Drp1-Dependent Mitochondrial Autophagy Plays a Protective Role Against Pressure Overload-Induced Mitochondrial Dysfunction and Heart Failure.
ABSTRACT: Mitochondrial autophagy is an important mediator of mitochondrial quality control in cardiomyocytes. The occurrence of mitochondrial autophagy and its significance during cardiac hypertrophy are not well understood.Mice were subjected to transverse aortic constriction (TAC) and observed at multiple time points up to 30 days. Cardiac hypertrophy developed after 5 days, the ejection fraction was reduced after 14 days, and heart failure was observed 30 days after TAC. General autophagy was upregulated between 1 and 12 hours after TAC but was downregulated below physiological levels 5 days after TAC. Mitochondrial autophagy, evaluated by electron microscopy, mitochondrial content, and Keima with mitochondrial localization signal, was transiently activated at ?3 to 7 days post-TAC, coinciding with mitochondrial translocation of Drp1. However, it was downregulated thereafter, followed by mitochondrial dysfunction. Haploinsufficiency of Drp1 abolished mitochondrial autophagy and exacerbated the development of both mitochondrial dysfunction and heart failure after TAC. Injection of Tat-Beclin 1, a potent inducer of autophagy, but not control peptide, on day 7 after TAC, partially rescued mitochondrial autophagy and attenuated mitochondrial dysfunction and heart failure induced by overload. Haploinsufficiency of either drp1 or beclin 1 prevented the rescue by Tat-Beclin 1, suggesting that its effect is mediated in part through autophagy, including mitochondrial autophagy.Mitochondrial autophagy is transiently activated and then downregulated in the mouse heart in response to pressure overload. Downregulation of mitochondrial autophagy plays an important role in mediating the development of mitochondrial dysfunction and heart failure, whereas restoration of mitochondrial autophagy attenuates dysfunction in the heart during pressure overload.
Project description:The receptor for advanced glycation end products (RAGE) is involved in heart failure (HF) by mediating diverse pathologic processes, including the promotion of inflammation and autophagy. However, the role of RAGE in pressure overload-induced HF is not well understood. We found that stimulation of RAGE triggered the death of neonatal rat ventricular myocytes (NRVMs), while cell death was alleviated by ATG5 knockdown. Using transverse aortic constriction (TAC) in mice as a model of pressure overload-induced HF, we demonstrated that RAGE knockout or RAGE blockade attenuated cardiac hypertrophy and fibrosis as well as cardiac dysfunction at 8 weeks after TAC. Importantly, RAGE knockout reversed upregulation of autophagy related proteins (LC3BII/I and Beclin 1) and reduced cardiomyocyte death, indicating that excessive autophagy after TAC was inhibited. Moreover, RAGE knockout or blockade reduced the upregulation of pp65-NF?B and BNIP3, which mediate autophagy. Taken together, these results suggest that RAGE plays an important role in the progression of HF by regulating autophagy. Therefore, inhibition of the RAGE-autophagy axis could be a promising new strategy for treatment of heart failure.
Project description:AIM: Aliskiren (ALK) is a renin inhibitor that has been used in the treatment of hypertension. The aim of this study was to determine whether ALK could ameliorate pressure overload-induced heart hypertrophy and fibrosis, and to elucidate the mechanisms of action. METHODS: Transverse aortic constriction (TAC) was performed in mice to induce heart pressure overload. ALK (150 mg·kg(-1)·d(-1), po), the autophagy inhibitor 3-methyladenine (10 mg·kg(-1) per week, ip) or the PKC?I inhibitor LY333531 (1 mg·kg(-1)·d-(1), po) was administered to the mice for 4 weeks. Heart hypertrophy, fibrosis and function were evaluated based on echocardiography, histological and biochemical measurements. Mechanically stretched cardiomyocytes of rats were used for in vitro experiments. The levels of signaling proteins were measured using Western blotting, while the expression of the relevant genes was analyzed using real-time QRT-PCR. RESULTS: TAC induced marked heart hypertrophy and fibrosis, accompanied by high levels of Ang II in plasma and heart, and by PKC?I/? and ERK1/2 phosphorylation in heart. Meanwhile, TAC induced autophagic responses in heart, i.e. increases in autophagic structures, expression of Atg5 and Atg16 L1 mRNAs and LC3-II and Beclin-1 proteins. These pathological alterations in TAC-mice were significantly ameliorated or blocked by ALK administration. In TAC-mice, 3-methyladenine administration also ameliorated heart hypertrophy, fibrosis and dysfunction, while LY333531 administration inhibited ERK phosphorylation and autophagy in heart. In mechanically stretched cardiomyocytes, CGP53353 (a PKC?I inhibitor) prevented ERK phosphorylation and autophagic responses, while U0126 (an ERK inhibitor) blocked autophagic responses. CONCLUSION: ALK ameliorates heart hypertrophy, fibrosis and dysfunction in the mouse model in setting of chronic pressure overload, via suppressing Ang II-PKC?I-ERK1/2-regulated autophagy.
