Increasing HCCS function in vivo mediates restoring heme integrity, limiting superoxide overproduction, and conferring reconditioning mitochondrial function in the acute postischemic heart

Abstract

Background: A serious consequence of acute myocardial ischemia-reperfusion injury (I/R) is heme impairment which causes mitochondrial dysfunction, which contributes to cell death. Such acute I/R-induced mitochondrial dysfunction is characterized by impaired respiration and overproducing superoxide. The cascading ROS propagates heme damage in the array of mitochondrial electron transport chain, adding insult to myocardial injury. Mitochondrial holocytochrome c synthetase (HCCS) catalyzes attaching heme to apocytochrome c or apocytochrome c1 by forming thioether bonds. We reasoned that overexpression of HCCS would ameliorate I/R injury by stabilization of heme moieties in key mitochondrial enzymes. Methods: A mouse model with overexpression of HCCS in the myocytes (HCCS-tg) was generated and echocardiography was used to evaluate cardiac function. The HCCS-tg mouse was subjected to 30-min of coronary ligation and 24-h of reperfusion. Mitochondria were isolated from the myocardium of the risk region and assessed by OCR. The profile of heme from the cardiac mitochondria was assessed by differential UV/VIS spectral analysis. EPR spin-trapping and ubiquinol (Q2H2)-mediated cytochrome c reduction were used to assay superoxide generation by CxI (complex I) and CxIII (complex III) in mitochondria. Results: We detected gaining HCCS function in vivo (one-fold increase) marginally increases the fractional shortening of the mouse heart. There is no significant difference in heart rate and LVIDs/LVIDd between wild type control and HCCS-tg. Increasing function of HCCS in the myocytes further enhances mitochondrial function of HCCS-tg via increasing state-3 OCR, decreasing state-4 OCR, and alleviating superoxide generation by CxI (NADH-linked) and CxIII (Q2H2-linked). Increased HCCS function significantly decreases the infarct size assessed by IF/AAR and IF/LV when the mouse hearts were subjected to acute I/R injury. Overexpressing HCCS in myocytes further protected the heme integrity and the associated activity of electron transport chain in the mitochondria from acute I/R injury. Acute I/R also mediates downregulation of Cox10 and Cox15 required for mitochondrial a-type heme (heme a) biosynthesis and integrity of Cx4 (complex IV) activity, while increasing HCCS function in vivo preserves the integrity of heme a and protects the levels of Cox10, Cox15, and Cx4 activity after acute I/R injury. Conclusion: Acute I/R mediates mitochondrial heme impairment and associated mitochondrial dysfunction via impairing HCCS function and the thioether bonds of c-type heme, whereas overexpression of HCCS in the myocytes mediates protecting integrity of heme from I/R injury, limiting overproducing superoxide, and conferring reconditioning mitochondrial function in the post-ischemic heart. None. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.