Alectinib is the first-line therapy for anaplastic lymphoma kinase rearranged non-small cell lung cancer globally. Sinus bradycardia, as the major adverse cardiac events of alectinib, still widely impact patient's quality of life. However, its underlying mechanism remains elusive. The aim of this study was to reveal the pathogenesis of the alectinib induced sinus bradycardia (AISB) in a rat model, including the electrophysiology alterations and the molecular mechanism. SD rats were administered alectinib (10 mg/kg/day) by gavage for 7-10 days to mimic the clinical AISB. 3-days alectinib treatment did not change heart rate and sinus node recovery time (SNRT) as assessed through in vivo electrophysiology study. Also, alectinib didn't influence automaticity in isolated heart or single sinus node cardiomyocytes, indicating alectinib cannot decrease sinus node function rapidly. The decreased heart rate and prolonged SNRT was found after 7-days alectinib treatment. The inducibility of atrial fibrillation was not affected under the same condition. The RNA-seq assay revealed the transcriptomic alterations in sinus node of alectinib treated rats, and the dysregulation of genes in cardiac function were observed. The decreased expression of L-type calcium channel Cacna1d was confirmed among the channel candidates identified from RNA-seq assay. Subsequently, the patch-clamp test revealed the reduction in the corresponding L-type calcium current density in 7-days alectinib treated rats. These findings revealed that the AISB was caused by the reduction in Cacna1d expression which resulted in the electro-dysfunction mediated by the suppressed ICaL.

Pituitary cells are specialized cells located within the pituitary gland, a small, pea-sized gland situated at the base of the brain. Through the use of cellular electrophysiological techniques, the electrical properties of these cells have been revealed. This review paper aims to introduce the ion currents that are known to be functionally expressed in pituitary cells. These currents include a voltage-gated Na+ current (INa), erg-mediated K+ current (IK(erg)), M-type K+ current (IK(M)), hyperpolarization-activated cation current (Ih), and large-conductance Ca2+-activated K+ (BKCa) channel. The biophysical characteristics of the respective ion current were described. Additionally, we also provide explanations for the effect of various drugs or compounds on each of these currents. GH3-cell exposure to GV-58 can increase the magnitude of INa with a concurrent rise in the inactivation time constant of the current. The presence of esaxerenone, an antagonist of the aldosterone receptor, directly suppresses the magnitude of peak and late INa. Risperidone, an atypical antipsychotic agent, is effective at suppressing the IK(erg) amplitude directly, and di(2-ethylhexyl)-phthalate suppressed IK(erg). Solifenacin and kynurenic acid can interact with the KM channel to stimulate IK(M), while carisbamate and cannabidiol inhibit the Ih amplitude activated by sustained hyperpolarization. Moreover, the presence of either rufinamide or QO-40 can enhance the activity of single BKCa channels. To summarize, alterations in ion currents within native pituitary cells or pituitary tumor cells can influence their functional activity, particularly in processes like stimulus-secretion coupling. The effects of small-molecule modulators, as demonstrated here, bear significance in clinical, therapeutic, and toxicological contexts.

Cardiac arrhythmias pose a major public health problem, and pharmacologic intervention remains key to their therapy. The 1970 landmark Vaughan Williams (VW) classification utilizing known actions of then available antiarrhythmic drugs (AADs) became and remains central to management, but it requires revision in response to extensive subsequent advances. Our modernized AAD classification reflected and sought to facilitate such fundamental physiological and clinical development. Here we respond to requests for an adaptation of our scheme specifically focused on clinical practice. (1) This adaptation improves the accessibility of our original scheme to clinical practice, focusing on key AADs in clinical use rather than investigational new drugs (INDs) while conserving and encompassing the classic VW scheme. (2) We preserve a rational conceptual framework based on current understanding of the relevant electrophysiological events, their underlying cellular or molecular cardiomyocyte targets, and the functional mechanisms they mediate. (3) The adopted subclasses within each AAD class parallel clinical practice by including only subclasses containing established AADs, or approved potential off-label drugs, as opposed to those only including INDs. (4) The simplified scheme remains flexible, permitting drugs to be placed in multiple classes where required, and the addition of classes and subclasses in light of future investigations and clinical approvals. Thus, we derive from our comprehensive modernized AAD classification a more focused and simpler scheme for clinical use. This both modernizes yet preserves the classic VW classification and remains flexible, thus accommodating future developments.

Heart rate (HR) affects heart failure outcomes, via uncertain mechanisms that may include left ventricular remodeling. However, in human ventricular myocardium, HR change has not been associated with a particular remodeling molecular phenotype.

Patients with nonischemic dilated cardiomyopathy (N=22) in sinus rhythm and refractory to β-blockade for both HR lowering and reverse remodeling were randomized 2:1 double-blind to the HCN4 (hyperpolarization-activated cyclic nucleotide-gated potassium channel 4) channel inhibitor ivabradine or placebo for 24 weeks treatment while maintaining target doses of β-blockers. Reverse remodeling was measured by left ventricular ejection fraction (LVEF), and myocardial gene expression by sequencing RNA extracted from endomyocardial biopsies. The primary statistical analysis was between HR change categories divided at the median, which resulted in Decreased HR (N=90) and Unchanged HR (N=8) groups.

