Benoît Giannesini

Non-invasive investigations of muscular fatigue: metabolic and electromyographic components

Muscle fatigue, which is defined as the decline in muscle performance during exercise, may occur at different sites along the pathway from the central nervous system through to the intramuscular contractile machinery. Historically, both impairment of neuromuscular transmission and peripheral alterations within the muscle have been proposed to be involved in the development of fatigue. However, according to the more recent studies, muscle energetics would have a key role in this process. Intramyoplasmic accumulation of inorganic phosphate (P(i)) and limitation in ATP availability are frequently proposed as the causative factors of fatigue development. Although attractive, these hypotheses have been elaborated on the basis of experimental results obtained in vitro and their physiological relevance has never been clearly demonstrated in vivo. In that context, non-invasive methods such as 31-phosphorus magnetic resonance spectroscopy ((31)P MRS) and electromyographic (EMG) recordings have been employed to understand both metabolic and electrical aspects of muscle fatigue under physiological condition. The main results of these studies are reviewed in the present paper.

Vitamin D protects against diet-induced obesity by enhancing fatty acid oxidation

Prospective studies reported an inverse correlation between 25-hydroxyvitamin D [25(OH)D] plasma levels and prevalence of obesity and type 2 diabetes. In addition, 25(OH)D status may be a determinant of obesity onset. However, the causality between these observations is not yet established. We studied the preventive effect of vitamin D3 (VD3) supplementation (15,000 IU/kg of food for 10 weeks) on onset of obesity in a diet-induced obesity mouse model. We showed that the VD3 supplementation limited weight gain induced by high-fat diet, which paralleled with an improvement of glucose homeostasis. The limitation of weight gain could further be explained by an increased lipid oxidation, possibly due to an up-regulation of genes involved in fatty acid oxidation and mitochondrial metabolism, leading to increased energy expenditure. Altogether, these data show that VD3 regulates energy expenditure and suggest that VD3 supplementation may represent a strategy of preventive nutrition to fight the onset of obesity and associated metabolic disorders.

Triadin Deletion Induces Impaired Skeletal Muscle Function

Triadin is a multiple proteins family, some isoforms being involved in muscle excitation-contraction coupling, and some having still unknown functions. To obtain clues on triadin functions, we engineered a triadin knock-out mouse line and characterized the physiological effect of triadin ablation on skeletal muscle function. These mice presented a reduced muscle strength, which seemed not to alter their survival and has been characterized in the present work. We first checked in these mice the expression level of the different proteins involved in calcium homeostasis and observed in fast muscles an increase in expression of dihydropyridine receptor, with a large reduction in calsequestrin expression. Electron microscopy analysis of KO muscles morphology demonstrated the presence of triads in abnormal orientation and a reduction in the sarcoplasmic reticulum terminal cisternae volume. Using calcium imaging on cultured myotubes, we observed a reduction in the total amount of calcium stored in the sarcoplasmic reticulum. Physiological studies have been performed to evaluate the influence of triadin deletion on skeletal muscle function. Muscle strength has been measured both on the whole animal model, using hang test or electrical stimulation combined with NMR analysis and strength measurement, or on isolated muscle using electrical stimulation. All the results obtained demonstrate an important reduction in muscle strength, indicating that triadin plays an essential role in skeletal muscle function and in skeletal muscle structure. These results indicate that triadin alteration leads to the development of a myopathy, which could be studied using this new animal model.

Functional investigations of exercising muscle: a noninvasive magnetic resonance spectroscopy-magnetic resonance imaging approach

Muscle fatigue, which is defined as the decline in muscle performance during exercise, may occur at different sites along the pathway from the central nervous system through to the intramuscular contractile machinery. Historically, both impairment of neuromuscular transmission and peripheral alterations within the muscle have been proposed as causative factors of fatigue development. However, according to more recent studies, muscle energetics play a key role in this process. Intramyoplasmic accumulation of inorganic phosphate (Pi) and limitation in ATP availability have been frequently evoked as the main mechanisms leading to fatigue. Although attractive, these hypotheses have been elaborated on the basis of experimental results obtained in vitro, and their physiological relevance has never been clearly demonstrated in vivo. In that context, noninvasive methods such as 31-phosphorus magnetic resonance spectroscopy and surface electromyography have been employed to understand both metabolic and electrical aspects of muscle fatigue under physiological conditions. Mapping of muscles activated during exercise is another interesting issue which can be addressed using magnetic resonance imaging (MRI). Exercise-induced T2 changes have been used in order to locate activated muscles and also as a quantitative index of exercise intensity. The main results related to both issues, i.e. the metabolic and electrical aspects of fatigue and the MRI functional investigation of exercising muscle, are discussed in the present review.

