Date of Award
Fall 2012
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Biological Sciences
First Advisor
Eddinger, Thomas J.
Second Advisor
Anderson, James T.
Third Advisor
Fitts, Robert H.
Abstract
The contractile properties of smooth muscle (SM) are broadly classified as tonic and phasic. Among the hypothesized underlying regulatory mechanisms for this difference is the different actomyosin ATPase kinetic properties of SM SMA/SMB myosin heavy chain (MHC) isoforms and the preferential expression of SMA and SMB MHC isoforms in tonic and phasic SM respectively. Thus, we hypothesized that SM SMA/B MHC expression determines tonic and phasic contractile patterns in SM.
To test the this hypothesis, the role of SMA and SMB MHC isoforms in tonic and phasic contractions was studied in phasic (longitudinal ileum and stomach circular antrum) and tonic (stomach circular fundus) smooth muscle tissues of SMB knockout mice. Knocking out the SMB MHC gene eliminated SMB MHC protein expression and resulted in up-regulation of the SMA MHC protein without altering the total MHC protein level. Switching from SMB to SMA MHC protein expression decreased the rate of the force transient and increased the sustained tonic force in SMB(-/-) antrum with high potassium (KPSS) or Carbachol (CCh) stimulation. The sustained tonic force in SMB(-/-) ileum was also significantly increased with KPSS stimulation but not with CCh. The increased tonic contraction under depolarized condition was not through changes in second messenger signaling pathways (PKC/CPI-17 or Rho/ROCK signaling pathway) or LC20 phosphorylation. Biochemical analyses showed that the expression of contractile regulatory proteins (MLCK, MLCP, PKC&alpha, and CPI-17) did not change significantly in tissues tested except for PKCá protein expression being significantly decreased in the SMB(-/-) antrum. However, specifically activating PKC&alpha with phorbol dibutyrate (PDBu) was not significantly different in knockout and wild type tissues, with total force being a fraction of the force generation with KPSS or CCh stimulation in SMB(-/-) ileum and antrum. Taken together, these data show inhibiting SMB MHC protein expression results in a compensatory increase in the SMA MHC protein expression and enhanced sustained tonic contraction with a reduced rate of force generation in these phasic tissues. These results are consistent with SMA and SMB MHC regulating tonic and phasic contraction in SM.