Increase of Cardiotrophin‐1 immunoreactivity in regenerating and overloaded but not denervated muscles of rats

The original report by Pennica et al. on Cardiotrophin‐1 (CT‐1) states that it markedly stimulates hypertrophy in cardiac myocytes both in vitro and in vivo and is predominantly expressed in the early mouse embryonic heart tube. CT‐1 is a member of the interleukin‐6 superfamily and past studies have shown that it exerts trophic effects on neurons, glial cells and their precursors, and is expressed during myogenesis. Thus CT‐1 is associated with physical and pathological changes in skeletal muscle. In this study, we examined whether CT‐1 is expressed in mechanically overloaded, regenerating, and denervated muscles of rats using immunohistochemistry. In the overloaded plantaris muscles at 1 and 3 days postsurgery, CT‐1 immunoreactivity was detected in the mononuclear cells that had infiltrated the extracellular space. CT‐1 immunoreactivity was also observed in the mononuclear cells invading the extracellular space at 2, 4, and 6 days after a bupivacaine injection and in degenerative and necrotic muscle fibers at 2 days postinjection. In the denervated muscles, the CT‐1 immunoreactivity did not change in intensity during the entire period of the denervation (2, 7, and 14 days postsurgery). The cells invading extracellular space and in necrotic muscle fibers possessing CT‐1 immunoreactivity might be muscle precursor cells (satellite cells) or migrating macrophages undergoing phagocytosis. Using double‐immunostainings for anti‐CT‐1/antic‐met, anti‐CT‐1/ anti‐M‐cadherin, and anti‐CT‐1/anti‐ED1, we found that satellite cells and macrophages exhibited CT‐1 immunoreactivity in the damaged muscles after bupivacaine injection. We therefore believe that CT‐1 plays a key role in regeneration and hypertrophy in the skeletal muscle of rats.     

Nerve‐dependent changes in skeletal muscle myosin heavy chain after experimental denervation and cross‐reinnervation and in a demyelinating mouse model of Charcot–Marie–Tooth disease type 1A

Innervation regulates the contractile properties of vertebrate muscle fibers, in part through the effect of electrical activity on expression of distinct myosins. Herein we analyze the role of innervation in regulating the accumulation of the general, maturational, and adult forms of rodent slow myosin heavy chain (MyHC) that are defined by the presence of distinct antigenic epitopes. Denervation increases the number of fibers that express general slow MyHC, but it decreases the adult slow MyHC epitope. Cross‐reinnervation of slow muscle by a fast nerve leads to an increase in the number of fibers that express fast MyHC. In both cases, there is an increase in the number of fibers that express slow and fast IIA MyHCs, but without the adult slow MyHC epitope. The data suggest that innervation is required for maturation and maintenance of diversity of both slow and fast fibers. The sequence of slow MyHC epitope transitions is a useful biomarker, and it may play a significant role during nerve‐dependent changes in muscle fiber function. We applied this detailed muscle analysis to a transgenic mouse model of human motor and sensory neuropathy IA, also known as Charcot–Marie–Tooth disease type 1A (CMT1A), in which electrical conduction in some motor nerves is poor due to demyelination. The mice display atrophy of some muscle fibers and changes in slow and fast MyHC epitope expression, suggestive of a progressive increase in innervation of muscle fibers by fast motor neurons, even at early stages. The potential role of these early changes in disease pathogenesis is assessed. Muscle Nerve 38: 1572–1584, 2008     

Myosin light and heavy chains in muscle regenerating in absence of the nerve: Transient appearance of the embryonic light chain

We examined myosin of fast and slow skeletal rat muscles regenerating after ischemia and bupivacaine injection in denervated limbs. Four days after injury two-dimensional gel electrophoresis revealed the presence of the embryonic light chain in the myosin isolated from the portion of muscle showing a homogeneous population of new small fibers by histological examination. Two weeks after injury this subunit was absent, whereas the two light chains, LC1F and LC2F, became prominent. One month after injury the still denervated soleus muscle maintained this light chain pattern. Gel electrophoresis in native condition of the myosin and peptide mapping of electrophoretically purified heavy chains confirmed that the muscle regenerating in absence of the nerve accumulated a myosin that had the general features of a fast, not slow, myosin but contained definite differences from the former.     

