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The isolated muscle fibre as a model of disuse atrophy: characterization using PhAct, a method to quantify f-actin.

ABSTRACT: Research into muscle atrophy and hypertrophy is hampered by limitations of the available experimental models. Interpretation of in vivo experiments is confounded by the complexity of the environment while in vitro models are subject to the marked disparities between cultured myotubes and the mature myofibres of living tissues. Here we develop a method (PhAct) based on ex vivo maintenance of the isolated myofibre as a model of disuse atrophy, using standard microscopy equipment and widely available analysis software, to measure f-actin content per myofibre and per nucleus over two weeks of ex vivo maintenance. We characterize the 35% per week atrophy of the isolated myofibre in terms of early changes in gene expression and investigate the effects on loss of muscle mass of modulatory agents, including Myostatin and Follistatin. By tracing the incorporation of a nucleotide analogue we show that the observed atrophy is not associated with loss or replacement of myonuclei. Such a completely controlled investigation can be conducted with the myofibres of a single muscle. With this novel method we can distinguish those features and mechanisms of atrophy and hypertrophy that are intrinsic to the muscle fibre from those that include activities of other tissues and systemic agents.

SUBMITTER: Duddy WJ

PROVIDER: S-EPMC3148270 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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The isolated muscle fibre as a model of disuse atrophy: characterization using PhAct, a method to quantify f-actin.

Duddy William J WJ Cohen Tatiana T Duguez Stephanie S Partridge Terence A TA

Experimental cell research 20110520 14

Research into muscle atrophy and hypertrophy is hampered by limitations of the available experimental models. Interpretation of in vivo experiments is confounded by the complexity of the environment while in vitro models are subject to the marked disparities between cultured myotubes and the mature myofibres of living tissues. Here we develop a method (PhAct) based on ex vivo maintenance of the isolated myofibre as a model of disuse atrophy, using standard microscopy equipment and widely availab ...[more]

PMID: 21635888

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Project description:Sarcopenia, a critical loss of muscle mass and function because of the physiological process of aging, contributes to disability and mortality in older adults. It increases the incidence of pathologic fractures, causing prolonged periods of hospitalization and rehabilitation. The molecular mechanisms underlying sarcopenia are poorly understood, but recent evidence suggests that increased transforming growth factor-? (TGF-?) signaling contributes to impaired satellite cell function and muscle repair in aged skeletal muscle. We therefore evaluated whether antagonism of TGF-? signaling via losartan, an angiotensin II receptor antagonist commonly used to treat high blood pressure, had a beneficial impact on the muscle remodeling process of sarcopenic mice. We demonstrated that mice treated with losartan developed significantly less fibrosis and exhibited improved in vivo muscle function after cardiotoxin-induced injury. We found that losartan not only blunted the canonical TGF-? signaling cascade but also modulated the noncanonical TGF-? mitogen-activated protein kinase pathway. We next assessed whether losartan was able to combat disuse atrophy in aged mice that were subjected to hindlimb immobilization. We showed that immobilized mice treated with losartan were protected against loss of muscle mass. Unexpectedly, this protective mechanism was not mediated by TGF-? signaling but was due to an increased activation of the insulin-like growth factor 1 (IGF-1)/Akt/mammalian target of rapamycin (mTOR) pathway. Thus, blockade of the AT1 (angiotensin II type I) receptor improved muscle remodeling and protected against disuse atrophy by differentially regulating the TGF-? and IGF-1/Akt/mTOR signaling cascades, two pathways critical for skeletal muscle homeostasis. Thus, losartan, a Food and Drug Administration-approved drug, may prove to have clinical benefits to combat injury-related muscle remodeling and provide protection against disuse atrophy in humans with sarcopenia.

