ROLE OF DYSTROPHIN IN CALCIUM REGULATION AND MECHANICAL PROPERTIES OF LYMPHATIC MUSCLE IN DUCHENNE MUSCULAR DYSTROPHY - PROJECT SUMMARY DUCHENNE MUSCULAR DYSTROPHY (DMD) LEADS TO PROGRESSIVE DISABILITY IN 1 OF EVERY 5000 MALES DUE TO AN X-LINKED MUTATION OF THE DMD GENE. DMD IS THE ABSENCE OF FUNCTIONAL DYSTROPHIN PROTEIN CHARACTERIZED BY PERSISTENT INFLAMMATION, DISRUPTED CALCIUM HOMEOSTASIS, MUSCLE DEGENERATION AND FIBROSIS. THE EARLY ONSET OF INFLAMMATION NOT ONLY EXACERBATES TISSUE DAMAGE BUT ALSO PLAYS A PIVOTAL ROLE IN THE PROGRESSION OF THE DISEASE. THE LYMPHATIC SYSTEM PLAYS A CRITICAL ROLE IN REGULATING INFLAMMATION AND FIBROSIS DURING PATHOGENESIS OF DISEASES. LYMPHATIC MUSCLE CELLS (LMC) IN THE COLLECTING LYMPHATIC VESSELS ENABLE PHASIC AND TONIC CONTRACTIONS THROUGH CA2+ REGULATION. THIS PROPOSAL BRINGS A NOVEL PERSPECTIVE FOR HOW THE LOSS OF DYSTROPHIN INFLUENCES THE LYMPHATIC SYSTEM DURING DMD PATHOGENESIS. PRELIMINARY DATA DEMONSTRATES: I) LYMPHATIC MUSCLE EXPRESSES FULL-LENGTH DYSTROPHIN (DP427KDA) AND TRUNCATED DYSTROPHIN PROTEIN (DP71KDA); II) DECREASE IN PHASIC AND INCREASE IN TONIC CONTRACTIONS IN COLLECTING LYMPHATIC VESSELS; III) INCREASE IN INFLAMMATORY-ASSOCIATED LYMPHANGIOGENESIS IN SKELETAL MUSCLES OF D2.MDX MICE; IV) DISORGANIZED LYMPHATIC MUSCLE STRUCTURE; AND V) DECREASE IN SERCA2A EXPRESSION IN LYMPHATIC MUSCLE OF D2.MDX MICE. HENCE THE CENTRAL HYPOTHESES ARE: 1) LOSS OF DYSTROPHIN IMPAIRS MECHANICAL CHARACTERISTICS OF LMCS, WHICH LEADS TO 2) DECREASED SERCA EXPRESSION/ACTIVITY IN LYMPHATIC MUSCLE, INCREASING INTRACELLULAR CA2+ AND IMPAIRING LYMPHATIC FUNCTION IN DMD. TO GAIN ADDITIONAL INSIGHT INTO HOW THE LOSS OF DYSTROPHIN PLAYS A ROLE IN THE LYMPHATIC FUNCTION, TWO RELATED, BUT INDEPENDENT AIMS ARE PROPOSED. AIM 1 WILL DETERMINE THE ROLE OF DYSTROPHIN IN LMCS MECHANICAL PROPERTIES AND CALCIUM REGULATORY PROTEINS. HERE, ATOMIC FORCE MICROSCOPY AND SPATIAL TRANSCRIPTOMICS APPROACHES WILL BE USED TO ASSESS CELL STIFFNESS AND ADHESION FORCE AND CALCIUM-REGULATORY PROTEINS IN CONTROL AND D2.MDX MICE. AIM 2 WILL INVESTIGATE HOW SERCA REGULATES LYMPHATIC FUNCTION IN DMD. HERE, IN VITRO AND EX VIVO CALCIUM IMAGING AND PRESSURE MYOGRAPHY WILL BE EMPLOYED. THIS RESEARCH WILL FUNDAMENTALLY ADVANCE THE UNDERSTANDING OF LYMPHATIC FUNCTION AND PROVIDE NOVEL INFORMATION IN TARGETING THE LYMPHATIC SYSTEM IN IMPROVING MUSCLE HEALTH IN DMD.