MYOSIN ISOFORMS & CALCIUM REGULATION OF ACTOMYOSIN ATPASE IN DETRUSOR

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We propose to use smooth muscle tissue from the rabbit model for outlet obstruction and human biopsy samples from patients with outlet obstruction (from Core B) to elucidate the cellular and molecular mechanisms underlying the changes in contractility associated with bladder smooth muscle hypertrophy and remodeling in outlet obstruction and it's reversal. Our preliminary data indicate that the expression of myosin isoforms, regulation of actomyosin ATPase, and the organization of myofilaments in different stages of bladder remodeling are important factors that affect the ability of smooth muscle to generate the active force need to empty the bladder. Based on these data, we hypothesize that altered contractility of the bladder smooth muscle associated with outlet obstruction is due to either changes in the composition of myosin isoforms, organization or myosin into the contractile apparatus. and/or the Ca2+ regulation of actomyosin. We will test this hypothesis using the rabbit model for partial outlet obstruction and human detrusor muscle from patients undergoing surgery for outlet obstruction. Specially, our experiments will address the splicing of the myosin mRNA at the 3' end (SM1 and SM2) or the 5' end (insert near the ATP-binding region on the head of the myosin molecules), different in smooth muscles from decompensated bladder walls as compared to normal and compensated bladders (during outlet obstruction and after reversal)? Is there a difference in the expression of these myosin isoforms at transcriptional and translational levels in bladder wall smooth muscle at different stages of compensation and decompensation? (3) What effect does exchanging LC17 isoforms have on the function and the regulation of myosin isolated from normal and hypertrophied smooth muscle? (4) Is the smooth muscle myosin isolated from the detrusor of decompensated bladder functionally different as compared to normal and compensated bladders? (5) Is the expression of myosin light chain kinase (MLCK), which plays a role in the regulation of actomyosin ATPase and force generation, altered during decompensation as compared with the normal and compensation stage? (6) What is the effect of addition of MLCK on the myosin light chain phosphorylation and force in chemically skinned smooth muscle fiber preparations made from normal and compensated bladder (during outlet obstruction and after the reversal of outlet obstruction) detrusor smooth muscles? Alterations in the proteins that form the contractile apparatus will be correlated with changes in the regulation of the entry of Ca2+ into the cytosol (Project 1) and velocity of force generation and regulation of cross-bridge cycling (Project 3). Together, the data from experiments outlined in the Urology Research Center proposal will identify the molecular mechanisms that are responsible for the contractile dysfunction in outlet obstruction.