Liraglutide alleviates diabetic cardiomyopathy in streptozotocin-induced diabetic rats by enhancing mitophagy mediated by the AMPK-Parkin signaling pathway

Abstract

BACKGROUND

Recent studies have shown that liraglutide, a glucagon-like peptide-1 receptor agonist, has unexpected cardioprotective effects. However, the distinctive effects of liraglutide on diabetic cardiomyopathy (DCM), particularly its effect on mitophagy, have not been fully elucidated.

AIM

To investigate the effects of liraglutide on cardiac damage and mitophagy in DCM rats.

METHODS

A high-fat diet and streptozotocin were used to induce DCM in rats. After 12 weeks of liraglutide treatment, rats underwent assessments of cardiac function, serum biochemical parameters, histological changes, apoptosis index, and protein levels. Furthermore, neonatal rat cardiomyocytes (NRCMs) were exposed to 25 mmol/L glucose plus 250 μmol/L palmitate (high glucose + palmitic acid), with or without 200 nmol/L liraglutide, to investigate the effects of liraglutide on cardiomyocyte injury and the underlying mechanisms.

RESULTS

Liraglutide improved myocardial function and ameliorated cardiac damage in DCM rats, as indicated by reduced myocardial apoptosis, hypertrophy, and interstitial fibrosis (P < 0.05). In NRCMs, Liraglutide alleviated mitochondrial morphological and functional damage as well as oxidative stress, improved mitophagic defects, and reduced cell apoptosis (P < 0.05). Mechanistically, liraglutide alleviated NRCMs damage by enhancing mitophagy mediated by the adenosine monophosphate-activated protein kinase (AMPK)-Parkin signaling pathway, which was evidenced by the reversal of its effects upon compound C treatment.

CONCLUSION

Liraglutide exerted cardioprotective effects in DCM rats by inhibiting cardiomyocyte apoptosis and promoting mitophagy mediated by the AMPK-Parkin signaling pathway.

Key Words: Diabetic cardiomyopathy; Liraglutide; Apoptosis; Mitochondria; Mitophagy; Parkin; AMPK signaling pathway

Core Tip: This study investigates the cardioprotective effects of liraglutide, a glucagon-like peptide-1 receptor agonist, in a rat model of diabetic cardiomyopathy (DCM) and in neonatal rat cardiomyocytes exposed to high glucose and palmitate. Liraglutide significantly improved cardiac function, alleviated cardiac damage—reducing myocardial apoptosis, hypertrophy, and interstitial fibrosis, and inhibited cardiomyocyte apoptosis by restoring mitochondrial function and promoting mitophagy through activation of the adenosine monophosphate-activated protein kinase-Parkin signaling pathway. These findings provide insight into the role of liraglutide in DCM and support its potential as a therapeutic strategy to mitigate cardiac injury in DCM.

INTRODUCTION

Diabetes mellitus (DM) is a growing healthcare challenge and imposes a heavy burden on global public health budget[1]. According to the International Diabetes Federation, 588.7 million individuals worldwide had diabetes in 2024 and 852.5 million will have the disease by 2050[2]. Diabetic cardiomyopathy (DCM) is a major complication of DM and is characterized by structural and functional dysfunction in the absence of hypertension, coronary artery disease, valvular heart disease, or other cardiac pathologies[3]. Studies consistently show that DCM primarily manifests as myocardial apoptosis, hypertrophy, and fibrosis, as well as cardiac systolic and diastolic dysfunction, leading to an increased risk of heart failure (HF) and sudden death in patients with diabetes[4,5]. Currently, there is a lack of effective therapeutic strategies beyond strict diabetes control to prevent progression to HF, particularly once the stigmata of DCM and consequent diastolic dysfunction have been established. Therefore, identifying specific molecular targets and treatments that address both glycemic control and preservation of cardiac function is particularly important.

Mitochondria are the primary generators of energy, mainly through glucose utilization and fatty acid oxidation. The heart, a highly energy-demanding tissue, is rich in mitochondria to maintain normal contractile function. Diabetic hearts shift away from glucose utilization and depend almost entirely on fatty acids as an energy source, which increases oxidative stress, leading to mitochondrial dysfunction and subsequent release of death-inducing factors, thereby increasing cardiomyocyte injury[6]. Mitophagy is a selective form of autophagy that targets and clears damaged mitochondria[7]. Damaged mitochondria trigger mitophagy to remove defective organelles and recycle essential components through encapsulation in a double-membrane structure. Effective elimination of damaged mitochondria via upregulated mitophagy protects cardiomyocytes against diabetic injury and alleviates cardiac dysfunction[8]. Emerging evidence indicates that defects in mitophagy and mitochondrial homeostasis are common features of aging and DCM[9]. PTEN-induced putative kinase 1 (PINK1)/E3 ubiquitin ligase Parkin-mediated mitophagy is the most well-established protective mechanism in mammals[10]. In response to mitochondrial stress, PINK1 is selectively stabilized on the outer mitochondrial membrane (OMM) and then activated via autophosphorylation. Activated PINK1 recruits Parkin to the OMM, phosphorylates it at the Ser65 site, and activates its E3 ubiquitin ligase activity, which promotes further ubiquitination of OMM proteins[11]. Polyubiquitination catalyzed by Parkin leads to the recruitment of sequestosome-1 (SQSTM1, also referred to as p62), a ubiquitin- and light chain 3 (LC3)-binding adaptor protein[12]. Autophagosomes then engulf damaged mitochondria and transfer them to lysosomes for degradation[7]. A previous study showed that Parkin expression was downregulated and mitophagy was reduced in DCM mice and in cardiomyocytes injured by high glucose (HG)[13]. Additionally, nicorandil treatment suppressed mitochondria-associated ferroptosis and the development of cardiac microvascular dysfunction by promoting Pink1/Parkin-dependent mitophagy in DCM[14]. Canagliflozin activated PINK1-Parkin-dependent mitophagy and improved mitochondrial function via increased phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) under high-glucose challenge in rat cardiomyocytes in vitro[15]. PINK1/Parkin-dependent mitophagy was enhanced by L-carnitine treatment, which reversed mitochondrial dysfunction and cardiac microvascular injury in DCM[16]. Therefore, PINK1/Parkin-mediated mitophagy is critical for cardiac repair in many cardiovascular diseases.

