Effects of chronic administration of clenbuterol on contractile properties and calcium homeostasis in rat extensor digitorum longus muscle.

Clenbuterol, a ß2-agonist, induces skeletal muscle hypertrophy and a shift from slow-oxidative to fast-glycolytic muscle fiber type profile. However, the cellular mechanisms of the effects of chronic clenbuterol administration on skeletal muscle are not completely understood. As the intracellular Ca2+ concentration must be finely regulated in many cellular processes, the aim of this study was to investigate the effects of chronic clenbuterol treatment on force, fatigue, intracellular calcium (Ca2+) homeostasis and Ca2+-dependent proteolysis in fast-twitch skeletal muscles (the extensor digitorum longus, EDL, muscle), as they are more sensitive to clenbuterol-induced hypertrophy. Male Wistar rats were chronically treated with 4 mg.kg-1 clenbuterol or saline vehicle (controls) for 21 days. Confocal microscopy was used to evaluate sarcoplasmic reticulum Ca2+ load, Ca2+-transient amplitude and Ca2+ spark properties. EDL muscles from clenbuterol-treated animals displayed hypertrophy, a shift from slow to fast fiber type profile and increased absolute force, while the relative force remained unchanged and resistance to fatigue decreased compared to control muscles from rats treated with saline vehicle. Compared to control animals, clenbuterol treatment decreased Ca2+-transient amplitude, Ca2+ spark amplitude and frequency and the sarcoplasmic reticulum Ca2+ load was markedly reduced. Conversely, calpain activity was increased by clenbuterol chronic treatment. These results indicate that chronic treatment with clenbuterol impairs Ca2+ homeostasis and this could contribute to the remodeling and functional impairment of fast-twitch skeletal muscle.

Modelling training response in elite female gymnasts and optimal strategies of overload training and taper.

The aim of the study is the modelling of training responses with a variable dose-response model in a sport discipline that requires highly complex coordination. We propose a method to optimise the training programme plan using the potential maximal performance gain associated with overload and tapering periods. Data from five female elite gymnasts were collected over a 3-month training period. The relationship between training amounts and performance was then assessed with a non-linear model. The optimal magnitude of training load reduction and its duration were investigated with and without an overload period using simulation procedures based on individual responses to training. The correlation between actual and modelled performances was significant (R² = 0.81 ± 0.02, P < 0.01). The standard error was 2.7%. Simulations revealed that taper preceded by an overload period allows a higher performance to be achieved compared to an absence of overload period (106.3 ± 0.3% vs. 105.1 ± 0.3%). With respect to the pre-taper load, the model predicts that optimal load reductions during taper were 48.4 ± 0.7% and 42.5 ± 1.0% for overloading and non-overloading strategies, respectively. Moreover, optimal durations of the taper period were 34 ± 0.5 days and 22 ± 0.5 days for overloading and non-overloading strategies, respectively. In conclusion, the study showed that the variable dose-response model describes precisely the training response in gymnasts.

Early activation of rat skeletal muscle IL-6/STAT1/STAT3 dependent gene expression in resistance exercise linked to hypertrophy.

Cytokine interleukin-6 (IL-6) is an essential regulator of satellite cell-mediated hypertrophic muscle growth through the transcription factor signal transducer and activator of transcription 3 (STAT3). The importance of this pathway linked to the modulation of myogenic regulatory factors expression in rat skeletal muscle undergoing hypertrophy following resistance exercise, has not been investigated. In this study, the phosphorylation and nuclear localization of STAT3, together with IL-6/STAT3-responsive gene expression, were measured after both a single bout of resistance exercise and 10 weeks of training. Flexor Digitorum Profundus muscle samples from Wistar rats were obtained 2 and 6 hours after a single bout of resistance exercise and 72 h after the last bout of either 2, 4, or 10 weeks of resistance training. We observed an increase in IL-6 and SOCS3 mRNAs concomitant with phosphorylation of STAT1 and STAT3 after 2 and 6 hours of a single bout of exercise (p<0.05). STAT3-dependent early responsive genes such as CyclinD1 and cMyc were also upregulated whereas MyoD and Myf5 mRNAs were downregulated (p<0.05). BrdU-positive satellite cells increased at 2 and 6 hours after exercise (p<0.05). Muscle fiber hypertrophy reached up to 100% after 10 weeks of training and the mRNA expression of Myf5, c-Myc and Cyclin-D1 decreased, whereas IL-6 mRNA remained upregulated. We conclude that the IL-6/STAT1/STAT3 signaling pathway and its responsive genes after a single bout of resistance exercise are an important event regulating the SC pool and behavior involved in muscle hypertrophy after ten weeks of training in rat skeletal muscle.

Calpastatin overexpression in the skeletal muscle of mice prevents clenbuterol-induced muscle hypertrophy and phenotypic shift.

