Heat‐Stress effects on the myosin heavy chain phenotype of rat soleus fibers during the early stages of regeneration

Introduction: We investigated heat‐stress effects on the adult myosin heavy chain (MyHC) profile of soleus muscle fibers at an early stage of regeneration. Methods: Regenerating fibers in adult rats were analyzed 2, 4, or 6 days after bupivacaine injection. Rats were heat stressed by immersion in water (42 ± 1°C) for 30 minutes 24 hours after bupivacaine injection and every other day thereafter. Results: No adult MyHC isoforms were observed after 2 days, whereas some fibers expressed only fast MyHC after 4 days. Heat stress increased fast and slow MyHC in regenerating fibers after 6 days. Regenerating fibers expressing only slow MyHC were observed only in heat‐stressed muscles. Bupivacaine injection increased the number of Pax7⁺ and MyoD⁺ satellite cells in regenerating fibers, more so in heat‐stressed rats. Conclusion: The results indicate that heat stress accelerates fast‐to‐slow MyHC phenotype conversion in regenerating fibers via activation of satellite cells. Muscle Nerve 52 : 1047–1056, 2015     

Effect of Agonist‐Antagonist Electrical Stimulation on Muscle Afferent Recordings in Anesthetized Rabbits

Objective. Sensory feedback extracted from muscle afferents is an approach to achieve closed‐loop control of paralyzed muscles using functional electrical stimulation (FES). The objective of the present study was to characterize the effect of agonist‐antagonist electrical stimulation on nerve cuff recordings of muscle afferent activity. Methods. Cuff electrodes were implanted around the tibial and peroneal nerve branches in five acute rabbit experiments. Two wires were implanted in each of the tibialis anterior (TA) and the lateral gastrocnemius (LG) muscles to obtain bipolar, intramuscular stimulation. Electroneurograms (ENG) were recorded during trapezoidal rotations of the ankle joint and compared during periods (25%, 50% and 100% of maximal force) with and without electrical stimulation of the muscle. Results. The activity from a stretched and electrically stimulated muscle showed the same pattern as the recordings from a matched nonstimulated muscle. The background afferent activity increased with increasing level of muscle stimulation. The static and dynamic sensitivities were not found to be different, except in one case (peroneal nerve at 100% TA recruitment). Discussion. The main contribution to the tibial activity was believed to originate from muscle afferents in nonstimulated, synergist muscles. The main contribution to the peroneal activity was believed to be from muscle afferents within the muscle being stimulated. It was suggested that the increased background activity could be attributed to the increased activity of the Golgi tendon organs. Conclusions. Sensory information about joint flexion and joint extension are preserved in muscle afferent recordings from electrically activated muscles at low and intermediate stimulation levels, but it still has to be shown whether muscle afferent information can be useful as sensory feedback in FES control.     

Neuromuscular transmission and muscle fatigue changes by nanostructured oxygen

Introduction: Oxygen (O₂) nanobubbles offer a new method for tissue oxygenation. The effects of O₂ nanobubbles on transmission at neuromuscular junctions (NMJs) and muscle function were explored in murine diaphragm. Methods: Electrophysiological parameters, NMJ ultrastructure, muscle force, and muscle fatigue were studied during superfusion with solutions with different oxygen levels or oxygen nanobubbles. Results: High frequency nerve stimulation of muscles superfused with O₂ nanobubble solution slowed neurotransmission decline over those with either control or hyperoxic solution. O₂ nanobubble solution increased the amplitude of evoked end plate potentials and quantal content but did not affect spontaneous activity. Electron microscopy of stimulated O₂ nanobubble treated NMJs showed accumulation of large synaptic vesicles and endosome‐like structures. O₂ nanobubble solution had no effects on isometric muscle force, but it significantly decreased fatigability and maximum force recovery time in nerve stimulated muscles. Conclusions: O₂ nanobubbles increase neurotransmission and reduce the probability of neurotransmission failure in muscle fatigue. Muscle Nerve 55 : 555–563, 2017     

Twitch and M‐wave potentiation induced by intermittent maximal voluntary quadriceps contractions: Differences between direct quadriceps and femoral nerve stimulation

