GUCY1A1-LDHA Axis Suppresses Ferroptosis in Cardiac Ischemia-Reperfusion Injury

BACKGROUND: Ischemia-reperfusion injury compromises revascularization strategies for myocardial infarction and contributes to cardiac microvascular disorders. This study aimed to investigate the role of the sGC (soluble guanylate cyclase)–cGMP–PKG (protein kinase G) pathway in cardiac microvascular reperfusion injury with a focus on ferroptosis. METHODS: Key genes in the sGC-cGMP-PKG pathway were analyzed at different reperfusion times using bulk and single-cell mRNA sequencing. Endothelial cell (EC) specific conditional GUCY1A1 (guanylate cyclase soluble subunit alpha 1) knockout mice (GUCY1A1 flox/flox /-CreERT2) and adeno-associated virus transfer-induced EC-specific GUCY1A1-overexpressing mice were assessed for cardiac microvascular reperfusion injury. LDHA (lactate dehydrogenase A) and GPX4 (glutathione peroxidase 4) phosphorylation sites were identified by mass spectrometry and mutationally inactivated via the CRISPR–Cas9 (clustered regularly interspaced short palindromic repeats and their associated protein 9) system. Protein interactions and chaperone-mediated autophagy of GPX4 were detected using coimmunoprecipitation assays. RESULTS: GUCY1A1 was decreased in the EC group after cardiac ischemia-reperfusion injury. EC-specific knockout of GUCY1A1 further reduced microvascular perfusion, increased the no-reflow area, and enlarged the infarction area in the acute phase of ischemia-reperfusion injury, ultimately aggravating cardiac dysfunction and structural remodeling in the chronic phase. In contrast, GUCY1A1 overexpression or its activator, vericiguat, alleviated microvascular dysfunction via the suppression of endothelial ferroptosis; the effects were majorly dependent on PKG activity. Mechanistically, PKG phosphorylated LDHA at threonine 95 and activated the LDHA moonlighting kinase function to phosphorylate GPX4, resulting in reduced chaperone-mediated autophagy-dependent degradation of GPX4 and ferroptosis. In human ischemic cardiomyopathy, GUCY1A1 expression, LDHA phosphorylation at threonine 95, and GPX4 phosphorylation at serine 131 were negatively associated with lipid peroxidation and cardiac fibrosis, suggesting that this pathway was involved in the pathogenesis of cardiac ischemia-reperfusion injury. CONCLUSIONS: These findings indicate that the compromise of the sGC-cGMP-PKG pathway is associated with reduced phosphorylation of LDHA and GPX4, and that GUCY1A1 activation may be considered as a strategy to alleviate endothelial ferroptosis and cardiac microvascular reperfusion injury.