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

Abstract

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

Assessing tissue metabolism by phosphorous-31 magnetic resonance spectroscopy and imaging: a methodology review

“…Continuous acquisition of 31 P spectra during exercise-recovery or ischemia-reperfusion provides the potential to assess mitochondrial oxidative capacity by quantifying the kinetics of PCr depletion and replenishment. Dynamic 31 P-MRS has been applied to assess skeletal muscle bioenergetics under physiological conditions (60,61,87,(96)(97)(98)(99), as well as to evaluate mitochondrial function in skeletal muscle in both diabetic patients and animal models of diabetes (8,(100)(101)(102). Figure 3 shows dynamic 31 P spectra and the changes of phosphate metabolites and pH during muscle contraction and recovery in human skeletal muscle (103).…”

“…However, baseline ATP concentration may change in chronic diseases (77)(78)(79), limiting the use of this approach in diseased tissues. Alternatively, many studies have used metabolite concentration ratios such as PCr-to-P i and PCrto-ATP ratios to evaluate metabolic changes in a variety of diseased conditions (4,(80)(81)(82)(83)(84)(85)(86)(87)(88). Reduced PCr-to-ATP ratio has been reported as a consequence of reduced CK activity or a loss of the total Cr content in heart diseases (82,(89)(90)(91).…”

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

“…Mechanical performance, muscular volume, and energy production from the net breakdown of phosphocreatine (PCr) via CK reaction, oxidative phosphorylation, and anaerobic glycolysis were assessed strictly noninvasively in electrostimulated gastrocnemius muscle of Mstn Ϫ/Ϫ and wild-type (Mstn ϩ/ϩ ) mice using an original experimental setup (14) Animal care and feeding. Thirteen 4-mo-old female Mstn ϩ/ϩ (n ϭ 7) and Mstn Ϫ/Ϫ mice (n ϭ 6) generated from a colony maintained on a C57Bl/6 background (Centre d'Expérimentation Fonctionnelle, Faculté de Médecine Pitié Salpêtrière, Paris, France) were used for these experiments.…”

Mitochondrial impairment induced by postnatal ActRIIB blockade does not alter function and energy status in exercising mouse glycolytic muscle in vivo

“…The left hindlimb was shaved before an electrode cream was applied at the knee and heel regions to optimize transcutaneous electrostimulation. An anesthetized animal was placed supine in a home-built cradle that had been especially designed for the strictly noninvasive MR investigation of gastrocnemius muscle function (20). This muscle was chosen because it is easily accessible for MR measurements and preferentially activated using our experimental methodology.…”

“…Absolute force production (expressed as force-time integral, in N·s) was calculated by integrating the isometric force (in N) with respect to time. During the force-frequency protocol, gastrocnemius muscle was electrostimulated with trains (0.75 s duration, rest interval of 30 s) of incremental frequencies (1,10,20,30,50,75, 100, and 150 Hz), and the corresponding force data were processed as follows. The isometric peak force was measured for each train, and the overall dataset was fitted to the Hill equation for calculating f50, i.e., the electrostimulation frequency for which 50% of the maximal force was exerted.…”