David J. Parry

CULTURING SATELLITE CELLS FROM LIVING SINGLE MUSCLE FIBER EXPLANTS

SummaryConventional methods for isolating myogenic (satellite) cells are inadequate when only small quantities of muscle, the tissue in which satellite cells reside, are available. We have developed a tissue culture system that reliably permits isolation of intact, living, single muscle fibers with associated satellite cells from predominantly fast and slow muscles of rat and mouse; maintenance of the isolated fibers in vitro; dissociation, proliferation, and differentiation of satellite cells from each fiber; and removal of the fiber from culture for analysis.

Trophic effect of ciliary neurotrophic factor on denervated skeletal muscle

The actions and receptor for ciliary neurotrophic factor (CNTF) are largely restricted to cells of the nervous system, although one of the CNTF receptor components, CNTFR alpha, is expressed by skeletal muscle. Here we show that the other CNTF receptor components, LIFR beta and gp130, are also expressed by skeletal muscle and that expression of all three CNTF receptor components is greatly increased in denervated muscle. In vivo, administration of CNTF activates these receptors on skeletal muscle by inducing receptor phosphorylation and immediate-early gene responses. Furthermore, CNTF reduces the denervation-induced atrophy of muscle and attenuates the reduced twitch and tetanic tensions that result from muscle denervation. Our findings reveal that, in addition to its known neurotrophic actions, CNTF exerts myotrophic effects by attenuating the morphological and functional changes associated with denervation of rat skeletal muscle.

Adaptation of rat extensor digitorum longus muscle to gamma irradiation and overload

The right extensor digitorum longus (EDL) muscle of growing male rats was overloaded by ablation of its synergist tibialis anterior (TA) muscle. Four weeks later, the overloaded muscle was heavier and contained larger type IIA, IIX and IIB fibres than either untreated contralateral muscle or control muscle from an untreated animal. The myonuclear-to-myoplasmic volume ratio was maintained in the overloaded muscle. Overloaded EDL muscle, previously subjected to a dose of irradiation sufficient to sterilise satellite cells, and EDL muscle which had been only irradiated, were significantly lighter and contained significantly smaller fibres than controls, though a significant amount of normal EDL muscle growth did occur following either treatment. The myonuclear-to-myoplasmic volume ratio of the irradiated muscles was smaller than in controls. Overloaded muscle, with or without prior irradiation, possessed a smaller proportion of fibres containing IIB myosin heavy chain (MHC) and a larger proportion of fibres containing IIA and IIX MHC; a significant percentage of these fibres coexpressed either type IIA and IIX MHC or type IIX and IIB MHC. Thus in the absence of satellite cell mitosis, muscles of young rats possess a limited capacity for normal growth but not for compensatory hypertrophy. Adaptations in MHC gene expression to chronic overload are completely independent of satellite cell activity.

Regulation of myosin heavy chain expression in adult rat hindlimb muscles during short-term paralysis: comparison of denervation and tetrodotoxin-induced neural inactivation

The extent to which myosin profiles within adult fast and slow muscles are altered by short‐term paralysis remains equivocal. We used an array of specific antibodies to identify adult and developmental MHC isoforms within EDL and soleus muscle fibers, and show a marked multiple expression of MHCs with a general shift towards slower and more energy efficient MHC profiles after 2 weeks of denervation or TTX nerve conduction block. Paralysis also induced marked expression of an embryonic MHC within most EDL cell types, and a subtle, paralysis‐sensitive, expression of α‐cardiac MHC within specific EDL and soleus extrafusal fibers. Comparison of treatment groups also permitted assessment of the relative influence of neural activity versus trophic factors on these isoforms, and confirmed activity as a major, but not sole, regulator of MHC expression.

