Christophe Vilmen

In vivo pH in metabolic-defective Ras-transformed fibroblast tumors: key role of the monocarboxylate transporter, MCT4, for inducing an alkaline intracellular pH.

We present an investigation of tumor pH regulation, designed to support a new anticancer therapy concept that we had previously proposed. Our study uses a tumor model of ras‐transformed hamster fibroblasts, CCL39, xenografted in the thighs of nude mice. We demonstrate, for the first time, that genetic modifications of specific mechanisms of proton production and/or proton transport result in distinct, reproducible changes in intracellular and extracellular tumor pH that can be detected and quantified noninvasively in vivo, simultaneously with determinations of tumor energetic status and necrosis in the same experiment. The CCL39 variants used were deficient in the sodium/proton exchanger, NHE‐1, and/or in the monocarboxylate transporter, MCT4; further, variants were deficient in glycolysis or respiration. MCT4 expression markedly increased the gradient between intracellular and extracellular pH from 0.14 to 0.43 when compared to CCL39 wild‐type tumors not expressing MCT4. The other genetic modifications studied produced smaller but significant increases in intracellular and decreases in extracellular pH. In general, increased pH gradients were paralleled by increased tumor growth performance and diminished necrotic regions, and 50% of the CCL39 variant expressing neither MCT4 nor NHE‐1, but possessing full genetic capacity for glycolysis and oxidative phosphorylation, underwent regression before reaching a 1‐cm diameter. Except for CCL39 wild‐type tumors, no significant HIF‐1α expression was detected. Our in vivo results support a multipronged approach to tumor treatment based on minimizing intracellular pH by targeting several proton production and proton transport processes, among which the very efficient MCT4 proton/lactate co‐transport deserves particular attention.

Reproducibility assessment of metabolic variables characterizing muscle energetics in Vivo: A 31P-MRS study

The purpose of the present study was to assess the reliability of metabolic parameters measured using 31P magnetic resonance spectroscopy (31P MRS) during two standardized rest‐exercise‐recovery protocols. Twelve healthy subjects performed the standardized protocols at two different intensities; i.e., a moderate intensity (MOD) repeated over a two‐month period and heavy intensity (HEAVY) repeated over a years time. Test‐retest reliability was analyzed using coefficient of variation (CV), limits of agreement (LOA), and intraclass correlation coefficients (ICC). During exercise and recovery periods, most of the metabolic parameters exhibited a good reliability. The CVs of individual concentration of phosphocreatine ([PCr]), concentration of adenosine diphosphate ([ADP]), and pH values recorded at end of the HEAVY exercise were lower than 15%. The CV calculated for the rate of PCr resynthesis and the maximal oxidative capacity were less than 13% during the HEAVY protocol. Inferred parameters such as oxidative and total adenosine triphosphate (ATP) production rates exhibited a good reliability (ICC ≈ 0.7; CV < 15% during the HEAVY protocol). Our results demonstrated that measurement error using 31P‐MRS during a standardized exercise was low and that biological variability accounted for the vast majority of the measurement variability. In addition, the corresponding metabolic measurements can reliably be used for longitudinal studies performed even over a long period of time. Magn Reson Med, 2009.

Muscle energetics changes throughout maturation: a quantitative 31P-MRS analysis.

We quantified energy production in 7 prepubescent boys (11.7 ± 0.6 yr) and 10 men (35.6 ± 7.8 yr) using (31)P-magnetic resonance spectroscopy to investigate whether development affects muscle energetics, given that resistance to fatigue has been reported to be larger before puberty. Each subject performed a finger flexions exercise at 0.7 Hz against a weight adjusted to 15% of their maximal voluntary strength for 3 min, followed by a 15-min recovery period. The total energy cost was similar in both groups throughout the exercise bout, whereas the interplay of the different metabolic pathways was different. At the onset of exercise, children exhibited a higher oxidative contribution (50 ± 15% in boys and 25 ± 8% in men, P < 0.05) to ATP production, whereas the phosphocreatine breakdown contribution was reduced (40 ± 10% in boys and 53 ± 12% in men, P < 0.05), likely as a compensatory mechanism. The anaerobic glycolysis activity was unaffected by maturation. The recovery phase also disclosed differences regarding the rates of proton efflux (6.2 ± 2.5 vs. 3.8 ± 1.9 mM · pH unit(-1) · min(-1), in boys and men, respectively, P < 0.05), and phosphocreatine recovery, which was significantly faster in boys than in men (rate constant of phosphocreatine recovery: 1.3 ± 0.5 vs. 0.7 ± 0.4 min(-1); V(max): 37.5 ± 14.5 vs. 21.1 ± 12.2 mM/min, in boys and men, respectively, P < 0.05). Our results obtained in vivo clearly showed that maturation affects muscle energetics. Children relied more on oxidative metabolism and less on creatine kinase reaction to meet energy demand during exercise. This phenomenon can be explained by a greater oxidative capacity, probably linked to a higher relative content in slow-twitch fibers before puberty.