Blockade of ActRIIB Signaling Triggers Muscle Fatigability and Metabolic Myopathy

Myostatin regulates skeletal muscle size via the activin receptor IIB (ActRIIB). However, its effect on muscle energy metabolism and energy-dependent muscle function remains largely unexplored. This question needs to be solved urgently since various therapies for neuromuscular diseases based on blockade of ActRIIB signaling are being developed. Here, we show in mice, that 4-month pharmacological abrogation of ActRIIB signaling by treatment with soluble ActRIIB-Fc triggers extreme muscle fatigability. This is associated with elevated serum lactate levels and a severe metabolic myopathy in the mdx mouse, an animal model of Duchenne muscular dystrophy. Blockade of ActRIIB signaling downregulates porin, a crucial ADP/ATP shuttle between cytosol and mitochondrial matrix leading to a consecutive deficiency of oxidative phosphorylation as measured by in vivo Phosphorus Magnetic Resonance Spectroscopy ((31)P-MRS). Further, ActRIIB blockade reduces muscle capillarization, which further compounds the metabolic stress. We show that ActRIIB regulates key determinants of muscle metabolism, such as Pparβ, Pgc1α, and Pdk4 thereby optimizing different components of muscle energy metabolism. In conclusion, ActRIIB signaling endows skeletal muscle with high oxidative capacity and low fatigability. The severe metabolic side effects following ActRIIB blockade caution against deploying this strategy, at least in isolation, for treatment of neuromuscular disorders.

In vivo reduction in ATP cost of contraction is not related to fatigue level in stimulated rat gastrocnemius muscle

1 We tested whether the reduction in ATP cost of contraction during in vivo stimulation of rat gastrocnemius muscle was related to fatigue level. 2 Muscles (n= 44) were electrically stimulated to perform 6 min repeated isometric contractions at different frequencies; one non‐fatiguing protocol (stimulation at 0.8 Hz) and five fatiguing protocols (2, 3.2, 4, 5.2 and 7.6 Hz) were used. Anaerobic and oxidative ATP turnover rates were measured non‐invasively using 31P‐magnetic resonance spectroscopy. 3 At the onset of the stimulation period, no signs of fatigue were measured in the six protocols and ATP cost of contraction did not differ significantly (P= 0.45) among protocols (mean value of 1.76 ± 0.11 mm (N s)−1). 4 For the six protocols, ATP cost of contraction was significantly reduced (P < 0.05) at the end of the stimulation period when compared with the initial value. This reduction did not differ significantly (P= 0.61) among the five fatiguing protocols (averaging 35 ± 3 % of initial value), whereas isometric force decreased significantly as stimulation frequency increased. No significant correlation (P= 0.87, r2= 0.01) was observed between isometric force and ATP cost of contraction at the end of the stimulation period. In addition, this reduction was significantly lower (P < 0.05) for the non‐fatiguing protocol (67 ± 9 % of initial value) when compared with the fatiguing protocols. 5 These results demonstrate that (i) the reduction in ATP cost of contraction during in vivo stimulation of rat gastrocnemius muscle is not related to the fatigue level; (ii) surprisingly, this reduction was significantly larger during the fatiguing protocols compared with the non‐fatiguing protocol.

A strictly noninvasive MR setup dedicated to longitudinal studies of mechanical performance, bioenergetics, anatomy, and muscle recruitment in contracting mouse skeletal muscle

MR techniques have proven their ability to investigate skeletal muscle function in situ. Their benefit in terms of noninvasiveness is, however, lost in animal research, given that muscle stimulation and force output measurements are usually achieved using invasive surgical procedures, thereby excluding repeated investigations in the same animal. This study describes a new setup allowing strictly noninvasive investigations of mouse gastrocnemius muscle function using 1H‐MRI and 31P‐MR spectroscopy. Its originality is to integrate noninvasive systems for inducing muscle contraction through transcutaneous stimulation and for measuring mechanical performance with a dedicated ergometer. In order to test the setup, muscle function was investigated using a fatiguing stimulation protocol (6 min of repeated isometric contractions at 1.7 Hz). T2‐weighted imaging demonstrated that transcutaneous stimulation mainly activated the gastrocnemius. Moreover, investigations repeated twice with a 7‐day interval between bouts did show a high reproducibility in measurements with regard to changes in isometric force and energy metabolism. In conclusion, this setup enables us for the first time to access mechanical performance, energy metabolism, anatomy, and physiology strictly noninvasively in contracting mouse skeletal muscle. The possibility for implementing longitudinal studies opens up new perspectives in many research areas, including ageing, pharmaceutical research, and gene and cell therapy. Magn Reson Med, 2010.