Respective HRs at baseline and 24 weeks were as follows: Decreased HR, 82.9±6.8 and 69.7±8.0 beats per minute (P=0.0005) and Unchanged HR, 80.8±5.7 and 79.2±11.6 beats per minute (P=0.58). All completing Decreased HR subjects were treated with ivabradine, whereas in the Unchanged HR group, 3 received ivabradine and 5 placebo. In Decreased HR, LVEF increased from 29.4±8.8% at baseline to 44.2±9.4% at 24 weeks (P=0.0003), compared with respective values of 26.6±11.4% and 29.2±12.0% (P=0.28) in Unchanged HR. HR and LVEF changes were not different from a previously conducted β-blocker nonischemic dilated cardiomyopathy study subdivided into LVEF responders and nonresponders. However, differentially expressed genes (N=151) in the Decreased versus Unchanged HR groups were >99% nonconcordant and therefore individually unique compared with β-blocker HR/LVEF responders versus nonresponders (2 shared differentially expressed genes). Multiple unique differentially expressed genes in Decreased HR including NRG1 upregulation are considered cardioprotective or involved in cardiac development.

In patients with nonischemic dilated cardiomyopathy in sinus rhythm, HR lowering per se (1) is associated with substantial left ventricular reverse remodeling; (2) its absence can cause β-blocker reverse remodeling nonresponse; and (3) when from HCN4 channel inhibition, results in a unique molecular phenotype.

URL: https://www.clinicaltrials.gov; Unique identifier: NCT02973594.

Tissue elasticity is essential to a broad spectrum of cell biology and organ function including the heart. Routine cell culture models on rigid polystyrene dishes are limited in studying the impact of tissue elasticity in distinct regions of the myocardium such as the cardiac conduction system. Gelatin, a derivative of collagen, is a simple and tunable platform for modeling tissue elasticity. We sought to study the effects of increasing tissue stiffness on cardiac pacemaker cell function by using transcription factor-reprogrammed pacemaker cells cultured on gelatin hydrogels with specific elasticity. Our data indicate that automaticity of the pacemaker cells, measured in rhythmic contractions and oscillating intracellular Ca2+ transients, was enhanced when cultured on a stiffer matrix of 14 kPa. This was accompanied by increased expression of cardiac pacemaker ion channel, Hcn4, and a reciprocal decrease in Cx43 expression compared with control conditions. Propagation of Ca2+ transients was slower in the pacemaker cell monolayers compared with control, which recapitulates a hallmark feature in the native pacemaker tissue. Ca2+ transient propagation of pacemaker cell monolayer was slower on stiffer than on softer hydrogel, and this was dependent on enhanced proliferation of cardiac fibroblasts rather than differences in gap junctional coupling. Culturing the pacemaker cells on rigid plastic plates led to irregular or loss of synchronous contractions as well as unusually long Ca2+ transient durations. Taken together, our data demonstrate that automaticity of pacemaker cells is augmented by stiffer extracellular matrix substrates within the elasticity range of the healthy myocardium. This simple approach presents a physiological in vitro model to study mechanoelectric feedback of cardiomyocytes including the conduction system cells.NEW & NOTEWORTHY The major achievement of this work is development of a robust and straightforward approach to model cardiac conduction system cells with a range of cardiac tissue elasticity with a goal to understand the impact of tissue stiffness on cardiac pacing. Our data provide a framework for further investigation of the heart rhythm in health and disease in the context of fibrosis.

Negative emotions can have a significant impact on individuals, which then influences their cardiovascular system. However, the underlying pathophysiological mechanisms and clinical implications of this association remain inadequately defined. A narrative review of pertinent literature was conducted to examine the pathophysiology, clinical manifestations, and treatment related to the interplay between emotions and conditions such as takotsubo cardiomyopathy, atherosclerosis, acute plaque rupture, and cardiac arrhythmias. Negative emotions can instigate a chronic stress response, which in turn heightens sympathetic nervous system activity and increases vulnerability to cardiovascular diseases. This intricate relationship between emotional states and cardiovascular health underscores the necessity for targeted lifestyle interventions and clinical strategies aimed at mitigating the adverse effects of negative emotions.

Hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) drives the funny current in cardiac pacemaker regions. Its involvement in sinoatrial node pacemaker generation is well known, but its function in the atrioventricular (AV) node (AVN) has not intensively been studied. HCN4 is expressed in the AVN, and its expression within the AVN seems similar across mammalian species with HCN4 presence in the inferior nodal extensions, compact node, and AV bundle. The main direct regulators of HCN4 are cAMP and protein kinase A. In addition, indirect regulators may affect HCN4 via trafficking and localization. However, these effects are underexplored in the AVN. AVN-specific effects in knockout and knockin mice include reduced funny current density and increased AV block. HCN4 expression in the AVN could be affected by aging, exercise, heart failure, and diabetes. This could underlie changes in PR interval, atria-His interval, Wenckebach cycle length, and AVN effective refractory period. Clinical reports link the HCN4 variant G1097W to AV block. Other clinical data come from studies assessing ivabradine, an HCN4 inhibitor. In animals, ivabradine resulted in prolonged PR and atrial-his intervals. To date, uncertainty regarding the role of HCN4 in the AVN remains. However, AVN-focused studies suggest HCN4's importance for AVN function. This review summarizes recent findings and highlights the involvement of HCN4 in normal and pathological AVN function.