Introduction: The aim of this study was to investigate differences in twitch and M‐wave potentiation in the quadriceps femoris when electrical stimulation is applied over the quadriceps muscle belly versus the femoral nerve trunk. Methods: M‐waves and mechanical twitches were evoked using direct quadriceps muscle and femoral nerve stimulation between 48 successive isometric maximal voluntary contractions (MVC) from 10 young, healthy subjects. Potentiation was investigated by analyzing the changes in M‐wave amplitude recorded from the vastus medialis (VM) and vastus lateralis (VL) muscles and in quadriceps peak twitch force. Results: Potentiation of twitch, VM M‐wave, and VL M‐wave were greater for femoral nerve than for direct quadriceps stimulation (P < 0.05). Despite a 50% decrease in MVC force, the amplitude of the M‐waves increased significantly during exercise. Conclusions: In addition to enhanced electrogenic Na⁺‐K⁺ pumping, other factors (such as synchronization in activation of muscle fibers and muscle architectural properties) may significantly influence the magnitude of M‐wave enlargement. Muscle Nerve 48 : 920–929, 2013     

Functional electrical stimulation following nerve injury in a large animal model

Introduction: Controversy exists over the effects of functional electrical stimulation (FES) on reinnervation. We hypothesized that intramuscular FES would not delay reinnervation after recurrent laryngeal nerve (RLn) axonotmesis. Methods: RLn cryo-injury and electrode implantation in ipsilateral posterior cricoarytenoid muscle (PCA) were performed in horses. PCA was stimulated for 20 weeks in eight animals; seven served as controls. Reinnervation was monitored through muscle response to hypercapnia, electrical stimulation and exercise. Ultimately, muscle fiber type proportions and minimum fiber diameters, and RLn axon number and degree of myelination were determined. Results: Laryngeal function returned to normal in both groups within 22 weeks. FES improved muscle strength and geometry, and induced increased type I:II fiber proportion (p = 0.038) in the stimulated PCA. FES showed no deleterious effects on reinnervation. Discussion: Intramuscular electrical stimulation did not delay PCA reinnervation after axonotmesis. FES can represent a supportive treatment to promote laryngeal functional recovery after RLn injury. Muscle Nerve 59:717–725, 2019     

Impaired regeneration in LGMD2A supported by increased PAX7‐positive satellite cell content and muscle‐specific microrna dysregulation

Introduction: Recent in vitro studies suggest that CAPN3 deficiency leads initially to accelerated myofiber formation followed by depletion of satellite cells (SC). In normal muscle, up‐regulation of miR‐1 and miR‐206 facilitates transition from proliferating SCs to differentiating myogenic progenitors. Methods: We examined the histopathological stages, Pax7 SC content, and muscle‐specific microRNA expression in biopsy specimens from well‐characterized LGMD 2A patients to gain insight into disease pathogenesis. Results: Three distinct stages of pathological changes were identified that represented the continuum of the dystrophic process from prominent inflammation with necrosis and regeneration to prominent fibrosis, which correlated with age and disease duration. Pax7‐positive SCs were highest in the fibrotic group and correlated with down‐regulation of miR‐1, miR‐133a, and miR‐206. Conclusions: These observations, and other published reports, are consistent with microRNA dysregulation leading to inability of Pax7‐positive SCs to transit from proliferation to differentiation. This results in impaired regeneration and fibrosis. Muscle Nerve 47: 731–739, 2013     

Tongue muscle plasticity following hypoglossal nerve stimulation in aged rats

Introduction: Age‐related decreases in tongue muscle mass and strength have been reported. It may be possible to prevent age‐related tongue muscle changes using neuromuscular electrical stimulation (NMES). Our hypothesis was that alterations in muscle contractile properties and myosin heavy chain composition would be found after NMES. Methods: Fifty‐four young, middle‐aged, and old 344/Brown Norway rats were included in this study. Twenty‐four rats underwent bilateral electrical stimulation of the hypoglossal nerves for 8 weeks and were compared with control or sham rats. Muscle contractile properties and myosin heavy chain (MHC) in the genioglossus (GG), styloglossus (SG), and hyoglossus (HG) muscles were examined. Results: Compared with unstimulated control rats, we found reduced muscle fatigue, increased contraction and half‐decay times, and increased twitch and tetanic tension. Increased type I MHC was found, except for in GG in old and middle‐aged rats. Conclusion: Transitions in tongue muscle contractile properties and phenotype were found after NMES. Muscle Nerve, 2013     

