Mechanisms of cardiac ischemia-reperfusion injury and cardioprotection

National Heart, Lung, And Blood Institute

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Abstract

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. 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. Mitochondrial calcium regulates bioenergetics but also serves as a trigger for cell death. With a sustained increase in catecholamine or a large increase in cytosolic calcium as occurs with ischemia, mitochondrial calcium can rise to high levels leading to activation of the mitochondrial permeability transition pore, thus initiating cell death. Calcium uptake into mitochondria occurs via the mitochondrial calcium uniporter (MCU), which is regulated by three EF-hand proteins, mitochondrial calcium uptake (MICU) 1, 2, and 3. MICU1/MCU ratios vary in different tissues, and alterations in substrate have been shown to regulate MICU1 levels altering MCU-mediated calcium uptake. Mitochondrial Ca2+ overload is proposed to regulate cell death via opening of the mitochondrial permeability transition pore. It is hypothesized that inhibition of the mitochondrial Ca2+ uniporter (MCU) will prevent Ca2+ accumulation during ischemia/reperfusion and thereby reduce cell death. To address this, we evaluate mitochondrial Ca2+ in ex vivo perfused hearts from germline MCU-KO and WT mice using transmural spectroscopy. Matrix Ca2+ levels are measured with the genetically encoded, red fluorescent Ca2+ indicator (R-GECO1) using an adeno-associated viral vector (AAV9) for delivery. Due to the pH sensitivity of R-GECO1 and known fall in pH during ischemia, hearts are glycogen depletion to decrease the ischemic fall in pH. At 20 mins of ischemia, there is significantly less mitochondrial Ca2+ in MCU-KO hearts compared to MCU-WT controls. However, an increase in mitochondrial Ca2+ is present in MCU-KO hearts suggesting that mitochondrial Ca2+ overload during ischemia is not solely dependent on MCU. MICU3 is a regulator of MCU which has generally been thought to function primarily in neuronal tissue where it is highly expressed, and thus has been largely ignored in other tissues such as the heart. We performed quantitative proteomics using Tandem Mass Tag labelling coupled to liquid chromatography and tandem mass spectrometry to analyze MCU and MCU regulators in cardiac and hepatic mitochondria. Normalizing MICU levels to MCU in heart and liver mitochondria, we found that the MICU1/MCU ratio was 0.75 in liver and 0.25 in heart which is consistent with previous studies3; however, the MICU3/MCU ratio in heart is more than three-fold higher than that found in liver. To confirm that the MICU3 we observed in heart mitochondria was not due to contamination by mitochondria from nerve tissue in heart, we isolated cardiomyocytes and compared the ratio of MICU3 to MCU in cardiomyocytes and heart mitochondria and found similar ratios. These data suggest that MICU3 might play a role in regulating MCU in heart and skeletal muscle. We generated a mouse model of systemic MICU3 ablation and examined its physiological role in skeletal muscle. We found that loss of MICU3 led to impaired exercise capacity. When the muscles were directly stimulated there was a decrease in time to fatigue. MICU3 ablation significantly increased the maximal force of the KO muscle and altered fiber type composition with an increase in the ratio of type IIb (low oxidative capacity) to type IIa (high oxidative capacity) fibers. Furthermore, MICU3-KO mitochondria have reduced uptake of Ca2+ and increased phosphorylation of pyruvate dehydrogenase (PDH), indicating that KO animals contain less Ca2+ in their mitochondria. Skeletal muscle from MICU3-KO mice exhibit a net oxidation of NADH during electrically stimulated muscle contractions, These data demonstrate that MICU3 plays an essential role in skeletal muscle physiology by setting the proper threshold for Ca2+m uptake, which is important for matching energy demand and supply in muscle. We also measured Ca2+m in MICU3-KO and WT perfused hearts loaded with Rhod-2-AM using optical transmural spectroscopy in an integrating sphere as described above. We perfused hearts for 5 min with Isoproterenol (Iso) to increase Ca2+m uptake. Transmural Rhod-2 fluorescence, a measure of Ca2+m, was collected by a spectrometer. Data were analyzed by subtraction of tissue background and correction for inner filter effects. MICU3-/- hearts exhibited significantly less increase in Ca2+m during the 5 minute stimulation with Iso. WT hearts showed a 2.500.11 fold increase in fluorescence with Iso treatment, whereas MICU3-/- hearts showed a 1.250.05 fold increase. As cardiac injury after ischemia-reperfusion is thought to be associated with increased mitochondrial calcium leading to mitochondria-initiated cell death, we tested whether Micu3-/- hearts were protected against ischemia-reperfusion injury. Ex vivo Langendorff-perfused hearts from WT and Micu3-/- mice were subjected to 20 minutes of global ischemia followed by 90 minutes of reperfusion. Micu3-/- hearts had reduced infarct size normalized to the entire LV and improved contractile function following reperfusion. Protection in ex vivo Micu3-/- hearts strongly supports a role for MICU3 in cardiac mitochondria. Interestingly, germline ablation of MCU or EMRE was not cardioprotective, likely due to adaptations in these germline knockout mice. We speculate that loss of MICU3 does not lead to adaptation and under conditions of prolonged elevated calcium, the loss of MICU3 is protective. We also performed some studies addressing the role of transcriptional regulation in cardiovascular disease. Transcriptional changes in heart failure have been the focus of numerous studies. Disparities between transcript and protein abundances highlight the need to better understand translation in heart failure. One mechanism for achieving proteomic diversity through protein synthesis and stability is through the activity of prolyl hydroxylases. 2-oxoglutarate- and Fe2+-dependent dioxygenases modify target proteins to regulate protein stability, turnover, and activity. A lesser-known member of this family is 2-oxoglutarate- and Fe2+-dependent oxygenase domain-containing protein 1 (OGFOD1). OGFOD1 regulates translation by catalyzing prolyl hydroxylation of ribosomal protein S23 (RPS23), but its cardiac functions are largely unexplored. To define the role of OGFOD1 in heart disease, we measured OGFOD1 RNA and protein in failing and non-failing human hearts, and found that OGFOD1 was significantly higher in failing hearts. Because heart failure can result from many pathologies, including hypertrophy, we investigated the role of OGFOD1 in cardiac hypertrophy. We induced hypertrophy via pharmacological -adrenergic stimulation by treating wildtype (WT) and OGFOD1-knockout (KO) mice with isoproterenol (ISO). In an independent study, we induced pressure-overload-mediated hypertrophy using transverse aortic constriction (TAC). KO mice showed greater than 10% reduction in hypertrophy, regardless the mode of hypertrophy induction (P = 0.0132 for TAC, P = 0.0101 for ISO). Future studies will utilize ribosome profiling to identify transcript pools whose synthesis are regulated by OGFOD1 to confer protection in cardiac hypertrophy. Results from these studies will provide important mechanistic insight into the therapeutic potential of OGFOD1 in human disease.