Project description:Muscle disuse produces severe atrophy and a slow-to-fast phenotype transition in the postural Soleus (Sol) muscle of rodents. Antioxidants, amino-acids and growth factors were ineffective to ameliorate muscle atrophy. Here we evaluate the effects of nandrolone (ND), an anabolic steroid, on mouse skeletal muscle atrophy induced by hindlimb unloading (HU). Mice were pre-treated for 2-weeks before HU and during the 2-weeks of HU. Muscle weight and total protein content were reduced in HU mice and a restoration of these parameters was found in ND-treated HU mice. The analysis of gene expression by real-time PCR demonstrates an increase of MuRF-1 during HU but minor involvement of other catabolic pathways. However, ND did not affect MuRF-1 expression. The evaluation of anabolic pathways showed no change in mTOR and eIF2-kinase mRNA expression, but the protein expression of the eukaryotic initiation factor eIF2 was reduced during HU and restored by ND. Moreover we found an involvement of regenerative pathways, since the increase of MyoD observed after HU suggests the promotion of myogenic stem cell differentiation in response to atrophy. At the same time, Notch-1 expression was down-regulated. Interestingly, the ND treatment prevented changes in MyoD and Notch-1 expression. On the contrary, there was no evidence for an effect of ND on the change of muscle phenotype induced by HU, since no effect of treatment was observed on the resting gCl, restCa and contractile properties in Sol muscle. Accordingly, PGC1α and myosin heavy chain expression, indexes of the phenotype transition, were not restored in ND-treated HU mice. We hypothesize that ND is unable to directly affect the phenotype transition when the specialized motor unit firing pattern of stimulation is lacking. Nevertheless, through stimulation of protein synthesis, ND preserves protein content and muscle weight, which may result advantageous to the affected skeletal muscle for functional recovery.

Project description:BackgroundSuccessful strategies to halt or reverse sarcopenia require a basic understanding of the factors that cause muscle loss with age. Acute periods of muscle loss in older individuals have an incomplete recovery of muscle mass and strength, thus accelerating sarcopenic progression. The purpose of the current study was to further understand the mechanisms underlying the failure of old animals to completely recover muscle mass and function after a period of hindlimb unloading.MethodsHindlimb unloading was used to induce muscle atrophy in Fischer 344-Brown Norway (F344BN F1) rats at 24, 28, and 30 months of age. Rats were hindlimb unloaded for 14 days and then reloaded at 24 months (Reloaded 24), 28 months (Reloaded 28), and 24 and 28 months (Reloaded 24/28) of age. Isometric torque was determined at 24 months of age (24 months), at 28 months of age (28 months), immediately after 14 days of reloading, and at 30 months of age (30 months). During control or reloaded conditions, rats were labelled with deuterium oxide (D2 O) to determine rates of muscle protein synthesis and RNA synthesis.ResultsAfter 14 days of reloading, in vivo isometric torque returned to baseline in Reloaded 24, but not Reloaded 28 and Reloaded 24/28. Despite the failure of Reloaded 28 and Reloaded 24/28 to regain peak force, all groups were equally depressed in peak force generation at 30 months. Increased age did not decrease muscle protein synthesis rates, and in fact, increased resting rates of protein synthesis were measured in the myofibrillar fraction (Fractional synthesis rate (FSR): %/day) of the plantaris (24 months: 2.53 ± 0.17; 30 months: 3.29 ± 0.17), and in the myofibrillar (24 months: 2.29 ± 0.07; 30 months: 3.34 ± 0.11), collagen (24 months: 1.11 ± 0.07; 30 months: 1.55 ± 0.14), and mitochondrial (24 months: 2.38 ± 0.16; 30 months: 3.20 ± 0.10) fractions of the tibialis anterior (TA). All muscles increased myofibrillar protein synthesis (%/day) in Reloaded 24 (soleus: 3.36 ± 0.11, 5.23 ± 0.19; plantaris: 2.53 ± 0.17, 3.66 ± 0.07; TA: 2.29 ± 0.14, 3.15 ± 0.12); however, in Reloaded 28, only the soleus had myofibrillar protein synthesis rates (%/day) >28 months (28 months: 3.80 ± 0.10; Reloaded 28: 4.86 ± 0.19). Across the muscles, rates of protein synthesis were correlated with RNA synthesis (all muscles combined, R2 = 0.807, P < 0.0001).ConclusionsThese data add to the growing body of literature that indicate that changes with age, including following disuse atrophy, differ by muscle. In addition, our findings lead to additional questions of the underlying mechanisms by which some muscles are maintained with age while others are not.

Dataset Information

The isolated muscle fibre as a model of disuse atrophy: characterization using PhAct, a method to quantify f-actin.

Publications

The isolated muscle fibre as a model of disuse atrophy: characterization using PhAct, a method to quantify f-actin.

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