Weakness and fatigability are typical features of Duchenne muscular dystrophy patients and are aggravated in dystrophic mdx mice by chronic treadmill exercise. Mechanical activity modulates gene expression and muscle plasticity. Here, we investigated the outcome of 4 (T4, 8 weeks of age) and 12 (T12, 16 weeks of age) weeks of either exercise or cage-based activity on a large set of genes in the gastrocnemius muscle of mdx and wild-type (WT) mice using quantitative real-time PCR. Basal expression of the exercise-sensitive genes peroxisome-proliferator receptor γ coactivator 1α (Pgc-1α) and Sirtuin1 (Sirt1) was higher in mdx versus WT mice at both ages. Exercise increased Pgc-1α expression in WT mice; Pgc-1α was downregulated by T12 exercise in mdx muscles, along with Sirt1, Pparγ and the autophagy marker Bnip3. Sixteen weeks old mdx mice showed a basal overexpression of the slow Mhc1 isoform and Serca2; T12 exercise fully contrasted this basal adaptation as well as the high expression of follistatin and myogenin. Conversely, T12 exercise was ineffective in WT mice. Damage-related genes such as gp91-phox (NADPH-oxidase2), Tgfβ, Tnfα and c-Src tyrosine kinase were overexpressed in mdx muscles and not affected by exercise. Likewise, the anti-inflammatory adiponectin was lower in T12-exercised mdx muscles. Chronic exercise with minor adaptive effects in WT muscles leads to maladaptation in mdx muscles with a disequilibrium between protective and damaging signals. Increased understanding of the pathways involved in the altered mechanical-metabolic coupling may help guide appropriate physical therapies while better addressing pharmacological interventions in translational research.

Slow-twitch muscles, devoted to postural maintenance, experience atrophy and weakness during muscle disuse due to bed-rest, aging or spaceflight. These conditions impair motion activities and can have survival implications. Human and animal studies demonstrate the anabolic role of IGF-1 on skeletal muscle suggesting its interest as a muscle disuse countermeasure. Thus, we tested the role of IGF-1 overexpression on skeletal muscle alteration due to hindlimb unloading (HU) by using MLC/mIgf-1 transgenic mice expressing IGF-1 under the transcriptional control of MLC promoter, selectively activated in skeletal muscle. HU produced atrophy in soleus muscle, in terms of muscle weight and fiber cross-sectional area (CSA) reduction, and up-regulation of atrophy gene MuRF1. In parallel, the disuse-induced slow-to-fast fiber transition was confirmed by an increase of the fast-type of the Myosin Heavy Chain (MHC), a decrease of PGC-1α expression and an increase of histone deacetylase-5 (HDAC5). Consistently, functional parameters such as the resting chloride conductance (gCl) together with ClC-1 chloride channel expression were increased and the contractile parameters were modified in soleus muscle of HU mice. Surprisingly, IGF-1 overexpression in HU mice was unable to counteract the loss of muscle weight and the decrease of fiber CSA. However, the expression of MuRF1 was recovered, suggesting early effects on muscle atrophy. Although the expression of PGC-1α and MHC were not improved in IGF-1-HU mice, the expression of HDAC5 was recovered. Importantly, the HU-induced increase of gCl was fully contrasted in IGF-1 transgenic mice, as well as the changes in contractile parameters. These results indicate that, even if local expression does not seem to attenuate HU-induced atrophy and slow-to-fast phenotype transition, it exerts early molecular effects on gene expression which can counteract the HU-induced modification of electrical and contractile properties. MuRF1 and HDAC5 can be attractive therapeutic targets for pharmacological countermeasures and then deserve further investigations.

