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-1a 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.Over the past decade, the study of tumor metabolism has gained new momentum, as the importance of biological processes that are closely linked to the phenotype is widely recognized in the current postgenomic era.1-3 Moreover, the crucial role of the local extracellular environment in tumorigenesis, cancer diagnosis and cancer treatment is increasingly being investigated, in addition to the behavior of cancer cells. 4 The communication between cancer cells and their microenvironment is complex, and involves metabolic events, blood perfusion, reactions of the immune system, cooperation with stromal cells and close interaction with the extracellular matrix.4 Among the multitude of factors influencing the formation, survival and growth of cancer cells, pH regulation occupies a central place.5 Indeed, the metabolism of rapidly growing cells is generating enormous amounts of acid. Mammalian cells can only survive at neutral or slightly alkaline intracellular pH values (pH i ). Therefore, cells need to tightly regulate their pH i to allow optimal functioning of the metabolic pathways. Furthermore, normal cells usually exist in an environment that is somewhat more alkaline (physiological value 7.35-7.45 6 ) than the intracellular space and are easily...

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 1 H-MRI and 31 P-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). T 2 -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. Key words: phosphorus MR spectroscopy; functional magnetic resonance imaging; skeletal muscle fatigue; ergometer; in vivo muscle stimulation; muscle contraction During the past two decades, transgenic mouse models have been used in order to identify the roles of genes in skeletal muscle development, physiology, and disease, thereby improving our knowledge in biology and medicine. The potential of these experimental models can be enhanced by the utilization of noninvasive techniques such as MR spectroscopy (MRS) and MRI in order to assess muscle function and metabolism in vivo (1-3). These techniques have indeed made possible the transition from in vitro biology to the integrative investigation of skeletal muscle function (4). In that respect, 31 P-MRS allows characterization of muscular energy metabolism through the measurement of intracellular pH and the concentration of the major phosphorylated compounds in contracting muscle. These measurements can also be used in order to quantify the relative aerobic and anaerobic contributions to energy production (5,6

The purpose of the present study was to assess the reliability of metabolic parameters measured using 31 P magnetic resonance spectroscopy ( 31 P MRS) during two standardized rest-exerciserecovery 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 year's time. Test-retest reliability was analyzed using coefficient of variation ( Key words: magnetic resonance spectroscopy; reliability; oxidative capacity; PCr kinetics; exercise Phosphorus-31 nuclear magnetic resonance spectroscopy ( 31 P-MRS) is now widely accepted as the "gold standard" method for noninvasive measurements of energy metabolism in exercising muscle. The analysis of changes in phosphorylated compounds concentrations and intracellular pH during rest-exercise-recovery protocols has provided significant advance in the understanding of underlying mechanisms of muscle energy production (1-4). Given the noninvasiveness of the method, 31 P-MRS enables repeated measurements and has been successfully used in a wide range of situations such as the assessment of therapeutic interventions in patients with metabolic disorders (5-7) and the characterization of training-induced metabolic changes (8 -11). More recently, the dynamics of phosphocreatine (PCr) has been used to evaluate the control of mitochondrial respiration in skeletal muscle (12) or the mechanism underlying the V O 2p slow component (13) typically observed during a high-intensity exercise; i.e., above the lactate threshold. In addition, the rate constant of postexercise recovery kinetics of [PCr] has been used as an index of mitochondrial function (14 -17). In addition to these traditional variables, more sophisticated analyses have been put forth in order to quantify the contribution of oxidative and anaerobic pathways to energy production in exercising muscle (2,18,19).Despite the widespread use of 31 P MRS for muscle energetics investigation, the reproducibility of the corresponding variables has been scarcely investigated. It has actually been investigated through repeated measurements performed 1 week (5) and 4 weeks apart (20). Bendahan et al. (5) reported a good reliability in metabolic parameters when investigated using analysis of variance (ANOVA). However, detection of systematic bias might have been affected by large random error between tests as suggested previously (21). observed large test-retest variability in metabolic parameters measured during steady state phases of moderate isometric plantar flexion exercise. However, kinetics parameters of recovery exhibited a small coefficient of variation (CV Ͻ 9%). In this line, van den Broek et al. (22) reported a low variability (CV Ͻ 11.5%) for the parameters describing the recovery kinetics of PCr and adenosine diphosphate (ADP) in a single subject who repeated the same exercise protocol 10 times. Based on a visual inspection of the kinetics of phosphorylated compounds and pH i...

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.

Isometric contractions induced by neuromuscular electrostimulation (NMES) have been shown to result in a prolonged force decrease but the time course of the potential central and peripheral factors have never been investigated. This study examined the specific time course of central and peripheral factors after isometric NMES-induced muscle damage. Twenty-five young healthy men were subjected to an NMES exercise consisting of 40 contractions for both legs. Changes in maximal voluntary contraction force of the knee extensors (MVC), peak evoked force during double stimulations at 10 Hz (Db10) and 100 Hz (Db100), its ratio (10∶100), voluntary activation, muscle soreness and plasma creatine kinase activity were assessed before, immediately after and throughout four days after NMES session. Changes in knee extensors volume and T2 relaxation time were also assessed at two (D2) and four (D4) days post-exercise. MVC decreased by 29% immediately after NMES session and was still 19% lower than the baseline value at D4. The decrease in Db10 was higher than in Db100 immediately and one day post-exercise resulting in a decrease (−12%) in the 10∶100 ratio. On the contrary, voluntary activation significantly decreased at D2 (−5%) and was still depressed at D4 (−5%). Muscle soreness and plasma creatine kinase activity increased after NMES and peaked at D2 and D4, respectively. T2 was also increased at D2 (6%) and D4 (9%). Additionally, changes in MVC and peripheral factors (e.g., Db100) were correlated on the full recovery period, while a significant correlation was found between changes in MVC and VA only from D2 to D4. The decrease in MVC recorded immediately after the NMES session was mainly due to peripheral changes while both central and peripheral contributions were involved in the prolonged force reduction. Interestingly, the chronological events differ from what has been reported so far for voluntary exercise-induced muscle damage.

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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 O 2 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 31 P-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 minfor 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.

Overall, the present ergometer allows quadriceps exercise in a MR system and should be useful for future metabolic studies for which reliable and absolute quantification of power output is warranted.