Protein kinase C theta (PKCθ) modulates the ClC-1 chloride channel activity and skeletal muscle phenotype: a biophysical and gene expression study in mouse models lacking the PKCθ

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

Statin-induced myotoxicity is exacerbated by aging: A biophysical and molecular biology study in rats treated with atorvastatin

Statin-induced skeletal muscle damage in rats is associated to the reduction of the resting sarcolemmal chloride conductance (gCl) and ClC-1 chloride channel expression. These drugs also affect the ClC-1 regulation by increasing protein kinase C (PKC) activity, which phosphorylate and close the channel. Also the intracellular resting calcium (restCa) level is increased. Similar alterations are observed in skeletal muscles of aged rats, suggesting a higher risk of statin myotoxicity. To verify this hypothesis, we performed a 4–5-weeks atorvastatin treatment of 24-months-old rats to evaluate the ClC-1 channel function by the two-intracellular microelectrodes technique as well as transcript and protein expression of different genes sensitive to statins by quantitative real-time-PCR and western blot analysis. The restCa was measured using FURA-2 imaging, and histological analysis of muscle sections was performed. The results show a marked reduction of resting gCl, in agreement with the reduced ClC-1 mRNA and protein expression in atorvastatin-treated aged rats, with respect to treated adult animals. The observed changes in myocyte-enhancer factor-2 (MEF2) expression may be involved in ClC-1 expression changes. The activity of PKC was also increased and further modulate the gCl in treated aged rats. In parallel, a marked reduction of the expression of glycolytic and mitochondrial enzymes demonstrates an impairment of muscle metabolism. No worsening of restCa or histological features was found in statin-treated aged animals. These findings suggest that a strong reduction of gCl and alteration of muscle metabolism coupled to muscle atrophy may contribute to the increased risk of statin-induced myopathy in the elderly

Cold stress in mice requires Nerve Growth Factor activity in brown fat and increases Osteocalcin expression in bone

Brown adipose tissue (BAT) is controlled by the sympathetic nervous system (SNS) and has the ability to dissipate energy through uncoupling protein-1 (UCP-1), influencing energy expenditure. Besides BAT, the SNS influences bone, and recent studies have demonstrated a positive correlation between BAT activity and bone. Nerve Growth Factor (NGF) genes are expressed in brain, white adipose tissue (WAT), BAT and bone where it coordinates brain and body reactions to challenges. Similarly Osteocalcin besides its role in bone metabolism acts as an hormone regulating glucose metabolism and brain. We previously showed that NGF and its receptor p75NTR genes are highly expressed in BAT versus brain in mice, suggesting that NGF may act as a regulator of energy. To further investigate the role of NGF and osteocalcin in bone and energy regulation we analyzed NGF and its receptor p75NTR and Osteocalcin mRNA from 3 months old mice after cold exposure. UCP-1 was used as positive control. Mice were divided into three groups: room temperature (RT), cold stress for 6h and 5 days (n = 5 for each group). Mice as control group were all placed at RT (23 °C) for 5 days, while the cold groups were placed at 4 °C for the abovementioned times. The mice were subsequently sacrificed and the interscapular BAT, bone and brain were analyzed for mRNA extraction. The exposure of 6 h to cold stress enhanced mRNA levels of UCP-1 and NGF genes by 3 and 2.5 fold vs controls, respectively, reducing mRNA of p75NTR by 19 fold. The UCP-1 gene was still up-regulated after 5 days of cold stress, the NGF gene was not affected and mRNA of the p75NTR gene was reduced by 7 fold vs controls. The mRNA NGF/NGFR genes were not affected in bone or brain following cold stress. The mRNA levels of osteocalcin in bone were upregulated following 6h and 5 days cold exposure vs controls. Osteocalcin gene in brain was downregulated following cold stress. Our study shows that NGF mRNA expression significantly increases after 6 hours of cold stress however with minor extent with respect to UCP-1. The enhanced mRNA level of NGF is observed in parallel with the decrease of NGF receptor gene expression. We found no change in NGF and p75NTR expression in brain or bone, but osteocalcin genes were upregulated in bone following cold stress. These results suggest that during cold stress when BAT-dependent thermogenesis is required, NGF activity is required, and osteocalcin may exert a local protective effect on bone mass

Cisplatin-Induced Cachexia in rats Causes Alterations in Skeletal Muscle Calcium Homeostasis

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Risk of Myopathy in Patients in Therapy with Statins: Identification of Biological Markers in a Pilot Study