Expression of MyoD and myogenin in dystrophic mice, mdx and dy, during regeneration

Expression of two myogenic regulatory factors, MyoD and myogenin, was studied in regenerating muscles of dystrophic mice and compared to a chemically induced regeneration process. First, the distribution of the two proteins was determined immunohistochemically at various time points after single administrations of a local anaesthetic, bupivacaine hydrochloride, which causes myonecrosis followed by regeneration. Detectable levels of MyoD appeared at 18 h and the expression reached their maximum levels at 48 h after the injection, which coincide with the stage when satellite cells are activated and start to proliferate. Myogenin became detectable in 24 h and its expression reached its highest level at 72 h after injection when newly formed myotubes appeared. The two genes were also expressed in the dystrophic muscles from dy and mdx mice which exhibit dystrophic pathological features but are associated with different phenotypes. In mdx mice the two genes were expressed at reasonably high levels in parallel with the active regenerating process, whereas in dy mice MyoD and myogenin expressions decreased as fibrosis progressed. However, MyoD was relatively more strongly expressed in the larger mature myotubes of dy mice than in those of mdx mice, suggesting prolonged regenerative activity. In dy and mdx mice, MyoD and myogenin were expressed in different quantities, indicating that these animals have distinct regenerating activities. Our findings confirm that expression of both MyoD and myogenin genes is necessary in the regenerative process for the proliferation of satellite cells (myoblasts) and for the development of early regenerating fibers (myotubes) even in dystrophic muscles. Received: 9 March 1999 / Revised: 30 August 1999 / Accepted: 6 September 1999     

Notexin causes greater myotoxic damage and slower functional repair in mouse skeletal muscles than bupivacaine

Although the myotoxins bupivacaine and notexin are employed for studying processes that regulate muscle regeneration after injury, no studies have compared their efficacy in causing muscle damage or assessing functional regeneration in mouse skeletal muscles. Bupivacaine causes extensive injury in rat muscles but its effects on mouse muscles are variable. We compared functional and morphological properties of regenerating mouse extensor digitorum longus (EDL) muscles after notexin or bupivacaine injection and tested the hypothesis that muscle damage would be more extensive and functional repair less complete after notexin injection. Bupivacaine caused degeneration of 45% of fibers and reduced maximum force (Po) to 42% of control after 3 days. In contrast, notexin caused complete fiber breakdown and loss of functional capacity after 3 days (P < 0.05). At 7 and 10 days after bupivacaine, Po was restored to 65% and 71% of control, respectively, whereas Po of notexin-injected muscles was only 10% and 39% of control at these time-points, respectively (P < 0.05). At 7 and 10 days after bupivacaine, approximately 30% of fibers were centrally nucleated (regenerating), whereas notexin-injected muscles were comprised entirely of regenerating fibers (P < 0.05). The results demonstrate that notexin causes a more extensive and complete injury than bupivacaine, and is a useful model for studying muscle regeneration in mice.     

Induced shift in myosin heavy chain expression in myosin myopathy by endurance training

Abstract. We recently described a new autosomal dominant myopathy (OMIM #605637) associated with a missense mutation in the myosin heavy chain (MyHC) IIa gene (MYH2), which encodes for the fast myosin isoform that is expressed in type 2A muscle fibers. There was a correlation between muscle pathology and expression of MyHC IIa. Low expression of the mutation was associated with a milder phenotype. Since physical activity influences MyHC isoform expression in normal individuals, we investigated whether endurance training can alter the expression of MyHC isoforms in patients with the MYH2 mutation.The expression of MyHC I, IIa and IIx was analysed in muscle specimens from six patients before and after an eight-week endurancetraining program by SDS-polyacrylamide gel electrophoresis and immuno-histochemistry.There was a clear and consistent shift from fast to slow MyHC isoform expression, but the training program did not result in the desired reduction of MyHC IIa, which may be due to the limited time period of training. Fiber type transition was further illustrated by the appearance of hybrid muscle fibers expressing more than one MyHC isoform after the training period. All patients showed an increase in maximal workload but no significant change in isometric muscle strength.We conclude that endurance training in patients with myosin myopathy may be an important way to alter the expression of defective MyHC isoforms.     

