.)
| | - Masami Sugihara
- Department of Cellular and Molecular Pharmacology (M.T., M.K., T.M., T.S., N.K.) and Department of Clinical Laboratory Medicine (M.S.), Juntendo University Graduate School of Medicine, Tokyo, Japan; Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan (M.I.-Y., Sh.M., R.I., H.K.); Department of Physiology, University of California San Francisco, San Francisco, California (J.S.); Department of Physiology, Nihon University School of Medicine, Tokyo, Japan (K.K., M.I.); Department of Legal Medicine, Hyogo Medical University, Nishinomiya, Japan (A.M., H.N.); and Department of Health Sciences at Fukuoka, International University of Health and Welfare, Fukuoka, Japan (Sa.M.)
| | - Masamitsu Iino
- Department of Cellular and Molecular Pharmacology (M.T., M.K., T.M., T.S., N.K.) and Department of Clinical Laboratory Medicine (M.S.), Juntendo University Graduate School of Medicine, Tokyo, Japan; Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan (M.I.-Y., Sh.M., R.I., H.K.); Department of Physiology, University of California San Francisco, San Francisco, California (J.S.); Department of Physiology, Nihon University School of Medicine, Tokyo, Japan (K.K., M.I.); Department of Legal Medicine, Hyogo Medical University, Nishinomiya, Japan (A.M., H.N.); and Department of Health Sciences at Fukuoka, International University of Health and Welfare, Fukuoka, Japan (Sa.M.)
| | - Aya Miura
- Department of Cellular and Molecular Pharmacology (M.T., M.K., T.M., T.S., N.K.) and Department of Clinical Laboratory Medicine (M.S.), Juntendo University Graduate School of Medicine, Tokyo, Japan; Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan (M.I.-Y., Sh.M., R.I., H.K.); Department of Physiology, University of California San Francisco, San Francisco, California (J.S.); Department of Physiology, Nihon University School of Medicine, Tokyo, Japan (K.K., M.I.); Department of Legal Medicine, Hyogo Medical University, Nishinomiya, Japan (A.M., H.N.); and Department of Health Sciences at Fukuoka, International University of Health and Welfare, Fukuoka, Japan (Sa.M.)
| | - Hajime Nishio
- Department of Cellular and Molecular Pharmacology (M.T., M.K., T.M., T.S., N.K.) and Department of Clinical Laboratory Medicine (M.S.), Juntendo University Graduate School of Medicine, Tokyo, Japan; Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan (M.I.-Y., Sh.M., R.I., H.K.); Department of Physiology, University of California San Francisco, San Francisco, California (J.S.); Department of Physiology, Nihon University School of Medicine, Tokyo, Japan (K.K., M.I.); Department of Legal Medicine, Hyogo Medical University, Nishinomiya, Japan (A.M., H.N.); and Department of Health Sciences at Fukuoka, International University of Health and Welfare, Fukuoka, Japan (Sa.M.)
| | - Sachio Morimoto
- Department of Cellular and Molecular Pharmacology (M.T., M.K., T.M., T.S., N.K.) and Department of Clinical Laboratory Medicine (M.S.), Juntendo University Graduate School of Medicine, Tokyo, Japan; Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan (M.I.-Y., Sh.M., R.I., H.K.); Department of Physiology, University of California San Francisco, San Francisco, California (J.S.); Department of Physiology, Nihon University School of Medicine, Tokyo, Japan (K.K., M.I.); Department of Legal Medicine, Hyogo Medical University, Nishinomiya, Japan (A.M., H.N.); and Department of Health Sciences at Fukuoka, International University of Health and Welfare, Fukuoka, Japan (Sa.M.)
| | - Hiroyuki Kagechika
- Department of Cellular and Molecular Pharmacology (M.T., M.K., T.M., T.S., N.K.) and Department of Clinical Laboratory Medicine (M.S.), Juntendo University Graduate School of Medicine, Tokyo, Japan; Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan (M.I.-Y., Sh.M., R.I., H.K.); Department of Physiology, University of California San Francisco, San Francisco, California (J.S.); Department of Physiology, Nihon University School of Medicine, Tokyo, Japan (K.K., M.I.); Department of Legal Medicine, Hyogo Medical University, Nishinomiya, Japan (A.M., H.N.); and Department of Health Sciences at Fukuoka, International University of Health and Welfare, Fukuoka, Japan (Sa.M.)
| | - Takashi Sakurai
- Department of Cellular and Molecular Pharmacology (M.T., M.K., T.M., T.S., N.K.) and Department of Clinical Laboratory Medicine (M.S.), Juntendo University Graduate School of Medicine, Tokyo, Japan; Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan (M.I.-Y., Sh.M., R.I., H.K.); Department of Physiology, University of California San Francisco, San Francisco, California (J.S.); Department of Physiology, Nihon University School of Medicine, Tokyo, Japan (K.K., M.I.); Department of Legal Medicine, Hyogo Medical University, Nishinomiya, Japan (A.M., H.N.); and Department of Health Sciences at Fukuoka, International University of Health and Welfare, Fukuoka, Japan (Sa.M.)
| | - Nagomi Kurebayashi
- Department of Cellular and Molecular Pharmacology (M.T., M.K., T.M., T.S., N.K.) and Department of Clinical Laboratory Medicine (M.S.), Juntendo University Graduate School of Medicine, Tokyo, Japan; Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan (M.I.-Y., Sh.M., R.I., H.K.); Department of Physiology, University of California San Francisco, San Francisco, California (J.S.); Department of Physiology, Nihon University School of Medicine, Tokyo, Japan (K.K., M.I.); Department of Legal Medicine, Hyogo Medical University, Nishinomiya, Japan (A.M., H.N.); and Department of Health Sciences at Fukuoka, International University of Health and Welfare, Fukuoka, Japan (Sa.M.)
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Barber KR, Vizcarra VS, Zilch A, Majuta L, Diezel CC, Culver OP, Hughes BW, Taniguchi M, Streicher JM, Vanderah TW, Riegel AC. The Role of Ryanodine Receptor 2 in Drug-Associated Learning. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.10.03.560743. [PMID: 37873212 PMCID: PMC10592901 DOI: 10.1101/2023.10.03.560743] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/25/2023]
Abstract
Type-2 ryanodine receptor (RyR2) ion channels facilitate the release of Ca 2+ from stores and serve an important function in neuroplasticity. The role for RyR2 in hippocampal-dependent learning and memory is well established and chronic hyperphosphorylation of RyR2 (RyR2P) is associated with pathological calcium leakage and cognitive disorders, including Alzheimer's disease. By comparison, little is known about the role of RyR2 in the ventral medial prefrontal cortex (vmPFC) circuitry important for working memory, decision making, and reward seeking. Here, we evaluated the basal expression and localization of RyR2 and RyR2P in the vmPFC. Next, we employed an operant model of sucrose, cocaine, or morphine self-administration (SA) followed by a (reward-free) recall test, to reengage vmPFC neurons and reactivate reward-seeking and re-evaluated the expression and localization of RyR2 and RyR2P in vmPFC. Under basal conditions, RyR2 was expressed in pyramidal cells but not regularly detected in PV/SST interneurons. On the contrary, RyR2P was rarely observed in PFC somata and was restricted to a different subcompartment of the same neuron - the apical dendrites of layer-5 pyramidal cells. Chronic SA of drug (cocaine or morphine) and nondrug (sucrose) rewards produced comparable increases in RyR2 protein expression. However, recalling either drug reward impaired the usual localization of RyR2P in dendrites and markedly increased its expression in somata immunoreactive for Fos, a marker of highly activated neurons. These effects could not be explained by chronic stress or drug withdrawal and instead appeared to require a recall experience associated with prior drug SA. In addition to showing the differential distribution of RyR2/RyR2P and affirming the general role of vmPFC in reward learning, this study provides information on the propensity of addictive drugs to redistribute RyR2P ion channels in a neuronal population engaged in drug-seeking. Hence, focusing on the early impact of addictive drugs on RyR2 function may serve as a promising approach to finding a treatment for substance use disorders.
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Golatkar V, Bhatt LK. mAKAPβ signalosome: A potential target for cardiac hypertrophy. Drug Dev Res 2023; 84:1072-1084. [PMID: 37203301 DOI: 10.1002/ddr.22081] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2022] [Revised: 03/05/2023] [Accepted: 05/06/2023] [Indexed: 05/20/2023]
Abstract
Pathological cardiac hypertrophy is the result of a prolonged increase in the workload of the heart that activates various signaling pathways such as MAPK pathway, PKA-dependent cAMP signaling, and CaN-NFAT signaling pathway which further activates genes for cardiac remodeling. Various signalosomes are present in the heart that regulates the signaling of physiological and pathological cardiac hypertrophy. mAKAPβ is one such scaffold protein that regulates signaling pathways involved in promoting cardiac hypertrophy. It is present in the outer nuclear envelope of the cardiomyocytes, which provides specificity of the target toward the heart. In addition, nuclear translocation of signaling components and transcription factors such as MEF2D, NFATc, and HIF-1α is facilitated due to the localization of mAKAPβ near the nuclear envelope. These factors are required for activation of genes promoting cardiac remodeling. Downregulation of mAKAPβ improves cardiac function and attenuates cardiac hypertrophy which in turn prevents the development of heart failure. Unlike earlier therapies for heart failure, knockout or silencing of mAKAPβ is not associated with side effects because of its high specificity in the striated myocytes. Downregulating expression of mAKAPβ is a favorable therapeutic approach toward attenuating cardiac hypertrophy and hence preventing heart failure. This review discusses mAKAPβ signalosome as a potential target for cardiac hypertrophy intervention.
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Affiliation(s)
- Vaishnavi Golatkar
- Department of Pharmacology, SVKM's Dr. Bhanuben Nanavati College of Pharmacy, Vile Parle (W), Mumbai, India
| | - Lokesh K Bhatt
- Department of Pharmacology, SVKM's Dr. Bhanuben Nanavati College of Pharmacy, Vile Parle (W), Mumbai, India
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Dries E, Gilbert G, Roderick HL, Sipido KR. The ryanodine receptor microdomain in cardiomyocytes. Cell Calcium 2023; 114:102769. [PMID: 37390591 DOI: 10.1016/j.ceca.2023.102769] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2023] [Revised: 06/11/2023] [Accepted: 06/12/2023] [Indexed: 07/02/2023]
Abstract
The ryanodine receptor type 2 (RyR) is a key player in Ca2+ handling during excitation-contraction coupling. During each heartbeat, RyR channels are responsible for linking the action potential with the contractile machinery of the cardiomyocyte by releasing Ca2+ from the sarcoplasmic reticulum. RyR function is fine-tuned by associated signalling molecules, arrangement in clusters and subcellular localization. These parameters together define RyR function within microdomains and are subject to disease remodelling. This review describes the latest findings on RyR microdomain organization, the alterations with disease which result in increased subcellular heterogeneity and emergence of microdomains with enhanced arrhythmogenic potential, and presents novel technologies that guide future research to study and target RyR channels within specific microdomains.
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Affiliation(s)
- Eef Dries
- Lab of Experimental Cardiology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium.
| | - Guillaume Gilbert
- Lab of Experimental Cardiology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium; Laboratoire ORPHY EA 4324, Université de Brest, Brest, France
| | - H Llewelyn Roderick
- Lab of Experimental Cardiology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium
| | - Karin R Sipido
- Lab of Experimental Cardiology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium
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Wei J, Guo W, Wang R, Paul Estillore J, Belke D, Chen YX, Vallmitjana A, Benitez R, Hove-Madsen L, Chen SRW. RyR2 Serine-2030 PKA Site Governs Ca 2+ Release Termination and Ca 2+ Alternans. Circ Res 2023; 132:e59-e77. [PMID: 36583384 DOI: 10.1161/circresaha.122.321177] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
BACKGROUND PKA (protein kinase A)-mediated phosphorylation of cardiac RyR2 (ryanodine receptor 2) has been extensively studied for decades, but the physiological significance of PKA phosphorylation of RyR2 remains poorly understood. Recent determination of high-resolution 3-dimensional structure of RyR2 in complex with CaM (calmodulin) reveals that the major PKA phosphorylation site in RyR2, serine-2030 (S2030), is located within a structural pathway of CaM-dependent inactivation of RyR2. This novel structural insight points to a possible role of PKA phosphorylation of RyR2 in CaM-dependent inactivation of RyR2, which underlies the termination of Ca2+ release and induction of cardiac Ca2+ alternans. METHODS We performed single-cell endoplasmic reticulum Ca2+ imaging to assess the impact of S2030 mutations on Ca2+ release termination in human embryonic kidney 293 cells. Here we determined the role of the PKA site RyR2-S2030 in a physiological setting, we generated a novel mouse model harboring the S2030L mutation and carried out confocal Ca2+ imaging. RESULTS We found that mutations, S2030D, S2030G, S2030L, S2030V, and S2030W reduced the endoplasmic reticulum luminal Ca2+ level at which Ca2+ release terminates (the termination threshold), whereas S2030P and S2030R increased the termination threshold. S2030A and S2030T had no significant impact on release termination. Furthermore, CaM-wild-type increased, whereas Ca2+ binding deficient CaM mutant (CaM-M [a loss-of-function CaM mutation with all 4 EF-hand motifs mutated]), PKA, and Ca2+/CaMKII (CaM-dependent protein kinase II) reduced the termination threshold. The S2030L mutation abolished the actions of CaM-wild-type, CaM-M, and PKA, but not CaMKII, in Ca2+ release termination. Moreover, we showed that isoproterenol and CaM-M suppressed pacing-induced Ca2+ alternans and accelerated Ca2+ transient recovery in intact working hearts, whereas CaM-wild-type exerted an opposite effect. The impact of isoproterenol was partially and fully reversed by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide and the CaMKII inhibitor N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide individually and together, respectively. S2030L abolished the impact of CaM-wild-type, CaM-M, and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide-sensitive component, but not the N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide-sensitive component, of isoproterenol.
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Affiliation(s)
- Jinhong Wei
- Department of Physiology and Pharmacology, Libin Cardiovascular Institute, University of Calgary, Alberta T2N 4N1, Canada (J.W., W.G., R.W., J.P.E., D.B., Y.-X.C., S.R.W.C.).,School of Medicine, Northwest University, Xi 'an, China (J.W.)
| | - Wenting Guo
- Department of Physiology and Pharmacology, Libin Cardiovascular Institute, University of Calgary, Alberta T2N 4N1, Canada (J.W., W.G., R.W., J.P.E., D.B., Y.-X.C., S.R.W.C.)
| | - Ruiwu Wang
- Department of Physiology and Pharmacology, Libin Cardiovascular Institute, University of Calgary, Alberta T2N 4N1, Canada (J.W., W.G., R.W., J.P.E., D.B., Y.-X.C., S.R.W.C.)
| | - John Paul Estillore
- Department of Physiology and Pharmacology, Libin Cardiovascular Institute, University of Calgary, Alberta T2N 4N1, Canada (J.W., W.G., R.W., J.P.E., D.B., Y.-X.C., S.R.W.C.)
| | - Darrell Belke
- Department of Physiology and Pharmacology, Libin Cardiovascular Institute, University of Calgary, Alberta T2N 4N1, Canada (J.W., W.G., R.W., J.P.E., D.B., Y.-X.C., S.R.W.C.)
| | - Yong-Xiang Chen
- Department of Physiology and Pharmacology, Libin Cardiovascular Institute, University of Calgary, Alberta T2N 4N1, Canada (J.W., W.G., R.W., J.P.E., D.B., Y.-X.C., S.R.W.C.)
| | | | - Raul Benitez
- Department of Automatic Control, Universitat Politècnica de Catalunya, 08034 Barcelona, Spain (A.V., R.B.)
| | - Leif Hove-Madsen
- Biomedical Research Institute Barcelona IIBB-CSIC, IIB Sant Pau and CIBERCV, Hospital de Sant Pau, 08025, Barcelona, Spain (L.H.-M.)
| | - S R Wayne Chen
- Department of Physiology and Pharmacology, Libin Cardiovascular Institute, University of Calgary, Alberta T2N 4N1, Canada (J.W., W.G., R.W., J.P.E., D.B., Y.-X.C., S.R.W.C.)
