Overload-induced Skeletal Muscle Hypertrophy Research Articles

Making the case for resistance training in improving vascular function and skeletal muscle capillarization.

Through decades of empirical data, it has become evident that resistance training (RT) can improve strength/power and skeletal muscle hypertrophy. Yet, until recently, vascular outcomes have historically been underemphasized in RT studies, which is underscored by several exercise-related reviews supporting the benefits of endurance training on vascular measures. Several lines of evidence suggest large artery diameter and blood flow velocity increase after a single bout of resistance exercise, and these events are mediated by vasoactive substances released from endothelial cells and myofibers (e.g., nitric oxide). Weeks to months of RT can also improve basal limb blood flow and arterial diameter while lowering blood pressure. Although several older investigations suggested RT reduces skeletal muscle capillary density, this is likely due to most of these studies being cross-sectional in nature. Critically, newer evidence from longitudinal studies contradicts these findings, and a growing body of mechanistic rodent and human data suggest skeletal muscle capillarity is related to mechanical overload-induced skeletal muscle hypertrophy. In this review, we will discuss methods used by our laboratories and others to assess large artery size/function and skeletal muscle capillary characteristics. Next, we will discuss data by our groups and others examining large artery and capillary responses to a single bout of resistance exercise and chronic RT paradigms. Finally, we will discuss RT-induced mechanisms associated with acute and chronic vascular outcomes.

Mechanisms of mechanical overload-induced skeletal muscle hypertrophy: current understanding and future directions.

Mechanisms underlying mechanical overload-induced skeletal muscle hypertrophy have been extensively researched since the landmark report by Morpurgo (1897) of "work-induced hypertrophy" in dogs that were treadmill trained. Much of the preclinical rodent and human resistance training research to date supports that involved mechanisms include enhanced mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling, an expansion in translational capacity through ribosome biogenesis, increased satellite cell abundance and myonuclear accretion, and postexercise elevations in muscle protein synthesis rates. However, several lines of past and emerging evidence suggest that additional mechanisms that feed into or are independent of these processes are also involved. This review first provides a historical account of how mechanistic research into skeletal muscle hypertrophy has progressed. A comprehensive list of mechanisms associated with skeletal muscle hypertrophy is then outlined, and areas of disagreement involving these mechanisms are presented. Finally, future research directions involving many of the discussed mechanisms are proposed.

CCN2 participates in overload-induced skeletal muscle hypertrophy

The regulation of skeletal muscle growth following pro-hypertrophic stimuli requires a coordinated response by different cell types that leads to extracellular matrix (ECM) remodeling and increases in muscle cross-sectional area. Indeed, matricellular proteins serve a key role as communication vehicles that facilitate the propagation of signaling stimuli required for muscle adaptation to environmental challenges. We found that the matricellular protein cellular communication network factor 2 (CCN2), also known as connective tissue growth factor (CTGF), is induced during a time course of overload-driven skeletal muscle hypertrophy in mice. To elucidate the role of CCN2 in mediating the hypertrophic response, we utilized genetically engineered mouse models for myofiber-specific CCN2 gain- and loss-of-function and then examined their response to mechanical stimuli through muscle overload. Interestingly, myofiber-specific deletion of CCN2 blunted muscle's hypertrophic response to overload without interfering with ECM deposition. On the other hand, when in excess through transgenic CCN2 overexpression, CCN2 was efficient in promoting overload-induced aberrant ECM accumulation without affecting myofiber growth. Altogether, our genetic approaches highlighted independent ECM and myofiber stress adaptation responses, and positioned CCN2 as a central mediator of both. Mechanistically, CCN2 acts by regulating focal adhesion kinase (FAK) mediated transduction of overload-induced extracellular signals, including interleukin 6 (IL6), and their regulatory impact on global protein synthesis in skeletal muscle. Overall, our study highlights the contribution of muscle-derived extracellular matrix factor CCN2 for proper hypertrophic muscle growth.

AMPK is indispensable for overload-induced muscle glucose uptake and glycogenesis but dispensable for inducing hypertrophy in mice.

