PHOSPHOINOSITIDE-3 KINASE IN CARDIAC HYPERTROPHY

该项目的长期目标是使用转基因和基因靶向小鼠确定磷酸肌醇-3激酶（PI 3 K）通路在心脏肥大中的作用，重点是其在正常生长和病理条件下对细胞大小确定的作用。 关于体内控制肌细胞大小的关键机制知之甚少。 虽然生长激素胰岛素样生长因子-1（IGF 1）途径在决定哺乳动物的整体大小方面发挥着关键作用，但IGF 1和PI 3 K途径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PI 3 K活性受到干扰的转基因小鼠。 有趣的是，表达组成性活性PI 3 K的小鼠发生中度心脏肥大而无心肌功能障碍。 形态学分析表明，心肌肥大主要是由于单个肌细胞的细胞大小增加。 相比之下，显性负性PI 3 K的心脏表达产生了较小的心脏，伴随着单个肌细胞的细胞大小的减少。 我们还创造了心肌细胞中IGF 1受体过表达的转基因小鼠。与表达组成性活性PI 3 K的小鼠相似，IGF 1受体小鼠也发生中度心脏肥大，而无心功能障碍体征。 这些结果提高了IGF 1-PI 3 K通路可能通过调节正常生长期间心肌细胞的大小在确定心脏大小方面发挥关键作用的可能性。 PI 3 K的两个已知下游靶标是Akt（蛋白激酶B）和p70/p85核糖体S6激酶（S6 K）。 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S6 K在PI 3 K激活的心脏肥大效应中的相对作用。 此外，为了检测PI 3 K、Akt和S6 K在压力超负荷诱导的肥大中的作用，我们将在表达这些激酶的组成型活性或显性阴性形式的转基因小鼠中产生主动脉带. 此外，我们将分别将PI 3 K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小鼠和PKC转基因小鼠杂交。 因此，我们的具体目标是：具体目标1：通过条件转基因系统确认PI 3 K在决定成年动物心脏大小中的作用。具体目的2：检查在体内心脏中PI 3 K是否是IGF 1/IGF 1受体的遗传下游。具体目标3：确定心脏肥大中PI 3 K通路、PKC通路和甲状腺激素受体信号传导之间的遗传相互作用。 具体目的4：确定在体外和体内确定肌细胞大小时，S6 K是否位于PI 3 K的下游。 具体目的5：确定Akt在体内和体外确定肌细胞大小和存活中的功能。具体目标6：确定PI 3 K、Akt和S6 K在调节压力超负荷性肥大中的作用。 这些研究将导致更好地理解PI 3 K通路在体内生理和病理条件下决定心脏和肌细胞大小的作用。

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.