Gravitational unloading inhibits the regenerative potential of atrophied soleus muscle in mice

Aim: The present study was performed to investigate the influence of unloading on the regeneration of atrophied and injured skeletal muscle. Methods: Male mice (C57BL/6J), aged 8 weeks, were used. Cardiotoxin (CTX) was injected into soleus muscles bilaterally. Gravitational unloading on soleus muscle was performed by hind limb suspension for 2 weeks before and additionally 6 weeks after CTX injection in one group. Soleus muscles in the remaining groups were loaded keeping the mice in the cages and were dissected 14, 28 and 42 days after the injection. Results: Recovery of the wet weight and protein content of soleus in the CTX‐injected group was inhibited by unloading. Increase in satellite cell number, induced by CTX injection and loading, was also inhibited by unloading. Disappearance of infiltration of mononucleated cells into the necrotic area was also delayed. This phenomenon suggests that regeneration, which is indicated by the appearance of fibres with central nuclei, was inhibited by unloading. Conclusion: Results suggested that loading plays an important role in the activation of the regenerating potential of injured skeletal muscle.     

Seven days of muscle re-loading and voluntary wheel running following hindlimb suspension in mice restores running performance,muscle morphology and metrics of fatigue but not muscle strength

In this study, we examined the effects of 2-week hindlimb un-loading in mice followed by re-ambulation with voluntary access to running wheels. The recovery period was terminated at a time point when physical performance—defined by velocity, time, and distance ran per day—of the suspended group matched that of an unsuspended group. Mice were assigned to one of four groups: unsuspended non-exercise (Control), 14 days of hindlimb suspension (HS), 7 days of access to running wheels (E7), or 14 days of HS plus 7 days access to running wheels (HSE7). HS resulted in significant decreases in body and muscle mass, hindlimb strength, soleus force, soleus specific force, fatigue resistance, and fiber cross sectional area (CSA). Seven days of re-ambulation with access to running wheels following HS recovered masses to Control values, increased fiber CSA, increased resistance to fatigue and improved recovery from fatigue in the soleus. HS resulted in a myosin heavy chain (MHC) phenotype shift from slow toward fast-twitch fibers, though running alone did not influence the expression of MHC fibers. Compared to the Control group, HSE7 mice did not recover functional hindlimb strength as assessed through measurements either in vivo or ex vivo. Results from this study demonstrate that 7 days of muscle re-loading with access to wheel-running following HS can stimulate muscle to regain mass and fiber CSA and exhibit improved metrics of fatigue resistance and recovery, yet muscles remain impaired in regard to strength. Understanding this mismatch between muscle morphology and strength may prove of value in designing effective exercise protocols for disuse muscle atrophy rehabilitation.     

Dietary astaxanthin supplementation attenuates disuse-induced muscle atrophy and myonuclear apoptosis in the rat soleus muscle

Extended periods of skeletal muscle disuse results in muscle atrophy and weakness. Currently, no therapeutic treatment is available for the prevention of this problem. Nonetheless, growing evidence suggests that prevention of disuse-induced oxidative stress in inactive muscle fibers can delay inactivity-induced muscle wasting. Therefore, this study tested the hypothesis that dietary supplementation with the antioxidant astaxanthin would protect against disuse muscle atrophy, in part, by prevention of myonuclear apoptosis. Wistar rats (8 weeks old) were divided into control (CT, n = 9), hindlimb unloading (HU, n = 9), and hindlimb unloading with astaxanthin (HU + AX, n = 9) groups. Following 2 weeks of dietary supplementation, rats in the HU and HU + AX groups were exposed to unloading for 7 days. Seven-day unloading resulted in reduced soleus muscle weight and myofiber cross-sectional area (CSA) by ~30 and ~47 %, respectively. Nonetheless, relative muscle weights and CSA of the soleus muscle in the HU + AX group were significantly greater than those of the HU group. Moreover, astaxanthin prevented disuse-induced increase in the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive nuclei. We conclude that astaxanthin supplementation prior to and during hindlimb unloading attenuates soleus muscle atrophy, in part, by suppressing myonuclear apoptosis.     

