We will use an integrative and multidisciplinary approach to investigate how the S1 head domain of the myosin heavy chain (MHC) protein drives muscle function. Myosin is the molecular motor of muscle and the major component of myofibrillar thick filaments. Its ATP-dependent interaction with actin-containing thin filaments powers muscle contraction. Our studies use the model organism Drosophila melanogaster, which has a single muscle Mhc gene but produces multiple forms of the protein (isoforms) by alternative RNA splicing. Using MHC null mutants in conjunction with germline transformation, we express engineered versions of the protein and employ them to test basic and novel hypotheses that predict structural, biochemical, fiber mechanical, physiological and locomotory properties imparted by specific myosin domains and amino acid residues. An innovative aspect of our system is that functions will be tested in vitro, in skeletal and cardiac muscle and in intact organisms. Therefore, we can determine directly and to what degree a specific biochemical property defines a physiological or locomotory characteristic. To this end, we will utilize a battery of in vitro and in vivo assays: ATPase, actin and nucleotide affinity, in vitro motility, x-ray crystallography, molecular modeling, electron microscopy, isolated fiber mechanics, video-based cardiac imaging and organismal locomotion.
Our first aim i s to elucidate the role of a critical communication element of the myosin motor called the relay domain. We will determine the importance of specific transient interactions of key amino acid residues of the relay that we hypothesize to interact with the converter domain or with the SH1-SH2 helix region during the mechanochemical cycle. For this, we will combine the transgenic approach with classical genetics to introduce and suppress mutations.
Our second aim will test predicted isoform-specific interactions during the mechanochemical cycle. To this end, we will exploit the Drosophila system to express flight and embryonic muscle myosin isoforms that will be crystallized and compared in multiple nucleotide binding states. This approach will also be used for structural analysis of human beta-cardiac myosin, which will be the first mammalian striated muscle myosin analyzed at atomic resolution.
Our third aim will test our hypotheses about the effects of a mutation in myosin that is known to cause restrictive cardiomyopathy. We will create a Drosophila model of this human disease by mutating the invariant proline at the myosin head-rod junction. We will define the biochemical, biophysical, mechanical and locomotory defects engendered by the myosin mutation. We will also examine whether the mutation affects the flexibility of the myosin head and determine how it influences Drosophila heart (dorsal vessel) structure and function. Overall, our novel integrative analyses will permit testing of models for the transduction of chemical energy into movement and will yield insight into how myosin functions in muscle. Further, we will directly address the role of myosin in human muscle disease, by defining the molecular basis of a restrictive cardiomyopathy.

Public Health Relevance

We study the structural and functional differences among alternative forms of the myosin motor protein in order to elucidate how these isoforms differentially regulate contraction of various muscle types during normal locomotion. We will also examine the role of myosin mutation in muscle disease initiation and progression by developing a genetic model for myosin-based restrictive cardiomyopathy. This is relevant to human health in that mutations in myosin cause heart diseases such as dilated, restrictive and hypertrophic cardiomyopathy, as well as skeletal muscle diseases such as inclusion body myopathy, central core disease, early onset distal myopathy and myosin storage myopathy.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032443-27
Application #
8274811
Study Section
Skeletal Muscle and Exercise Physiology Study Section (SMEP)
Program Officer
Gindhart, Joseph G
Project Start
1983-07-01
Project End
2013-05-31
Budget Start
2012-05-01
Budget End
2013-05-31
Support Year
27
Fiscal Year
2012
Total Cost
$496,023
Indirect Cost
$141,931
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
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
Kooij, Viola; Viswanathan, Meera C; Lee, Dong I et al. (2016) Profilin modulates sarcomeric organization and mediates cardiomyocyte hypertrophy. Cardiovasc Res 110:238-48
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
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
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
Wang, Yang; Melkani, Girish C; Suggs, Jennifer A et al. (2012) Expression of the inclusion body myopathy 3 mutation in Drosophila depresses myosin function and stability and recapitulates muscle inclusions and weakness. Mol Biol Cell 23:2057-65

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