Integrating biomedical electronic devices holds profound promise for advancements in healthcare and enhancing individuals' quality of life. However, the persistent challenges associated with the traditional batteries' limited lifespan and bulkiness hinder these devices' long-term functionality and consistent power supply. Here, we delve into the biology and material interfaces in self-powered medical devices by summarizing the intrinsic electric demands in humans, analyzing material and biological mechanisms for electricity generation and storage, and discussing the pathways toward self-chargeable powering. As a result, the current challenges in material designs and biological integrations emerged to shape the future directions in advancing self-powered medical devices. This paper calls on the community to integrate biology and material science to develop self-powering medical devices and improve their clinical prospects.

In the human sinoatrial node (SAN), HCN4 is the primary subtype among the four HCN (hyperpolarization activated cyclic nucleotide-gated) family subtypes. A tetramer of HCN subunits forms the ion channel conducting the hyperpolarization-activated "funny" current (If), which plays an important regulatory role in maintaining the pacemaker activity of the SAN. With the advancement of detection technologies over the past 20 years, the relationship between base mutations in the HCN4 gene encoding the HCN4 protein and arrhythmias has been continuously elucidated. The expression and kinetic changes of mutated channels were investigated in COS-7, CHO, HEK-293T cells, and Xenopus oocytes, but their functional changes were not elucidated in human myocardial cells. New genome editing methods, such as Base editor and Prime editor, use components of the CRISPR system and other enzymes to directly install single-gene mutation into cellular DNA without causing double-stranded DNA breaks, which reproduce and correct base mutations. In this review, we summarize all base mutations of the HCN4 gene, discuss the clinical characteristics and function of some base mutations, and combine base editors to explore the establishment of disease models and the potential for future gene correction.

In mammals, the four subunit isoforms HCN1-4 assemble to form functional homotetrameric and heterotetrameric hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channels. Despite the outstanding relevance of HCN channels for organisms, including generating electrical rhythmicity in cardiac pacemaker cells and diverse types of brain neurons, key channel properties are still elusive. In particular, the unitary conductance, γ, of HCN channels is highly controversial. We analyzed the unitary conductance at femtosiemens resolution of all four homotetrameric channels of the mouse, mHCN1-4. All conductance values are in the range of 1 pS which is exceptionally small compared to most other ion channels. Surprisingly, the conductance among the isoforms differs up to threefold (γmHCN2 = 1.54 pS > γmHCN1 = 0.84 pS > γmHCN3 = 0.54 pS ≈ γmHCN4 = 0.51 pS) though the residues in the two narrow parts of the pore, the selectivity filter and the inner gate, are conserved. Mutagenesis and all-atom molecular dynamics simulations demonstrate that the differences in the conductance are generated by different amounts of negative charges in the outer channel vestibule, which control ion accumulation. In line with these results, heterotetrameric channels exhibit intermediate unitary conductance values with respect to the homotetrameric channels. Our approach demonstrates how HCN channels can be functionally differentiated at the single-channel level, paving the way to target specific channels with selective drugs.

Cardiac arrhythmias stemming from abnormal sinoatrial node (SAN) function can lead to sudden death. Developing a biological pacemaker device for treating sick sinus syndrome (SSS) could offer a potential cure. Understanding SAN differentiation is crucial, yet its regulatory mechanism remains unclear. We reanalyzed published RNA-seq data and identified Odz4 as a SAN-specific candidate. In situ hybridization revealed Odz4 expression in the cardiac crescent and throughout the cardiac conduction system (CCS). To assess the role of Odz4 in CCS differentiation, we utilized a Tet-Off inducible system for its intracellular domain (ICD). Embryonic bodies (EBs) exogenously expressing Odz4-ICD exhibited an increased propensity to develop into pacemaker-like cells with enhanced automaticity and upregulated expression of SAN-specific genes. CellChat and GO analyses unveiled SAN-specific enrichment of ligand-receptor sets, especially Ptn-Ncl, and extracellular matrix components in the group exogenously expressing Odz4-ICD. Our findings underscore the significance of Odz4 in SAN development and offer fresh insights into biological pacemaker establishment.

What physiological role does a slow hyperpolarization-activated ion channel with mixed cation selectivity play in the fast world of neuronal action potentials that are driven by depolarization? That puzzling question has piqued the curiosity of physiology enthusiasts about the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are widely expressed across the body and especially in neurons. In this review, we emphasize the need to assess HCN channels from the perspective of how they respond to time-varying signals, while also accounting for their interactions with other co-expressing channels and receptors. First, we illustrate how the unique structural and functional characteristics of HCN channels allow them to mediate a slow negative feedback loop in the neurons that they express in. We present the several physiological implications of this negative feedback loop to neuronal response characteristics including neuronal gain, voltage sag and rebound, temporal summation, membrane potential resonance, inductive phase lead, spike triggered average, and coincidence detection. Next, we argue that the overall impact of HCN channels on neuronal physiology critically relies on their interactions with other co-expressing channels and receptors. Interactions with other channels allow HCN channels to mediate intrinsic oscillations, earning them the "pacemaker channel" moniker, and to regulate spike frequency adaptation, plateau potentials, neurotransmitter release from presynaptic terminals, and spike initiation at the axonal initial segment. We also explore the impact of spatially non-homogeneous subcellular distributions of HCN channels in different neuronal subtypes and their interactions with other channels and receptors. Finally, we discuss how plasticity in HCN channels is widely prevalent and can mediate different encoding, homeostatic, and neuroprotective functions in a neuron. In summary, we argue that HCN channels form an important class of channels that mediate a diversity of neuronal functions owing to their unique gating kinetics that made them a puzzle in the first place.