Chronic muscle loading (overload) induces skeletal muscles to undergo hypertrophy and to increase glucose uptake. Although AMP-activated protein kinase (AMPK) reportedly serves as a negative regulator of hypertrophy and a positive regulator of glucose uptake, its role in overload-induced skeletal muscle hypertrophy and glucose uptake is unclear. This study aimed to determine whether AMPK regulates overload-induced hypertrophy and glucose uptake in skeletal muscles. To this end, skeletal muscle overload was induced through unilateral synergist ablations in wild-type (WT) and transgenic mice, expressing the dominant-negative mutation of AMPK (AMPK-DN). After 14days, parameters, including muscle fiber cross-sectional area (CSA), glycogen level, and in vivo [3 H]-2-deoxy-D-glucose uptake, were assessed. No significant difference was observed in body weight or blood glucose level between the WT and AMPK-DN mice. However, the 14-day muscle overload activated the AMPK pathway in WT mice skeletal muscle, whereas this response was impaired in the AMPK-DN mice. Despite a normal CSA gain in each fiber type, the AMPK-DN mice demonstrated a significant impairment of overload-induced muscle glucose uptake and glycogenesis, compared to WT mice. Moreover, 14-day overload-induced changes in GLUT4 and HKII expression levels were reduced in AMPK-DN mice, compared to WT mice. This study demonstrated that AMPK activation is indispensable for overload-induced muscle glucose uptake and glycogenesis; however, it is dispensable for the induction of hypertrophy in AMPK-DN mice. Furthermore, the AMPK/GLUT4 and HKII axes may regulate overload-induced muscle glucose uptake and glycogenesis.

Glutamine supplementation versus functional overload in extensor digitorum longus muscle hypertrophy

• We compared glutamine supplementation and overload inducing EDL muscle hypertrophy. • Glutamine and overload raised muscle fiber cross-sectional area to a similar extent. • Glutamine potentiated the overload-induced increase in the resistance to fatigue. Abstract Glutamine levels directly associate with total protein content in cultured skeletal muscle cells, whereas glutamine supplementation enhances skeletal muscle mass in catabolic experimental conditions. We compared the effect of glutamine administration on Extensor Digitorum Longus muscle (EDL) weight, fiber cross-sectional area (CSA), contractile activity, and protein metabolism signaling with a functional overload-induced skeletal muscle hypertrophy protocol. Glutamine supplementation raised the predominance of EDL muscle fibers with CSA between 1001 and 2000 μm2 (49.7 %), the p-4E-BP1/total 4E-BP1 ratio, and the effect of overload on resistance to fatigue. The proportion of the EDL muscle fiber CSA distribution for the combination of both treatments was similar to that induced by overload or glutamine separately; 54.3 % muscle fibers with CSA between 1001 and 2000 μm². Glutamine supplementation did not markedly affect the changes induced by overload on protein synthesis signaling pathways, except for a further increase of the p-4E-BP1/total 4E-BP1 ratio. The effect of glutamine on EDL muscle fiber CSA distribution and protein synthesis signaling mimicked the response to overload. The association of glutamine and overload induced EDL muscle hypertrophy further increased the resistance to fatigue.

Effect of prior chronic aerobic exercise on overload-induced skeletal muscle hypertrophy in mice.

This study aimed to examine how regular aerobic training can affect the muscle hypertrophy induced by overloading. Male C57BL/6J mice were randomly divided into three groups: rest group, low-intensity aerobic exercise group, and high-intensity aerobic exercise group. Mice in the exercise groups were assigned to run at a speed of 10 m/min (low-intensity) or 25 m/min (high-intensity) for 30 min/day, five days/week, for four weeks. Then, the right hind leg gastrocnemius muscles were surgically removed to overload the plantaris and soleus muscles, while the left hind leg was subjected to a sham-operation. Both the plantaris and soleus muscles grew larger in the overloaded legs than those in the sham-operated legs. Muscle growth increased in the plantaris muscles in the low-intensity exercise group compared to that in the rest or high-intensity exercise groups at one and two weeks after overloading. This enhancement was not observed in the soleus muscles. Consistently, we observed changes in the expression of proteins involved in anabolic intracellular signaling, including Akt, mechanistic target of rapamycin (mTOR), and p70S6K, in the plantaris muscles. Our data showed for the first time that chronic low-intensity aerobic exercise precipitates overload-induced muscle growth.