New experimental setup for studying strictly noninvasively skeletal muscle function in rat using 1H-magnetic resonance (MR) imaging and 31P-MR spectroscopy.

Traditional setups for in situ MR investigation of skeletal muscle function in animals use invasive systems for muscle stimulation and force measurement. These systems require surgical preparation and therefore exclude repetitive investigations on the same animal. This article describes a new experimental setup allowing strictly noninvasive MR investigations of muscle function in contracting rat gastrocnemius muscle using 1H‐MR imaging and 31P‐MR spectroscopy. The novelty of this setup is the integration of two noninvasive systems allowing muscle contraction by transcutaneous stimulation and force measurement with a dedicated ergometer. Muscle function was investigated in 20 rats (275–300 g) through a fatiguing stimulation protocol, either with this noninvasive setup (n = 10) or with a traditional MR setup (n = 10). T2‐weighted images demonstrated that transcutaneous stimulation activated mainly the gastrocnemius muscle. Moreover, the changes in force development and in energy metabolism obtained with the noninvasive setup were qualitatively and quantitatively similar to those obtained with the traditional setup. This noninvasive setup is thus suitable for investigating skeletal muscle function in situ. It offers the possibility to repeat investigations in the same animal, avoiding individual variability and enabling longitudinal follow‐up studies. This opens up new perspectives in various research areas including pharmaceutical research. Magn Reson Med, 2005.

Lack of myostatin impairs mechanical performance and ATP cost of contraction in exercising mouse gastrocnemius muscle in vivo

Although it is well established that the lack of myostatin (Mstn) promotes skeletal muscle hypertrophy, the corresponding changes regarding force generation have been studied mainly in vitro and remain conflicting. Furthermore, the metabolic underpinnings of these changes are very poorly documented. To clarify this issue, we have investigated strictly noninvasively in vivo the impact of the lack of Mstn on gastrocnemius muscle function and energetics in Mstn-targeted knockout (Mstn-/-) mice using ¹H-magnetic resonance (MR) imaging and ³¹P-MR spectroscopy during maximal repeated isometric contractions induced by transcutaneous electrostimulation. In Mstn-/- animals, although body weight, gastrocnemius muscle volume, and absolute force were larger (+38, +118, and +34%, respectively) compared with wild-type (Mstn+/+) mice, specific force (calculated from MR imaging measurements) was significantly lower (-36%), and resistance to fatigue was decreased. Besides, Mstn deficiency did not affect phosphorylated compound concentrations and intracellular pH at rest but caused a large increase in ATP cost of contraction (up to +206% compared with Mstn+/+) throughout the stimulation period. Further, Mstn deficiency limits the shift toward oxidative metabolism during muscle activity despite the fact that oxidative ATP synthesis capacity was not altered. Our data demonstrate in vivo that the absence of Mstn impairs both mechanical performance and energy cost of contraction in hypertrophic muscle. These findings must be kept in mind when considering Mstn as a potential therapeutic target for increasing muscle mass in patients suffering from muscle-wasting disorders.

Citrulline malate supplementation increases muscle efficiency in rat skeletal muscle.

Citrulline malate (CM; CAS 54940-97-5, Stimol®) is known to limit the deleterious effect of asthenic state on muscle function, but its effect under healthy condition remains poorly documented. The aim of this longitudinal double-blind study was to investigate the effect of oral ingestion of CM on muscle mechanical performance and bioenergetics in normal rat. Gastrocnemius muscle function was investigated strictly non-invasively using nuclear magnetic resonance techniques. A standardized rest-stimulation- (5.7 min of repeated isometric contractions electrically induced by transcutaneous stimulation at a frequency of 3.3 Hz) recovery-protocol was performed twice, i.e., before (t(0)-24 h) and after (t(0)+48 h) CM (3 g/kg/day) or vehicle treatment. CM supplementation did not affect PCr/ATP ratio, [PCr], [Pi], [ATP] and intracellular pH at rest. During the stimulation period, it lead to a 23% enhancement of specific force production that was associated to significant decrease in both PCr (28%) and oxidative (32%) costs of contraction, but had no effect on the time-courses of phosphorylated compounds and intracellular pH. Furthermore, both the rate of PCr resynthesis during the post-stimulation period (VPCr(rec)) and the oxidative ATP synthesis capacity (Q(max)) remained unaffected by CM treatment. These data demonstrate that CM supplementation under healthy condition has an ergogenic effect associated to an improvement of muscular contraction efficiency.