The sinoatrial node (SN) generates the heart rate (HR). Its spontaneous activity is regulated by a complex interplay between the modulation by the autonomic nervous system (ANS) and intrinsic factors including ion channels in SN cells. However, the systemic and intrinsic regulatory mechanisms are still poorly understood. This study aimed to elucidate the sex-specific differences in heart morphology and SN function, particularly focusing on basal HR, expression and function of hyperpolarization-activated HCN4 and HCN1 channels and mRNA abundance of ion channels and mRNA abundance of ion channels contributing to diastolic depolarization (DD) and spontaneous action potentials (APs).

Body weight, heart weight and tibia length of 2- to 3-month-old male and female mice were measured. Conscious in-vivo HR of male and female mice was recorded via electrocardiography (ECG). Unconscious ex-vivo HR, stroke volume (SV) and ejection fraction (EF) were recorded via echocardiography. Ex-vivo HR was measured via Langendorff apparatus. Volume of atria, ventricles and whole hearts were measured from the ex-vivo hearts by microcomputed tomography (micro-CT). Immunohistochemistry targeting HCN4 and HCN1 was conducted in the SN and RA tissues from both male and female hearts. The funny current (I f) of SN cells in 1 nM and following wash-on of 1 μM isoproterenol (ISO) were recorded via whole cell patch clamp. The APs of SN tissue were recorded via sharp microelectrode and optical mapping of membrane voltage. The relative abundance of mRNAs was measured in male and female mice by qPCR.

Heart weight to tibia length ratio and heart volume of females were significantly smaller than males. Unconscious in-vivo HR in male mice was higher than that in females. Conscious in-vivo HR, ex-vivo HR, SV, and EF showed no notable difference between male and female mice. Immunohistochemistry revealed HCN4, HCN1, and the sum of HCN4 and HCN1, expression in the SN was notably elevated compared with the RA in both male and females, but there was no sex difference in these channels expression. There were also no significant sex differences in the V 0.5 of I f in SN cells in the presence of 1 nM ISO, however wash-on 1 μM ISO in the same cells induced a significantly increased shift of V 0.5 to more positive voltages in males than in females. The expression of mRNA coding for adrenergic receptor beta-1 (Adrb1) and cholinergic receptors muscarinic 2 (chrm2) in male mice was higher compared with that in female mice. Early diastolic depolarization (EDD) rate in APs from peripheral SN (pSN) from male mice were higher than these in female mice. Mice of both sexes showed equivalent frequency of SN APs and spatial localization of the leading site in control, and similar significant response to ISO 100 nM superfusion.

Males display faster in-vivo HR, but not ex-vivo HR, than females associated with increased expression of Adrb1 in male versus female. This suggests a possible difference in the β-adrenergic modulation in males and females, possibly related to the greater ISO response of I f observed in cells from males. The role of hormonal influences or differential expression of other ion channels may explain these sex-specific variations in HR dynamics. Further investigations are necessary to pinpoint the precise molecular substrates responsible for these differences.

Shenxian-Shengmai (SXSM) is a Chinese patent medicine used in the treatment of sick sinus syndrome (SSS). However, its active chemical compounds and the underlying molecular mechanisms remain unclear. In this study, we researched the underlying mechanisms of SXSM in treating SSS. We conducted network analysis and molecular docking to identify the small molecules and core targets responsible for the therapeutic efficacy of SXSM on SSS. In vitro experiments were performed to verify the potential therapeutic mechanism. Network pharmacological analysis identified 17 core targets. Among these, BMP4, KCNH2, KCNMA1, and KCNQ1 were identified to be involved in various biological processes, such as the formation and regulation of the cardiac pacemaking system and potassium ion transmembrane transport. The experimental analysis revealed that SXSM could upregulate the expression of the Bmp4/Tbx3/Hcn4 pathway and the expression of Kcnh2, Kcnma1, and Kcnq1 channels, which protected and improved the pacemaking function of pacemaker cells (P cells) and increased the heart rate. These findings provide a scientific basis in the study of the mechanism of traditional Chinese medicine in the treatment of SSS.

Pharmaceutical companies routinely screen compounds for hemodynamics related safety risk. In vitro secondary pharmacology is initially used to prioritize compounds while in vivo studies are later used to quantify and translate risk to humans. This strategy has shown limitations but could be improved via the incorporation of molecular findings in the animal-based toxicological risk assessment. The aim of this study is to develop a mathematical model for rat and dog species that can integrate secondary pharmacology modulation and therefore facilitate the overall pre-clinical safety translation assessment. Following an extensive literature review, we built two separate models recapitulating known regulation processes in dogs and rats. We describe the resulting models and show that they can reproduce a variety of interventions in both species. We also show that the models can incorporate the mechanisms of action of a pre-defined list of 50 pharmacological mechanisms whose modulation predict results consistent with known pharmacology. In conclusion, a mechanistic model of hemodynamics regulations in rat and dog species has been developed to support mechanism-based safety translation in drug discovery and development.