Statin therapy may induce skeletal muscle damage ranging from myalgia to severe rhabdomyolysis. Our previous preclinical studies showed that statin treatment in rats involves the reduction of skeletal muscle ClC-1 chloride channel expression and related chloride conductance (gCl). An increase of the activity of protein kinase C theta (PKC theta) isoform, able to inactivate ClC-1, may contribute to destabilize sarcolemma excitability. These effects can be detrimental for muscle function leading to drug-induced myopathy. Our goal is to study the causes of statin-induced muscle side effects in patients at the aim to identify biological markers useful to prevent and counteract statin-induced muscle damage. We examined 10 patients, who experienced myalgia and hyper-CK-emia after starting statin therapy compared to 9 non-myopathic subjects not using lipid-lowering drugs. Western Blot (WB) analysis showed a 40% reduction of ClC-1 protein and increased expression of phosphorylated PKC in muscle biopsies of statin-treated patients with respect to untreated subjects, independently from their age and statin type. Real-time PCR analysis showed that despite reduction of the protein, the ClC-1 mRNA was not significantly changed, suggesting post-transcriptional modification. The mRNA expression of a series of genes was also evaluated. MuRF-1 was increased in accord with muscle atrophy, MEF-2, calcineurin (CN) and GLUT-4 transporter were reduced, suggesting altered transcription, alteration of glucose homeostasis and energy deficit. Accordingly, the phosphorylated form of AMPK, measured by WB, was increased, suggesting cytoprotective process activation. In parallel, mRNA expression of Notch-1, involved in muscle cell proliferation, was highly expressed in statin-treated patients, indicating active regeneration. Also, PGC-1-alpha and isocitrate-dehydrogenase increased expression together with increased activity of mitochondrial citrate-synthase, measured by spectrophotometric assay, suggests mitochondrial biogenesis. Thus, the reduction of ClC-1 protein and consequent sarcolemma hyperexcitability together with energy deficiency appear to be among the most important alterations to be associated with statin-related risk of myopathy in humans. Thus, it may be important to avoid statin treatment in pathologies characterized by energy deficit and chloride channel malfunction. This study validates the measure of ClC-1 expression as a reliable clinical test for assessing statin-dependent risk of myopathy

Effects of Nandrolone in the counteraction of skeletal muscle atrophy in a mouse model of muscle disuse: molecular biology and functional evaluation

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

Multidisciplinary study of a new ClC-1 mutation causing myotonia congenita: a paradigm to understand and treat ion channelopathies

Myotonia congenita is an inherited disease that is characterized by impaired muscle relaxation after contraction caused by loss-of-function mutations in the skeletal muscle ClC-1 channel. We report a novel ClC-1 mutation, T335N, that is associated with a mild phenotype in 1 patient, located in the extracellular I-J loop. The purpose of this study was to provide a solid correlation between T335N dysfunction and clinical symptoms in the affected patient as well as to offer hints for drug development. Our multidisciplinary approach includes patch-clamp electrophysiology on T335N and ClC-1 wild-type channels expressed in tsA201 cells, Western blot and quantitative PCR analyses on muscle biopsies from patient and unaffected individuals, and molecular dynamics simulations using a homology model of the ClC-1 dimer. T335N channels display reduced chloride currents as a result of gating alterations rather than altered surface expression. Molecular dynamics simulations suggest that the I-J loop might be involved in conformational changes that occur at the dimer interface, thus affecting gating. Finally, the gene expression profile of T335N carrier showed a diverse expression of K(+) channel genes, compared with control individuals, as potentially contributing to the phenotype. This experimental paradigm satisfactorily explained myotonia in the patient. Furthermore, it could be relevant to the study and therapy of any channelopathy

Effects of Nandrolone (ND) treatment on gene expression level in Soleus muscle of HU mice.

<p>Histograms show quantification of transcript levels performed with real time PCR, for <i>mTO</i>R, <i>Eif2ak3</i>, <i>Pgc1α</i>, <i>Lc3- β</i>, <i>Cathepsin</i>, <i>Atrogin-1 and Murf-1</i> genes normalized by the <i>Hprt1</i> gene, in the 4 experimental groups (Ctrl, HU, HU-V, HU-ND). Each bar represents the mean ± S.E.M. from the number of animals as indicated in the brackets above the bars. Statistical analysis was performed for each muscle type using ANOVA followed by Fisher t-test *vs Ctrl (at least P<0.05).</p

Effects of Nandrolone (ND) treatment on muscle total protein content of HU mice.

<p>Each bar represents the mean ± S.E.M. calculated from <b>(A)</b> 6 control (CTRL) Soleus (Sol) muscles; 6 hindlimb unloaded (HU) Sol muscles; 5 vehicle-treated hindlimb unloaded (HU-V) Sol muscles; 6 nandrolone-treated hindlimb unloaded (HU-ND) Sol muscles and <b>(B)</b> 6 control (CTRL) Extensor Digitorum Longus (EDL) muscle samples; 6 hindlimb unloaded (HU) EDL muscles; 6 vehicle-treated hindlimb unloaded (HU-V) EDL muscles; 6 nandrolone-treated hindlimb unloaded (HU-ND) EDL muscles. Statistical analysis was performed for each muscle type using ANOVA followed by Bonferroni’s t-test (F = 3.25, dF = 3/19, P<0.05 for Sol muscle) (F = 1.78, dF = 3/20, N.S. for EDL muscle). Significantly different (at least P<0.05) versus (*) CTRL, versus (#) HU and versus (°) HU-V.</p