Mechanism of shear stress-induced ATP release in ventricular myocytes: roles of pannexin and mitochondria.

Shear stress induces atrial Ca2+ waves via connexin43 (Cx43)-mediated ATP release. Here, we examined whether ventricular myocytes release ATP under shear stress, and the underlying and regulatory mechanisms. A bioluminescence assay and the 'sniffer patch' were used to measure ATP release from multiple and single murine ventricular myocytes, respectively, in combination with laminar flow or micro-puffing. Shear stress (∼16 dyn/cm2) induced transient ATP release from myocyte batches, peaking at ~7 mol/cm2-membrane within 2 s. This response was abolished by the application of La3+ but was not affected by Gap19 treatment or zero external Ca2+. In addition, shear-induced ATP release was not different between control and cardiac-specific Cx43-conditional knockout (Cx43-cKO) ventricular cells, suggesting no role for Cx43. Shear-induced ATP release was suppressed by probenecid, low concentrations of carbenoxolone, or the pannexin (Panx) 1 inhibitory peptide 10Panx1 by 60-75%, suggesting a major role for Panx. Pharmacological screening further revealed a partial (30-40%) role of 9-anthracenecarboxylic acid-sensitive, tamoxifen-insensitive Cl- channels in ATP release. The sniffer patch, using a P2X7 receptor-overexpressing HEK293 cell positioned at a local region of the ventricular cell, showed a rapid development of shear-induced currents, approximated to be ∼1-10 μM ATP. The Cx43-cKO did not affect these sniffer cell currents. This shear response was suppressed by mitochondrial uncoupling or inhibition of the electron transport chain by 70-80% but was rather enhanced by Mito-TEMPO. Our data suggest that shear stress induces ATP release from murine ventricle cells mainly via Panx and that mitochondrial oxidative phosphorylation may be required for this response.