Cardiac chloride and pH regulation in health and disease

心脏病是美国人死亡的主要原因，它比所有癌症造成的死亡人数都多 加在一起。冠心病(或缺血性心脏病，IHD)，是最常见的心脏病类型， 并伴有心肌局部pH值显著下降。然而，pH调节的机制和 心肌细胞中H+中和缓冲液如HCO3-和Cl-的动态平衡仍不完全 理解，使设计针对pH调节的治疗策略变得困难。最近，我们有 从心肌细胞中鉴定并克隆了溶质载体SLc26a6的不同亚型。Slc26a6是 心脏中主要的Cl-/HCO3-交换。我们证明了SLc26a6介导生电性Cl-/HCO3- 心房和心室肌细胞的交换活动。我们的发现增加了SLC26a6可能 代表心脏中主要的Cl-/HCO3-调节机制。我们已经获得了令人振奋的数据 支持SLc26a6在心脏兴奋性和收缩能力中的关键作用。我们记录了空删除 SLC26a6致小鼠动作电位缩短、窦性心动过缓、QRS波群碎裂 与野生型窝产仔相比，心功能受损。我们已经确定并描述了两个 人SLC26A6在人心脏中的亚型，也是电产生的，类似于小鼠心脏的SLC26A6。在……里面 此外，我们最近发现并报道了一种动态的搏动细胞内pH(Phi)调节系统， 其与流行的三种已知的动力系统相吻合，即电的， Ca2+和机械系统。然而，关键问题仍然没有得到回答。SLC26a6活动如何影响 不仅是Phi，还有心脏的AP和收缩能力？研究的目标是确定机械环节 SLc26a6活性与心脏AP和收缩功能之间的关系。SLC26a6介导的Cl-/HCO3-的贡献 也将测试接近Phi的瞬变。综上所述，我们假设SLc26a6在 PHI将通过Na+/HCO3-共转运体(NBCe)和 Na+/H+交换器(NHE)，进而通过肌膜Na+-调节细胞内钙离子浓度。 钙离子交换器(NCX)。因此，SLc26a6的消融将导致细胞内Na+和Ca2+的减少 分别通过NHE/NBCe和NCX的作用。我们进一步假设SLC26a6在 心脏动态phi调节中的作用，调节心脏起搏活动和收缩能力。我们会 使用包括功能电生理记录在内的多学科方法来测试我们的假设， 成像、生化、分子和遗传学方法以及体外和体内功能研究。野性 类型和心脏特异的Slc26a6基因敲除小鼠模型以及人类心肌细胞将被测试。三 明确SLc26a6对心脏PHi和心脏功能的调节机制。我们会 测试Slc26a6如何调节动态心肌phi、Na+和Ca~(2+)动态平衡，从而调节兴奋性和收缩能力。 PHI和心功能之间的关系将被直接测试，以获得对 SLC26a6在心脏中的功能作用。我们将使用包括多模式二次谐波在内的新技术 世代(SHG)显微镜和我们最近建立的动态pH记录技术。2.确定 SLC26a6在心肌缺血/再灌注中的作用机制我们将测试Slc26a6的贡献 对I/R小鼠模型心功能的影响。将测试SLc26a6在心脏I/R损伤中的机制作用 体外共聚焦成像测定pH值、细胞内Na+和Ca~(2+)浓度。将采用I/R损伤作为对照 和Slc26a6-/-小鼠。3.确定sLc26a6在心脏功能中的作用及其调控机制 起搏活动。我们将测试SLC26a6在调节AP放电频率中的机制作用， 窦房结细胞的起搏电流、钙信号和phi。此外，心电遥测将用于测试 Slc26a6在意识控制和SAN特异性slc26a6-/-小鼠中的作用。我们的研究将揭开一个失踪的 PHI调节与心脏Na+、Ca2+动态平衡之间的分子联系。预期的结果将是 为SLc26a6在心脏PHI调节、心脏兴奋性和心脏功能中的作用提供新的见解 生理和病理条件。在翻译水平上，SLC26a6可能代表了一种新的治疗方法 心脏缺血和心律失常的心脏保护靶点。

Heart disease is the leading cause of mortality in the United States and causes more deaths than all cancers combined. Coronary heart disease (or ischemic heart disease, IHD), the most common type of heart disease, is accompanied by a major decline of local pH in myocardium. However, the mechanisms of pH regulation and the homeostasis of H+ neutralizing buffers, such as HCO3- and Cl- in cardiomyocytes remain incompletely understood, making it difficult to design therapeutic strategies targeting pH regulation. Recently, we have identified and cloned different isoforms of a solute carrier, Slc26a6, from cardiac myocytes. Slc26a6 is the predominant Cl-/HCO3- exchanger in the heart. We demonstrated that Slc26a6 mediates electrogenic Cl-/HCO3- exchange activities in both atrial and ventricular myocytes. Our findings raise the possibility that Slc26a6 may represent the predominant Cl-/HCO3- regulatory mechanism in the heart. We have obtained exciting data to support the critical roles of Slc26a6 in cardiac excitability and contractility. We documented that null deletion of Slc26a6 in mice results in shortened action potentials (APs), sinus bradycardia, fragmented QRS complexes and impaired cardiac function compared to wild type littermates. We have identified and characterized two isoforms of human SLC26A6 in human heart, which are also electrogenic, akin to mouse cardiac Slc26a6. In addition, we recently identified and reported a dynamic beat-to-beat intracellular pH (pHi) regulation system, termed “pHi transients”, which dovetails