Abstract 9711: Cardioprotection by Endogenous Cardiac Myocyte P2x4; Receptors in Pressure Overload and Ischemic Heart Failure

Abstract

Despite progress in its therapy, heart failure is a leading cause of mortality and morbidity. In defining a biological role of the ligand-gated ion channel P2X4 receptor, we uncovered a protective role of this channel in heart failure. P2X4 gene was flanked by loxP sites bwteen exons 2 and 4. The second loxP site together with the Frt-PGKneo-Frt cassette was inserted in intron 1 in a direction opposite to P2X4 transcription. After targeting the ES cells, PGKneo was removed by breeding chimeric males with ROSA26-Flpe females to generate P2X4 flox/+ F1 pups. To create cardiac myocyte-specific knockout of P2X4 receptors, homozygous P2X4 floxed/floxed mice were bred with Tg mice (Myh6-cre/Esr1*, Jax stock no: 005650) containing a tamoxifen responsive Cre-estrogen receptor chimeric recombinase driven by the α-myosin heavy chain promoter. Knockout of P2X4 receptors (KO) was confirmed by lack of detectable P2X4 with immunoblotting and by lack of contractile response to the P2X agonist 2-methylthioATP. KO animals showed normal basal cardiac function but depressed contractile performance in both left coronary artery ligation and pressure overload models of heart failure by echocardiography and isolated working heart parameters. By contrast, cardiac myocyte-specific overexpression of the P2X4 receptor protected against pressure overload-induced and post-infarct heart failure. P2X4 receptors co-immunoprecipitated with nitric oxide synthase 3 (NOS3 or eNOS). Administration of eNOS inhibitor L-NIO blocked the salutary effect of cardiac P2X4 receptor overexpression in post-infarct heart failure as did eNOS knockout. The present study uncovered a protective role of endogenous cardiac myocyte P2X4 receptors in heart failure and demonstrated an important role of eNOS in mediating this protection.