Associated with the debilitating loss of motor function of paralyzed muscles after spinal cord injury (SCI) are adaptive changes in muscle phenotype (from slow fatigue resistant to fast fatigable), which limit muscle function during rehabilitation, ultimately, limiting the ability to retrain the spinal cord neural circuitry below the level of the lesion. An understanding of the mechanisms regulating muscle adaptations after SCI would facilitate the development of therapies designed to counteract the muscle phenotypic adaptations. The archetypal markers for skeletal muscle phenotype are the isoforms of the myosin heavy chain (MHC), slow (MHC-I) and fast (MHC-IIa, -IIx, and -IIb), which regulate contractile velocity and ATP utilization rates, in the order of fastest to slowest (highest to lowest ATP utilization rate) IIb>llx>IIa>I. We have demonstrated that muscle paralysis results in significant shifts in MHC isoform content in mammalian muscle, resulting in altered physiological performance. The hallmarks of these adaptations are reductions in MHC-I (slow) and increases in MHC-IIx (fast) protein expression. However, the cellular and molecular mechanisms that regulate the changes remain unknown. The overall goal of this project is to elucidate the molecular mechanisms involved in regulating the expression of MHC isoforms in paralyzed muscles following SCI and to assess the impact of locomotor rehabilitation.
Four specific aims will be addressed. 1) To characterize the adaptations in MHC isoform expression that occur during the early dynamic 15 day period after ST. To identify the adaptations during the early period after ST, hindlimb muscles from rats completely transected for periods up to 15 days post-ST will be analyzed for MHC isoform expression at the levels of transcription, post-transcription/pre-translation, and post-translation. To assess adaptations in transcription, the muscles will be analyzed for pre-mRNA; for adaptations at the post-transcriptional/pre-translational level, the muscles will be analyzed for mRNA; and for adaptations at the post-translational level, MHC isoform proteins will be quantified during this highly dynamic period of muscle phenotype transformation. 2) To fully characterize the important sequences in the promoter regions of the MHC isoform genes. We are presently performing a general characterization of the MHC promoters and identifying which general regions are important for ST-induced alterations in gene expression. Therefore, this specific aim will identify the specific transcription factor binding sites important for ST-induced alterations in gene expression. For this aim we will utilize site-directed mutagenesis to delete or mutate specific DNA binding motifs in the MHC isoform gene promoters known to act as binding sites for specific DNA transcription factors. 3) To determine the protein factors and protein complexes that bind to the DNA elements identified in Specific Aim 2. Electrophoretic mobility shift assays will be performed to determine the specific proteins and protein complexes that bind to the important DNA sequences in the promoter regions of the MHC isoform genes. The levels of important transcription factors present in myonuclei of control and ST rat muscle will be quantified. 4) To determine the influence of body weight support treadmill step training (BWST) on the expression of the MHC isoforms at a molecular level in ST rats. The effectiveness of three different BWST training programs will be determined by analysis of the MHC isoform protein, mRNA, and pre-mRNA content, as well as muscle mass. In this way, the specific types of training required to maintain the muscle in a normal phenotypic state can be determined. These experiments will allow us to determine the level of phenotype adaptation that can be induced in paralyzed muscles by specific rehabilitation programs.
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