Project description:Fast skeletal myosin binding protein-C (fMyBP-C) is one of three MyBP-C paralogs and is predominantly expressed in fast skeletal muscle. Mutations in the gene that encodes fMyBP-C, MYBPC2, is associated with distal arthrogryposis, while fMyBP-C protein is reduced in diseased muscle. However, the functional and structural roles of fMyBP-C in skeletal muscle remain unclear. To address this gap, we generated a homozygous fMyBP-C knockout mouse (C2-/-) and characterized it, both in vivo and in vitro. Ablation of fMyBP-C was benign in terms of muscle weight, fiber type, cross-sectional area, and sarcomere ultrastructure. However, grip strength and plantar-flexor muscle strength were significantly decreased in C2-/- compared to WT. Peak isometric tetanic force (Po) and isotonic speed of contraction were significantly reduced in isolated extensor digitorum longus (EDL) from C2-/- mice. In EDL muscles of C2-/- mice, small-angle X-ray diffraction revealed significant increase in equatorial intensity ratio (I1.1/I1.0) during contraction, indicating a greater degree of myosin head shift towards actin while MLL4 layer-line intensity was decreased at rest, indicating less ordered myosin heads at rest. Interfilament lattice spacing was also significantly increased in C2-/- EDL muscle compared to WT. Consistent with these findings, we observed a significant reduction of steady-state isometric force during Ca2+ activations, myofilament calcium sensitivity, sinusoidal stiffness in skinned EDL muscle fibers from C2-/- mice. Finally, C2-/- muscles displayed disruption of inflammatory and regenerative genes and increased muscle damage upon mechanical overload. Together, our data suggest that fMyBP-C is essential for maximal speed and force of contraction, sarcomere integrity, and calcium sensitivity in fast twitch muscle.
Project description:Myosin-binding protein C (MyBP-C) is a thick filament regulatory protein found exclusively in the C-zone of the A band in the sarcomeres of vertebrate striated muscle. Cardiac, slow skeletal and fast skeletal MyBP-C (fMyBP-C) paralogs perform different functions. However, the functional role of fMyBP-C in fast skeletal muscle is completely unknown. Genetic mutations in human fMyBP-C lead to skeletal myopathies. All three isoforms share similar protein structures, but likely differ substantially in terms of expression and function, which may serve the distinct physiologies of fast and slow muscle fibers. In the present study, we developed a novel fMyBP-C global knockout (KO) mouse model (C2-/-) to investigate the structural, functional, molecular, cellular and physiological roles of fMyBP-C in skeletal muscle.
Project description:Amyotrophic lateral sclerosis (ALS) is a lethal motor neuron disease that progressively debilitates neuronal cells that control voluntary muscle activity. In a mouse model of ALS that expresses mutated human superoxide dismutase 1 (SOD1-G93A) skeletal muscle is one of the tissues affected early by mutant SOD1 toxicity. Fast-twitch and slow-twitch muscles are differentially affected in ALS patients and in the SOD1-G93A model, fast-twitch muscles being more vulnerable. We used miRNA microarrays to investigate miRNA alterations in fast-twitch (EDL) and slow-twitch (soleus) skeletal muscles of symptomatic SOD1-G93A animals and their age-matched wild type littermates.
Project description:Amyotrophic lateral sclerosis (ALS) is a lethal motor neuron disease that progressively debilitates neuronal cells that control voluntary muscle activity. In a mouse model of ALS that expresses mutated human superoxide dismutase 1 (SOD1-G93A) skeletal muscle is one of the tissues affected early by mutant SOD1 toxicity. Fast-twitch and slow-twitch muscles are differentially affected in ALS patients and in the SOD1-G93A model, fast-twitch muscles being more vulnerable. We used miRNA microarrays to investigate miRNA alterations in fast-twitch (EDL) and slow-twitch (soleus) skeletal muscles of symptomatic SOD1-G93A animals and their age-matched wild type littermates. At age of 90 days RNA was extracted from extensor digitorum longus (EDL) and soleus (SOL) muscles of male SOD1-G93A animals and their age-matched wild type male littermates. RNA was hybridized on Affymetrix Multispecies miRNA-2_0 Array.