Satellite cells in slow and fast rat muscles differ in respect to acetylcholinesterase regulation mechanisms they convey to their descendant myofibers during regeneration

The hypothesis of satellite cell diversity in slow and fast mammalian muscles was tested by examining acetylcholinesterase (AChE) regulation in muscles regenerating (1) under conditions of muscle disuse (tenotomy, leg immobilization) in which the pattern of neural stimulation is changed, and (2) after cross-transplantation when the regenerating muscle develops under a foreign neural stimulation pattern. Soleus (SOL) and extensor digitorum longus (EDL) muscles of the rat were allowed to regenerate after ischemic-toxic injury either in their own sites or had been cross-transplanted to the site of the other muscle. Molecular forms of AChE in regenerating muscles were analyzed by velocity sedimentation in linear sucrose gradients. Neither tenotomy nor limb immobilization significantly affected the characteristic pattern of AChE molecular forms in regenerating SOL muscles, suggesting that the neural stimulation pattern is probably not decisive for its induction. During an early phase of regeneration, the general pattern of AChE molecular forms in the cross-transplanted regenerating muscle was predominantly determined by the type of its muscle of origin, and much less by the innervating nerve which exerted only a modest modifying effect. However, alkali-resistant myofibrillar ATPase activity on which the separation of muscle fibers into type I and type II is based, was determined predominantly by the motor nerve innervating the regenerating muscle. Mature regenerated EDL muscles (13 weeks after injury) which had been innervated by the SOL nerve became virtually indistinguishable from the SOL muscles in regard to their pattern of AChE molecular forms. However, AChE patterns of mature regenerated SOL muscles that had been innervated by the EDL nerve still displayed some features of the SOL pattern. In regard to AChE regulation, muscle satellite cells from slow or fast rat muscles convey to their descendant myotubes the information shifting their initial development in the direction of either slow or fast muscle, respectively. The satellite cells in fast or slow muscles are, therefore, intrinsically different. Intrinsic information is expressed mostly during an early phase of regeneration whereas later on the regulatory influence of the motor nerve more or less predominates. © 1994 Wiley-Liss, Inc.     

Muscle Weakness, Paralysis, and Atrophy after Human Cervical Spinal Cord Injury

Muscle weakness and failure of central motor drive were assessed in triceps brachii muscles of individuals with chronic cervical spinal cord injury (SCI) and able-bodied controls. Electrical stimuli were applied to the radial nerve during rest and during triceps submaximal and maximal voluntary contractions (MVCs). The mean forces and integrated EMGs generated by SCI subjects during MVCs were significantly less than those produced by controls (P < 0.01), with 74 and 71% of muscles generating <10% control force and EMG, respectively. There was an inverse linear relationship between the evoked and voluntary forces (n = 32 muscles of SCI subjects) which, when extrapolated to zero evoked force, also showed significant whole muscle weakness for SCI compared to control subjects (P < 0.01). Severe muscle atrophy was revealed which might reflect disuse and/or muscle denervation subsequent to motoneuron loss. Many triceps muscles of SCI subjects showed no force occlusion (n = 41) or were impossible to stimulate selectively (n = 61). Force was always evoked when the radial nerve was stimulated during MVCs of SCI subjects. The force elicited by single magnetic shocks applied to the motor cortex at Cz′ during voluntary contractions of SCI subjects was also inversely related to the voluntary triceps force exerted (n = 18), but usually no force could be elicited during MVCs. Thus central motor drive was probably maximal to these muscles, and the force evoked during MVCs by below-lesion stimulation must come from activation of paralyzed muscle. SCI subjects also had significantly longer mean central nervous system (CNS) conduction times to triceps (P < 0.01) suggesting that the measured deficits reflect CNS rather than peripheral nervous system factors. Thus, the weak voluntary strength of these partially paralyzed muscles is not due to submaximal excitation of higher CNS centers, but results mainly from reduction of this input to triceps motoneurons.     