Glucagone-like peptide-1 (GLP-1) is a member of the proglucagon incretin family and is mainly secreted by intestinal L cells in response to food intake, helping maintain glucose homeostasis[17]. GLP-1 receptor agonists (GLP-1RAs) are a cornerstone of type 2 diabetes treatment and have favorable effects on cardiovascular outcomes[18]. Liraglutide, a GLP-1RA, has been widely recognized to reduce cardiovascular mortality and preserve cardiac function in patients with diabetes[19], and its cardioprotective effects are independent of its antihyperglycemic action. Previous studies have suggested that liraglutide’s cardiovascular protection may relate to anti-inflammatory effects, increased stability of atherosclerotic plaques, and improved vascular endothelial function[20,21]. Recent studies also reported that liraglutide upregulated mitophagy—helping restore mitochondrial function and protect pancreatic β-cell damage from oxidative stress[22]; ameliorated delirium-like behaviors in aged mice undergoing cardiac surgery by mitigating microglial activation through promotion of mitophagy[23]; and suppressed nod-like receptor protein 3 (NLRP3) inflammasome-induced hepatocyte pyroptosis by augmenting mitophagy to slow progression of nonalcoholic steatohepatitis[24]. A key pathway that controls diabetes is the AMPK signaling pathway[25]. In several studies, AMPK activation enhanced cellular glucose uptake, inhibited intracellular glucose production, and promoted cytoprotection by conserving energy through suppression of protein translation and stimulation of autophagy[15,25]. Impaired AMPK activity is present in diabetes, and drugs used to treat diabetes, such as metformin, are known to act through the regulation of AMPK[26,27]. Thus, drugs that regulate and activate the AMPK pathway are potential candidates for the management of DM and its complications. Despite these findings, the effects of liraglutide on mitophagy in DCM and the signaling pathways involved remain largely unknown. Therefore, this study aimed to investigate the beneficial effect of liraglutide on cardiac injury in DCM rats and elucidate potentially protective mechanisms related to mitophagy in cardiomyocytes.

MATERIALS AND METHODS

Reagents

Liraglutide was purchased from Novo Nordisk (Bagsværd, Denmark). Streptozotocin (STZ), bromodeoxyuridine, and a Masson trichrome stain kit were obtained from Sigma-Aldrich (St. Louis, MO, United States). Wheat germ agglutinin (WGA) stain kit, mitochondria isolation kit, MitoProbe JC-1, MitoTracker Red dye, LysoTracker Green dye, BCA protein assay kit, and electrochemiluminescence (ECL) kit were purchased from Thermo Fisher Scientific (Waltham, MA, United States). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, United States). CCK-8 was obtained from Dojindo Laboratories (Kumamoto, Japan). ATP assay kit, MitoSOX Red kit and one-step terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) apoptosis assay kit were purchased from Beyotime Biotechnology (Shanghai, China). Alexa Fluor 488 secondary antibody was purchased from Invitrogen (Carlsbad, CA, United States). Anti-NDUF88, anti-SDHB, anti-UQCRC2, anti-MTCO2, anti-ATP5A1, anti-OPA1, anti-FIS1, anti-Parkin, anti-PINK1, and anti-LC3 antibodies were obtained from ProteinTech (Wuhan, Hubei Province, China). Anti-MFN1, anti-Drp1, and anti-cytochrome c antibodies were obtained from Abcam (Cambridge, United Kingdom). Anti-caspase 3, anti-cleaved caspase 3, anti-MFN2, anti-AMPKα, anti-phosphorylated AMPKα, anti-VDAC, anti-β-actin, and anti-GAPDH antibodies were obtained from Cell Signaling Technology (Danvers, MA, United States). Horse radish peroxidase-conjugated secondary antibodies were obtained from ProteinTech (Wuhan, Hubei Province, China). Compound C (AMPK inhibitor) was purchased from MedChemExpress (Monmouth Junction, NJ, United States).

Animals and treatments

Sprague-Dawley rats were fed a high-fat diet (HFD; 60% kcal from fat and 1% kcal from cholesterol)[28] or normal chow (ND) for 18 weeks, as previously described. After four weeks of high-fat feeding, rats were injected intraperitoneally with STZ (30 mg/kg/day in citrate buffer) for seven consecutive days to induce insulin deficiency. Control rats received an equal volume of citrate buffer intraperitoneally at the same time. Blood glucose levels were measured with a One-Touch blood glucose meter (New Brunswick, NJ, United States) one-week postinjection. Rats with blood glucose levels greater than 16.7 mmol/L for two consecutive days were considered diabetic, and further experiments for the construction of a DCM model were performed. Diabetic rats were randomly divided into three groups (n = 8 per group) and treated daily for 12 consecutive weeks while maintained on HFD: (1) DCM group (0.9% saline administered subcutaneously, DCM); (2) Low-dose liraglutide group (Lira 100 µg/kg/day administered subcutaneously, Lira 100); and (3) High-dose liraglutide group (Lira 200 µg/kg/day administered subcutaneously, Lira 200). The liraglutide dose was adjusted according to the body weight (BW) of each rat. Rats fed ND and injected subcutaneously with an equal volume of 0.9% saline for 12 weeks served as the control group (n = 8). DCM-induced cardiac dysfunction was verified by echocardiography in diabetic rats assigned to the DCM group after six consecutive weeks of HFD.

At the end of the 12-week treatment with liraglutide or 0.9% saline, all rats were euthanized with 1% sodium pentobarbital (50 mg/kg) after a 4-hour fast for assessment of cardiac function. Eyes were enucleated, and blood samples were collected for the determination of blood glucose and lipid parameters. Hearts were then removed and washed with ice-cold saline for further analysis. The Sino Animal (Beijing) Science and Technology Development Co., Ltd approved the animal experimental research project (No. 20240234YZH-3R).

Fasting blood glucose and serum lipid analysis

Serum was prepared from each blood sample by centrifugation at 3500 rpm for 10 minutes. Fasting blood glucose (FBG) was measured in tail vein blood using an Accu-Chek glucose meter with matched blood glucose strips (Roche, Germany). Lipid parameters, including total cholesterol (TC), blood glucose, triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C), were examined using an automatic biochemistry analyzer (Hitachi 917, Tokyo, Japan).

Echocardiography

Echocardiography was performed using a small-animal, high-resolution ultrasound imaging system (Vevo 2100, VisualSonics, Canada). Rats were placed supine on a heating table and anesthetized with 3%-5% isoflurane. Transthoracic echocardiography was performed with a 30-MHz transducer to record B-mode echocardiograms, M-mode echocardiograms, and color Doppler flow images. The left ventricular (LV) internal end-diastolic diameter (LVIDd) and LV internal dimension systole (LVIDs) were measured. LV ejection fraction (LVEF) and LV fractional shortening (LVFS) were calculated with VevoLAB software (version 2.2.3, VisualSonics).

