The role of cystic fibrosis transmembrane conductance regulator in the regulation of ATP release in heart and skeletal muscle

Abstract

ATP is released in response to hypoxia, ischaemia and catecholamine stimulation in rat ventricular myocytes. Cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in the heart, but the exact role of CFTR in ATP release remains unknown. CFTR is reported to be involved in regulating a separate ATP release pore in many tissues, but the identity of the pore and its underlying regulatory mechanisms are unclear. This study investigated the role of CFTR in regulating ATP release from cardiomyocytes, during simulated ischemia produced by lactic acid treatment. We mimicked the ischemic condition by treating primary cultured cardiomyocytes with 10 mM lactic acid. Lactic acid treatment depressed pH and enhanced ATP release from cardiomyocytes. Both specific CFTR inhibitors, CFTRinh-172 and GlyH-101, as well as CFTR siRNA abolished acidosis-induced ATP release, suggesting that CFTR is involved in this process. Forskolin and IBMX, agents that activate CFTR by elevating intracellular cAMP, also increased ATP release, further confirming the role of CFTR in acidosis-induced ATP release. Lactic acid entry into the cells stimulated H+ extrusion by the Na+/H+ exchanger (NHE), thus driving Na+ extrusion by the Na+/Ca2+ exchanger (NCX), which elevated near-membrane calcium as visualized under TIRF microscopy. Proximity ligation assays confirmed NHE and NCX colocalization with CFTR. Inhibition of either NHE with amiloride, or NCX with SN6, abolished acidosis-induced ATP release. BAPTA, a calcium chelator, suppressed acidosis- or Forskolin and IBMX-induced-ATP release. The increased near-membrane calcium was responsible for CFTR activation, involving cAMP, PKA, FAK, Pyk2 and Src. Decreased CFTR dephosphorylation appeared to be functionally more important than increased CFTR phosphorylation in this process. CFTR-dependent bicarbonate entry was responsible for cardiomyocyte ATP release through mitochondrial signaling. cAMP was generated within mitochondria by bicarbonate-regulated soluble adenylyl cyclase (sAC), which activated mitochondrial PKA. KH7, an sAC-specific inhibitor, decreased ATP formation and release, both in cardiomyocytes and isolated mitochondria. Bicarbonate, which entered myocytes during CFTR opening, activated both ATP production in isolated mitochondria and ATP release to the extracellular space. Bicarbonate treatment of isolated cardiomyocyte mitochondria increased cytochrome c expression, as well as cytochrome c and ATP release. High bicarbonate doses (40 mM) induced mitochondrial oxidative phosphorylation whereas CFTR inhibition reduced the oxygen consumption rate of intact cardiomyocytes and extracellular bicarbonate removal abolished ATP release. Cytochrome c release from mitochondria subsequently increased caspase activity and opened Panx1. Live cell caspase assay showed that lactic acid increased cardiomyocyte caspase activity, whereas both cyclosporin A, a specific mitochondrial permeability transition pore inhibitor, and V5, a Bax pore inhibitor, induced cytochrome c accumulation in isolated mitochondria. Immunofluorescence imaging and proximity ligation assay suggested that CFTR was co-localized with Pannexin1, and cardiomyocyte ATP release was abolished by caspase and Panx1 inhibitors, as well as Panx1 siRNA. In summary, CFTR regulates oxidative phosphorylation and ATP release in cardiomyocytes. CFTR-dependent bicarbonate entry initiates mitochondrial signaling, resulting in increased ATP and cytochrome c release into the cytoplasm. Furthermore, Cytochrome c activates caspase 3, in turn opening Panx1, through which ATP is released.