Electrical stimulation impairs early functional recovery and accentuates skeletal muscle atrophy after sciatic nerve crush injury in rats

Neuromuscular recovery after peripheral nerve lesion depends on the regeneration of severed axons that re‐establish their functional connection with the denervated muscle. The aim of this study was to determine the effects of electrical stimulation (ES) on the neuromuscular recovery after nerve crush injury in rats. Electrical stimulation was carried out on the tibialis anterior (TA) muscle after sciatic nerve crush injury in a rat model. Six ES sessions were administered every other day starting from day 3 postinjury until the end of the experiment (day 14). The sciatic functional index was calculated. Muscle excitability, neural cell adhesion molecule (N‐CAM) expression, and muscle fiber cross‐sectional area (CSA) were accessed from TA muscle. Regenerated sciatic nerves were analyzed by light and confocal microscopy. Both treated (crush+ES) and untreated (crush) groups had their muscle weight and CSA decreased compared with the normal group (P < 0.05). Electrical stimulation accentuated muscle fiber atrophy more in the crush+ES than in the crush group (P < 0.05). N‐CAM expression increased in both crush and crush+ES groups compared with the normal group (P < 0.05). Regenerated nerves revealed no difference between the crush and crush+ES groups. Nevertheless, functional recovery at day 14 post‐injury was significantly lower in crush+ES group compared with the crush group. In addition, the crush+ES group had chronaxie values significantly higher on days 7 and 13 compared with the crush group, which indicates a decrease in muscle excitability in the crush+ES animals. The results of this study do not support a benefit of the tested protocol of ES during the period of motor nerve recovery following injury. Muscle Nerve, 2010     

Contractile properties of human Thenar muscles paralyzed by spinal cord injury

The electrical and mechanical properties of paralyzed human thenar muscles were measured in response to supramaximal stimulation of the median nerve in individuals with chronic cervical spinal cord injury. These data were compared to those recorded from control muscles. Spontaneous motor unit activity was common in paralyzed muscles. There was significantly more variance in the twitch and tetanic forces, twitch/tetanus force ratios, twitch and tetanic half-relaxation times, and the stimulus frequencies which generated half-maximal force in paralyzed versus control muscles. Approximately half the paralyzed thenar muscles were significantly weaker than control muscles and their compound action potential amplitudes were reduced significantly. Paralyzed muscles had significantly higher twitch/tetanus force ratios. The mean stimulus frequency which generated half-maximal force was also reduced significantly. Thus for rehabilitation purposes, lower stimulation rates are required to elicit any given submaximal force from chronically paralyzed thenar muscles. © 1997 John Wiley & Sons, Inc. Muscle Nerve 20: 788–799, 1997     

Surface‐distributed low‐frequency asynchronous stimulation delays fatigue of stimulated muscles

Introduction: One important reason why functional electrical stimulation (FES) has not gained widespread clinical use is the limitation imposed by rapid muscle fatigue due to non‐physiological activation of the stimulated muscles. We aimed to show that asynchronous low‐pulse‐rate (LPR) electrical stimulation applied by multipad surface electrodes greatly postpones the occurrence of muscle fatigue compared with conventional stimulation (high pulse rate, HPR). Methods: We compared the produced force vs. time of the forearm muscles responsible for finger flexion in 2 stimulation protocols, LPR (f_L = 10 Hz ) and HPR (f_H = 40 Hz ). Results: Surface‐distributed low‐frequency asynchronous stimulation (sDLFAS) doubles the time interval before the onset of fatigue (104 ± 80%) compared with conventional synchronous stimulation. Conclusions: Combining the performance of multipad electrodes (increased selectivity and facilitated positioning) with sDLFAS (decreased fatigue) can improve many FES applications in both the lower and upper extremities. Muscle Nerve 48 : 930–937, 2013     

