Connexin-43-Hemichannel-Mediated ATP Efflux Triggers Arrhythmogenic CA2+ Waves via P2X Purinoceptor Current in Atrial Myocytes

Abstract

Myocardium is subjected to mechanical stresses and adapts to them. Atrial myocytes are exposed to high shear stress during hemodynamic overload and blood regurgitation. We have previously shown evidence that shear stress (∼16 dyn/cm2)-induced atrial global Ca2+ waves are abolished by the blockades of ATP release, gap junction hemichannel or P2 purinergic signaling. In this study, we assessed activation of gap junction hemichannels by shear stress, and its role in induction of arrhythmogenic currents and in proarrhythmic Ca2+ waves in rat atrial myocytes. Calcein dye efflux, but not oregon flux, was accelerated by shear application. The shear-induced calcein efflux was enhanced by zero external Ca2+, and was suppressed by La3+, but not by probenecid, suggesting activation of connexins by shear stress. Shear stress produced inward cation (Cs+) currents at resting potentials, and this current was completely suppressed by La3+ or carbenoxolone, and was enhanced by quinine. The current was also partly suppressed by P2X receptor inhibition (by ∼50%) or by blockade of P2Y1 receptor/transient receptor potential melastatin subfamily 4 (by ∼30%). Shear-induced NMDG+ current was one-fifth of the Cs+ current, showed linear voltage-dependence with a reversal at 0 mV and was eliminated by introduction of anti-connexin-43 antibodies. Shear-induced ATP release from a monolayer of atrial cells, assessed by chemiluninescence, was abolished by either connexin-43 hemichannel inhibitor Gap 19 or connexin-43 knock-down. Simultaneous measurement of ATP release and Ca2+ images using a sniffer patch clamp and two-dimensional confocal microscopy, respectively, further revealed that ATP releases occurred at 200-300-ms prior to the onset of the Ca2+ waves under shear stress and were sustained under prolonged shear stimulus. Our data suggest that shear stress induces connexin-43-hemichannel-mediated ATP release, thereby initiating P2X purinoceptor-dependent triggered Ca2+ waves in atrial myocytes.