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. As mitochondrial Ca2+ has been postulated to regulate cell energetics and cell death; pathways that are best studied in an intact organ. We develop a method to optically measure mitochondrial Ca2+ and demonstrate its validity for mitochondrial Ca2+ and metabolism using hearts from wild type mice and mice with germline knockout of the mitochondria calcium uniporter (MCU-KO). To determine whether this method was suitable for measuring physiological changes in mitochondrial Ca2+ we treated ex vivo wild type hearts with 20 nM isoproterenol. The data show a clear increase in Rhod-2 fluorescence in WT hearts consistent with an increase in mitochondrial Ca2+. We previously reported that germline MCU-KO hearts do not show an impaired response to adrenergic stimulation. To test whether this lack of impairment in germline MCU-KO mice was due to an alternative Ca2+ uptake mechanism we measured mitochondrial Ca2+ in WT and MCU-KO hearts (n=5 for each genotype) following treatment with 20 nM isoproterenol. The WT and MCU-KO hearts exhibited a similar increase in heart rate, flow rate and left ventricular developed pressure (LVDP) confirming our previous results in which we found little or no difference in contractility following adrenergic stimulation in an in vivo model in the germline MCU-KO mice. Concurrent with the measurement of flow rate and heart rate we measured changes in mitochondrial Ca2+ following isoproterenol addition in WT and MCU-KO hearts. We observed a 3-4 fold increase in Rhod-2 fluorescence in WT hearts with addition of isoproterenol, but consistent with the lack of MCU we observed no increase in Rhod-2 fluorescence in the MCU-KO hearts. Taken together these data do not support an alternative Ca2+ entry mechanism into the mitochondria in the MCU-KO hearts, at least during 5 min of isoproterenol stimulation. Furthermore, the lack of increase in Rhod-2 fluorescence following isoproterenol treatment in the MCU-KO hearts confirms the mitochondrial localization of the Rhod-2 as the increase in heart rate and flow with isoproterenol demonstrate the cytosolic Ca2+ did increase without any detected changes in corrected Rhod-2 fluorescence. 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. We 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.