Expression of utrophin and its mRNA in denervated mdx mouse muscle

Utrophin is a large cytoskeletal protein which shows high homology to dystrophin. In contrast to the sarcolemmal distribution of dystrophin. utrophin accumulates at the postsynaptic membrane of the neuromuscular junction. Because of its localization within this compartment of muscle fibers, expression of utrophin may be significantly influenced by the presence of the motor nerve. We tested this hypothesis by denervating muscles of mdx mouse and monitoring levels of utrophin and its mRNA by immunofluorescence, immunoblotting and RT‐PCR. A significant increase in the number of utrophin positive fibers was observed by immunofluorescence 3 to 21 days after sectioning of the sciatic nerve. Quantitative analyses of utrophin and its transcripts in hindlimb muscles denervated for two weeks showed only a moderate increase in the levels of both utrophin (∼ 2‐fold) and its transcript (∼60 to 90%). The present data suggest that although utrophin is a component of the postsynaptic membrane, its neural regulation is distinct from that of the acetylcholine receptor.

Ciliary neurotrophic factor: regulation of acetylcholinesterase in skeletal muscle and distribution of messenger RNA encoding its receptor in synaptic versus extrasynaptic compartments

Several recent studies have shown that the ciliary neurotrophic factor exerts myotrophic effects in addition to its well-characterized neurotrophic actions on various neuronal populations. Since expression of acetylcholinesterase in skeletal muscle has been shown to be regulated by putative yet unknown nerve-derived trophic factors, we tested the hypothesis that the ciliary neurotrophic factor is a neurotrophic agent capable of influencing expression of acetylcholinesterase in adult rat skeletal muscle in vivo. To this end, we first determined the impact of daily ciliary neurotrophic factor administration on expression of acetylcholinesterase in both intact and denervated rat soleus muscles. The results of our experiments indicate that although chronic administration of ciliary neurotrophic factor partially counteracted the atrophic response of soleus muscles to surgical denervation, thus confirming its myotrophic effects, it failed to either increase acetylcholinesterase expression in intact muscles or prevent the decrease normally occurring in seven-day denervated muscles. In fact, acetylcholinesterase messenger RNA and enzyme levels were further reduced by ciliary neurotrophic factor treatment in denervated muscles without significant modifications in the pattern of acetylcholinesterase molecular forms. Conversely, transcript levels of the epsilon subunit of the acetylcholine receptor in intact and denervated soleus muscles treated with the ciliary neurotrophic factor were similar to those observed in their respective counterparts from vehicle-treated animals. In addition, we also determined whether transcripts encoding the receptor for the ciliary neurotrophic factor selectively accumulate in junctional domains of rat skeletal muscle fibres. In contrast to the preferential localization of transcripts encoding acetylcholinesterase and the epsilon subunit of the acetylcholine receptor within the postsynaptic sarcoplasm, messenger RNAs for the ciliary neurotrophic factor receptor appeared homogeneously distributed between junctional and extra-junctional compartments of both diaphragm and extensor digitorum longus muscle fibres, with no compelling evidence for a selective accumulation within the postsynaptic sarcoplasm. These data show that the ciliary neurotrophic factor exerts an inhibitory influence on expression of acetylcholinesterase in muscle fibres. Furthermore, the lack of an effect on expression of the epsilon acetylcholine receptor transcripts indicates that treatment with ciliary neurotrophic factor does not lead to general adaptations in the expression of all synaptic proteins. Given the distribution of transcripts encoding the ciliary neurotrophic factor receptor along multinucleated muscle fibres, we propose a model whereby the ciliary neurotrophic factor, or a related unknown molecule that also utilizes the receptor for the ciliary neurotrophic factor, contributes to the maintenance of low levels of enzyme activity in extrajunctional regions of muscle fibres by acting as a repressor of acetylcholinesterase expression that functions directly or indirectly via a pretranslational regulatory mechanism. Accordingly, these results further highlight the complexity of the regulatory mechanisms presiding over acetylcholinesterase expression in vivo.