Perioperative myocardial injury after noncardiac surgery is associated with postoperative mortality. Heart rate (HR) is an independent risk factor for perioperative myocardial injury. In this pilot trial we tested the feasibility of a randomised, placebo-controlled trial of personalised HR-targeted perioperative ivabradine.

This was a single-centre, randomised, placebo-controlled, double-blind, parallel group, feasibility pilot trial conducted at Geneva University Hospitals. We included patients ≥75 yr old or ≥45 yr old with cardiovascular risk factors planned for intermediate- or high-risk surgery. Patients were randomised to receive ivabradine (2.5, 5.0, or 7.5 mg) or placebo according to their HR, twice daily, from the morning of surgery until postoperative day 2. Primary outcomes were appropriate dosage and blinding success rates.

Between October 2020 and January 2022, we randomised 78 patients (recruitment rate of 1.3 patients week-1). Some 439 of 444 study drug administrations were adequate (99% appropriate dosage rate). The blinding success rate was 100%. There were 137 (31%) administrations of Pill A (placebo in both groups for HR ≤70 beats min-1). Nine (11.5%) patients had a high-sensitive cardiac troponin T elevation ≥14 ng L-1 between any two measurements. The number of bradycardia episodes was eight in the placebo group and nine in the ivabradine group.

This pilot study demonstrates the feasibility of, and provides guidance for, a future trial testing the efficacy of personalised perioperative ivabradine. Future studies should include patients at higher risk of cardiac complications.

NCT04436016.

The cardiac conduction system, composed of pacemaker cells and conducting cardiomyocytes, orchestrates the propagation of electrical activity to synchronize heartbeats. The conduction system plays a crucial role in the development of cardiac arrhythmias. In the embryo, the cells of the conduction system derive from the same cardiac progenitors as the contractile cardiomyocytes and and the key question is how this choice is made during development.

This review focuses on recent advances in developmental biology using the mouse as animal model to better understand the cellular origin and molecular regulations that control morphogenesis of the cardiac conduction system, including the latest findings in single-cell transcriptomics. The conducting cell fate is acquired during development starting with pacemaking activity and last with the formation of a complex fast-conducting network. Cardiac conduction system morphogenesis is controlled by complex transcriptional and gene regulatory networks that differ in the components of the cardiac conduction system.

The adenovirus-mediated somatic transfer of the embryonic T-box transcription factor 18 (TBX18) gene can convert chamber cardiomyocytes into induced pacemaker cells. However, the translation of therapeutic TBX18-induced cardiac pacing faces safety challenges. Here we show that the myocardial expression of synthetic TBX18 mRNA in animals generates de novo pacing and limits innate and inflammatory immune responses. In rats, intramyocardially injected mRNA remained localized, whereas direct myocardial injection of an adenovirus carrying a reporter gene resulted in diffuse expression and in substantial spillover to the liver, spleen and lungs. Transient expression of TBX18 mRNA in rats led to de novo automaticity and pacemaker properties and, compared with the injection of adenovirus, to substantial reductions in the expression of inflammatory genes and in activated macrophage populations. In rodent and clinically relevant porcine models of complete heart block, intramyocardially injected TBX18 mRNA provided rate-adaptive cardiac pacing for one month that strongly correlated with the animal's sinus rhythm and physical activity. TBX18 mRNA may aid the development of biological pacemakers.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) show tremendous promise for cardiac regeneration following myocardial infarction (MI), but their transplantation gives rise to transient ventricular tachycardia (VT) in large-animal MI models, representing a major hurdle to translation. Our group previously reported that these arrhythmias arise from a focal mechanism whereby graft tissue functions as an ectopic pacemaker; therefore, we hypothesized that hPSC-CMs engineered with a dominant negative form of the pacemaker ion channel HCN4 (dnHCN4) would exhibit reduced automaticity and arrhythmogenic risk following transplantation.

We used CRISPR/Cas9-mediated gene-editing to create transgenic dnHCN4 hPSC-CMs, and their electrophysiological behavior was evaluated in vitro by patch-clamp recordings and optical mapping. Next, we transplanted WT and homozygous dnHCN4 hPSC-CMs in a pig MI model and compared post-transplantation outcomes including the incidence of spontaneous arrhythmias and graft structure by immunohistochemistry.

In vitro dnHCN4 hPSC-CMs exhibited significantly reduced automaticity and pacemaker funny current (I f ) density relative to wildtype (WT) cardiomyocytes. Following transplantation with either dnHCN4 or WT hPSC-CMs, all recipient hearts showed transmural infarct scar that was partially remuscularized by scattered islands of human myocardium. However, in contrast to our hypothesis, both dnHCN4 and WT hPSC-CM recipients exhibited frequent episodes of ventricular tachycardia (VT).

While genetic silencing of the pacemaker ion channel HCN4 suppresses the automaticity of hPSC-CMs in vitro, this intervention is insufficient to reduce VT risk post-transplantation in the pig MI model, implying more complex mechanism(s) are operational in vivo.