Histological analysis

Rat hearts were isolated, imaged, weighed, and measured after euthanasia at the end of the study. Hearts were fixed in 4% paraformaldehyde, dehydrated, cleared, embedded in paraffin, and sectioned at 4-µm at the papillary muscle level for subsequent experiments. Haematoxylin and eosin (H&E) staining was used to assess structural changes in myocardial tissue. Masson trichrome staining and WGA staining were performed on paraffin sections to measure cardiac fibrosis and cardiomyocyte cross-sectional area. Quantitative analysis of fibrosis deposition and cardiomyocyte cross-sectional area was performed using ImageJ software (version 1.53c; NIH, Bethesda, MD, United States).

TUNEL assay

Cardiomyocyte apoptosis was assessed by TUNEL staining according to the instructions provided by the manufacturer. Briefly, cardiac sections were incubated with proteinase K at 37 °C for 20 minutes. The TUNEL reaction mixture was then added and incubated at 37 °C for 1 hour, followed by counterstaining with DAPI. The number of TUNEL-positive cells was quantified with ImageJ software to identify apoptotic cells. For in vitro experiments, cells were seeded in confocal dishes and stained with the same reagents to detect apoptosis.

Cell culture of neonatal rat cardiomyocytes

Neonatal rat cardiomyocytes (NRCMs) were cultured from hearts of 1-3-day-old Sprague-Dawley rats as described previously[29]. NRCMs were plated at a density of 5 × 10⁴ cells/cm² in DMEM containing 5.5 mmol/L D-glucose, supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin, and maintained in an incubator at 37 °C with 5% (v/v) CO₂ to explore underlying mechanisms of DCM in the present study. After 12 hours of serum starvation, NRCMs were treated with 25 mmol/L D-glucose plus 250 μmol/L palmitate [HG + palmitic acid (PA) group] with or without different concentrations (50 nmol/L, 100 nmol/L, 200 nmol/L, or 300 nmol/L) of liraglutide (the Lira group) for 24 hours. Cardiomyocytes cultured in DMEM supplemented with 5.5 mmol/L D-glucose served as the control group. To investigate signaling pathways involved in the protective effects of liraglutide, NRCMs were treated with the AMPK inhibitor compound C (5 μg/mL) for 24 hours under HG + PA and liraglutide conditions.

Cell viability assay

The viability of NRCMs was evaluated with a CCK-8 assay performed according to the protocol provided by the manufacturer. When NRCMs in 96-well plates reached subconfluence (70%-80%), they were treated with different concentrations of liraglutide for 24 hours under HG + PA conditions. After treatment, 10 μL of CCK-8 solution was added to each well and incubated for 1 hour at 37 °C. Absorbance values (optical density) were determined with a microplate reader (TECAN Infinite F200 PRO, Switzerland) at 450 nm.

Western blotting

Heart tissues from rats and cultured NRCMs were homogenized on ice in lysis buffer containing protease and phosphatase inhibitors. Homogenates were centrifuged at 12000 × g for 15 minutes, and supernatants were collected. To evaluate the change in cytochrome C during HG + PA-induced apoptosis of NRCMs, a mitochondria isolation kit was used to separate mitochondria from cytosolic components, and proteins were extracted for subsequent Western blot analysis. Protein concentrations were determined using a BCA protein assay kit. A total of 10-30 μg protein from each sample was resolved by SDS-PAGE on 12% polyacrylamide gels and transferred to 0.22 µm polyvinylidene difluoride (PVDF) membranes, followed by blocking with 5% skim milk or bovine serum albumin for 1 hour at room temperature. PVDF membranes were incubated separately with primary antibodies at 4 °C overnight and then with secondary antibodies for 2 hours at room temperature. Protein expression was detected using an ECL kit and semi-quantified with ImageJ software.

Determination of mitochondrial reactive oxygen species generation

To detect mitochondrial reactive oxygen species (ROS) in NRCMs, Mito-SOX Red staining was performed at 37 °C for 10 minutes. Mito-SOX Red is a selective probe that targets mitochondria in live cells, where it undergoes rapid oxidation by mitochondria-derived superoxide, resulting in a distinct red fluorescence. Images were acquired with a confocal microscope, and fluorescence intensities of Mito-SOX, indicative of mitochondrial ROS (mitoROS) levels, for each group were quantified using ImageJ software.

Measurement of the mitochondrial membrane potential

JC-1, a fluorescent dye dependent on the mitochondrial membrane potential (MMP), was used to assess changes in MMP. JC-1 forms aggregates that emit red fluorescence in healthy cells with high MMP, whereas JC-1 monomers emit green fluorescence in apoptotic cells with low MMP. Cardiomyocytes were cultured in confocal dishes and, after treatment, incubated with JC-1 working solution for 20 minutes at 37 °C, then imaged with a confocal microscope. Rapid-exposure confocal images were quantified to determine mean fluorescence intensities in arbitrary regions using ImageJ software.

ATP content assay

To assess the energetic and functional status of mitochondria, an ATP bioluminescent assay kit was used to measure ATP levels in cardiomyocytes after treatment. ATP concentration was calculated using an ATP standard curve and expressed as nmol per mg protein.

Mitochondrial morphology and mitophagy assessment

Cultured NRCMs were seeded in confocal dishes at 70% confluence for subsequent treatment. Cells were then stained with 100 nM MitoTracker Red to assess mitochondrial morphology. To evaluate colocalization of mitochondria and lysosomes, cells were prestained with 100 nM MitoTracker Red to visualize mitochondria and subsequently stained with 1 μmol/L LysoTracker Green to visualize lysosomes. Colocalization of mitochondria and lysosomes was used to assess mitophagy. Fluorescence images were acquired on a confocal microscope and analyzed using ImageJ software.

Transmission electron microscopy

Transmission electron microscopy (TEM) was used to visualize mitochondrial morphology and typical autophagosomes engulfing damaged mitochondria. After treatment, NRCMs were digested with trypsin and washed twice with PBS (centrifuged at 1000 rpm × 5 minutes). A cell pellet containing approximately 5 × 10⁶ cells per sample was fixed in electron microscopy-grade fixative. After washing with 0.1 mol/L phosphate buffer (pH = 7.4), cells underwent secondary fixation, graded alcohol dehydration, and acetone treatment. Samples were infiltrated with Epon 812 embedding medium and polymerized at 60 °C for 48 hours. Ultrathin sections (60-80 nm) were cut, stained with 2% uranyl acetate and 2.6% lead citrate, and examined using a transmission electron microscope.