Overload-induced skeletal muscle hypertrophy is not impaired in STZ-diabetic rats.

The aim of this study was to evaluate the effect of overload-induced hypertrophy on extensor digitorum longus (EDL) and soleus muscles of streptozotocin-induced diabetic rats. The overload-induced hypertrophy and absolute tetanic and twitch forces increases in EDL and soleus muscles were not different between diabetic and control rats. Phospho-Akt and rpS6 contents were increased in EDL muscle after 7 days of overload and returned to the pre-overload values after 30 days. In the soleus muscle, the contents of total and phospho-Akt and total rpS6 were increased in both groups after 7 days. The contents of total Akt in controls and total rpS6 and phospho-Akt in the diabetic rats remained increased after 30 days. mRNA expression after 7 days of overload in the EDL muscle of control and diabetic animals showed an increase in MGF and follistatin and a decrease in myostatin and Axin2. The expression of FAK was increased and of MuRF-1 and atrogin-1 decreased only in the control group, whereas Ankrd2 expression was enhanced only in diabetic rats. In the soleus muscle caused similar changes in both groups: increase in FAK and MGF and decrease in Wnt7a, MuRF-1, atrogin-1, and myostatin. Differences between groups were observed only in the increased expression of follistatin in diabetic animals and decreased Ankrd2 expression in the control group. So, insulin deficiency does not impair the overload-induced hypertrophic response in soleus and EDL muscles. However, different mechanisms seem to be involved in the comparable hypertrophic responses of skeletal muscle in control and diabetic animals.

The transcriptional coactivator PGC-1α is dispensable for chronic overload-induced skeletal muscle hypertrophy and metabolic remodeling

Skeletal muscle mass loss and dysfunction have been linked to many diseases. Conversely, resistance exercise, mainly by activating mammalian target of rapamycin complex 1 (mTORC1), promotes skeletal muscle hypertrophy and exerts several therapeutic effects. Moreover, mTORC1, along with peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), regulates skeletal muscle metabolism. However, it is unclear whether PGC-1α is required for skeletal muscle adaptations after overload. Here we show that although chronic overload of skeletal muscle via synergist ablation (SA) strongly induces hypertrophy and a switch toward a slow-contractile phenotype, these effects were independent of PGC-1α. In fact, SA down-regulated PGC-1α expression and led to a repression of energy metabolism. Interestingly, however, PGC-1α deletion preserved peak force after SA. Taken together, our data suggest that PGC-1α is not involved in skeletal muscle remodeling induced by SA.

Vitamin C administration attenuates overload‐induced skeletal muscle hypertrophy in rats

This study aimed to investigate the effects of vitamin C administration on skeletal muscle hypertrophy induced by mechanical overload in rats. Male Wistar rats were randomly assigned to three groups: (i) sham-operated group (n = 8), (ii) placebo-administered group (n = 8) and (iii) vitamin C-administered group (n = 8). In the placebo-administered and vitamin C-administered groups, the gastrocnemius and soleus muscles of the right hindlimb were surgically removed to overload the plantaris muscle. Vitamin C (500 mg kg(-1)) was orally administered to the vitamin C-administered group once a day for 14 days. Synergist muscle ablation caused significant increases in wet weight and protein concentration of the plantaris muscle in both the placebo-administered (P < 0.01) and vitamin C-administered groups (P < 0.01) compared with the sham-operated group (SHA). However, the magnitude of plantaris muscle hypertrophy (expressed as a percentage of the contralateral plantaris muscle) was significantly smaller (P < 0.01) in the vitamin C-administered group (141%) than in the placebo-administered group (PLA) (152%). Compared with the SHA, only the PLA showed higher expressions of phosphorylated p70s6k and Erk1/2 (positive regulators of muscle protein synthesis) and a lower expression of atrogin-1 (a muscle atrophy marker). Concentrations of vitamin C and oxidative stress markers in the overloaded muscle were similar between the placebo-administered and vitamin C-administered groups. Oral vitamin C administration can attenuate overload-induced skeletal muscle hypertrophy, which may have implications for antioxidant supplementation during exercise training.