Effects of a prior high‐intensity knee‐extension exercise on muscle recruitment and energy cost: a combined local and global investigation in humans

The effects of a priming exercise bout on both muscle energy production and the pattern of muscle fibre recruitment during a subsequent exercise bout are poorly understood. The purpose of the present study was to determine whether a prior exercise bout which is known to increase O2 supply and to induce a residual acidosis could alter energy cost and muscle fibre recruitment during a subsequent heavy‐intensity knee‐extension exercise. Fifteen healthy subjects performed two 6 min bouts of heavy exercise separated by a 6 min resting period. Rates of oxidative and anaerobic ATP production, determined with 31P‐magnetic resonance spectroscopy, and breath‐by‐breath measurements of pulmonary oxygen uptake were obtained simultaneously. Changes in muscle oxygenation and muscle fibre recruitment occurring within the quadriceps were measured using near‐infrared spectroscopy and surface electromyography. The priming heavy‐intensity exercise increased motor unit recruitment (P < 0.05) in the early part of the subsequent exercise bout but did not alter muscle energy cost. We also observed a reduced deoxygenation time delay, whereas the deoxygenation amplitude was increased (P < 0.01). These changes were associated with an increased oxidative ATP cost after ∼50 s (P < 0.05) and a slight reduction in the overall anaerobic rate of ATP production (0.11 ± 0.04 mm min−1 W−1 for bout 1 and 0.06 ± 0.11 mm min−1 W−1 for bout 2; P < 0.05). We showed that a priming bout of heavy exercise led to an increased recruitment of motor units in the early part of the second bout of heavy exercise. Considering the increased oxidative cost and the unaltered energy cost, one could suggest that our results illustrate a reduced metabolic strain per fibre.

Comparative determination of energy production rates and mitochondrial function using different 31P MRS quantitative methods in sedentary and trained subjects.

Muscle energetics has been largely and quantitatively investigated using 31P MRS. Various methods have been used to estimate the corresponding rate of oxidative ATP synthesis (ATPox); however, potential differences among methods have not been investigated. In this study, we aimed to compare the rates of ATP production and energy cost in two groups of subjects with different training status using four different methods: indirect method (method 1), ADP control model (method 2) and phosphate potential control model (method 3). Method 4 was a modified version of method 3 with the introduction of a correction factor allowing for similar values to be obtained for the end‐exercise oxidative ATP synthesis rate inferred from exercise measurements and the initial recovery phosphocreatine resynthesis rate. Seven sedentary and seven endurance‐trained subjects performed a dynamic standardised rest–exercise–recovery protocol. We quantified the rates of ATPox and anaerobic ATP synthesis (ATPana) using 31P MRS data recorded at 1.5 T. The rates of ATPox over the entire exercise session were independent of the method used, except for method 4 which provided significantly higher values in both groups (p < 0.01). In addition, methods 1–3 were cross‐correlated, thereby confirming their statistical agreement. The rate of ATPana was significantly higher with method 1 (p < 0.01) and lower with method 4 (p < 0.01). As a result of the higher rate of ATPox, EC (method 4) calculated over the entire exercise session was higher and initial EC (method 1) was lower in both groups compared with the other methods. We showed in this study that the rate of ATPox was independent of the calculation method, as long as no corrections (method 4) were performed. In contrast, results related to the rates of ATPana were strongly affected by the calculation method and, more exactly, by the estimation of protons generated by ATPox. Although the absolute EC values differed between the methods, within‐ or between‐subject comparisons are still valid given the tight relationships between them. Copyright

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.

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.