Statistical analysis

All experiments were repeated at least three times. Quantitative data are expressed as mean ± SEM. An unpaired 2-tailed Student's t-test was used for comparisons between two groups, and one-way ANOVA with Tukey post hoc test was used to analyze differences among multiple groups. SPSS 19.0 (SPSS Inc.) and GraphPad Prism 8.0 (GraphPad) were used for statistical analysis. A P < 0.05 was considered statistically significant.

RESULTS

Liraglutide improves cardiac function and biochemical profiles in STZ-induced DCM rats

Successful establishment of diabetic rats by intraperitoneal injection of STZ at 30 mg/kg BW daily for seven consecutive days was confirmed by elevated FBG concentrations (≥ 16.7 mmol/L). Subsequently, diabetic rats were subcutaneously administered liraglutide or 0.9% saline for 12 weeks as indicated. In addition, DCM was verified by echocardiography in rats assigned to the DCM group after six consecutive weeks of HFD feeding, which revealed cardiac dysfunction. To confirm the cardioprotective effects of liraglutide in DCM rats, echocardiography was performed. The results showed a significant decline in cardiac function in DCM rats compared with control rats, primarily reflected in LVEF, LVFS, LVIDd, and LVIDs. In contrast, significant recovery of cardiac function was observed after treatment with liraglutide at both 100 and 200 µg/kg/day for 12 weeks (Figure 1A-E). These findings indicate that liraglutide (100 µg/kg/day and 200 µg/kg/day) effectively ameliorated cardiac dysfunction in DCM rats.

Figure 1 Liraglutide improves cardiac function and biochemical profiles in streptozotocin-induced diabetic cardiomyopathy rats. A: Representative echocardiographic images for four groups of rats at the end of the experiment. B mode represents a two-dimensional echocardiogram showing left ventricular (LV) long-axis view. M mode represents M-mode echocardiogram showing LV dimensions; B-E: Quantification of LV ejection fraction, LV fractional shortening, LV internal diastolic dimension, LV internal diameter in systole in four groups of rats, n = 8 per group; F: Serum level of fasting blood glucose in the four groups of rats, n = 8 per group; G-J: Serum levels of triglyceride, total cholesterol, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol in the four groups of rats, n = 8 per group. Values are presented as the mean ± SD, n = 8. ^aP < 0.05; ^bP < 0.01; ^CP < 0.001. Con: Control group; DCM: Diabetic cardiomyopathy; LVEF: Left ventricular ejection fraction; LVFS: Left ventricular fractional shortening; LVIDd: Left ventricular internal end-diastolic diameter; LVIDs: Left ventricular internal dimension systole; FBG: Fasting blood glucose; TG: Triglyceride; TC: Total cholesterol; LDL-C: Low-density lipoprotein cholesterol; HDL-C: High-density lipoprotein cholesterol.

After echocardiographic assessment, blood samples were collected to determine blood glucose and lipid parameters. Compared with control rats, FBG in DCM rats was significantly increased, and FBG in rats treated with both doses of liraglutide was decreased, with a more pronounced reduction in the high-dose group (Figure 1F). Levels of TC, TG, and LDL-C were significantly higher, whereas HDL-C was lower, in DCM rats than in control rats, and liraglutide treatment clearly reversed these parameters in a dose-dependent manner (Figure 1G-J).

Liraglutide alleviates cardiac damage in DCM rats

The BWs of rats in the four groups were measured at the end of the experiments. Both heart weight (HW) and heart size were greater in DCM rats than in control rats. Compared with DCM rats, liraglutide-treated rats had decreased HW and size. The HW-to-BW ratio, an index of cardiac hypertrophy, was higher in DCM rats than in control rats; this effect was reversed by liraglutide treatment (Figure 2A and B).

Figure 2 Liraglutide alleviates myocardial injury in diabetic cardiomyopathy rats. A: Representative images of the hearts from four groups of rats (the first row, scale bar = 5mm), haematoxylin and eosin staining (n = 6 per group, scale bar = 2000 μm as indicated in upper panel, scale bar = 200 μm as indicated in low panel), Masson’s trichrome staining (n = 6 per group, scale bar = 2000 μm as indicated in upper panel, scale bar = 200 μm as indicated in low panel). Wheat germ agglutinin (WGA) staining (n = 6 per group, scale bar = 50 μm), and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining (n = 4 per group, scale bar = 75 μm); B: The ratio of heart weight to body weight in four groups of rats (n = 8 per group); C: Quantification of Masson’s trichrome staining (n = 6 per group); D: Quantification of WGA staining (n = 6 per group); E: Quantification of TUNEL staining (n = 6 per group); F: Representative Western blotting images of pro-caspase 3, cleaved caspase 3, PTEN-induced putative kinase 1 (PINK1), Parkin, p-AMPKα and AMPKα in the hearts of the four groups of rats; G-J: Quantification of the protein expression including pro-caspase 3, cleaved caspase 3, PINK1, Parkin, p-AMPKα and AMPKα in F. Values are presented as the mean ± SD. ^aP < 0.05; ^bP < 0.01; ^CP < 0.001. Con: Control group; DCM: Diabetic cardiomyopathy; H&E: Haematoxylin and eosin; WGA: Wheat germ agglutinin; HW: Heart weight; BW: Body weight.

Myocardial tissues from each experimental group were stained with H&E and examined using a light microscope. In the control group, myocardial cells were arranged neatly and densely with a normal histological structure of cardiac muscle fibers. In the DCM group, the myocardium was damaged and fragmented, with disordered myocardial fibers and markedly widened inter-fiber gaps. Notably, liraglutide treatment significantly reduced cardiomyocyte vacuolization and inhibited myocardial structural abnormalities, indicating improvement of pathological structural changes in the myocardium of DCM rats. Myocardial interstitial fibrosis is a crucial factor in the cardiopathogenesis of DCM; therefore, Masson’s trichrome staining was used to quantify and assess the effects of liraglutide on myocardial fibrotic tissue in DCM rats. Compared with control rats, DCM hearts exhibited significant myocardial interstitial fibrosis with an increased collagen volume fraction. After 12 weeks of liraglutide at both doses, cardiac fibrosis was markedly decreased compared with the DCM group (Figure 2A and C). In addition, WGA labeling of the cell membrane to determine the cross-sectional area of cardiomyocytes revealed obvious cardiomyocyte hypertrophy in DCM rats compared with control rats, which was strongly alleviated by liraglutide (Figure 2A and D).

