It has long been known that cardiac myocytes release ATP, and that the release of ATP is increased in response to hypoxia and ischaemia.1 The mechanisms underlying that release and its regulation, however, remain unclear. In this issue of Acta Physiologica , Wang and colleagues from the Ballard laboratory present intriguing new evidence of a role for the cystic fibrosis transmembrane conductance regulator (CFTR) in the release of ATP from cardiac myocytes in response to simulated ischaemia.2 CFTR is a phosphorylation‐dependent anion channel perhaps best known for its role in cyclic AMP‐dependent Cl⁻ and HCO₃⁻ transport and mucous secretion by epithelial tissues.3 Mutations in CFTR cause cystic fibrosis, the most common life‐threatening inherited disorder, which is associated with thickened mucous production in the airway leading to lung infections and the diagnostic high‐salt sweat.3 While cystic fibrosis is a multiorgan disorder, the primary defect is considered to be a reduction in Cl⁻ and HCO₃⁻ transport across the apical membrane of epithelial cells.3 Other than right ventricular dysfunction arising from cor pulmonale , an indirect consequence of lung disease, cystic fibrosis has not been considered a direct cause of cardiomyopathy.4 In this regard, the report by Wang et al2 is timely in that recent advances in echocardiography, together with the improved lifespan of cystic fibrosis patients, have provided evidence that mutations in CFTR may cause a primary defect in the heart.4

The role of CFTR in the heart, particularly in the human, has been the subject of much debate. Within a few years of its cloning, CFTR was shown to be present in cardiac myocytes where it was suggested to underlie an anion‐selective cyclic AMP‐dependent channel current activated by β‐adrenoceptor agonists or stimulation of adenylyl cyclase (see Hume and colleagues5 for a comprehensive review of the early work on CFTR and other anion channels in the heart). Measurements of the equilibrium potential for Cl⁻ (E _Cl) in cardiac myocytes vary between studies, partly depending on the presence or absence of bicarbonate buffer systems as the chloride conductances of the sarcolemma operate in parallel with other anion transporters, particularly the Cl⁻/HCO₃⁻ exchanger. Nevertheless, E _Cl is generally accepted to be between −65 and −40 mV, which can be either negative or positive to the membrane potential during the cardiac cycle.5 Cardiac CFTR was therefore suggested to play an important role in the regulation of action potential duration and resting membrane potential by the neurotransmitters and hormones.5 Importantly, together with sarcolemmal anion transporters, CFTR can also play a role in the regulation of intracellular pH and cell volume so that multiple facets to the function of this ion channel in the heart are likely.5 However, while β‐adrenoceptor‐dependent currents were found in adult cardiac myocytes of some of the species investigated, this was not true of all species. Currents were absent from some species in which RT‐PCR or immuno‐detection methods indicated the presence of CFTR: using standard whole‐cell anion conductance‐selective recording conditions (eg K⁺‐free solutions, use of HEPES rather than HCO₃⁻ buffer, a high concentration of Ca²⁺ chelator in the intracellular pipette solution, the presence of extracellular Ca²⁺ channel blocker), cyclic AMP‐dependent currents could reliably be demonstrated in ventricular myocytes from rabbit, guinea pig, pig and simian monkey, but not in myocytes from dog, mouse or, notably for this study, rat hearts.5, 6 Of particular importance, the existence of a CFTR current in human cardiac myocytes has been controversial.5 Reports of the activation of CFTR‐like currents in mouse ventricular myocytes by stimulation of purinoceptors, but not β‐adrenoceptors, through pathways involving concomitant activation of both protein kinase A and protein kinase C hinted at there being species differences in the signalling mechanisms coupled to CFTR activation in cardiac myocytes.5 The demonstration by Wang et al2 of the existence of a CFTR‐like current in rat ventricular myocytes for the first time adds valuable new evidence in support of this conjecture. While the explanation of what accounts for the difference between this and previous studies remains unclear, it is worth noting that, in contrast to previous studies, HCO₃⁻ was included in the external recording solution in this study.2, 6

CFTR has been suggested to be involved in ATP release in a number of cell types, although it is now generally accepted that CFTR does not represent the conductive pathway for ATP extrusion.3, 5 Using a luciferin‐luciferase bioluminescence assay, Wang et al show that release of ATP by rat cardiac myocytes in culture could be increased by either acid‐loading with lactic acid or by direct increase in intracellular cyclic AMP concentration using the adenylyl cyclase activator, forskolin (FSK), in combination with the general phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX).2 The increase in ATP release, whether by lactic acid or FSK/IBMX treatment, could be prevented by the inhibitors of CFTR, CFTR_inh172 or GlyH‐101, or by knockdown of CFTR expression using siRNA. Evidence of a role for pannexin 1 (Panx1) in the ATP conductive pathway was provided by inhibition of Panx1 and by knockdown of Panx1 expression using siRNA. A proximity ligation assay indicated colocalization of Panx1 with CFTR. CFTR was clearly placed upstream of Panx1 as treatment of the cells with CFTR siRNA caused a reduction in Panx1 protein expression. Whole‐cell recordings showed that either acidosis or FSK/IBMX activated chloride‐dependent currents sensitive to CFTR_inh172 in rat ventricular myocytes.