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9
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Chai-Hu-San-Shen Capsule Ameliorates Ventricular Arrhythmia Through Inhibition of the CaMKII/FKBP12.6/RyR2/Ca 2+ Signaling Pathway in Rats with Myocardial Ischemia. EVIDENCE-BASED COMPLEMENTARY AND ALTERNATIVE MEDICINE 2022; 2022:2670473. [PMID: 36225189 PMCID: PMC9550443 DOI: 10.1155/2022/2670473] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/07/2022] [Accepted: 08/18/2022] [Indexed: 11/18/2022]
Abstract
Ventricular arrhythmia is one of the main causes of sudden cardiac death, especially after myocardial ischemia. Previous studies have shown that Chai-Hu-San-Shen capsule (CHSSC) can reduce the incidence of ventricular arrhythmias following myocardial ischemia, however, the mechanisms of it are unclear. In present study, we explored the mechanism of CHSSC ameliorates ventricular arrhythmia following myocardial ischemia via inhibiting the CaMKII/FKBP12.6/RyR2/Ca2+ signaling pathway. In vivo, a myocardial ischemia rat model was established and treated with CHSSC to evaluate the therapeutic effect of CHSSC. In vitro, we established an ischemia model in H9C2 cells and treated with CHSSC, KN-93, or H-89. Then, intracellular Ca2+ content, the expression of RyR2, and the interaction between FKBP12.6 and RyR2 were detected. The results showed that CHSSC could delay the occurrence of ventricular arrhythmias and shorten the duration of ventricular arrhythmias. After myocardial ischemia, the intracellular Ca2+ content was increased, and CHSSC treatment mitigated this increase, down-regulated the levels of p-CaMKII, CaMKII, p-RyR2, and RyR2, and up-regulated the levels of p-RyR2 (Ser2808) and p-RyR2 (Ser2814). Co-immunoprecipitation showed an interaction between FKBP12.6 and RyR2, and CHSSC up-regulated the content of the FKBP12.6-RyR2 complex in ischemic cells. In conclusion, our study showed that CaMKII activation led to hyperphosphorylation of RyR2 (Ser2814) and RyR2 (Ser2808) during cardiomyocyte ischemia, which resulted in dissociation of the FKBP12.6-RyR2 complex, and increased intracellular Ca2+ content, which may contribute to the development of ventricular arrhythmias. CHSSC may reduce the incidence of ventricular arrhythmias following myocardial ischemia through inhibition of the CaMKII/RyR2/FKBP12.6/Ca2+ signaling pathway.
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10
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Zheng J, Dooge HC, Pérez-Hernández M, Zhao YT, Chen X, Hernandez JJ, Valdivia CR, Palomeque J, Rothenberg E, Delmar M, Valdivia HH, Alvarado FJ. Preserved cardiac performance and adrenergic response in a rabbit model with decreased ryanodine receptor 2 expression. J Mol Cell Cardiol 2022; 167:118-128. [PMID: 35413295 PMCID: PMC9610860 DOI: 10.1016/j.yjmcc.2022.04.004] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/20/2021] [Revised: 03/11/2022] [Accepted: 04/06/2022] [Indexed: 11/19/2022]
Abstract
Ryanodine receptor 2 (RyR2) is an ion channel in the heart responsible for releasing into the cytosol most of the Ca2+ required for contraction. Proper regulation of RyR2 is critical, as highlighted by the association between channel dysfunction and cardiac arrhythmia. Lower RyR2 expression is also observed in some forms of heart disease; however, there is limited information on the impact of this change on excitation-contraction (e-c) coupling, Ca2+-dependent arrhythmias, and cardiac performance. We used a constitutive knock-out of RyR2 in rabbits (RyR2-KO) to assess the extent to which a stable decrease in RyR2 expression modulates Ca2+ handling in the heart. We found that homozygous knock-out of RyR2 in rabbits is embryonic lethal. Remarkably, heterozygotes (KO+/-) show ~50% loss of RyR2 protein without developing an overt phenotype at the intact animal and whole heart levels. Instead, we found that KO+/- myocytes show (1) remodeling of RyR2 clusters, favoring smaller groups in which channels are more densely arranged; (2) lower Ca2+ spark frequency and amplitude; (3) slower rate of Ca2+ release and mild but significant desynchronization of the Ca2+ transient; and (4) a significant decrease in the basal phosphorylation of S2031, likely due to increased association between RyR2 and PP2A. Our data show that RyR2 deficiency, although remarkable at the molecular and subcellular level, has only a modest impact on global Ca2+ release and is fully compensated at the whole-heart level. This highlights the redundancy of RyR2 protein expression and the plasticity of the e-c coupling apparatus.
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Affiliation(s)
- Jingjing Zheng
- Department of Medicine and Cardiovascular Research Center, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI, USA; Department of Forensic Pathology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China
| | - Holly C Dooge
- Department of Medicine and Cardiovascular Research Center, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI, USA
| | - Marta Pérez-Hernández
- Leon H Charney Division of Cardiology, New York University Grossman School of Medicine,. New York, NY, United States of America
| | - Yan-Ting Zhao
- Center for Arrhythmia Research, University of Michigan, Ann Arbor, MI, United States of America
| | - Xi Chen
- Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, United States of America
| | - Jonathan J Hernandez
- Department of Pediatrics, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI, United States of America
| | - Carmen R Valdivia
- Department of Medicine and Cardiovascular Research Center, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI, USA
| | - Julieta Palomeque
- Centro de Investigaciones Cardiovasculares, CCT-La Plata-CONICET, Facultad de Ciencias Médicas, UNLP, La Plata, Argentina
| | - Eli Rothenberg
- Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, New York University Grossman School of Medicine, New York, NY, United States of America
| | - Mario Delmar
- Leon H Charney Division of Cardiology, New York University Grossman School of Medicine,. New York, NY, United States of America
| | - Héctor H Valdivia
- Department of Medicine and Cardiovascular Research Center, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI, USA
| | - Francisco J Alvarado
- Department of Medicine and Cardiovascular Research Center, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI, USA.
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11
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Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase G. Curr Res Physiol 2022; 5:171-178. [PMID: 35356048 PMCID: PMC8958330 DOI: 10.1016/j.crphys.2022.03.004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2021] [Revised: 02/20/2022] [Accepted: 03/21/2022] [Indexed: 11/21/2022] Open
Abstract
Background The cGMP-dependent protein kinase G (PKG) phosphorylates the cardiac ryanodine receptor (RyR2) in vitro. We aimed to determine whether modulation of endogenous PKG alters RyR2-mediated spontaneous Ca2+ release and whether this effect is linked to a change in RyR2 phosphorylation. Methods & Results: Human embryonic kidney (HEK293) cells with inducible RyR2 expression were treated with the cGMP analogue 8-Br-cGMP (100 μM) to activate endogenous PKG. In cells transfected with luminal Ca2+ sensor, D1ER, PKG activation significantly reduced the threshold for RyR2-mediated spontaneous Ca2+ release (93.9 ± 0.4% of store size with vehicle vs. 91.7 ± 0.8% with 8-Br-cGMP, P = 0.04). Mutation of the proposed PKG phosphorylation sites, S2808 and S2030, either individually or as a combination, prevented the decrease in Ca2+ release threshold induced by endogenous PKG activation. Interestingly, despite a functional dependence on expression of RyR2 phosphorylation sites, 8-Br-cGMP activation of PKG did not promote a detectable change in S2808 phosphorylation (P = 0.9). Paradoxically, pharmacological inhibition of PKG with KT 5823 (1 μM) also reduced the threshold for spontaneous Ca2+ release through RyR2 without affecting S2808 phosphorylation. Silencing RNA knockdown of endogenous PKG expression also had no quantifiable effect on RyR2 S2808 phosphorylation (P = 0.9). However, unlike PKG inhibition with KT 5823, PKG knockdown did not alter spontaneous Ca2+ release propensity or luminal Ca2+ handling. Conclusion In an intact cell model, activation of endogenous PKG reduces the threshold for RyR2-mediated spontaneous Ca2+ release in a manner dependent on the RyR2 phosphorylation sites S2808 and S2030. This study clarifies the regulation of RyR2 Ca2+ release by endogenous PKG and functionally implicates the role of RyR2 phosphorylation.
PKG regulation of RyR2 has been under-researched relative to other kinases. Endogenous PKG activation reduced the threshold for spontaneous RyR2 Ca2+ release. Regulation of RyR2 by PKG required expression of serine residues 2808 and 2030. Knockdown of PKG found minimal basal regulation of RyR2 by PKG.
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12
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The function and regulation of calsequestrin-2: implications in calcium-mediated arrhythmias. Biophys Rev 2022; 14:329-352. [PMID: 35340602 PMCID: PMC8921388 DOI: 10.1007/s12551-021-00914-6] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2021] [Accepted: 11/14/2021] [Indexed: 01/09/2023] Open
Abstract
Cardiac arrhythmias are life-threatening events in which the heart develops an irregular rhythm. Mishandling of Ca2+ within the myocytes of the heart has been widely demonstrated to be an underlying mechanism of arrhythmogenesis. This includes altered function of the ryanodine receptor (RyR2)-the primary Ca2+ release channel located to the sarcoplasmic reticulum (SR). The spontaneous leak of SR Ca2+ via RyR2 is a well-established contributor in the development of arrhythmic contractions. This leak is associated with increased channel activity in response to changes in SR Ca2+ load. RyR2 activity can be regulated through several avenues, including interactions with numerous accessory proteins. One such protein is calsequestrin-2 (CSQ2), which is the primary Ca2+-buffering protein within the SR. The capacity of CSQ2 to buffer Ca2+ is tightly associated with the ability of the protein to polymerise in response to changing Ca2+ levels. CSQ2 can itself be regulated through phosphorylation and glycosylation modifications, which impact protein polymerisation and trafficking. Changes in CSQ2 modifications are implicated in cardiac pathologies, while mutations in CSQ2 have been identified in arrhythmic patients. Here, we review the role of CSQ2 in arrhythmogenesis including evidence for the indirect and direct regulation of RyR2 by CSQ2, and the consequences of a loss of functional CSQ2 in Ca2+ homeostasis and Ca2+-mediated arrhythmias. Supplementary Information The online version contains supplementary material available at 10.1007/s12551-021-00914-6.
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13
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Arslanova A, Shafaattalab S, Ye K, Asghari P, Lin L, Kim B, Roston TM, Hove-Madsen L, Van Petegem F, Sanatani S, Moore E, Lynn F, Søndergaard M, Luo Y, Chen SRW, Tibbits GF. Using hiPSC-CMs to Examine Mechanisms of Catecholaminergic Polymorphic Ventricular Tachycardia. Curr Protoc 2021; 1:e320. [PMID: 34958715 DOI: 10.1002/cpz1.320] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal inherited cardiac arrhythmia condition, triggered by physical or acute emotional stress, that predominantly expresses early in life. Gain-of-function mutations in the cardiac ryanodine receptor gene (RYR2) account for the majority of CPVT cases, causing substantial disruption of intracellular calcium (Ca2+ ) homeostasis particularly during the periods of β-adrenergic receptor stimulation. However, the highly variable penetrance, patient outcomes, and drug responses observed in clinical practice remain unexplained, even for patients with well-established founder RyR2 mutations. Therefore, investigation of the electrophysiological consequences of CPVT-causing RyR2 mutations is crucial to better understand the pathophysiology of the disease. The development of strategies for reprogramming human somatic cells to human induced pluripotent stem cells (hiPSCs) has provided a unique opportunity to study inherited arrhythmias, due to the ability of hiPSCs to differentiate down a cardiac lineage. Employment of genome editing enables generation of disease-specific cell lines from healthy and diseased patient-derived hiPSCs, which subsequently can be differentiated into cardiomyocytes. This paper describes the means for establishing an hiPSC-based model of CPVT in order to recapitulate the disease phenotype in vitro and investigate underlying pathophysiological mechanisms. The framework of this approach has the potential to contribute to disease modeling and personalized medicine using hiPSC-derived cardiomyocytes. © 2021 Wiley Periodicals LLC.
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Affiliation(s)
- Alia Arslanova
- Cellular and Regenerative Medicine Centre, British Columbia Children's Hospital Research Institute, Vancouver, British Columbia, Canada.,Molecular Cardiac Physiology Group, Departments of Biomedical Physiology and Kinesiology and Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada
| | - Sanam Shafaattalab
- Cellular and Regenerative Medicine Centre, British Columbia Children's Hospital Research Institute, Vancouver, British Columbia, Canada.,Molecular Cardiac Physiology Group, Departments of Biomedical Physiology and Kinesiology and Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada
| | - Kevin Ye
- Cellular and Regenerative Medicine Centre, British Columbia Children's Hospital Research Institute, Vancouver, British Columbia, Canada.,Molecular Cardiac Physiology Group, Departments of Biomedical Physiology and Kinesiology and Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada
| | - Parisa Asghari
- Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada
| | - Lisa Lin
- Cellular and Regenerative Medicine Centre, British Columbia Children's Hospital Research Institute, Vancouver, British Columbia, Canada.,Molecular Cardiac Physiology Group, Departments of Biomedical Physiology and Kinesiology and Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada
| | - BaRun Kim
- Molecular Cardiac Physiology Group, Departments of Biomedical Physiology and Kinesiology and Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada
| | - Thomas M Roston
- British Columbia Children's Hospital Heart Center, Vancouver, British Columbia, Canada
| | - Leif Hove-Madsen
- Cardiac Rhythm and Contraction Group, IIBB-CSIC, CIBERCV, IIB Sant Pau, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
| | - Filip Van Petegem
- Department of Biochemistry and Molecular Biology, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada
| | - Shubhayan Sanatani
- British Columbia Children's Hospital Heart Center, Vancouver, British Columbia, Canada
| | - Edwin Moore
- Cardiac Rhythm and Contraction Group, IIBB-CSIC, CIBERCV, IIB Sant Pau, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
| | - Francis Lynn
- Cellular and Regenerative Medicine Centre, British Columbia Children's Hospital Research Institute, Vancouver, British Columbia, Canada
| | | | - Yonglun Luo
- Department of Biomedicine, Aarhus University, Aarhus, Denmark
| | - S R Wayne Chen
- Department of Physiology and Pharmacology, Libin Cardiovascular Institute, University of Calgary, Calgary, Alberta, Canada
| | - Glen F Tibbits
- Cellular and Regenerative Medicine Centre, British Columbia Children's Hospital Research Institute, Vancouver, British Columbia, Canada.,Molecular Cardiac Physiology Group, Departments of Biomedical Physiology and Kinesiology and Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.,School of Biomedical Engineering, University of British Columbia, Vancouver, British Columbia, Canada
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14
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Sergienko NM, Donner DG, Delbridge LMD, McMullen JR, Weeks KL. Protein phosphatase 2A in the healthy and failing heart: New insights and therapeutic opportunities. Cell Signal 2021; 91:110213. [PMID: 34902541 DOI: 10.1016/j.cellsig.2021.110213] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2021] [Revised: 12/02/2021] [Accepted: 12/07/2021] [Indexed: 02/06/2023]
Abstract
Protein phosphatases have emerged as critical regulators of phosphoprotein homeostasis in settings of health and disease. Protein phosphatase 2A (PP2A) encompasses a large subfamily of enzymes that remove phosphate groups from serine/threonine residues within phosphoproteins. The heterogeneity in PP2A structure, which arises from the grouping of different catalytic, scaffolding and regulatory subunit isoforms, creates distinct populations of catalytically active enzymes (i.e. holoenzymes) that localise to different parts of the cell. This structural complexity, combined with other regulatory mechanisms, such as interaction of PP2A heterotrimers with accessory proteins and post-translational modification of the catalytic and/or regulatory subunits, enables PP2A holoenzymes to target phosphoprotein substrates in a highly specific manner. In this review, we summarise the roles of PP2A in cardiac physiology and disease. PP2A modulates numerous processes that are vital for heart function including calcium handling, contractility, β-adrenergic signalling, metabolism and transcription. Dysregulation of PP2A has been observed in human cardiac disease settings, including heart failure and atrial fibrillation. Efforts are underway, particularly in the cancer field, to develop therapeutics targeting PP2A activity. The development of small molecule activators of PP2A (SMAPs) and other compounds that selectively target specific PP2A holoenzymes (e.g. PP2A/B56α and PP2A/B56ε) will improve understanding of the function of different PP2A species in the heart, and may lead to the development of therapeutics for normalising aberrant protein phosphorylation in settings of cardiac remodelling and dysfunction.
