Mechanism for Obstruction-Induced Detrusor Remodeling: Role of Hypoxia & Stretch (PI, Samuel K. Chacko, D.V.M., Ph.D.).
This project is concerned with specific smooth muscle phenomena that are altered in bladder outlet obstruction. The degree of detrusor remodeling which results in impaired contractility is unknown. It is also unclear exactly what factors control these changes, and there is no way at present to predict which of the scenarios (no change, impaired contractility, hyperactivity/impaired contractility) will occur. Likewise, there is no way to predict whether partial or complete recovery will occur following relief of the obstruction in the impaired contractility scenario, either alone or with hyperactivity. Recent studies on the generation of active force and its regulation in smooth muscle reveal an important role for calcium sensitization (shown schematically in Project 3). The major players involved in the mechanism for calcium sensitization pathway has been shown to be the G-protein-coupled Rho-kinase (ROK) and CPI-17 (which itself is regulated by PKC (7) and ROK) (8;13) Although the major mechanism that regulates smooth muscle contraction is the elevation of cytosolic free Ca2+ (5), several other regulatory mechanisms could modify the sensitivity of the contractile and regulatory proteins to [Ca2+]. The RhoA/ROK cascade affects the level of phosphorylation of the myosin regulatory light chain (MLC20) by inhibition of the myosin phosphatase through phosphorylation of its regulatory subunit (MYPT1) (6). The factors that initiate the molecular, biochemical, and structural changes in the detrusor myocytes during PBOO are not understood, although stretch and hypoxia are all possible mechanisms. Our preliminary data on remodeling DSM shows an overexpression of transcription factors (TFs) for gene expression of proteins involved in the calcium sensitization pathway compared to that of the normal DSM. Obstruction-induced overdistension of the bladder wall is thought to cause stretch and hypoxia of the DSM layer. The hypothesize that obstruction-induced hypoxia and stretch in the urinary bladder wall cause overexpression of transcription factors for gene activation of proteins (Rho A, ROK, and CPI-17) involved in the calcium sensitization pathway in the DSM will be tested using the detrusor from partially obstructed mice and by exposing smooth muscle cell cultures to hypoxia, stretch, or both. Our proposed experiments would provide the data to elucidate the molecular mechanism for alteration of the expression of TFs that regulate gene expression of proteins involved in the calcium sensitization pathway, which modulates the MLC20 phosphorylation-mediated regulatory mechanisms for actin-myosin interaction and contraction in the DSM.