Mechanisms of cardiac ischemia-reperfusion injury and cardioprotection

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The long-term goals of this project are to understanding mechanisms involved in cardioprotection. We have focused on the role of mitochondrial calcium and the permeability transition pore. We have also initiated studies examining the role of proline hydroxylation in cardiac disease. Ischemia-reperfusion (I/R) injury is mediated in large part by opening of the mitochondrial permeability transition pore (PTP). Consequently, inhibitors of the PTP hold great promise for the treatment of a variety of cardiovascular disorders. At present, PTP inhibition is obtained only through the use of drugs such as cyclosporine (CsA) which target cyclophilin D (CypD) a key modulator, but not a structural component of the PTP. This limitation might explain controversial findings in clinical studies. Therefore, we investigated the protective effects against I/R injury of small-molecule inhibitors of the PTP (Compound 63 and TR002) that do not target CypD. These compounds were identified by a combination of high throughput screening and follow-up medicinal chemistry to discover novel small-molecule inhibitors of the PTP. Both compounds exhibited a dose-dependent inhibition of PTP opening in isolated mitochondria and were more potent than CsA. Notably, PTP inhibition with these novel inhibitors was observed also in mitochondria devoid of CypD. Compounds 63 and TR002 prevented PTP opening and mitochondrial depolarization induced by calcium overload. Cardioprotection was observed also in adult mouse ventricular myocytes as well as ex vivo in perfused hearts treated with these PTP inhibitors. Thus, this study demonstrates that compounds 63 and TR002 represent novel cardioprotective agents that inhibit PTP opening independent of CypD targeting. An increase in mitochondrial calcium is a well-known trigger of cell death. We were therefore interested in investigating the mitochondrial calcium uniport (MCU) complex, which is responsible for mitochondrial calcium uptake. In previous years we have studied the MCU protein. EMRE is another component of MCU complex. We investigated the role of EMRE in regulating mitochondrial calcium. The uniporter subunit EMRE interacts with the channel-forming protein MCU and is essential for mitochondrial calcium uptake in cells, but EMREs impact on organismal physiology is less well-characterized. To understand the role of EMRE in physiology, we generated the first mouse model of EMRE deletion and showed that mitochondria lacking EMRE are unable to take up calcium. We also measured mitochondrial calcium in situ in an isolated flexor digitorum brevis muscle fiber loaded with the fluorescent calcium-sensitive dye Rhod-2 AM; the mitochondria were stained with MitoTracker Green, and the ratio of Rhod-2 AM to MitoTracker Green wasmeasured. Using this method, we found significantly lower mitochondrial calcium in the EMRE deleted muscle fibers, and obtained similar results using other methods to measure mitochondrial calcium in liver and heart mitochondria. Furthermore, in a blue native gel we showed that in EMRE-/- mitochondria the MCU complex has a lower molecular weight, suggesting that EMRE is needed for correct assembly of the MCU complex. EMRE-/- mice are born less frequently, suggesting an important role for EMRE during development. However, the mice which are born are viable, healthy, and do not manifest overt metabolic impairment, at rest or with exercise. We examined the role of the uniporter in a disease model of muscular dystrophy exhibiting calcium overload and found an increase in EMRE, suggesting that EMRE contributes to modulating uniporter activity in response to cellular stress. We have also studied the role of another post-translational modification which can be regulated by hypoxia and appears to play a role in cardioprotection. Prolyl hydroxylation is a post-translational modification that regulates protein stability, turnover, and activity. The proteins that catalyze prolyl hydroxylation belong to the 2-oxoglutarate- and iron-dependent oxygenase family of enzymes. A newly-described member of this family is 2-oxoglutarate- and iron-dependent oxygenase domain-containing protein 1 (Ogfod1). We isolated hearts from wild type (WT) and Ogfod1 knockout (KO) mice and performed quantitative proteomics using Tandem Mass Tag labelling coupled to Liquid Chromatography and tandem Mass Spectrometry (LC-MS/MS) to identify protein changes. Ingenuity Pathway Analysis identified Urate Biosynthesis/Inosine 5-phosphate Degradation and Purine Nucleotides Degradation II (Aerobic) to be the most significantly-enriched pathways among up-regulated proteins. To determine whether these proteomic changes translated to metabolic changes, we analyzed metabolites. Inosine 5-monophosphate (IMP) was 3.5x higher in KO hearts (P = 0.011) further supporting a role for Ogfod1 in regulating purine nucleotide metabolism. Enhancing purine nucleotide salvage has been shown to increase ATP pools and improve cardiac function in hearts where ATP has been depleted as a result of an ischemic episode. Therefore, we challenged Ogfod1-knockout mice with ischemia and reperfusion (I/R), or increased cardiac work, and found that Ogfod1 ablation protecteds the myocardium against I/R injury and hypertrophy against these ischemia-reperfusion injury. Taken together, these data show that Ogfod1 deletion alters purine degradation, leading to protection against obesity and ischemia-reperfusion injury and hypertrophic remodeling, and making OGFOD1 a potential therapeutic target. In summary, using these multiple approaches we have define new targets of cardioprotection.