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Affiliation(s)
- Nicola M Sergienko
- Baker Heart and Diabetes Institute, Melbourne VIC 3004, Australia; Central Clinical School, Monash University, Clayton VIC 3800, Australia
| | - Daniel G Donner
- Baker Heart and Diabetes Institute, Melbourne VIC 3004, Australia; Baker Department of Cardiometabolic Health, The University of Melbourne, Parkville VIC 3010, Australia
| | - Lea M D Delbridge
- Department of Anatomy and Physiology, The University of Melbourne, Parkville VIC 3010, Australia
| | - Julie R McMullen
- Baker Heart and Diabetes Institute, Melbourne VIC 3004, Australia; Baker Department of Cardiometabolic Health, The University of Melbourne, Parkville VIC 3010, Australia; Department of Physiology and Department of Medicine Alfred Hospital, Monash University, Clayton VIC 3800, Australia; Department of Physiology, Anatomy and Microbiology, La Trobe University, Bundoora VIC 3086, Australia; Department of Diabetes, Central Clinical School, Monash University, Clayton VIC 3800, Australia.
| | - Kate L Weeks
- Baker Heart and Diabetes Institute, Melbourne VIC 3004, Australia; Department of Anatomy and Physiology, The University of Melbourne, Parkville VIC 3010, Australia; Baker Department of Cardiometabolic Health, The University of Melbourne, Parkville VIC 3010, Australia; Department of Diabetes, Central Clinical School, Monash University, Clayton VIC 3800, Australia.
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15
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Gaitán-González P, Sánchez-Hernández R, Arias-Montaño JA, Rueda A. Tale of two kinases: Protein kinase A and Ca 2+/calmodulin-dependent protein kinase II in pre-diabetic cardiomyopathy. World J Diabetes 2021; 12:1704-1718. [PMID: 34754372 PMCID: PMC8554373 DOI: 10.4239/wjd.v12.i10.1704] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/25/2021] [Revised: 07/28/2021] [Accepted: 08/30/2021] [Indexed: 02/06/2023] Open
Abstract
Metabolic syndrome is a pre-diabetic state characterized by several biochemical and physiological alterations, including insulin resistance, visceral fat accumulation, and dyslipidemias, which increase the risk for developing cardiovascular disease. Metabolic syndrome is associated with augmented sympathetic tone, which could account for the etiology of pre-diabetic cardiomyopathy. This review summarizes the current knowledge of the pathophysiological consequences of enhanced and sustained β-adrenergic response in pre-diabetes, focusing on cardiac dysfunction reported in diet-induced experimental models of pre-diabetic cardiomyopathy. The research reviewed indicates that both protein kinase A and Ca2+/calmodulin-dependent protein kinase II play important roles in functional responses mediated by β1-adrenoceptors; therefore, alterations in the expression or function of these kinases can be deleterious. This review also outlines recent information on the role of protein kinase A and Ca2+/calmodulin-dependent protein kinase II in abnormal Ca2+ handling by cardiomyocytes from diet-induced models of pre-diabetic cardiomyopathy.
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Affiliation(s)
- Pamela Gaitán-González
- Department of Biochemistry, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Ciudad de México 07360, Mexico
| | - Rommel Sánchez-Hernández
- Department of Physiology, Biophysics and Neurosciences, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Ciudad de México 07360, Mexico
| | - José-Antonio Arias-Montaño
- Department of Physiology, Biophysics and Neurosciences, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Ciudad de México 07360, Mexico
| | - Angélica Rueda
- Department of Biochemistry, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Ciudad de México 07360, Mexico
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16
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Desch K, Langer JD, Schuman EM. Dynamic bi-directional phosphorylation events associated with the reciprocal regulation of synapses during homeostatic up- and down-scaling. Cell Rep 2021; 36:109583. [PMID: 34433048 PMCID: PMC8411114 DOI: 10.1016/j.celrep.2021.109583] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2021] [Revised: 06/15/2021] [Accepted: 07/29/2021] [Indexed: 01/17/2023] Open
Abstract
Homeostatic synaptic scaling allows for bi-directional adjustment of the strength of synaptic connections in response to changes in their input. Protein phosphorylation modulates many neuronal processes, but it has not been studied on a global scale during synaptic scaling. Here, we use liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses to measure changes in the phosphoproteome in response to up- or down-scaling in cultured cortical neurons over minutes to 24 h. Of ~45,000 phosphorylation events, ~3,300 (associated with 1,285 phosphoproteins) are regulated by homeostatic scaling. Activity-sensitive phosphoproteins are predominantly located at synapses and involved in cytoskeletal reorganization. We identify many early phosphorylation events that could serve as sensors for the activity offset as well as late and/or persistent phosphoregulation that could represent effector mechanisms driving the homeostatic response. Much of the persistent phosphorylation is reciprocally regulated by up- or down-scaling, suggesting that mechanisms underlying these two poles of synaptic regulation make use of a common signaling axis.
Global proteome and phosphoproteome dynamics following homeostatic synaptic scaling Approximately 3,300 activity-sensitive, synapse-associated phospho-events Persistent signaling of ~25% of initial phospho-events (min to 24 h) Persistent and reciprocal phosphoregulation links synaptic up- and down-scaling
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Affiliation(s)
- Kristina Desch
- Max Planck Institute for Brain Research, Max von Laue Strasse 4, 60438 Frankfurt, Germany
| | - Julian D Langer
- Max Planck Institute for Brain Research, Max von Laue Strasse 4, 60438 Frankfurt, Germany.
| | - Erin M Schuman
- Max Planck Institute for Brain Research, Max von Laue Strasse 4, 60438 Frankfurt, Germany.
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17
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Woll KA, Van Petegem F. Calcium Release Channels: Structure and Function of IP3 Receptors and Ryanodine Receptors. Physiol Rev 2021; 102:209-268. [PMID: 34280054 DOI: 10.1152/physrev.00033.2020] [Citation(s) in RCA: 130] [Impact Index Per Article: 32.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Ca2+-release channels are giant membrane proteins that control the release of Ca2+ from the endoplasmic and sarcoplasmic reticulum. The two members, ryanodine receptors (RyRs) and inositol-1,4,5-trisphosphate Receptors (IP3Rs), are evolutionarily related and are both activated by cytosolic Ca2+. They share a common architecture, but RyRs have evolved additional modules in the cytosolic region. Their massive size allows for the regulation by tens of proteins and small molecules, which can affect the opening and closing of the channels. In addition to Ca2+, other major triggers include IP3 for the IP3Rs, and depolarization of the plasma membrane for a particular RyR subtype. Their size has made them popular targets for study via electron microscopic methods, with current structures culminating near 3Å. The available structures have provided many new mechanistic insights int the binding of auxiliary proteins and small molecules, how these can regulate channel opening, and the mechanisms of disease-associated mutations. They also help scrutinize previously proposed binding sites, as some of these are now incompatible with the structures. Many questions remain around the structural effects of post-translational modifications, additional binding partners, and the higher-order complexes these channels can make in situ. This review summarizes our current knowledge about the structures of Ca2+-release channels and how this informs on their function.
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Affiliation(s)
- Kellie A Woll
- Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada
| | - Filip Van Petegem
- Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada
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18
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Paterek A, Oknińska M, Chajduk E, Polkowska-Motrenko H, Mączewski M, Mackiewicz U. Systemic iron deficiency does not affect the cardiac iron content and progression of heart failure. J Mol Cell Cardiol 2021; 159:16-27. [PMID: 34139233 DOI: 10.1016/j.yjmcc.2021.06.005] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/09/2021] [Revised: 05/18/2021] [Accepted: 06/08/2021] [Indexed: 10/21/2022]
Abstract
Chronic heart failure (HF) is often accompanied by systemic iron deficiency (ID). However, effects of ID on cardiac iron status and progression of HF are unknown. To investigate these effects rats underwent LAD ligation to induce post-myocardial infarction HF or sham operation. After 3 weeks the animals from both groups were randomized into three subgroups: control, moderate ID and severe ID+anemia (IDA) by a combination of phlebotomy and low iron diet for 5 weeks. Serum and hepatic iron content were reduced by 55% and 70% (ID) and by 80% and 77% (IDA), respectively, while cardiac iron content was unchanged in HF rats. Changes in expression of all cardiomyocyte iron handling proteins indicating preserved cardiomyocytes iron status in HF and ID/IDA. Contractile function of LV cardiomyocytes, Ca2+ transient amplitude, sarcoplasmic reticulum Ca2+ release and SERCA2a function was augmented by ID and IDA and it was accompanied by an increase in serum catecholamines. Neither ID nor IDA affected left ventricular (LV) systolic or diastolic function or dimensions. To sum up, systemic ID does not result in cardiac ID and does not affect progression of HF and even improves contractile function and Ca2+ handling of isolated LV cardiomyocytes, however, at the cost of increased catecholamine level. This suggests that intravenous iron therapy should be considered as an additional therapeutic option in HF, preventing the increase of catecholaminergic drive with its well-known long-term adverse effects.
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Affiliation(s)
- Aleksandra Paterek
- Department of Clinical Physiology, Centre of Postgraduate Medical Education, Warsaw, Poland
| | - Marta Oknińska
- Department of Clinical Physiology, Centre of Postgraduate Medical Education, Warsaw, Poland
| | - Ewelina Chajduk
- Centre for Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology, Warsaw, Poland
| | - Halina Polkowska-Motrenko
- Centre for Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology, Warsaw, Poland
| | - Michał Mączewski
- Department of Clinical Physiology, Centre of Postgraduate Medical Education, Warsaw, Poland
| | - Urszula Mackiewicz
- Department of Clinical Physiology, Centre of Postgraduate Medical Education, Warsaw, Poland.
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19
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Complex functionality of protein phosphatase 1 isoforms in the heart. Cell Signal 2021; 85:110059. [PMID: 34062239 DOI: 10.1016/j.cellsig.2021.110059] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2021] [Revised: 05/21/2021] [Accepted: 05/28/2021] [Indexed: 02/04/2023]
Abstract
Protein phosphatase 1(PP1) is a key regulator of cardiac function through dephosphorylating serine/threonine residues within target proteins to oppose the function of protein kinases. Studies from failing hearts of animal models and human patients have demonstrated significant increase of PP1 activity in myocardium, while elevated PP1 activity in transgenic mice leads to cardiac dysfunction, suggesting that PP1 might be a therapeutic target to ameliorate cardiac dysfunction in failing hearts. In fact, cardiac overexpression of inhibitor 1, the endogenous inhibitor of PP1, increases cardiac contractility and suppresses heart failure progression. However, this notion of PP1 inhibition for heart failure treatment has been challenged by recent studies on the isoform-specific roles of PP1 in the heart. PP1 is a holoenzyme composed of catalytic subunits (PP1α, PP1β, or PP1γ) and regulatory proteins that target them to distinct subcellular locations for functional specificity. This review will summarize how PP1 regulates phosphorylation of some of the key cardiac proteins involved in Ca2+ handling and cardiac contraction, and the potential role of PP1 isoforms in controlling cardiac physiology and pathophysiology.
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20
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Safabakhsh S, Panwar P, Barichello S, Sangha SS, Hanson PJ, Van Petegem F, Laksman Z. THE ROLE OF PHOSPHORYLATION IN ATRIAL FIBRILLATION: A FOCUS ON MASS SPECTROMETRY APPROACHES. Cardiovasc Res 2021; 118:1205-1217. [PMID: 33744917 DOI: 10.1093/cvr/cvab095] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/18/2020] [Accepted: 03/16/2021] [Indexed: 11/14/2022] Open
Abstract
Atrial fibrillation (AF) is the most common arrhythmia worldwide. It is associated with significant increases in morbidity in the form of stroke and heart failure, and a doubling in all-cause mortality. The pathophysiology of AF is incompletely understood, and this has contributed to a lack of effective treatments and disease-modifying therapies. An important cellular process that may explain how risk factors give rise to AF includes post-translational modification (PTM) of proteins. As the most commonly occurring PTM, protein phosphorylation is especially relevant. Although many methods exist for studying protein phosphorylation, a common and highly resolute technique is mass spectrometry (MS). This review will discuss recent evidence surrounding the role of protein phosphorylation in the pathogenesis of AF. MS-based technology to study phosphorylation and uses of MS in other areas of medicine such as oncology will also be presented. Based on these data, future goals and experiments will be outlined that utilize MS technology to better understand the role of phosphorylation in AF and elucidate its role in AF pathophysiology. This may ultimately allow for the development of more effective AF therapies.
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Affiliation(s)
- Sina Safabakhsh
- Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada
| | - Pankaj Panwar
- AbCellera Biologicals Inc., Vancouver, British Columbia, Canada
| | - Scott Barichello
- Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada
| | - Sarabjit S Sangha
- Cellular and Regenerative Medicine Centre, BC Children's Hospital Research Institute, 950 West 28th Avenue, Vancouver, British Columbia, Canada.,Molecular Cardiac Physiology Group, Departments of Biomedical Physiology and Kinesiology and Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, Canada
| | - Paul J Hanson
- UBC Heart Lung Innovation Centre, Vancouver, British Columbia, Canada.,UBC Department of Pathology and Laboratory Medicine, Vancouver, British Columbia, Canada
| | - Filip Van Petegem
- Department of Biochemistry and Molecular Biology, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada
| | - Zachary Laksman
- Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada
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21
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Wu Y, Zhao W, Ye F, Huang S, Chen H, Zhou R, Jiang W. Tetrandrine attenuates left ventricular dysfunction in rats with myocardial infarction. Exp Ther Med 2020; 21:119. [PMID: 33335582 PMCID: PMC7739846 DOI: 10.3892/etm.2020.9551] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2020] [Accepted: 09/16/2020] [Indexed: 12/01/2022] Open
Abstract
The present study aimed to determine whether tetrandrine could attenuate left ventricular dysfunction and remodeling in rats with myocardial infarction. Sprague-Dawley rats were randomly divided into six groups (n=5/group) as follows: i) Healthy control group; ii) sham operation group; iii) myocardial infarction model group; iv) myocardial infarction + low-dose tetrandrine group (10 mg/kg); v) myocardial infarction + medium-dose tetrandrine group (50 mg/kg); and vi) myocardial infarction + high-dose tetrandrine group (80 mg/kg). Left ventricular end-diastolic diameter (LVIDd), left ventricular end-systolic diameter (LVIDs), ejection fraction (EF%) and left ventricular fractional shortening rate (FS%) were measured using ultrasonography. The pathological changes were observed by hematoxylin and eosin (H&E) staining. Left ventricular tissue section TUNEL staining was also performed. Furthermore, the triglyceride (TG), total cholesterol (TC), high density lipoprotein (HDL) and low-density lipoprotein (LDL) in the arterial blood were examined by biochemical testing. Expression levels of intracellular Ca2+ homeostasis-related proteins including ryanodine receptor calmodulin, CaM-dependent protein kinase IIδ, protein kinase A, FK506 binding protein 12.6 were measured using western blot analysis. Ultrasonography results showed that in the myocardial infarction model rats, the levels of LVIDd and LVIDs were significantly higher; however, the levels of EF% and FS% were lower compared with those in the sham operation group, which was alleviated by tetrandrine. H&E results showed that tetrandrine alleviated the pathological characteristics of myocardial infarction model rats. Furthermore, tetrandrine significantly inhibited myocardial cell apoptosis in rats with myocardial infarction. Tetrandrine significantly inhibited the levels of TG, TC and LDL and increased the levels of HDL in the arterial blood of rats with myocardial infarction. These findings revealed that tetrandrine could attenuate left ventricular dysfunction in rats with myocardial infarction, which might be associated with intracellular Ca2+ homeostasis.
