(Verbatim from the application): The long-term goal of this proposal is to understand the role of smooth muscle myosin isoforms in vascular smooth muscle function and response to pathological conditions. The hypothesis is that the population of myosin heavy chain isoforms expressed in smooth muscle is a major contributor to the contractile properties of phasic and tonic vascular smooth muscles. To test this hypothesis transgenic mouse lines have been developed harboring the transgene for the myosin isoforms SMaSM1 and SMaSM2 targeted to smooth muscle by the SMP8-alpha-actin promoter. Additional transgenic mice will be developed carrying SMbSM1 and SMbSM2 transgenes using similar constructs. These transgenic mice have the potential to over-express different combinations of both C-terminal (SM1 and SM2) and N-terminal (SMa and SMb) myosin isoforms in vascular smooth muscle. These mice provide us with the means to elucidate the functional role of both the N- and C-terminal isoforms of smooth muscle myosin in vivo.
Specific Aim 1 characterizes the expression of myosin isoforms from the transgenes in aortic smooth muscle and determines if expression of the transgenes alters the morphology of the tissue.
Specific Aim 2 compares the functional differences in smooth muscle between expression of SMaSM1, SMaSM2 and SMbSM1, SMbSM2 transgenes and evaluates the impact of SMa versus SMb isoform expression on in situ cardiac function.
In Specific Aim 3 we propose to generate tail-less SM1 constructs or SM1 constructs with serine1953mutated to alanine. These constructs will provide insight into the mechanism by which the C-terminal tail affects contractile function in vivo.
In Specific Aim 4 we correlate the functional properties of both tonic (aorta) and phasic (pulmonary vein sic) smooth muscle with altered myosin isoform composition. We will use permeabilized preparations to test the hypothesis that myosin isoforms are major determinants of Ca2+-sensitivity and shortening velocity. The approach to these objectives includes recombinant DNA technology, analysis of transgene expression by Southern and Northern blots, in situ hybridization to determine cellular localization of transgene mRNA expression, SDS-PAGE and Western blots to determine protein transgene expression. Measurement of contractility, including concentration-force-relaxation studies, contraction velocity and energetics will be correlated with the distribution of myosin isoforms. These experiments will provide crucial information on the role of myosin isoforms in cardiovascular function in both physiological and pathological circumstances.