Na+ currents near and away from endplates on human fast and slow twitch muscle fibers

Fast and slow twitch muscle fibers have distinct contractile properties. Here we determined that membrane excitability also varies with fiber type. Na⁺ currents (I_NA) were studied with the loose-patch voltage clamp technique on 29 histochemically classified human intercostal skeletal muscle fibers at the endplate border and <200 μm from the endplate (extrajunctional). Fast and slow twitch fibers showed slow inactivation of endplate border and extrajunctional I_NA and had increased I_NA at the endplate border compared to extrajunctional membrane. The voltage dependencies of I_NA were similar on the endplate border and extrajunctional membrane, which suggests thatboth regions have physiclogically similar channels. Fast twitch fibers had larger I_NA on the endplate border and extrajunctional membrane and manifest fast and slow inactivation of I_NA at more negative potentials than slow twitch fibers. For normal muscle, the differences between I_NA on fast and slow twitch fibers might: (1) enable fast twitch fibers to operate at high firing frequencies for brief periods; and (2) enable slow twitch fibers to operate at low firing frequencies for prolonged times. Disorders of skeletal membrane excitability, such as the periodic paralyses and myotonias, may impact fast and slow twitch fibers differently due to the distinctive Na⁺ channel properties of each fiber type. © 1993 John Wiley & Sons, Inc.     

Alterations of intrinsic tongue muscle properties with aging

Introduction: Age‐related decline in the intrinsic lingual musculature could contribute to swallowing disorders, yet the effects of age on these muscles is unknown. We hypothesized there is reduced muscle fiber size and shifts to slower myosin heavy chain (MyHC) fiber types with age. Methods: Intrinsic lingual muscles were sampled from 8 young adult (9 months) and 8 old (32 months) Fischer 344/Brown Norway rats. Fiber size and MyHC were determined by fluorescent immunohistochemistry. Results: Age was associated with a reduced number of rapidly contracting muscle fibers, and more slowly contracting fibers. Decreased fiber size was found only in the transverse and verticalis muscles. Discussion: Shifts in muscle composition from faster to slower MyHC fiber types may contribute to age‐related changes in swallowing duration. Decreasing muscle fiber size in the protrusive transverse and verticalis muscles may contribute to reductions in maximum isometric tongue pressure found with age. Differences among regions and muscles may be associated with different functional demands. Muscle Nerve 56 : E119–E125, 2017     

Innervation of dystrophic muscle after muscle stem cell therapy

Introduction: Duchenne muscular dystrophy (DMD) is caused by loss of the structural protein, dystrophin, resulting in muscle fragility. Muscle stem cell (MuSC) transplantation is a potential therapy for DMD. It is unknown whether donor‐derived muscle fibers are structurally innervated. Methods: Green fluorescent protein (GFP)–expressing MuSCs were transplanted into the tibials anterior of adult dystrophic mdx/mTR mice. Three weeks later the neuromuscular junction was labeled by immunohistochemistry. Results: The percent overlap between pre‐ and postsynaptic immunolabeling was greater in donor‐derived GFP⁺ myofibers, and fewer GFP⁺ myofibers were identified as denervated compared with control GFP^– fibers (P = 0.001 and 0.03). GFP⁺ fibers also demonstrated acetylcholine receptor fragmentation and expanded endplate area, indicators of muscle reinnervation (P = 0.008 and 0.033). Conclusion: It is unclear whether GFP⁺ fibers are a result of de novo synthesis or fusion with damaged endogenous fibers. Either way, donor‐derived fibers demonstrate clear histological innervation. Muscle Nerve 54 : 763–768, 2016     