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Affiliation(s)
- Youyang Wu
- Department of Cardiology, The Third Clinical Institute Affiliated to Wenzhou Medical University, Wenzhou, Zhejiang 325000, P.R. China
| | - Wei Zhao
- Department of Cardiology, The Third Clinical Institute Affiliated to Wenzhou Medical University, Wenzhou, Zhejiang 325000, P.R. China
| | - Fanhao Ye
- Department of Cardiology, The Third Clinical Institute Affiliated to Wenzhou Medical University, Wenzhou, Zhejiang 325000, P.R. China
| | - Shiwei Huang
- Department of Cardiology, The Third Clinical Institute Affiliated to Wenzhou Medical University, Wenzhou, Zhejiang 325000, P.R. China
| | - Hao Chen
- Department of Cardiology, The Third Clinical Institute Affiliated to Wenzhou Medical University, Wenzhou, Zhejiang 325000, P.R. China
| | - Rui Zhou
- Department of Cardiology, The Third Clinical Institute Affiliated to Wenzhou Medical University, Wenzhou, Zhejiang 325000, P.R. China
| | - Wenbing Jiang
- Department of Cardiology, The Third Clinical Institute Affiliated to Wenzhou Medical University, Wenzhou, Zhejiang 325000, P.R. China
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22
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DiNello E, Bovo E, Thuo P, Martin TG, Kirk JA, Zima AV, Cao Q, Kuo IY. Deletion of cardiac polycystin 2/PC2 results in increased SR calcium release and blunted adrenergic reserve. Am J Physiol Heart Circ Physiol 2020; 319:H1021-H1035. [PMID: 32946258 DOI: 10.1152/ajpheart.00302.2020] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Transient receptor potential proteins (TRPs) act as nonselective cation channels. Of the TRP channels, PC2 (also known as polycystin 2) is localized to the sarcoplasmic reticulum (SR); however, its contribution to calcium-induced calcium release and overall cardiac function in the heart is poorly understood. The goal of this study was to characterize the effect of cardiac-specific PC2 deletion in adult cardiomyocytes and in response to chronic β-adrenergic challenge. We used a temporally inducible model to specifically delete PC2 from cardiomyocytes (Pkd2 KO) and characterized calcium and contractile dynamics in single cells. We found enhanced intracellular calcium release after Pkd2 KO, and near super-resolution microscopy analysis suggested this was due to close localization of PC2 to the ryanodine receptor. At the organ level, speckle-tracking echocardiographical analysis showed increased dyssynchrony in the Pkd2 KO mice. In response to chronic adrenergic stimulus, cardiomyocytes from the Pkd2 KO had no reserve β-adrenergic calcium responses and significantly attenuated wall motion in the whole heart. Biochemically, without adrenergic stimulus, there was an overall increase in PKA phosphorylated targets in the Pkd2 KO mouse, which decreased following chronic adrenergic stimulus. Taken together, our results suggest that cardiac-specific PC2 limits SR calcium release by affecting the PKA phosphorylation status of the ryanodine receptor, and the effects of PC2 loss are exacerbated upon adrenergic challenge.NEW & NOTEWORTHY Our goal was to characterize the role of the transient receptor potential channel polycystin 2 (PC2) in cardiomyocytes following adult-onset deletion. Loss of PC2 resulted in decreased cardiac shortening and cardiac dyssynchrony and diminished adrenergic reserve. These results suggest that cardiac-specific PC2 modulates intracellular calcium signaling and contributes to the maintenance of adrenergic pathways.
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Affiliation(s)
- Elisabeth DiNello
- Department of Cell and Molecular Physiology, Loyola University Chicago, Chicago, Illinois.,Cardiovascular Research Institute, Loyola University Chicago, Chicago, Illinois
| | - Elisa Bovo
- Department of Cell and Molecular Physiology, Loyola University Chicago, Chicago, Illinois.,Cardiovascular Research Institute, Loyola University Chicago, Chicago, Illinois
| | - Paula Thuo
- Department of Cell and Molecular Physiology, Loyola University Chicago, Chicago, Illinois.,Cardiovascular Research Institute, Loyola University Chicago, Chicago, Illinois
| | - Thomas G Martin
- Graduate School, Loyola University Chicago, Chicago, Illinois
| | - Jonathan A Kirk
- Department of Cell and Molecular Physiology, Loyola University Chicago, Chicago, Illinois.,Cardiovascular Research Institute, Loyola University Chicago, Chicago, Illinois
| | - Aleksey V Zima
- Department of Cell and Molecular Physiology, Loyola University Chicago, Chicago, Illinois.,Cardiovascular Research Institute, Loyola University Chicago, Chicago, Illinois
| | - Quan Cao
- Department of Cell and Molecular Physiology, Loyola University Chicago, Chicago, Illinois.,Cardiovascular Research Institute, Loyola University Chicago, Chicago, Illinois
| | - Ivana Y Kuo
- Department of Cell and Molecular Physiology, Loyola University Chicago, Chicago, Illinois.,Cardiovascular Research Institute, Loyola University Chicago, Chicago, Illinois.,Department of Pharmacology, Yale University, New Haven, Connecticut
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23
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Multisite phosphorylation of the cardiac ryanodine receptor: a random or coordinated event? Pflugers Arch 2020; 472:1793-1807. [PMID: 33078311 DOI: 10.1007/s00424-020-02473-3] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2020] [Revised: 09/03/2020] [Accepted: 10/02/2020] [Indexed: 10/23/2022]
Abstract
Many proteins are phosphorylated at more than one phosphorylation site to achieve precise tuning of protein function and/or integrate a multitude of signals into the activity of one protein. Increasing the number of phosphorylation sites significantly broadens the complexity of molecular mechanisms involved in processing multiple phosphorylation sites by one or more distinct kinases. The cardiac ryanodine receptor (RYR2) is a well-established multiple phospho-target of kinases activated in response to β-adrenergic stimulation because this Ca2+ channel is a critical component of Ca2+ handling machinery which is responsible for β-adrenergic enhancement of cardiac contractility. Our review presents a selective overview of the extensive, often conflicting, literature which focuses on identifying reliable lines of evidence to establish if multiple RYR2 phosphorylation is achieved randomly or in a specific sequence, and whether phosphorylation at individual sites is functionally specific and additive or similar and can therefore be substituted.
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24
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Blackwell DJ, Knollmann BC. SPEG Kinase: Hitting the Brake in Atrial Fibrillation. Circulation 2020; 142:1173-1175. [PMID: 32955933 PMCID: PMC7523464 DOI: 10.1161/circulationaha.120.050226] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Affiliation(s)
- Daniel J Blackwell
- Vanderbilt Center for Arrhythmia Research and Therapeutics, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN
| | - Björn C Knollmann
- Vanderbilt Center for Arrhythmia Research and Therapeutics, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN
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25
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Dridi H, Kushnir A, Zalk R, Yuan Q, Melville Z, Marks AR. Reply to 'Mechanisms of ryanodine receptor 2 dysfunction in heart failure'. Nat Rev Cardiol 2020; 17:749-750. [PMID: 32901149 DOI: 10.1038/s41569-020-00444-w] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Affiliation(s)
- Haikel Dridi
- Department of Physiology and Cellular Biophysics, Clyde and Helen Wu Center for Molecular Cardiology, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA
| | - Alexander Kushnir
- Department of Physiology and Cellular Biophysics, Clyde and Helen Wu Center for Molecular Cardiology, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA
| | - Ran Zalk
- The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer Sheva, Israel
| | - Qi Yuan
- Department of Physiology and Cellular Biophysics, Clyde and Helen Wu Center for Molecular Cardiology, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA
| | - Zephan Melville
- Department of Physiology and Cellular Biophysics, Clyde and Helen Wu Center for Molecular Cardiology, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA
| | - Andrew R Marks
- Department of Physiology and Cellular Biophysics, Clyde and Helen Wu Center for Molecular Cardiology, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA.
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26
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Grogan A, Coleman A, Joca H, Granzier H, Russel MW, Ward CW, Kontrogianni-Konstantopoulos A. Deletion of obscurin immunoglobulin domains Ig58/59 leads to age-dependent cardiac remodeling and arrhythmia. Basic Res Cardiol 2020; 115:60. [PMID: 32910221 PMCID: PMC9302192 DOI: 10.1007/s00395-020-00818-8] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/16/2020] [Accepted: 08/06/2020] [Indexed: 12/23/2022]
Abstract
Obscurin comprises a family of giant modular proteins that play key structural and regulatory roles in striated muscles. Immunoglobulin domains 58/59 (Ig58/59) of obscurin mediate binding to essential modulators of muscle structure and function, including canonical titin, a smaller splice variant of titin, termed novex-3, and phospholamban (PLN). Importantly, missense mutations localized within the obscurin-Ig58/59 region that affect binding to titins and/or PLN have been linked to the development of myopathy in humans. To elucidate the pathophysiological role of this region, we generated a constitutive deletion mouse model, Obscn-ΔIg58/59, that expresses obscurin lacking Ig58/59, and determined the consequences of this manipulation on cardiac morphology and function under conditions of acute stress and through the physiological process of aging. Our studies show that young Obscn-ΔIg58/59 mice are susceptible to acute β-adrenergic stress. Moreover, sedentary Obscn-ΔIg58/59 mice develop left ventricular hypertrophy that progresses to dilation, contractile impairment, atrial enlargement, and arrhythmia as a function of aging with males being more affected than females. Experiments in ventricular cardiomyocytes revealed altered Ca2+ cycling associated with changes in the expression and/or phosphorylation levels of major Ca2+ cycling proteins, including PLN, SERCA2, and RyR2. Taken together, our work demonstrates that obscurin-Ig58/59 is an essential regulatory module in the heart and its deletion leads to age- and sex-dependent cardiac remodeling, ventricular dilation, and arrhythmia due to deregulated Ca2+ cycling.
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MESH Headings
- Action Potentials
- Age Factors
- Animals
- Arrhythmias, Cardiac/enzymology
- Arrhythmias, Cardiac/genetics
- Arrhythmias, Cardiac/pathology
- Arrhythmias, Cardiac/physiopathology
- Calcium Signaling
- Calcium-Binding Proteins/metabolism
- Female
- Gene Deletion
- Heart Rate
- Hypertrophy, Left Ventricular/enzymology
- Hypertrophy, Left Ventricular/genetics
- Hypertrophy, Left Ventricular/pathology
- Hypertrophy, Left Ventricular/physiopathology
- Immunoglobulin Domains
- Male
- Mice, Inbred C57BL
- Mice, Knockout
- Myocytes, Cardiac/enzymology
- Myocytes, Cardiac/pathology
- Phosphorylation
- Protein Serine-Threonine Kinases/deficiency
- Protein Serine-Threonine Kinases/genetics
- Rho Guanine Nucleotide Exchange Factors/deficiency
- Rho Guanine Nucleotide Exchange Factors/genetics
- Ryanodine Receptor Calcium Release Channel/metabolism
- Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism
- Sedentary Behavior
- Sex Factors
- Ventricular Dysfunction, Left/enzymology
- Ventricular Dysfunction, Left/genetics
- Ventricular Dysfunction, Left/pathology
- Ventricular Dysfunction, Left/physiopathology
- Ventricular Function, Left
- Ventricular Remodeling
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Affiliation(s)
- Alyssa Grogan
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, 21201, USA
| | - Andrew Coleman
- Department of Orthopedics, University of Maryland School of Medicine, Baltimore, MD, 21201, USA
| | - Humberto Joca
- Department of Orthopedics, University of Maryland School of Medicine, Baltimore, MD, 21201, USA
| | - Henk Granzier
- Department of Physiology, University of Arizona College of Medicine, Tucson, AZ, 85724, USA
| | - Mark W Russel
- Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, MI, 48109, USA
| | - Christopher W Ward
- Department of Orthopedics, University of Maryland School of Medicine, Baltimore, MD, 21201, USA
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27
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Baine S, Thomas J, Bonilla I, Ivanova M, Belevych A, Li J, Veeraraghavan R, Radwanski PB, Carnes C, Gyorke S. Muscarinic-dependent phosphorylation of the cardiac ryanodine receptor by protein kinase G is mediated by PI3K-AKT-nNOS signaling. J Biol Chem 2020; 295:11720-11728. [PMID: 32580946 PMCID: PMC7450129 DOI: 10.1074/jbc.ra120.014054] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2020] [Revised: 06/20/2020] [Indexed: 12/30/2022] Open
Abstract
Post-translational modifications of proteins involved in calcium handling in myocytes, such as the cardiac ryanodine receptor (RyR2), critically regulate cardiac contractility. Recent studies have suggested that phosphorylation of RyR2 by protein kinase G (PKG) might contribute to the cardioprotective effects of cholinergic stimulation. However, the specific mechanisms underlying these effects remain unclear. Here, using murine ventricular myocytes, immunoblotting, proximity ligation as-says, and nitric oxide imaging, we report that phosphorylation of Ser-2808 in RyR2 induced by the muscarinic receptor agonist carbachol is mediated by a signaling axis comprising phosphoinositide 3-phosphate kinase, Akt Ser/Thr kinase, nitric oxide synthase 1, nitric oxide, soluble guanylate cyclase, cyclic GMP (cGMP), and PKG. We found that this signaling pathway is compartmentalized in myocytes, as it was distinct from atrial natriuretic peptide receptor-cGMP-PKG-RyR2 Ser-2808 signaling and independent of muscarinic-induced phosphorylation of Ser-239 in vasodilator-stimulated phosphoprotein. These results provide detailed insights into muscarinic-induced PKG signaling and the mediators that regulate cardiac RyR2 phosphorylation critical for cardiovascular function.
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Affiliation(s)
- Stephen Baine
- College of Pharmacy, Ohio State University, Columbus, Ohio, USA
| | - Justin Thomas
- College of Pharmacy, Ohio State University, Columbus, Ohio, USA
| | - Ingrid Bonilla
- Department of Physiology and Cell Biology, Ohio State University, Columbus, Ohio, USA
| | - Marina Ivanova
- Department of Physiology and Cell Biology, Ohio State University, Columbus, Ohio, USA
| | - Andriy Belevych
- Department of Physiology and Cell Biology, Ohio State University, Columbus, Ohio, USA
| | - Jiaoni Li
- Department of Biomedical Engineering, Ohio State University, Columbus, Ohio, USA
| | | | | | - Cynthia Carnes
- College of Pharmacy, Ohio State University, Columbus, Ohio, USA
| | - Sandor Gyorke
- Department of Biomedical Engineering, Ohio State University, Columbus, Ohio, USA
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28
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Romero-García T, Landa-Galvan HV, Pavón N, Mercado-Morales M, Valdivia HH, Rueda A. Autonomous activation of CaMKII exacerbates diastolic calcium leak during beta-adrenergic stimulation in cardiomyocytes of metabolic syndrome rats. Cell Calcium 2020; 91:102267. [PMID: 32920522 DOI: 10.1016/j.ceca.2020.102267] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2020] [Revised: 08/05/2020] [Accepted: 08/05/2020] [Indexed: 02/06/2023]
Abstract
Autonomous Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation induces abnormal diastolic Ca2+ leak, which leads to triggered arrhythmias in a wide range of cardiovascular diseases, including diabetic cardiomyopathy. In hyperglycemia, Ca2+ handling alterations can be aggravated under stress conditions via the β-adrenergic signaling pathway, which also involves CaMKII activation. However, little is known about intracellular Ca2+ handling disturbances under β-adrenergic stimulation in cardiomyocytes of the prediabetic metabolic syndrome (MetS) model with obesity, and the participation of CaMKII in these alterations. MetS was induced in male Wistar rats by administering 30 % sucrose in drinking water for 16 weeks. Fluo 3-loaded MetS cardiomyocytes exhibited augmented diastolic Ca2+ leak (in the form of spontaneous Ca2+ waves) under basal conditions and that Ca2+ leakage was exacerbated by isoproterenol (ISO, 100 nM). At the molecular level, [3H]-ryanodine binding and basal phosphorylation of cardiac ryanodine receptor (RyR2) at Ser2814, a CaMKII site, were increased in heart homogenates of MetS rats with no changes in RyR2 expression. These alterations were not further augmented by Isoproterenol. SERCA pump activity was augmented 48 % in MetS hearts before β-adrenergic stimuli, which is associated to augmented PLN phosphorylation at T17, a target of CaMKII. In MetS hearts. CaMKII auto-phosphorylation (T287) was increased by 80 %. The augmented diastolic Ca2+ leak was prevented by CaMKII inhibition with AIP. In conclusion, CaMKII autonomous activation in cardiomyocytes of MetS rats with central obesity significantly contributes to abnormal diastolic Ca2+ leak, increasing the propensity for β-adrenergic receptor-driven lethal arrhythmias.
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Affiliation(s)
- Tatiana Romero-García
- Department of Biochemistry, Centro de Investigación y de Estudios Avanzados del IPN, Cinvestav-IPN, Mexico City, 07300 Mexico
| | - Huguet V Landa-Galvan
- Department of Biochemistry, Centro de Investigación y de Estudios Avanzados del IPN, Cinvestav-IPN, Mexico City, 07300 Mexico
| | - Natalia Pavón
- Department of Pharmacology, Instituto Nacional de Cardiología "Ignacio Chavez", Mexico City, Mexico
| | - Martha Mercado-Morales
- Department of Biochemistry, Centro de Investigación y de Estudios Avanzados del IPN, Cinvestav-IPN, Mexico City, 07300 Mexico
| | - Héctor H Valdivia
- Department of Medicine, Division of Cardiovascular Medicine, University of Wisconsin School of Medicine and Public Health, Madison, WI, 53705, USA
| | - Angélica Rueda
- Department of Biochemistry, Centro de Investigación y de Estudios Avanzados del IPN, Cinvestav-IPN, Mexico City, 07300 Mexico.