Effect of support stimulation on unloaded soleus in rat

We studied the efficacy of plantar support in the prevention of atrophy in a disused soleus muscle during hindlimb suspension (HS). The 14-day investigation involved three groups of hindlimb-suspended male Wistar rats and a group of control rats (C). In all HS groups, the left hindlimbs (L) of the animals were left free. As for the right hindlimbs (R), they were either provided support by an adjustable platform (Sup), or immobilized at the ankle joint in a neutral position (Im), or both supported by the platform and immobilized (Sup+Im). Mass, cross-sectional area (CSA) and slow twitch (ST) fiber percentage (ST%) in the R soleus muscle were similar in the Sup and control groups. In the Sup+Im group, these parameters were significantly lower than in the Sup R and C groups. However, the CSA of ST fibers in the Sup+Im R soleus was significantly higher than in those hindlimbs that were left hanging free. Succinate dehydrogenase activity in ST fibers, and α-glycerophosphate dehydrogenase activity in fast-twitch fibers had decreased in the Sup R as compared with the controls. The maximal rate of ADP-stimulated mitochondrial respiration was increased in the free-hanging Sup L hindlimb in comparison with the control. In conclusion, during HS: (1) hindlimb support prevents slow-to-fast fiber transformation and losses in muscle mass and fiber CSA, but brings about a decrease in metabolic enzyme activity, and (2) hindlimb plantar support attenuates but does not fully prevent ST fiber atrophy in the immobilized soleus. Electronic Publication     

A rat resistance exercise regimen attenuates losses of musculoskeletal mass during hindlimb suspension

Exposure to microgravity and/or spaceflight causes dramatic losses in both muscle and bone mass. In normal gravity, resistance exercise has been effectively used to increase muscle and bone mass. We tested a novel form of resistance exercise training using flywheel technology as a countermeasure to offset the loss of musculoskeletal mass during 4 weeks of adult rat hindlimb suspension (HS), an unloading model of microgravity. Male, Sprague-Dawley rats (6-month old) were operantly conditioned to perform resistance exercise, and then randomly assigned to groups of sedentary control (CON), HS, and HS with resistance exercise training (HSRT; 2 sets of approximately 21 repetitions, 3 days week(-1) for 4 weeks during suspension). In soleus, HS resulted in lower (P < 0.05) muscle mass to body mass ratio (approximately 50% of controls) and rates of protein synthesis. HSRT significantly attenuated the loss of muscle mass in soleus muscle, and rates of protein synthesis for soleus were similar for HSRT and controls. There were no differences among groups for mass or rates of protein synthesis in extensor digitorum longus. In cancellous regions of the distal femur, HS resulted in significant reductions of bone mineral density (BMD), but this was restored to control levels with HSRT. Cortical regions of the femur were not different among HS, HSRT or control groups. Together, these data suggest that resistance training using flywheel technology may be a promising tool to attenuate losses of the musculoskeletal system during periods of hindlimb unloading.     

Effects of voluntary wheel running and amino acid supplementation on skeletal muscle of mice

The aims of the present study were as follows: (1) to examine the adaptational changes to chronic endurance voluntary exercise and (2) to investigate the effects of amino acid supplementation on the adaptational changes induced by endurance training in hindlimb (gastrocnemius, tibialis, soleus) and respiratory (diaphragm) muscles of mice. Male C57Bl6 mice were divided in four groups: control sedentary, sedentary supplemented with amino acid mixture (BigOne, 1.5 mg g day^–1 in drinking water for 8 weeks), running (free access to running wheels for 8 weeks), and running supplemented with amino acid mixture. Myosin heavy chain (MHC) isoform distribution was determined in all muscles considered. Fiber cross-sectional area (CSA) was measured in the soleus muscle. In all muscles except the tibialis, endurance training was associated with an overall shift towards the expression of slower MHC isoforms. Amino acid supplementation produced a shift towards the expression of faster MHC isoforms in the soleus and diaphragm muscles, and partially antagonized the effects of training. Immunohistochemical analysis of CSA of individual muscle fibers from the soleus muscle suggests that voluntary running produced a decrease in the size of type 1 fibers, and amino acid supplementation during training resulted in an increase in size in both type 1 and type 2A fibers. Collectively, these results suggest that the endurance adaptations induced by voluntary running depend on the muscle type, and that amino acid supplementation is able to modulate both fiber size and MHC isoform composition of skeletal muscles in sedentary and exercised mice.     

