MyoD, a master regulator of myogenesis, exhibits a circadian rhythm in its mRNA and protein levels, suggesting a possible role in the daily maintenance of muscle phenotype and function. We report that MyoD is a direct target of the circadian transcriptional activators CLOCK and BMAL1, which bind in a rhythmic manner to the core enhancer of the MyoD promoter. Skeletal muscle of Clock Δ19 and Bmal1 −/− mutant mice exhibited ∼30% reductions in normalized maximal force. A similar reduction in force was observed at the single-fiber level. Electron microscopy (EM) showed that the myofilament architecture was disrupted in skeletal muscle of Clock Δ19 , Bmal1 −/− , and MyoD −/− mice. The alteration in myofilament organization was associated with decreased expression of actin, myosins, titin, and several MyoD target genes. EM analysis also demonstrated that muscle from both Clock Δ19 and Bmal1 −/− mice had a 40% reduction in mitochondrial volume. The remaining mitochondria in these mutant mice displayed aberrant morphology and increased uncoupling of respiration. This mitochondrial pathology was not seen in muscle of MyoD −/− mice. We suggest that altered expression of both Pgc-1α and Pgc-1β in Clock Δ19 and Bmal1 −/− mice may underlie this pathology. Taken together, our results demonstrate that disruption of CLOCK or BMAL1 leads to structural and functional alterations at the cellular level in skeletal muscle. The identification of MyoD as a clock-controlled gene provides a mechanism by which the circadian clock may generate a muscle-specific circadian transcriptome in an adaptive role for the daily maintenance of adult skeletal muscle.circadian clock | myofilaments | mitochondria A fundamental, evolutionarily conserved property of most organisms, from cyanobacteria to plants and animals, is the daily cycling of their internal physiology as well as certain behaviors in animals, such as sleep and feeding (1). The timing of these circadian rhythms is synchronized to the environment by external cues, with light and nutrient availability being two of the most salient entrainment cues (2). The synchronization of endogenous circadian rhythms with the daily cycles in the environment provides an adaptive mechanism for organisms to anticipate cyclic demands on cellular physiology and behavior (3, 4). At the molecular level, the circadian clock represents a well-defined gene regulatory network composed of transcriptional-translational feedback loops (5). The positive arm of the loop is composed of the transcription factors CLOCK and BMAL1, which heterodimerize and bind to E-box elements on target genes such as Per1 to drive the negative part of the feedback loop (5). More recently, the same molecular clock factors that have been identified in the central clock in the suprachiasmatic nucleus have been found to exist in most peripheral tissues (reviewed in ref. 6).We recently characterized the circadian transcriptome of adult skeletal muscle. These mRNAs exhibit significant oscillation in gene expression with a repeating period len...

Skeletal muscle is a major source of circulating insulin growth factor‐1 (IGF‐1), particularly during exercise. It expresses two main isoforms. One of the muscle IGF‐1 isoforms (muscle L.IGF‐1) is similar to the main liver IGF‐1 and presumably has an endocrine action. The other muscle isoform as a result of alternative splicing has a different 3′ exon sequence and is apparently designed for an autocrine/paracrine action (mechano‐growth factor, MGF). Using RNase protection assays with a probe that distinguishes these differently spliced forms of IGF‐1, their expression and also the expression of two structural genes was measured in rabbit extensor digitorum longus muscles subjected to different mechanical signals. Within 4 days, stretch using plaster cast immobilization with the limb in the plantar flexed position resulted in marked upregulation of both forms of IGF‐1 mRNA. Electrical stimulation at 10 Hz combined with stretch (overload) resulted in an even greater increase of both types of IGF‐1 transcript, whereas electrical stimulation alone, i.e. without stretch, resulted in no significant increase over muscle from sham‐operated controls. Previously, it was shown that stretch combined with electrical stimulation of the dorsiflexor muscles in the adult rabbit results in a marked increase in muscle mass involving increases in both length and girth, within a few days. The expression of both systemic and autocrine IGF‐1 growth factors provides a link between the mechanical signal and the marked increase in the structural gene expression involved in tissue remodelling and repair. The expression of the β actin gene was seen to be markedly upregulated in the stretched and stretched/stimulated muscles. It was concluded that the increased expression of this cytoskeletal protein gene is an indication that the production of IGF‐1 may initially be a response to local damage. Switches in muscle fibre phenotype were studied using a specific gene probe for the 2X myosin heavy chain gene. Type 2X expression was found to decrease markedly with stimulation alone and when electrical stimulation was combined with stretch. Unlike the induction of IGF‐1 and β actin, the decreased expression of the 2X myosin mRNA was less marked in the ‘stretch only’ muscles. This indicates that the interconversion of fibre type 2X to 2A may in some situations be commensurate with, but not under the control of IGF‐1.