HCN1-4 channels are the molecular determinants of the If/Ih current that crucially regulates cardiac and neuronal cell excitability. HCN dysfunctions lead to sinoatrial block (HCN4), epilepsy (HCN1), and chronic pain (HCN2), widespread medical conditions awaiting subtype-specific treatments. Here, we address the problem by solving the cryo-EM structure of HCN4 in complex with ivabradine, to date the only HCN-specific drug on the market. Our data show ivabradine bound inside the open pore at 3 Å resolution. The structure unambiguously proves that Y507 and I511 on S6 are the molecular determinants of ivabradine binding to the inner cavity, while F510, pointing outside the pore, indirectly contributes to the block by controlling Y507. Cysteine 479, unique to the HCN selectivity filter (SF), accelerates the kinetics of block. Molecular dynamics simulations further reveal that ivabradine blocks the permeating ion inside the SF by electrostatic repulsion, a mechanism previously proposed for quaternary ammonium ions.

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) have found utility for conducting in vitro drug screening and disease modelling to gain crucial insights into pharmacology or disease phenotype. However, diseases such as atrial fibrillation, affecting >33 M people worldwide, demonstrate the need for cardiac subtype-specific cells. Here, we sought to investigate the base characteristics and pharmacological differences between commercially available chamber-specific atrial or ventricular hiPSC-CMs seeded onto ultra-thin, flexible PDMS membranes to simultaneously measure contractility in a 96 multi-well format. We investigated the effects of GPCR agonists (acetylcholine and carbachol), a Ca2+ channel agonist (S-Bay K8644), an HCN channel antagonist (ivabradine) and K+ channel antagonists (4-AP and vernakalant). We observed differential effects between atrial and ventricular hiPSC-CMs on contractile properties including beat rate, beat duration, contractile force and evidence of arrhythmias at a range of concentrations. As an excerpt of the compound analysis, S-Bay K8644 treatment showed an induced concentration-dependent transient increase in beat duration of atrial hiPSC-CMs, whereas ventricular cells showed a physiological increase in beat rate over time. Carbachol treatment produced marked effects on atrial cells, such as increased beat duration alongside a decrease in beat rate over time, but only minimal effects on ventricular cardiomyocytes. In the context of this chamber-specific pharmacology, we not only add to contractile characterization of hiPSC-CMs but propose a multi-well platform for medium-throughput early compound screening. Overall, these insights illustrate the key pharmacological differences between chamber-specific cardiomyocytes and their application on a multi-well contractility platform to gain insights for in vitro cardiac liability studies and disease modelling.

Lymphoid restricted membrane protein (LRMP) is a specific regulator of the hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channel. LRMP prevents cAMP-dependent potentiation of HCN4, but the interaction domains, mechanisms of action, and basis for isoform-specificity remain unknown. Here, we identify the domains of LRMP essential for this regulation, show that LRMP acts by disrupting the intramolecular signal transduction between cyclic nucleotide binding and gating, and demonstrate that multiple unique regions in HCN4 are required for LRMP isoform-specificity. Using patch clamp electrophysiology and Förster resonance energy transfer (FRET), we identified the initial 227 residues of LRMP and the N-terminus of HCN4 as necessary for LRMP to associate with HCN4. We found that the HCN4 N-terminus and HCN4-specific residues in the C-linker are necessary for regulation of HCN4 by LRMP. Finally, we demonstrated that LRMP-regulation can be conferred to HCN2 by addition of the HCN4 N-terminus along with mutation of five residues in the S5 region and C-linker to the cognate HCN4 residues. Taken together, these results suggest that LRMP inhibits HCN4 through an isoform-specific interaction involving the N-terminals of both proteins that prevents the transduction of cAMP binding into a change in channel gating, most likely via an HCN4-specific orientation of the N-terminus, C-linker, and S4-S5 linker.

In patients with septic shock, compensatory tachycardia initially serves to maintain adequate cardiac output and tissue oxygenation but may persist despite appropriate fluid and vasopressor resuscitation. This sustained elevation in heart rate and altered heart rate variability, indicative of autonomic dysfunction, is a well-established independent predictor of adverse outcomes in critical illness. Elevated heart rate exacerbates myocardial oxygen demand, reduces ventricular filling time, compromises coronary perfusion during diastole, and impairs the isovolumetric relaxation phase of the cardiac cycle, contributing to ventricular-arterial decoupling. This also leads to increased ventricular and atrial filling pressures, with a heightened risk of arrhythmias. Ivabradine, a highly selective inhibitor of the sinoatrial node's pacemaker current (If or "funny" current), mitigates heart rate by modulating diastolic depolarization slope without affecting contractility. By exerting a selective chronotropic effect devoid of negative inotropic properties, ivabradine shows potential for improving hemodynamics in septic shock patients with cardiac dysfunction. This review evaluates the plausible mechanisms and existing evidence regarding the utility of ivabradine in managing patients with septic shock.