EFFECTS OF STIMULATION FREQUENCY AND PULSE DURATION ON FATIGUE AND METABOLIC COST DURING A SINGLE BOUT OF NEUROMUSCULAR ELECTRICAL STIMULATION

We have investigated the effects of stimulation frequency and pulse duration on fatigue and energy metabolism in rat gastrocnemius muscle during a single bout of neuromuscular electrical stimulation (NMES). Electrical pulses were delivered at 100 Hz (1‐ms pulse duration) and 20 Hz (5‐ms pulse duration) for the high (HF) and low (LF) frequency protocols, respectively. As a standardization procedure, the averaged stimulation intensity, the averaged total charge, the initial peak torque, the duty cycle, the contraction duration and the torque‐time integral were similar in both protocols. Fatigue was assessed using two testing trains delivered at a frequency of 100 Hz and 20 Hz before and after each protocol. Metabolic changes were investigated in vivo using 31P‐magnetic resonance spectroscopy (31P‐MRS) and in vitro in freeze‐clamped muscles. Both LF and HF NMES protocols induced the same decrease in testing trains and metabolic changes. We conclude that, under carefully controlled and comparable conditions, the use of low stimulation frequency and long pulse duration do not minimize the occurrence of muscle fatigue or affect the corresponding stimulation‐induced metabolic changes so that this combination of stimulation parameters would not be adequate in the context of rehabilitation. Muscle Nerve, 2010

Multimodal MRI and 31P-MRS Investigations of the ACTA1(Asp286Gly) Mouse Model of Nemaline Myopathy Provide Evidence of Impaired In Vivo Muscle Function, Altered Muscle Structure and Disturbed Energy Metabolism

Nemaline myopathy (NM), the most common non-dystrophic congenital disease of skeletal muscle, can be caused by mutations in the skeletal muscle α-actin gene (ACTA1) (~25% of all NM cases and up to 50% of severe forms of NM). Muscle function of the recently generated transgenic mouse model carrying the human Asp286Gly mutation in the ACTA1 gene (Tg(ACTA1)Asp286Gly) has been mainly investigated in vitro. Therefore, we aimed at providing a comprehensive picture of the in vivo hindlimb muscle function of Tg(ACTA1)Asp286Gly mice by combining strictly noninvasive investigations. Skeletal muscle anatomy (hindlimb muscles, intramuscular fat volumes) and microstructure were studied using multimodal magnetic resonance imaging (Dixon, T2, Diffusion Tensor Imaging [DTI]). Energy metabolism was studied using 31-phosphorus Magnetic Resonance Spectroscopy (31P-MRS). Skeletal muscle contractile performance was investigated while applying a force-frequency protocol (1–150 Hz) and a fatigue protocol (6 min–1.7 Hz). Tg(ACTA1)Asp286Gly mice showed a mild muscle weakness as illustrated by the reduction of both absolute (30%) and specific (15%) maximal force production. Dixon MRI did not show discernable fatty infiltration in Tg(ACTA1)Asp286Gly mice indicating that this mouse model does not reproduce human MRI findings. Increased T2 values were observed in Tg(ACTA1)Asp286Gly mice and might reflect the occurrence of muscle degeneration/regeneration process. Interestingly, T2 values were linearly related to muscle weakness. DTI experiments indicated lower λ2 and λ3 values in Tg(ACTA1)Asp286Gly mice, which might be associated to muscle atrophy and/or the presence of histological anomalies. Finally 31P-MRS investigations illustrated an increased anaerobic energy cost of contraction in Tg(ACTA1)Asp286Gly mice, which might be ascribed to contractile and non-contractile processes. Overall, we provide a unique set of information about the anatomic, metabolic and functional consequences of the Asp286Gly mutation that might be considered as relevant biomarkers for monitoring the severity and/or the progression of NM and for assessing the efficacy of potential therapeutic interventions.

In vivo and in vitro investigations of heterozygous nebulin knock-out mice disclose a mild skeletal muscle phenotype

Nemaline myopathy is the most common congenital skeletal muscle disease, and mutations in the nebulin gene account for 50% of all cases. Recent studies suggest that the disease severity might be related to the nebulin expression levels. Considering that mutations in the nebulin gene are typically recessive, one would expect that a single functional nebulin allele would maintain nebulin protein expression which would result in preserved skeletal muscle function. We investigated skeletal muscle function of heterozygous nebulin knock-out (i.e., nebulin(+/-)) mice using a multidisciplinary approach including protein and gene expression analysis and combined in vivo and in vitro force measurements. Skeletal muscle anatomy and energy metabolism were studied strictly non-invasively using magnetic resonance imaging and 31P-magnetic resonance spectroscopy. Maximal force production was reduced by around 16% in isolated muscle of nebulin(+/-) mice while in vivo force generating capacity was preserved. Muscle weakness was associated with a shift toward a slower proteomic phenotype, but was not related to nebulin protein deficiency or to an impaired energy metabolism. Further studies would be warranted in order to determine the mechanisms leading to a mild skeletal muscle phenotype resulting from the expression of a single nebulin allele.