Project description:Alpha-lipoic acid (?-LA), a well-known antioxidant, was proved to active ALDH2 in nitrate tolerance and diabetic animal model. However, the therapeutic advantage of ?-LA for heart failure and related signaling pathway have not been explored. This study was designed to examine the role of ?-LA-ALDH2 in heart failure injury and mitochondrial damage. ALDH2 knockout (ALDH2-/-) mice and primary neonatal rat cardiomyocytes (NRCMs) were subjected to assessment of myocardial function and mitochondrial autophagy. Our data demonstrated ?-LA significantly reduced the degree of TAC-induced LV hypertrophy and dysfunction in wild-type mice, not in ALDH2-/- mice. In molecular level, ?-LA significantly restored ALDH2 activity and expression as well as increased the expression of a novel mitophagy receptor protein FUNDC1 in wild-type TAC mice. Besides, we confirmed that ALDH2 which was activated by ?-LA governed the activation of Nrf1-FUNDC1 cascade. Our data suggest that ?-LA played a positive role in protecting the heart against adverse effects of chronic pressure overload.
Project description:<h4>Rationale</h4>Lipid overload-induced heart dysfunction is characterized by cardiomyocyte death, myocardial remodeling, and compromised contractility, but the impact of excessive lipid supply on cardiac function remains poorly understood.<h4>Objective</h4>To investigate the regulation and function of the mitochondrial fission protein Drp1 (dynamin-related protein 1) in lipid overload-induced cardiomyocyte death and heart dysfunction.<h4>Methods and results</h4>Mice fed a high-fat diet (HFD) developed signs of obesity and type II diabetes mellitus, including hyperlipidemia, hyperglycemia, hyperinsulinemia, and hypertension. HFD for 18 weeks also induced heart hypertrophy, fibrosis, myocardial insulin resistance, and cardiomyocyte death. HFD stimulated mitochondrial fission in mouse hearts. Furthermore, HFD increased the protein level, phosphorylation (at the activating serine 616 sites), oligomerization, mitochondrial translocation, and GTPase activity of Drp1 in mouse hearts, indicating that Drp1 was activated. Monkeys fed a diet high in fat and cholesterol for 2.5 years also exhibited myocardial damage and Drp1 activation in the heart. Interestingly, HFD decreased nicotinamide adenine dinucleotide (oxidized) levels and increased Drp1 acetylation in the heart. In adult cardiomyocytes, palmitate increased Drp1 acetylation, phosphorylation, and protein levels, and these increases were abolished by restoration of the decreased nicotinamide adenine dinucleotide (oxidized) level. Proteomics analysis and in vitro screening revealed that Drp1 acetylation at lysine 642 (K642) was increased by HFD in mouse hearts and by palmitate incubation in cardiomyocytes. The nonacetylated Drp1 mutation (K642R) attenuated palmitate-induced Drp1 activation, its interaction with voltage-dependent anion channel 1, mitochondrial fission, contractile dysfunction, and cardiomyocyte death.<h4>Conclusions</h4>These findings uncover a novel mechanism that contributes to lipid overload-induced heart hypertrophy and dysfunction. Excessive lipid supply created an intracellular environment that facilitated Drp1 acetylation, which, in turn, increased its activity and mitochondrial translocation, resulting in cardiomyocyte dysfunction and death. Thus, Drp1 may be a critical mediator of lipid overload-induced heart dysfunction as well as a potential target for therapy.