TUNEL staining detects breaks in nuclear DNA during apoptosis by labeling dUTP, which binds to the 3'-OH ends of fragmented DNA in apoptotic cells. In our TUNEL assay, cardiac apoptosis was significantly increased in DCM rats. In contrast, blue fluorescence, which represents apoptosis, was markedly weaker after liraglutide treatment, indicating inhibition of apoptosis in cardiomyocytes from DCM rats (Figure 2A and E). Caspase-3, an aspartate-specific cysteine protease, is a central effector of apoptosis. To further evaluate the protective effect of liraglutide in DCM rats, we examined procaspase-3 and cleaved caspase-3 by western blotting. Liraglutide significantly reduced the expression of cleaved caspase-3 (Figure 2F and G). Moreover, the mitophagy-related proteins Parkin and PINK1, as well as p-AMPK and AMPK, which regulate energy balance and metabolism, were markedly decreased in heart tissue of DCM model rats; these changes were partially reversed by liraglutide (Figure 2F and H-J). These findings suggest that mitophagy and the AMPK pathway may contribute to liraglutide-mediated cardiac protection. Overall, the results indicate that liraglutide exerts protective effects against cardiac tissue damage in DCM rats.

Liraglutide ameliorates cardiomyocyte apoptosis induced by HG+PA

To investigate the protective effects of liraglutide on cardiomyocyte apoptosis, NRVMs were incubated with liraglutide under HG + PA culture conditions. A CCK-8 assay was used to evaluate cell viability, and liraglutide at 100 nmol/L, 200 nmol/L, and 300 nmol/L significantly increased NRVM survival in a concentration-dependent manner (Figure 3A). Consistently, liraglutide (200 nmol/L) ameliorated HG + PA-induced cardiomyocyte apoptosis, as quantified by TUNEL staining (Figure 3B and C). In addition, immunoblotting showed that liraglutide decreased the expression of cleaved caspase 3 compared with HG + PA-induced cardiomyocytes (Figure 3D and E). Loss of mitochondrial membrane integrity contributes to the release of cytochrome C from mitochondria, which is directly linked to the mitochondrial apoptotic pathway. To determine whether liraglutide prevents cytochrome C release from mitochondria, NRCMs were fractionated, and cytosolic and mitochondrial fractions were subjected to western blot analysis. HG + PA-treated cells exhibited a significant increase in cytosolic cytochrome C, accompanied by a marked reduction in mitochondrial cytochrome C. Notably, liraglutide inhibited HG + PA-induced cytochrome C release, and the decrease in cytosolic cytochrome C correlated with its increase in the mitochondrial fraction (Figure 3F-I). These findings collectively suggest that HG + PA triggers the release of mitochondrial cytochrome C into the cytosol, indicative of mitochondrial dysfunction and subsequent apoptosis in NRCMs, whereas liraglutide counteracts these pathological alterations.

Figure 3 Liraglutide ameliorate cardiomyocyte apoptosis induced by high glucose + palmitic acid. A: Representative CCK8 images for optimum treatment dose of liraglutide (Lira: 200 nmol/L); B and C: Representative and quantitative analysis images of cell terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining for the three groups (scale bar: 50 μm); D, F and G: Representative Western blotting images of pro-caspase 3 and cleaved caspase 3, cytosolic-cytochrome C and mitosolic-cytochrome C in the three groups [control group (Con), high glucose (HG) + palmitic acid (PA; 25 mmol/L D-glucose plus 250 μmol/L palmitate), Lira (HG + PA with 200 nmol/L liraglutide)]; E, H and I: Quantification of the expression of the proteins including cleaved caspase 3, cytosolic-cytochrome C and mitosolic-cytochrome C. Values are presented as the mean ± SD. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. Con: Control group; HG: High glucose; PA: Palmitic acid.

Liraglutide alleviates mitochondrial damage in HG + PA-induced cardiomyocytes

Mitochondrial dysfunction and morphological defects are implicated in the pathogenesis of DCM[7]. Mitochondria regulate cell survival and death through balanced homeostasis and normal morphology, which are governed primarily by fission and fusion. Mitochondria with clear networks were classified as tubular mitochondria; wholly fragmented or permeabilized mitochondria were classified as fragmented mitochondria; and other morphologies were classified as intermediate mitochondria. In this study, the MitoTracker Red probe was used to assess the role of liraglutide in maintaining mitochondrial morphology (Figure 4A and B). As expected, a normal mitochondrial network characterized by tubular mitochondria was observed in control cardiomyocytes. In contrast, mitochondria in HG + PA-induced NRCMs were fragmented, whereas liraglutide markedly rescued mitochondrial morphology. We then measured proteins related to mitochondrial dynamics and found significant increases in mitochondrial fission-related proteins (FIS1 and DRP1) and decreases in mitochondrial fusion-related proteins (MFN1, MFN2, and OPA1) in HG + PA-treated NRCMs; these changes were notably ameliorated by liraglutide (Figure 4C-H).

Figure 4 Liraglutide rescues mitochondrial damage in neonatal rat cardiomyocytes induced by high glucose + palmitic acid. A: Representative images Mito Tracker Red of the three groups (scale bar = 25 μm as indicated in upper panel, scale bar = 5 μm as indicated in low panel); B: Quantification of the mitochondrial mean length (white double arrow); C: Representative western blotting images of mitochondrial fission and fusion related proteins including Fis1, DRP1, OPA1, MFN1 and MFN2 in the three groups; D-H: Quantification of the expression of the proteins including Fis1, DRP1, OPA1, MFN1 and MFN2 in B; I: Mito-SOX Red staining representing mitochondrial reactive oxygen species (mitoROS) levels in the three groups (scale bar: 25 μm); J: Quantification of mitoROS production as measured with mito-SOX Red staining in I; K: Representative fluorescence staining images of JC-1 aggregates (red) and JC-1 monomer (green, bar = 25 μm); L: Relative fluorescence intensity of aggregate/monomeric JC-1 in K; M: Quantification of ATP production level in the three groups; N: Representative western blotting images of mitochondrial respiratory chain proteins including NUDFB8, SDHB, UQRC2, MTCO2, ATPA1; O-S: Quantification of the expression of mitochondrial respiratory chain related proteins including NUDFB8, SDHB, UQRC2, MTCO2, ATPA1 in L. Values are presented as the mean ± SD. ^aP < 0.001; ^bP < 0.01. Con: Control group; HG: High glucose; PA: Palmitic acid.

