Striated muscle contraction is dependent upon highly dynamic processes that rely on coordinated communication among, and relative movement of, individual thin filament components. The goal of this application is to understand how human cardiomyopathy mutations located at conserved interfaces between thin filament subunits lead to disease. Drosophila melanogaster, the fruit fly, benefits from robust experimental tools that permit efficient tissue-specific expression of disease alleles in cardiac or skeletal muscle and relatively rapid genetic interaction screens. The fly represents a powerful in vivo system to scrutinize the most proximal events caused by thin filament lesions to facilitate our effort to understand the molecular basis of contractile regulation and, importantly, of myopathic responses in humans. A remarkably integrative approach will be employed that relies upon several new Drosophila models of actin and troponin T (TnT)-based cardiomyopathies. Animal models do not currently exist for six of the seven mutations under investigation here, minimizing our comprehension of the pathological effects of these disease alleles in the physiological context of muscle. Using a unique combination of imaging techniques that includes high-speed live video, confocal, atomic force and electron microscopy we will define, for the first time, the structural and functional effects of the cardiomyopathy mutations from the tissue to the molecular level. The studies will involve pioneering strategies to evaluate Drosophila systolic and diastolic molecular mechanics in vivo.
Aim 1 will rely on multiple hypertrophic cardiomyopathy (HCM) models that express one of three ?-cardiac actin missense mutations. We will test the hypothesis that the HCM actin variants induce similar cardiac and skeletal pathology in flies due to equivalently disturbed tropomyosin (Tm)-based contractile regulation that leads to excessive contractile activity.
For Aim 2 the hierarchical effects of several TnT cardiomyopathy mutations will be delineated. We will test the hypothesis that the mutations differentially influence TnT-Tm interaction, which distinctly affects contractile regulation and activity and consequently prompts diverse cardiac remodeling in flies.
For Aim 3 "second-site" actin mutations will be used to improve cardiac pathology initiated by aberrant TnT, in vivo. Using Drosophila we identified specific actin lesions that suppress troponin-mediated skeletal myopathy. We will now test the hypothesis that these second-site actin mutations can ameliorate TnT-based cardiomyopathies in our fly models. Overall this work is significant since it will provide critical structural-functional information necessary to better comprehend how the thin filament machine functions normally and during disease. Additionally, our efforts will yield genotype-phenotype information in a less complex model system that limits genetic modifiers and environmental factors to help establish paradigms and treatment strategies for pathological processes involved in cardiac remodeling.

Public Health Relevance

We propose to use a transgenic animal model system, Drosophila melanogaster (the fruit fly), to define the mechanisms by which mutations in various thin filament components lead to human cardiac disease. We will produce several new models of actin and troponin-T-based cardiomyopathies to determine the molecular defects that drive diverse and complex tissue remodeling. Finally, in vivo genetic suppression experiments, designed to ameliorate cardiac decline during troponin-T-mediated disease, will resolve novel interactions among thin filament components involved in regulating muscle contraction.

Agency
National Institute of Health (NIH)
Type
High Priority, Short Term Project Award (R56)
Project #
1R56HL124091-01
Application #
8903521
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Wang, Lan-Hsiang
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Baltimore
State
MD
Country
United States
Zip Code
21218