The signalling pathways involved in ATP release were investigated. The pH‐sensitive fluorophore, BCECF‐AM and total internal reflection fluorescence (TIRF) microscopy were used to show that treatment with lactic acid produced a transient, near‐membrane intracellular acidosis. The sensitivity of ATP release to cyanohydroxycinnamic acid provided evidence that lactic acid (comprising both lactate and proton moieties) entered the cells via a monocarboxylate transporter. The localized intracellular acidosis is proposed to result in Na⁺/H⁺ exchange and, indirectly through reverse‐mode Na⁺/Ca²⁺ exchange, a near‐membrane increase in intracellular [Ca²⁺] (detected using fluo‐4‐AM and TIRF). It is suggested that the increase in intracellular [Ca²⁺] concentration resulted in CFTR gating through a combination of Ca²⁺‐sensitive adenylyl cyclase activation and inhibition of CFTR dephosphorylation through Ca²⁺‐dependent tyrosine kinase phosphorylation of protein phosphatase 2A. Proximity ligation assay demonstrated the colocalization of Na⁺/H⁺ and Na⁺/Ca²⁺ exchangers with CFTR in rat ventricular myocytes. The increased release of ATP depended on the presence of external HCO₃⁻. A scheme is proposed by which HCO₃⁻ influx, either via activated CFTR directly or through Cl⁻/HCO₃⁻ exchange dependent on CFTR activity, and Ca²⁺ entry via reverse‐mode Na⁺/Ca²⁺ exchange caused release of cytochrome c and ATP from mitochondria. Activation of caspase 3 led to Panx1 gating and release of ATP from the cells. The authors argue that it is unlikely that the levels of cytochrome c released in this study would trigger apoptosis.

The authors propose that CFTR may play an important role in protection against acidosis/ischaemia.2 First, they suggest that CFTR‐mediated HCO₃⁻ entry would stimulate mitochondrial ATP synthesis. Second, the release of ATP would likely enhance O₂ supply‐to‐demand ratio through coronary vasodilation.1 The authors do not comment on a possible autocrine pathway by which ATP release would stimulate CFTR through purinoceptor activation. Nevertheless, it is important to note that data from CFTR knockout mice have provided evidence of a cardioprotective role for CFTR.7-9 In demonstrating the involvement of CFTR in ATP release from cardiac myocytes, in this study Wang et al have provided invaluable data regarding the multiple facets of CFTR channel function in the heart.2 While further work is required, this paper adds to the evidence calling for a re‐evaluation of the role of CFTR in the heart.4

早就知道心肌细胞会释放ATP，而ATP的释放会随着缺氧和局部缺血而增加。1然而，释放的基本机制及其调控仍不清楚。在本期《生理生理学》中，来自巴拉德实验室的Wang和同事提出了有趣的新证据，表明囊性纤维化跨膜电导调节剂（CFTR）在对模拟缺血的反应中从心肌细胞释放ATP的作用。2 CFTR是其在环AMP依赖性氯作用也许是最公知的磷酸化依赖性阴离子通道^-和HCO ₃ ^-由上皮组织运输和粘液分泌。3CFTR突变会导致囊性纤维化，这是最常见的威胁生命的遗传性疾病，与气道粘液产生增厚，导致肺部感染和诊断性高盐汗有关。3虽然囊性纤维化是一种多器官病症，主缺陷被认为是在氯的减少^-和HCO ₃ ^-跨越上皮细胞的顶膜运输。3除了肺心病引起的肺心病引起的右心室功能障碍外，囊性纤维化还没有被认为是心肌病的直接原因。4在这方面，Wang等人的报告2超声心动图的最新进展以及囊性纤维化患者寿命的改善是很及时的，这提供了证据表明CFTR的突变可能导致心脏的主要缺陷。4

