Non-Selective Cation Currents Mediated by Cx43 Hemichannel-P2X4 Receptor Signaling Pathway in Rat Atrial Myocytes under Shear Stress

Abstract

Cardiac myocytes are exposed to shear stress under physiological conditions and hemodynamic disturbances. Shear stress has been thought to trigger two distinct global Ca2+ waves in atrial myocytes through autocrine activation of P2X4 receptor (P2X4R) and P2Y1-receptor (P2Y1R) via connexin 43 (Cx43)-mediated ATP release. In this study, we examined whether such shear stress-triggered Cx43-ATP-P2 receptor signaling generates depolarizing currents in atrial myocytes. Whole-cell patch clamp and dye-flux assays were used with genetic and pharmacological interventions in rat atrial myocytes and HL-1 cells. We found that shear stress activated a non-selective inward cation (Cs+) current (IS,Cs) at resting potential which was eliminated by Cx43 blockade using siRNA, antibodies, carbenoxolone or La3+. IS,Cs was enhanced by removal of external Ca2+, whereas internal Ca2+ buffering suppressed IS,Cs. Hemichannel-mediated dye (calcein red-orange) flux was induced by shear stress. This response was augmented by removing external Ca2+ but was not affected by pannexin blocker (probenecid). The shear-activated current was significantly reduced when Cs+ was replaced with NMDG+, and this was enhanced by quinine and suppressed by La3+. P2X4R antagonists (5-BDBD and PSB-12054), P2XR-blocker (iso-PPADS) and anti-P2X4R antibodies each blocked IS,Cs by approximately 50%. About 25% of IS,Cs was suppressed by P2Y1R inhibitor (MRS2179) and/or transient receptor potential melastatin 4 blocker (9-phenanthrol). P2X4R, but not P2X5R, was co-localized with Cx43 at the cell ends. P2X4R-dependent IS,Cs was augmented in atrial myocytes from hypertrophied rat hearts by short-term aortic constriction. Our data suggest that shear stress triggers Cx43 hemichannels to gate P2X4Rs in their vicinity. This shear stress signaling may play an important role in membrane depolarization at negative potentials and in atrial remodeling under increased afterload.