Mechanisms of cardiac ischemia-reperfusion injury and cardioprotection

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

Cardiovascular disease is the leading cause of death in the US. It is characterized by inappropriate cardiomyocyte death, and as adult cardiomyocytes undergo limited cell division, cardiomyocyte death has detrimental consequences. In spite of considerable effort, the mechanisms responsible for cardiomyocyte death are poorly understood. This lack of mechanistic understanding is a major contributor to the failure to translate cardioprotective drugs to the clinic. An elevation in mitochondrial Ca2+ and ROS are reported to be the primary initiators for cell death in cardiomyocytes by activation of a non-specific channel in the inner mitochondrial membrane known as the permeability transition pore (PTP). Because the identity of the PTP is debated, we have focused on understanding the mechanisms regulating the increase in mitochondrial Ca2+ and ROS during ischemia and reperfusion. To better understand how Ca2+ regulates cardiac function, it is important to have robust methods to measure mitochondrial Ca2+ in models such as perfused heart. We developed a novel method for measuring mitochondria Ca2+ in a beating perfused heart. To better understand the mechanisms responsible for regulating mitochondrial Ca2+ we studied several mouse models with genetic deletion of key mitochondrial transport proteins and their regulators. We tested the hypothesis that mitochondrial calcium uniporter (MCU)-dependent Ca2+ entry into the mitochondria during ischemia and/or reperfusion causes an increase in mitochondrial Ca2+ that initiates cell death. Using the newly developed methods, we demonstrated that loss of MCU reduces but does not eliminate the rise in mitochondrial Ca2+ during ischemia. We also identified that MICU3 a regulator of MCU plays and important role in regulating mitochondrial Ca2+. MICU3 knock out hearts and cardiomyocytes exhibited a significantly smaller increase in [Ca2+]m than wild-type hearts following acute isoproterenol infusion. In contrast, heart with overexpression of MICU3 exhibited an enhanced increase in [Ca2+]m compared with control hearts. Echocardiography analysis showed no significant difference in cardiac function in knock out MICU3 mice relative to wild-type mice at baseline. However, mice with overexpression of MICU3 exhibited significantly reduced ejection fraction and fractional shortening compared with control mice. We observed a significant increase in the ratio of heart weight to tibia length in hearts with overexpression of MICU3 compared with controls, consistent with hypertrophy. We also found a significant decrease in MICU3 protein and expression in failing human hearts. Our results indicate that increased and decreased expression of MICU3 enhances and reduces, respectively, the uptake of [Ca2+]m in the heart. We conclude that MICU3 plays an important role in regulating [Ca2+]m physiologically, and overexpression of MICU3 is sufficient to induce cardiac hypertrophy, making MICU3 a possible therapeutic target. We also examined the role of mitochondrial Ca2+ efflux pathways. Transmembrane protein 65 (Tmem65), a mitochondrial inner membrane protein, has been shown to be required for Na+-dependent mitochondrial Ca2+ extrusion. A Tmem65 cardiac-specific knockout (Tmem65 cKO) mice generated by crossing Tmem65fl/fl mice with Myh6-Cre mice were used to investigate the loss function of Tmem65 in the heart. Using isolated cardiac mitochondria we demonstrated that ablation of Tmem65 resulted in a loss of Na+-dependent mitochondrial Ca2+ extrusion. Using transmural spectroscopy, mitochondrial matrix free Ca2+ was measured in ex vivo perfused hearts expressing a genetically encoded red fluorescent Ca2+ indicator (R-GECO1). No-flow global ischemia significantly increased mitochondrial Ca2+ in WT hearts, while there was no detectable ischemia-induced mitochondrial Ca2+ increase in Tmem65 cKO hearts. Interestingly, there was no differences in post-ischemic cardiac functional recovery and myocardial infarction between WT control and Tmem65 cKO hearts subjected to ischemia reperfusion. These findings demonstrate that Tmem65 plays an important role in regulating Na+-dependent mitochondrial Ca2+ efflux, which might have an impact on mitochondrial Ca2+ level at baseline and during disease states. Mitochondria are considered to be a primary source of reactive oxygen species (ROS) during cardiac ischemia-reperfusion injury. There is evidence that succinate, accumulated during ischemia, is oxidized by succinate dehydrogenase (SDH) upon tissue reperfusion which may facilitate the production of ROS by reverse electron transport (RET) at complex I. This mechanism requires that mitochondrial membrane potential, the primary component of the proton motive force (Delta p), is substantial upon reperfusion, however, until now real-time assessment of mitochondrial membrane potential has not been possible in intact heart models. To address this limitation, we interrogated the relationship between mitochondrial membrane potential and the reduction state of mitochondrial b haems which sensitively and rapidly respond to mitochondrial membrane potential. Multi-wavelength absorbance spectroscopy and spectral analysis enabled the continuous monitoring of redox-sensitive mitochondrial haems bL and bH in isolated mitochondria and the isolated perfused mouse heart. Calibration of b haem absorbance against triphenylmethylphosphonium (TPMP+) distribution in isolated mitochondria revealed the parameter fbL, calculated as the proportion of reduced bL relative to total reduced b haem (fbL = bL/bL+bH), exhibited a sigmoidal relationship with mitochondrial membrane potential. To determine how mitochondrial membrane potential responds during cardiac ischemia-reperfusion injury, b haem absorbance was determined in the isolated perfused heart by transmural absorbance spectroscopy and mitochondrial membrane potential calculated using the standard curve generated in isolated mitochondria. We found that mitochondrial membrane potential was 166 ± 18 mV (n=25, mean ± SD) in the isolated heart at normoxia and could be modulated by cardioactive agents including uncoupler. When hearts were subjected to global ischemia-reperfusion, mitochondrial membrane potential declined during ischemia but was rapidly re-established upon reperfusion. The re-establishment of mitochondrial membrane potential upon reperfusion was blocked by Atpenin A5, an SDH inhibitor, and BAM15, an uncoupler, confirming that proton pumping by the respiratory chain was responsible for the repolarization observed. Here we describe a non-invasive approach for the real-time determination of mitochondrial membrane potential in the isolated heart. Together these data show that mitochondrial membrane potential is rapidly re-established upon reperfusion consistent with the possibility that RET at complex I contributes to ischemia-reperfusion injury. To establish a clear link between succinate accumulation and mitochondrial-derived ROS in cardiac ischemia-reperfusion injury, future work will focus on real-time detection of ROS using the genetically-encoded mitochondrial-targeted fluorescent ROS indicator, HyPer7.