Satellite cells and myonuclei in long-term denervated rat muscles

Background: The percentage of satellite cells rapidly decreases in aneurally regenerating soleus muscles of rat. Also denervation of intact muscles causes fiber loss and regeneration, but the fate of satellite cells is unknown; myonuclei have been suggested to undergo changes resembling those in apoptotic cells. Methods: Rat soleus and extensor digitorum longus (EDL) muscles were denervated at birth or at age 5 weeks and investigated after periods of up to 38 weeks. At least 400 myonuclei in each muscle were assessed by electron microscopy, and satellite cell nuclei were counted. In sity nick translation and tailing were performed after 30 weeks denervation in order to demonstrate DNA breaks associated with apoptosis. Results: Myotubes indicating regeneration were prominent in the adult denervated soleus and deep layers of EDL muscles after 7 weeks and in the superficial parts of EDL muscle after 16 weeks. The percentage of satellite cell nuclei slowly decreased to less than one fifth of normal after 20–30 weeks. Almost all satellite cells had vanished 10 weeks after neonatal denervation. Degenerating myonuclei in adult, but not in neonatally denervated muscles, remotely resembled apoptotic nuclei of lymphocytes, but no evidence of DNA breaks was found. Conclusion: Denervation of rat skeletal muscles causes, in addition to fiber atrophy, loss of fibers with subsequent regeneration. Proliferation of satellite cells under aneural conditions may lead to exhaustion of the satellite cell pool. This process is more rapid in growing than in adult muscles. Myonuclei in denervated muscles do not show DNA breaks which can be demonstrated by in situ nick translation. © 1995 Wiley-Liss, Inc.     

Skeletal muscle mass recovery from atrophy in IL-6 knockout mice

Aim: Skeletal muscle interleukin‐6 (IL‐6) expression is induced by continuous contraction, overload‐induced hypertrophy and during muscle regeneration. The loss of IL‐6 can alter skeletal muscle’s growth and extracellular matrix remodelling response to overload‐induced hypertrophy. Insulin‐like growth factor‐1 (IGF‐1) gene expression and related signalling through Akt/mTOR is a critical regulator of muscle mass. The significance of IL‐6 expression during the recovery from muscle atrophy is unclear. This study’s purpose was to determine the effect of IL‐6 loss on mouse gastrocnemius (GAS) muscle mass during recovery from hindlimb suspension (HS)‐induced atrophy. Methods: Female C57BL/6 [wild type (WT)] and IL‐6 knockout (IL‐6 KO) mice at 10 weeks of age were assigned to control, HS or HS followed by normal cage ambulation groups. Results: GAS muscle atrophy was induced by 10 days of HS. HS induced a 20% loss of GAS mass in both WT and IL‐6 KO mice. HS+7 days of recovery restored WT GAS mass to cage‐control values. GAS mass from IL‐6 KO mice did not return to cage‐control values until HS+14 days of recovery. Both IGF‐1 mRNA expression and Akt/mTOR signalling were increased in WT muscle after 1 day of recovery. In IL‐6 KO muscle, IGF‐1 mRNA expression was decreased and Akt/mTOR signalling was not induced after 1 day of recovery. MyoD and myogenin mRNA expression were both induced in WT muscle after 1 day of recovery, but not in IL‐6 KO muscle. Conclusion: Muscle IL‐6 expression appears important for the initial growth response during the recovery from disuse.     

Functional overloading facilitates the regeneration of injured soleus muscles in mice

The effect of functional overloading on the regenerating process of injured skeletal muscle was investigated in 10-week-old male mice (C57BL/6J). Functional overloading on soleus of both hindlimbs was performed by cutting the distal tendons of plantaris and gastrocnemius muscles for 2 weeks before cardiotoxin (CTX) injection as the preconditioning and also during 10 weeks of recovery. To activate the necrosis-regeneration cycle, 0.1 ml of 10-microM CTX was injected into soleus muscle. The mean values of absolute muscle weight and the percentage of Pax7-positive nuclei in soleus were increased by the preconditioning. These values, as well as total muscle protein content, in the group with CTX injection plus overloading were larger than in the group with CTX injection alone. Fibers with central nucleus were noted in the group with CTX injection with or without overloading. The rate of disappearance of fibers having central nucleus during recovery was stimulated by overloading. Histological analyses revealed that the regeneration of injured soleus muscle with overloading proceeded more rapidly than the muscle without overloading. These results, in combination with previous lines of evidence, strongly suggest that functional overloading may facilitate the regeneration of injured skeletal muscles.     