Muscle spindles in the human bulbospongiosus and ischiocavernosus muscles

Introduction: Muscle spindles are crucial for neuronal regulation of striated muscles, but their presence and involvement in the superficial perineal muscles is not known. Methods: Bulbospongiosus and ischiocavernosus muscle specimens were obtained from 31 human cadavers. Serial sections were stained with hematoxylin and eosin, Sirius red, antibodies against Podocalyxin, myosin heavy chain isoforms (MyHC‐slow tonic, S46; MyHC‐2a/2x, A4.74), and neurofilament for the purpose of muscle spindle screening, counting, and characterization. Results: A low but consistent number of spindles were detected in both muscles. The muscles contained few intrafusal fibers, but otherwise showed normal spindle morphology. The extrafusal fibers of both muscles were small in diameter. Conclusions: The presence of muscle spindles in bulbospongiosus and ischiocavernosus muscles supports physiological models of pelvic floor regulation and may provide a basis for further clinical observations regarding sexual function and micturition. The small number of muscle spindles points to a minor level of proprioceptive regulation. Muscle Nerve 52 : 55–62, 2015     

Perineuronal nets affect parvalbumin expression in GABAergic neurons of the mouse hippocampus

Recent studies have suggested that the perineuronal net (PNN), a specialised extracellular matrix structure, and parvalbumin (PV), an EF‐hand calcium‐binding protein, are involved in the regulation of plasticity of neural circuits. Here, we aimed to quantitatively estimate the relationship between the two plasticity regulators, PV and PNNs, in the hippocampus of young adult mice. Dual fluorescence staining for PV and Wisteria floribunda agglutinin (a broad PNN marker) showed that a substantial population of PV‐expressing (PV⁺) GABAergic neurons lacked PNNs. Optical disector analysis demonstrated that there were fewer PNN⁺ neurons than PV⁺ neurons. The ratio of PNN expression in PV⁺ neurons was generally lower in the dendritic layers than in the principal cell layers, whereas the ratio of PV expression in PNN⁺ neurons was effectively 100%. The mean PV fluorescence was significantly higher in PNN⁺/PV⁺ neurons than in PNN^?/PV⁺ neurons. Cumulative frequencies for single‐cell PV fluorescence indicated that intensely stained PV⁺ neurons tend to be enwrapped by PNNs, whereas weakly stained PV⁺ neurons are likely to lack PNNs. We digested the PNNs by a unilateral injection of chondroitinase ABC (chABC) into the dorsal CA1 region. Although the densities of PV⁺ neurons remained unchanged, the PV fluorescence declined 7 days after chABC injection. Quantitative real‐time polymerase chain reaction analysis demonstrated a reduction in PV mRNA expression following chABC injection. These findings indicate that the presence or absence of PNNs affects the relative PV expression in GABAergic neurons in the hippocampus.     

Development of contractile properties of minced muscle regenerates in the rat

To test functional properties of regenerating skeletal muscle, the triceps surae muscles of 1-month-old rats were minced into 1-mm³ pieces and replaced. Myogenic cells originating from the degenerating implanted muscle fragments form a model of the original muscle and contractile activity is first detected after 7–8 days. Early regenerates exhibited contractile properties of very slow muscles. Contraction time speeded up until by 30–40 days it approached those of the normal fast muscles. The development of contractile function closely resembles that of normal muscles during the first postnatal weeks and it thus appears that the regenerating muscle undergoes a functional “developmental recapitulation.” It is concluded that, despite a quantitative deficiency in the mass of regenerating muscle, those fibers which are present rapidly approach the normal condition in functional as well as morphological characteristics.     

Human leukocyte antigen class I in polymyositis: Leukocyte infiltrates,regeneration, and impulse block

In polymyositis (PM), T-cell mediated myocytotoxicity is directed against strongly human leukocyte antigen class I positive (HLA-I⁺) muscle fibers. Fiber regeneration probably is partly responsible for this HLA-I up-regulation. We have evaluated regeneration, denervation/impulse blockade, and focal leukocyte infiltrates as possible HLA-I inducing factors in PM. Distinctive patterns of HLA-I, nerve cell adhesion molecule (NCAM), and vimentin expression accompany denervation and regeneration. Regenerating fibers also have centralized nuclei. Using semiquantitative methods, we examined strongly HLA-I⁺ fibers in PM muscle biopsies for these markers. Sarcoplasmic HLA-I levels were related to the presence of leukocyte infiltrates and invasion of fibers. Strongly HLA-I⁺ fibers were frequently invaded, and regeneration-associated changes were usually observed at sites of fiber damage. Sarcoplasmic HLA-I levels were stable along intact fibers, also adjacent to leukocyte infiltrates. A majority of the strongly HLA-I⁺ fibers were nonregenerating (NCAM⁺ only). Though other mechanisms cannot be excluded, this suggests that impulse blockade or denervation may contribute to extra HLA-I up-regulation in these fibers. © 1997 John Wiley & Sons, Inc. Muscle Nerve 20: 1534–1540, 1997     

