Aldehyde Dehydrogenase-2Deficiency Aggravates Cardiac Dysfunction Elicited By Endoplasmic Reticulum Sress Induction

The endoplasmic reticulum (ER) is a multifunctional intracellular organelle involved in multiprocesses within the cell including synthesis and folding of secretory and membrane proteins, Ca2+homeostasis, and lipid biosynthesis. ER stress develops in response to a wide variety of cellular stressors, such as heat, hypoxia, metabolic starvation, angiotensin II, tumor necrosis and change in lipid metabolism. These stressors reduce the protein folding capacity and disrupt the homeostatic functions of the ER, leading to the accumulation and aggregation of unfolded and misfolded protein in ER, a condition referred to as the ER stress. To date, it has been well established that three ER-resident transmembrane proteins are activated to initiate and mediate ER stress:pancreatic ER kinase-like ER kinase (PERK), activating transcription factor6(ATF6) and inositol-requiring enzyme1(IRE1). Compelling evidence indicated that ER stress-induced apoptosis is an important factor in pathophysiological condition. Recently, ER stress received some significant attention by cardiovascular community as it has been implicated in an array of human diseases including ischemia/reperfusion injury of the brain and heart, atherosclerosis, cardiac hypertrophy and heart failure. Thus, ER stress appears to be a candidate instigator for pathological cell death and functional change intimately involved in the maintenance of cardiovascular homeostasis, indicating an important role as a therapeutic target for the management of cardiovascular diseases.Aldehyde dehydrogenase2(ALDH2) is emerging as a key enzyme of endogenous cytoprotection in the heart. It is involved in the detoxification of reactive aldehydes such as4-hydroxy-2-nonenal (4-HNE). Increasing evidence has revealed a beneficial role of ALDH2against tissue and cellular injuries originated alcohol, acetaldehyde and toxic aldehyde-induced reactive oxygen species (ROS) formation.4-HNE is a highly cytoxic aldehyde that gets accumulated in the heart in response to ischemia/reperfusion insult which in turn inactivates ALDH2by protein adducts formation. Pharmacological enhancement of ALDH2activity can protect the heart against ischemic injury. Transgenic overexpression of ALDH2was found to prevent ethanol exposure-induced myocardial anomalies. To the contrary, inhibition of ALDH2by oxidative stress leads to cardiac dysfunction in diabetes mellitus. These results further revealed a possible antioxidant property of this enzyme. Nevertheless, the association between ALDH2and myocardial injury mediated by ER stress has not been elucidated.Based on previous observations in the animal model challenged with tunicamycin, the aim of this study was to determine whether ALDH2knockout (KO) triggers deterioration of cardiac function after ER stress induction in ALDH2KO mice and whether ALDH2overexpression may prevent tunicamycin-induced ER stress and apoptosis.Part Ⅰ The Effect of ALDH2Knockout on Cardiac Dysfunction Induced by Endoplasmic Reticulum StressObjective This study was designed to examine the role of ALDH2knockout in the regulation of cardiac function and apoptosis following endoplasmic reticulum stress (ERS).Methods:Wild-type and ALDH2knockout mice were subjected to tunicamycin (3mg/kg) challenge for48h before assessment of echocardiography. Protein levels of Grp78, p-eIF2α and CHOP were determined using Western blot analysis. Apoptosis in the heart was detected using TUNEL assay.Results:To elicit ERS, mice were challenged with tunicamycin (3mg/kg, i.p.) prior to the assessment of echocardiographic properties. Tunicamycin overtly decreased ejection fraction and fractional shortening, indicating cardiac dysfunction following ERS. ALDH2deficiency exacerbated cardiac dysfunction following ERS induction, manifested by the change of ejection fraction and fractional shortening. Western blot analysis indicated that tunicamycin significantly increased the protein levels of Grp78, p-eIF2α and CHOP. These proteins were significantly elevated in ALDH2KO mice, compared with WT mice. TUNEL assay revealed significantly greater apoptotic cell death following tunicamycin challenge, with a more pronounced effect in ALDH2KO mice.Conclusions:ALDH2deficiency exacerbated promoted ERS and apoptotic cell death in the heart, which was implicated in the