Long‐term regeneration of fast and slow murine skeletal muscles after induced injury by ACL myotoxin isolated from Agkistrodon contortrix laticinctus (broad‐banded copperhead) venom

The aim of the present work was to analyze the regenerated muscle types I and II fibers of the soleus and gastrocnemius muscles of mice, 8 months after damage induced by ACL myotoxin (ACLMT). Animals received 5 mg/kg of ACLMT into the subcutaneous lateral region of the right hind limb, near the Achilles tendon; contralateral muscles received saline. Longitudinal and cross sections (10 μm) of frozen muscle tissue were evaluated. Eight months after ACLMT injection, both muscle types I and II fibers of soleus and gastrocnemius muscles still showed centralized nuclei and small regenerated fibers. Compared with the left muscle, the incidence of type I fibers increased in the right muscle (21% ± 03% versus 12% ± 06%, P = 0.009), whereas type II fibers decreased (78% ± 02% versus 88% ± 06%, P = 0.01). The incidence of type IIC fibers was normal. These results confirm that ACLMT induced muscle type fiber transformation from type II to type I, through type IIC. The area analysis of types I and II fibers of the gastrocnemius revealed that injured right muscles have a higher percentage of small fibers in both types I and II fibers (0–1,500 μm²) than left muscles, which have larger normal type I and II fibers (1,500–3,500 μm²). These results indicate that ACLMT can be used as an excellent model to study the rearrangement of motor units and the transformation of muscle fiber types during regeneration. Anat Rec 254:521–533, 1999. © 1999 Wiley‐Liss, Inc.     

Clenbuterol-induced fiber type transition in the soleus of adult rats

This study examined the effects of 6 weeks of treatment with theβ ₍₂₎-adrenoceptor agonist, clenbuterol, on the soleus muscle of adult female Sprague-Dawley rats. Animals (4 months old) were divided into two groups: clenbuterol treated (CL,n=7) (2 mg kg⁻¹ body mass injected subcutaneously every other day), and control (CON,n=7) (injected with isotonic saline). Post-treatment body weights were ≈ 5% greater in the CL group compared to CON (P<0.05). Polyacrylamide gel electrophoresis (SDS-PAGE) of soleus myofibrillar protein indicated a clenbuterol-induced decrease (P<0.05) in the relative percentage of type I myosin heavy chain (MHC) with a concomitant increase (P<0.05) in type IIdx MHC, while the proportion of type IIa MHC was unaffected. ATPase fiber typing revealed increases (P<0.05) in the proportion of type II fibers expressed both as a percentage of total fiber number and total cross-sectional area (CSA). Finally, mean type II fiber CSA was ≈25% greater (P<0.05) in the CL groups as compared to the CON group. These data indicate that clenbuterol treatment results in alterations in the MHC phenotype and an increased proportion of type II fiber CSA in the soleus of adult rats. These observations were due to an increase in the total number of type II fibers, as well as hypertrophy of these fibers. Thus, the relative increase in the number of histochemically determined type II fibers and the emergence of the normally unexpressed type IIdx MHC isoform in the soleus suggest a clenbuterol-induced transition of muscle fiber phenotype as well as selective hypertrophy of the type 11 fibers.     

Reduced expression of MyHC slow isoform in rat soleus during unloading is accompanied by alterations of endogenous inhibitors of calcineurin/NFAT signaling pathway

