Mapping SCA1 regional vulnerabilities reveals neural and skeletal muscle contributions to disease.

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an expanded polyglutamine tract in the widely expressed ataxin-1 (ATXN1) protein. To elucidate anatomical regions and cell types that underlie mutant ATXN1-induced disease phenotypes, we developed a floxed conditional knockin mouse (f-ATXN1146Q/2Q) with mouse Atxn1 coding exons replaced by human ATXN1 exons encoding 146 glutamines. f-ATXN1146Q/2Q mice manifested SCA1-like phenotypes including motor and cognitive deficits, wasting, and decreased survival. Central nervous system (CNS) contributions to disease were revealed using f-ATXN1146Q/2Q;Nestin-Cre mice, which showed improved rotarod, open field, and Barnes maze performance by 6-12 weeks of age. In contrast, striatal contributions to motor deficits using f-ATXN1146Q/2Q;Rgs9-Cre mice revealed that mice lacking ATXN1146Q/2Q in striatal medium-spiny neurons showed a trending improvement in rotarod performance at 30 weeks of age. Surprisingly, a prominent role for muscle contributions to disease was revealed in f-ATXN1146Q/2Q;ACTA1-Cre mice based on their recovery from kyphosis and absence of muscle pathology. Collectively, data from the targeted conditional deletion of the expanded allele demonstrated CNS and peripheral contributions to disease and highlighted the need to consider muscle in addition to the brain for optimal SCA1 therapeutics.

Retention of stress susceptibility in the mdx mouse model of Duchenne muscular dystrophy after PGC-1α overexpression or ablation of IDO1 or CD38.

Duchenne muscular dystrophy (DMD) is a lethal degenerative muscle wasting disease caused by the loss of the structural protein dystrophin with secondary pathological manifestations including metabolic dysfunction, mood and behavioral disorders. In the mildly affected mdx mouse model of DMD, brief scruff stress causes inactivity, while more severe subordination stress results in lethality. Here, we investigated the kynurenine pathway of tryptophan degradation and the nicotinamide adenine dinucleotide (NAD+) metabolic pathway in mdx mice and their involvement as possible mediators of mdx stress-related pathology. We identified downregulation of the kynurenic acid shunt, a neuroprotective branch of the kynurenine pathway, in mdx skeletal muscle associated with attenuated peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) transcriptional regulatory activity. Restoring the kynurenic acid shunt by skeletal muscle-specific PGC-1α overexpression in mdx mice did not prevent scruff -induced inactivity, nor did abrogating extrahepatic kynurenine pathway activity by genetic deletion of the pathway rate-limiting enzyme, indoleamine oxygenase 1. We further show that reduced NAD+ production in mdx skeletal muscle after subordination stress exposure corresponded with elevated levels of NAD+ catabolites produced by ectoenzyme cluster of differentiation 38 (CD38) that have been implicated in lethal mdx response to pharmacological ß-adrenergic receptor agonism. However, genetic CD38 ablation did not prevent mdx scruff-induced inactivity. Our data do not support a direct contribution by the kynurenine pathway or CD38 metabolic dysfunction to the exaggerated stress response of mdx mice.

Delineating regional vulnerability in the neurodegenerative disease SCA1 using a conditional mutant ATXN1 mouse.

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an expanded polyglutamine tract in the widely expressed ATXN1 protein. To elucidate anatomical regions and cell types that underlie mutant ATXN1-induced disease phenotypes, we developed a floxed conditional knockout mouse model ( f-ATXN1 146Q/2Q ) having mouse Atxn1 coding exons replaced by human exons encoding 146 glutamines. F-ATXN1 146Q/2Q mice manifest SCA1-like phenotypes including motor and cognitive deficits, wasting, and decreased survival. CNS contributions to disease were revealed using ATXN1 146Q/2Q ; Nestin-Cre mice, that showed improved rotarod, open field and Barnes maze performances. Striatal contributions to motor deficits were examined using f-ATXN1 146Q/2Q ; Rgs9-Cre mice. Mice lacking striatal ATXN1 146Q/2Q had improved rotarod performance late in disease. Muscle contributions to disease were revealed in f-ATXN1 146Q/2Q ; ACTA1-Cre mice which lacked muscle pathology and kyphosis seen in f-ATXN1 146Q/2Q mice. Kyphosis was not improved in f-ATXN1 146Q/2Q ;Nestin - Cre mice. Thus, optimal SCA1 therapeutics will require targeting mutant ATXN1 toxic actions in multiple brain regions and muscle.

Lifelong Ulk1-Mediated Autophagy Deficiency in Muscle Induces Mitochondrial Dysfunction and Contractile Weakness.

The accumulation of damaged mitochondria due to insufficient autophagy has been implicated in the pathophysiology of skeletal muscle aging. Ulk1 is an autophagy-related kinase that initiates autophagosome assembly and may also play a role in autophagosome degradation (i.e., autophagy flux), but the contribution of Ulk1 to healthy muscle aging is unclear. Therefore, the purpose of this study was to investigate the role of Ulk1-mediated autophagy in skeletal muscle aging. At age 22 months (80% survival rate), muscle contractile and metabolic function were assessed using electrophysiology in muscle-specific Ulk1 knockout mice (MKO) and their littermate controls (LM). Specific peak-isometric torque of the ankle dorsiflexors (normalized by tibialis anterior muscle cross-sectional area) and specific force of the fast-twitch extensor digitorum longus muscles was reduced in MKO mice compared to LM mice (p < 0.03). Permeabilized muscle fibers from MKO mice had greater mitochondrial content, yet lower mitochondrial oxygen consumption and greater reactive oxygen species production compared to fibers from LM mice (p ≤ 0.04). Alterations in neuromuscular junction innervation patterns as well as changes to autophagosome assembly and flux were explored as possible contributors to the pathological features in Ulk1 deficiency. Of primary interest, we found that Ulk1 phosphorylation (activation) to total Ulk1 protein content was reduced in older muscles compared to young muscles from both human and mouse, which may contribute to decreased autophagy flux and an accumulation of dysfunctional mitochondria. Results from this study support the role of Ulk1-mediated autophagy in aging skeletal muscle, reflecting Ulk1's dual role in maintaining mitochondrial integrity through autophagosome assembly and degradation.