Project description:Common acute injuries to skeletal muscle can lead to significant pain and disability. The current therapeutic approaches for treating muscle injuries are dependent on the clinical severity but not on the type of injury. The aim of this study was to compare the molecular events accompanying the degeneration and repair phases of contraction- and trauma-induced muscle injuries by applying DNA microarray methodology to two well-characterized mouse models of skeletal muscle injury, i.e., eccentric contraction-induced injury (CI) and traumatic injury induced by freezing (FI). Histopathological evaluation and measurements of muscle strength were accompanied by analyses of expression for 12,488 known genes at four time points ranging from 6 hours to 7 days post-injury. Real-time RT-PCR was used to confirm some of the gene expression temporal profiles. While both types of injury cause early induction of transcription, myogenic, and stress-responsive factors, they also induce injury type-specific gene expression profiles. CI only activates a set of genes associated with the protection and repair of protein and structural integrity while FI activates gene sets which result in extensive inflammatory responses, tissue remodeling, angiogenesis, and myofibre and extracellular matrix synthesis. This study identified genes that are candidates for therapeutic manipulation following two disparate types of muscle injury. Keywords: time course, comparative genomic hybridization
Project description:Purpose of this study was to compare the effect of c-myc over expression and acute high intensity muscle contraction on mRNA transcriptome in skeletal muscle
Project description:Study to examine the effect of a demanding 15 minute whole hindlimb isometric contraction protocol in vivo on changes in gene expression, 4 hours post-contraction. Keywords = skeletal muscle Keywords = isometric contractions
Project description:Assessment of gene expression after muscle contraction with the hypothesis that the lack of LKB1 will alter gene expression, especially inflammation-related genes.
Project description:The basic helix-loop-helix factor Myod initiates skeletal muscle differentiation by directly and sequentially activating sets of muscle differentiation genes, including those encoding muscle contractile proteins. We hypothesize that Pbx homeodomain proteins direct Myod to a subset of its transcriptional targets, in particular fast twitch muscle differentiation genes, thereby regulating the competence of muscle precursor cells to differentiate. We have previously shown that Pbx proteins bind with Myod on the promoter of the zebrafish fast muscle gene mylpfa and that Pbx proteins are required for Myod to activate mylpfa expression and the fast-twitch muscle-specific differentiation program in zebrafish embryos. Here we have investigated the interactions of Pbx with another muscle fiber-type regulator, Prdm1a, a SET-domain DNA-binding factor that directly represses mylpfa expression and fast muscle differentiation. The prdm1a mutant phenotype, early and increased fast muscle differentiation, is the opposite of the Pbx-null phenotype, delayed and reduced fast muscle differentiation. To determine whether Pbx and Prdm1a have opposing activities on a common set of genes, we used RNA-seq analysis to globally assess gene expression in zebrafish embryos with single- and double-losses-of-function for Pbx and Prdm1a. We find that the levels of expression of certain fast muscle genes are increased or approximately wild type in pbx2/4-MO;prdm1a-/- embryos, suggesting that Pbx activity normally counters the repressive action of Prdm1a for a subset of the fast muscle program. However, other fast muscle genes require Pbx but are not regulated by Prdm1a. Thus, our findings reveal that subsets of the fast muscle program are differentially regulated by Pbx and Prdm1a. Our findings provide an example of how Pbx homeodomain proteins act in a balance with other transcription factors to regulate subsets of a cellular differentiation program. Total RNA samples were genotyped and pooled for 4 sample types: control-MO;prdm1+/+; control-MO;prdm1-/-; pbx2/4-MO;prdm1+/+; and pbx2/4-MO;prdm1-/- embryos at the 10 somite (s) stage from three independent sets of egg collections/injections.