Shox2 plays a vital role in the morphogenesis and physiological function of the sinoatrial node (SAN), the primary cardiac pacemaker, manifested by the formation of a hypoplastic SAN and failed differentiation of pacemaker cells in Shox2 mutants. Shox2 and Nkx2-5 are co-expressed in the developing SAN and regulate the fate of the pacemaker cells through a Shox2-Nkx2-5 antagonistic mechanism. Here we show that simultaneous inactivation of Nkx2-5 in the SAN of Shox2 mutants (dKO) rescued the pacemaking cell fate but not the hypoplastic defects, indicating uncoupling of SAN cell fate determination and morphogenesis. Single-cell RNA-seq revealed that the presumptive SAN cells of Shox2-/- mutants failed to activate pacemaking program but remained in a progenitor state preceding working myocardium, while both wildtype and dKO SAN cells displayed normal pacemaking cell fate with similar cellular state. Shox2 thus acts as a safeguard but not a determinant to ensure the pacemaking cell fate through the Shox2-Nkx2-5 antagonistic mechanism, which is segregated from its morphogenetic regulatory function in SAN development.

The heart is the first organ to form during embryonic development, establishing the circulatory infrastructure necessary to sustain life and enable downstream organogenesis. Critical to the heart's function is its ability to initiate and propagate electrical impulses that allow for the coordinated contraction and relaxation of its chambers, and thus, the movement of blood and nutrients. Several specialized structures within the heart, collectively known as the cardiac conduction system (CCS), are responsible for this phenomenon. In this review, we discuss the discovery and scientific history of the mammalian cardiac conduction system as well as the key genes and transcription factors implicated in the formation of its major structures. We also describe known human diseases related to CCS development and explore existing challenges in the clinical context.

Preclinical data have confirmed that human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can remuscularize the injured or diseased heart, with several clinical trials now in planning or recruitment stages. However, because ventricular arrhythmias represent a complication following engraftment of intramyocardially injected PSC-CMs, it is necessary to provide treatment strategies to control or prevent engraftment arrhythmias (EAs). Here, we show in a porcine model of myocardial infarction and PSC-CM transplantation that EAs are mechanistically linked to cellular heterogeneity in the input PSC-CM and resultant graft. Specifically, we identify atrial and pacemaker-like cardiomyocytes as culprit arrhythmogenic subpopulations. Two unique surface marker signatures, signal regulatory protein α (SIRPA)+CD90-CD200+ and SIRPA+CD90-CD200-, identify arrhythmogenic and non-arrhythmogenic cardiomyocytes, respectively. Our data suggest that modifications to current PSC-CM-production and/or PSC-CM-selection protocols could potentially prevent EAs. We further show that pharmacologic and interventional anti-arrhythmic strategies can control and potentially abolish these arrhythmias.

Lymphoid restricted membrane protein (LRMP) is a specific regulator of the hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channel. LRMP prevents cAMP-dependent potentiation of HCN4 but the interaction domains, mechanisms of action, and basis for isoform-specificity remain unknown. Here we identify the domains of LRMP essential for regulation. We show that LRMP acts by disrupting the intramolecular signal transduction between cyclic nucleotide binding and gating. And we demonstrate that multiple unique regions in HCN4 are required for LRMP isoform-specificity. Using patch clamp electrophysiology and Förster resonance energy transfer (FRET), we showed that the initial 227 residues of LRMP and the N-terminus of HCN4 are necessary for LRMP to interact with HCN4. We found that the HCN4 N-terminus and HCN4-specific residues in the C-linker are necessary for regulation of HCN4 by LRMP. And we demonstrate that LRMP-regulation can be conferred to HCN2 by addition of the HCN4 N-terminus along with mutation of 5 residues in the S5 region and C-linker to the cognate HCN4 residues. Taken together, these results suggest that LRMP inhibits HCN4 through an isoform-specific interaction involving the N-terminals of both proteins that prevents the transduction of cAMP binding into a change in channel gating via an HCN4-specific orientation of the N-terminus, C-linker, and S4-S5 linker.

Our study was conducted to investigate whether cadherin-5 (CDH5), a vascular endothelial cell adhesion glycoprotein, could facilitate the differentiation of human induced pluripotent stem cells (hiPSCs) into sinoatrial node-like pacemaker cells (SANLPCs), following previous findings of silk-fibroin hydrogel-induced direct conversion of quiescent cardiomyocytes into pacemaker cells in rats through the activation of CDH5. In this study, the differentiating hiPSCs were treated with CDH5 (40 ng/mL) between Day 5 and 7 during cardiomyocytes differentiation. The findings in the present study demonstrated that CDH5 stimulated the expression of pacemaker-specific markers while suppressing markers associated with working cardiomyocytes, resulting in an increased proportion of SANLPCs among hiPSCs-derived cardiomyocytes (hiPSC-CMs) population. Moreover, CDH5 induced typical electrophysiological characteristics resembling cardiac pacemaker cells in hiPSC-CMs. Further mechanistic investigations revealed that the enriched differentiation of hiPSCs into SANLPCs induced by CDH5 was partially reversed by iCRT14, an inhibitor of β-catenin. Therefore, based on the aforementioned findings, it could be inferred that the regulation of β-catenin by CDH5 played a crucial role in promoting the enriched differentiation of hiPSCs into SANLPCs, which presents a novel avenue for the construction of biological pacemakers in forthcoming research.

The sympathetic nervous system increases HR by activating β-adrenergic receptors (β-ARs) and increasing cAMP in sinoatrial node (SAN) myocytes while phosphodiesterases (PDEs) degrade cAMP. Chronotropic incompetence, the inability to regulate heart rate (HR) in response to sympathetic nervous system activation, is common in hypertensive heart disease; however, the basis for this is poorly understood. The objective of this study was to determine the mechanisms leading to chronotropic incompetence in mice with angiotensin II (AngII)-induced hypertensive heart disease.

