Project Grant K08HL173569

MULTI-SCALE ANALYSIS OF THE ROLE OF SKELETAL MUSCLE ACTIN MUTATIONS IN DILATED CARDIOMYOPATHY - PROJECT ABSTRACT THERE ARE TWO MAIN OBJECTIVES FOR THIS 5-YEAR CAREER DEVELOPMENT PLAN: 1) DEFINE THE ROLE AND MECHANISTIC BASIS OF SKELETAL MUSCLE ACTIN MUTATIONS IN DILATED CARDIOMYOPATHY (DCM), AND 2) TRANSITION THE PRINCIPAL INVESTIGATOR (PI) TO INDEPENDENCE THROUGH PROFESSIONAL AND SCIENTIFIC MENTORSHIP. DESPITE CURRENT THERAPIES, MANY PATIENTS WITH DCM PROGRESS TO END STAGE HEART FAILURE POSSIBLY DUE TO A LACK OF TREATING CHANGES IN CONTRACTILITY WHICH NEEDS FURTHER EXPLORATION. THE BASIS OF ALL CONTRACTION IN CARDIAC AND SKELETAL MUSCLES IS MYOSIN PULLING ON ACTIN FILAMENTS. SKELETAL MUSCLE ACTIN IS THE MINOR ACTIN ISOFORM IN THE HEART, AND IT HAS NEVER BEEN MECHANISTICALLY EXPLORED IN DCM. IN THIS PROPOSAL, THE PI ESTABLISHES A MULTISCALE EXPERIMENTAL PLATFORM THAT UTILIZES BIOCHEMICAL, BIOPHYSICAL, AND IN VIVO METHODS TO ROBUSTLY STUDY THE EFFECTS OF THE SKELETAL MUSCLE ACTIN MUTATION R256H ON HEART AND SKELETAL MUSCLE. THE PI PREVIOUSLY FOUND THAT R256H EXCEPTIONALLY ASSOCIATES WITH DCM WITHOUT SKELETAL MYOPATHY UNLIKE ALL OTHER SKELETAL MUSCLE ACTIN MUTATIONS. HIS PRELIMINARY DATA DEMONSTRATES THAT R256H HAS A DOMINANT NEGATIVE EFFECT ON CONTRACTILITY ONLY IN THE PRESENCE OF THE ACTIN-BINDING PROTEINS TROPONIN AND TROPOMYOSIN WHICH IS A NOVEL MECHANISM THAT WAS DISCOVERED USING A NEW TECHNIQUE OF RECOMBINANT ACTIN PURIFICATION CREATED BY THE PI. HE ALSO ESTABLISHES THAT R256H CAUSES HYPCONTRACTILITY IN HUMAN CARDIOMYOCYTES AND MOUSE HEARTS. HE HYPOTHESIZES THAT R256H CAUSES DCM WITHOUT SKELETAL MYOPATHY DUE TO TISSUE-SPECIFIC EXPRESSION OF DIFFERENT ISOFORMS OF TROPONIN AND TROPOMYOSIN. THIS HYPOTHESIS WILL BE TESTED BY ELUCIDATING THE BIOCHEMICAL AND STRUCTURAL EFFECTS OF R256H IN THE CONTEXT OF CARDIAC AND SKELETAL MUSCLE TROPONIN AND TROPOMYOSIN (AIM 1), DEFINING THE EFFECTS OF R256H ON CARDIAC AND SKELETAL MUSCLE CELLS AND ENGINEERED TISSUES (AIM 2), AND ANALYZING THE EFFECTS OF R256H ON CARDIAC AND SKELETAL MUSCLES OF MICE (AIM 3). COMPLETING THESE AIMS WILL ESTABLISH THE ROLE OF SKELETAL MUSCLE ACTIN MUTATIONS IN DCM FOR THE FIRST TIME AND CREATE A MULTISCALE PLATFORM TO STUDY ADDITIONAL SKELETAL MUSCLE ACTIN MUTATIONS IN CARDIOMYOPATHY FOR THE PI'S FIRST R01. MOREOVER, WITH GUIDANCE OF A FORMAL MENTORING COMMITTEE, THE PI WILL COMPLETE A CURRICULUM THAT WILL BUILD HIS BIOCHEMISTRY AND BIOPHYSICS KNOWLEDGE AND TECHNIQUES, TEACH HIM CRYOEM STRUCTURAL ANALYSIS TO CONNECT STRUCTURE TO FUNCTION, AND EXPAND HIS UNDERSTANDING OF CARDIAC AND SKELETAL MUSCLE DISEASE MODELING IN ANIMALS. FINALLY, HE WILL IMPROVE HIS PROFESSIONAL, LOGISTICAL, AND EDUCATIONAL SKILLS TO FULLY TRANSITION TO AN INDEPENDENT INVESTIGATOR AND PRODUCTIVE MEMBER OF THE SCIENTIFIC COMMUNITY CAPABLE OF TRAINING FUTURE PHYSICIAN-SCIENTISTS.