Hydrogen Sulfide Preserves Endothelial Nitric Oxide Synthase Function by Inhibiting Proline-Rich Kinase 2: Implications for Cardiomyocyte Survival and Cardioprotection

Abstract

Hydrogen sulfide (H₂S) exhibits beneficial effects in the cardiovascular system, many of which depend on nitric oxide (NO). Proline-rich tyrosine kinase 2 (PYK2), a redox-sensitive tyrosine kinase, directly phosphorylates and inhibits endothelial NO synthase (eNOS). We investigated the ability of H₂S to relieve PYK2-mediated eNOS inhibition and evaluated the importance of the H₂S/PYK2/eNOS axis on cardiomyocyte injury in vitro and in vivo. Exposure of H9c2 cardiomyocytes to H₂O₂ or pharmacologic inhibition of H₂S production increased PYK2 (Y402) and eNOS (Y656) phosphorylation. These effects were blocked by treatment with Na₂S or by overexpression of cystathionine γ-lyase (CSE). In addition, PYK2 overexpression reduced eNOS activity in a H₂S-reversible manner. The viability of cardiomyocytes exposed to Η₂Ο₂ was reduced and declined further after the inhibition of H₂S production. PYK2 downregulation, l-cysteine supplementation, or CSE overexpression alleviated the effects of H₂O₂ on H9c2 cardiomyocyte survival. Moreover, H₂S promoted PYK2 sulfhydration and inhibited its activity. In vivo, H₂S administration reduced reactive oxygen species levels, as well as PYK2 (Y402) and eNOS (Y656) phosphorylation. Pharmacologic blockade of PYK2 or inhibition of PYK2 activation by Na₂S reduced myocardial infarct size in mice. Coadministration of a PYK2 inhibitor and Na₂S did not result in additive effects on infarct size. We conclude that H₂S relieves the inhibitory effect of PYK2 on eNOS, allowing the latter to produce greater amounts of NO, thereby affording cardioprotection. Our results unravel the existence of a novel H₂S-NO interaction and identify PYK2 as a crucial target for the protective effects of H₂S under conditions of oxidative stress.