C57BL/6 mice were infused with saline or AngII (2.5 mg/kg/day for 3 weeks) to induce hypertensive heart disease. HR and SAN function in response to the β-AR agonist isoproterenol (ISO) were studied in vivo using telemetry and electrocardiography, in isolated atrial preparations using optical mapping, in isolated SAN myocytes using patch-clamping, and using molecular biology. AngII-infused mice had smaller increases in HR in response to physical activity and during acute ISO injection. Optical mapping of the SAN in AngII-infused mice demonstrated impaired increases in conduction velocity and altered conduction patterns in response to ISO. Spontaneous AP firing responses to ISO in isolated SAN myocytes from AngII-infused mice were impaired due to smaller increases in diastolic depolarization (DD) slope, hyperpolarization-activated current (If), and L-type Ca2+ current (ICa,L). These changes were due to increased localization of PDE4D surrounding β1- and β2-ARs in the SAN, increased SAN PDE4 activity, and reduced cAMP generation in response to ISO. Knockdown of PDE4D using a virus-delivered shRNA or inhibition of PDE4 with rolipram normalized SAN sensitivity to β-AR stimulation in AngII-infused mice.

AngII-induced hypertensive heart disease results in impaired HR responses to β-AR stimulation due to up-regulation of PDE4D and reduced effects of cAMP on spontaneous AP firing in SAN myocytes.

Aims: The behavior of pacemaker cardiomyocytes (PCs) in the sinoatrial node (SAN) is modulated by neurohormonal and paracrine factors, many of which signal through G-protein coupled receptors (GPCRs). The aims of the present study are to catalog GPCRs that are differentially expressed in the mammalian SAN and to define the acute physiological consequences of activating the cholecystokinin-A signaling system in isolated PCs. Methods and results: Using bulk and single cell RNA sequencing datasets, we identify a set of GPCRs that are differentially expressed between SAN and right atrial tissue, including several whose roles in PCs and in the SAN have not been thoroughly characterized. Focusing on one such GPCR, Cholecystokinin-A receptor (CCKAR), we demonstrate expression of Cckar mRNA specifically in mouse PCs, and further demonstrate that subsets of SAN fibroblasts and neurons within the cardiac intrinsic nervous system express cholecystokinin, the ligand for CCKAR. Using mouse models, we find that while baseline SAN function is not dramatically affected by loss of CCKAR, the firing rate of individual PCs is slowed by exposure to sulfated cholecystokinin-8 (sCCK-8), the high affinity ligand for CCKAR. The effect of sCCK-8 on firing rate is mediated by reduction in the rate of spontaneous phase 4 depolarization of PCs and is mitigated by activation of beta-adrenergic signaling. Conclusion: (1) PCs express many GPCRs whose specific roles in SAN function have not been characterized, (2) Activation of the cholecystokinin-A signaling pathway regulates PC automaticity.

Genetic predisposition to cardiac arrhythmias has been a field of intense investigation. Research initially focused on rare hereditary arrhythmias, but over the last two decades, the role of genetic variation (single nucleotide polymorphisms) in heart rate, rhythm, and arrhythmias has been taken into consideration as well. In particular, genome-wide association studies have identified hundreds of genomic loci associated with quantitative electrocardiographic traits, atrial fibrillation, and less common arrhythmias such as Brugada syndrome. A significant number of associated variants have been found to systematically localize in non-coding regulatory elements that control the tissue-specific and temporal transcription of genes encoding transcription factors, ion channels, and other proteins. However, the identification of causal variants and the mechanism underlying their impact on phenotype has proven difficult due to the complex tissue-specific, time-resolved, condition-dependent, and combinatorial function of regulatory elements, as well as their modest conservation across different model species. In this review, we discuss research efforts aimed at identifying and characterizing-trait-associated variant regulatory elements and the molecular mechanisms underlying their impact on heart rate or rhythm.

In a family with inappropriate sinus tachycardia (IST), we identified a mutation (p.V240M) of the hyperpolarization-activated cyclic nucleotide-gated type 4 (HCN4) channel, which contributes to the pacemaker current (If) in human sinoatrial node cells. Here, we clinically study fifteen family members and functionally analyze the p.V240M variant. Macroscopic (IHCN4) and single-channel currents were recorded using patch-clamp in cells expressing human native (WT) and/or p.V240M HCN4 channels. All p.V240M mutation carriers exhibited IST that was accompanied by cardiomyopathy in adults. IHCN4 generated by p.V240M channels either alone or in combination with WT was significantly greater than that generated by WT channels alone. The variant, which lies in the N-terminal HCN domain, increased the single-channel conductance and opening frequency and probability of HCN4 channels. Conversely, it did not modify the channel sensitivity for cAMP and ivabradine or the level of expression at the membrane. Treatment with ivabradine based on functional data reversed the IST and the cardiomyopathy of the carriers. In computer simulations, the p.V240M gain-of-function variant increases If and beating rate and thus explains the IST of the carriers. The results demonstrate the importance of the unique HCN domain in HCN4, which stabilizes the channels in the closed state.