with the prevailing three known dynamic systems, namely electrical, Ca2+, and mechanical systems. However, critical questions remain unanswered. How do Slc26a6 activities affect not only pHi, but also cardiac AP and contractility? The goal of study is to determine the mechanistic links between the Slc26a6 activities and cardiac AP and contractility. Contributions of Slc26a6-mediated Cl-/HCO3- towards the pHi transients will also be tested. Taken together, we hypothesize that the activities of Slc26a6 on pHi will directly contribute towards intracellular Na+ homeostasis, through Na+/HCO3- cotransporter (NBCe) and Na+/H+ exchanger (NHE), and subsequently regulate intracellular Ca2+ concentration through sarcolemmal Na+- Ca2+ exchanger (NCX). Therefore, ablation of Slc26a6 will result in a reduction in intracellular Na+ and Ca2+ through the actions of NHE/NBCe and NCX, respectively. We further hypothesize that Slc26a6 plays important roles in the dynamic pHi regulation in the heart regulating cardiac pacemaking activities and contractility. We will test our hypothesis using multidisciplinary approaches including functional electrophysiological recordings, imaging, biochemical, molecular and genetic approaches as well as ex vivo and in vivo functional studies. Wild type and cardiac-specific Slc26a6 knockout mouse model as well as human cardiomyocytes will be tested. Three specific aims are: 1. To determine the regulatory mechanisms of Slc26a6 on cardiac pHi and function. We will test how Slc26a6 regulates dynamic cardiac pHi, Na+ and Ca2+ homeostasis, hence, excitability and contractility. The relationship between pHi and cardiac function will be directly tested to gain mechanistic insights into the functional roles of Slc26a6 in the heart. We will use novel techniques including multimodal second harmonic generation (SHG) microscopy and our recently established dynamic pH recording techniques. 2. To determine the mechanistic roles of Slc26a6 in cardiac ischemia/reperfusion (I/R). We will test the contributions of Slc26a6 to cardiac function in the I/R mouse model. Mechanistic roles of Slc26a6 in cardiac I/R injury will be tested using ex vivo confocal imaging of pHi, intracellular Na+ and Ca2+ concentrations. I/R injury will be employed in control and Slc26a6-/- mice. 3. To determine the functional roles and regulatory mechanisms of Slc26a6 in cardiac pacemaking activities. We will test the mechanistic roles of Slc26a6 in the regulation of AP firing frequency, pacemaker currents, Ca2+ signaling, and pHi in SAN cells. Additionally, ECG telemetry will be used to test the roles of Slc26a6 in conscious control and SAN-specific Slc26a6-/- mice. Our studies will unravel a missing molecular link between pHi regulation and Na+, and Ca2+ homeostasis in the heart. The anticipated results will provide novel insights into the roles of Slc26a6 in cardiac pHi regulation, cardiac excitability, and function under physiological and pathological conditions. At the translational level, Slc26a6 may represent a novel therapeutic target for cardioprotection in cardiac ischemia and arrhythmia.