Project Grant R01AR084437

NUCLEAR SKELETAL MUSCLE ALPHA-ACTIN AND INTRANUCLEAR ROD MYOPATHY - ABSTRACT PATHOGENIC VARIANTS IN ACTA1, WHICH ENCODES SKELETAL MUSCLE-SPECIFIC A-ACTIN (SKA), CAUSE A RANGE OF CONGENITAL MYOPATHIES; AT THE SEVERE END OF THE DISEASE SPECTRUM ARE PATIENTS DIAGNOSED WITH NEMALINE MYOPATHY WITH INTRANUCLEAR RODS, OR INTRANUCLEAR ROD MYOPATHY. INTRANUCLEAR ROD MYOPATHY IS CHARACTERIZED BY THE FORMATION OF SKA-CONTAINING ROD-LIKE STRUCTURES WITHIN THE NUCLEUS OF MYOCYTES, AND IS ASSOCIATED WITH INFANTILE-ONSET MUSCLE WEAKNESS AND DEATH IN EARLY CHILDHOOD DUE TO RESPIRATORY FAILURE, FOR WHICH THERE ARE NO CURRENT TARGETED THERAPIES. RECURRENT VARIANTS AFFECTING VAL163 IN SKA ARE ASSOCIATED WITH INTRANUCLEAR ROD FORMATION, BUT THE PATHOGENIC MECHANISMS DRIVING THE SEVERITY OF DISEASE IN THESE PATIENTS ARE UNKNOWN. WE WERE THE FIRST TO SHOW THAT THE SMOOTH MUSCLE-SPECIFIC A-ACTIN HAS A CRITICAL FUNCTION IN THE NUCLEUS: LOSS OF NUCLEAR SMOOTH MUSCLE-SPECIFIC A-ACTIN PREVENTS COMPLETE DIFFERENTIATION OF SMOOTH MUSCLE CELLS. MUSCLE BIOPSIES FROM PATIENTS WITH INTRANUCLEAR ROD MYOPATHY SHOW IMMATURE MUSCLE FIBERS AND INCREASED NUMBERS OF PROGENITOR-LIKE SATELLITE CELLS, SUGGESTING THAT INCOMPLETE DIFFERENTIATION OF SKELETAL MYOCYTES MAY BE A PATHOLOGIC FEATURE OF THIS DISEASE. WE THEREFORE HYPOTHESIZE THAT SKA FUNCTIONS IN THE NUCLEUS TO PROMOTE SKELETAL MYOCYTE DIFFERENTIATION AND THAT DISRUPTION OF THE NUCLEAR FUNCTIONS OF SKA IN PATIENTS WITH INTRANUCLEAR RODS CAUSES INCOMPLETE SKELETAL MYOCYTE DIFFERENTIATION, WHICH UNDERLIES THE SEVERE AND EARLY-ONSET DISEASE. WE WILL TEST OUR HYPOTHESIS BY THE FOLLOWING SPECIFIC AIMS: 1) IN THE C2C12 MOUSE MYOBLAST CELL LINE, WE WILL IDENTIFY INTRANUCLEAR LOCALIZATION, PROTEIN BINDING PARTNERS, AND TARGET GENOMIC LOCI FOR NUCLEAR SKA WITH AND WITHOUT P.VAL163 VARIANTS. 2) WE WILL ASSESS WHETHER THE INTRANUCLEAR ROD-ASSOCIATED P.VAL163LEU VARIANT PREVENTS COMPLETE DIFFERENTIATION IN VITRO USING A MODEL OF HUMAN INDUCED PLURIPOTENT STEM CELL (IPSCS) DIFFERENTIATION TO SKELETAL MYOCYTES. WE WILL DIFFERENTIATE CRISPR/CAS9-EDITED ACTA1 P.VAL163LEU IPSCS ALONGSIDE ISOGENIC CONTROLS. 3) WE WILL GENERATE A CONDITIONAL KNOCK-IN MOUSE MODEL FOR THE ACTA1 P.VAL163LEU VARIANT AND INDUCE KNOCK-IN IN SKELETAL MYOCYTES EITHER DURING DEVELOPMENT WITH A CONSTITUTIVE ACTA1-CRE OR AFTER DEVELOPMENT WITH AN INDUCIBLE ACTA1- MERCREMER. WE WILL ASSESS WHETHER INTRODUCTION OF THE VARIANT DURING DEVELOPMENT RESULTS IN A MORE SEVERE PHENOTYPE OF INCOMPLETELY DIFFERENTIATED MUSCLE. TAKEN TOGETHER, THESE DATA WILL IDENTIFY A NOVEL ROLE FOR SKA IN THE NUCLEUS DURING HEALTHY SKELETAL MYOCYTE DEVELOPMENT AND WILL IDENTIFY A KEY PATHOGENIC MECHANISM FOR INTRANUCLEAR ROD MYOPATHY. IDENTIFYING A DISEASE MECHANISM MAY LEAD TO DEVELOPMENT OF THERAPEUTIC STRATEGIES FOR THESE PATIENTS.