Accumulating evidence indicates that mitochondrial oxidative damage and mitochondrial dysfunction occur in diabetic hearts and contribute to the development of DCM[30]. We measured mitoROS, primarily of mitochondrial origin, and found that mitoROS production was dramatically elevated in HG + PA-induced cardiomyocytes, whereas liraglutide significantly reduced the mitoROS content (Figure 4I and J). The MMP was assessed by JC-1 staining; the MMP (ratio of red to green fluorescence) was markedly lower than in control cardiomyocytes but was significantly increased after liraglutide treatment (Figure 4K and L). Because ATP generation via the respiratory chain is a principal function of mitochondria, we measured ATP content and the expression of respiratory chain-related proteins (NDUFB8, SDHB, MTCO2, UQCR2, and ATP5A1). HG + PA-induced NRCMs showed significant reductions in ATP production and in the expression of these proteins; however, both were markedly increased after liraglutide intervention (Figure 4M-S).

Overall, liraglutide significantly ameliorated mitochondrial damage by maintaining the balance of mitochondrial fission and fusion, restoring normal mitochondrial morphology, and improving mitochondrial function in HG + PA-induced NRCMs.

Liraglutide attenuates mitochondrial damage and cardiomyocyte apoptosis via AMPK-Parkin pathway-mediated mitophagy

Mitophagy, which selectively removes damaged mitochondria, may be a protective mechanism in DCM. To investigate the effect of liraglutide on mitophagy in HG + PA-treated cardiomyocytes, we assessed mitophagy as indicated by increased colocalization of mitochondria (labeled with MitoTracker Red) with lysosomes (tagged with LysoTracker Green). Our results showed that liraglutide alleviated HG + PA-induced mitochondrial damage by increasing the colocalization of mitochondria with lysosomes and enhancing mitophagy in NRCMs (Figure 5A and B). Because mitophagy is regulated by multiple proteins, including LC3, Parkin, and PINK1, the expression levels of these proteins were reduced under HG + PA stimulation but increased with liraglutide treatment. Western blotting revealed significant reductions in the LC3-II/I ratio and in Parkin expression in HG + PA-induced NRCMs compared with normal cardiomyocytes, whereas PINK1 expression was not altered. Liraglutide treatment reversed the reductions in the LC3-II/I ratio and Parkin expression (Figure 5C-F). These data suggest that liraglutide inhibits mitochondrial damage by promoting mitophagy in cardiomyocytes injured by HG + PA.

Figure 5 Liraglutide attenuates mitophagy by activating AMPK-Parkin pathway in neonatal rat cardiomyocytes induced by high glucose + palmitic acid. A: Representative colocalization images of lysosomes (LysoTracker Green) and mitochondria (Mito Tracker Red; scale bar: 10 µm); B: Percentage of cells with lysosome (LysoTracker Green) and mitochondria (Mito Tracker Red) colocalization; C: Representative western blotting images of LC3 I, LC3 II, PTEN-induced putative kinase 1 (PINK1) and Pakin in the four groups; D-F: Quantification of the expression of the proteins including LC3 I and LC3 II, PINK1 and Pakin in C; G: Representative western blotting images of p-AMPKα and AMPKα in the four groups; H: Quantification of the expression levels of p-AMPKα and AMPKα in G. Values are presented as the mean ± SD. ^aP < 0.05; ^bP < 0.001. Con: Control group; HG: High glucose; PA: Palmitic acid.

AMPK is an important mediator in the protection of the heart against diabetes-induced cardiomyopathy[14,31]. A reduction in AMPK phosphorylation was detected in HG + PA-induced NRCMs (Figure 5G and H). In addition to findings in DCM rats, we hypothesized that liraglutide-mediated AMPK activation would significantly increase mitophagy. We therefore tested whether the protective effects of liraglutide on cardiac and mitochondrial function occur by enhancing AMPK-dependent mitophagy. First, compound C, an AMPK inhibitor, was used to examine the relationship between AMPK signaling and mitochondrial oxidative stress; compound C markedly reversed the liraglutide-induced reduction in mitoROS in HG + PA-treated NRCMs (Figure 6A and B). Next, the MMP restored by liraglutide was significantly decreased by compound C (Figure 6C and D). Compound C also worsened mitochondrial morphology that had been improved by liraglutide (Figure 6E and F). TEM further showed that HG + PA increased the fraction of swollen mitochondria and reduced relative mitochondrial crista density; these effects were partially counteracted by liraglutide. By contrast, after compound C intervention, mitochondrial morphology again showed partial loss of cristae and vacuolization, similar to the HG + PA group, suggesting that compound C inhibited the beneficial effects of liraglutide on mitochondria (Figure 6G-I). In addition, TUNEL assays and western blotting were performed to evaluate the effects of compound C on HG + PA-induced cardiomyocyte apoptosis. The number of TUNEL-positive cells and the levels of cleaved caspase-3 and cytoplasmic cytochrome C were significantly higher in the compound C group than in the liraglutide group, whereas mitochondrial cytochrome C was markedly lower (Figure 6J-Q). These results suggest that inhibition of AMPK signaling reverses the anti-oxidative stress effects, the improvements in mitochondrial morphology, and the anti-apoptotic actions induced by liraglutide. Furthermore, compared with liraglutide, compound C inhibited mitophagy by decreasing the colocalization of mitochondria with lysosomes and decreasing the LC3-II/I ratio and Parkin levels in NRCMs (Figure 6R-V).

Figure 6 Liraglutide attenuates mitochondrial damage and cardiomyocytes apoptosis via AMPK-Parkin pathway mediated mitophagy in neonatal rat cardiomyocytes induced by high glucose + palmitic acid. A: Mito-SOX Red staining representing mitochondrial reactive oxygen species (mitoROS) levels in the four groups (scale bar: 25 μm); B: Quantification of mitoROS production as measured with mito-SOX Red staining in B; C: Representative fluorescence staining images of JC-1 aggregates (red) and JC-1 monomer (scale bar: 25 μm); D: Relative fluorescence intensity of aggregate/monomeric JC-1 in D; E: Representative images Mito Tracker Red of the four groups (scale bar = 25 μm as indicated in upper panel, scale bar = 5 μm as indicated in low panel); F: Quantification of the mitochondrial mean length (white double arrow); G: Transmission electron microscopic images showing mitochondria in cardiomyocytes. Normal mitochondria were denoted by white arrows, while the damaged mitochondria (edema, cristae rupture and vacuolized) were indicated by yellow arrows (scale bar: 1 µm); H and I: Fraction of swollen mitochondria and mitochondrial loss of cristae in the four groups; J and K: Representative and quantitative analysis images of cell terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining for the three groups (scale bar: 50 μm); L-N: Representative western blotting images of pro-caspase 3, cleaved caspase 3, cytosolic-cytochrome C and mytosolic-cytochrome C in the the four groups; O-Q: Quantification of the expression of the proteins including pro-caspase 3, cleaved caspase3, cytosolic-cytochrome C and mytosolic-cytochrome C in L, N and P; R: Representative colocalization images of lysosomes (LysoTracker Green) and mitochondria (Mito Tracker Red; scale bar: 10 µm); S: The percentage of cells with lysosome (LysoTracker Green) and mitochondria (MitoTracker Red) colocalization; T: Representative Western blotting images of LC3 I, LC3 II and Pakin in the four groups; U and V: Quantification of the expression of the proteins including LC3I, LC3II and Pakin in T. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. Con: Control group; HG: High glucose; PA: Palmitic acid.