Protein synthesis in bupivacaine (Marcaine)-treated,regenerating skeletal muscle

Skeletal muscle regeneration has been induced by injection of the myotoxic drug bupivacaine (Marcaine) into the rat tibialis anterior muscle. Doses of 1.5 and 1.0% wt/vol produce significant levels of muscle regeneration, but these doses also produce large regions of ischemic muscle. Doses of 0.75 and 0.5% bupivacaine are also effective in inducing regeneration and produce little or no ischemia. Regenerating muscle is significantly more active in the incorporation of ³⁵S-methionine into protein than is control muscle, and the activity increase is directly proportional to the bupivacaine dose injected. Polyribosomes were isolated in greater yield from bupivacaine-treated muscles, as compared with control muscles, 5 days postinjection, and were also more active in cell-free protein synthesis than control polysomes. Again, the yield and activity of the muscle polysomes was directly proportional to the bupivacaine concentration used for injection. Polyacrylamide gel electrophoresis of polysomal cell-free reaction mixtures demonstrated the synthesis of a number of myofibrillar proteins.     

Ca2+‐dependent ion channels underlying spontaneous activity in insect circadian pacemaker neurons

Electrical activity in the gamma frequency range is instrumental for temporal encoding on the millisecond scale in attentive vertebrate brains. Surprisingly, also circadian pacemaker neurons in the cockroach Rhyparobia maderae (Leucophaea maderae) employ fast spontaneous rhythmic activity in the gamma band frequency range (20–70 Hz) together with slow rhythmic activity. The ionic conductances controlling this fast spontaneous activity are still unknown. Here, Ca²⁺ imaging combined with pharmacology was employed to analyse ion channels underlying spontaneous activity in dispersed circadian pacemakers of the adult accessory medulla, which controls circadian locomotor activity rhythms. Fast spontaneous Ca²⁺ transients in circadian pacemakers accompany tetrodotoxin (TTX)‐blockable spontaneous action potentials. In contrast to vertebrate pacemakers, the spontaneous depolarisations from rest appear to be rarely initiated via TTX‐sensitive sustained Na⁺ channels. Instead, they are predominantly driven by mibefradil‐sensitive, low‐voltage‐activated Ca²⁺ channels and DK‐AH269‐sensitive hyperpolarisation‐activated, cyclic nucleotide‐gated cation channels. Rhythmic depolarisations activate voltage‐gated Na⁺ channels and nifedipine‐sensitive high‐voltage‐activated Ca²⁺ channels. Together with Ca²⁺ rises, the depolarisations open repolarising small‐conductance but not large‐conductance Ca²⁺‐dependent K⁺ channels. In contrast, we hypothesise that P/Q‐type Ca²⁺ channels coupled to large‐conductance Ca²⁺‐dependent K⁺ channels are involved in input‐dependent activity.     

Distribution of [3H]RNA in goldfish optic tectum following intraocular or intracranial injection of [3H]uridine. Evidence of axonal migration of RNA in regenerating optic fibers.

