REGULATION OF SMOOTH MUSCLE CONTRACTILE PROPERTIES

平滑肌的收缩特性大致分为相性（快）和强直性（慢）。相位平滑肌的特点是快速的力激活、力松弛和Vmax，而强直平滑肌的特点是缓慢的力激活、力松弛和Vmax。然而，调节平滑肌收缩特性的分子机制尚不清楚。本次资助的总体目标是确定平滑肌收缩特性的分子机制，并阐明快速和缓慢收缩特性的机制。我们的假设是，收缩蛋白的剪接变异同种异构体决定了平滑肌的收缩特性。验证这一假设的具体目的是确定MLC17 （specific Aim 1）、MHC （specific Aim 2）和MLC磷酸酶（specific Aim 3）的剪接变体是否是平滑肌收缩特性的分子决定因素。我们还将确定MLC磷酸酶的剪接变异体是否影响激动剂诱导的力增强的大小或敏感性（Specific Aim 3）。为了验证这些特异性目标，我们将在培养的胚胎主动脉和沙囊平滑肌细胞中表达MLC17、MHC和MLC磷酸酶的剪接变体异构体。我们将确定在培养的主动脉和砂囊平滑肌细胞中正常存在或不表达的同种异构体的表达对培养的平滑肌细胞机械性能的影响。在强制表达单个收缩蛋白后，我们将测定单个培养平滑肌细胞的最大力、力激活和力松弛率、Vmax和MLC20磷酸化，并将结果与未转染的对照组进行比较。这些实验将阐明单个收缩蛋白的表达对培养平滑肌细胞力学特性的影响。这些研究的结果将阐明决定平滑肌收缩特性的机制，并为未来研究平滑肌收缩能力如何随疾病状态而改变奠定基础。

The contractile properties of smooth muscle are broadly classified as phasic (fast) and tonic (slow). Phasic smooth muscle is characterized by a rapid rates of force activation, force relaxation and Vmax, whereas tonic smooth muscle is characterized by slow rates of force activation, force relaxation and Vmax. However, the molecular mechanism that regulates the contractile properties of smooth muscle is unknown. The overall goal of this grant is to determine the molecular mechanism for the contractile properties of smooth muscle and to elucidate the mechanism for fast and slow contractile properties. Our hypothesis is that splice variant isoforms of contractile proteins determine the contractile properties of smooth muscle. The specific aims to test this hypothesis are to determine if splice variants of MLC17 (Specific Aim 1), MHC (Specific Aim 2) and MLC phosphatase (Specific Aim 3) are molecular determinants of the contractile properties of smooth muscle. We will also determine if splice variants isoforms of MLC phosphatase effect either the magnitude or sensitivity of agonist induced force enhancement (Specific Aim 3). To test these Specific Aims we will force the expression of both splice variant isoforms of MLC17, MHC and MLC phosphatase in cultured embryonic aortic and gizzard smooth muscle cells. We will determine the effects of the expression of the isoform normally present or not expressed in the cultured aortic and gizzard smooth muscle cells on the mechanical properties of cultured smooth muscle cells. After forcing the expression of a single contractile protein, we will determine the maximum force, the rates of force activation and force relaxation, Vmax, and MLC20 phosphorylation of single cultured smooth muscle cells and compare the results to those obtained in the non-transfected controls. These experiments will elucidate the effects of the expression of a single contractile protein, in isolation, on the mechanical properties of cultured smooth muscle cells. The results of these studies should elucidate the mechanism that determines the contractile properties of smooth muscle, and form a foundation for future investigation of how smooth muscle contractility is altered by disease states.