Stem cells have the ability to differentiate into specific cell types. The two defining characteristics of a stem cell are perpetual self-renewal and the ability to differentiate into a specialized adult cell type. There are two major classes of stem cells: pluripotent that can become any cell in the adult body, and multipotent that are restricted to becoming a more limited population of cells. Cell sources, characteristics, differentiation and therapeutic applications are discussed. Stem cells have great potential in tissue regeneration and repair but much still needs to be learned about their biology, manipulation and safety before their full therapeutic potential can be achieved.

Cysteine-rich protein 3 or muscle LIM protein (MLP) is thought to be a mechanosensor in cardiac myocytes. Therefore, the subcellular location of MLP may have functional implications in health and disease. Our hypothesis is that MLP becomes mislocalized after prolonged overload, resulting in impaired mechanosensing in cardiac myocytes. Using the techniques of biochemical subcellular fractionation and immunocytochemistry, we found MLP exhibits oligomerization in the membrane and cytoskeleton of cultured cardiac rat neonatal myocytes. Nuclear MLP was always monomeric. MLP translocated to the nucleolus in response to 10% cyclic stretch at 1 Hz for 48 h. This was associated with a threefold increase in S6 ribosomal protein (P Ͻ 0.01; n ϭ 3 cultures). Adenoviral overexpression of MLP also resulted in a twofold increase in S6 protein, suggesting that MLP can activate ribosomal protein synthesis in the nucleolus. In ventricles from aortic-banded and myocardially infarcted rat hearts, nuclear MLP increased by twofold (P Ͻ 0.01; n ϭ 7) along with a significant decrease in the nonnuclear oligomeric fraction. The ratio of nuclear to nonnuclear MLP increased threefold in both groups (P Ͻ 0.01; n ϭ 7). In failing human hearts, there was almost a complete loss of oligomeric MLP. Using a flag-tagged adenoviral MLP, we demonstrate that the COOH terminus is required for oligomerization and that this is a precursor to stretch sensing and subsequent nuclear translocation. Therefore, reduced oligomeric MLP in the costamere and cytoskeleton may contribute to impaired mechanosensing in heart failure.hypertrophy; mechanosensing; cytoskeleton; nucleocytoplasmic shuttling PROLONGED HEMODYNAMIC OVERLOAD results in cardiac hypertrophy with detrimental changes in myocardial gene expression and morphology (7,8,19,20,24,32) that predict morbidity and mortality (9, 28). Stretch sensors, necessary for myocyte enlargement, are thought to become impaired as a result of prolonged overload (11). However, the mechanisms by which chronic overload leads to decompensation and failure are poorly understood.Many proteins shuttle from subcellular compartments to the nucleus and may act as molecular sensors (4). In myocytes, some of these nucleocytoplasmic proteins are located in costameric focal adhesions and the cytoskeleton where mechanical forces are transmitted. One such protein, muscle LIM protein (MLP) or cysteine-rich protein 3, contains two zinc finger LIM domains. MLP is known to interact with many proteins of the cytoskeleton, including ␣-actinin and the titin-binding protein telethonin (34). MLP is thought to be part of a mechanosensing mechanism in myocytes, and this is likely to occur through two main mechanisms. First, the LIM domains enable the protein to interact with a variety of other cellular proteins, many of which are associated with known signaling pathways. The potential role of MLP as a sensor of mechanical activity through its interactions with other proteins has been thoroughly reviewed recently (18). Second, the protein contains...