CFTR在心脏，特别是在人类中的作用一直是许多争论的主题。在克隆的几年内，CFTR被证明存在于心肌细胞中，提示它是由β肾上腺素受体激动剂激活或刺激腺苷酸环化酶激活的阴离子选择性环AMP依赖性通道电流的基础（参见Hume和同事5全面审查CFTR和心脏中其他阴离子通道的早期工作）。的氯离子平衡电位的测量^- （ê_氯在心肌细胞）的研究之间变化，这部分取决于碳酸氢盐缓冲液系统的存在或不存在作为肌膜的氯化物电导并联与其他阴离子转运，特别是氯操作^- / HCO ₃ ^-热交换器。尽管如此，通常公认的E _Cl在-65至-40 mV之间，在心动周期中，其对膜电位可能为负或正。5因此，建议心脏CFTR在神经递质和激素对动作电位持续时间和静息膜电位的调节中起重要作用。5重要的是，CFTR与肌膜上的阴离子转运蛋白一起，还可在细胞内pH和细胞体积的调节中发挥作用，因此心脏中该离子通道的功能可能有多个方面。5然而，尽管在某些被调查物种的成年心肌细胞中发现了依赖于β-肾上腺素受体的电流，但并非所有物种都如此。电流是缺席一些物种中，RT-PCR或免疫检测方法指示CFTR的存在：使用标准的全细胞电导阴离子选择性记录条件（例如ķ ⁺ -free解决方案，使用HEPES的而非HCO ₃ ^-缓冲，细胞内移液器溶液中高浓度的Ca ²⁺螯合剂，细胞外Ca ²⁺的存在通道阻滞剂），可以可靠地在兔，豚鼠，猪和猿猴的心室肌细胞中证实依赖于环AMP的电流，但在狗，小鼠或本研究中特别是大鼠心脏的心肌细胞中却没有。5，6特别重要的是，人类心肌细胞中CFTR电流的存在一直存在争议。5关于通过刺激嘌呤受体而非β肾上腺素受体的刺激，通过涉及蛋白激酶A和蛋白激酶C的同时激活的途径，在小鼠心室肌细胞中激活CFTR样电流的报道提示，在耦合的信号传导机制中存在物种差异心肌细胞的CFTR活化。5 Wang等人的示范2首次在大鼠心室肌细胞中存在CFTR样电流的发现，为支持这一推测提供了有价值的新证据。虽然，这和以前的研究之间的差异如何解释的解释仍不清楚，但值得注意的是，相比于以往的研究，HCO ₃ ^-被列入本研究中的外部记录解决方案。2 6

尽管现在已经普遍接受CFTR并不代表ATP挤出的传导途径，但已建议CFTR参与许多细胞类型的ATP释放。3，5使用萤光素-萤光素酶生物发光测定法，Wang等人表明，培养物中大鼠心肌细胞的ATP释放可以通过用乳酸酸加载或通过使用腺苷酸环化酶激活剂直接增加细胞内环状AMP浓度来增加，佛司可林（FSK）与一般的磷酸二酯酶抑制剂异丁基甲基黄嘌呤（IBMX）组合。2 CFTR，CFTR _inh抑制剂可阻止通过乳酸或FSK / IBMX处理提高ATP释放172或GlyH-101，或使用siRNA敲低CFTR表达。Pannexin 1（Panx1）在ATP传导途径中的作用的证据是通过抑制Panx1和使用siRNA抑制Panx1表达来提供的。邻近结扎试验表明Panx1与CFTR共定位。CFTR显然位于Panx1的上游，因为用CFTR siRNA处理细胞会导致Panx1蛋白表达降低。全细胞记录显示，酸中毒或FSK / IBMX激活了对大鼠心室肌细胞CFTR _inh 172敏感的氯化物依赖性电流。

研究了与ATP释放有关的信号通路。pH敏感的荧光团，BCECF-AM和全内反射荧光（TIRF）显微镜用于显示乳酸处理会导致短暂的近膜细胞内酸中毒。ATP对氰基羟基肉桂酸的敏感性表明，乳酸（包括乳酸和质子部分）是通过单羧酸盐转运蛋白进入细胞的。有人认为局部细胞内酸中毒会导致Na ⁺ / H ⁺交换，并通过反向模式Na ⁺ / Ca ²⁺交换间接导致细胞内[Ca ²⁺]（使用Fluo-4-AM和TIRF检测到）。提示细胞内[Ca ²⁺ ]浓度的增加是通过结合Ca ²⁺敏感的腺苷酸环化酶激活和通过蛋白磷酸酶2A的Ca ²⁺依赖酪氨酸激酶磷酸化抑制CFTR去磷酸化而导致CFTR门控的。邻近结扎实验表明，Na ⁺ / H ⁺和Na ⁺ / Ca ²⁺交换子与CFTR在大鼠心室肌细胞中共定位。ATP的释放增加依赖于外部HCO存在₃ ^- 。一个方案是建议由HCO ₃ ^-涌入，或者通过直接激活CFTR或通过氯^- / HCO ₃ ^-交换依赖于CFTR活性和Ca ²⁺通过反向模式的Na条目⁺ /钙²⁺从线粒体细胞色素c和ATP的交换引起的释放。caspase 3的激活导致Panx1门控和从细胞释放ATP。作者认为，这项研究中释放的细胞色素c的水平不太可能触发细胞凋亡。

作者认为CFTR可能在预防酸中毒/缺血方面发挥重要作用。2首先，他们认为，CFTR介导的HCO ₃ ^-进入将刺激线粒体ATP合成。其次，ATP的释放可能会通过冠状血管舒张增加O ₂供需比。1作者没有评论ATP释放通过嘌呤受体激活而刺激CFTR的可能的自分泌途径。尽管如此，重要的是要注意，来自CFTR基因敲除小鼠的数据已经为CFTR的心脏保护作用提供了证据。7-9在证明CFTR参与心肌细胞ATP释放的过程中，Wang等人已经提供了关于CFTR通道功能在心脏多个方面的宝贵数据。2尽管还需要进一步的工作，但本文增加了证据，要求对CFTR在心脏中的作用进行重新评估。4