One of the factors that has spurred renewed interest in myofibril formation and maintenance in cardiac muscles is the genetic analyses linking diseases with mutations in sarcomeric proteins. The emphasis in this proposal focuses on the formation and maintenance of the A-band, a key region of the myofibril for the stability and contraction of cardiac sarcomeres. The proposed experiments will combine analyses of the temporal and spatial organization and dynamic properties of selected A-band proteins in live cells undergoing myofibrillogenesis. We have proposed a three-step model for the formation of myofibrils in muscle cells: premyofibrils to nascent myofibrils to mature myofibrils. Our first working hypothesis is that as muscle myosin II molecules in nascent myofibrils realign from an overlapping array to form A-bands in mature myofibrils, their exchange with a cytoplasmic pool of myosin is reduced due to increased binding interactions between the assembling proteins. Proteins that are mutated may have altered dynamics that lead to myofibril instability. Our second working hypothesis is that one of titin's roles in the formation of A-bands is to prevent non-muscle myosin II from co-polymerizing with muscle myosin II. There are three specific aims in this proposal. The first specific aim is to analyze the dynamics of four A-band proteins (muscle myosin II heavy chains, essential and regulatory light chains, C-Proteins) in live cells as myofibrils assemble de novo in living avian cardiomyocytes. The second specific Aim is to test the hypothesis that expression of mutated molecules of muscle myosin II heavy chains, and G-protein, that are known to be involved in Hypertrophic Cardiomyopathies, will have different dynamic properties from wild type A-band proteins and will alter A-band stability or induce myofibril disarray in transacted cardiomyocytes. The third specific aim is;to test the hypothesis that titin and A-band regions of titin prevent the co-assembly of non-muscle myosin II and muscle myosin ll. A minimal domain of titin effective in preventing copolymerization of the two myosin isoforms will be identified with recombinant titin fragments derived from the A-band domains of titin that are responsible for its binding to the light meromyosin regions of muscle myosin II heavy chains. The advanced imaging methods coupled with molecular biological and biochemical techniques should yield new insights into basic and pathologic processes in myofibril assembly in living cardiomyocytes.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Cardiovascular Differentiation and Development Study Section (CDD)
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Evans, Frank
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Upstate Medical University
Anatomy/Cell Biology
Schools of Medicine
United States
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Dube, Dipak K; Dube, Syamalima; Shrestha, Runa et al. (2018) Qualitative and quantitative evaluation of TPM transcripts and proteins in developing striated chicken muscles indicate TPM4? is the major sarcomeric cardiac tropomyosin from early embryonic life to adulthood. Cytoskeleton (Hoboken) 75:437-449
Dube, Syamalima; Abbott, Lynn; Randhawa, Samender et al. (2018) Sarcomeric TPM3? in developing chicken. Cytoskeleton (Hoboken) 75:174-182
Wang, Jushuo; Fan, Yingli; Sanger, Jean M et al. (2018) Nonmuscle myosin II in cardiac and skeletal muscle cells. Cytoskeleton (Hoboken) 75:339-351
Dube, Dipak K; Dube, Syamalima; Abbott, Lynn et al. (2017) Identification, characterization, and expression of sarcomeric tropomyosin isoforms in zebrafish. Cytoskeleton (Hoboken) 74:125-142
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Wang, Jushuo; Dube, Dipak K; White, Jennifer et al. (2012) Clock is not a component of Z-bands. Cytoskeleton (Hoboken) 69:1021-31
Wang, Jushuo; Dube, Dipak K; Mittal, Balraj et al. (2011) Myotilin dynamics in cardiac and skeletal muscle cells. Cytoskeleton (Hoboken) 68:661-70
Sanger, Jean M; Wang, Jushuo; Gleason, Lisa M et al. (2010) Arg/Abl-binding protein, a Z-body and Z-band protein, binds sarcomeric, costameric, and signaling molecules. Cytoskeleton (Hoboken) 67:808-23
Sanger, Joseph W; Wang, Jushuo; Holloway, Beth et al. (2009) Myofibrillogenesis in skeletal muscle cells in zebrafish. Cell Motil Cytoskeleton 66:556-66
Du, Aiping; Sanger, Jean M; Sanger, Joseph W (2008) Cardiac myofibrillogenesis inside intact embryonic hearts. Dev Biol 318:236-46

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