Early activation of mTORC1 signalling in response to mechanical overload is independent of phosphoinositide 3‐kinase/Akt signalling

The mammalian target of rapamycin complex 1 (mTORC1) functions as a central integrator of a wide range of signals that modulate protein metabolism and cell growth. However, the contributions of individual pathways regulating mTORC1 activity in skeletal muscle are poorly defined. The purpose of this study was to determine the regulatory mechanisms that contribute to mTORC1 activation during mechanical overload-induced skeletal muscle hypertrophy. Consistent with previous studies, mechanical overload induced progressive hypertrophy of the plantaris muscle which was associated with significant increases in total RNA content and protein metabolism. mTORC1 was activated after a single day of overload as indicated by a significant increase in S6K1 phosphorylation at T389 and T421/S424. In contrast, Akt activity, as assessed by Akt phosphorylation status (T308 and S473), phosphorylation of direct downstream targets (glycogen synthase kinase 3 β, proline-rich Akt substrate 40 kDa and tuberous sclerosis 2 (TSC2)) and a kinase assay, was not significantly increased until 2–3 days of overload. Inhibition of phosphoinositide 3-kinase (PI3K) activity by wortmannin was sufficient to block insulin-dependent signalling but did not prevent the early activation of mTORC1 in response to overload. We identified that the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)-dependent pathway was activated at day 1 after overload. In addition, a target of MEK/ERK signalling, phosphorylation of TSC2 at S664, was also increased at this early time point. These observations demonstrate that in vivo, mTORC1 activation at the early phase of mechanical overload in skeletal muscle occurs independently of PI3K/Akt signalling and provide evidence that the MEK/ERK pathway may contribute to mTORC1 activation through phosphorylation of TSC2.

Pitfalls in target mRNA quantification for real-time quantitative RT-PCR in overload-induced skeletal muscle hypertrophy

Quantifying target mRNA using real-time quantitative reverse transcription-polymerase chain reaction requires an accurate normalization method. Determination of normalization factors (NFs) based on validated reference genes according to their relative stability is currently the best standard method in most usual situations. This method controls for technical errors, but its physiological relevance requires constant NF values for a fixed weight of tissue. In the functional overload model, the increase in the total RNA concentration must be considered in determining the NF values. Here, we pointed out a limitation of the classical geNorm-derived normalization. geNorm software selected reference genes despite that the NF values extensively varied under experiment. Only the NF values calculated from four intentionally selected genes were constant between groups. However, a normalization based on these genes is questionable. Indeed, three out of four genes belong to the same functional class (negative regulator of muscle mass), and their use is physiological nonsense in a hypertrophic model. Thus, we proposed guidelines for optimizing target mRNA normalization and quantification, useful in models of muscle mass modulation. In our study, the normalization method by multiple reference genes was not appropriate to compare target mRNA levels between overloaded and control muscles. A solution should be to use an absolute quantification of target mRNAs per unit weight of tissue, without any internal normalization. Even if the technical variations will stay present as a part of the intergroup variations, leading to less statistical power, we consider this method acceptable because it will not generate misleading results.

Early Activation of mTOR in Response to Mechanical Overload Occurs Independent of PI3K/Akt Signaling

