MuRF1 is a muscle fiber-type II associated factor and together with MuRF2 regulates type-II fiber trophicity and maintenance

MuRF1 is a member of the RBCC (RING, B-box, coiled-coil) superfamily that has been proposed to act as an atrogin during muscle wasting. Here, we show that MuRF1 is preferentially induced in type-II muscle fibers after denervation. Fourteen days after denervation, MuRF1 protein was further elevated but remained preferentially expressed in type-II muscle fibers. Consistent with a fiber-type dependent function of MuRF1, the tibialis anterior muscle (rich in type-II muscle fibers) was considerably more protected in MuRF1-KO mice from muscle wasting when compared to soleus muscle with mixed fiber-types. We also determined fiber-type distributions in MuRF1/MuRF2 double-deficient KO (dKO) mice, because MuRF2 is a close homolog of MuRF1. MuRF1/MuRF2 dKO mice showed a profound loss of type-II fibers in soleus muscle. As a potential mechanism we identified the interaction of MuRF1/MuRF2 with myozenin-1, a calcineurin/NFAT regulator and a factor required for maintenance of type-II muscle fibers. MuRF1/MuRF2 dKO mice had lost myozenin-1 expression in tibialis anterior muscle, implicating MuRF1/MuRF2 as regulators of the calcineurin/NFAT pathway. In summary, our data suggest that expression of MuRF1 is required for remodeling of type-II fibers under pathophysiological stress states, whereas MuRF1 and MuRF2 together are required for maintenance of type-II fibers, possibly via the regulation of myozenin-1. (C) 2010 Elsevier Inc. All rights reserved.

Properties of slow- and fast-twitch muscle fibres in a mouse model of amyotrophic lateral sclerosis

This investigation was undertaken to determine if there are altered histological, pathological and contractile properties in presymptomatic or endstage diseased muscle fibres from representative slow-twitch and fast-twitch muscles of SOD1 G93A mice in comparison to wildtype mice. In presymptomatic SOD1 G93A mice, there was no detectable peripheral dysfunction, providing evidence that muscle pathology is secondary to motor neuronal dysfunction. At disease endstage however, single muscle fibre contractile analysis demonstrated that fast-twitch muscle fibres and neuromuscular junctions are preferentially affected by amyotrophic lateral sclerosis-induced denervation, being unable to produce the same levels of force when activated by calcium as muscle fibres from their age-matched controls. The levels of transgenic SOD1 expression, aggregation state and activity were also examined in these muscles but there no was no preference for muscle fibre type. Hence, there is no simple correlation between SOD1 protein expression/activity, and muscle fibre type vulnerability in SOD1 G93A mice.

Interaction of contractile activity and training history on mRNA abundance in skeletal muscle from trained athletes

Skeletal muscle displays enormous plasticity to respond to contractile activity with muscle from strength- (ST) and endurance-trained (ET) athletes representing diverse states of the adaptation continuum. Training adaptation can be viewed as the accumulation of specific proteins. Hence, the altered gene expression that allows for changes in protein concentration is of major importance for any training adaptation. Accordingly, the aim of the present study was to quantify acute subcellular responses in muscle to habitual and unfamiliar exercise. After 24-h diet/exercise control, 13 male subjects (7 ST and 6 ET) performed a random order of either resistance (8 x 5 maximal leg extensions) or endurance exercise (1 h of cycling at 70% peak O2 uptake). Muscle biopsies were taken from vastus lateralis at rest and 3 h after exercise. Gene expression was analyzed using real-time PCR with changes normalized relative to preexercise values. After cycling exercise, peroxisome proliferator-activated receptor- coactivator-1 (ET 8.5-fold, ST 10-fold, P < 0.001), pyruvate dehydrogenase kinase-4 (PDK-4; ET 26-fold, ST 39-fold), vascular endothelial growth factor (VEGF; ET 4.5-fold, ST 4-fold), and muscle atrophy F-box protein (MAFbx) (ET 2-fold, ST 0.4-fold) mRNA increased in both groups, whereas MyoD (3-fold), myogenin (0.9-fold), and myostatin (2-fold) mRNA increased in ET but not in ST (P < 0.05). After resistance exercise PDK-4 (7-fold, P < 0.01) and MyoD (0.7-fold) increased, whereas MAFbx (0.7-fold) and myostatin (0.6-fold) decreased in ET but not in ST. We conclude that prior training history can modify the acute gene responses in skeletal muscle to subsequent exercise.

