Molecular mechanisms affecting utrophin expression in fast versus slow skeletal muscles; possible therapeutic targets for Duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy is a fatal X-linked myopathy characterized by the absence of the cytoskeletal protein dystrophin. The absence of dystrophin leads to impaired sarcolemmal integrity, and inefficient muscle regeneration. Currently, there is no effective therapy to significantly improve the quality of life for DMD patients. One therapeutic approach is to express, at therapeutically relevant levels, a homologous protein that can compensate for dystrophin. Utrophin is one such protein, primarily expressed at the neuromuscular junction, which can compensate for dystrophin function. Therefore, it becomes imperative to identify pathways that regulate the expression of utrophin. Such efforts could identify targets that may be manipulated in attempts to promote utrophin expression along the entire length of dystrophin-deficient myofibers. In this thesis, we investigate the mechanisms that promote higher levels of utrophin expression in extra-synaptic regions of slow, versus fast muscles. We determined through the use of isoform specific antibodies and primers that the A isoform of utrophin is highly expressed in slow, relative to fast muscles. Direct plasmid injections, and in vitro transfection assays with A-utrophin promoter-reporter constructs determined that peroxisome proliferators activated receptor gamma co-factor 1 alpha (PGC-1alpha) and calcineurin can affect the transcription of A-utrophin via the transcription factors GABPalpha and NFATc1, respectively. Similar techniques using utrophin 3'UTR reporter constructs and in vitro stability assays, also determined that calcineurin activity could affect the stability of A-utrophin transcripts via an AU-rich element (ARE) in the utrophin 3'UTR. We also interbred transgenic mice with stimulated calcineurin signaling or impaired calmodulin signaling in skeletal muscles, with dystrophin-deficient mdx mice. We found that stimulation of calcineurin activity improved dystrophic pathology, and increased A-utrophin expression. In contrast, inhibition of calmodulin signaling exacerbated dystrophic pathology, impaired pathways that promote the slow myofiber program, decreased GABPalpha and PGC-1alpha expression and reduced A-utrophin levels. Collectively, these observations demonstrate that Ca+2/calmodulin regulated effectors such as calcineurin and PGC-1alpha can regulate the expression of utrophin, and can serve as potential therapeutic targets for DMD.