Mammalian skeletal muscle comprises different fiber types, whose identity is first established during embryonic development by intrinsic myogenic control mechanisms and is later modulated by neural and hormonal factors. The relative proportion of the different fiber types varies strikingly between species, and in humans shows significant variability between individuals. Myosin heavy chain isoforms, whose complete inventory and expression pattern are now available, provide a useful marker for fiber types, both for the four major forms present in trunk and limb muscles and the minor forms present in head and neck muscles. However, muscle fiber diversity involves all functional muscle cell compartments, including membrane excitation, excitation-contraction coupling, contractile machinery, cytoskeleton scaffold, and energy supply systems. Variations within each compartment are limited by the need of matching fiber type properties between different compartments. Nerve activity is a major control mechanism of the fiber type profile, and multiple signaling pathways are implicated in activity-dependent changes of muscle fibers. The characterization of these pathways is raising increasing interest in clinical medicine, given the potentially beneficial effects of muscle fiber type switching in the prevention and treatment of metabolic diseases.

Fiber types in mammalian skeletal muscles.

Sarcopenia is a loss of muscle mass and function in the elderly that reduces mobility, diminishes quality of life, and can lead to fall-related injuries, which require costly hospitalization and ex...

/pdf/sarcopenia-aging-related-loss-of-muscle-mass-and-function-2u9nyjfrxt.pdf

Sarcopenia: Aging-Related Loss of Muscle Mass and Function.

Plasmid pRSVL persisted and expressed luciferase for at least 19 months in mouse skeletal muscle after intramuscular injection. Other injected plasmids also stably expressed long-term suggesting that any plasmid DNA could stably persist and express in muscle. Plasmid DNA was demonstrated by quantitative PCR in some of the muscle DNA samples for at least 19 months after injection. The methylation pattern of the plasmid DNA remained in its bacterial form indicating that the foreign DNA did not replicate in the muscle cells. The electroporation of total cellular DNA from injected muscles into bacteria indicated that the plasmid DNA was extrachromosomal. Chromosomal integration of plasmid DNA was searched for by electroporating the injected muscle DNA into bacteria after restriction enzyme digestion and ligation. No plasmids containing plasmid/chromosome junctions were observed in over 1800 colonies examined. Lack of integration increases the theoretical safety of this gene transfer technique. Long-term stability of plasmid DNA in muscle indicates that muscle is an attractive target tissue for the introduction of extrachromosomal plasmid or viral DNA for the purpose of gene therapy.

Very long term persistence of plasmid dna and foreign gene expression in mouse muscle

Function of the myogenic regulatory factors Myf5, MyoD, Myogenin and MRF4 in skeletal muscle, satellite cells and regenerative myogenesis.

The fusion of myoblasts into multinucleate syncytia plays a fundamental role in muscle function, as it supports the formation of extended sarcomeric arrays, or myofibrils, within a large volume of cytoplasm. Principles learned from the study of myoblast fusion not only enhance our understanding of myogenesis, but also contribute to our perspectives on membrane fusion and cell-cell fusion in a wide array of model organisms and experimental systems. Recent studies have advanced our views of the cell biological processes and crucial proteins that drive myoblast fusion. Here, we provide an overview of myoblast fusion in three model systems that have contributed much to our understanding of these events: the Drosophila embryo; developing and regenerating mouse muscle; and cultured rodent muscle cells.

Myoblast fusion: lessons from flies and mice

We present here a new technique with which to visualize nuclei in living muscle fibres in the intact animal, involving injection of labelled DNA into single cells. This approach allowed us to determine the position of all of nuclei within a sarcolemma without labelling satellite cells. In contrast to what has been reported in tissue culture, we found that the nuclei were immobile, even when observed over several days. Nucleic density was uniform along the fibre except for the endplate and some myotendinous junctions, where the density was higher. The perijunctional region had the same number of nuclei as the rest of the fibre. In the extensor digitorum longus (EDL) muscle, the extrajunctional nuclei were elongated and precisely aligned to the long axis of the fibre. In the soleus, the nuclei were rounder and not well aligned. When comparing small and large fibres in the soleus, the number of nuclei varied approximately in proportion to cytoplasmic volume, while in the EDL the number was proportional to surface area. Statistical analysis revealed that the nuclei were not randomly distributed in either the EDL or the soleus. For each fibre, actual distributions were compared with computer simulations in which nuclei were assumed to repel each other, which optimizes the distribution of nuclei with respect to minimizing transport distances. The simulated patterns were regular, with clear row-like structures when the density of nuclei was low. The non-random and often row-like distribution of nuclei observed in muscle fibres may thus reflect regulatory mechanisms whereby nuclei repel each other in order to minimize transport distances.

Number and spatial distribution of nuclei in the muscle fibres of normal mice studied in vivo.

