Role of Rho-kinase signaling in heart and its involvement of cardiac diseases

研究了RhoA/Rho-Kinase/Myosin磷酸酶(MP)信号在心脏中的作用及其在心脏病中的作用。肌球蛋白磷酸酶靶标亚基2(MYPT2)可与I型磷酸酶δ亚型的催化亚单位(PP1cδ)和HS-M_&lt；21&gt；形成心肌肌球蛋白磷酸酶全酶。MYPT2被确定为RhoA活性形式的靶点。Rho-Kinase磷酸化的MYPT2及其硫代磷酸化可引起MP活性的抑制。高表达MYPT2-PP1cδ的新生大鼠心肌细胞表现出较低的肌球蛋白轻链2(MLC2)磷酸化水平，并对Anitensin II诱导的肌节组织具有抵抗作用，提示MYPT2-PP1cδ可作为MP参与肌小球的组织形成。心脏特异表达MYPT2-PP1cδ转基因小鼠(TG)的心脏表现为扩张型心肌病样表型，包括左心室内径增大，左室壁变薄，Sh…减少更令人振奋。甘油三酯心脏乳头肌的收缩敏感性降低，MLC2的磷酸化水平明显低于野生型。这些结果表明，MP对MLC2的磷酸化具有调节作用，MLC2的磷酸化水平对维持活体正常的心功能很重要。为了分析Rho-Kinase在心脏中的功能，我们建立了过量表达Rho-Kinase的显性阴性片段或成分活性片段的条件性转基因小鼠。然而，每个转基因小鼠和心脏特异性Cre小鼠的双转基因小鼠未能诱导足够数量的每个片段，也没有观察到表型。因此，我们建立了过表达白血病相关Rho鸟嘌呤核苷酸交换因子活性片段(LARG-TG)的条件性转基因小鼠，以研究Rho/Rho-Kinase在心脏中的信号转导。我们将继续制作LARG-TG和Cre双转基因小鼠，研究其表型，以了解Rho/Rho-Kinase信号在心脏中的作用。较少

The roles of RhoA/Rho-kinase/myosin phosphatase (MP) signaling in heart and its involvement in cardiac diseases had been investigated.Myosin phosphatase target subunit 2 (MYPT2) could interact with a catalytic subunit of type 1 phosphatase δ isoform (PP1cδ) and HS-M_<21> to form a cardiac myosin phosphatase holoenzyne. MYPT2 was identified to be a target of the active form of RhoA. Rho-kinase phosphorylated MYPT2 and its thiophosphorylation could induce the inhibition of MP activity. The cultured rat neonatal cardiomyocytes overexpressing MYPT2-PP1cδ showed the lower levels of myosin light chain 2 (MLC2) phosphorylation and were resistant to the sarcomere organization induced by angitensin II, indicating that MYPT2-PP1cδ could act as MP and was involved in sarcomere organization.The heart of the cardiac-specific MYPT2-PP1cδ transgenic mice (Tg) showed the dilated cardiomyopathy-like phenotypes, including the enlargement of LV dimension, thinning of LV wall and the reduced fractional sh … More ortening. The papillary muscle of Tg heart showed the reduced Ca^<2+> sensitivity of contraction and the phosphorylation levels of MLC2 were signfficantly lower as compared with wild type. These results suggested that MP play a role to regulate MLC2 phosphorylation and MLC2 phosphorylation level is important to maintain normal cardiac functions in vivo.To analyze the functions of Rho-kinase in heart, the conditional transgenic mice overexpressing the dominant negative or the constitutively active fragment of Rho-kinase were generated. However, the double transgenic mice of each Tg and heart-specific Cre mice failed to induce an enough amount of each fragment and no phenotypes were observed. Therefore, the conditional transgenic mice overexpressing the active fragment of leukemia-associated Rho guanine nucleotide exchange factors (LARG-Tg) were generated to investigate the Rho/Rho-kinase signaling in heart. We will continue to make the double transgenic mice of LARG-Tg and Cre mice to investigate its phenotypes to understand the role of Rho/Rho-kinase signaling in heart. Less