CYTOSKELETON AND CONTRACTILE DYSFUNCTION IN HYPERTROPHY

该项目的最初目标是确定细胞骨架的改变是否在肥厚心肌的收缩功能障碍中起作用。已取得的重要成果是：1)显示了肥厚和衰竭的右室和左室的心肌细胞中基于微管的收缩功能障碍，2)将这些发现扩展到离体组织和完整的心脏，3)对基于微管的收缩功能障碍的病因进行了生物物理表征，4)发现这种机制严格限制在压力超负荷肥厚中，其中壁应力持续增加，5)证明微管是唯一受影响的主要肌丝外细胞骨架蛋白，6)这一现象是基于微管蛋白的增加，从而微管的增加，以及微管一旦形成后稳定性的增加，7)发现MAP4，即微管 主要的心脏微管稳定蛋白在压力超负荷时显著上调 肥厚，以及8)发现肥厚过程中两个微小的β-微管蛋白亚型转录上调，我们发现这两个亚型模拟了这些基因的发育调节，解释了β-微管蛋白的增加。这项工作的第一个目标是使用心脏受限表达突变的β-微管蛋白的转基因小鼠，这些突变的β-微管蛋白导致微管稳定性降低或增加，以便直接验证这样的假设，即当肥厚的心肌中发生这些心肌细胞微管的变化时，微管网络稳定性增强，从而密度增加会导致收缩功能障碍。这第一个目标将直接关注微管本身的稳定性，而不是伴随着肥厚的心脏环境的许多复杂性。第二个目标是在心脏受限表达MAP4的转基因小鼠中寻找压力超负荷肥大的外科模型，以及 在体外培养的心肌细胞中腺病毒介导的MAP4表达增加的基础 微管网络的稳定性和密度取决于MAP4微管亲和力的增加，MAP4是心脏的主要微管相关蛋白。由于我们已经发现MAP4在肥大中的表达增加，而且我们刚刚发现MAP4被去磷酸化，从而增加了MAP4与微管的亲和力，所以第二个目标将集中在肥厚MAP4去磷酸化的机制上。

The initial goal of this project was to determine whether cytoskeletal alterations have any role in the contractile dysfunction of hypertrophied myocardium. Important accomplishments have been 1) the demonstration of microtubule-based contractile dysfunction in cardiocytes from the hypertrophied and failing RV and LV, 2) extension of these findings to isolated tissue and to the intact heart, 3) biophysical characterization of the etiology of microtubule-based contractile dysfunction, 4) the finding that this mechanism is tightly restricted to pressure overload hypertrophy in which wall stress is persistently increased, 5) the demonstration that microtubules are the only major extra-myofilament cytoskeletal protein so affected, 6) the finding that this phenomenon is based both on increased tubulin, and thus microtubules, and on increased stability of the microtubules once formed, 7) the finding that MAP4, the major cardiac microtubule-stabilizing protein, is markedly upregulated in pressure overload hypertrophy, and 8) the finding that transcriptional upregulation of two minor beta -tubulin isoforms during hypertrophy, which we found to mimic the developmental regulation of these genes, accounts for the increase in beta-tubulin. The first goal for the work proposed is to use transgenic mice having cardiac-restricted expression of mutant beta-tubulins that cause reduced or increased microtubule stability in order to directly test the hypothesis that enhanced microtubule network stability and thus density cause contractile dysfunction when these alterations of cardiocyte microtubules occur in hypertrophied myocardium. This first goal will focus directly on microtubule stability per se, absent the many attendant complexities of the hypertrophic cardiac environment. The second goal is to seek in surgical models of pressure overload hypertrophy, in transgenic mice having cardiac-restricted expression of MAP4, and in isolated cardiocytes with adenovirus-mediated expression of MAP4 the basis for increased microtubule network stability and density in terms of increased microtubule affinity of MAP4, the major microtubule-associated protein of the heart. Since we have found increased MAP4 expression in hypertrophy, and we have just discovered that this MAP4 is dephosphorylated, thus increasing MAP4 -microtubule affinity, this second goal will focus on the mechanisms of hypertrophic MAP4 dephosphorylation.