Elucidating the molecular pathways linking electrical activity to gene expression is necessary for understanding the effects of exercise on muscle. Fast muscles express higher levels of MyoD and lower levels of myogenin than slow muscles, and we have previously linked myogenin to expression of oxidative enzymes. We here report that in slow muscles, compared with fast, 6 times as much of the MyoD is in an inactive form phosphorylated at T115. In fast muscles, 10 h of slow electrical stimulation had no effect on the total MyoD protein level, but the fraction of phosphorylated MyoD was increased 4-fold. Longer stimulation also decreased the total level of MyoD mRNA and protein, while the level of myogenin protein was increased. Fast patterned stimulation did not have any of these effects. Overexpression of wild type MyoD had variable effects in active slow muscles, but increased expression of fast myosin heavy chain in denervated muscles. In normally active soleus muscles, MyoD mutated at T115 (but not at S200) increased the number of fibres containing fast myosin from 50% to 85% in mice and from 13% to 62% in rats. These data establish de-phosphorylated active MyoD as a link between the pattern of electrical activity and fast fibre type in adult muscles.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.2007.141457

De‐phosphorylation of MyoD is linking nerve‐evoked activity to fast myosin heavy chain expression in rodent adult skeletal muscle

Muscle is a permanent tissue, and in the adult pronounced changes can occur in pre-existing fibres without the formation of new fibres. Thus, the mechanisms responsible for phenotype transformation in the adult might be distinct from mechanisms regulating muscle differentiation during muscle formation and growth. Myogenin is a muscle-specific, basic helix-loop-helix transcription factor that is important during early muscle differentiation. It is also expressed in the adult, where its role is unknown. In this study we have overexpressed myogenin in glycolytic fibres of normal adult mice by electroporation and single-cell intracellular injection of expression vectors. Myogenin had no effects on myosin heavy chain fibre type, but induced a considerable increase in succinate dehydrogenase and NADH dehydrogenase activity, with some type IIb fibres reaching the levels observed histochemically in normal type IIx and IIa fibres. mRNA levels for malate dehydrogenase were similarly altered. The size of the fibres overexpressing myogenin was reduced by 30–50 %. Thus, the transfected fibres acquired a phenotype reminiscent of the phenotype obtained by endurance training in man and other animals, with a higher oxidative capacity and smaller size. We conclude that myogenin can alter pre-existing glycolytic fibres in the intact adult animal.

Myogenin induces higher oxidative capacity in pre-existing mouse muscle fibres after somatic DNA transfer

The effects of exercise on skeletal muscle are mediated by a coupling between muscle electrical activity and gene expression. Several activity correlates, such as intracellular Ca2+, hypoxia and metabolites like free fatty acids (FFAs), might initiate signalling pathways regulating fibre-type-specific genes. FFAs can be sensed by lipid-dependent transcription factors of the peroxisome proliferator-activated receptor (PPAR) family. We found that the mRNA for the predominant muscle isoform, PPARδ, was three-fold higher in the slow/oxidative soleus compared to the fast/glycolytic extensor digitorum longus (EDL) muscle. In histological sections of the soleus, the most oxidative fibres display the highest levels of PPARδ protein. When the soleus muscle was stimulated electrically by a pattern mimicking fast/glycolytic IIb motor units, the mRNA level of PPARδ was reduced to less than half within 24 h. In the EDL, a three-fold increase was observed after slow type I-like electrical stimulation. When a constitutively active form of PPARδ was overexpressed for 14 days in normally active adult fibres after somatic gene transfer, the number of I/IIa hybrids in the EDL more than tripled, IIa fibres increased from 14% to 25%, and IIb fibres decreased from 55% to 45%. The level of succinate dehydrogenase activity increased and size decreased, also when compared to normal fibres of the same type. Thus PPARδ can change myosin heavy chain, oxidative enzymes and size locally in muscle cells in the absence of general exercise. Previous studies on PPARδ in muscle have been performed in transgenic animals where the transgene has been present during muscle development. Our data suggest that PPARδ can mediate activity effects acutely in pre-existing adult fibres, and thus is an important link in excitation–transcription coupling.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.2007.133025

PPARδ expression is influenced by muscle activity and induces slow muscle properties in adult rat muscles after somatic gene transfer

Aim: Muscle is perhaps the most frequently considered tissue for non-viral gene therapy, in particular after gene transfer by electroporation. Expression in muscle is stable, but since the cell turnover is so slow incorporation in the host genome is not required. This raises interesting practical and theoretical questions related to the behaviour of the transgenic DNA under such conditions.



Methods:  We have investigated expression of reporter genes from plasmid mixtures electroporated into the extensor digitorum longus (EDL) muscle in mice in order to assess the degree of coexpression.



Results:  Under conditions where the reporter is easily identified the coexpression rate was 100%, as none of 287 fibres from five different muscles expressing blue fluorescent protein (BFP) failed to express green fluorescent protein (GFP). With other reporter combinations the rate was lower, but this we attribute to marginal sensitivity for fluorescent proteins, or from reporter protein degradation for β-galactosidase.



Conclusions:  The high degree of coexpression suggests that a large copy number takes part in the final transcription with this system. The finding also enhances the usefulness of muscle and electroporation for gene therapy and experimental biology.

Coexpression after electroporation of plasmid mixtures into muscle in vivo

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