School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong.
Department of Anaesthesiology, The University of Hong Kong, Pokfulam, Hong Kong.
Acta Physiol (Oxf). 2020 Sep;230(1):e13495. doi: 10.1111/apha.13495. Epub 2020 Jun 19.
Cystic 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.
Proteins 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.
Lactic 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.
During 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.
囊性纤维化跨膜电导调节因子（CFTR）在心脏中表达，但在心脏中的功能尚不清楚。CFTR 调节许多组织中的 ATP 释放孔，但孔的身份和调节机制尚不清楚。我们研究了 CFTR 在模拟缺血（乳酸处理）期间从原代心肌细胞和心室壁中释放 ATP 的作用。
使用抑制剂和 siRNA 研究了参与 ATP 释放信号通路的蛋白；通过共免疫荧光和临近连接测定鉴定了共定位；使用全内反射显微镜鉴定了近膜 pH 和钙的变化；使用大鼠心脏的间质微透析研究了体内 ATP 释放。
乳酸诱导培养的心肌细胞和体内左心室 CFTR 依赖性 ATP 释放。乳酸进入增加了近膜钙，这涉及与 CFTR 共定位的 Na/H-和 Na/Ca-交换器。钙进入诱导 CFTR 激活，涉及 cAMP、蛋白激酶 A、FAK、Pyk2 和 Src。去除细胞外碳酸氢根消除了乳酸或 CFTR 激活剂诱导的心肌细胞 ATP 释放。碳酸氢根刺激细胞色素 c 表达、细胞色素 c 释放和分离的心肌细胞线粒体中的 ATP 释放。连接蛋白 1（Panx1）与 CFTR 共定位。乳酸增加心肌细胞半胱天冬酶活性：半胱天冬酶抑制剂或 Panx1 siRNA 消除了心肌细胞 ATP 释放，而 Panx1 抑制消除了体内心脏 ATP 释放。
在模拟缺血期间，CFTR 依赖性碳酸氢根进入刺激了线粒体中 ATP 和细胞色素 c 的释放；在细胞质中，细胞色素 c 激活了半胱天冬酶 3，进而激活了 Panx1，ATP 通过打开的 Panx1 通道释放。