Metabolic and nonmetabolic components of fatigue monitored with 31P-NMR

The goal of this study was to determine the roles of metabolic and nonmetabolic factors in muscle fatigue. Rat gastrocnemius muscles were fatigued by stimulation of the nerve (n = 6) or muscle (n = 4, after 2 days of denervation). ³¹Phosphorus nuclear magnetic resonance spectroscopy was used to measure levels of intracellular inorganic phosphate (Pi) and hydrogen ions (H⁺) (which are thought to inhibit contraction) and the high-energy phosphates, phosphocreatine (PCr), and ATP. For both indirect and direct stimulation, with fatigue to ≈60% initial tetanic force, [Pi] increased from ≈3.5 mmol/L to ≈20 mmol/L and [PCr] decreased from ≈27 mmol/L to ≈9 mmol/L. However, with continued fatigue to 25–35% initial tetanic force, neither [Pi] or [PCr] changed further. [ATP] and pH changed only slightly during fatigue. The results are consistent with early fatigue arising from metabolic inhibition of contraction; but later fatigue arising independent of metabolites, due to impaired activation beyond the neuromuscular junction. © 1994 John Wiley & Sons, Inc.     

Expression of myogenic regulatory factors and myo-endothelial remodeling in sporadic inclusion body myositis

Muscle repair relies on coordinated activation and differentiation of satellite cells, a process that is unable to counterbalance progressive degeneration in sporadic inclusion body myositis (s-IBM). To explore features of myo regeneration, the expression of myogenic regulatory factors Pax7, MyoD and Myogenin and markers of regenerating fibers was analyzed by immunohistochemistry in s-IBM muscle compared with polymyositis, dermatomyositis, muscular dystrophy and age-matched controls. In addition, the capillary density and number of interstitial CD34⁺ hematopoietic progenitor cells was determined by double-immunoflourescence staining. Satellite cells and regenerating fibers were significantly increased in s-IBM similar to other inflammatory myopathies and correlated with the intensity of inflammation (R > 0.428). Expression of MyoD, visualizing activated satellite cells and proliferating myoblasts, was lower in s-IBM compared to polymyosits. In contrast, Myogenin a marker of myogenic cell differentiation was strongly up-regulated in s-IBM muscle. The microvascular architecture in s-IBM was distorted, although the capillary density was normal. Notably, CD34⁺ hematopoietic cells were significantly increased in the interstitial compartment. Our findings indicate profound myo-endothelial remodeling of s-IBM muscle concomitant to inflammation. An altered expression of myogenic regulatory factors involved in satellite cell activation and differentiation, however, might reflect perturbations of muscle repair in s-IBM.     

Physical exercise during muscle regeneration improves recovery of the slow/oxidative phenotype

Introduction: As skeletal muscle mass recovery after extensive injury is improved by contractile activity, we explored whether concomitant exercise accelerates recovery of the contractile and metabolic phenotypes after muscle injury. Methods: After notexin‐induced degeneration of a soleus muscle, Wistar rats were assigned to active (running exercise) or sedentary groups. Myosin heavy chains (MHC), metabolic enzymes, and calcineurin were studied during muscle regeneration at different time points. Results: The mature MHC profile recovered earlier in active rats (21 days after injury) than in sedentary rats (42 days). Calcineurin was higher in the active degenerated than in the sedentary degenerated muscles at day 14. Citrate synthase and total lactate dehydrogenase (LDH) activity decreased after injury and were similarly recovered in both active and sedentary groups at 14 or 42 days, respectively. H‐LDH isozyme activity recovered earlier in the active rats. Conclusions: Exercise improved recovery of the slow/oxidative phenotype after soleus muscle injury. Muscle Nerve 55 : 91–100, 2017     

Effect of voluntary physical activity initiated at age 7 months on skeletal hindlimb and cardiac muscle function in mdx mice of both genders

Introduction: The effects of voluntary activity initiated in adult mdx (C57BL/10ScSc‐DMD^mdx/J) mice on skeletal and cardiac muscle function have not been studied extensively. Methods: We studied the effects of 3 months of voluntary wheel running initiated at age 7 months on hindlimb muscle weakness, increased susceptibility to muscle contraction?induced injury, and left ventricular function in mdx mice. Results: We found that voluntary wheel running did not worsen the deficit in force‐generating capacity and the force drop after lengthening contractions in either mdx mouse gender. It increased the absolute maximal force of skeletal muscle in female mdx mice. Moreover, it did not affect left ventricular function, structural heart dimensions, cardiac gene expression of inflammation, fibrosis, or remodeling markers. Conclusion: These results indicate that voluntary activity initiated at age 7 months had no detrimental effects on skeletal or cardiac muscles in either mdx mouse gender. Muscle Nerve 52 : 788–794, 2015     