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29
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Wang Y, Li C, Shi L, Chen X, Cui C, Huang J, Chen B, Hall DD, Pan Z, Lu M, Hong J, Song LS, Zhao S. Integrin β1D Deficiency-Mediated RyR2 Dysfunction Contributes to Catecholamine-Sensitive Ventricular Tachycardia in Arrhythmogenic Right Ventricular Cardiomyopathy. Circulation 2020; 141:1477-1493. [PMID: 32122157 DOI: 10.1161/circulationaha.119.043504] [Citation(s) in RCA: 38] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
BACKGROUND Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a hereditary heart disease characterized by fatty infiltration, life-threatening arrhythmias, and increased risk of sudden cardiac death. The guideline for management of ARVC in patients is to improve quality of life by reducing arrhythmic symptoms and to prevent sudden cardiac death. However, the mechanism underlying ARVC-associated cardiac arrhythmias remains poorly understood. METHODS Using protein mass spectrometry analyses, we identified that integrin β1 is downregulated in ARVC hearts without changes to Ca2+-handling proteins. As adult cardiomyocytes express only the β1D isoform, we generated a cardiac specific β1D knockout mouse model and performed functional imaging and biochemical analyses to determine the consequences of integrin β1D loss on function in the heart in vivo and in vitro. RESULTS Integrin β1D deficiency and RyR2 Ser-2030 hyperphosphorylation were detected by Western blotting in left ventricular tissues from patients with ARVC but not in patients with ischemic or hypertrophic cardiomyopathy. Using lipid bilayer patch clamp single channel recordings, we found that purified integrin β1D protein could stabilize RyR2 function by decreasing RyR2 open probability, mean open time, and increasing mean close time. Also, β1D knockout mice exhibited normal cardiac function and morphology but presented with catecholamine-sensitive polymorphic ventricular tachycardia, consistent with increased RyR2 Ser-2030 phosphorylation and aberrant Ca2+ handling in β1D knockout cardiomyocytes. Mechanistically, we revealed that loss of DSP (desmoplakin) induces integrin β1D deficiency in ARVC mediated through an ERK1/2 (extracellular signal-regulated kinase 1 and 2)-fibronectin-ubiquitin/lysosome pathway. CONCLUSIONS Our data suggest that integrin β1D deficiency represents a novel mechanism underlying the increased risk of ventricular arrhythmias in patients with ARVC.
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Affiliation(s)
- Yihui Wang
- State Key Laboratory of Cardiovascular Disease, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing (Y.W., C.L., X.C., C.C., M.L., S.Z.)
| | - Chunyan Li
- State Key Laboratory of Cardiovascular Disease, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing (Y.W., C.L., X.C., C.C., M.L., S.Z.)
| | - Ling Shi
- Department of Pharmacology, College of Pharmacy, and State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education, Harbin Medical University, Heilongjiang, China (L.S., Z.P.)
| | - Xiuyu Chen
- State Key Laboratory of Cardiovascular Disease, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing (Y.W., C.L., X.C., C.C., M.L., S.Z.)
| | - Chen Cui
- State Key Laboratory of Cardiovascular Disease, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing (Y.W., C.L., X.C., C.C., M.L., S.Z.)
| | | | - Biyi Chen
- Division of Cardiovascular Medicine, Department of Internal Medicine, Abboud Cardiovascular Research Center, Carver College of Medicine, University of Iowa, Iowa City (B.C., D.D.H., L.-S.S.)
| | - Duane D Hall
- Division of Cardiovascular Medicine, Department of Internal Medicine, Abboud Cardiovascular Research Center, Carver College of Medicine, University of Iowa, Iowa City (B.C., D.D.H., L.-S.S.)
| | - Zhenwei Pan
- Department of Pharmacology, College of Pharmacy, and State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education, Harbin Medical University, Heilongjiang, China (L.S., Z.P.)
| | - Minjie Lu
- State Key Laboratory of Cardiovascular Disease, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing (Y.W., C.L., X.C., C.C., M.L., S.Z.)
| | - Jiang Hong
- Department of Cardiology, Fujian Institute of Coronary Heart Disease, Fujian Medical University Union Hospital, China (J.H.)
- Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, China (J.H.)
| | - Long-Sheng Song
- Division of Cardiovascular Medicine, Department of Internal Medicine, Abboud Cardiovascular Research Center, Carver College of Medicine, University of Iowa, Iowa City (B.C., D.D.H., L.-S.S.)
- Fraternal Order of Eagles Diabetes Research Center, Carver College of Medicine, University of Iowa, Iowa City (L.-S.S.)
- Department of Veterans Affairs Medical Center, Iowa City, IA (L.-S.S.)
| | - Shihua Zhao
- State Key Laboratory of Cardiovascular Disease, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing (Y.W., C.L., X.C., C.C., M.L., S.Z.)
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30
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Potenza DM, Janicek R, Fernandez-Tenorio M, Niggli E. Activation of endogenous protein phosphatase 1 enhances the calcium sensitivity of the ryanodine receptor type 2 in murine ventricular cardiomyocytes. J Physiol 2020; 598:1131-1150. [PMID: 31943206 DOI: 10.1113/jp278951] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2019] [Accepted: 01/10/2020] [Indexed: 01/23/2023] Open
Abstract
KEY POINTS Increased protein phosphatase 1 (PP-1) activity has been found in end stage human heart failure. Although PP-1 has been extensively studied, a detailed understanding of its role in the excitation-contraction coupling mechanism, in normal and diseased hearts, remains elusive. The present study investigates the functional effect of the PP-1 activity on local Ca2+ release events in ventricular cardiomyocytes, by using an activating peptide (PDP3) for the stimulation of the endogenous PP-1 protein. We report that acute de-phosphorylation may increase the sensitivity of RyR2 channels to Ca2+ in situ, and that the RyR2-serine2808 phosphorylation site may mediate such a process. Our approach unmasks the functional importance of PP-1 in the regulation of RyR2 activity, suggesting a potential role in the generation of a pathophysiological sarcoplasmic reticulum Ca2+ leak in the diseased heart. ABSTRACT Changes in cardiac ryanodine receptor (RyR2) phosphorylation are considered to be important regulatory and disease related post-translational protein modifications. The extent of RyR2 phosphorylation is mainly determined by the balance of the activities of protein kinases and phosphatases, respectively. Increased protein phosphatase-1 (PP-1) activity has been observed in heart failure, although the regulatory role of this enzyme on intracellular Ca2+ handling remains poorly understood. To determine the physiological and pathophysiological significance of increased PP-1 activity, we investigated how the PP-1 catalytic subunit (PP-1c) alters Ca2+ sparks in permeabilized cardiomyocytes and we also applied a PP-1-disrupting peptide (PDP3) to specifically activate endogenous PP-1, including the one anchored on the RyR2 macromolecular complex. We compared wild-type and transgenic mice in which the usually highly phosphorylated site RyR2-S2808 has been ablated to investigate its involvement in RyR2 modulation (S2808A+/+ ). In wild-type myocytes, PP-1 increased Ca2+ spark frequency by two-fold, followed by depletion of the sarcoplasmic reticulum Ca2+ store. Similarly, PDP3 transiently increased spark frequency and decreased sarcoplasmic reticulum Ca2+ load. RyR2 Ca2+ sensitivity, which was assessed by Ca2+ spark recovery analysis, was increased in the presence of PDP3 compared to a negative control peptide. S2808A+/+ cardiomyocytes did not respond to both PP-1c and PDP3 treatment. Our results suggest an increased Ca2+ sensitivity of RyR2 upon de-phosphorylation by PP-1. Furthermore, we have confirmed the S2808 site as a target for PP-1 and as a potential link between RyR2s modulation and the cellular response.
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Affiliation(s)
| | | | | | - Ernst Niggli
- Department of Physiology, University of Bern, Bern, Switzerland
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31
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Munro ML, Jones PP. Too much of a good thing? Establishing a role of excessive RyR2 dephosphorylation in heart disease. J Physiol 2020; 598:1115-1116. [PMID: 31994730 DOI: 10.1113/jp279569] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2020] [Accepted: 01/27/2020] [Indexed: 01/06/2023] Open
Affiliation(s)
- Michelle L Munro
- Department of Physiology and HeartOtago, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand
| | - Peter P Jones
- Department of Physiology and HeartOtago, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand
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32
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Federico M, Valverde CA, Mattiazzi A, Palomeque J. Unbalance Between Sarcoplasmic Reticulum Ca 2 + Uptake and Release: A First Step Toward Ca 2 + Triggered Arrhythmias and Cardiac Damage. Front Physiol 2020; 10:1630. [PMID: 32038301 PMCID: PMC6989610 DOI: 10.3389/fphys.2019.01630] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2019] [Accepted: 12/24/2019] [Indexed: 12/19/2022] Open
Abstract
The present review focusses on the regulation and interplay of cardiac SR Ca2+ handling proteins involved in SR Ca2+ uptake and release, i.e., SERCa2/PLN and RyR2. Both RyR2 and SERCA2a/PLN are highly regulated by post-translational modifications and/or different partners' proteins. These control mechanisms guarantee a precise equilibrium between SR Ca2+ reuptake and release. The review then discusses how disruption of this balance alters SR Ca2+ handling and may constitute a first step toward cardiac damage and malignant arrhythmias. In the last part of the review, this concept is exemplified in different cardiac diseases, like prediabetic and diabetic cardiomyopathy, digitalis intoxication and ischemia-reperfusion injury.
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Affiliation(s)
- Marilén Federico
- Centro de Investigaciones Cardiovasculares "Dr. Horacio E. Cingolani", CCT-La Plata/CONICET, Facultad de Cs. Médicas, Universidad Nacional de La Plata, La Plata, Argentina
| | - Carlos A Valverde
- Centro de Investigaciones Cardiovasculares "Dr. Horacio E. Cingolani", CCT-La Plata/CONICET, Facultad de Cs. Médicas, Universidad Nacional de La Plata, La Plata, Argentina
| | - Alicia Mattiazzi
- Centro de Investigaciones Cardiovasculares "Dr. Horacio E. Cingolani", CCT-La Plata/CONICET, Facultad de Cs. Médicas, Universidad Nacional de La Plata, La Plata, Argentina
| | - Julieta Palomeque
- Centro de Investigaciones Cardiovasculares "Dr. Horacio E. Cingolani", CCT-La Plata/CONICET, Facultad de Cs. Médicas, Universidad Nacional de La Plata, La Plata, Argentina.,Centro de Altos Estudios en Ciencias Humanas y de la Salud, Universidad Abierta Interamericana, Buenos Aires, Argentina
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33
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Al Kury LT. Calcium Homeostasis in Ventricular Myocytes of Diabetic Cardiomyopathy. J Diabetes Res 2020; 2020:1942086. [PMID: 33274235 PMCID: PMC7683117 DOI: 10.1155/2020/1942086] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/08/2020] [Revised: 10/24/2020] [Accepted: 10/29/2020] [Indexed: 12/12/2022] Open
Abstract
Diabetes mellitus (DM) is a chronic metabolic disorder commonly characterized by high blood glucose levels, resulting from defects in insulin production or insulin resistance, or both. DM is a leading cause of mortality and morbidity worldwide, with diabetic cardiomyopathy as one of its main complications. It is well established that cardiovascular complications are common in both types of diabetes. Electrical and mechanical problems, resulting in cardiac contractile dysfunction, are considered as the major complications present in diabetic hearts. Inevitably, disturbances in the mechanism(s) of Ca2+ signaling in diabetes have implications for cardiac myocyte contraction. Over the last decade, significant progress has been made in outlining the mechanisms responsible for the diminished cardiac contractile function in diabetes using different animal models of type I diabetes mellitus (TIDM) and type II diabetes mellitus (TIIDM). The aim of this review is to evaluate our current understanding of the disturbances of Ca2+ transport and the role of main cardiac proteins involved in Ca2+ homeostasis in the diabetic rat ventricular cardiomyocytes. Exploring the molecular mechanism(s) of altered Ca2+ signaling in diabetes will provide an insight for the identification of novel therapeutic approaches to improve the heart function in diabetic patients.
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Affiliation(s)
- Lina T. Al Kury
- Department of Health Sciences, College of Natural and Health Sciences, Zayed University, Abu Dhabi 144534, UAE
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34
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Huang JH, Chen YC, Lu YY, Lin YK, Chen SA, Chen YJ. Arginine vasopressin modulates electrical activity and calcium homeostasis in pulmonary vein cardiomyocytes. J Biomed Sci 2019; 26:71. [PMID: 31530276 PMCID: PMC6747756 DOI: 10.1186/s12929-019-0564-3] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2019] [Accepted: 09/10/2019] [Indexed: 02/06/2023] Open
Abstract
Background Atrial fibrillation (AF) frequently coexists with congestive heart failure (HF) and arginine vasopressin (AVP) V1 receptor antagonists are used to treat hyponatremia in HF. However, the role of AVP in HF-induced AF still remains unclear. Pulmonary veins (PVs) are central in the genesis of AF. The purpose of this study was to determine if AVP is directly involved in the regulation of PV electrophysiological properties and calcium (Ca2+) homeostasis as well as the identification of the underlying mechanisms. Methods Patch clamp, confocal microscopy with Fluo-3 fluorescence, and Western blot analyses were used to evaluate the electrophysiological characteristics, Ca2+ homeostasis, and Ca2+ regulatory proteins in isolated rabbit single PV cardiomyocytes incubated with and without AVP (1 μM), OPC 21268 (0.1 μM, AVP V1 antagonist), or OPC 41061 (10 nM, AVP V2 antagonist) for 4–6 h. Results AVP (0.1 and 1 μM)-treated PV cardiomyocytes had a faster beating rate (108 to 152%) than the control cells. AVP (1 μM) treated PV cardiomyocytes had higher late sodium (Na+) and Na+/Ca2+ exchanger (NCX) currents than control PV cardiomyocytes. AVP (1 μM) treated PV cardiomyocytes had smaller Ca2+i transients, and sarcoplasmic reticulum (SR) Ca2+ content as well as higher Ca2+ leak. However, combined AVP (1 μM) and OPC 21268 (0.1 μM) treated PV cardiomyocytes had a slower PV beating rate, larger Ca2+i transients and SR Ca2+ content, smaller late Na+ and NCX currents than AVP (1 μM)-treated PV cardiomyocytes. Western blot experiments showed that AVP (1 μM) treated PV cardiomyocytes had higher expression of NCX and p-CaMKII, and a higher ratio of p-CaMKII/CaMKII. Conclusions AVP increases PV arrhythmogenesis with dysregulated Ca2+ homeostasis through vasopressin V1 signaling.
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Affiliation(s)
- Jen-Hung Huang
- Division of Cardiovascular Medicine, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, 111 Hsin-Lung Road, Sec. 3, Taipei, 116, Taiwan.,Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
| | - Yao-Chang Chen
- Department of Biomedical Engineering, and Institute of Physiology, National Defense Medical Center, Taipei, Taiwan
| | - Yen-Yu Lu
- Division of Cardiology, Department of Internal Medicine, Sijhih Cathay General Hospital, New Taipei City, Taiwan.,School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan
| | - Yung-Kuo Lin
- Division of Cardiovascular Medicine, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, 111 Hsin-Lung Road, Sec. 3, Taipei, 116, Taiwan.,Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
| | - Shih-Ann Chen
- Heart Rhythm Center and Division of Cardiology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
| | - Yi-Jen Chen
- Division of Cardiovascular Medicine, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, 111 Hsin-Lung Road, Sec. 3, Taipei, 116, Taiwan. .,Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan. .,Cardiovascular Research Center, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan.