Neurotrophins Differentially Regulate Expression of Cholinergic Enzymes in Cultured Spinal Cord Motoneurons

The neurotransmitter enzymes acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) are known to be co-expressed in mature cholinergic neurons. However, their pattern of expression has previously been shown to be independently regulated under several experimental conditions. In non-cholinergic neurons for example, AChE can be expressed in the absence of detectable levels of ChAT activity (1,2). In addition, recent studies have shown that muscle-derived trophic factors are capable of modulating expression of ChAT (3,4) but the effects of these molecules on AChE have yet to be determined. Since motoneurons are known to be subjected to the influences of muscle-derived trophic factors and since they express high levels of both AChE and ChAT, we have therefore initiated a series of experiments to examine the developmental and trophic regulation of both AChE and ChAT using the motoneuronal cell line NSC-34. This cell line is derived from mouse-mouse neural hybrid cell lines obtained from the fusion of neuroblastoma with motor neuron-containing embryonic day 12–14 spinal cord cells (5). NSC-34 neurons reproduce several aspects of neuronal development and differentiation in addition to expressing many of the morphological and physiological characteristics of primary motoneurons. Histochemical and biochemical analyses of NSC-34 spinal cord motoneurons showed that they express high levels of AChE enzyme activity. Differentiation of these cells, as evidenced for example by the growth of neuntes, resulted in a significant increase in both cell-associated and secreted AChE. This increase resulted primarily from an increase in the content of the molecular form G4. Quantitative RT-PCR assays revealed that AChE mRNA levels increased by approximately 4-fold following differentiation.

Ridotta capacità rigenerativa del muscolo distrofico di topo DY2J/DY2J in rapporto all’età

La progressiva degenerazione del tessuto muscolare nelle distrofie musculari riflette l’incapacita della rigenerazione muscolare di compensare la continua distruzione delle fibre. Tuttavia manca una dimostrazione diretta di un declino con l’eta della capacita rigenerativa muscolare nel muscolo distrofico. In questo studio abbiamo confrontato la risposta rigenerativa di muscoli di topo normale e distrofico (ceppo dy2J/dy2J) in rapporto all’eta dell’animale. Le fibre rigeneranti, identificate mediante anticorpi specifici per la catena pesante della miosina embrionale, sono numerose nei muscoli di topi distrofici giovani mentre sono rare nei muscoli di topi distrofici vecchi. Lesioni da congelamento inducono la comparsa di un gran numero di fibre rigeneranti nei topi normali indipendentemente dall’eta, mentre la risposta rigenerativa dei topi distrofici diminuisce drasticamente con l’eta. Questo effetto dipende verosimilmente da una progressiva diminuzione della capacita replicativa dei mioblasti satelliti.The progressive wasting of muscle tissue in muscular dystrophies is apparently due to an inability of muscle regeneration to keep pace with the continued degeneration of fibers. However, these is no direct evidence for an age-related decline in the regenerative capacity of dystrophic muscle in response to noxious stimuli. We have compared the regenerative response of skeletal muscles from young (2 month old) and old (12 month old) normal and dystrophic (dy2J/dy2J) mice. Regenerating fibers, identified using antibodies specific for embryonic myosin heavy chain, were numerous in muscles from young dystrophic mice but rare in muscles from old dystrophic animals. Five days after a freezing injury a large number of regenerating fibers were identified at the site of the lesion in both young and old normal mice. In contrast, the regenerative response of dystrophic muscles varied strikingly with age and an abortive muscle regeneration was seen in old dystrophic animals. These results can be accounted for by a progressive age-related decline in the replicative capacity of dystrophic satellite myoblasts.RiassuntoLa progressiva degenerazione del tessuto muscolare nelle distrofie musculari riflette l’incapacità della rigenerazione muscolare di compensare la continua distruzione delle fibre. Tuttavia manca una dimostrazione diretta di un declino con l’età della capacità rigenerativa muscolare nel muscolo distrofico. In questo studio abbiamo confrontato la risposta rigenerativa di muscoli di topo normale e distrofico (ceppo dy2J/dy2J) in rapporto all’età dell’animale. Le fibre rigeneranti, identificate mediante anticorpi specifici per la catena pesante della miosina embrionale, sono numerose nei muscoli di topi distrofici giovani mentre sono rare nei muscoli di topi distrofici vecchi. Lesioni da congelamento inducono la comparsa di un gran numero di fibre rigeneranti nei topi normali indipendentemente dall’età, mentre la risposta rigenerativa dei topi distrofici diminuisce drasticamente con l’età. Questo effetto dipende verosimilmente da una progressiva diminuzione della capacità replicativa dei mioblasti satelliti.