Mechanisms of Cu-binding factors to promote myogenic gene expression - PROJECT SUMMARY Copper (Cu) is an essential trace element required for mitochondrial function, antioxidant defense, and gene regulation. While its systemic roles are well established, the mechanisms by which Cu controls gene expression and tissue regeneration, particularly through Cu-binding proteins (Cu-BPs), remain poorly understood. Our lab has identified Cu-responsive regulators, including the transcription factor Mtf1 and LIM- domain proteins Crip2 and Csrp2, as key modulators of skeletal muscle cell proliferation and differentiation. These proteins respond to Cu availability and appear to be also involved in skeletal muscle regeneration. Our published and preliminary work in cultured primary myoblasts shows that Mtf1 governs myogenic and homeostatic genes via SWI/SNF chromatin remodelers, while Crip2 and Csrp2 exhibit Cu-dependent chromatin binding and control distinct biological pathways. Crip2 regulates metal homeostasis and protein synthesis, while Csrp2 modulates Wnt signaling and mitochondrial gene expression. In Menkes (Atp7a-/-) and Wilson (Atp7b-/-) disease models, we observed impaired muscle regeneration, suggesting that Cu homeostasis is essential for effective repair. This project will define how Cu-BPs coordinate gene expression and epigenetic regulation during muscle regeneration, and how these processes are disrupted in Menkes and Wilson disease. Our central hypothesis is that Crip2 and Csrp2 orchestrate temporally distinct transcriptional programs during myogenesis, and that Cu imbalance impairs their function, leading to deficits in muscle stem cell activity, fiber contractility, and tissue repair. To test this, we will: (1) Define how Cu homeostasis and the Cu-BPs Crip2 and Csrp2 regulate muscle regeneration in healthy and disease states using in vivo injury models, RNA-seq, CUT&Tag, and ATAC-seq to examine Cu-dependent gene regulation; (2) Characterize Cu-dependent mechanisms of Crip2 and Csrp2 in primary myoblast proliferation and differentiation from Menkes and Wilson models, focusing on their stability, localization, chromatin occupancy, and functional roles; and (3) Assess muscle fiber dysfunction and therapeutic response in Menkes and Wilson disease using patch-clamp electrophysiology, contractility assays, and mitochondrial imaging to determine how Cu dysregulation impairs excitability. We will also use these three models to test whether Cu- modulating therapies (Cu-histidinate, elesclomol, D-penicillamine, trientine) can restore function. This research will define how Cu regulates muscle regeneration, myoblast proliferation and differentiation and myofiber function, providing a mechanistic foundation for therapies targeting muscle dysfunction in disorders of Cu metabolism. These insights may also apply to broader muscle-wasting conditions, including aging and metabolic disease, where trace metal homeostasis is disrupted.
