Satellite cells of the rat soleus muscle in the process of compensatory hypertrophy combined with denervation

Compensatory hypertrophy was induced in the rat soleus muscle by sectioning the tendon of the ipsilateral gastrocnemius and plantaris muscle. Seven days after tenotomy of synergistic muscles, when soleus hypertrophy attains about 40%, the number of satellite cells (expressed as percentage of all muscle nuclei found in the same cross-sections) as revealed by electron microscopy, was increased from 5.8+/-0.06% in the normal soleus muscle to 16.6+/-1.26%. After four days' denervation of the soleus muscle the percentage of satellite cells was increased to 7.2+/-0.62%. In experiments where hypertrophy of the soleus muscle was combined with denervation three days after tenotomy of synergists, and examined after another four days (during which time it loses, as has previously been shown, over 40% of its predenervation weight), the number of satellite cells was greatly increased to 29.9+/-3.42%. This increase is apparently due to two independent processes which take place during the first postoperative period: a) mitotic division of satellite cells during the early stages of compensatory hypertrophy and b) pinching off of muscle nuclei from rapidly atrophying muscle fibres due to subsequent denervation. Activation of satellite cells was mainly manifested by expansion of smooth and especially of rough endoplasmic reticulum, a rich Golgi complex, high pinocytotic activity, increased number of ribosomes and by nuclear changes. Concomitantly with the increased number of satellite cells, proliferation of fibroblasts, macrophages and mast cells could be observed.     

Cooperative control of striated muscle mass and metabolism by MuRF1 and MuRF2

The muscle-specific RING finger proteins MuRF1 and MuRF2 have been proposed to regulate protein degradation and gene expression in muscle tissues. We have tested the in vivo roles of MuRF1 and MuRF2 for muscle metabolism by using knockout (KO) mouse models. Single MuRF1 and MuRF2 KO mice are healthy and have normal muscles. Double knockout (dKO) mice obtained by the inactivation of all four MuRF1 and MuRF2 alleles developed extreme cardiac and milder skeletal muscle hypertrophy. Muscle hypertrophy in dKO mice was maintained throughout the murine life span and was associated with chronically activated muscle protein synthesis. During ageing (months 4-18), skeletal muscle mass remained stable, whereas body fat content did not increase in dKO mice as compared with wild-type controls. Other catabolic factors such as MAFbox/atrogin1 were expressed at normal levels and did not respond to or prevent muscle hypertrophy in dKO mice. Thus, combined inhibition of MuRF1/MuRF2 could provide a potent strategy to stimulate striated muscles anabolically and to protect muscles from sarcopenia during ageing.     

Urokinase-type plasminogen activator and macrophages are required for skeletal muscle hypertrophy in mice

Adult skeletal muscle possesses remarkable potential for growth in response to mechanical loading; however, many of the cellular and molecular mechanisms involved remain undefined. The hypothesis of this study was that the extracellular serine protease, urokinase-type plasminogen activator (uPA), is required for muscle hypertrophy, in part by promoting macrophage accumulation in muscle subjected to increased mechanical loading. Compensatory muscle hypertrophy was induced in mouse plantaris (PLT) muscles by surgical ablation of synergist muscles. Following synergist ablation, PLT muscles in wild-type mice demonstrated edema and infiltration of neutrophils and macrophages but an absence of overt muscle fiber damage. Sham procedures resulted in no edema or accumulation of inflammatory cells. In addition, synergist ablation was associated with a large increase in activity of uPA in the PLT muscle. uPA-null mice demonstrated complete abrogation of compensatory hypertrophy associated with reduced macrophage accumulation, indicating that uPA is required for hypertrophy. Macrophages isolated from wild-type PLT muscle during compensatory hypertrophy expressed uPA and IGF-I, both of which may contribute to hypertrophy. To determine whether macrophages are required for muscle hypertrophy, clodronate liposomes were administered to deplete macrophages in wild-type mice; this resulted in reduced muscle hypertrophy. Decreased macrophage accumulation was associated with reduced cell proliferation but did not alter signaling through the mammalian target of rapamycin pathway. These data indicate that uPA and macrophages are required for muscle hypertrophy following synergist ablation.     