Regulation of STARS and its downstream target suggest a novel pathway involved in human skeletal hypertrophy and atrophy

Skeletal muscle atrophy is a severe consequence of ageing, neurological disorders and chronic disease. Identifying the intracellular signalling pathways controlling changes in skeletal muscle size and function is vital for the future development of potential therapeutic interventions. Striated activator of Rho signalling (STARS), an actin-binding protein, has been implicated in rodent cardiac hypertrophy; however its role in human skeletal muscle has not been determined. This study aimed to establish if STARS, as well as its downstream signalling targets, RhoA, myocardin-related transcription factors A and B (MRTF-A/B) and serum response factor (SRF), were increased and decreased respectively, in human quadriceps muscle biopsies taken after 8 weeks of both hypertrophy-stimulating resistance training and atrophy-stimulating de-training. The mRNA levels of the SRF target genes involved in muscle structure, function and growth, such as α-actin, myosin heavy chain IIa (MHCIIa) and insulin-like growth factor-1 (IGF-1), were also measured. Following resistance training, STARS, MRTF-A, MRTF-B, SRF, α-actin, MHCIIa and IGF-1 mRNA, as well as RhoA and nuclear SRF protein levels were all significantly increased by between 1.25- and 3.6-fold. Following the de-training period all measured targets, except for RhoA, which remained elevated, returned to base-line. Our results show that the STARS signalling pathway is responsive to changes in skeletal muscle loading and appears to play a role in both human skeletal muscle hypertrophy and atrophy.

The bone morphogenetic protein axis is a positive regulator of skeletal muscle mass

Although the canonical transforming growth factor β signaling pathway represses skeletal muscle growth and promotes muscle wasting, a role in muscle for the parallel bone morphogenetic protein (BMP) signaling pathway has not been defined. We report, for the first time, that the BMP pathway is a positive regulator of muscle mass. Increasing the expression of BMP7 or the activity of BMP receptors in muscles induced hypertrophy that was dependent on Smad1/5-mediated activation of mTOR signaling. In agreement, we observed that BMP signaling is augmented in models of muscle growth. Importantly, stimulation of BMP signaling is essential for conservation of muscle mass after disruption of the neuromuscular junction. Inhibiting the phosphorylation of Smad1/5 exacerbated denervation-induced muscle atrophy via an HDAC4-myogenin–dependent process, whereas increased BMP–Smad1/5 activity protected muscles from denervation-induced wasting. Our studies highlight a novel role for the BMP signaling pathway in promoting muscle growth and inhibiting muscle wasting, which may have significant implications for the development of therapeutics for neuromuscular disorders.

Androgenic and estrogenic regulation of Atrogin-1, MuRF1 and myostatin expression in different muscle types of male mice

The molecular factors targeted by androgens and estrogens on muscle mass are not fully understood. The current study aimed to explore gene and protein expression of Atrogin-1, MuRF1, and myostatin in an androgen deprivation-induced muscle atrophy model.

Statin-induced increases in atrophy gene expression occur independently of changes in PGC1α protein and mitochondrial content

One serious side effect of statin drugs is skeletal muscle myopathy. Although the mechanism(s) responsible for statin myopathy remains to be fully determined, an increase in muscle atrophy gene expression and changes in mitochondrial content and/or function have been proposed to play a role. In this study, we examined the relationship between statin-induced expression of muscle atrophy genes, regulators of mitochondrial biogenesis, and markers of mitochondrial content in slow- (ST) and fast-twitch (FT) rat skeletal muscles. Male Sprague Dawley rats were treated with simvastatin (60 or 80 mg·kg(-1)·day(-1)) or vehicle control via oral gavage for 14 days. In the absence of overt muscle damage, simvastatin treatment induced an increase in atrogin-1, MuRF1 and myostatin mRNA expression; however, these were not associated with changes in peroxisome proliferator gamma co-activator 1 alpha (PGC-1α) protein or markers of mitochondrial content. Simvastatin did, however, increase neuronal nitric oxide synthase (nNOS), endothelial NOS (eNOS) and AMPK α-subunit protein expression, and tended to increase total NOS activity, in FT but not ST muscles. Furthermore, simvastatin induced a decrease in β-hydroxyacyl CoA dehydrogenase (β-HAD) activity only in FT muscles. These findings suggest that the statin-induced activation of muscle atrophy genes occurs independent of changes in PGC-1α protein and mitochondrial content. Moreover, muscle-specific increases in NOS expression and possibly NO production, and decreases in fatty acid oxidation, could contribute to the previously reported development of overt statin-induced muscle damage in FT muscles.

