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 × 10^-18 mols/µm²-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.<b>NEW & NOTEWORTHY</b> In this study, we demonstrated shear stress-induced ATP release from ventricular myocytes for the first time using the sniffer patch technique and a bioluminescence assay, incorporating cardiac connexin 43 conditional knockout and pharmacological interventions. Our data also provide new evidence that pannexin primarily mediates ATP release in ventricular myocytes under shear stress and that mitochondrial oxidative phosphorylation is a prerequisite for this shear stress-mediated ATP release.