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.
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.
|Suggs, Jennifer A; Melkani, Girish C; Glasheen, Bernadette M et al. (2017) A Drosophila model of dominant inclusion body myopathy type 3 shows diminished myosin kinetics that reduce muscle power and yield myofibrillar defects. Dis Model Mech 10:761-771|
|Cannon, Leah; Zambon, Alexander C; Cammarato, Anthony et al. (2017) Expression patterns of cardiac aging in Drosophila. Aging Cell 16:82-92|
|Bloemink, Marieke J; Melkani, Girish C; Bernstein, Sanford I et al. (2016) The Relay/Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II. J Biol Chem 291:1763-73|
|Achal, Madhulika; Trujillo, Adriana S; Melkani, Girish C et al. (2016) A Restrictive Cardiomyopathy Mutation in an Invariant Proline at the Myosin Head/Rod Junction Enhances Head Flexibility and Function, Yielding Muscle Defects in Drosophila. J Mol Biol 428:2446-61|
|Kooij, Viola; Viswanathan, Meera C; Lee, Dong I et al. (2016) Profilin modulates sarcomeric organization and mediates cardiomyocyte hypertrophy. Cardiovasc Res 110:238-48|
|Kaushik, Gaurav; Spenlehauer, Alice; Sessions, Ayla O et al. (2015) Vinculin network-mediated cytoskeletal remodeling regulates contractile function in the aging heart. Sci Transl Med 7:292ra99|
|Kronert, William A; Melkani, Girish C; Melkani, Anju et al. (2015) A Failure to Communicate: MYOSIN RESIDUES INVOLVED IN HYPERTROPHIC CARDIOMYOPATHY AFFECT INTER-DOMAIN INTERACTION. J Biol Chem 290:29270-80|
|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|
|Iwamoto, Hiroyuki; Trombitás, Károly; Yagi, Naoto et al. (2014) X-ray diffraction from flight muscle with a headless myosin mutation: implications for interpreting reflection patterns. Front Physiol 5:416|
|Melkani, Girish C; Trujillo, Adriana S; Ramos, Raul et al. (2013) Huntington's disease induced cardiac amyloidosis is reversed by modulating protein folding and oxidative stress pathways in the Drosophila heart. PLoS Genet 9:e1004024|
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