Taken together, these data strongly indicate that liraglutide protects cardiomyocytes against HG + PA-induced mitochondrial damage and apoptosis by enhancing mitophagy mediated by the AMPK-Parkin signaling pathway.

DISCUSSION

In this study, we investigated the effects of liraglutide on cardiac function and mitophagy in DCM rats and NRCMs. The results show that liraglutide significantly improved cardiac function, modified FBG levels and lipid profiles, and alleviated cardiac eccentric hypertrophy and fibrosis. Moreover, liraglutide inhibited cardiomyocyte apoptosis and mitigated mitochondrial damage by promoting mitophagy, mediated by activation of the AMPK-Parkin signaling pathway (Figure 7).

Figure 7 The molecular mechanism of the improvement of liraglutide in diabetic cardiomyopathy. Graphic summary demonstrating liraglutide could be a novel AMPK-Parkin activator to ameliorate myocardial dysfunction in high-fat diet and streptozotocin induced rat. In diabetes, liraglutide increases phosphorylation of AMPK, upregulates the expression of PTEN-induced putative kinase 1 and Parkin, and then, recruits and phosphorylates more Parkin, activates mitophagy, and improves mitochondrial function (mitochondrial membrane potential recovery, mitochondrial reactive oxygen species level decrease and ATP content increase). HFD: High-fat diet; STZ: Streptozotocin; HG: High glucose; PA: Palmitic acid; ∆Ψm: Mitochondrial membrane potential. Created in BioRender (Supplementary material).

DM is a well-known risk factor for HF in the form of DCM, defined as myocardial dysfunction in patients with DM in the absence of coronary artery disease and hypertension[3,32]. The underlying pathological mechanisms of DCM are not fully understood, but accumulating evidence implicates oxidative stress, increased myocardial fibrosis and hypertrophy, metabolic derangements, inflammation, apoptosis, impaired intracellular calcium handling, activation of the renin-angiotensin-aldosterone system, mitochondrial dysfunction, and aberrant autophagy flux, among other factors[33,34]. Current therapeutic strategies primarily target glycemic control but show limited efficacy in reversing established myocardial damage, highlighting the urgent need for mechanism-driven interventions[3,30]. Despite certain limitations, animal models have been crucial for exploring DCM pathophysiology and identifying potential therapeutic targets. Various models of type 1 DM (T1DM) and type 2 DM (T2DM) have been developed to evaluate the effects of diabetes on the heart. These models are established through genetic manipulations, dietary interventions, and treatment with pancreatic toxins, each mimicking several aspects of DM and DCM[32,35]. STZ, a glucosamine-nitrosourea antibiotic toxic to pancreatic β-cells, has been widely used to generate experimental models of both T1DM and T2DM[36]. High-dose STZ protocols, which cause severe and extensive β-cell necrosis with near-total loss of pancreatic insulin secretion, are used primarily to study T1DM[37]. As previously shown, a single high dose of STZ (200 mg/kg) injected into C57BL/6J mice induces near-complete depletion of pancreatic β-cells, producing T1DM mice[38]. In another study, mice received a single high dose of STZ (150 mg/kg) followed by HFD feeding for eight weeks to induce DCM, and the results demonstrated severe cardiac dysfunction and myocardial damage, manifested as significant decreases in LVEF and LVFS and marked increases in LVIDd and LVIDs, accompanied by severe interstitial fibrosis and cardiac hypertrophy[39]. By contrast, administration of a low dose of STZ with HFD chow has been used to induce DCM in rats or mice, modeling T2DM characterized by insufficient insulin production due to partial β-cell destruction[40,41]. A recent study showed that an HFD combined with one or more intraperitoneal injections of low-dose STZ (30 mg/kg) is a straightforward and reliable method for inducing DCM in rats; the resulting model exhibited persistent hyperglycemia and insulin resistance and developed histopathological changes indicating cardiac hypertrophy, myocardial fibrosis, and diastolic dysfunction[42]. In this study, rats were injected with STZ (30 mg/kg/day for seven consecutive days) combined with HDF for 18 weeks to induce DCM, resulting in cardiac dysfunction characterized by decreased LVEF and LVFS and increased LVIDd and LVIDs, accompanied by higher TG, TC, and LDL-C and lower HDL-C, as well as more severe myocardial apoptosis, cardiac hypertrophy, and interstitial fibrosis than in normal control rats. These results align with previous studies and represent typical characteristics of DCM rats[43,44].

