Dilated cardiomyopathy (DCM), the most common cardiomyopathy form, can result from mutations in contractile proteins (e.g. myosin). However, the structural, molecular, and physiological origins leading to cardiac dilation in myosin-based DCM are not well understood. We will take advantage of the powerful genetic tools available in Drosophila to generate the first fly models of myosin-based DCM and determine the mechanistic basis of disease. Multidisciplinary approaches will be implemented to determine how single amino acid changes in myosin disrupt intramolecular interactions and cause biochemical, structural and physiological defects in striated muscles.
In Aim 1, we will generate the first X-ra crystal structures for myosin harboring mutations known to cause DCM in humans and predicted to modulate actin binding. Mutant His-tagged myosin will be expressed in indirect flight muscles (IFMs), purified, and use for crystallography. We will test the hypothesis that: DCM mutations in myosin that disrupt intramolecular interactions near or within the actin- binding site re-orient key residues important for actin binding.
Aim 2 will implement a variety of approaches to better understand the biochemical, cell biological, and functional defects associated with human myosin DCM mutations. We will express and purify mutant myosin from IFMs for biochemical/biophysical assays (actin co- sedimentation, ATPase, in vitro motility) to determine the molecular basis of DCM due to myosin mutations. Ultrastructural analyses of IFMs will provide insight into the defects in myofibrillar assembly and maintenance induced by the mutations. Furthermore, we will determine if expression of such mutations cause skeletal muscle dysfunction using flight and jump tests. We will test the hypothesis that: mutations in myosin can weaken actin affinity, reduce enzymatic activity of myosin, and cause structural and functional defects in indirect flight muscles.
In Aim 3, we will assess remodeling events that occur in the Drosophila heart due to expression of myosin DCM mutations. Although it is known that the Drosophila heart can remodel into a dilated phenotype, it is unknown if it dilates in response to myosin mutations known to cause DCM in humans. We will perform cardiac physiological and ultrastructural analyses of micro-dissected heart preparations to test the hypothesis that: expression of DCM-associated myosin mutations causes defects in cardiac contractility and leads to pathological remodeling akin to the human condition, i.e. cardiac dilation, arrhythmias, and ultrastructural defects. Overall, our project will provide detailed and comprehensive analyses to better understand how myosin dysfunction causes DCM and to determine the feasibility of using Drosophila as an assessment tool for human DCM. These studies will offer outstanding training in structural biology, biochemistry, and cell and molecular biology aimed at studying protein dysfunction related to heart disease to prepare the applicant for a related career in academia.

Public Health Relevance

We propose to generate the first fruit fly (Drosophila melanogaster) models of dilated cardiomyopathy (DCM) due to mutations in myosin. We will implement multidisciplinary approaches to better understand how changes within the structure of the molecule lead to biochemical defects, and how these abnormalities relate to structural and physiological decline of striated muscles. These studies will provide novel insight into the mechanistic basis of disease and potential applications of modeling other human myosin DCM mutations in Drosophila.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
1F31HL128118-01
Application #
8911613
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Meadows, Tawanna
Project Start
2015-08-19
Project End
2019-08-18
Budget Start
2015-08-19
Budget End
2016-08-18
Support Year
1
Fiscal Year
2015
Total Cost
Indirect Cost
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