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35
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Aguilar-Sanchez Y, Rodriguez de Yurre A, Argenziano M, Escobar AL, Ramos-Franco J. Transmural Autonomic Regulation of Cardiac Contractility at the Intact Heart Level. Front Physiol 2019; 10:773. [PMID: 31333477 PMCID: PMC6616252 DOI: 10.3389/fphys.2019.00773] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2019] [Accepted: 06/03/2019] [Indexed: 01/14/2023] Open
Abstract
The relationship between cardiac excitability and contractility depends on when Ca2+ influx occurs during the ventricular action potential (AP). In mammals, it is accepted that Ca2+ influx through the L-type Ca2+ channels occurs during AP phase 2. However, in murine models, experimental evidence shows Ca2+ influx takes place during phase 1. Interestingly, Ca2+ influx that activates contraction is highly regulated by the autonomic nervous system. Indeed, autonomic regulation exerts multiple effects on Ca2+ handling and cardiac electrophysiology. In this paper, we explore autonomic regulation in endocardial and epicardial layers of intact beating mice hearts to evaluate their role on cardiac excitability and contractility. We hypothesize that in mouse cardiac ventricles the influx of Ca2+ that triggers excitation–contraction coupling (ECC) does not occur during phase 2. Using pulsed local field fluorescence microscopy and loose patch photolysis, we show sympathetic stimulation by isoproterenol increased the amplitude of Ca2+ transients in both layers. This increase in contractility was driven by an increase in amplitude and duration of the L-type Ca2+ current during phase 1. Interestingly, the β-adrenergic increase of Ca2+ influx slowed the repolarization of phase 1, suggesting a competition between Ca2+ and K+ currents during this phase. In addition, cAMP activated L-type Ca2+ currents before SR Ca2+ release activated the Na+-Ca2+ exchanger currents, indicating Cav1.2 channels are the initial target of PKA phosphorylation. In contrast, parasympathetic stimulation by carbachol did not have a substantial effect on amplitude and kinetics of endocardial and epicardial Ca2+ transients. However, carbachol transiently decreased the duration of the AP late phase 2 repolarization. The carbachol-induced shortening of phase 2 did not have a considerable effect on ventricular pressure and systolic Ca2+ dynamics. Interestingly, blockade of muscarinic receptors by atropine prolonged the duration of phase 2 indicating that, in isolated hearts, there is an intrinsic release of acetylcholine. In addition, the acceleration of repolarization induced by carbachol was blocked by the acetylcholine-mediated K+ current inhibition. Our results reveal the transmural ramifications of autonomic regulation in intact mice hearts and support our hypothesis that Ca2+ influx that triggers ECC occurs in AP phase 1 and not in phase 2.
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Affiliation(s)
- Yuriana Aguilar-Sanchez
- Department of Physiology and Biophysics, School of Medicine, Rush University Medical Center, Chicago, IL, United States
| | - Ainhoa Rodriguez de Yurre
- Laboratorio de Cardio Inmunologia, Instituto de Biofisica Carlos Chagas Filho, Rio de Janeiro, Brazil
| | - Mariana Argenziano
- Division of Human Genetics, Children's Hospital of Philadelphia, Philadelphia, PA, United States
| | - Ariel L Escobar
- Department of Bioengineering, School of Engineering, University of California, Merced, Merced, CA, United States
| | - Josefina Ramos-Franco
- Department of Physiology and Biophysics, School of Medicine, Rush University Medical Center, Chicago, IL, United States
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36
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Fernández-Miranda G, Romero-Garcia T, Barrera-Lechuga TP, Mercado-Morales M, Rueda A. Impaired Activity of Ryanodine Receptors Contributes to Calcium Mishandling in Cardiomyocytes of Metabolic Syndrome Rats. Front Physiol 2019; 10:520. [PMID: 31114513 PMCID: PMC6503767 DOI: 10.3389/fphys.2019.00520] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2018] [Accepted: 04/11/2019] [Indexed: 01/11/2023] Open
Abstract
Metabolic syndrome (MetS) has become a global epidemic. MetS is a serious health problem because of its related cardiovascular complications, which include hypertension and delayed heart rate recovery after exercise. The molecular bases of cardiac dysfunction in MetS are still under scrutiny and may be related to anomalies in the activity and expression of key proteins involved in the cardiac excitation-contraction coupling (ECC). The cardiac Ca2+ channel/ryanodine receptor (RyR2) participates in releasing Ca2+ from internal stores and plays a key role in the modulation of ECC. We examined alterations in expression, phosphorylation status, Ca2+ sensitivity, and in situ function (by measuring Ca2+ sparks and Ca2+ transients) of RyR2; alterations in these characteristics could help to explain the Ca2+ handling disturbances in MetS cardiomyocytes. MetS was induced in rats by adding commercially refined sugar (30% sucrose) to their drinking water for 24 weeks. Cardiomyocytes of MetS rats displayed decreased Ca2+ transient amplitude and cell contractility at all stimulation frequencies. Quiescent MetS cardiomyocytes showed a decrease in Ca2+ spark frequency, amplitude, and spark-mediated Ca2+ leak. The [3H]-ryanodine binding data showed that functionally active RyRs are significantly diminished in MetS heart microsomes; and exhibited rapid Ca2+-induced inactivation. The phosphorylation of corresponding Ser2814 (a preferential target for CaMKII) of the hRyR2 was significantly diminished. RyR2 protein expression and Ser2808 phosphorylation level were both unchanged. Further, we demonstrated that cardiomyocyte Ca2+ mishandling was associated with reduced SERCA pump activity due to decreased Thr17-PLN phosphorylation, suggesting a downregulation of CaMKII in MetS hearts, though the SR Ca2+ load remained unchanged. The reduction in the phosphorylation level of RyR2 at Ser2814 decreases RyR2 availability for activation during ECC. In conclusion, the impaired in situ activity of RyR2 may also account for the poor overall cardiac outcome reported in MetS patients; hence, the SERCA pump and RyR2 are both attractive potential targets for future therapies.
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Affiliation(s)
- Gaudencio Fernández-Miranda
- Departamento de Bioquímica, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV), Mexico City, Mexico
| | - Tatiana Romero-Garcia
- Departamento de Bioquímica, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV), Mexico City, Mexico
| | - Tarín P Barrera-Lechuga
- Departamento de Bioquímica, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV), Mexico City, Mexico
| | - Martha Mercado-Morales
- Departamento de Bioquímica, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV), Mexico City, Mexico
| | - Angélica Rueda
- Departamento de Bioquímica, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV), Mexico City, Mexico
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37
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Sheard TD, Hurley ME, Colyer J, White E, Norman R, Pervolaraki E, Narayanasamy KK, Hou Y, Kirton HM, Yang Z, Hunter L, Shim JU, Clowsley AH, Smith AJ, Baddeley D, Soeller C, Colman MA, Jayasinghe I. Three-Dimensional and Chemical Mapping of Intracellular Signaling Nanodomains in Health and Disease with Enhanced Expansion Microscopy. ACS NANO 2019; 13:2143-2157. [PMID: 30715853 PMCID: PMC6396323 DOI: 10.1021/acsnano.8b08742] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/16/2018] [Accepted: 02/04/2019] [Indexed: 05/08/2023]
Abstract
Nanodomains are intracellular foci which transduce signals between major cellular compartments. One of the most ubiquitous signal transducers, the ryanodine receptor (RyR) calcium channel, is tightly clustered within these nanodomains. Super-resolution microscopy has previously been used to visualize RyR clusters near the cell surface. A majority of nanodomains located deeper within cells have remained unresolved due to limited imaging depths and axial resolution of these modalities. A series of enhancements made to expansion microscopy allowed individual RyRs to be resolved within planar nanodomains at the cell periphery and the curved nanodomains located deeper within the interiors of cardiomyocytes. With a resolution of ∼ 15 nm, we localized both the position of RyRs and their individual phosphorylation for the residue Ser2808. With a three-dimensional imaging protocol, we observed disturbances to the RyR arrays in the nanometer scale which accompanied right-heart failure caused by pulmonary hypertension. The disease coincided with a distinct gradient of RyR hyperphosphorylation from the edge of the nanodomain toward the center, not seen in healthy cells. This spatial profile appeared to contrast distinctly from that sustained by the cells during acute, physiological hyperphosphorylation when they were stimulated with a β-adrenergic agonist. Simulations of RyR arrays based on the experimentally determined channel positions and phosphorylation signatures showed how the nanoscale dispersal of the RyRs during pathology diminishes its intrinsic likelihood to ignite a calcium signal. It also revealed that the natural topography of RyR phosphorylation could offset potential heterogeneity in nanodomain excitability which may arise from such RyR reorganization.
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Affiliation(s)
- Thomas
M. D. Sheard
- School
of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom
| | - Miriam E. Hurley
- School
of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom
| | - John Colyer
- School
of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom
| | - Ed White
- School
of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom
| | - Ruth Norman
- School
of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom
| | - Eleftheria Pervolaraki
- School
of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom
| | - Kaarjel K. Narayanasamy
- School
of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom
| | - Yufeng Hou
- Institute
of Experimental Medical Research, Oslo University
Hospital Ullevål, Oslo 0407, Norway
| | - Hannah M. Kirton
- School
of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom
| | - Zhaokang Yang
- School
of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom
| | - Liam Hunter
- School
of Physics and Astronomy, Faculty of Mathematics and Physical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom
| | - Jung-uk Shim
- School
of Physics and Astronomy, Faculty of Mathematics and Physical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom
| | | | - Andrew J. Smith
- School
of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom
| | - David Baddeley
- Auckland
Bioengineering Institute, University of
Auckland, UniServices
House, Level, 6/70 Symonds St, Grafton, Auckland 1010, New Zealand
| | - Christian Soeller
- Living
Systems Institute, University of Exeter, Devon EX4 4QL, United Kingdom
| | - Michael A. Colman
- School
of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom
| | - Izzy Jayasinghe
- School
of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom
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38
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Hegyi B, Bers DM, Bossuyt J. CaMKII signaling in heart diseases: Emerging role in diabetic cardiomyopathy. J Mol Cell Cardiol 2019; 127:246-259. [PMID: 30633874 DOI: 10.1016/j.yjmcc.2019.01.001] [Citation(s) in RCA: 95] [Impact Index Per Article: 15.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/27/2018] [Accepted: 01/04/2019] [Indexed: 02/07/2023]
Abstract
Calcium/calmodulin-dependent protein kinase II (CaMKII) is upregulated in diabetes and significantly contributes to cardiac remodeling with increased risk of cardiac arrhythmias. Diabetes is frequently associated with atrial fibrillation, coronary artery disease, and heart failure, which may further enhance CaMKII. Activation of CaMKII occurs downstream of neurohormonal stimulation (e.g. via G-protein coupled receptors) and involve various posttranslational modifications including autophosphorylation, oxidation, S-nitrosylation and O-GlcNAcylation. CaMKII signaling regulates diverse cellular processes in a spatiotemporal manner including excitation-contraction and excitation-transcription coupling, mechanics and energetics in cardiac myocytes. Chronic activation of CaMKII results in cellular remodeling and ultimately arrhythmogenic alterations in Ca2+ handling, ion channels, cell-to-cell coupling and metabolism. This review addresses the detrimental effects of the upregulated CaMKII signaling to enhance the arrhythmogenic substrate and trigger mechanisms in the heart. We also briefly summarize preclinical studies using kinase inhibitors and genetically modified mice targeting CaMKII in diabetes. The mechanistic understanding of CaMKII signaling, cardiac remodeling and arrhythmia mechanisms may reveal new therapeutic targets and ultimately better treatment in diabetes and heart disease in general.
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Affiliation(s)
- Bence Hegyi
- Department of Pharmacology, University of California Davis, Davis, CA, USA
| | - Donald M Bers
- Department of Pharmacology, University of California Davis, Davis, CA, USA.
| | - Julie Bossuyt
- Department of Pharmacology, University of California Davis, Davis, CA, USA
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Potenza DM, Janicek R, Fernandez-Tenorio M, Camors E, Ramos-Mondragón R, Valdivia HH, Niggli E. Phosphorylation of the ryanodine receptor 2 at serine 2030 is required for a complete β-adrenergic response. J Gen Physiol 2018; 151:131-145. [PMID: 30541771 PMCID: PMC6363414 DOI: 10.1085/jgp.201812155] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2018] [Revised: 08/12/2018] [Accepted: 10/22/2018] [Indexed: 11/20/2022] Open
Abstract
Phosphorylation is thought to play a role in modulation of the ryanodine receptor 2 channel. Using a S2030A knock-in mouse model, Potenza et al. reveal that phosphorylation of RyR2-S2030 mediates channel regulation during the β-adrenergic response. During physical exercise or stress, the sympathetic system stimulates cardiac contractility via β-adrenergic receptor (β-AR) activation, resulting in protein kinase A (PKA)–mediated phosphorylation of the cardiac ryanodine receptor RyR2. PKA-dependent “hyperphosphorylation” of the RyR2 channel has been proposed as a major impairment that contributes to progression of heart failure. However, the sites of PKA phosphorylation and their phosphorylation status in cardiac diseases are not well defined. Among the known RyR2 phosphorylation sites, serine 2030 (S2030) remains highly controversial as a site of functional impact. We examined the contribution of RyR2-S2030 to Ca2+ signaling and excitation–contraction coupling (ECC) in a transgenic mouse with an ablated RyR2-S2030 phosphorylation site (RyR2-S2030A+/+). We assessed ECC gain by using whole-cell patch–clamp recordings and confocal Ca2+ imaging during β-ARs stimulation with isoproterenol (Iso) and consistent SR Ca2+ loading and L-type Ca2+ current (ICa) triggering. Under these conditions, ECC gain is diminished in mutant compared with WT cardiomyocytes. Resting Ca2+ spark frequency (CaSpF) with Iso is also reduced by mutation of S2030. In permeabilized cells, when SR Ca2+ pump activity is kept constant (using 2D12 antibody against phospholamban), cAMP does not change CaSpF in S2030A+/+ myocytes. Using Ca2+ spark recovery analysis, we found that mutant RyR Ca2+ sensitivity is not enhanced by Iso application, contrary to WT RyRs. Furthermore, ablation of RyR2-S2030 prevents acceleration of Ca2+ waves and increases latency to the first spontaneous Ca2+ release after a train of stimulations during Iso treatment. Together, these results suggest that phosphorylation at S2030 may represent an important step in the modulation of RyR2 activity during β-adrenergic stimulation and a potential target for the development of new antiarrhythmic drugs.
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Affiliation(s)
| | | | | | - Emmanuel Camors
- Center for Arrhythmia Research, Department of Medicine, University of Michigan, Ann Arbor, MI
| | - Roberto Ramos-Mondragón
- Center for Arrhythmia Research, Department of Medicine, University of Michigan, Ann Arbor, MI
| | - Héctor H Valdivia
- Department of Medicine, Wisconsin Institutes for Medical Research, University of Wisconsin, Madison, WI.,Center for Arrhythmia Research, Department of Medicine, University of Michigan, Ann Arbor, MI
| | - Ernst Niggli
- Department of Physiology, University of Bern, Bern, Switzerland
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Zhu W, Wang C, Hu J, Wan R, Yu J, Xie J, Ma J, Guo L, Ge J, Qiu Y, Chen L, Liu H, Yan X, Liu X, Ye J, He W, Shen Y, Wang C, Mohler PJ, Hong K. Ankyrin-B Q1283H Variant Linked to Arrhythmias Via Loss of Local Protein Phosphatase 2A Activity Causes Ryanodine Receptor Hyperphosphorylation. Circulation 2018; 138:2682-2697. [PMID: 30571258 PMCID: PMC6276866 DOI: 10.1161/circulationaha.118.034541] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/23/2018] [Accepted: 08/10/2018] [Indexed: 11/16/2022]
Abstract
BACKGROUND Human loss-of-function variants of ANK2 (ankyrin-B) are linked to arrhythmias and sudden cardiac death. However, their in vivo effects and specific arrhythmogenic pathways have not been fully elucidated. METHODS We identified new ANK2 variants in 25 unrelated Han Chinese probands with ventricular tachycardia by whole-exome sequencing. The potential pathogenic variants were validated by Sanger sequencing. We performed functional and mechanistic experiments in ankyrin-B knockin (KI) mouse models and in single myocytes isolated from KI hearts. RESULTS We detected a rare, heterozygous ANK2 variant (p.Q1283H) in a proband with recurrent ventricular tachycardia. This variant was localized to the ZU5C region of ANK2, where no variants have been previously reported. KI mice harboring the p.Q1283H variant exhibited an increased predisposition to ventricular arrhythmias after catecholaminergic stress in the absence of cardiac structural abnormalities. Functional studies illustrated an increased frequency of delayed afterdepolarizations and Ca2+ waves and sparks accompanied by decreased sarcoplasmic reticulum Ca2+ content in KI cardiomyocytes on isoproterenol stimulation. The immunoblotting results showed increased levels of phosphorylated ryanodine receptor Ser2814 in the KI hearts, which was further amplified on isoproterenol stimulation. Coimmunoprecipitation experiments demonstrated dissociation of protein phosphatase 2A from ryanodine receptor in the KI hearts, which was accompanied by a decreased binding of ankyrin-B to protein phosphatase 2A regulatory subunit B56α. Finally, the administration of metoprolol or flecainide decreased the incidence of stress-induced ventricular arrhythmias in the KI mice. CONCLUSIONS ANK2 p.Q1283H is a disease-associated variant that confers susceptibility to stress-induced arrhythmias, which may be prevented by the administration of metoprolol or flecainide. This variant is associated with the loss of protein phosphatase 2A activity, increased phosphorylation of ryanodine receptor, exaggerated delayed afterdepolarization-mediated trigger activity, and arrhythmogenesis.