Under muscle disuse conditions decrease of expression of MyHC of slow type, and sometimes of type IIa, as well as upregulation of expression of IIb and IId/x isoforms were observed. Through dephosphorylation and entry of NFAT molecules to the nucleus calcineurin/NFATc1 signaling pathway promotes upregulation of the slow MyHC expression. We supposed that downregulation of calcineurin pathway took place during unloading. The study was aimed to analyze the states of the myonuclear NFAT inhibitors calsarcin I (CSI) and calsarcin II (CSII) (also referred to as myozenin II and I) and GSK3β in rat soleus during hindlimb suspension (HS). Male Wistar rats were subjected to 3, 7 and 14 day of HS. We found that after 3 days of HS the content of CSII mRNA twofold increased in soleus as compared to the controls. This level was increased by more than fivefold (as compared to control) after 2 weeks of HS. The increase of CSII mRNA expression may be explained as the mechanism of stabilization of fast phenotype. We found that from the 3 day till 14 day of HS the content of MuRF-1 and MuRF-2 in the nuclear fraction fourfold to fivefold increased in HS soleus. We supposed that nuclear import of the MuRFs allows to promote CSII expression during unloading. We also observed the decline of the phosphorylated GSK3β content in the nuclear extract of the soleus tissue. Thus decline of slow MyHC expression characteristic for the unloading conditions is accompanied with the increased expression and activation of the factors known to prevent NFAT accumulation in the myonuclei.     

Effects of 20-week intermittent cold-water-immersion on phenotype and myonuclei in single fibers of rat hindlimb muscles

The effects of 20 weeks of intermittent cold-water-immersion on myosin heavy chain (MHC) expression,cross-sectional area (CSA), myonuclear number, and myonuclear domain size in isolated single fiber of soleus and extensor digitorum longus (EDL) muscles were studied in male Wistar rats. Cold exposure was accomplished by submerging the rats in shoulder-deep water, maintained at approximately 18 degrees C, for 1 hour/day, 5 days/week and for 20 weeks. Cold exposure resulted in a significant inhibition of body and soleus muscle weight gain. The percent type IIa MHC fibers of EDL muscle was increased, whereas that of type IIa + b MHC fibers was less in cold-exposed group than controls (p < 0.05). The mean CSA and myonuclear number in type I MHC fibers of soleus muscle in cold-exposed group were significantly less than controls. Myonuclear domain in type IIa fibers of EDL in the cold-exposed group was greater than controls (p < 0.05). It is suggested that prolonged cold exposure causes the fiber-type-specific adaptation in rat hindlimb muscles. It is further indicated that cold-exposure-related modulation of myonuclear number was closely related to reduction of fiber CSA, not the shift of fiber phenotype.     

Author index

This study examined the time course change of the capillary luminal diameter and the number of capillaries in the rat soleus muscle during hind‐limb suspension. Male Wistar rats were divided into 1 and 3 weeks of hind‐limb suspension (HS) groups (HS‐1 and HS‐3). The HS groups were compared with age‐matched control groups. All morphometric parameters with respect to capillary and muscle fibre cross‐sectional area were determined in perfusion‐fixed soleus muscles. After 1 and 3 weeks of hind‐limb suspension, the mean muscle fibre cross‐sectional area was significantly decreased in HS‐1 (–32.0%) and HS‐3 (–59.3%) compared with age‐matched control groups. Despite a lower capillary‐to‐fibre ratio (HS‐1, –19.3%; HS‐3, –21.2%), the capillary density was unchanged in HS‐1 and significantly increased in HS‐3 compared with age‐matched control groups. The mean capillary luminal diameter was significantly smaller in HS‐1 (–19.9%) and HS‐3 (–21.9%) than in the age‐matched control groups. The capillary‐to‐fibre perimeter ratio which indicates the capillary surface area available for gas exchange between blood and tissue did not significantly differ between control groups and HS groups. In conclusion, the morphometrical adaptations in rat soleus with the suspension involved changes in both the capillary luminal diameter and number of capillaries, and the change in capillary surface area was proportional to the degree of muscle atrophy in HS groups.     

Effects of running exercise during recovery from hindlimb unloading on soleus muscle fibers and their spinal motoneurons in rats

The effects of hindlimb unloading and recovery with or without running exercise on morphological and metabolic properties of soleus muscle fibers and their spinal motoneurons in rats were investigated. Ten-week-old rats were hindlimb suspended for 2 weeks and thereafter were rehabilitated with or without voluntary running exercise for 2 weeks. A decreased percentage of type I fibers and atrophy of all types of fibers were observed after hindlimb unloading. In addition, decreased oxidative enzyme activity of all types of fibers was observed after hindlimb unloading. In contrast, an improvement in the decreased percentage of type I fibers, decreased fiber cross-sectional area, and decreased fiber oxidative enzyme activity was observed after recovery with running exercise, but not without running exercise. There were no changes in the number, cell body size, or oxidative enzyme activity of motoneurons innervating the soleus muscle after hindlimb unloading or recovery with or without running exercise. These results indicate that running exercise is beneficial for the recovery of the decreased percentage of type I fibers and the atrophy and decreased oxidative enzyme activity of all types of fibers in the soleus muscle induced by hindlimb unloading and that there are no changes in morphological or metabolic properties of spinal motoneurons innervating the soleus muscle following decreased or increased neuromuscular activity.     

