Regulation of Vascular Smooth Muscle Contraction
The contractile properties of smooth muscle are broadly classified as phasic (fast) and tonic (slow). Phasic smooth muscle is characterized by relatively rapid rates of force activation, force relaxation and Vmax, whereas tonic smooth muscle is characterized by slower rates of force activation, force relaxation and Vmax However, the molecular mechanism that regulates the contractile properties of smooth muscle is unknown. Others and we have demonstrated that the mechanism for this diversity in contractile properties between phasic and tonic smooth muscle lies at the level of the contractile filaments, and differences in the kinetics of the actomyosin ATPase (AMATPase). The overall goal of this grant is to investigate the molecular mechanism for the differences in the kinetics of the AMATPase in tonic and phasic smooth muscle. Our hypothesis is that differences in the kinetics of the AMATPase are due to splice variant expression of the myosin heavy chain (MHC) and the 17-kDa myosin light chain (MLC17). The Specific Aims to test this hypothesis are to determine if splice variants of MHC and MLC17 are molecular determinants for the kinetics of the AMATPase of smooth muscle (Specific Aims 1-3). In addition, we will determine if non-muscle myosin is responsible for force maintenance in smooth muscle (Specific Aim 4). To test Specific Aims 1-3, we will force the expression of both splice variant isoforms of MHC and MLC17 in cultured embryonic aortic and gizzard smooth muscle cells. We will over-express both the endogenous and the alternative isoform of MHC and MLC17 in cultured aortic and gizzard smooth muscle cells and tissue strips. After forcing the expression of a single contractile protein, we will determine the effect of elevations of inorganic Pi and MgADP on steady state force and stiffness of single cultured smooth muscle cells, as well as in smooth muscle strips 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 kinetics of the AMATPase of smooth muscle. In addition, to determine if non-muscle myosin participates in the molecular mechanism for force maintenance (Specific Aim 4), we will determine the properties of force maintenance in a transgenic mouse line, which lacks non-muscle myosin IIB. The results of these studies should elucidate the mechanism that determines the kinetics of the AMATPase of smooth muscle, and form a foundation for future investigation of how smooth muscle contractility is altered by disease states.
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