Project description:Pressure overload cardiac hypertrophy, a risk factor for heart failure, is associated with reduced mitochondrial fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS) proteins that correlate in rodents with reduced PGC-1? expression.To determine the role of PGC-1? in maintaining mitochondrial energy metabolism and contractile function in pressure overload hypertrophy.PGC-1? deficient (KO) mice and wildtype (WT) controls were subjected to transverse aortic constriction (TAC). Although LV function was modestly reduced in young KO hearts, there was no further decline with age so that LV function was similar between KO and WT when TAC was performed. WT-TAC mice developed relatively compensated LVH, despite reduced mitochondrial function and repression of OXPHOS and FAO genes. In nonstressed KO hearts, OXPHOS gene expression and palmitoyl-carnitine-supported mitochondrial function were reduced to the same extent as banded WT, but FAO gene expression was normal. Following TAC, KO mice progressed more rapidly to heart failure and developed more severe mitochondrial dysfunction, despite a similar overall pattern of repression of OXPHOS and FAO genes as WT-TAC. However, in relation to WT-TAC, PGC-1? deficient mice exhibited greater degrees of oxidative stress, decreased cardiac efficiency, lower rates of glucose metabolism, and repression of hexokinase II protein.PGC-1? plays an important role in maintaining baseline mitochondrial function and cardiac contractile function following pressure overload hypertrophy by preserving glucose metabolism and preventing oxidative stress.
Project description:Energic deficiency of cardiomyocytes is a dominant cause of heart failure. An antianginal agent, trimetazidine improves the myocardial energetic supply. We presumed that trimetazidine protects the cardiomyocytes from the pressure overload-induced heart failure through improving the myocardial metabolism. C57BL/6 mice were subjected to transverse aortic constriction (TAC). After 4 weeks of TAC, heart failure was observed in mice manifested by an increased left ventricular (LV) chamber dimension, an impaired LV ejection fraction evaluated by echocardiography analysis, which were significantly restrained by the treatment of trimetazidine. Trimetazidine restored the mitochondrial morphology and function tested by cardiac transmission electron microscope and mitochondrial dynamic proteins analysis. Positron emission tomography showed that trimetazidine significantly elevated the glucose uptake in TAC mouse heart. Trimetazidine restrained the impairments of the insulin signaling in TAC mice and promoted the translocation of glucose transporter type IV (GLUT4) from the storage vesicle to membrane. However, these cardioprotective effects of trimetazidine in TAC mice were notably abolished by compound C (C.C), a specific AMPK inhibitor. The enlargement of neonatal rat cardiomyocyte induced by mechanical stretch, together with the increased expression of hypertrophy-associated proteins, mitochondria deformation and dysfunction were significantly ameliorated by trimetazidine. Trimetazidine enhanced the isolated cardiomyocyte glucose uptake <i>in vitro</i>. These benefits brought by trimetazidine were also removed with the presence of C.C. In conclusion, trimetazidine attenuated pressure overload-induced heart failure through improving myocardial mitochondrial function and glucose uptake via AMPK.
Project description:Cardiorenal syndrome type 2 is characterized by kidney failure as a consequence of heart failure that affects >50% of heart failure patients. Murine transverse aortic constriction (TAC) is a heart failure model, where pressure overload is induced on the heart without any systemic hypertension or its consequences. Whether renal function is altered in this model is debated, and if so, at which time post-TAC renal dysfunction starts to contribute to worsening of cardiac function. We therefore studied the effects of progressive heart failure development on kidney function in the absence of chronically elevated systemic blood pressure and renal perfusion pressure. C57BL/6J mice (N = 129) were exposed to TAC using a minimally invasive technique and followed from 3 to 70 days post-TAC. Cardiac function was determined with 3D ultrasound and showed a gradual decrease in stroke volume over time. Renal renin expression and plasma renin concentration increased with progressive heart failure, suggesting hypoperfusion of the kidney. In addition, plasma urea concentration, a surrogate marker for renal dysfunction, was increased post-TAC. However, no structural abnormalities in the kidney, nor albuminuria were present at any time-point post-TAC. Progressive heart failure is associated with increased renin expression, but only mildly affected renal function without inducing structural injury. In combination, these data suggest that heart failure alone does not contribute to kidney dysfunction in mice.