It had been established that mammalian target of rapamycin complex 1 (TORC1) functions as a central integrator of a wide range of signals that modulate protein metabolism and cell growth, however, the regulatory mechanisms which determine mTOR activity in skeletal muscle are poorly defined. PURPOSE: The aim of this study was to determine the upstream signaling mechanisms that contribute to mTOR activation during mechanical overload-induced skeletal muscle hypertrophy in vivo. METHODS: C57BL/6J mice (male, 12-14 wk of age) were used in this study. The bilateral synergist ablation model was used to induce hypertrophy of the plantaris muscle by functional overload. Muscle samples were collected at days 1, 2, 3, 5, 7 and 10 after synergist ablation surgery. Insulin (0.75 Unit/kg), wortmannin (3 mg/kg) and rapamycin (1.5 mg/kg) were administered by intraperitoneal injection. RESULTS: Consistent with previous studies, mechanical overload induced progressive hypertrophy of the plantaris muscle (Ex. 1.7-fold increase in total muscle mass, 14.2-fold increase in rates of protein synthesis and 2.1-fold increase in total RNA contents following 7 days of mechanical overload). Phosphorylation states of both Akt and mTOR signaling pathways were determined. Activity of mTOR signaling was monitored by analysis of Thr389 phosphorylation of S6 kinase 1 (S6K1). We found a significant increase in Thr389 phosphorylation of S6K1 by day 1 following mechanical overload. In contrast we saw no change in Akt phosphorylation, no change in Akt kinase activity and we found no change in the phosphorylation of known downstream targets of Akt including glycogen synthase kinase 3, proline-rich Akt substrate 40 kDa and tuberous sclerosis 2 at day 1 of mechanical overload. These results were surprising and indicated that activation of phosphoinositide 3-kinase (PI3K)/Akt signaling was not required for mTOR activation in vivo. To determine whether PI3K-dependent signaling was necessary for the early activation of mTOR, we treated mice with the PI3K-specific inhibitor wortmannin, which was sufficient to inhibit insulin-dependent signal inputs, but did not prevent overload-induced mTOR activation in vivo. CONCLUSIONS: These observations demonstrate that mTOR activation at the early phase of mechanical overload occurs independent of PI3K/Akt regulation in skeletal muscle. This suggests that other signal inputs including mechanical strain and/or nutrient status are important for this initial signaling event during overload-induced hypertrophic growth. Supported by NIH Grant ROI AR45617 to KAE and AHA postdoctoral fellowship (0825668D) to MM.

Intercellular adhesion molecule‐1 is expressed by skeletal muscle cells in hypertrophying muscle of mice

Our laboratory recently demonstrated that β2 integrins contribute to overload‐induced skeletal muscle hypertrophy. One potential mechanism by which β2 integrin expressing leukocytes influence the hypertrophic response of skeletal muscle involves intercellular adhesion molecule‐1 (ICAM‐1), the major ligand for β2 integrins. The objective of our current work is to examine ICAM‐1 expression and localization to skeletal muscle cells in hypertrophying muscle and to determine if β2 integrins influence ICAM‐1 expression and/or its localization.Plantaris muscles of wild type and β2 integrin deficient mice were exposed to 3, 7, or 14 days of mechanical overload via synergist ablation. Real‐time PCR and western blotting analysis revealed that ICAM‐1 gene and protein expression was significantly elevated in overloaded muscle. Experiments using flow cytometry and multi‐photon laser scanning confocal microscopy revealed that ICAM‐1 was expressed by mononuclear cells (satellite cells/myoblasts and leukocytes) as well as mature muscle fibers following mechanical loading.We speculate that expression of ICAM‐1 by skeletal muscle cells after mechanical loading serves as a mechanism by which neutrophils and macrophages can bind to and directly communicate with skeletal muscle cells to promote hypertrophy.This work was partially supported by The University of Toledo Interdisciplinary Grant Program.

Tax sugar, save muscle?

debates are currently raging in the United States over healthcare reform. Certain political circles are advocating the implementation of a tax on sugary products to reign in ballooning costs associated with treating the one in three individuals in the United States now classified as obese. Now, this

The expression patterns of Pax7 in satellite cells during overload‐induced rat adult skeletal muscle hypertrophy

Activated satellite cells (SCs) have the ability to reacquire a quiescent, undifferentiated state. Pax7 plays a crucial role in allowing activated SCs to undergo self-renewal. Because the increase in the SC population is induced during overload-induced skeletal muscle hypertrophy, it is possible that Pax7-regulated SC self-renewal is involved in the modulation of the SC population during the functional overload of skeletal muscles. However, the characteristics of the expression patterns of Pax7 in SCs during the functional overload of adult skeletal muscles are poorly understood. Using immunohistochemical approaches, we examined the temporal and spatial expression patterns of Pax7 expressed in SCs during the functional overloading of rat skeletal muscles. The time course of Pax7 expression in SCs was similar to that of the expression of the differentiation regulatory factor myogenin during the early stage of functional overload. However, the percentage of SCs that expressed Pax7 was markedly higher than that of the SCs that expressed myogenin. Coexpression of Pax7 and myogenin was not detected in SCs. In addition, the expression of cyclin-dependent kinase inhibitor p21, which regulates cell cycle arrest and differentiation, was not detected in Pax7-positive SCs. These results suggest that Pax7-regulated self-renewal of SCs may be induced during the early stage of functional overload and may contribute to modulating the SC population in hypertrophied muscles. Furthermore, it was suggested that the numbers of SCs which underwent self-renewal may be higher than that of SCs which were provided as the additional myonuclei for hypertrophying myofibres.