Accumulating evidence suggests that the calpain/calpastatin system is involved in skeletal muscle remodelling induced by ß(2) -adrenoceptor agonist treatment. In addition to other pathways, the Akt/mammalian target of rapamycin (mTOR) pathway, controlling protein synthesis, and the calcium/calmodulin-dependent protein kinase 2 (CamK2) and AMP-activated protein kinase (AMPK) pathways, recently identified as calpain substrates, could be relevant in ß(2) -adrenoceptor agonist-induced skeletal muscle remodelling. In the present study we investigated muscle hypertrophy and phenotypic shifts, as well as the molecular response of components of the Akt/mTOR pathway (i.e. Akt, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), ribosomal protein S6 (rpS6), CamK2 and AMPK), in response to calpastatin overexpression in the skeletal muscle of mice treated with 1 mg/kg per day clenbuterol for 21 days. Using gene electrotransfer of a calpastatin expression vector into the tibialis anterior of adult mice, we found that calpastatin overexpression attenuates muscle hypertrophy and phenotypic shifts induced by clenbuterol treatment. At the molecular level, calpastatin overexpression markedly decreased calpain activity, but was ineffective in altering the phosphorylation of Akt, 4E-BP1 and rpS6. In contrast, calpastatin overexpression increased the protein expression of both total AMPK and total CamK2. In conclusion, the results support the contention that the calpain/calpastatin system plays a crucial role in skeletal muscle hypertrophy and phenotypic shifts under chronic clenbuterol treatment, with AMPK and CamK2 probably playing a minor role. Moreover, the calpastatin-induced inhibition of hypertrophy under clenbuterol treatment was not related to a decreased mTOR-dependent initiation of protein translation.

Time course in calpain activity and autolysis in slow and fast skeletal muscle during clenbuterol treatment.

Calpains are Ca2+ cysteine proteases that have been proposed to be involved in the cytoskeletal remodeling and wasting of skeletal muscle. Cumulative evidence also suggests that ß2-agonists can lead to skeletal muscle hypertrophy through a mechanism probably related to calcium-dependent proteolytic enzyme. The aim of our study was to monitor calpain activity as a function of clenbuterol treatment in both slow and fast phenotype rat muscles. For this purpose, for 21 days we followed the time course of the calpain activity and of the ubiquitous calpain 1 and 2 autolysis, as well as muscle remodeling in the extensor digitorum longus (EDL) and soleus muscles of male Wistar rats treated daily with clenbuterol (4 mg·kg-1). A slow to fast fiber shift was observed in both the EDL and soleus muscles after 9 days of treatment, while hypertrophy was observed only in EDL after 9 days of treatment. Soleus muscle but not EDL muscle underwent an early apoptonecrosis phase characterized by hematoxylin and eosin staining. Total calpain activity was increased in both the EDL and soleus muscles of rats treated with clenbuterol. Moreover, calpain 1 autolysis increased significantly after 14 days in the EDL, but not in the soleus. Calpain 2 autolysis increased significantly in both muscles 6 hours after the first clenbuterol injection, indicating that clenbuterol-induced calpain 2 autolysis occurred earlier than calpain 1 autolysis. Together, these data suggest a preferential involvement of calpain 2 autolysis compared with calpain 1 autolysis in the mechanisms underlying the clenbuterol-induced skeletal muscle remodeling.

N-acetylcysteine protects against bupivacaine-induced myotoxicity caused by oxidative and sarcoplasmic reticulum stress in human skeletal myotubes.

BACKGROUND: Local anesthetics offer the benefits of extended analgesia with greater patient satisfaction and faster rehabilitation compared with intravenous morphine. These benefits, however, can be offset by adverse iatrogenic muscle pain. Here, the authors investigate the mechanisms of local anesthetic-induced myotoxicity and assess the protective effect of N-acetylcysteine. METHODS: The authors used primary cell cultures of human skeletal muscle myoblasts to study local anesthetic adverse effects. Production of reactive oxygen species was investigated in human skeletal myotubes by fluorescence microscopy. Expression of sarcoplasmic/endoplasmic reticulum stress markers and induction of apoptosis were followed by immunofluorescence and Western blot analysis. Finally, the effect of N-acetylcysteine on bupivacaine-induced myotoxicity was investigated in vitro. RESULTS: Bupivacaine sequentially induced reactive oxygen species production, oxidative stress, sarcoplasmic/endoplasmic reticulum stress, and activation of caspases 9 and 7 in human differentiated myoblasts. These iatrogenic effects were prevented by N-acetylcysteine. CONCLUSIONS: The authors demonstrated a protective effect of N-acetylcysteine against bupivacaine-induced sarcoplasmic/endoplasmic reticulum stress and apoptosis in primary human skeletal muscle cell.

Age-dependent bupivacaine-induced muscle toxicity during continuous peripheral nerve block in rats.

