Contraction of heart muscle results from the cyclic interaction of myosin with actin under the regulation of sarcomeric thin and thick filament proteins. Based on results from skinned fibers and cells in culture it has been proposed that phosphorylation of the regulatory light chain (RLC) of sarcomeric myosin plays a role in enhancing ventricular contraction while phosphorylation of either sarcomeric or cytoplasmic myosin-IIB promotes sarcomere assembly. Functional changes in the expression and activity of Ca2+/calmodulin- dependent myosin light chain kinase (MLCK) may lead to cardiomyopathy. Although cardiac myocytes contain smooth (sm) muscle MLCK, it is not clear that this is the only kinase responsible for RLC phosphorylations. We propose to identify the kinase that phosphorylates the respective RLCs in addition to physiological roles for RLC phosphorylations in cardiac contraction and sarcomere assembly.
Specific Aim 1. Determine effects of overexpression and knockout of smMLCK in ventricular and atrial myocytes. Transgenic mice will be made with MLCK expressed specifically in cardiac myocytes. The gene for smooth muscle MLCK will be ablated by crossing mice with the floxed smooth muscle MLCK gene and mice expressing Cre specifically in cardiac myocytes. Other kinases that phosphorylate RLC will be identified. RLC phosphorylations will be measured in cardiac tissues as well as isolated myocytes.
Specific Aim 2. Determine if functional consequences are associated with changes in expression of MLCK isoforms. Anatomical properties of hearts from transgenic and knockout mice will be assessed. Functional properties including contractile indices will be measured in perfused hearts and in vivo by invasive and noninvasive measurements. Responsiveness to stresses that lead to cardiac hypertrophy will also be evaluated.
Specific Aim 3. Determine if sarcomere assembly is affected by overexpression or knockout of smMLCK. Sarcomere formation in response to hypertrophic agents will be measured in myocytes from newborn transgenic or knockout mice. Morphometric analyses will be combined with RLC phosphorylation measurements. Development of myofibrillar structure will be examined in hearts from embryonic knockout mice. Results from these studies will provide novel biochemical insights into the physiological regulation and functional importance of cardiac myosin light chain phosphorylation.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL080536-04
Application #
7564721
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Evans, Frank
Project Start
2006-02-01
Project End
2011-01-31
Budget Start
2009-02-01
Budget End
2010-01-31
Support Year
4
Fiscal Year
2009
Total Cost
$381,118
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Physiology
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
Chang, Audrey N; Gao, Ning; Liu, Zhenan et al. (2018) The dominant protein phosphatase PP1c isoform in smooth muscle cells, PP1c?, is essential for smooth muscle contraction. J Biol Chem 293:16677-16686
Chang, Audrey N; Mahajan, Pravin; Knapp, Stefan et al. (2016) Cardiac myosin light chain is phosphorylated by Ca2+/calmodulin-dependent and -independent kinase activities. Proc Natl Acad Sci U S A 113:E3824-33
Cowley, Patrick M; Wang, Guanying; Chang, Audrey N et al. (2015) The ?1A-adrenergic receptor subtype mediates increased contraction of failing right ventricular myocardium. Am J Physiol Heart Circ Physiol 309:H888-96
Chang, Audrey N; Battiprolu, Pavan K; Cowley, Patrick M et al. (2015) Constitutive phosphorylation of cardiac myosin regulatory light chain in vivo. J Biol Chem 290:10703-16
Chang, Audrey N; Huang, Jian; Battiprolu, Pavan K et al. (2013) The effects of neuregulin on cardiac Myosin light chain kinase gene-ablated hearts. PLoS One 8:e66720
Stull, James T; Kamm, Kristine E; Vandenboom, Rene (2011) Myosin light chain kinase and the role of myosin light chain phosphorylation in skeletal muscle. Arch Biochem Biophys 510:120-8
Kamm, Kristine E; Stull, James T (2011) Signaling to myosin regulatory light chain in sarcomeres. J Biol Chem 286:9941-7
Ding, Peiguo; Huang, Jian; Battiprolu, Pavan K et al. (2010) Cardiac myosin light chain kinase is necessary for myosin regulatory light chain phosphorylation and cardiac performance in vivo. J Biol Chem 285:40819-29
Chang, Audrey N; Chen, Guohua; Gerard, Robert D et al. (2010) Cardiac myosin is a substrate for zipper-interacting protein kinase (ZIPK). J Biol Chem 285:5122-6
He, Wei-Qi; Peng, Ya-Jing; Zhang, Wen-Cheng et al. (2008) Myosin light chain kinase is central to smooth muscle contraction and required for gastrointestinal motility in mice. Gastroenterology 135:610-20

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