Angiotensin-converting enzyme inhibition attenuates myonuclear addition in overloaded slow-twitch skeletal muscle

Because optimal overload-induced skeletal muscle hypertrophy requires ANG II, we aimed to determine the effects of blocking ANG II production [via angiotensin-converting enzyme (ACE) inhibition] on potential mediators of hypertrophy in overloaded skeletal muscle, namely, myonuclear addition and fibroblast content. In a 2 x 2 design, adult (200-225 g) female Sprague-Dawley rats were placed into one of four groups (n = 8/group): 7-day skeletal muscle overload, sham operation, 7-day skeletal muscle overload with ACE inhibition, or sham operation with ACE inhibition. Functional overloads of the plantaris and soleus muscles were produced via bilateral surgical ablation of the synergistic gastrocnemius muscle, and ACE inhibition was accomplished by the addition of the ACE inhibitor enalapril maleate to the animals' daily drinking water (0.3 mg/ml). Myonuclear addition and extrasarcolemmal nuclear proliferation, as measured by in vivo 5-bromo-2'-deoxyuridine labeling, were significantly (P < or = 0.05) increased by overload in both the slow-twitch soleus and fast-twitch plantaris muscles. Furthermore, ACE inhibition attenuated these overload-induced increases in the soleus muscle but not in the plantaris muscle. However, the effect of ACE inhibition on soleus extrasarcolemmal nuclei was not likely due to differences in fibroblast content because overload elicited significant increases in vimentin-positive areas in soleus and plantaris muscles, and these areas were unaffected by ACE inhibition in either muscle. There was no effect of ACE inhibition on any measure in sham-operated muscles. Collectively, these data indicate that ANG II may mediate the satellite cell response to overload in slow-twitch soleus but not in fast-twitch plantaris muscles and that this effect may occur independently of changes in fibroblast content.

Wnt/β-catenin signaling activates growth-control genes during overload-induced skeletal muscle hypertrophy

Beta-catenin is a transcriptional activator shown to regulate the embryonic, postnatal, and oncogenic growth of many tissues. In most research to date, beta-catenin activation has been the unique downstream function of the Wnt signaling pathway. However, in the heart, a Wnt-independent mechanism involving Akt-mediated phosphorylation of glycogen synthase kinase (GSK)-3beta was recently shown to activate beta-catenin and regulate cardiomyocyte growth. In this study, results have identified the activation of the Wnt/beta-catenin pathway during hypertrophy of mechanically overloaded skeletal muscle. Significant increases in beta-catenin were determined during skeletal muscle hypertrophy. In addition, the Wnt receptor, mFrizzled (mFzd)-1, the signaling mediator disheveled-1, and the transcriptional co-activator, lymphocyte enhancement factor (Lef)-1, are all increased during hypertrophy of the overloaded mouse plantaris muscle. Experiments also determined an increased association between GSK-3beta and the inhibitory frequently rearranged in advanced T cell-1 protein with no increase in GSK-3beta phosphorylation (Ser9). Finally, skeletal muscle overload resulted in increased nuclear beta-catenin/Lef-1 expression and induction of the transcriptional targets c-Myc, cyclin D1, and paired-like homeodomain transcription factor 2. Thus this study provides the first evidence that the Wnt signaling pathway induces beta-catenin/Lef-1 activation of growth-control genes during overload induced skeletal muscle hypertrophy.

Involvement of nitric oxide synthase in skeletal muscle adaptation to chronic overload.