The distribution of [³H]RNA in the goldfish optic tectum following eitherintra-ocular orintracranial injection of [³H]uridine during optic fiber regeneration has been studied by light (LMA) and electron (EMA) microscopic autoradiography.In one group of 4 fish both optic nerves were crushed, and 18 days later [³H]uridine was injected into the right eye. A second group of 5 fish, in which only one optic nerve had been crushed, received intracranial injections of [³H]uridine 18 or 22 days after the crush. All fish were sacrificed 24 days after crushing the optic nerves, a time when regenerating optic fibers have entered the tectum and are establishing functional reconnections. Tecta were fixed in situ with glutaraldehyde, dissected out, and samples were processed for LMA and EMA. Controls were carried out to ensure that [³H]RNA was the only radioactive component present in the tissue after fixation.The distribution of silver grains related to [³H]RNA in intraocularly injected goldfish was different from that following intracranial injection. Following intraocular injection virtually all the [³H]RNA was located in the layers of the left optic tectum (contralateral to the side of intraocular injection) where the regenerating optic fibers course and terminate, whereas virtually no radioactivity was present in the right optic tectum. EMA quantitative analysis of the labeled layers of the left optic tectum revealed that perikarya of cells, most of which are glial cells, had a density of grains related to [³H]RNA of 20–28 g/100 sq.μm; axonal growth cones had a density of 14–24 g/100 sq.μm. Grain densities over non-axonal cell structures were markedly lower, ranging between 3 and 6 g/sq.μm. Grains located over axons and growth cones accounted for 50–60% of all counted grains.Inintracranially injected goldfish, either 2 or 6 days after injection, silver grains were clustered over leptomeninges as well as vessels and parenchymal cells of the tectal strata containing the regenerating optic fibers. In the stratum opticum a high grain density was seen over glial cells, whereas virtually no grains were present over the fascicles of regenerating axons. EMA quantitative analysis revealed a grain density over glial and other parenchymal cells of the stratum opticum of 67 g/100 sq.μm, whereas densities over growth cones and regenerating axons were 1.3 g/100 sq.μm and 1.8 g/100 sq.μm respectively. Grains located over axons and growth cones accounted for 3.3% of all counted grains.On the basis of the present and previous findings it is suggested that followingintraocular injection of [³H]uridine the [³H]RNA present inside regenerating optic axons is transported from the ganglion cells of the retina; on the other hand, the [³H]RNA present in surrounding glial cells is the result of local utilization of [³H]RNA precursors which also migrate from the retina along with the [³H]RNA.It is also concluded that 2 and 6 days followingintracranial injection of [³H]uridine no substantial tranfer of [³H]RNA from glial cells to regenerating optic fibers occurs in the goldfish optic tectum.     

Electrical stimulation influences satellite cell differentiation after sciatic nerve crush injury in rats

Introduction: Electrical stimulation is often used to prevent muscle atrophy and preserve contractile function, but its effects on the satellite cell population after nerve injury are not well understood. In this study we aimed to determine whether satellite cell differentiation is affected by electrical stimulation after nerve crush. Methods: The sciatic nerves of Sprague‐Dawley (SD) rats were crushed. Half of the injured rats received daily electrical stimulation of the gastrocnemius muscle, and the others did not. Tests for detecting paired box protein 7 (Pax7), myogenic differentiation antigen (MyoD), embryonic myosin heavy chain (eMyHC), and force production were performed 2, 4, and 6 weeks after injury. Results: More Pax7⁺/MyoD⁺ nuclei in stimulated muscles were observed than in non‐stimulated muscles. eMyHC expression was elevated in stimulated muscles and correlated positively with enhanced force production. Conclusions: Increased satellite cell differentiation is correlated with preserved muscle function in response to electrical stimulation after nerve injury. Muscle Nerve 51: 400–411, 2015     

Dystrophin-positive muscle fibers following C2 myoblast transplantation into mdx nude mice

To determine when and how the dystrophin-positive muscle fibers are formed after myoblast transplantation into dystrophin-negative muscles, the tibialis anterior (TA) muscle from mdx nude mouse was chronologically examined after C2 myoblast transplantation by immunohistochemical and glucose 6-phosphate isomerase (GPI) isoenzyme analyses. The host TA muscle transplanted with C2 myoblasts became necrotic with accumulation of basic fibroblast growth factor in the necrotic areas. This may stimulate concomitant proliferation of the host satellite cells and C2 myoblasts. Small dystrophinpositive muscle fibers appeared in the necrotic areas 3 days after transplantation. This TA muscle contained two different kinds of homodimer GPI isoenzymes but did not contain the heterodimer, suggesting rare fusion of host and donor cells. The dystrophin-positive muscle fibers in the necrotic areas rapidly increased in number and in size by 7 days, but they were smaller than the original host muscle fibers. They had central nuclei, indicating that they were regenerating fibers. The presence of heterodimer GPI isoenzyme in these muscles indicated that the regenerating fibers were mosaic host/donor muscle fibers. The dystrophin-positive muscle fibers are probably formed first by fusion of donor cells with each other and then later by the fusion of host satellite and donor cells.