During the clinical syndrome of congestive heart failure (CHF), the vasculature is characterized by resting vasoconstriction, abnormal responses to stress, and resistance to nitric oxide (NO) mediated vasodilatation. The mechanism that leads to these changes in the vasculature is unknown. Previous studies have not addressed whether changes in the contractile phenotype of vascular smooth muscle cause these abnormalities. The goal of this application is to determine the mechanism for the vascular abnormalities associated with CHF. Smooth muscle activation is dependent on 20 kDa myosin light chain (MLC20) phosphorylation levels, which depend on the relative activities of MLC kinase and MLC phosphatase. The MLC phosphatase is a holoenzyme consisting of three subunits; a approximately -20 kDa subunit, a approximately -38 kDa catalytic subunit and a myosin binding subunit (MBS). Four distinct isoforms of the MBS are characterized by the presence (133 kDa) or absence (130 kDa) of a central insert and a leucine zipper. Preliminary data demonstrate that the 130 kDa isoform has an increased activity compared to the 133 kDa isoform, and that the central insert determines the sensitivity of smooth muscle to agonist induced force enhancement. In addition, our data suggest that splice variant expression of the leucine zipper determines the sensitivity to NO mediated vasodilatation. This application is based on the hypothesis that vascular abnormalities associated with CHF are due to changes in splice variant expression of the MBS. We suggest that the resting vasoconstriction is due to changes in splice variant expression of the central insert, while the resistance to NO mediated vasodilatation is due to changes in splice variant expression of the leucine zipper. To test this hypothesis, we will determine (1) the sensitivity of smooth muscle to agonist induced force enhancement, NO mediated vasodilatation, and the expression of splice variant isoforms of the MBS prior to and after the development of CHF; and (2) if forced expression of the splice variant isoforms of the MBS alters the sensitivity to agonist induced force enhancement mad NO mediated smooth muscle relaxation. We will use several different vascular smooth muscles of control and a rat infarct model of CHF, as well as cultured smooth muscle cells to test our hypothesis. These studies will elucidate the molecular mechanism underlying the tissue specific differences in the sensitivity to agonist induced force enhancement and NO mediated smooth muscle relaxation.
