This application seeks to understand mechanistic elements of the myosin motor of striated muscle and to determine how specific human mutations disrupt myosin function and lead to skeletal muscle disease or cardiomyopathy. We will employ a multidisciplinary approach that takes advantage of the powerful genetic tools available for Drosophila melanogaster along with a broad range of expertise that allows us to study myosin in an integrative manner, from its crystal structure and kinetic function through its effects upon muscle ultrastructure, fiber mechanical properties and locomotion.
For Aim 1, we will build the first models of human distal arthrogryposis syndromes, which cause skeletal muscle contractures of varying severity. Using transgenic Drosophila, we will assess effects upon myosin structure, ATPase activity, in vitro motility, indirect flight and jump muscle ultrastructure, fiber mechanics and organism locomotion. We will test the hypothesis that distal arthrogryposis mutations cause abnormal interactions with nucleotides yielding enhanced ADP binding, reduced sliding velocity, slowed relaxation dynamics and hypercontraction and that the severity of the defects correlates with the severity of the human syndromes.
For Aim 2, we will examine the mechanistic basis of myosin dysfunction caused by two hypertrophic cardiomyopathy mutations. We will construct organisms expressing point mutations that change the charge of the disease-causing residues and then test predicted suppressor mutations for functional rescue in vitro and in vivo. This will reveal interactions altered by the initial mutatins and provide direct insight into protein-protein interactions that are critical for myosin function. We will test the hypothesis that mutations in residues that cause human hypertrophic cardiomyopathy alter contacts between the strands of the central ?-sheet of myosin or interactions between the N-terminal domain of myosin and the lever arm, resulting in increases in ATPase activity, actin sliding velocity, fiber power levels and myofibrillar disarray.
For Aim 3 we will examine the ability of the Drosophila heart to hypertrophy as a result of expressing myosin mutations known to cause human hypertrophic cardiomyopathy. This will be the first attempt to determine how the simple Drosophila heart tube, which is known to exhibit dilated, constricted or hypertrophic phenotypes, reacts to human contractile protein mutations that cause hypertrophy. We will monitor effects upon RNA transcription, calcium handling, ultrastructure and cardiac physiology and determine whether manipulating signaling pathways by gene knockdown or pharmacological intervention ameliorates observed defects. We will test the hypothesis that myosin mutations that cause human hypertrophic cardiomyopathy result in significant wall thickening, abnormal myofibrillar arrays, arrhythmias, disrupted calcium signaling, and transcript profiles that mimic the human condition, and that these pathologies can be suppressed by pharmacological treatment or genetic intervention. Overall our project will take advantage of an innovative experimental system to test significant questions regarding basic myosin function and disease.

Public Health Relevance

We propose to use a transgenic model system, the fruit fly Drosophila melanogaster, to examine the mechanism by which the myosin molecular motor functions in striated muscle. We will produce models of human myosin-based muscle disease (distal arthrogryposis) and heart disease (hypertrophic cardiomyopathy) to determine the molecular defects that cause abnormal skeletal and cardiac muscle function. We will use genetic suppression and drug treatment to better understand the bases of myosin malfunction and to develop our model system as a means of testing potential therapeutic approaches.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM032443-28
Application #
8518982
Study Section
Special Emphasis Panel (ZRG1-MOSS-A (02))
Program Officer
Gindhart, Joseph G
Project Start
1983-07-01
Project End
2017-04-30
Budget Start
2013-06-01
Budget End
2014-04-30
Support Year
28
Fiscal Year
2013
Total Cost
$353,245
Indirect Cost
$116,961
Name
San Diego State University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
073371346
City
San Diego
State
CA
Country
United States
Zip Code
92182
Kronert, William A; Melkani, Girish C; Melkani, Anju et al. (2014) Mapping interactions between myosin relay and converter domains that power muscle function. J Biol Chem 289:12779-90
Caldwell, James T; Melkani, Girish C; Huxford, Tom et al. (2012) Transgenic expression and purification of myosin isoforms using the Drosophila melanogaster indirect flight muscle system. Methods 56:25-32
Kronert, William A; Melkani, Girish C; Melkani, Anju et al. (2012) Alternative relay and converter domains tune native muscle myosin isoform function in Drosophila. J Mol Biol 416:543-57
Kaushik, Gaurav; Zambon, Alexander C; Fuhrmann, Alexander et al. (2012) Measuring passive myocardial stiffness in Drosophila melanogaster to investigate diastolic dysfunction. J Cell Mol Med 16:1656-62
Bloemink, Marieke J; Melkani, Girish C; Dambacher, Corey M et al. (2011) Two Drosophila myosin transducer mutants with distinct cardiomyopathies have divergent ADP and actin affinities. J Biol Chem 286:28435-43
Cammarato, Anthony; Ahrens, Christian H; Alayari, Nakissa N et al. (2011) A mighty small heart: the cardiac proteome of adult Drosophila melanogaster. PLoS One 6:e18497
Cammarato, Anthony; Li, Xiaochuan Edward; Reedy, Mary C et al. (2011) Structural basis for myopathic defects engendered by alterations in the myosin rod. J Mol Biol 414:477-84
Kronert, William A; Melkani, Girish C; Melkani, Anju et al. (2010) Mutating the converter-relay interface of Drosophila myosin perturbs ATPase activity, actin motility, myofibril stability and flight ability. J Mol Biol 398:625-32
Bloemink, Marieke J; Dambacher, Corey M; Knowles, Aileen F et al. (2009) Alternative exon 9-encoded relay domains affect more than one communication pathway in the Drosophila myosin head. J Mol Biol 389:707-21
Kronert, William A; Dambacher, Corey M; Knowles, Aileen F et al. (2008) Alternative relay domains of Drosophila melanogaster myosin differentially affect ATPase activity, in vitro motility, myofibril structure and muscle function. J Mol Biol 379:443-56

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