BACKGROUND: Regional blocks improve postoperative analgesia and postoperative rehabilitation in children and adult patients. Continuous peripheral nerve blocks have been proposed as safe and effective techniques for postoperative pain relief and chronic pain therapy, particularly in small children. Few clinical reports have described myotoxicity induced by bupivacaine in these young patients, in contrast with a larger number of observations in adults. Here, the authors addressed this issue by a comparative evaluation of bupivacaine-induced myotoxicity in young versus adult rats. METHODS: Femoral nerve block catheters were inserted in male Wistar rats. Young (3-week-old) and adult (12-week-old) rats were randomly assigned to received seven injections (1 ml/kg) of 0.25% bupivacaine (n = 6 per experiment) or isotonic saline (n = 6 per experiment) at 8-h intervals. Rats were killed 8 h after the last injection. Psoas muscle adjacent to the femoral nerve was quickly dissected. Oxygen consumption rates were measured in saponin-skinned fibers, mitochondrial adenosine triphosphate synthesis rates were determined by bioluminescence, and citrate synthase activity was determined by spectrophotometry. Muscle ultrastructural damage was also examined and scored as normal, focal disruption, moderate disruption, or extreme disruption of the sarcomeres. RESULTS: Bupivacaine caused a reduction of mitochondrial adenosine triphosphate synthesis rate, a decrease of citrate synthase activity, and muscle ultrastructural damages. Young rats treated with bupivacaine showed more severe alterations of mitochondrial bioenergetics and muscle ultrastructure. CONCLUSIONS: These findings demonstrate that bupivacaine-induced myotoxicity can be explained by mitochondrial bioenergetics alterations, which are more severe in young rats.

Erythropoietin protects against local anesthetic myotoxicity during continuous regional analgesia.

BACKGROUND: Local anesthetics offer the benefits of extended analgesia with greater patient satisfaction and faster rehabilitation compared with intravenous morphine. These benefits, however, can be offset by adverse iatrogenic muscle pain caused by bupivacaine. Here, the authors describe the mechanisms of local anesthetic-induced myotoxicity and a partial protective effect of recombinant human erythropoietin (rhEPO). METHODS: The authors developed a rat analgesia model with femoral nerve catheter and a cell culture model of human skeletal muscle myoblasts to study local anesthetic effects. Rats were randomly assigned to four different groups: daily intraperitoneal injection with 5,000 U/kg rhEPO or saline coupled to a perineural catheter injection with 1 ml/kg bupivacaine, 0.25%, or saline. In psoas rat muscle, oxygen consumption rates were measured using a Clark-type electrode in saponin-skinned fibers. Mitochondrial adenosine triphosphate synthesis rates were determined by bioluminescence. Enzymatic activity of mitochondrial respiratory chain complexes was measured on tissue homogenates using spectrophotometric procedures, and mitochondrial morphology was analyzed by transmission electron microscopy. In addition, the interaction between bupivacaine and rhEPO was investigated on human skeletal muscle myoblasts by fluorescence microscopy using mitotracker green and using the lipophilic cation JC-1. RESULTS: Bupivacaine caused impairment of mitochondrial structure and bioenergetics in rats. Human myoblasts treated with bupivacaine showed a dose-dependent decrease in mitochondrial membrane potential associated with unusual morphologies. Impairment of mitochondrial bioenergetics was prevented partially by the use of rhEPO coadministered with bupivacaine. CONCLUSIONS: The authors demonstrated a dose- and time-dependent protective effect of rhEPO against bupivacaine-induced myotoxicity in regional analgesia.

Contrasting effect of exercise and angiotensin II hypertension on in vivo and in vitro cardiac angiogenesis in rats.

Cardiac vessel density (beta-actin immunolabeling) and angiogenic capacity of coronary artery explants (culture in collagen gel) was determined in hypertrophied heart obtained by exercise training (10 wk) or ANG II infusion for 10 days. A group of rats received ANG II the last 10 days of training. The heart weight index was similarly elevated after exercise, and ANG II-hypertension compared with controls (3.16 +/- 0.09 and 3.11 +/- 0.11 vs. 2.68 +/- 0.08 mg/g, respectively), whereas tail cuff pressure (TCP) increased only in sedentary rats infused with ANG II. Vessel density was increased by 36% in trained rats and reduced by 30% in ANG II-infused rats. The number of sprouts generated by coronary rings was reduced by 50% in ANG II-infused rats and increased by 50% in exercise trained rats compared with controls (35 +/- 4 and 113 +/- 5 vs. 71 +/- 1 sprouts per ring, respectively). Exercise-training partly prevented the hypertensive effect of ANG II (TCP of 141 +/- 5 mmHg), whereas heart weight index (3.66 +/- 0.06 mg/g body wt) was not lowered. Myocardial vessel density was normalized, and sprouting from coronary rings increased by 50% in trained rats infused with ANG II compared with sedentary normotensive rats. Cardiac VEGF (Western blot analysis) was higher in hypertensive rats and not affected by exercise. Facing a similar increase in cardiac mass, intense training, but not ANG II hypertension, is accompanied by an increase in vascular density of the heart. The effect of training is unlikely related to changes in resting VEGF and may represent enhanced angiogenic capacity of the coronary vascular bed.