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. We examine the role of mitochondrial calcium in cardiac cell death and cardioprotection by studying hearts lacking the mitochondrial calcium uniporter (MCU). Knockout (KO) of the mitochondrial Ca2+ uniporter (MCU) abrogates rapid mitochondrial Ca2+ uptake and permeability transition pore (PTP) opening. However, hearts from global MCU-KO mice were not protected from ischemic injury. Furthermore, MCU-KO hearts were resistant to protection afforded by cyclosporin A (CsA), a pore desensitizer that inhibits binding of cyclophilin D (CypD) to the PTP. This study investigates the hypothesis that the lack of protection in MCU-KO may be explained by alterations in PTP opening due to compensatory changes in CypD signaling. To investigate whether pore opening can occurs in MCU-KO, Ca2+ uptake and swelling were measured in isolated mitochondria in the presence of the Ca2+ ionophore ETH129 to permit Ca2+ entry into the matrix. With ETH129, MCU-KO mitochondria were able to take up Ca2+ and underwent pore opening similar to WT. To investigate the Ca2+ sensitivity of PTP in MCU-KO, basal Ca2+ was set to the same level in mitochondria from KO and WT prior to a Ca2+ uptake assay. MCU-KO underwent PTP opening before WT, suggesting that PTP Ca2+ sensitivity is altered in the absence of MCU. To determine whether CypD-mediated regulation of PTP opening may be different following global MCU deletion, experiments were performed to examine the interaction between CypD and the proposed PTP component ATP synthase. Mitochondria isolated from WT and MCU KO hearts were incubated with an immunocapture antibody to pulldown ATP synthase. Interestingly, preliminary results suggest that there was more CypD associated with ATP synthase in MCU KO in comparison to WT (n=5, P=0.088). As phosphorylation of CypD has been proposed to enhance PTP opening, immunoprecipitation experiments were performed using an antibody for phosphorylated proteins. MCU KO mitochondria had an increase in the amount of phosphorylated CypD (n=7, P=0.058). These results suggest that absence of MCU may alter PTP opening such that less Ca2+ is required to trigger PTP, which may be due to compensatory changes in CypD-mediated pore regulation. The subunit EMRE is thought to be critical for uniporter activity. 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. We developed a novel mouse model of EMRE deletion and determine that loss of EMRE prevents calcium uptake in isolated mitochondria. However, EMRE-/- mice, though born less frequently, are viable, healthy, and do not manifest overt metabolic impairment, at rest or with exercise. While we show that EMRE is indeed essential for mitochondrial calcium uniporter function, uniporter loss appears well-tolerated physiologically. Finally, to investigate the role of EMRE in disease processes, we examine the role of EMRE deletion in a muscular dystrophy model associated with mitochondrial calcium overload and found an increase in EMRE, suggesting that EMRE contributes to modulating uniporter activity in response to cellular stress. We also examined the role of cyclophilin D (CyPD) and post-translational modifications of CyPD in regulating cardiac cell death and protection. Our previous study in mouse embryonic fibroblasts showed that cysteine 202 of cyclophilin D (CyPD) is necessary for redox stress-induced activation of the mitochondrial permeability transition pore (mPTP). To further investigate the essential function of this cysteine residue in situ, we used CRISPR to develop a knock-in mouse model (C57BL/6N stain), where CyPD cysteine 202 was mutated to a serine (C202S-KI). The amount of total CyPD expressed in the CyPD C202S-KI did not differ compared to the wild-type (WT). However, the CyPD C202S-KI mouse hearts elicit a significant cardioprotective effect against ischemia-reperfusion (I/R) injury in the Langendorff perfused heart model. After 20 min of global ischemia followed by 90 min of reperfusion, the post-ischemic recovery of rate pressure product (RPP= heart rate x LVDP) was 45.04.2% in CyPD WT and 59.64.0% in CyPD C202S-KI mice. Myocardial infarct size was decreased in CyPD C202S-KI mouse hearts versus CyPD WT mice (24.54.7% vs 49.82.7%). Isolated heart mitochondria from CyPD C202-KI mice had a higher calcium retention capacity compared to CyPD WT mice (140.020.82 vs 213.316.67 umol Ca+2/g protein). However, in contrast to CyPD knockout mice which exhibit more pronounced cardiac hypertrophy in response to pressure overload stimulation than control mice, CyPD C202S-KI mice developed a comparable level of hypertrophy to their WT littermate in an angiotensin II-induced hypertrophy model delivered by implanted osmotic minipumps. In conclusion, these results show that mutated CyPD C202S affords cardioprotection against I/R injury, suggesting that the redox-modification of cysteine 202 might play an important role in the regulation of CyPD and its downstream targets such as mPTP. Although changes in translation and altered splicing play an important role in differentiation and disease processes such as cancer and heart failure, the precise mechanisms regulating them are not well understood. In this manuscript we provide insight into a new regulator of translation and splicing, OGFOD1 (oxoglutarate, glucose and iron dependent protein 1). Protein prolyl-hydroxylases are a family of oxygen and -ketoglutarate-dependent enzymes that catalyze hydroxylation of amino acid residues such as prolines or asparagines. We examined the role of a newly described ribosomal prolyl-hydroxylase, OGFOD1. OGFOD1 has been shown to hydroxylate a proline (P62) in the ribosomal protein S23 (Rps23) which regulates translation, and inhibition of OGFOD1 has been reported to lead to a decrease in translation and an increase in stress granule formation. The precise role of OGFOD1 in cardiomyocytes is unclear. In addition, inhibitors of the HIF prolyl-hydroxylase (PHD2; prolyl-hydroxylase domain containing protein 2) which are currently in clinical trials to treat anemia have also been recently shown to inhibit OGFOD1, adding to the importance of understanding the role of OGFOD1. To better define the specific role of OGFOD1 we deleted OGFOD1 and compared protein synthesis, stability and protein levels in isogenic WT and OGFOD1-knockout induced pluripotent stem cell-derived cardiomyocytes (OGFOD1-KO iPSC-CM). We used a previously developed in vitro model of human iPSC-derived cardiomyocytes in which we used CRISPR-Cas9 to delete OGFOD1. In vitro models of human stem cell-derived cardiomyocytes have become important for a wide range of biomedical applications such as disease modeling or drug screening. We found that loss of OGFOD1 led to a decrease in protein synthesis in proteins enriched in ribosomal proteins and splicing factors, consistent with an important role for OGFOD1 in regulating protein translation. When comparing the proteomic profiles of OGFOD1-KO to WT we made the surprising finding that loss of OGFOD1 led to an increase in a number of cardiac specific proteins. These data are consistent with the hypothesis that OGFOD1 alters translation of specific proteins leading to an altered protein landscape enhancing the levels of cardiac proteins.