Some hormonal factors (hypophysectomy, castration and testosterone administration) modifying the course of "compensatory" muscle hypertrophy in the rat.

1. Maximum compensatory hypertrophy of the soleus and plantaris muscle in male rats is attained seven days after tenotomy of the gastrocnemius muscle (39% and 9% respectively). When tenotomy of the gastrocnemius was performed seven days ater hypophysectomy, hypertrophy in these two muscles was aproximately half that found in control animals. 2. After 81-day castration of young male rats the weight of the saleus and plantaris was reduced and hypertrophy following tenotomy of the gastrocneumius muscle did not develop. 3. Chronically castrated rats received testosterone two weeks prior to tenotomy of the gastrocnemius and a week during the muscle hypertrophy phase. Hypertrophy of the soleus in castrated rats which had received testosterone seven days after tenotomy of the gastrocnemius was 25% as compared with muscles of castrated animals. The corresponding value in the plantaris muscle was 10%. 4. These results indicate that even calf muscles of the rat, namely the soleus and plantaris muscles, are significantly affected by testosterone under these conditions, although it is not, as yet, clear whether its action is direct or indirect.     

Casein kinase 2 inactivation by Mg2+, Mn2+ and Co2+ ions

In order to elucidate the relationship between hypertension and hypertrophy in the production of heat shock proteins, we studied the induction of the HSP72 synthesis by the heart and gracilis muscles of normo (WKY) and hypertensive (SHR) rats subjected to hyperthermia (42°C±0.5 for 15 min). Two age groups were investigated in each strain: young (2 months, with developing cardiac hypertrophy) and old (18 months, with fully developed chronic cardiac hypertrophy). The gracilis muscle never developed hypertrophy, independently of hypertension or aging. 72 kDa inducible protein was determined by Western blot analysis using a specific monoclonal antibody. We also used a commercial standard, loaded on each blot, to quantitate densitometrically the signal.The heart of young SHR responds to heat shock more than their normotensive age-matched control (298.8±24.7% vs 88.3 ±8.5%, p<0.001). This response is not maintained during aging as we did not find any significant difference between normo-and hypertensive old rats after exposure to hyperthermia (43.6±5.3% vs 65.3±10.4%).Unlike the heart, the gracilis muscle shows a basal spontaneous HSP72 synthesis in both the SHR (71.4±10.8%) and WKY (40.6±11.7%) animals. There was a significant increase in HSP72 synthesis in the gracilis muscle of young SHR with respect to their control (186.2±18.7% vs 115.8±9.9%, p<0.02) which was maintained also during aging (171.9±17.3% vs 95.2±10.5%, p<0.01).In conclusion, these data show that hypertension results in an increased synthesis of HSP72 both in cardiac and gracilis muscle in response to heat shock. This abnormal response is attenuated by aging in the heart but not in the gracilis muscle. Thus, the abnormality seems to be independent from hypertrophy and linked to genetic determination of the disease.     

Satellite cell regulation following myotrauma caused by resistance exercise

It is generally accepted that the primary mechanisms governing skeletal muscle hypertrophy are satellite cell activation, proliferation, and differentiation. Specific growth factors and hormones modulate satellite cell activity during normal muscle growth, but as a consequence of resistance exercise additional regulators may stimulate satellite cells to contribute to gains in myofiber size and number. Present knowledge of the regulation of the cellular, biochemical and molecular events accompanying skeletal muscle hypertrophy after resistance exercise is incomplete. We propose that resistance exercise may induce satellite cells to become responsive to cytokines from the immune system and to circulating hormones and growth factors. The purpose of this paper is to review the role of satellite cells and growth factors in skeletal muscle hypertrophy that follows resistance exercise.     