Differential atrophy of the lower-limb musculature during prolonged bed-rest

Patients with medical, orthopaedic and surgical conditions are often assigned to bed-rest and/or immobilised in orthopaedic devices. Although such conditions lead to muscle atrophy, no studies have yet considered differential atrophy of the lower-limb musculature during inactivity to enable the development of rehabilitative exercise programmes. Bed-rest is a model used to simulate the effects of spaceflight and physical inactivity. Ten male subjects underwent 56-days of bed-rest. Magnetic resonance imaging of the lower-limbs was performed at 2-weekly intervals during bed-rest. Volume of individual muscles of the lower-limb and subsequently, rates of atrophy were calculated. Rates of atrophy differed (F = 7.4, p < 0.0001) between the muscles with the greatest rates of atrophy seen in the medial gastrocnemius, soleus and vastii (p < 0.00000002). The hamstring muscles were also affected (p < 0.00015). Atrophy was less in the ankle dorsiflexors and anteromedial hip muscles (p > 0.081). Differential rates of atrophy were seen in synergistic muscles (e.g. adductor magnus > adductor longus, p = 0.009; medial gastrocnemius > lateral gastrocnemius, p = 0.002; vastii > rectus femoris, p = 0.0002). These results demonstrate that muscle imbalances can occur after extended periods of reduced postural muscle activity, potentially hampering recovery on return to full upright body position. Such deconditioned patients should be prescribed "closed-chain" simulated resistance exercises, which target the lower-limb antigravity extensor muscles which were most affected in bed-rest.

Differential atrophy of the postero-lateral hip musculature during prolonged bedrest and the influence of exercise countermeasures

As part of the 2nd Berlin BedRest Study (BBR2-2), we investigated the pattern of muscle atrophy of the postero-lateral hip and hamstring musculature during prolonged inactivity and the effectiveness of two exercise countermeasures. Twenty-four male subjects underwent 60 days of head-down tilt bedrest and were assigned to an inactive control (CTR), resistive vibration exercise (RVE), or resistive exercise alone (RE) group. Magnetic resonance imaging (MRI) of the hip and thigh was taken before, during, and at end of bedrest. Volume of posterolateral hip and hamstring musculature was calculated, and the rate of muscle atrophy and the effect of countermeasure exercises were examined. After 60 days of bedrest, the CTR group showed differential rates of muscle volume loss (F = 21.44; P ≤ 0.0001) with fastest losses seen in the semi-membranosus, quadratus femoris and biceps femoris long head followed by the gluteal and remaining hamstring musculature. Whole body vibration did not appear to have an additional effect above resistive exercise in preserving muscle volume. RE and RVE prevented and/or reduced muscle atrophy of the gluteal, semi-membranosus, and biceps femoris long head muscles. Some muscle volumes in the countermeasure groups displayed faster recovery times than the CTR group. Differential atrophy occurred in the postero-lateral hip musculature following a prolonged period of unloading. Short-duration high-load resistive exercise during bedrest reduced muscle atrophy in the mono-articular hip extensors and selected hamstring muscles. Future countermeasure design should consider including isolated resistive hamstring curls to target this muscle group and reduce the potential for development of muscle imbalances.

Heterogeneous atrophy occurs within individual lower limb muscles during 60 days of bed rest

To better understand disuse muscle atrophy, via magnetic resonance imaging, we sequentially measured muscle cross-sectional area along the entire length of all individual muscles from the hip to ankle in nine male subjects participating in 60-day head-down tilt bed rest (2nd Berlin BedRest Study; BBR2-2). We hypothesized that individual muscles would not atrophy uniformly along their length such that different regions of an individual muscle would atrophy to different extents. This hypothesis was confirmed for the adductor magnus, vasti, lateral hamstrings, medial hamstrings, rectus femoris, medial gastrocnemius, lateral gastrocnemius, tibialis posterior, flexor hallucis longus, flexor digitorum longus, peroneals, and tibialis anterior muscles (P ≤ 0.004). In contrast, the hypothesis was not confirmed in the soleus, adductor brevis, gracilis, pectineus, and extensor digitorum longus muscles (P ≥ 0.20). The extent of atrophy only weakly correlated (r = -0.30, P < 0.001) with the location of greatest cross-sectional area. The rate of atrophy during bed rest also differed between muscles (P < 0.0001) and between some synergists. Most muscles recovered to their baseline size between 14 and 90 days after bed rest, but flexor hallucis longus, flexor digitorum longus, and lateral gastrocnemius required longer than 90 days before recovery occurred. On the basis of findings of differential atrophy between muscles and evidence in the literature, we interpret our findings of intramuscular atrophy to reflect differential disuse of functionally different muscle regions. The current work represents the first lower-limb wide survey of intramuscular differences in disuse atrophy. We conclude that intramuscular differential atrophy occurs in most, but not all, of the muscles of the lower limb during prolonged bed rest.