Project description:Berberine has been verified to protect cardiac function in patients with heart failure (HF). However, the mechanism(s) involved in berberine-mediated cardioprotective effects has not been clearly elucidated. The aim of this study was to further investigate the mechanism(s) involved in the beneficial effects of berberine on transverse aortic contraction (TAC)-induced chronic HF. Mice were randomly divided into four groups. Berberine was administered at a dose of 50 mg/kg/day for 4 weeks via oral gavage. Our findings showed that TAC-induced pressure overload (PO) prompted cardiac dysfunction, cardiac hypertrophy, interstitial fibrosis, cardiomyocyte apoptosis and mitochondrial injury, accompanied with suppressed mitophagy, the effects of which were attenuated by berberine. Furthermore, mitophagy regulators PINK1 and mito-Parkin were downregulated in TAC-induced HF, while berberine upregulated PINK1/Parkin-mediated mitophagy. Notably, knockdown of PINK1 by small interfering RNA significantly suppressed Parkin-mediated mitochondrial ubiquitination and nullified the beneficial actions on HF exerted by berberine. Taken together, our results indicated that berberine plays a critical role in attenuating cardiac hypertrophy and preserving cardiac function from PO induced HF. The potential underlying mechanism is the activation of mitochondrial autophagy via PINK1/Parkin/Ubiquitination pathway.
Project description:Mitochondrial ATP synthase catalyzes the coupling of oxidative phosphorylation. Under pathological conditions, ATP synthase hydrolyzes ATP to replenish protons from the matrix into the intermembrane space, sustaining mitochondrial membrane potential. ATPase inhibitory factor 1 (IF1) is a nuclear-encoded, ATP synthase-interacting protein that selectively inhibits the hydrolysis activity of ATP synthase, which may render the protective role of IF1 in ischemic hearts. However, the in vivo cardiac function of IF1 and the potential therapeutic application targeting IF1 remain obscure. In the present study, we uncovered that IF1 is upregulated in mouse hearts with pressure overload-induced hypertrophy and in human hearts with dilated cardiomyopathy. IF1 knockout (KO) mice were protected against cardiac dysfunction and pathological development induced by transverse aortic constriction (TAC) or isoproterenol infusion. The reduced ATP hydrolysis activated AMPK activity in IF1 KO hearts, which together facilitated autophagy. These results suggest that IF1 upregulation in the failing heart may be a maladaptive response. Inhibiting IF1 in the hypertrophied heart not only prevents cell death from excessive mitochondrial depolarization but also activates AMPK signaling and increases autophagy. Therefore, IF1 inhibition may serve as a potential therapeutic target in treating pathological cardiac hypertrophy and heart failure.
Project description:Heart failure has high morbidity and mortality in the developed countries. Autophagy is important for the quality control of proteins and organelles in the heart. Rubicon (Run domain Beclin-1-interacting and cysteine-rich domain-containing protein) has been identified as a potent negative regulator of autophagy and endolysosomal trafficking. The aim of this study was to investigate the in vivo role of Rubicon-mediated autophagy and endosomal trafficking in the heart. We generated cardiomyocyte-specific Rubicon-deficient mice and subjected the mice to pressure overload by means of transverse aortic constriction. Rubicon-deficient mice showed heart failure with left ventricular dilatation, systolic dysfunction and lung congestion one week after pressure overload. While autophagic activity was unchanged, the protein amount of beta-1 adrenergic receptor was decreased in the pressure-overloaded Rubicon-deficient hearts. The increases in heart rate and systolic function by beta-1 adrenergic stimulation were significantly attenuated in pressure-overloaded Rubicon-deficient hearts. In isolated rat neonatal cardiomyocytes, the downregulation of the receptor by beta-1 adrenergic agonist was accelerated by knockdown of Rubicon through the inhibition of recycling of the receptor. Taken together, Rubicon protects the heart from pressure overload. Rubicon maintains the intracellular recycling of beta-1 adrenergic receptor, which might contribute to its cardioprotective effect.