IGF and myostatin pathways are respectively induced during the earlier and the later stages of skeletal muscle hypertrophy induced by clenbuterol,a β2‐adrenergic agonist

Clenbuterol, a β₂‐adrenergic agonist, increases the hypertrophy of skeletal muscle. Insulin‐like growth factor (IGF) is reported to work as a potent positive regulator in the clenbuterol‐induced hypertrophy of skeletal muscles. However, the precise regulatory mechanism for the hypertrophy of skeletal muscle induced by clenbuterol is unknown. Myostatin, a member of the TGFβ super family, is a negative regulator of muscle growth. The aim of the present study is to elucidate the function of myostatin and IGF in the hypertrophy of rat masseter muscle induced by clenbuterol. To investigate the function of myostatin and IGF in regulatory mechanism for the clenbuterol‐induced hypertrophy of skeletal muscles, we analysed the expression of myostatin and phosphorylation levels of myostatin and IGF signaling components in the masseter muscle of rat to which clenbuterol was orally administered for 21 days. Hypertrophy of the rat masseter muscle was induced between 3 and 14 days of oral administration of clenbuterol and was terminated at 21 days. The expression of myostatin and the phosphorylation of smad2/3 were elevated at 21 days. The phosphorylation of IGF receptor 1 (IGFR1) and akt1 was elevated at 3 and 7 days. These results suggest that myostatin functions as a negative regulator in the later stages in the hypertrophy of rat masseter muscle induced by clenbuterol, whereas IGF works as a positive regulator in the earlier stages. Copyright © 2012 John Wiley & Sons, Ltd.     

Regulation of atrial contraction by PKA and PKC during development and regression of eccentric cardiac hypertrophy

ANG II plays a major role in development of cardiac hypertrophy through its AT1 receptor subtype, whereas angiotensin-converting enzyme (ACE) inhibitors are effective in reversing effects of ANG II on the heart. The objective of this study was to investigate the role of PKA and PKC in the contractile response of atrial tissue during development and ACE inhibitor-induced regression of eccentric hypertrophy induced by aortocaval shunt. At 1 wk after surgery, sham and shunt rats were divided into captopril-treated and untreated groups for 2 wk. Then isometric contraction was assessed by electrical stimulation of isolated rat left atrial preparations superfused with Tyrode solution in the presence or absence of specific inhibitors KT-5720 (for PKA) and Ro-32-0432 (for PKC) and high Ca2+. Peak tension developed was greater in shunt than in sham hearts. However, when expressed relative to tissue mass, hypertrophied muscle showed weaker contraction than muscle from sham rats. In sham rats, peak tension developed was more affected by PKC than by PKA inhibition, whereas this differential effect was reduced in the hypertrophied heart. Treatment of shunt rats with captopril regressed left atrial hypertrophy by 67% and restored PKC-PKA differential responsiveness toward sham levels. In the hypertrophied left atria, there was an increase in the velocity of contraction and relaxation that was not evident when expressed in specific relative terms. Treatment with ACE inhibitor increased the specific velocity of contraction, as well as its PKC sensitivity, in shunt rats. We conclude that ACE inhibition during eccentric cardiac hypertrophy produces a negative trophic and a positive inotropic effect, mainly through a PKC-dependent mechanism.     

Mimecan is involved in aortic hypertrophy induced by sinoaortic denervation in rats

This study was designed to investigate whether mimecan was involved in aortic hypertrophy induced by sinoaortic denervation in rats. 8 weeks after sinoaortic denervation, when compared to sham-operated rats, sinoaortic denervated rats exhibited aortic hypertrophy and down-regulation of mimecan. Through classic univariate correlation analysis, it was found that mimecan mRNA was negatively related to extent of aortic hypertrophy. Treatment of primary cultured vascular smooth muscle cells with the Ang II (1 μM), which was found locally increased in the aortae of sinoaortic denervated rats, resulted in a reduction of mimecan expression. In vitro, knockdown of mimecan in vascular smooth muscle cells promoted cell proliferation induced by 15% of fetal bovine serum or Ang II (1 μM). We concluded that down-regulation of mimecan was involved in aortic hypertrophy induced by sinoaortic denervation in rats.     

Nifedipine does not impede clenbuterol-stimulated muscle hypertrophy.

