Abstract
Myocardial infarction is a leading cause of death worldwide and occurs due to blockage in blood supply to the heart. Re-establishment of blood flow after a brief period of ischemia leads to paradoxical exacerbation of the cardiomyocyte and its death. This phenomenon is known as ischemia-reperfusion injury. Major evidence in the pathogenesis of ischemia-reperfusion injury is due to oxidative stress, which is an imbalance between reactive oxygen species (ROS) and antioxidants. Membrane consisting of polyunsaturated fatty acid is attacked by ROS leading to lipid peroxidation and the generation of one of the toxic aldehydes 4-hydroxynonenal (4-HNE). Evidence suggests that 4-HNE increases during an ischemia-reperfusion injury in the heart. Apoptosis-inducing factor, mitochondrion-associated 2 (AIFM2) is a mitochondrial located oxidoreductase that participates in caspase-independent apoptosis. In this study, we sought to identify the role of 4-HNE in regulating AIFm2 translocation and cardiomyocyte death during an ischemia-reperfusion injury in the heart. Following ischemia both RNA and protein expression of AIFm2 significantly increased in the ischemic heart compared to sham. Also, 4-HNE adducted AIFm2 translocated from mitochondria to the nucleus shown by western blot analysis in ischemic heart. The mass spectrometric analysis was done to see the modification site on AIFm2 by 4-HNE and revealed that His 174 and Cys 187 are two sites on AIFm2 where 4-HNE adduction occurred. To identify the modification site responsible for AIFm2 translocation we performed site-direct mutagenesis in H9C2 cardiomyocyte, where Histidine 174 was replaced by arginine and Cys 187 was replaced by threonine. When the ischemia-reperfusion injury was induced, only Histidine 174 mutant failed to translocate to the nucleus indicating His 174 modification by 4-HNE was responsible for AIFm2 translocation. To further support the transport mechanism, protein expression of Importin; an adaptor protein responsible for the transfer of proteins to the nucleus was increased in the ischemic heart compared to sham. Collectively, those results for the first time identify the unique role of 4-HNE modification on AIFm2 protein during an ischemia-reperfusion injury in the heart.
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