P6283Downregulation of Tim44 exacerbates oxidative stress-induced ROS production and cardiomyocytes death by reducing mitochondrial SOD2

Abstract Background Mitochondrial dysfunction has been highlighted as a critical driver of cardiac remodeling and failure. Mitochondria contains about 1500 proteins, 99% of which are encoded in the nuclear genome. Therefore, protein import into mitochondria is essential to maintain mitochondrial function. Previous reports suggest that nuclear-encoded mitochondrial precursor proteins import into mitochondria by multiple complex; translocase of outer membrane (TOM), translocase of inner membrane (TIM), and protein associated motor (PAM). However, the role of these protein import machineries of mitochondria in cardiac remodeling remains to be elucidated. Objective The purpose of this study was to elucidate the role of TOM, TIM, and PAM complex in cardiac remodeling and cardiomyocyte death. Methods and results C57BL/6J mice were subjected to myocardial infarction (MI) by permanent ligation of left anterior descending artery. Four weeks after operation, MI-mice demonstrated left ventricular (LV) dilation (LV end-diastolic dimension: 3.91 vs. 5.54 mm, n=8–11, p<0.05) and dysfunction (LV fractional shortening: 33.3 vs. 7.7%, n=8–11, p<0.05). Tim44 protein levels, a component of PAM complex, in mitochondrial fraction from non-infarcted left ventricle were significantly decreased compared with those in the heart from sham-operated mice by 39% (p<0.05), whereas other proteins related to TOM, TIM and PAM complex such as Tom20, Tom22, Tom40, Tom70, Tim22, Tim23 and mtHSP70 were not altered between MI-mice and sham-mice. In addition, blue-native polyacrylamide gel electrophoresis revealed that a protein complex associated to Tim44 was significantly decreased in non-infarcted LV by 40% (p<0.05). Superoxide dismutase 2 (SOD2), a mitochondrial matrix protein, was decreased in mitochondrial fraction from non-infarcted LV by 20% (p<0.05), accompanied by enhancing protein carbonylation, a marker of oxidative stress, by 40% (p<0.05). To assess the role of Tim44, it was downregulated by small interfering RNA in cultured neonatal rat ventricular myocytes (NRVMs). Knockdown of Tim44 significantly decreased SOD2 protein levels in mitochondrial fractionation (22%, p<0.05), with no significant changes in its mRNA levels. Furthermore, knockdown of Tim44 significantly increased protein carbonylation (20%, p<0.05) and cleaved caspase 3 (47%, p<0.05) and decreased cell viability (69%, p<0.05), assessed by cell titer assay, in H2O2-treatred NRVMs. Conclusions Downregulation of Tim44 exacerbates oxidative stress-induced ROS production and cardiomyocytes death, which is associated with a decrease in mitochondrial SOD2. Endogenous Tim44 might play a protective role in cardiac remodeling by attenuating oxidative stress and cardiomyocyte death via SOD2 import into mitochondria.