Muscle injury in rats induces upregulation of inflammatory cytokines in injured muscle and calcitonin gene–related peptide in dorsal root ganglia innervating the injured muscle

Introduction: In this study we evaluated the relationships among the behavioral changes after muscle injury, histological changes, changes in inflammatory cytokines in the injured muscle, and changes in the sensory nervous system innervating the muscle in rats. Methods: We established a model of muscle injury in rats using a dropped weight. Behavior was assessed using the CatWalk system. Subsequently, bilateral gastrocnemius muscles and dorsal root ganglia (DRGs) were resected. Muscles were stained with hematoxylin and eosin, and inflammatory cytokines in injured muscles were assayed. DRGs were immunostained for calcitonin gene–related peptide (CGRP). Results: Changes of behavior and upregulation of inflammatory cytokines in injured muscles subsided within 2 days of injury. Repaired tissue was observed 3 weeks after injury. However, upregulation of CGRP in DRG neurons continued for 2 weeks after injury. Conclusion: These findings may explain in part the pathological mechanism of persistent muscle pain. Muscle Nerve 54 : 776–782, 2016     

Nonvolitional assessment of tibialis anterior force and architecture during critical illness

Introduction: Contemporaneous measures of muscle architecture and force have not previously been conducted during critical illness to examine their relationship with intensive care unit (ICU)‐acquired weakness. Methods: Ankle dorsiflexor muscle force (ADMF) with high‐frequency electrical peroneal nerve stimulation and skeletal muscle architecture via ultrasound were measured in 21 adult, critically ill patients, 16 at ICU admission. Results: Thirteen patients were measured on 2 occasions. Among these, 10 who were measured at ICU admission demonstrated muscle weakness. Despite significant reductions in tibialis anterior (Δ = ?88.5 ± 78.8 mm², P = 0.002) and rectus femoris (Δ = ?126.1 ± 129.1 mm², P = 0.006) cross‐sectional areas between occasions, ADMF did not change (100‐HZ ankle dorsiflexor force 9.8 [IQR, 8.0–14.4] kg vs. 8.6 (IQR, 6.7–19.2) kg, P = 0.9). Discussion: Muscle weakness was evident at ICU admission. No additional decrements were observed 7 days later despite significant reductions in muscle size. These data suggest that not all ICU weakness is truly “acquired” and questions our understanding of muscle function during critical illness. Muscle Nerve 57 : 964–972, 2018     

Pattern of myogenesis and vascular repair in early and advanced lesions of juvenile dermatomyositis

Regenerative processes that counteract perifascicular muscle atrophy and capillary loss in juvenile dermatomyositis (JDM) are not well characterized. We aimed to analyze the pattern of myo-regeneration in relation to vascular damage and repair in muscle specimens from JDM patients. Myogenic regulatory factors that are sequentially expressed during myogenesis were studied by immunohistochemistry. Capillary density, numbers of CD34⁺ endothelial progenitor cells within the endomysium and molecules implicated in angiogenesis were evaluated by double-immunofluorescence techniques. Myogenic regulatory factors were significantly up-regulated in JDM muscle exhibiting a different pattern in early and advanced lesions. In early lesions Pax7⁺ satellite cells and both MyoD⁺ and Myogenin⁺ myogenic cells were moderately increased. In lesions with advanced perifascicular atrophy Pax7⁺ satellite cells were numerous, but absence of MyoD⁺ in the context of increased Myogenin⁺ expression suggested a dysregulation of the myogenic regenerative pathway. The overall capillary density in JDM was decreased, but regions of capillary loss in advanced lesions alternated with focal increase of hyperplastic endothelial cells in early lesions. Up-regulation of endoglin in hyperplastic endothelial cells in conjunction with overexpression of TGF-β1 and VEGF suggested activation of neovascularization. Conversely, CD34⁺ endothelial progenitor cells were not increased arguing against relevant contribution to vascular repair. Our results demonstrate substantial induction of myogenesis in JDM. While the early phase of myogenesis appears to be associated with endothelial cell activation, an altered expression of MRFs in perifascicular regions with capillary depletion suggests an impairment of myogenic differentiation that may contribute to perifascicular muscle fiber atrophy in JDM.