Connexin Distribution in Physiological Versus Pathological Cardiac Hypertrophy

在过去的24年里，这笔赠款支持的研究建立在大量数据的基础上 表明心脏的结构、组成和功能都对心脏的变化做出快速和可逆的反应 血流动力学负荷。由这笔赠款支持的第一组研究使用了分离的细胞或心肌细胞，并且完好无损。 动物来证明负荷作为心脏细胞生长的中心调节器的作用。第二组研究 在这项拨款的支持下，我们发现了负载变化作为主要的实验变量 在严重的压力超负荷性心肌肥厚期间，致密的心肌细胞微管网络 与在这种情况下发生的收缩功能障碍有关。 对这种异常微管网络的后续研究的最初目标是确定它是如何 导致肥厚心肌的收缩功能障碍。主要的发现是：1)它是 基于增加的微管蛋白和微管，以及更大的微管稳定性，2)主要的Car- DIAC微管稳定微管相关蛋白MAP4在压力超负荷时表达显著上调 肥大并广泛与微管结合；3)收缩功能障碍是由粘性负荷引起的 通过致密的微管网络缩短肌丝。 然而，微管在间期细胞中最重要的正常作用是 不是为了确定细胞的流变性，而是为了减少大分子在细胞内的运输 和囊泡通过微管相关的运动蛋白和动力蛋白家族的马达蛋白。事实上，这是一种 在成人心肌细胞极度扩散受限的细胞质中起着绝对重要的作用。因为这个原因， 由于已知过度装饰抑制了微管依赖的细胞内转运 对于有MAP的微管，我们接下来询问激活的肾上腺素能受体是否基于微管的运输 和/或mRNA型核糖核蛋白复合体被MAP4与微管结合抑制。 超负荷肥大。事实上是这样的。就是这样。 在这项最新工作的基础上，我们建议在这里研究微管变化的潜在作用。 网络组织和MAP4结合导致连接蛋白43[Cx43]运输和定位异常， 已知的缝隙连接蛋白在数量和定位上经历了重要的功能变化 病理性心肌肥大。第一个目标中的基础研究将使用分离的细胞以及手术。 以确定MAP4修饰的微管和伴随的密度- 破坏微管网络，抑制Cx43向缝隙连接的正常转运，以及Cx43依赖于Cx43。 电生理功能受损。第二个和第三个目标中的翻译研究将进行比较 病理压力与生理性容量超负荷肥大的程度和持续时间相等。我们会 首先扩展第一个目标的发现，询问MAP4在致密微管网络中的修饰是否 病理性肥厚在Cx43转运和定位改变中起作用，这在临床上是重要的 形成致心律失常的底物。然后我们会问，在病理性肥厚中，受体是否被阻断， 早期数据表明，将防止微管表型异常，也将防止Cx43异常 在这个环境中的表型。 在第一个目标中，我们将使用小鼠模型，在第二个和第三个目标中，我们将使用我们的长期- 生理性肥大与病理性肥大的站立猫模型。虽然我们认识到最好是- 能够使用单一物种，在这项研究中，最初的机械部分只能在小鼠身上完成，但 后来的定量翻译部分需要非常可重复性的动物模型，可以可靠和可验证的 -梗阻，生理性肥厚与病理性肥厚的程度和持续时间相当， 在每种环境中紧张地表征细胞骨架特性。

Research supported by this grant during the previous twenty-four years has been built around extensive data showing that cardiac structure, composition, and function each respond rapidly and reversibly to changes in hemodynamic load. The first set of studies supported by this grant used isolated cells, or cardiocytes, and intact animals to demonstrate the role of load as a central regulator of cardiocyte growth. The second set of studies supported by this grant, which also used load change as the primary experimental variable, led to our discovery of a dense cardiocyte microtubule network during severe pressure-overload cardiac hypertrophy that contrib- utes to the contractile dysfunction which occurs in this setting. The initial goals for the subsequent studies of this abnormal microtubule network were to determine how it contributes to the contractile dysfunction of hypertrophied myocardium. Major findings have been that 1) it is based both on increased tubulin, and thus microtubules, and on greater microtubule stability, 2) the major car- diac microtubule-stabilizing microtubule-associated protein, MAP4, is greatly upregulated in pressure overload hypertrophy and binds extensively to microtubules, and 3) contractile dysfunction is caused by viscous loading imposed on shortening myofilaments by the dense microtubule network. However, the most important normal role of the microtubules in an interphase cell such as the cardiocyte is not to determine cellular rheological properties but rather to subserve intracellular transport of macromolecules and vesicles via the microtubule-associated kinesin and dynein families of motor proteins. Indeed, this is an absolutely essential role in the extremely diffusion-restricted cytoplasm of the adult cardiocyte. For this reason, and because of the known inhibition of microtubule-dependent intracellular transport by excessive decoration of microtubules with MAPs, we next asked if microtubule-based transport of the activated ¿-adrenergic receptor and/or mRNA - ribonucleoprotein complexes was inhibited by MAP4 binding to microtubules in pressure- overload hypertrophy. Such, in fact. was the case. Building on this most recent work, we propose to examine here the potential role of alterations in microtubule network organization and MAP4 binding in causing abnormal transport and localization of connexin43 [Cx43], a gap junction protein known to undergo functionally important alterations in quantity and localization during pathological cardiac hypertrophy. The basic research in the first objective will use isolated cells as well as oper- ated and transgenic mice to determine whether MAP4 decoration of microtubules, and the attendant densifica- tion of the microtubule network, inhibit the normal transport of Cx43 to gap junctions as well as Cx43-depen- dent electrophysiological function. The translational research in the second and third objectives will compare an equal degree & duration of pathological pressure vs. physiological volume overload hypertrophy. We will first extend the findings of the first objective to ask if MAP4 decoration of the dense microtubule network in pathological hypertrophy has a role in the altered Cx43 transport and localization that are important clinically in forming an arrhythmogenic substrate. We will then ask if ¿-receptor blockade in pathological hypertrophy, which early data indicates will prevent the abnormal microtubule phenotype, will also prevent the abnormal Cx43 phenotype in this setting. In the first objective we will use murine models, and in the second and third objectives we will use our long- standing feline models of physiological versus pathological hypertrophy. While we recognize that it is prefer- able to use a single species, in this research the initial mechanistic portion can only be done in the mouse, but the later quantitative translational portions require very reproducible animal models that can be reliably and verifiably ¿-blocked and have an equivalent degree and duration of physiological vs. pathological hypertrophy, with ex- tensively characterized cytoskeletal properties in each setting.