Protective role of HOXB5-orchestrated mitochondrial homeostasis and mitophagy in cardiomyocytes after ischemia-reperfusion via transcriptionally activating Sirt5.

Abstract

PURPOSE: While reperfusion is essential for restoring blood flow, it can paradoxically exacerbate myocardial injury by disrupting energy metabolism, leading to cell necrosis, apoptosis, and structural damage. Despite the significance of ischemia-reperfusion (I/R) injury, effective treatments remain scarce, highlighting the need for a deeper understanding of its underlying mechanisms to develop targeted therapies. METHODS: A rat model of myocardial I/R injury and a H9c2 cell-based hypoxia/reoxygenation (H/R) model were utilized for primary validation. HOXB5 was overexpressed several days prior to I/R or H/R induction. Gene and protein expression were assessed by RT-qPCR, Western blotting, immunohistochemistry, and immunofluorescence. Cell viability and apoptosis were evaluated using CCK-8 and flow cytometry. Mitochondrial function and ferroptosis were analyzed by commercial kits. The regulatory relationship among Lin28a, HOXB5, and Sirt5 was examined using RIP, ChIP-PCR, and dual-luciferase assays. RESULTS: HOXB5 expression was significantly reduced in infarcted myocardial tissue following I/R injury, of which overexpression attenuated H/R-trigged apoptosis to motivate H9c2 cell survival. Meanwhile, HOXB5 overexpression decreased mitochondrial ROS production, improved mitochondrial respiration, mitochondrial complexes (I, II, III, and V) activity, and reduced mitophagy under H/R conditions in H9c2 cells. Mechanistically, Lin28A was identified as a regulator of HOXB5, which in turn transcriptionally activated Sirt5. Furthermore, the protective effects of HOXB5 overexpression on myocardial histological damage and cardiac function through modulating ferroptosis in rats' I/R injury was abrogated by Sirt5 knockdown. CONCLUSION: Our study reveals the crucial role of HOXB5 in maintaining mitochondrial homeostasis and regulating mitophagy, which in turn protects cardiomyocytes from I/R injury through Sirt5. These findings underscore the HOXB5-Sirt5 pathway as a promising therapeutic target for mitigating I/R injury.