A Novel Mechanism For The Protective Role Of Mitochondrial Aldehyde Dehydrogenase (ALDH2) In Streptozotocin-induced Diabetic Cardiomyopathy

BackgroundDiabetes is a common metabolic disorder which brings heavily burden to society andeconomy. Diabetic complications can cause multi system damaging and has a poorprognosis. Epidemiology data shows that cardiovascular diseases are major complicationof diabetes mellitus. At present, reducing the diabetes-induced myocardial damage andimproving the quality of patients’ lives is a key point for the researchers. Mitochondrialaldehyde dehydrogenase2(ALDH2) is well known for detoxification of acetaldehydewith the role in the metabolism of acetaldehyde oxidation. ALDH2offers cardiacprotection against myopathic conditions including ischemia-reperfusion injury, alcoholismand diabetes mellitus although the precise mechanism is unclear. AMP-activatedprotein kinase (AMPK) as a downstream molecular of ALDH2in eukaryotic cells plays apivotal role in cellular energy metabolism homeostasis and regulation of autophagy. ALDH2has been reported as a pivotal cardioprotective factor through regulating AMPKsignalling pathway during ischemia-reperfusion injury. Recent study shows that ALDH2and its agonist Alda-1could effectively alleviate the alcohol-induced myocardial injury byregulating the AMPK signal pathway. However, the molecular mechanisms of ALDH2alleviates high glucose-induced cardiomyopathy injury remain incompletely understood.Recent study found that the autophagy marker proteins were decreased in STZ-induceddiabetic cardiomyopathy, which might the cause of high glucose-induced myocardialinjury. Previous research has been found that ALDH2plays a protect effect by regulatingthe autophagy upstream protein AMPK. At present, there is no study shown that the effectof ALDH2in high glucose-induced myocardial autophagy.Therefore, the aim of this study was to examine (1) whether ALDH2can protectsagainst streptozotocin-induced myocardial damage; if any,(2) the molecular mechanismof that ALDH2protects against diabetes-induced myocardial injury including AMPKdependent signal pathway and regulation of autophagy. It may provide a new strategyand target for prevention and treatment of diabetes-induced myocardial damage.ObjectivesThe purpose of this study was to explain the impact of ALDH2onstreptozotocin-induced diabetic cardiomyopathy. We examined the cell viability, apoptosis,and mitochondrial function, as well as the autophagy marker proteins expression in H9C2cells following high glucose exposured, and to explore the regulatory mechanismsinvolved in diabetes or high glucose-induced changes in autophagy with a focus on AMPKdependent signal pathway.MethodsThis study was designed to evaluate the impact of ALDH2on streptozotocin-induceddiabetic cardiomyopathy. Friendly virus B (FVB) and ALDH2transgenic mice weretreated with streptozotocin (intraperitoneal injection of200mg/kg) to induce diabetes.Then we examined the cell shortening and relengthening, intracellular Ca2+transient,histological examination. Western blotting was used to evaluate the expression of P62, Atg7, LC3B, phosphorylated of AMPK and FoxO3a. H9C2cells were incubated inDMEM with high doses (30mM) of glucose for48h, in the absence or presence of ALDH2activator Alda-1(20μM), the autophagy inhibitor3-MA (10mM), the autophagy inducerrapamycin (Rapa,100nM) and the AMPK inhibitor Compound C (CC,10mM). The cellviability was examined by MTT assay. The apoptosis was measured by terminaldeoxvnucleotidy1transferase-mediated dUTP nick-end labeling (TUNEL) assay. JC-1Assay Kit was used to examine the mitochondrial membrane potential. Western blottingwas used to evaluate the expression of P62, Atg7, LC3B, phosphorylated of AMPK,ULK1and FoxO3a.ResultsStreptozotocin led to cardiomyocyte contractile and intracellular Ca2+defects, includingdepressed peak shortening and maximal velocity of shortening and relengthening,increased cardiomyocyte area; prolonged duration of relengthening; and dampenedintracellular Ca2+rise and clearance. But these changes were effectively ablated byALDH2overexpression. Western blot analysis revealed disrupted phosphorylation ofAMPK, elevated phosphorylation of FoxO3a and downregulated expression of autophagy.Intriguingly, ALDH2attenuated or ablated streptozotocin-induced myocardial contractileand intracellular Ca2+anomalies, as well as changes the phosphorylation of FoxO3a,AMPK and the expression of autophagy. High glucose-induced cells death and apoptosiswere reversed by Alda-1. High glucose challenge dampened autophagy in H9C2cells asevidenced by enhanced p62, decreased Atg7and LC3B, the effect of which was alleviatedby the ALDH2activator Alda-1. Compared with NG group, the phosphorylation of AMPKand ULK1was significantly degraded, but the p-FoxO3a was markedly increased.Rapamycin effectively reduced the mitochondrial dysfunction. The autophagy inhibitor3-MA and the AMPK inhibitor compound C mitigated Alda-1-offered beneficial effectwhereas the autophagy inducer rapamycin mimicked or exacerbated high glucose-inducedcell injury. Moreover, compound C nullified Alda-1-induced protection againstSTZ-induced changes in autophagy and function. ConclusionBoth vitro and vivo data reveal that ALDH2plays a protective role on diabetes inducedcardiomyocyte injury, which effect may be mediated the AMPK signal pathway regulatethe level of autophagy.