The mechanism(s) responsible for beta2-adrenergic receptor-mediated skeletal muscle and cardiac hypertrophy remains undefined. This study examined whether calcium influx through L-type calcium channels contributed to the development of cardiac and skeletal muscle (plantaris; gastrocnemius; soleus) hypertrophy during an 8-day treatment with the beta2-adrenergic receptor agonist clenbuterol. Concurrent blockade of L-type calcium channels with nifedipine did not reverse the hypertrophic action of clenbuterol. Moreover, nifedipine treatment alone resulted in both cardiac and soleus muscle hypertrophy (6% and 7%, respectively), and this effect was additive to the clenbuterol-mediated hypertrophy in the heart and soleus muscles. The hypertrophic effects of nifedipine were not associated with increases in total beta-adrenergic receptor density, nor did nifedipine reverse clenbuterol-mediated beta-adrenergic receptor downregulation in either the left ventricle or soleus muscle. Both nifedipine and clenbuterol-induced hypertrophy increased total protein content of the soleus and left ventricle, with no change in protein concentration. In conclusion, our results support the hypothesis that beta2-adrenergic receptor agonist-induced muscle hypertrophy is mediated by mechanisms other than calcium influx through L-type calcium channels.     

IGF-1 induces human myotube hypertrophy by increasing cell recruitment

Insulin-like growth factor-1 (IGF-1) has been shown in rodents (i) in vivo to induce muscle fiber hypertrophy and to prevent muscle mass decline with age and (ii) in vitro to enhance the proliferative life span of myoblasts and to induce myotube hypertrophy. In this study, performed on human primary cultures, we have shown that IGF-1 has very little effect on the proliferative life span of human myoblasts but does delay replicative senescence. IGF-1 also induces hypertrophy of human myotubes in vitro, as characterized by an increase in the mean number of nuclei per myotube, an increase in the fusion index, and an increase in myosin heavy chain (MyHC) content. In addition, muscle hypertrophy can be triggered in the absence of proliferation by recruiting more mononucleated cells. We propose that IGF-1-induced hypertrophy can involve the recruitment of reserve cells in human skeletal muscle.     

Turnover of muscle protein in the fowl. Collagen content and turnover in cardiac and skeletal muscles of the adult fowl and the changes during stretch-induced growth

The collagen content and the rate of collagen synthesis were measured in the anterior and posterior latissimus dorsi muscles and in heart from fully grown fowl. This was done by measuring the proline/hydroxyproline ratios in the muscle and by a constant infusion of [¹⁴C]proline. These measurements were also made during the hypertrophy of the anterior muscle in response to the attachment of a weight to one wing of the fowl. In the non-growing muscles the collagen content was higher in the anterior muscle (22.8% of total protein) than in the posterior muscle (9.5% of total protein) and lowest in the heart (3.8% of total protein). In the two skeletal muscles a little over half of the collagen was accounted for by internal collagen (i.e. perimysium and endomysium). Collagen synthesis in these non-growing muscles occurred at 0.59%/day in each of the two skeletal muscles and at 0.88%/day in the cardiac muscle. During hypertrophy the collagen content of the anterior muscle increased, but not as fast as intracellular protein, so that after 58 days the concentration had fallen from 22.8 to 14.4% of total protein. This may have resulted from an incomplete production of the epimysial sheath, since the concentration of internal collagen did not fall and as a result accounted for over 80% of the total in the enlarged muscle. Collagen synthesis increased 8-fold during the first week of the hypertrophy, but never amounted to more than 4% of the total muscle protein synthesis. When the net accumulation of collagen is compared with the increased rate of synthesis it is concluded that between 30 and 70% of the newly synthesized collagen may have been degraded.     

