["\nAbstract\n\nAim\nCystic fibrosis transmembrane conductance regulator (CFTR) is expressed in the heart, but its function there is unclear. CFTR regulates an ATP release pore in many tissues, but the identity and regulatory mechanism of the pore are unknown. We investigated the role of CFTR in ATP release from primary cardiomyocytes and ventricular wall in vivo.\n\n\nMethods\nProteins involved in the signalling pathway for ATP release during simulated ischaemia (lactic acid treatment) were investigated using inhibitors and siRNA; colocalization was identified by coimmunofluorescence and proximity ligation assays; changes in near‐membrane pH and calcium were identified with total internal reflection microscopy; in vivo ATP release was investigated using interstitial microdialysis of rat heart.\n\n\nResults\nLactic acid‐induced CFTR‐dependent ATP release from cultured cardiomyocytes and left ventricle in vivo. Lactic acid entry elevated near‐membrane calcium, which involved Na/H‐ and Na/Ca‐exchangers colocalized with CFTR. Calcium entry‐induced CFTR activation, which involved cAMP, protein kinase A, FAK, Pyk2 and Src. Removal of extracellular bicarbonate abolished cardiomyocyte ATP release induced by lactic acid or CFTR activators. Bicarbonate stimulated cytochrome c expression, cytochrome c release and ATP release from isolated cardiomyocyte mitochondria. Pannexin 1 (Panx1) colocalized with CFTR. Lactic acid increased cardiomyocyte caspase activity: caspase inhibitors or Panx1 siRNA abolished cardiomyocyte ATP release, while pannexin inhibition abolished cardiac ATP release in vivo.\n\n\nConclusion\nDuring simulated ischaemia, CFTR‐dependent bicarbonate entry stimulated ATP and cytochrome c release from mitochondria; in the cytoplasm, cytochrome c‐activated caspase 3, which in turn activated Panx1, and ATP was released through the opened Panx1 channel.\n\n", "Acta Physiologica, Volume 230, Issue 1, September 2020. "]