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. MICU3 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. To examine the physiologic function of MICU3, we generated two independent mouse lines with global deletion of MICU3 (Micu3-/-) utilizing CRISPR-Cas9 methods. By co-microinjecting sgRNAs targeting Exon 1 and Exon 12, we created two mutant lines, one with a small frameshift deletion in Exon 1 and the other with the entire region between Exon 1 and Exon 12 deleted. The mice were viable, and there were no gross differences in adult Micu3-/- mice compared to wild-type (WT) littermates. MICU3 protein expression was not present in Micu3-/- cardiomyocytes. Preliminary results showed no significant differences between the two lines, thus subsequent studies were focused on the line with deletion of Exon 1-12. MCU, MICU1 and NCLX protein levels were similar between the WT and Micu3-/- hearts. Echocardiograms showed no significant baseline differences in heart rate, fractional shortening (FS) or ejection fraction (EF) between WT and Micu3-/- mice. MICU3 has been shown to facilitate MICU1-mediated progressive channel activation by calcium. As sustained mitochondria calcium overload associated with long term isoproterenol treatment has been suggested to lead to hypertrophy and cardiac dysfunction, we tested the hypothesis that Micu3-/- mice would have less dysfunction following isoproterenol-induced hypertrophy. Both sexes of adult (8-17 weeks) Micu3-/- and WT littermate mice were treated with continuous infusion of isoproterenol (15mg/kg/day) via an osmotic mini-pump for two weeks. Echocardiograms were performed at baseline and after treatment to assess changes in cardiac dimensions and function. Whereas WT mice exhibited depressed function (FS and EF) after 2 weeks of treatment with isoproterenol, Micu3-/- mice demonstrated normal function. Correspondingly, WT mice developed left ventricular (LV) dilation from baseline, whereas LV dimension remained stable in Micu3-/- mice. To determine whether loss of MICU3 protected hearts from isoproterenol-induced mitochondrial calcium overload, after 2 weeks of isoproterenol treatment we assessed levels of phosphorylated pyruvate dehydrogenase (PDH). Micu3-/- hearts exhibited elevated phosphorylated PDH, which has been demonstrated to be inversely proportional to mitochondrial calcium level, indicating lower mitochondrial calcium relative to WT hearts. 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. Our results show that loss of MICU3 confers cardioprotection against ischemia-reperfusion injury and isoproterenol-induced cardiac dysfunction. MICU3 levels relative to MCU and MICU1 are higher in heart than in liver, and loss of MICU3 reduces calcium overload during sustained isoproterenol treatment. Taken together these data support the novel finding that MICU3 plays an important role in regulating pathological calcium overload in the heart. 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). We previously reported that germline MCU-KO hearts do not show an impaired response to adrenergic stimulation. We tested whether these MCU-KO hearts have alternative Ca2+ uptake mechanisms and found a complete lack of increase in mitochondrial Ca2+ in the absence of MCU with little impact on function or metabolism following isoproterenol. 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. We have performed studies to investigate the mechanisms of cardioprotection in these mice.