Inhibition of Pyk2 prevents Cx43 remodeling and cardiomyocyte injury during hypoxic and adrenergic stress

Hypoxia induces pathological remodeling in cardiomyocytes, in part by disrupting gap junction communication through Connexin43 (Cx43) phosphorylation and internalization. Here, we identify Proline-rich tyrosine kinase 2 (Pyk2) as a critical mediator of hypoxia- and adrenergic-induced Cx43 remodeling and cardiomyocyte dysfunction. Neonatal rat ventricular myocytes exposed to hypoxia (1 % O₂) for 1 or 24 h exhibited increased expression of hypoxia-inducible factor 1α and activated Pyk2 (pY402 and pY579/580), without changes in the total level of Pyk2. Hypoxia also enhanced Cx43 phosphorylation at Y265, a Pyk2-specific site, and increased expression of GJA1-20 k, an alternatively translated Cx43 isoform that supports Cx43 trafficking and mitochondrial function. Pharmacological Pyk2 inhibition with PF4618433 prevented hypoxia-induced Pyk2 activation, Cx43 phosphorylation, and attenuated the increase in GJA1-20 k expression. Immunofluorescence analysis revealed that hypoxia reduced Cx43 localization at intercellular junctions, an effect reversed by PF4618433 treatment. Functionally, Pyk2 inhibition reduced cell damage, as reflected by lower lactate dehydrogenase release and improved contractile activity. Mechanistically, PF4618433 attenuated apoptosis, oxidative stress, and preserved mitochondrial membrane potential, consistent with improved mitochondrial function. Furthermore, norepinephrine-induced Pyk2 activation and Cx43 Y265 phosphorylation were comparably suppressed by PF4618433, linking Pyk2 to both hypoxic as well as adrenergic stress signaling in cardiomyocytes. These findings demonstrate that Pyk2 activation promotes Cx43 downregulation, contributing to cardiomyocyte injury under hypoxic and adrenergic stress. Inhibition of Pyk2 preserves gap junction integrity, maintains mitochondrial function, and enhances cell survival, supporting Pyk2 as a promising therapeutic target in cardiac injury.