In skeletal muscle, the resting chloride conductance (gCl), due to the ClC-1 chloride channel, controls the sarcolemma electrical stability. Indeed, loss-of-function mutations in ClC-1 gene are responsible of myotonia congenita. The ClC-1 channel can be phosphorylated and inactivated by protein kinases C (PKC), but the relative contribution of each PKC isoforms is unknown. Here, we investigated on the role of PKCθ in the regulation of ClC-1 channel expression and activity in fast- and slow-twitch muscles of mouse models lacking PKCθ. Electrophysiological studies showed an increase of gCl in the PKCθ-null mice with respect to wild type. Muscle excitability was reduced accordingly. However, the expression of the ClC-1 channel, evaluated by qRT-PCR, was not modified in PKCθ-null muscles suggesting that PKCθ affects the ClC-1 activity. Pharmacological studies demonstrated that although PKCθ appreciably modulates gCl, other isoforms are still active and concur to this role. The modification of gCl in PKCθ-null muscles has caused adaptation of the expression of phenotype-specific genes, such as calcineurin and myocyte enhancer factor-2, supporting the role of PKCθ also in the settings of muscle phenotype. Importantly, the lack of PKCθ has prevented the aging-related reduction of gCl, suggesting that its modulation may represent a new strategy to contrast the aging process

Hindlimb unloading (HU) in rats induces severe atrophy and a slow-to-fast phenotype transition in postural slow-twitch muscles, as occurs in human disuse conditions, such as spaceflight or bed rest. In rats, a reduction of soleus muscle weight and a decrease of cross-sectional area (CSA) were observed as signs of atrophy. An increased expression of the fast-isoform of myosin heavy chain (MHC) showed the phenotype transition. In parallel the resting cytosolic calcium concentration (restCa) was decreased and the resting chloride conductance (gCl), which regulates muscle excitability, was increased toward the values of the fast-twitch muscles. Here, we investigated the possible role of taurine, which is known to modulate calcium homeostasis and gCl, in the restoration of muscle impairment due to 14-days-HU. We found elevated taurine content and higher expression of the taurine transporter TauT in the soleus muscle as compared to the fast-twitch extensor digitorum longus (EDL) muscle of control rats. Taurine level was reduced in the HU soleus muscle, although, TauT expression was not modified. Taurine oral supplementation (5 g/kg) fully prevented this loss, and preserved resting gCl and restCa together with the slow MHC phenotype. Taurine supplementation did not prevent the HU-induced drop of muscle weight or fiber CSA, but it restored the expression of MURF-1, an atrophy-related gene, suggesting a possible early protective effect of taurine. In conclusion, taurine prevented the HU-induced phenotypic transition of soleus muscle and might attenuate the atrophic process. These findings argue for the beneficial use of taurine in the treatment of disuse-induced muscle dysfunction.

Nerve Growth Factor (NGF)/Brain-derived Neurotrophic Factor (BDNF) and osteocalcin share common effects regulating energy, bone mass, reproduction and neuronal functions. To investigate on the gene-relationship between NGF, BDNF, and Osteocalcin we compared by RT-PCR the transcript levels of Ngf, Bdnf and Osteocalcin as well as of their receptors p75NTR/NTRK1, NTRK2, and Gprc6a in brain, bone, white/brown adipose tissue (WAT/BAT) and reproductive organs of 3 months old female and male mice. Brain and bone were used as positive controls for NGF/BDNF and Osteocalcin respectively. The role of oxitocin(Oxt) and its receptor(Oxtr) was also investigated. Ngf expression shows an opposite trend compared to Bdnf. Ngf /p75NTR expression is 50% higher in BAT than brain, in both genders, but lower in bone. In contrast, Bdnf expression in bone is higher than in brain, but low in BAT/WAT. We found Osteocalcin gene expressed in brain in both genders, but Gprc6a expression is low in brain and BAT/WAT. As expected, Gprc6a gene is expressed in bone. Oxt gene was markedly expressed in brain, Oxtr in the ovaries and in fat and bone in both genders. Ngf is highly expressed in reproductive tissues and p75NTR mRNA levels are respectively 300, 100, and 50% higher in testis/ovaries/uterus than in brain. In contrast, BDNF genes are not expressed in reproductive tissues. As expected, Gprc6a is expressed in testis but not in the ovaries/uterus. A significant correlation was found between the expression levels of the gene ligands and their receptors in brain, BAT and testis suggesting a common pathway of different genes in these tissues in either male and female. Changes in the expression levels of osteocalcin, Ngf, or Bdnf genes may mutually affect the expression levels of the others. Moreover, it may be possible that different ligands may operate through different receptor subtypes. Oxt and Oxtr failed to show significant correlation. The up-regulation of Ngf /p75NTR in BAT is consistent with NGF as an energy regulator and with BDNF regulating bone.