following cardiac dysfunction. Part Ⅱ The Role of ALDH2in Endoplasmic Reticulum Stress and Apoptosis in CardiomyocytesObjective This study was aim to examine the role of ALDH2in the regulation of endoplasmic reticulum stress (ERS) and apoptosis following tunicamycin challenge in cardiomyocytesMethods:To induce ER stress, cardiomyocytes were incubated with tunicamycin (5μg/ml) for12hours. Cells were pre-incubated with N-acetyl-L cystein (NAC,5mmol/L) and LY294002(20μM) respectively for1hour before adding tunicamycin. Adenoviral vectors coding for ALDH2and ALDH2siRNA were used to elevate or knockdown the expression of ALDH2in cardiomyocytes, respectively. Protein levels of Grp78, p-eIF2a and CHOP were determined using Western blot analysis. Apoptosis was evaluated using caspase-3activity and MTT assay.Results:The presence of ALDH2in the adenoviral vectors was confirmed using DNA sequencing and restriction mapping. Western blot analysis indicated that tunicamycin significantly increased the protein levels of Grp78, p-eIF2α and CHOP in cardiomyocytes, which were abrogated by ALDH2overexpression and exacerbated by ALDH2knockdown. MTT assay depicted that tunicamycin-induced toxicity was promoted by siRNA silencing of ALDH2while it was prevented by ALDH2overexpression. Moreover, tunicamycin significantly promoted caspase-3activity, the effect of which was enhanced and attenuated, respectively, by ALDH2silencing and ALDH2overexpression.Conclusions:Data from in vitro study indicated that ALDH2is a key mediator in the regulation of ERS and ERS-induced apoptosis, indicating a protective role of ALDH2against ERS in cardiomyocytes. Part III The mechanism of ALDH2in protection against Endoplasmic Reticulum Stress and Apoptosis in CardiomyocytesObjective This study was aim to explore the mechanism of ALDH2in the protection against endoplasmic reticulum stress (ERS) and ERS-induced apoptosis following tunicamycin challenge in cardiomyocytesMethods:To induce ER stress, cardiomyocytes were incubated with tunicamycin (5μg/ml) for12hours. Cells were pre-incubated with LY294002(20μM) respectively for1hour before adding tunicamycin. Adenoviral vectors coding for ALDH2was used to elevate the expression of ALDH2in cardiomyocytes. Protein levels of Grp78, p-eIF2α, CHOP, p-Akt and p47phox NADPH oxidase were determined using Western blot analysis. Apoptosis was evaluated using caspase-3activity and MTT assay.Results:Western blot analysis indicated that phosphorylation of Akt was significantly dampened after ER stress induction in cardiomyocytes, which was aggravated by ALDH2knockdown. In contrast, the depressed AKT activation was restored by ALDH2overexpression and NAC treatment. Moreover, the protein levels of p47phox subunit was significantly increased by tunicamycin and augmented by ALDH2knockdown. ALDH2overexpression and treatment of NAC mitigated p47phox subunit upregulation induced by tunicamycin. Intriguingly, inhibition of PI3-K with LY294002did not affect the decreased ER stress markers conferred by ALDH2, whereas remarkably reversed the downregulation of p47phox elicited by ALDH2. The anti-apoptosis property of ALDH2, evaluated by MTT assay and caspase-3activity, was abrogated by LY294002.Conclusions:These results indicated that the protective role of ALDH2against ERS-induced cell death was probably mediated by Akt via a p47phox NADPH oxidase-dependent manner. Part IV The Role of4-HNE in ALDH2-regulated Endoplasmic Reticulum Stress in CardiomyocytesObjective This study was aim to investigate the role of4-HNE in ALDH2-regulated endoplasmic reticulum stress (ERS) following tunicamycin challenge in cardiomyocytesMethods:To induce ER stress, cardiomyocytes were incubated with tunicamycin (5μg/ml) for12hours. Adenoviral vectors coding for ALDH2and ALDH2siRNA were used to elevate or knockdown the expression of ALDH2in cardiomyocytes. Protein levels of4-HNE-adducts, Grp78and p-Akt were determined using Western blot analysis.Results:Western blot analysis indicated that4-HNE-protein adducts were obviously increased following tunicamycin treatment, the effect of which was obliterated by ALDH2overexpression while it was were augmented by ALDH2knockdown. In addition, we found that4-HNE increased the levels of GRP78and dephosphorylation of Akt in cardiomyocytesConclusions:These results indicated that the protective role of ALDH2against ERS was probably mediated through4-HNE scavenging.