PHOSPHOINOSITIDE-3 KINASE IN CARDIAC HYPERTROPHY

该项目的长期目标是利用转基因和基因靶向的小鼠来确定磷脂酰肌醇-3激酶(PI3K)通路在心肌肥厚中的作用，重点是它在正常生长和病理条件下决定细胞大小的作用。关于在体内控制心肌细胞大小的关键机制，人们知之甚少。众所周知，生长激素胰岛素样生长因子-1(IGF1)通路在决定哺乳动物整体动物大小方面起着关键作用，但IGF1和PI3K通路在决定单个细胞和器官大小方面的联系尚未建立。因此，我们最近创造了心脏中PI3K活性受到干扰的转基因小鼠。有趣的是，表达结构性活性PI3K的小鼠发展为中度心肌肥厚，没有心肌功能障碍。形态计量学分析表明，心肌肥厚主要是由于单个心肌细胞大小的增加。相反，显性阴性PI3K的心脏表达产生较小的心脏，伴随而来的是单个心肌细胞的细胞大小减少。我们还创造了在心肌细胞中过度表达IGF1受体的转基因小鼠。与表达构成活性PI3K的小鼠类似，IGF1受体小鼠也出现中度心肌肥大，没有心脏功能障碍的迹象。这些结果提出IGF1-PI3K通路可能通过调节正常生长过程中心肌细胞的大小，在决定心脏大小方面发挥关键作用。PI3K的两个已知下游靶点是Akt(蛋白激酶B)和p70/p85核糖体S6激酶(S6K)。然而，它们在体内心脏生长中的作用尚未确定。因此，我们将确定Akt和S6K在PI3K激活的心肌肥大效应中的相对作用。此外，为了研究PI3K、Akt和S6K在压力超负荷诱导的肥厚中的作用，我们将在表达这些激酶的结构性活性形式或显性-阴性形式的转基因小鼠中创建主动脉带。此外，我们将分别将PI3K转基因小鼠与Wondisford实验室和King实验室产生的甲状腺受体转基因和PKC转基因小鼠进行杂交。因此，我们的具体目标是：具体目标1：通过条件转基因系统证实PI3K在成年动物心脏大小确定中的作用。特定目的2：研究PI3K在活体心脏中是否位于IGF1/IGF1受体的下游。具体目的3：确定PI3K通路、PKC通路和甲状腺激素受体信号在心肌肥厚中的遗传相互作用。具体目的4：确定S6K是否位于PI3K下游，以决定体外和体内心肌细胞的大小。具体目的5：确定Akt在决定体内外心肌细胞大小和存活中的作用。具体目标6：确定PI3K、Akt和S6K在调节压力超负荷肥厚中的作用。这些研究将有助于更好地理解PI3K通路在体内生理和病理条件下决定心脏和心肌细胞大小的作用。

The longterm goal of this project is to define the role of the phosphoinositide-3 kinase (PI3K) pathway in cardiac hypertrophy using transgenic and gene-targeted mice, with a focus on its role on cell size determination during nomal growth and in pathological conditions. Little is known regarding the critical mechanism that controls the myocyte size in vivo. While the growth hormone insulin-like growth factor-1 (IGF1) pathway is well-known to play a critical role in determining overall animal size in mammals, the link between IGF1 and the PI3K pathway in determining individual cell and organ size has not been established. Accordingly, we have recently created transgenic mice with perturbed PI3K activity in the heart. Interestingly, the mice expressing constitutively active PI3K develop moderate cardiac hypertrophy without myocardial dysfunction. The morphometric analysis indicated that cardiac hypertrophy is primarily due to an increase in cell size of the individual myocytes. In contrast, cardiac expression of a dominant-negative PI3K yielded a smaller heart with a concomitant decrease in cell size of individual myocytes. We have also created transgenic mice overexpressing the IGF1 receptor in the cardiac myocyte. Similar to the mice expressing constitutively active PI3K, the IGF1 receptor mice also develop moderate cardiac hypertrophy without signs of cardiac dysfunction. These results raise the possibility that the IGF1-PI3K pathway may play a critical role in determining heart size by regulating the size of cardiomyocytes during normal growth. Two known downstream targets of PI3K are the Akt (protein kinase B) and the p70/p85 ribosomal S6 kinase (S6K). However, their role in the cardiac growth in vivo has not been established. Therefore, we will determine the relative role of Akt and S6K for the cardiac hypertrophic effect of PI3K activation. In addition, in order to examine the role of PI3K, Akt, and S6K in pressure overload- induced hypertrophy, we will create aortic banding in the transgenic mice that express constitutively active or dominant- negative forms of these kinases. Furthermore, we will cross the PI3K transgenic mice with thyroid receptor transgenics and PKC transgenics generated by the Wondisford lab and the King lab, respectively. Accordingly, our Specific Aims are: Specific Aim 1: To confirm the role of PI3K in determining the size of the heart in the adult animal by a conditional transgenic system. Specific Aim 2: To examine whether PI3K is genetically downstream of IGF1/IGF1 receptor in the in vivo heart. Specific Aim 3: To determine genetic interactions among the PI3K pathway, PKC pathway, and thyroid hormone receptor signaling in cardiac hypertrophy. Specific Aim 4: To determine whether S6K is downstream of PI3K in determining the myocyte cell size in vitro and in vivo. Specific Aim 5: To determine the function of Akt in determining myocyte cell size and survival in vivo and in vitro. Specific Aim 6: To determine the role of PI3K, Akt, and S6K in modulating pressure overload hypertrophy. These studies will lead to a better understanding of the role of the PI3K pathway in determining the heart and myocyte size in physiological and pathological conditions in vivo.