Ultrastructure of Skeletal Muscles in Mice Lacking Muscle‐Specific VEGF Expression

Vascular endothelial growth factor‐A (VEGF) influences several physiological processes including endothelial cell function, angiogenesis and maintenance of organ/tissue capillarity. While the functional aspects of VEGF were vigorously investigated, only little detail is known on structural integrity of skeletal muscle fibers and capillaries in mice lacking VEGF expression in their muscles. Therefore, we assessed systematically the architecture of the glycolytic plantaris and the oxidative soleus muscles obtained from muscle‐specific VEGF knockout (mVEGF‐KO, n = 7) mice and their wild‐type (WT, n = 7) littermates by morphometry after transmission electron microscopy. The capillary/fiber ratio was lower (plantaris: ?63.5%; soleus: ?54.8%; P ≤ 0.05) in mVEGF‐KO mice than in WT mice. In plantaris, quantification of volume density (Vv) of compartments revealed higher Vv of total mitochondria (+56.5%, P ≤ 0.05) as well as higher Vv‐values for both intrafibrillar (+39%; P ≤ 0.05) and subsarcolemmal (+220%; P ≤ 0.05) mitochondrial pools in mVEGF‐KO mice than WT mice. The capillary phenotype also differed (P ≤ 0.05) between the two mouse‐strains: Vv (–17.4%), absolute area size (–19.1%) and thickness (–19.6%) of the endothelium layer were lower and Vv of capillary lumen (+15.1%) was higher in mVEGF‐KO mice than in WT littermates. In soleus, mitochondrial Vv in fibers and the structural indicators specific to the capillary phenotype exhibited the same tendency in differences between the mouse strains without reaching statistical significance. Our morphometric analysis demonstrates that the lower capillary supply in plantaris of mVEGF‐KO mice is accompanied by higher mitochondrial Vv in muscle fibers as well as lumen dilation and endothelium thinning of capillaries. These structural alterations were more pronounced in a glycolytic than an oxidative muscle. Anat Rec, 300:2239–2249, 2017. © 2017 Wiley Periodicals, Inc.     

Contractile properties of rat soleus motor units following 14 days of hindlimb unloading

The purpose of this study was to compare the isometric contractile properties of rat soleus motor units after 14 days of hindlimb unloading (HU) to those under control conditions. The motor units (MU) were classified using two mechanical criteria: the presence or not of a sag during unfused tetani and the value of the twitch time-to-peak (TTP). Under control conditions, the soleus muscle was composed of 85% of slow-type (sag −, TTP > 20 ms) and 15% of fast-type (sag +, TTP < 20 ms) units. Following HU, these two populations were still present and results showed: (1) large decreases in their maximal tetanic tensions (of −67% and −60% for slow- and fast-type, respectively), and (2) changes in their relative proportions, i.e. a decrease in the percentage of slow-type units and a twofold increase in the percentage of fast-type units were observed. These latter changes might be the consequence of a complete transformation of slow-towards fast-type units. A third population appeared in the HU solei, 26% of the samples, combining the presence of a sag and speed-related properties between those of slow- and fast-type units. These slow-intermediate units might come from slow units partially transformed into a faster type during HU. Thus the present study showed that unloading conditions induced a reorganisation of the soleus motor unit profile. The complete or partial transformation of the motor units could be related to the changes in the electromyographical activity of the unloaded soleus. Received: 30 June 1995/Received after revision: 12 January 1996/Accepted: 22 January 1996     

The Effects of Age and Hindlimb Supension on the Levels of Expression of the Myogenic Regulatory Factors Myod and Myogenin in Rat Fast and Slow Skeletal Muscles