The initial manifestation of DCM is impaired diastolic relaxation, which ultimately leads to clinical HF, even in the absence of coronary artery disease, hypertension, or dyslipidemia[33]. Current medications aim to improve cardiac function in patients with diabetes by interrupting the progression of DCM[45]. Three newer targets for antidiabetic agents—GLP1R, dipeptidylpeptidase 4, and sodium-glucose cotransporter 2, have gained prominence because of their cardioprotective capacity and crucial roles in DCM[46]. However, the mechanisms underlying the attenuation of DCM remain unclear. Among these targets, GLP1Rs are expected to become preferred drug targets for patients with diabetes and DCM, surpassing current options owing to specificity, lower toxicity, and the ability to overcome drawbacks such as weight gain and hypoglycemia[47,48]. Recently, an increasing number of studies have shown that liraglutide exerts beneficial cardiovascular effects in both humans and animals[49,50]. Data from the LEADER trial revealed lower rates of hospitalization for congestive HF and reduced risks of cardiovascular mortality and all-cause death among patients with DCM receiving liraglutide therapy[51]. In a recent double-blind randomized controlled trial in European patients with T2DM, liraglutide significantly decreased LV diastolic filling, LV filling pressure, stroke volume, and ejection fraction, presumably through natriuresis and vasorelaxation that unload the left ventricle[49]. Recent animal studies have further highlighted the regulatory effects of liraglutide on cardiac function in DCM and diabetes[52,53]. Liraglutide can halt the development of DCM in rats by reducing cardiomyocyte apoptosis, oxidative stress, and inflammation when administered one week after the onset of DM[54]. Another study showed that liraglutide nearly preserved normal myocardial structure and significantly protected against myocardial inflammation and fibrosis in STZ-induced DCM rats[55]. Hussein et al[56] reported that liraglutide significantly reversed abnormal elevations of glucose and cardiac enzymes (CK-MB and LDH), improving insulin resistance and cardiomyocyte injury in T2DM rats induced by HFD + STZ. Liraglutide also exerted cardioprotective effects in DCM rats by alleviating cardiomyocyte apoptosis, reducing ROS accumulation, and ameliorating elevations in cardiac enzymes (troponin I and CK-MB)[53]. Our present study confirmed and extended previous findings, showing that liraglutide alleviated cardiac dysfunction, inhibited cardiomyocyte apoptosis and hypertrophy, and reduced interstitial fibrosis in DCM rats. Liraglutide also significantly reduced FBG, TG, TC, and LDL-C while increasing HDL-C, at both low and high doses.

Disrupted mitochondrial fission and fusion (the major processes maintaining mitochondrial dynamics) contribute substantially to mitochondrial dysfunction[57]. Excessive fission and impaired fusion lead to pronounced mitochondrial fragmentation, which in turn drives ROS accumulation, insufficient ATP production, and ultimately DCM[58]. Mitochondria are the primary sources of ROS in patients with diabetes[9]. The human heart has exceptionally high energy demands, with more than 95% of its energy derived from mitochondrial oxidative metabolism. Mitophagy functions as a cytoprotective mechanism by removing damaged or superfluous mitochondria[59]. Previous studies have reported mitophagy deficiency in the hearts of patients with DCM and in diabetic animal models[60-62]. A growing body of evidence supports the protective role of PINK1/Parkin-dependent mitophagy in preserving both mitochondrial morphology and function[63,64]. Zheng et al[65] reported that the traditional Chinese medicine JCYSTL formula activates PINK1/Parkin-mediated mitophagy to protect renal tubules from mitochondrial dysfunction and apoptosis by regulating mitochondrial respiratory chain complex activity in diabetic rats. A previous study showed that liraglutide suppressed NLRP3 inflammasome-induced activation of pyroptosis, attenuated mitochondrial dysfunction and ROS generation, and ultimately augmented mitophagy in hepatocytes stimulated with PA and lipopolysaccharide[24]. Additionally, Lin et al[66] reported that liraglutide ameliorated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity in a Parkinson’s disease mouse model by enhancing mitophagy flux, rebalancing mitochondrial dynamics, and reducing α-synuclein aggregation and oxidative stress. In the present study, liraglutide protected HG + PA-induced NRCMs from injury by reducing ROS levels, maintaining the MMP, increasing ATP content, restoring mitochondrial morphology, rescuing mitochondrial function, increasing the expression of respiratory chain-related proteins, decreasing the number of autophagosomes, and promoting mitophagy, which ultimately inhibited cardiomyocyte apoptosis. Our findings provide further evidence that liraglutide exerts a protective effect by enhancing mitophagy. On the other hand, another study has reported results inconsistent with previous studies, showing that liraglutide prevented HG-induced HUVECs dysfunction via inhibition of PINK1/Parkin-dependent mitophagy[67].

AMPK is an evolutionarily conserved serine/threonine-protein kinase that functions as an energy sensor in cells and plays key roles in regulating fatty acid and glucose metabolism, endoplasmic reticulum stress, and cardioprotective protein expression[25]. Under various physiological and pathological conditions, AMPK is phosphorylated by an upstream kinase and binds AMP or ADP rather than ATP, leading to activation[26]. Increasing evidence suggests a strong link between mitochondrial protection and AMPK signaling, including roles in mitochondrial dynamics, mitochondrial biogenesis, and mitophagy[68]. A recently published study reported that overexpression of mitochondrion-localized AMPKα1 suppressed endothelial ferroptosis and mitophagy mediated by the mitochondria-associated AMPK-Parkin-ACSL4 signaling pathway in DCM mice[14]. Han et al[69] found that the AMPK agonist metformin ameliorated renal oxidative stress and tubulointerstitial fibrosis in HFD/STZ-induced diabetic mice, activated AMPK phosphorylation, increased translocation of PINK1 from the cytoplasm to mitochondria, and promoted mitophagy through the p-AMPK-Pink1-Parkin pathway. Yang et al[15] also reported that canagliflozin mitigated DCM by activating PINK1-Parkin-dependent mitophagy and improving mitochondrial function with increased phosphorylation of AMPK in HFD/STZ-induced diabetic mice and in cardiomyocytes under high-glucose conditions. However, contrary to the widely held view that AMPK stimulates mitophagy, Longo et al[70] unexpectedly reported that MK-8722, an AMPK activator, inhibits NIX/BNIP3-dependent mitophagy triggered by hypoxia and the iron chelator DFP. The authors suggested that NIX and BNIP3 target functional mitochondria for degradation and that AMPK may block this process to preserve functional mitochondria. Our data showed that AMPK phosphorylation was increased after liraglutide treatment in both DCM rats and HG + PA-induced NRCMs, accompanied by increased expression of PINK1 and Parkin. Moreover, PINK1 and Parkin were reduced in HG + PA-treated NRCMs with concomitant attenuation of mitophagy, whereas liraglutide reversed these effects. Therefore, the AMPK inhibitor compound C was used to examine the relationship between AMPK signaling and mitophagy. Compound C markedly reversed the protective effects of liraglutide in HG + PA-induced NRCMs, indicating that liraglutide promotes AMPK-Parkin-mediated mitophagy and enhances mitochondrial function to achieve cardiovascular benefit.

CONCLUSION

This study demonstrated a significant cardioprotective effect of liraglutide in DCM, improving cardiac function and inhibiting excessive cardiomyocyte apoptosis, hypertrophy, and interstitial fibrosis by enhancing mitochondrial function. More importantly, the cardioprotective effect of liraglutide was achieved by promoting mitophagy mediated by activation of the AMPK-Parkin signaling pathway. These findings suggest that AMPK-Parkin-mediated mitophagy could be a potential therapeutic target for effective management of DCM or diabetes-related HF.