Identify a new non-canonical role of MEF2D⍺2 protein isoform in skeletal muscle metabolism

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Project Summary: Despite the widespread occurrence of alternative splicing in skeletal muscle, the role of very few muscle-specific protein isoforms produced by alternative splicing has been studied. In contrast, altered transcript splicing and splicing regulator expression is frequently found in muscle dystrophies and aging-associated decline in muscle function and metabolism. Skeletal muscle makes up to 40% of body weight in healthy human adults and plays a predominant role in regulating whole-body metabolism. Yet, the role of alternate protein products of alternative splicing in skeletal muscle function and metabolism is largely unknown. My recent work demonstrated that the Rbfox family of RNA-binding proteins is vital for regulating skeletal muscle homeostasis in adulthood. Inducible Rbfox knockout in adult mouse skeletal muscle caused ~50% reduction in muscle mass within four weeks, altered glucose metabolism, and splicing of >740 gene transcripts. Many RBFOX-regulated alternative exons are evolutionarily conserved, suggesting roles for the alternate protein isoforms in adult skeletal muscle function. RBFOX proteins regulate mutually exclusive ⍺1 and ⍺2 exons of the MEF2D transcription factor to produce the predominant adult skeletal muscle-specific isoform, MEF2D⍺2. The four MEF2 (MEF2A-D) members of the highly conserved family of transcription factors are important for embryonic muscle development, but their role in the adult skeletal muscle is not known. The ⍺2 exon inclusion increases to >75% after birth to produce the predominant MEF2D⍺2 isoform in adult skeletal muscle. To determine the role of MEF2D⍺2, I deleted the ⍺2-exon of Mef2d using CRISPR-Cas9 to generate, Mef2d⍺2 Eko mouse line. Compared to wild-type mice, Mef2d ⍺2 Eko mice displayed reduced running capacity and muscle fatty acid oxidation. Our preliminary data indicate minimal to no change in muscle transcriptome in muscles of Mef2d⍺2 Eko mice. We also found that most MEF2D is present in the cytosolic fraction of skeletal muscle and interacts with mitochondrial and muscle metabolic proteins. Given the reduced muscle fatty acid oxidation in skeletal muscles of Mef2d⍺2 Eko mice, we hypothesize that MEF2D⍺2 protein interacts with metabolic proteins in the cytosol to optimize fatty oxidation in adult skeletal muscle. In aim1, we will identify and validate proteins interacting with MEF2D⍺2 exclusively or preferentially in vivo. In aim2, we will determine the impact of the loss of MEF2D⍺2 on its interactors and muscle fatty acid oxidation and validate top MEF2D⍺2-protein interactions in human skeletal muscle tissues. A disruption in skeletal muscle glucose and fatty acid metabolism often manifests before the development of type II diabetes and obesity, one of the most prevalent lifestyle diseases of the modern world. Thus, our work will identify a new non-canonical role of MEF2D⍺2 in muscle metabolism, which we expect to be conserved across evolution as MEF2D⍺2 exon and splice sites are conserved from fish to humans.