Vibration mechanosignals superimposed to resistive exercise result in baseline skeletal muscle transcriptome profiles following chronic disuse in bed rest

Disuse-induced muscle atrophy is a major concern in aging, in neuromuscular diseases, post-traumatic injury and in microgravity life sciences affecting health and fitness also of crew members in spaceflight. By using a laboratory analogue to body unloading we perform for the first time global gene expression profiling joined to specific proteomic analysis to map molecular adaptations in disused (60 days of bed rest) human soleus muscle (CTR) and in response to a resistive exercise (RE) countermeasure protocol without and with superimposed vibration mechanosignals (RVE). Adopting Affymetrix GeneChip technology we identified 235 differently transcribed genes in the CTR group (end-vs. pre-bed rest). RE comprised 206 differentially expressed genes, whereas only 51 changed gene transcripts were found in RVE. Most gene transcription and proteomic changes were linked to various key metabolic pathways (glycolysis, oxidative phosphorylation, tricarboxylic acid (TCA) cycle, lipid metabolism) and to functional contractile structures. Gene expression profiling in bed rest identified a novel set of genes explicitly responsive to vibration mechanosignals in human soleus. This new finding highlights the efficacy of RVE protocol in reducing key signs of disuse maladaptation and atrophy, and to maintain a close-to-normal skeletal muscle quality outcome following chronic disuse in bed rest.

Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle

Skeletal muscle mitochondrial content and oxidative capacity are important determinants of muscle function and whole-body health. Mitochondrial content and function are enhanced by endurance exercise and impaired in states or diseases where muscle function is compromised, such as myopathies, muscular dystrophies, neuromuscular diseases, and age-related muscle atrophy. Hence, elucidating the mechanisms that control muscle mitochondrial content and oxidative function can provide new insights into states and diseases that affect muscle health. In past studies, we identified Perm1 (PPARGC1- and ESRR-induced regulator, muscle 1) as a gene induced by endurance exercise in skeletal muscle, and regulating mitochondrial oxidative function in cultured myotubes. The capacity of Perm1 to regulate muscle mitochondrial content and function in vivo is not yet known. In this study, we use adeno-associated viral (AAV) vectors to increase Perm1 expression in skeletal muscles of 4-wk-old mice. Compared to control vector, AAV1-Perm1 leads to significant increases in mitochondrial content and oxidative capacity (by 40-80%). Moreover, AAV1-Perm1-transduced muscles show increased capillary density and resistance to fatigue (by 33 and 31%, respectively), without prominent changes in fiber-type composition. These findings suggest that Perm1 selectively regulates mitochondrial biogenesis and oxidative function, and implicate Perm1 in muscle adaptations that also occur in response to endurance exercise.-Cho, Y., Hazen, B. C., Gandra, P. G., Ward, S. R., Schenk, S., Russell, A. P., Kralli, A. Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.

Glucocorticoids enhance muscle endurance and ameliorate Duchenne muscular dystrophy through a defined metabolic program

Classic physiology studies dating to the 1930s demonstrate that moderate or transient glucocorticoid (GC) exposure improves muscle performance. The ergogenic properties of GCs are further evidenced by their surreptitious use as doping agents by endurance athletes and poorly understood efficacy in Duchenne muscular dystrophy (DMD), a genetic muscle-wasting disease. A defined molecular basis underlying these performance-enhancing properties of GCs in skeletal muscle remains obscure. Here, we demonstrate that ergogenic effects of GCs are mediated by direct induction of the metabolic transcription factor KLF15, defining a downstream pathway distinct from that resulting in GC-related muscle atrophy. Furthermore, we establish that KLF15 deficiency exacerbates dystrophic severity and muscle GC-KLF15 signaling mediates salutary therapeutic effects in the mdx mouse model of DMD. Thus, although glucocorticoid receptor (GR)-mediated transactivation is often associated with muscle atrophy and other adverse effects of pharmacologic GC administration, our data define a distinct GR-induced gene regulatory pathway that contributes to therapeutic effects of GCs in DMD through proergogenic metabolic programming.