In this study we tested the hypothesis that, compared to young adult rats, senescent rats have a reduced ability to respond to muscle unloading. Unloading of the muscles was induced by hindlimb suspension (HS) of young adult and senescent rats for 21 days. Plantaris muscles from young adult rats had significantly higher levels of myogenin mRNA and protein (890 % and 314 %, respectively, P < 0.05) than plantaris muscles from senescent rats and also a higher MyoD mRNA level (280 %, P < 0.05), but ageing did not increase MyoD protein levels. Although HS did not increase plantaris mRNA or protein levels of myogenin or MyoD in senescent rats (P = 0.22), myogenin mRNA and protein levels increased by 850 % and 580 % respectively, and MyoD mRNA and protein levels by 235 % and 1600 %, respectively in young adult rats (P < 0.05). Soleus muscles from senescent rats had 150 % and 85 % greater myogenin and MyoD mRNA levels, respectively (P < 0.05), than soleus muscles from young adult rats, whereas protein levels of myogenin were similar (P > 0.05) and MyoD protein levels were 60 % lower in the muscle of senescent rats (P < 0.05). In young rats, soleus muscle mRNA levels of myogenin and MyoD were not altered by HS but myogenin protein levels decreased by 57 % (P < 0.05) whereas MyoD protein levels increased by 187 % (P < 0.05). In senescent rats, HS decreased soleus muscle myogenin mRNA and protein levels by 42 % and 26 % respectively (P < 0.05), but MyoD protein and mRNA levels were not changed. MRF4 levels were not affected by ageing in either muscle. These data suggest that ageing reduces the ability of fast muscles to increase myogenin protein levels, and prevents both fast and slow muscles from increasing MyoD protein levels during muscle unloading. Experimental Physiology (2001) 86.4, 509-517.     

Cardiotoxin-induced skeletal muscle injury elicits profound changes in anabolic and stress signaling,and muscle fiber type composition

To improve muscle healing upon injury, it is of importance to understand the interplay of key signaling pathways during muscle regeneration. To study this, mice were injected with cardiotoxin (CTX) or PBS in the Tibialis Anterior muscle and were sacrificed 2, 5 and 12 days upon injection. The time points represent different phases of the regeneration process, i.e. destruction, repair and remodeling, respectively. Two days upon CTX-injection, p-mTORC1 signaling and stress markers such as BiP and p-ERK1/2 were upregulated. Phospho-ERK1/2 and p-mTORC1 peaked at d5, while BiP expression decreased towards PBS levels. Phospho-FOXO decreased 2 and 5 days following CTX-injection, indicative of an increase in catabolic signaling. Furthermore, CTX-injection induced a shift in the fiber type composition, characterized by an initial loss in type IIa fibers at d2 and at d5. At d5, new type IIb fibers appeared, whereas type IIa fibers were recovered at d12. To conclude, CTX-injection severely affected key modulators of muscle metabolism and histology. These data provide useful information for the development of strategies that aim to improve muscle molecular signaling and thereby recovery.

     

Thermal preconditioning prevents fiber type transformation of the unloading induced-atrophied muscle in rats

Muscle atrophy is accompanied by a slow-to-fast transformation of the slow muscle, e.g., the soleus muscle, which is characterized by a decrease in the expression of the slow myosin heavy chain (MyHC) isoform. Heat stress before hindlimb unloading, i.e., thermal preconditioning, has been shown to reduce the rate of disuse-induced muscle atrophy. The present study examined whether thermal preconditioning could prevent a slow-to-fast transformation of the MyHC isoform through the induction of heat-shock protein (HSP) 72. Thermally preconditioned rats (Heat + HU) were individually placed in an environmentally controlled heat chamber for 1 h before hindlimb unloading for 2 weeks (HU). Although the mean fiber cross-sectional areas of the soleus muscle decreased in the HU and Heat + HU group, the loss of myofibrillar protein was attenuated in the Heat + HU group. Furthermore, a slow-to-fast transformation of MyHC isoform was inhibited in the Heat + HU group with the overexpression of HSP72. These results indicate that thermal preconditioning before hindlimb unloading attenuates the decrease of the slow MyHC isoform in the soleus muscle. Therefore, thermal preconditioning provides a new approach to prevent disuse-induced fiber type transformation of skeletal muscle.