Role of the Extracellular Space as a Modulator of the Cardiac Gap Junction - Conduction Velocity Relationship

项目摘要 虽然缝隙连接的研究经常提到它们对正常的电激活的重要性， 心脏，有很少的协议的程度，缝隙连接重塑所需的减缓电激活。 我们先前的工作提出了以下开创性的意见。首先，心脏水合作用的程度可以 调节间隙连接解偶联是否与可测量的传导减慢相关， 心律失常风险增加。第二，在心脏中，邻近缝隙连接斑块的微区丰富， 钠通道同种型Nav1.5标称长150 nm，宽10-20 nm。通过使用超- 分辨率显微镜，电子显微镜，光学映射，和尖端计算模拟，我们 提供的证据表明，perinexus是一个强有力的候选结构作为心脏ephapse。三是 证明用于进行离体全心脏研究的缓冲液可以调节perinexal宽度和 心脏传导和缝隙连接之间的关系。简而言之，改变细胞外钠和 钾可以补偿连接蛋白43的50%损失，使得传导看起来正常。这些发现 与分析传导速度与 和缝隙连接，以至于有可能通过事后文献分析来确定一组是否 将发现传导减慢继发于Cx43的损失，这仅仅是由于所使用的缓冲器。这项工作不仅重要 但它指出了一个更发人深省的问题： 离体和体外生物学的基础工具（缓冲液）可以简单地产生依赖于研究者的结果 基于离子缓冲剂组合物。在这个应用程序中，我们将测试三个新的目标。1.我们将测试独特的 计算预测缝隙连接和视神经耦合，以证明传导储备 假设是唯一连续的“电缆状”传播，且近场耦合可以自衰减 当细胞间的宽度非常窄时传导。2.我们将展示如何改变纤维间和 会阴分离分别改变心脏传导。这些研究结果将与以下模型进行比较： 为了进一步支持这一假设，即肝纤维化耦合是一种替代形式， 肌细胞之间的电耦合。3.我们提供的证据表明，改变灌注液离子浓度， 在酸中毒期间，将传导恢复到与基于间隙连接的治疗相同的程度。在这个最终目标中，我们将 确定灌注液组合物如何在无血流缺血期间调节电耦合， 冠脉结扎此外，我们将测试灌注液组合物是否可以使GAP的作用痛苦。 局部缺血时的连接治疗。

Project Abstract While studies of gap junctions often cite their fundamental importance to normal electrical activation of the heart, there is little agreement on the degree of gap junction remodeling required to slow electrical activation. Our prior work has made the following seminal observations. First, the degree of cardiac hydration can modulate whether gap junction uncoupling will be associated with measurable conduction slowing and increased arrhythmia risk. Second, the microdomain adjacent to gap junction plaques is rich in the cardiac sodium channel isoform Nav1.5, is nominally 150 nm long and 10-20 nm wide. Through the use of super- resolution microscopy, electron microscopy, optical mapping, and cutting edge computations simulations, we provided evidence that the perinexus is a strong candidate structure as the cardiac ephapse. Third, we demonstrate that the buffer used to perform ex vivo whole-heart studies can modulate both perinexal width and the relationship between cardiac conduction and gap junctions. In short, altering extracellular sodium and potassium can compensate for a 50% loss of connexin43 such that conduction appears normal. These findings are entirely consistent with the various groups that have analyzed the relationship between conduction velocity and gap junctions, so much so that it is possible by post-hoc literature analysis to determine whether a group will find conduction slowing secondary to loss of Cx43 simply by the buffer used. This work is not just important for understanding alternative modes of electrical communication, but it points out an even more sobering issue: the foundational tool of ex vivo and in vitro biology (buffers) may yield investigator dependent results simply based on ionic buffer composition. In this application we will test three new aims. 1. We will test unique computational predictions of gap junctional and ephaptic coupling to demonstrate that the conduction reserve hypothesis is unique to continuous “cable-like” propagation, and ephaptic coupling can self-attenuate conduction when intercellular widths are very narrow. 2. We will demonstrate how changing interfibrillar and perinexal separation separately alters cardiac conduction. These findings will be compared to models of ephaptic coupling in order to further support the hypothesis that ephaptic coupling is an alternative form of electrical coupling between myocytes. 3. We provide evidence that altering perfusate ion concentration can rescue conduction to the same degree as gap junction based therapy during acidosis. In this final aim, we will determine how perfusate composition modulates electrical coupling during no-flow ischemia and complete coronary artery ligation. Also, we will test whether perfusate composition can agonize the effects of gap junction therapy during ischemia.