The Identification and Characterization of MKRP, a Novel Kelch Related Protein

The cells of adult myofibers in mammals are terminally differentiated and are incapable of division and self-renewal. Regeneration of damaged skeletal muscle tissue is facilitated through the proliferation and differentiation of resident stem cells known as satellite cells . These satellite cells remain quiescent in uninjured tissue, occupying a sublaminar position between the sarcolemma and basal lamina of adult myofibers. In response to trauma, these cells become activated, and proliferate. The activated cells will reestablish the pool of reside quiescent satellite cells, while others will proliferate, migrate by chemotaxis to the area of injury, withdraw from the cell cycle, and differentiate into new myoblasts. These myoblasts will fuse with and repair the injured fibers, or align with each other and fuse to form new fibers. Genes involved in this process should exhibit an altered pattern of expression in skeletal muscle in response to injury by cardiotoxin, and the changes in expression level can be quantified though the measurement of mRNA levels at specified time points following injury. Utilizing transcriptome analysis, we identified a completely novel transcript that is induced in the myogenic progenitor cells following cardiotoxin injury. The novel transcript contained an open reading frame that coded for a protein belonging to the Kelch superfamily. Expression of the transcript was restricted to skeletal muscle lineages during development, and to myogenic progenitor cells and immature myotubes during injury regeneration. Because of its structural identity and restriction to skeletal muscle, the novel transcript was named the myogenic Kelch related protein (MKRP). Knockdown of MKRP expression using siRNA in C2C12 cells revealed an inhibition of both migration and differentiation in myogenic progenitor cells. A yeast two-hybrid screen identified calsarcin-2 as a potential interacting protein, indicating a possible role for MKRP in the calcineurin pathway during myogenic differentiation.