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Affiliation(s)
- Wengen Zhu
- Department of Cardiovascular Medicine (W.Z., C.W., J.H., J.Y., J.M., L.G., J.G., H.L., K.H.), Second Affiliated Hospital of Nanchang University, China
| | - Cen Wang
- Department of Cardiovascular Medicine (W.Z., C.W., J.H., J.Y., J.M., L.G., J.G., H.L., K.H.), Second Affiliated Hospital of Nanchang University, China
| | - Jinzhu Hu
- Department of Cardiovascular Medicine (W.Z., C.W., J.H., J.Y., J.M., L.G., J.G., H.L., K.H.), Second Affiliated Hospital of Nanchang University, China
| | - Rong Wan
- Jiangxi Key Laboratory of Molecular Medicine (R.W., J.X., X.Y., X.L., W.H., Y.S., K.H.), Second Affiliated Hospital of Nanchang University, China
| | - Jianhua Yu
- Department of Cardiovascular Medicine (W.Z., C.W., J.H., J.Y., J.M., L.G., J.G., H.L., K.H.), Second Affiliated Hospital of Nanchang University, China
| | - Jinyan Xie
- Jiangxi Key Laboratory of Molecular Medicine (R.W., J.X., X.Y., X.L., W.H., Y.S., K.H.), Second Affiliated Hospital of Nanchang University, China
| | - Jianyong Ma
- Department of Cardiovascular Medicine (W.Z., C.W., J.H., J.Y., J.M., L.G., J.G., H.L., K.H.), Second Affiliated Hospital of Nanchang University, China
| | - Linjuan Guo
- Department of Cardiovascular Medicine (W.Z., C.W., J.H., J.Y., J.M., L.G., J.G., H.L., K.H.), Second Affiliated Hospital of Nanchang University, China
| | - Jin Ge
- Department of Cardiovascular Medicine (W.Z., C.W., J.H., J.Y., J.M., L.G., J.G., H.L., K.H.), Second Affiliated Hospital of Nanchang University, China
| | - Yumin Qiu
- Department of General Surgery (Y.Q., L.C.), Second Affiliated Hospital of Nanchang University, China
| | - Leifeng Chen
- Department of General Surgery (Y.Q., L.C.), Second Affiliated Hospital of Nanchang University, China
| | - Hualong Liu
- Department of Cardiovascular Medicine (W.Z., C.W., J.H., J.Y., J.M., L.G., J.G., H.L., K.H.), Second Affiliated Hospital of Nanchang University, China
| | - Xia Yan
- Jiangxi Key Laboratory of Molecular Medicine (R.W., J.X., X.Y., X.L., W.H., Y.S., K.H.), Second Affiliated Hospital of Nanchang University, China
| | - Xiuxia Liu
- Jiangxi Key Laboratory of Molecular Medicine (R.W., J.X., X.Y., X.L., W.H., Y.S., K.H.), Second Affiliated Hospital of Nanchang University, China
| | - Jin Ye
- Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui (J.Y., C.W.)
| | - Wenfeng He
- Jiangxi Key Laboratory of Molecular Medicine (R.W., J.X., X.Y., X.L., W.H., Y.S., K.H.), Second Affiliated Hospital of Nanchang University, China
| | - Yang Shen
- Jiangxi Key Laboratory of Molecular Medicine (R.W., J.X., X.Y., X.L., W.H., Y.S., K.H.), Second Affiliated Hospital of Nanchang University, China
| | - Chao Wang
- Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui (J.Y., C.W.)
| | - Peter J. Mohler
- Department of Physiology and Cell Biology, The Ohio State University Wexner Medical Center, College of Medicine, The Dorothy M. Davis Heart and Lung Research Institute, Departments of Physiology and Cell Biology and Internal Medicine, Columbus (P.J.M.)
| | - Kui Hong
- Department of Cardiovascular Medicine (W.Z., C.W., J.H., J.Y., J.M., L.G., J.G., H.L., K.H.), Second Affiliated Hospital of Nanchang University, China
- Jiangxi Key Laboratory of Molecular Medicine (R.W., J.X., X.Y., X.L., W.H., Y.S., K.H.), Second Affiliated Hospital of Nanchang University, China
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Denham NC, Pearman CM, Caldwell JL, Madders GWP, Eisner DA, Trafford AW, Dibb KM. Calcium in the Pathophysiology of Atrial Fibrillation and Heart Failure. Front Physiol 2018; 9:1380. [PMID: 30337881 PMCID: PMC6180171 DOI: 10.3389/fphys.2018.01380] [Citation(s) in RCA: 106] [Impact Index Per Article: 15.1] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2018] [Accepted: 09/11/2018] [Indexed: 12/20/2022] Open
Abstract
Atrial fibrillation (AF) is commonly associated with heart failure. A bidirectional relationship exists between the two-AF exacerbates heart failure causing a significant increase in heart failure symptoms, admissions to hospital and cardiovascular death, while pathological remodeling of the atria as a result of heart failure increases the risk of AF. A comprehensive understanding of the pathophysiology of AF is essential if we are to break this vicious circle. In this review, the latest evidence will be presented showing a fundamental role for calcium in both the induction and maintenance of AF. After outlining atrial electrophysiology and calcium handling, the role of calcium-dependent afterdepolarizations and atrial repolarization alternans in triggering AF will be considered. The atrial response to rapid stimulation will be discussed, including the short-term protection from calcium overload in the form of calcium signaling silencing and the eventual progression to diastolic calcium leak causing afterdepolarizations and the development of an electrical substrate that perpetuates AF. The role of calcium in the bidirectional relationship between heart failure and AF will then be covered. The effects of heart failure on atrial calcium handling that promote AF will be reviewed, including effects on both atrial myocytes and the pulmonary veins, before the aspects of AF which exacerbate heart failure are discussed. Finally, the limitations of human and animal studies will be explored allowing contextualization of what are sometimes discordant results.
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Affiliation(s)
- Nathan C. Denham
- Unit of Cardiac Physiology, Division of Cardiovascular Sciences, Manchester Academic Health Science Centre, University of Manchester, Manchester, United Kingdom
| | | | | | | | | | | | - Katharine M. Dibb
- Unit of Cardiac Physiology, Division of Cardiovascular Sciences, Manchester Academic Health Science Centre, University of Manchester, Manchester, United Kingdom
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Laver DR. Regulation of the RyR channel gating by Ca 2+ and Mg 2. Biophys Rev 2018; 10:1087-1095. [PMID: 29926426 PMCID: PMC6082316 DOI: 10.1007/s12551-018-0433-4] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2018] [Accepted: 06/06/2018] [Indexed: 12/21/2022] Open
Abstract
Ryanodine receptors (RyRs) are the Ca2+ release channels in the sarcoplasmic reticulum in striated muscle which play an important role in excitation-contraction coupling and cardiac pacemaking. Single channel recordings have revealed a wealth of information about ligand regulation of RyRs from mammalian skeletal and cardiac muscle (RyR1 and RyR2, respectively). RyR subunit has a Ca2+ activation site located in the luminal and cytoplasmic domains of the RyR. These sites synergistically feed into a common gating mechanism for channel activation by luminal and cytoplasmic Ca2+. RyRs also possess two inhibitory sites in their cytoplasmic domains with Ca2+ affinities of the order of 1 μM and 1 mM. Magnesium competes with Ca2+ at these sites to inhibit RyRs and this plays an important role in modulating their Ca2+-dependent activity in muscle. This review focuses on how these sites lead to RyR modulation by Ca2+ and Mg2+ and how these mechanisms control Ca2+ release in excitation-contraction coupling and cardiac pacemaking.
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Affiliation(s)
- Derek R Laver
- School of Biomedical Sciences and Pharmacy, University of Newcastle and Hunter Medical Research Institute, Callaghan, NSW, 2308, Australia.
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43
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Denniss A, Dulhunty AF, Beard NA. Ryanodine receptor Ca 2+ release channel post-translational modification: Central player in cardiac and skeletal muscle disease. Int J Biochem Cell Biol 2018; 101:49-53. [PMID: 29775742 DOI: 10.1016/j.biocel.2018.05.004] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2018] [Revised: 05/10/2018] [Accepted: 05/13/2018] [Indexed: 12/21/2022]
Abstract
Calcium release from internal stores is a quintessential event in excitation-contraction coupling in cardiac and skeletal muscle. The ryanodine receptor Ca2+ release channel is embedded in the internal sarcoplasmic reticulum Ca2+ store, which releases Ca2+ into the cytoplasm, enabling contraction. Ryanodine receptors form the hub of a macromolecular complex extending from the extracellular space to the sarcoplasmic reticulum lumen. Ryanodine receptor activity is influenced by the integrated effects of associated co-proteins, ions, and post-translational phosphor and redox modifications. In healthy muscle, ryanodine receptors are phosphorylated and redox modified to basal levels, to support cellular function. A pathological increase in the degree of both post-translational modifications disturbs intracellular Ca2+ signalling, and is implicated in various cardiac and skeletal disorders. This review summarises our current understanding of the mechanisms linking ryanodine receptor post-translational modification to heart failure and skeletal myopathy and highlights the challenges and controversies within the field.
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Affiliation(s)
- Amanda Denniss
- Health Research Institute, Faculty of Science and Technology, University of Canberra, Bruce, ACT, Australia
| | - Angela F Dulhunty
- John Curtin School of Medical Research, The Australian National University, Acton, ACT, Australia
| | - Nicole A Beard
- Health Research Institute, Faculty of Science and Technology, University of Canberra, Bruce, ACT, Australia.
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Ranieri A, Kemp E, Burgoyne JR, Avkiran M. β-Adrenergic regulation of cardiac type 2A protein phosphatase through phosphorylation of regulatory subunit B56δ at S573. J Mol Cell Cardiol 2017; 115:20-31. [PMID: 29294329 PMCID: PMC5823843 DOI: 10.1016/j.yjmcc.2017.12.016] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/02/2017] [Revised: 12/19/2017] [Accepted: 12/29/2017] [Indexed: 11/18/2022]
Abstract
Background Type 2A protein phosphatase (PP2A) enzymes are serine/threonine phosphatases which comprise a scaffold A subunit, a regulatory B subunit and a catalytic C subunit, and have been implicated in the dephosphorylation of multiple cardiac phosphoproteins. B subunits determine subcellular targeting, substrate specificity and catalytic activity, and can themselves be regulated by post-translational modifications. We explored potential β-adrenergic regulation of PP2A in cardiomyocytes through phosphorylation of the regulatory B subunit isoform B56δ. Methods and results Phosphate affinity SDS-PAGE and immunoblot analysis revealed increased phosphorylation of B56δ in adult rat ventricular myocytes (ARVM) exposed to the β-adrenergic receptor (βAR) agonist isoprenaline (ISO). Phosphorylation of B56δ occurred at S573, primarily through stimulation of the β1AR subtype, and was dependent on PKA activity. The functional role of the phosphorylation was explored in ARVM transduced with adenoviruses expressing wild type (WT) or non-phosphorylatable (S573A) B56δ, fused to GFP at the N-terminus. C subunit expression was increased in ARVM expressing GFP-B56δ-WT or GFP-B56δ-S573A, both of which co-immunoprecipitated with endogenous C and A subunits. PP2A activity in cell lysates was increased in response to ISO in ARVM expressing GFP-B56δ-WT but not GFP-B56δ-S573A. Immunoblot analysis of the phosphoproteome in ARVM expressing GFP-B56δ-WT or GFP-B56δ-S573A with antibodies detecting (i) phospho-serine/threonine residues in distinct kinase substrate motifs or (ii) specific phosphorylated residues of functional importance in selected proteins revealed a comparable phosphorylation profile in the absence or presence of ISO stimulation. Conclusions In cardiomyocytes, βAR stimulation induces PKA-mediated phosphorylation of the PP2A regulatory subunit isoform B56δ at S573, which increases associated PP2A catalytic activity. This is likely to regulate the phosphorylation status of specific B56δ-PP2A substrates, which remain to be identified.
PP2A subunit B56δ is phosphorylated on β-adrenergic stimulation of cardiomyocytes. Phosphorylation occurs at Ser573 and increases B56δ-PP2A catalytic activity. Response is mediated by the β1-adrenoceptor subtype and protein kinase A. Phosphorylated B56δ abundance is increased in pathological cardiac hypertrophy.
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Affiliation(s)
- Antonella Ranieri
- School of Cardiovascular Medicine and Sciences, King's College London British Heart Foundation Centre of Research Excellence, The Rayne Institute, St Thomas' Hospital, London, United Kingdom
| | - Elizabeth Kemp
- School of Cardiovascular Medicine and Sciences, King's College London British Heart Foundation Centre of Research Excellence, The Rayne Institute, St Thomas' Hospital, London, United Kingdom
| | - Joseph R Burgoyne
- School of Cardiovascular Medicine and Sciences, King's College London British Heart Foundation Centre of Research Excellence, The Rayne Institute, St Thomas' Hospital, London, United Kingdom
| | - Metin Avkiran
- School of Cardiovascular Medicine and Sciences, King's College London British Heart Foundation Centre of Research Excellence, The Rayne Institute, St Thomas' Hospital, London, United Kingdom.
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45
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Andrei SR, Ghosh M, Sinharoy P, Dey S, Bratz IN, Damron DS. TRPA1 ion channel stimulation enhances cardiomyocyte contractile function via a CaMKII-dependent pathway. Channels (Austin) 2017; 11:587-603. [PMID: 28792844 DOI: 10.1080/19336950.2017.1365206] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
RATIONALE Transient receptor potential channels of the ankyrin subtype-1 (TRPA1) are non-selective cation channels that show high permeability to calcium. Previous studies from our laboratory have demonstrated that TRPA1 ion channels are expressed in adult mouse ventricular cardiomyocytes (CMs) and are localized at the z-disk, costamere and intercalated disk. The functional significance of TRPA1 ion channels in the modulation of CM contractile function have not been explored. OBJECTIVE To identify the extent to which TRPA1 ion channels are involved in modulating CM contractile function and elucidate the cellular mechanism of action. METHODS AND RESULTS Freshly isolated CMs were obtained from murine heart and loaded with Fura-2 AM. Simultaneous measurement of intracellular free Ca2+ concentration ([Ca2+]i) and contractility was performed in individual CMs paced at 0.3 Hz. Our findings demonstrate that TRPA1 stimulation with AITC results in a dose-dependent increase in peak [Ca2+]i and a concomitant increase in CM fractional shortening. Further analysis revealed a dose-dependent acceleration in time to peak [Ca2+]i and velocity of shortening as well as an acceleration in [Ca2+]i decay and velocity of relengthening. These effects of TRPA1 stimulation were not observed in CMs pre-treated with the TRPA1 antagonist, HC-030031 (10 µmol/L) nor in CMs obtained from TRPA1-/- mice. Moreover, we observed no significant increase in cAMP levels or PKA activity in response to TRPA1 stimulation and the PKA inhibitor peptide (PKI 14-22; 100 nmol/L) failed to have any effect on the TRPA1-mediated increase in CM contractile function. However, TRPA1 stimulation resulted in a rapid phosphorylation of Ca2+/calmodulin-dependent kinase II (CaMKII) (1-5 min) that correlated with increases in CM [Ca2+]i and contractile function. Finally, all aspects of TRPA1-dependent increases in CM [Ca2+]i, contractile function and CaMKII phosphorylation were virtually abolished by the CaMKII inhibitors, KN-93 (10 µmol/L) and autocamtide-2-related peptide (AIP; 20 µmol/L). CONCLUSIONS These novel findings demonstrate that stimulation of TRPA1 ion channels in CMs results in activation of a CaMKII-dependent signaling pathway resulting in modulation of intracellular Ca2+ availability and handling leading to increases in CM contractile function. Cardiac TRPA1 ion channels may represent a novel therapeutic target for increasing the inotropic and lusitropic state of the heart.