Stimulation of slow skeletal muscle fiber gene expression by calcineurin in vivo

Adult skeletal muscle fibers can be categorized into fast and slow twitch subtypes based on specialized contractile and metabolic properties and on distinctive patterns of muscle gene expression. Muscle fiber-type characteristics are dependent on the frequency of motor nerve stimulation and are thought to be controlled by calcium-dependent signaling. The calcium, calmodulin-dependent protein phosphatase, calcineurin, stimulates slow fiber-specific gene promoters in cultured skeletal muscle cells, and the calcineurin inhibitor, cyclosporin A, inhibits slow fiber gene expression in vivo, suggesting a key role of calcineurin in activation of the slow muscle fiber phenotype. Calcineurin has also been shown to induce hypertrophy of cardiac muscle and to mediate the hypertrophic effects of insulin-like growth factor-1 on skeletal myocytes in vitro. To determine whether activated calcineurin was sufficient to induce slow fiber gene expression and hypertrophy in adult skeletal muscle in vivo, we created transgenic mice that expressed activated calcineurin under control of the muscle creatine kinase enhancer. These mice exhibited an increase in slow muscle fibers, but no evidence for skeletal muscle hypertrophy. These results demonstrate that calcineurin activation is sufficient to induce the slow fiber gene regulatory program in vivo and suggest that additional signals are required for skeletal muscle hypertrophy.     

Role of damage and management in muscle hypertrophy: Different behaviors of muscle stem cells in regeneration and hypertrophy

Skeletal muscle is a dynamic tissue with two unique abilities; one is its excellent regenerative ability, due to the activity of skeletal muscle–resident stem cells named muscle satellite cells (MuSCs); and the other is the adaptation of myofiber size in response to external stimulation, intrinsic factors, or physical activity, which is known as plasticity. Low physical activity and some disease conditions lead to the reduction of myofiber size, called atrophy, whereas hypertrophy refers to the increase in myofiber size induced by high physical activity or anabolic hormones/drugs. MuSCs are essential for generating new myofibers during regeneration and the increase in new myonuclei during hypertrophy; however, there has been little investigation of the molecular mechanisms underlying MuSC activation, proliferation, and differentiation during hypertrophy compared to those of regeneration. One reason is that ‘degenerative damage’ to myofibers during muscle injury or upon hypertrophy (especially overloaded muscle) is believed to trigger similar activation/proliferation of MuSCs. However, evidence suggests that degenerative damage of myofibers is not necessary for MuSC activation/proliferation during hypertrophy. When considering MuSC-based therapy for atrophy, including sarcopenia, it will be indispensable to elucidate MuSC behaviors in muscles that exhibit non-degenerative damage, because degenerated myofibers are not present in the atrophied muscles. In this review, we summarize recent findings concerning the relationship between MuSCs and hypertrophy, and discuss what remains to be discovered to inform the development and application of relevant treatments for muscle atrophy.     

Protein synthesis, amino acid uptake, and pools during isoproterenol-induced hypertrophy of the rat heart and tibialis muscle

Chronic administration of isoproterenol (ISO) produces hypertrophy of the rat heart and tibialis muscle. With doses of 0.1, 0.3, and 0.6 mg/kg, hypertrophy of the heart is significantly by the 3rd day of treatment. Maximum cardiac enlargement attained with doses of 0.3 and 0.6 mg/kg occurs after 21 days and averages 40% above control values. ISO increases tibialis muscle weight by 15%. Incorporation of 14C-labeled amino acids into total heart and tibialis muscle protein is stimulated by ISO. Maximum stimulation occurs 2-3 h after the fifth daily injection of ISO. The stimulation of incorporation is greater during the first few days of treatment and decreases gradually thereafter. A single injection of ISO decreases the total amino acid concentration of the serum, heart and tibialis muscle whereas the rate of amino acid uptake by the heart and tibialis muscle is increased by ISO. The production of hypertrophy of the heart and tibialis muscle in diabetic or castrated animals by ISO suggests that insulin and testosterone are not essential in the mechanism of ISO-induced hypertrophy.     