The purpose of this study was to determine the necessity of nitric oxide (NO) for hypertrophy and fiber-type transition in overloaded (OL) skeletal muscle. Endogenous NO production was blocked by administering N(G)-nitro-L-arginine methyl ester (L-NAME; 0.75 mg/ml; approximately 100 mg x kg-1 x day-1) in drinking water. Thirty-eight female Sprague-Dawley rats (approximately 250 g) were randomly divided into four groups: control-nonoverloaded (Non-OL), control-OL, L-NAME-Non-OL, and L-NAME-OL. Chronic overload of the plantaris was induced bilaterally by surgical removal of the gastrocnemius and soleus. Rats in the Non-OL groups received sham surgeries. L-NAME treatment began 24 h before surgery and continued until the rats were killed 14 days postsurgery. Although OL induced hypertrophy in both control (+76%) and L-NAME (+39%) conditions (P < 0.05), mean plantaris-to-body mass ratio in the L-NAME-OL group was significantly lower (P < 0.05) than that in the control-OL group. Microphotometric analysis of histochemically determined fiber types revealed increases in cross-sectional area (P < 0.05) for all fiber types (types I, IIA, and IIB/X) in the OL plantaris from control rats, whereas L-NAME-OL rats exhibited increases only in type I and IIB/X fibers. SDS-PAGE analysis of myosin heavy chain (MHC) composition in the plantaris indicated a significant (P < 0.05) OL effect in the control rats. Specifically, the mean proportion of type I MHC increased 6% (P < 0.05), whereas the proportion of type IIb MHC decreased approximately 9% (P < 0.05). No significant OL effects on MHC profile were observed in the L-NAME rats. These data support a role of NO in overload-induced skeletal muscle hypertrophy and fiber-type transition.

ANG II is required for optimal overload-induced skeletal muscle hypertrophy.

ANG II mediates the hypertrophic response of overloaded cardiac muscle, likely via the ANG II type 1 (AT(1)) receptor. To examine the potential role of ANG II in overload-induced skeletal muscle hypertrophy, plantaris and/or soleus muscle overload was produced in female Sprague-Dawley rats (225-250 g) by the bilateral surgical ablation of either the synergistic gastrocnemius muscle (experiment 1) or both the gastrocnemius and plantaris muscles (experiment 2). In experiment 1 (n = 10/group), inhibiting endogenous ANG II production by oral administration of an angiotensin-converting enzyme (ACE) inhibitor during a 28-day overloading protocol attenuated plantaris and soleus muscle hypertrophy by 57 and 96%, respectively (as measured by total muscle protein content). ACE inhibition had no effect on nonoverloaded (sham-operated) muscles. With the use of new animals (experiment 2; n = 8/group), locally perfusing overloaded soleus muscles with exogenous ANG II (via osmotic pump) rescued the lost hypertrophic response in ACE-inhibited animals by 71%. Furthermore, orally administering an AT(1) receptor antagonist instead of an ACE inhibitor produced a 48% attenuation of overload-induced hypertrophy that could not be rescued by ANG II perfusion. Thus ANG II may be necessary for optimal overload-induced skeletal muscle hypertrophy, acting at least in part via an AT(1) receptor-dependent pathway.

Androgen cytosol binding during compensatory overload-induced skeletal muscle hypertrophy.

This study was undertaken to evaluate whether the increased androgen cytosol binding is an early or later event in the sequence of skeletal muscle hypertrophy induced by surgical overload. Following removal of the synergistic gastrocnemius and soleus muscles, plantaris muscle weights of overloaded hypophysectomized made rats were heavier than those in the control by 29% at 2 days, 41% at 7 days, 38% at 14 days, and 47% at 35 days. Androgen cytosol receptor binding capacities (femtomoles per milligram protein), determined using a synthetic androgen, [3H]methyltrienolone (R1881), were higher than observed in muscles of controls at all points of muscle enlargement. At high concentrations of labeled ligand, Scatchard analyses became nonlinear and were resolved using a two-component binding model. Receptor capacity of the higher affinity "androgenic component" for methyltrienolone binding in plantaris muscles was lower at 2 days but 60-80% higher at 7, 14, and 35 days in the hypertrophied group than in the control group. The lower affinity "glucocorticoid component" was higher in the overloaded group at all points following surgery. Several glucocorticoids and estradiol-17 beta competed equally with androgens for methyltrienolone binding. However, when cytosols were incubated with triamcinolone acetonide to block methyltrienolone binding to glucocorticoid receptors, the androgenic component was highly specific for androgens. These results show that total [3H]methyltrienolone cytosol concentrations increased in parallel with the muscle hypertrophy, yet the individual components of methyltrienolone binding attained greater concentrations in overloaded muscles by an apparently different sequence of events.