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Affiliation(s)
- Spencer R Andrei
- a Department of Medicine , Vanderbilt University Medical Center , Nashville , TN , USA
| | - Monica Ghosh
- b Department of Biological Sciences , Kent State University , Kent , OH , USA
| | - Pritam Sinharoy
- c Department of Anesthesia , Perioperative and Pain Medicine, Stanford School of Medicine , Stanford , CA , USA
| | - Souvik Dey
- b Department of Biological Sciences , Kent State University , Kent , OH , USA
| | - Ian N Bratz
- d Department of Integrated Medical Sciences , Northeast Ohio Medical University , Rootstown , OH , USA
| | - Derek S Damron
- b Department of Biological Sciences , Kent State University , Kent , OH , USA
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Jones PP, Guo W, Chen SRW. Control of cardiac ryanodine receptor by sarcoplasmic reticulum luminal Ca 2. J Gen Physiol 2017; 149:867-875. [PMID: 28798281 PMCID: PMC5583710 DOI: 10.1085/jgp.201711805] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2017] [Revised: 06/25/2017] [Accepted: 07/18/2017] [Indexed: 12/22/2022] Open
Abstract
Jones et al. propose that SR luminal Ca2+ regulates RyR2 activity via a luminal Ca2+ sensor distinct from the cytosolic Ca2+ sensor.
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Affiliation(s)
- Peter P Jones
- Department of Physiology, School of Biomedical Sciences, University of Otago, Dunedin, Otago, New Zealand .,HeartOtago, University of Otago, Dunedin, Otago, New Zealand
| | - Wenting Guo
- Libin Cardiovascular Institute of Alberta, Department of Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada
| | - S R Wayne Chen
- Libin Cardiovascular Institute of Alberta, Department of Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada
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Gonano LA, Jones PP. FK506-binding proteins 12 and 12.6 (FKBPs) as regulators of cardiac Ryanodine Receptors: Insights from new functional and structural knowledge. Channels (Austin) 2017. [PMID: 28636428 DOI: 10.1080/19336950.2017.1344799] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022] Open
Abstract
Ryanodine Receptors (RyRs) are intracellular Ca2+ channels that mediate Ca2+ flux from the sarco(endo)plasmic reticulum in many cell types. The interaction of RyRs with FK506-binding proteins (FKBPs) has been proposed as an important regulatory mechanism, where the loss of this interaction leads to channel dysfunction. In the heart, phosphorylation of RyR has been suggested to disrupt the RyR-FKBP interaction promoting altered Ca2+ signaling, heart failure and arrhythmias. However, the functional result of FKBP interaction with RyR and how this interaction is regulated remains highly controversial. Recently, high resolution structures of RyR have provided novel aspects to the ongoing debate. This review will discuss the most recent functional data in light of these new structures.
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Affiliation(s)
- Luis A Gonano
- a Department of Physiology , School of Biomedical Sciences and HeartOtago, University of Otago , Dunedin, Otago , New Zealand
| | - Peter P Jones
- a Department of Physiology , School of Biomedical Sciences and HeartOtago, University of Otago , Dunedin, Otago , New Zealand
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48
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The effect of PKA-mediated phosphorylation of ryanodine receptor on SR Ca 2+ leak in ventricular myocytes. J Mol Cell Cardiol 2017; 104:9-16. [PMID: 28131630 DOI: 10.1016/j.yjmcc.2017.01.015] [Citation(s) in RCA: 35] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/13/2016] [Revised: 11/22/2016] [Accepted: 01/24/2017] [Indexed: 01/08/2023]
Abstract
Functional impact of cardiac ryanodine receptor (type 2 RyR or RyR2) phosphorylation by protein kinase A (PKA) remains highly controversial. In this study, we characterized a functional link between PKA-mediated RyR2 phosphorylation level and sarcoplasmic reticulum (SR) Ca2+ release and leak in permeabilized rabbit ventricular myocytes. Changes in cytosolic [Ca2+] and intra-SR [Ca2+]SR were measured with Fluo-4 and Fluo-5N, respectively. Changes in RyR2 phosphorylation at two PKA sites, serine-2031 and -2809, were measured with phospho-specific antibodies. cAMP (10μM) increased Ca2+ spark frequency approximately two-fold. This effect was associated with an increase in SR Ca2+ load from 0.84 to 1.24mM. PKA inhibitory peptide (PKI; 10μM) abolished the cAMP-dependent increase of SR Ca2+ load and spark frequency. When SERCA was completely blocked by thapsigargin, cAMP did not affect RyR2-mediated Ca2+ leak. The lack of a cAMP effect on RyR2 function can be explained by almost maximal phosphorylation of RyR2 at serine-2809 after sarcolemma permeabilization. This high RyR2 phosphorylation level is likely the consequence of a balance shift between protein kinase and phosphatase activity after permeabilization. When RyR2 phosphorylation at serine-2809 was reduced to its "basal" level (i.e. RyR2 phosphorylation level in intact myocytes) using kinase inhibitor staurosporine, SR Ca2+ leak was significantly reduced. Surprisingly, further dephosphorylation of RyR2 with protein phosphatase 1 (PP1) markedly increased SR Ca2+ leak. At the same time, phosphorylation of RyR2 at serine 2031 did not significantly change under identical experimental conditions. These results suggest that RyR2 phosphorylation by PKA has a complex effect on SR Ca2+ leak in ventricular myocytes. At an intermediate level of RyR2 phosphorylation SR Ca2+ leak is minimal. However, complete dephosphorylation and maximal phosphorylation of RyR2 increases SR Ca2+ leak.
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Alvarado FJ, Chen X, Valdivia HH. Ablation of the cardiac ryanodine receptor phospho-site Ser2808 does not alter the adrenergic response or the progression to heart failure in mice. Elimination of the genetic background as critical variable. J Mol Cell Cardiol 2017; 103:40-47. [PMID: 28065668 DOI: 10.1016/j.yjmcc.2017.01.001] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/15/2016] [Accepted: 01/04/2017] [Indexed: 10/20/2022]
Abstract
BACKGROUND Phosphorylation of the cardiac ryanodine receptor (RyR2) phospho-site S2808 has been touted by the Marks group as a hallmark of heart failure (HF) and a critical mediator of the physiological fight-or-flight response of the heart. In support of this hypothesis, mice unable to undergo phosphorylation at RyR2-S2808 (S2808A) were significantly protected against HF and displayed a blunted response to adrenergic stimulation. However, the issue remains highly controversial because several groups have been unable to reproduce these findings. An important variable not considered before is the genetic background of the mice used to obtain these divergent results. METHODS AND RESULTS We backcrossed a RyR2-S2808A mouse into a congenic C57Bl/6 strain, the same strain used by the Marks group to conduct their experiments. We then performed several key experiments to confirm or discard the genetic background of the mouse as a relevant variable, including induction of HF by myocardial infarction and tests of integrity of adrenergic response. Congenic C57Bl/6 harboring the S2808A mutation showed similar echocardiographic parameters that indicated identical progression towards HF compared to wild type controls, and had a normal response to adrenergic stimulation in whole animal and cellular experiments. CONCLUSIONS The genetic background of the different mouse models is unlikely to be the source of the divergent results obtained by the Marks group in comparison to several other groups. Cardiac adrenergic response and progression towards HF proceed unaltered in mice harboring the RyR2-S2808A mutation. Preventing RyR2-S2808 phosphorylation does not preclude a normal sympathetic response nor mitigates the pathophysiological consequences of MI.
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Affiliation(s)
- Francisco J Alvarado
- Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, United States.
| | - Xi Chen
- Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, United States
| | - Héctor H Valdivia
- Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, United States; Center for Arrhythmia Research, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, United States.
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Ho HT, Belevych AE, Liu B, Bonilla IM, Radwański PB, Kubasov IV, Valdivia HH, Schober K, Carnes CA, Györke S. Muscarinic Stimulation Facilitates Sarcoplasmic Reticulum Ca Release by Modulating Ryanodine Receptor 2 Phosphorylation Through Protein Kinase G and Ca/Calmodulin-Dependent Protein Kinase II. Hypertension 2016; 68:1171-1178. [PMID: 27647848 DOI: 10.1161/hypertensionaha.116.07666] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2016] [Accepted: 08/21/2016] [Indexed: 01/01/2023]
Abstract
Although the effects and the underlying mechanism of sympathetic stimulation on cardiac Ca handling are relatively well established both in health and disease, the modes of action and mechanisms of parasympathetic modulation are poorly defined. Here, we demonstrate that parasympathetic stimulation initiates a novel mode of excitation-contraction coupling that enhances the efficiency of cardiac sarcoplasmic reticulum Ca store utilization. This efficient mode of excitation-contraction coupling involves reciprocal changes in the phosphorylation of ryanodine receptor 2 at Ser-2808 and Ser-2814. Specifically, Ser-2808 phosphorylation was mediated by muscarinic receptor subtype 2 and activation of PKG (protein kinase G), whereas dephosphorylation of Ser-2814 involved activation of muscarinic receptor subtype 3 and decreased reactive oxygen species-dependent activation of CaMKII (Ca/calmodulin-dependent protein kinase II). The overall effect of these changes in phosphorylation of ryanodine receptor 2 is an increase in systolic Ca release at the low sarcoplasmic reticulum Ca content and a paradoxical reduction in aberrant Ca leak. Accordingly, cholinergic stimulation of cardiomyocytes isolated from failing hearts improved Ca cycling efficiency by restoring altered ryanodine receptor 2 phosphorylation balance.
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Affiliation(s)
- Hsiang-Ting Ho
- From the Department of Physiology and Cell Biology (H.-T.H., A.E.B., B.L., P.B.R., S.G.), College of Pharmacy (I.M.B., P.B.R., C.A.C.), and College of Veterinary Medicine (K.S.), The Ohio State University, Columbus; Davis Heart and Lung Research Institute, Columbus, OH (H.-T.H., A.E.B., B.L., I.M.B., P.B.R., C.A.C., S.G.); Department of Medicine, Duke University, Durham, NC (H.-T.H.); Laboratory of Neuromuscular Physiology, I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Petersburg, Russia (I.V.K.); and Center for Arrhythmia Research, Cardiovascular Division of the Department of Internal Medicine, University of Michigan, Ann Arbor (H.H.V.)
| | - Andriy E Belevych
- From the Department of Physiology and Cell Biology (H.-T.H., A.E.B., B.L., P.B.R., S.G.), College of Pharmacy (I.M.B., P.B.R., C.A.C.), and College of Veterinary Medicine (K.S.), The Ohio State University, Columbus; Davis Heart and Lung Research Institute, Columbus, OH (H.-T.H., A.E.B., B.L., I.M.B., P.B.R., C.A.C., S.G.); Department of Medicine, Duke University, Durham, NC (H.-T.H.); Laboratory of Neuromuscular Physiology, I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Petersburg, Russia (I.V.K.); and Center for Arrhythmia Research, Cardiovascular Division of the Department of Internal Medicine, University of Michigan, Ann Arbor (H.H.V.)
| | - Bin Liu
- From the Department of Physiology and Cell Biology (H.-T.H., A.E.B., B.L., P.B.R., S.G.), College of Pharmacy (I.M.B., P.B.R., C.A.C.), and College of Veterinary Medicine (K.S.), The Ohio State University, Columbus; Davis Heart and Lung Research Institute, Columbus, OH (H.-T.H., A.E.B., B.L., I.M.B., P.B.R., C.A.C., S.G.); Department of Medicine, Duke University, Durham, NC (H.-T.H.); Laboratory of Neuromuscular Physiology, I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Petersburg, Russia (I.V.K.); and Center for Arrhythmia Research, Cardiovascular Division of the Department of Internal Medicine, University of Michigan, Ann Arbor (H.H.V.)
| | - Ingrid M Bonilla
- From the Department of Physiology and Cell Biology (H.-T.H., A.E.B., B.L., P.B.R., S.G.), College of Pharmacy (I.M.B., P.B.R., C.A.C.), and College of Veterinary Medicine (K.S.), The Ohio State University, Columbus; Davis Heart and Lung Research Institute, Columbus, OH (H.-T.H., A.E.B., B.L., I.M.B., P.B.R., C.A.C., S.G.); Department of Medicine, Duke University, Durham, NC (H.-T.H.); Laboratory of Neuromuscular Physiology, I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Petersburg, Russia (I.V.K.); and Center for Arrhythmia Research, Cardiovascular Division of the Department of Internal Medicine, University of Michigan, Ann Arbor (H.H.V.)
| | - Przemysław B Radwański
- From the Department of Physiology and Cell Biology (H.-T.H., A.E.B., B.L., P.B.R., S.G.), College of Pharmacy (I.M.B., P.B.R., C.A.C.), and College of Veterinary Medicine (K.S.), The Ohio State University, Columbus; Davis Heart and Lung Research Institute, Columbus, OH (H.-T.H., A.E.B., B.L., I.M.B., P.B.R., C.A.C., S.G.); Department of Medicine, Duke University, Durham, NC (H.-T.H.); Laboratory of Neuromuscular Physiology, I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Petersburg, Russia (I.V.K.); and Center for Arrhythmia Research, Cardiovascular Division of the Department of Internal Medicine, University of Michigan, Ann Arbor (H.H.V.)
| | - Igor V Kubasov
- From the Department of Physiology and Cell Biology (H.-T.H., A.E.B., B.L., P.B.R., S.G.), College of Pharmacy (I.M.B., P.B.R., C.A.C.), and College of Veterinary Medicine (K.S.), The Ohio State University, Columbus; Davis Heart and Lung Research Institute, Columbus, OH (H.-T.H., A.E.B., B.L., I.M.B., P.B.R., C.A.C., S.G.); Department of Medicine, Duke University, Durham, NC (H.-T.H.); Laboratory of Neuromuscular Physiology, I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Petersburg, Russia (I.V.K.); and Center for Arrhythmia Research, Cardiovascular Division of the Department of Internal Medicine, University of Michigan, Ann Arbor (H.H.V.)
| | - Héctor H Valdivia
- From the Department of Physiology and Cell Biology (H.-T.H., A.E.B., B.L., P.B.R., S.G.), College of Pharmacy (I.M.B., P.B.R., C.A.C.), and College of Veterinary Medicine (K.S.), The Ohio State University, Columbus; Davis Heart and Lung Research Institute, Columbus, OH (H.-T.H., A.E.B., B.L., I.M.B., P.B.R., C.A.C., S.G.); Department of Medicine, Duke University, Durham, NC (H.-T.H.); Laboratory of Neuromuscular Physiology, I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Petersburg, Russia (I.V.K.); and Center for Arrhythmia Research, Cardiovascular Division of the Department of Internal Medicine, University of Michigan, Ann Arbor (H.H.V.)
| | - Karsten Schober
- From the Department of Physiology and Cell Biology (H.-T.H., A.E.B., B.L., P.B.R., S.G.), College of Pharmacy (I.M.B., P.B.R., C.A.C.), and College of Veterinary Medicine (K.S.), The Ohio State University, Columbus; Davis Heart and Lung Research Institute, Columbus, OH (H.-T.H., A.E.B., B.L., I.M.B., P.B.R., C.A.C., S.G.); Department of Medicine, Duke University, Durham, NC (H.-T.H.); Laboratory of Neuromuscular Physiology, I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Petersburg, Russia (I.V.K.); and Center for Arrhythmia Research, Cardiovascular Division of the Department of Internal Medicine, University of Michigan, Ann Arbor (H.H.V.)
| | - Cynthia A Carnes
- From the Department of Physiology and Cell Biology (H.-T.H., A.E.B., B.L., P.B.R., S.G.), College of Pharmacy (I.M.B., P.B.R., C.A.C.), and College of Veterinary Medicine (K.S.), The Ohio State University, Columbus; Davis Heart and Lung Research Institute, Columbus, OH (H.-T.H., A.E.B., B.L., I.M.B., P.B.R., C.A.C., S.G.); Department of Medicine, Duke University, Durham, NC (H.-T.H.); Laboratory of Neuromuscular Physiology, I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Petersburg, Russia (I.V.K.); and Center for Arrhythmia Research, Cardiovascular Division of the Department of Internal Medicine, University of Michigan, Ann Arbor (H.H.V.)
| | - Sándor Györke
- From the Department of Physiology and Cell Biology (H.-T.H., A.E.B., B.L., P.B.R., S.G.), College of Pharmacy (I.M.B., P.B.R., C.A.C.), and College of Veterinary Medicine (K.S.), The Ohio State University, Columbus; Davis Heart and Lung Research Institute, Columbus, OH (H.-T.H., A.E.B., B.L., I.M.B., P.B.R., C.A.C., S.G.); Department of Medicine, Duke University, Durham, NC (H.-T.H.); Laboratory of Neuromuscular Physiology, I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Petersburg, Russia (I.V.K.); and Center for Arrhythmia Research, Cardiovascular Division of the Department of Internal Medicine, University of Michigan, Ann Arbor (H.H.V.).
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