Effect of hindlimb immobilization and recovery on compensatory hypertrophied rat plantaris muscle

Compensatory hypertrophy of the rat plantaris muscle (PLT) was induced by ipsilateral gastrocnemius muscle ablation. Following 8 weeks (wks) of hypertrophy, hindlimbs were cast immobilized (HI) for 4 weeks after which weight bearing was unrestricted for 8 wks (recovery). Compensatory hypertrophy increased PLT wet weight/body weight ratio (83%), muscle fiber cross-sectional areas (1.5 to 2 fold), and the percent of slow oxidative (% SO) fibers (2 fold) in the experimental compared to the contralateral sham control muscle. PLT protein content and maximal activities of phosphofructokinase (PFK), mitochondrial glycerol phosphate dehydrogenase, and succinate dehydrogenase were unaltered with muscle hypertrophy. HI produced significant decreases in PFK activity (50%) and muscle fiber cross-sectional areas (50%) but did not significantly change the histochemical myofibrillar ATPase profile. Following remobilization, muscle weight/body weight ratio and maximal enzyme activities recovered to that of aged matched controls. Muscle fiber areas returned to pre-immobilization sizes but were approximately 25% smaller than aged matched control hypertrophy muscles. The % SO fibers in the hypertrophied muscle remained higher than controls but did not return to pre-immobilization values. These results indicate that biochemical and histochemical characteristics of hypertrophied rat PLT recover from HI during 8 wks of normal weight bearing similar to that of normal control muscle. However, the recovery time period was insufficient to allow complete compensation of fiber size to that of the age-matched control animals.     

Effective fiber hypertrophy in satellite cell-depleted skeletal muscle

An important unresolved question in skeletal muscle plasticity is whether satellite cells are necessary for muscle fiber hypertrophy. To address this issue, a novel mouse strain (Pax7-DTA) was created which enabled the conditional ablation of >90% of satellite cells in mature skeletal muscle following tamoxifen administration. To test the hypothesis that satellite cells are necessary for skeletal muscle hypertrophy, the plantaris muscle of adult Pax7-DTA mice was subjected to mechanical overload by surgical removal of the synergist muscle. Following two weeks of overload, satellite cell-depleted muscle showed the same increases in muscle mass (approximately twofold) and fiber cross-sectional area with hypertrophy as observed in the vehicle-treated group. The typical increase in myonuclei with hypertrophy was absent in satellite cell-depleted fibers, resulting in expansion of the myonuclear domain. Consistent with lack of nuclear addition to enlarged fibers, long-term BrdU labeling showed a significant reduction in the number of BrdU-positive myonuclei in satellite cell-depleted muscle compared with vehicle-treated muscle. Single fiber functional analyses showed no difference in specific force, Ca(2+) sensitivity, rate of cross-bridge cycling and cooperativity between hypertrophied fibers from vehicle and tamoxifen-treated groups. Although a small component of the hypertrophic response, both fiber hyperplasia and regeneration were significantly blunted following satellite cell depletion, indicating a distinct requirement for satellite cells during these processes. These results provide convincing evidence that skeletal muscle fibers are capable of mounting a robust hypertrophic response to mechanical overload that is not dependent on satellite cells.     

EMG activity in "compensatory" muscle hypertrophy

The functional elimination of synergistic muscles leads to dramatic muscle hypertrophy. However, neither resting EMG activity recorded by an implanted electrode array, nor activity during locomotion have substantiated the assumption that the hypertrophy in the rat soleus muscle is caused by hyperactivity.     

Integrin signaling's potential for mediating gene expression in hypertrophying skeletal muscle.

Overloaded skeletal muscle undergoes dramatic shifts in gene expression, which alter both the phenotype and mass. Molecular biology techniques employing both in vivo and in vitro hypertrophy models have demonstrated that mechanical forces can alter skeletal muscle gene regulation. This review's purpose is to support integrin-mediated signaling as a candidate for mechanical load-induced hypertrophy. Research quantifying components of the integrin-signaling pathway in overloaded skeletal muscle have been integrated with knowledge regarding integrins role during development and cardiac hypertrophy, with the hope of demonstrating the pathway's importance. The role of integrin signaling as an integrator of mechanical forces and growth factor signaling during hypertrophy is discussed. Specific components of integrin signaling, including focal adhesion kinase and low-molecular-weight GTPase Rho are mentioned as downstream targets of this signaling pathway. There is a need for additional mechanistic studies capable of providing a stronger linkage between integrin-mediated signaling and skeletal muscle hypertrophy; however, there appears to be abundant justification for this type of research.