Mitochondria-cytokine crosstalk following skeletal muscle injury and disuse: a mini-review.

Skeletal muscle mitochondria are highly adaptable, highly dynamic organelles that maintain the functional integrity of the muscle fiber by providing ATP for contraction and cellular homeostasis (e.g., Na+/K+ ATPase). Emerging as early modulators of inflammation, mitochondria sense and respond to cellular stress. Mitochondria communicate with the environment, in part, by release of physical signals called mitochondrial-derived damage-associated molecular patterns (mito-DAMPs) and deviation from routine function (e.g., reduced ATP production, Ca2+ overload). When skeletal muscle is compromised, mitochondria contribute to an acute inflammatory response necessary for myofibril regeneration; however, exhaustive signaling associated with altered or reduced mitochondrial function can be detrimental to muscle outcomes. Here, we describe changes in mitochondrial content, structure, and function following skeletal muscle injury and disuse and highlight the influence of mitochondria-cytokine crosstalk on muscle regeneration and recovery. Although the appropriate therapeutic modulation following muscle stressors remains unknown, retrospective gene expression analysis reveals that interleukin-6 (IL-6), interleukin-1ß (IL-1ß), chemokine C-X-C motif ligand 1 (CXCL1), and monocyte chemoattractant protein 1 (MCP-1) are significantly upregulated following three unique muscle injuries. These cytokines modulate mitochondrial function and execute bona fide pleiotropic roles that can aid functional recovery of muscle, however, when aberrant, chronically disrupt healing partly by exacerbating mitochondrial dysfunction. Multidisciplinary efforts to delineate the opposing regulatory roles of inflammatory cytokines in the muscle mitochondrial environment are required to modulate regenerative behavior following skeletal muscle injury or disuse. Future therapeutic directions to consider include quenching or limited release of mito-DAMPs and cytokines present in cytosol or circulation.

Spatial frequency metrics for analysis of microscopic images of musculoskeletal tissues.

Purpose: Imaging-based metrics for analysis of biological tissues are powerful tools that can extract information such as shape, size, periodicity, and many other features to assess the requested qualities of a tissue. Muscular and osseous tissues consist of periodic structures that are directly related to their function, and so analysis of these patterns likely reflects tissue health and regeneration.Methods: A method for assessment of periodic structures is by analyzing them in the spatial frequency domain using the Fourier transform. In this paper, we present two filters which we developed in the spatial frequency domain for the purpose of analyzing musculoskeletal structures. These filters provide information about 1) the angular orientation of the tissues and 2) their periodicity. We explore periodic structural patterns in the mitochondrial network of skeletal muscles that are reflective of muscle metabolism and myogenesis; and patterns of collagen fibers in the bone that are reflective of the organization and health of bone extracellular matrix.Results: We present an analysis of mouse skeletal muscle in healthy and injured muscles. We used a transgenic mouse that ubiquitously expresses fluorescent protein in their mitochondria and performed 2-photon microscopy to image the structures. To acquire the collagen structure of the bone we used non-linear SHG microscopy of mouse flat bone. We analyze and compare juvenile versus adult mice, which have different structural patterns.Conclusions: Our results indicate that these metrics can quantify musculoskeletal tissues during development and regeneration.

Mitochondrial dysfunction in skeletal muscle of fukutin-deficient mice is resistant to exercise- and 5-aminoimidazole-4-carboxamide ribonucleotide-induced rescue.

NEW FINDINGS: What is the central question of this study? Does fukutin deficiency in skeletal muscle cause mitochondrial dysfunction, and if so, can AMP-activated protein kinase (AMPK) stimulation via 5-aminoimidazole-4-carboxamide ribonucleotide attenuate this through regulation of mitochondrial biogenesis and autophagy? What is the main finding and its importance? Mitochondrial dysfunction is associated with fukutin deficiency and AMPK stimulation may benefit muscle contractility to a greater extent than mitochondrial function. ABSTRACT: Disruptions in the dystrophin-glycoprotein complex (DGC) are clearly the primary basis underlying various forms of muscular dystrophies and dystroglycanopathies, but the cellular consequences of DGC disruption are still being investigated. Mitochondrial abnormalities are becoming an apparent consequence and contributor to dystrophy disease pathology. Herein, we demonstrate that muscle-specific deletion of the fukutin gene (Myf5/fktn-KO mice (Fktn KO)), a model of secondary dystroglycanopathy, results in ∼30% lower muscle strength (P < 0.001) and 16% lower mitochondrial respiratory function (P = 0.002) compared to healthy littermate controls (LM). We also observed ∼80% lower expression of the gene for peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) (P = 0.004), a primary transcription factor for mitochondrial biogenesis, in Fktn KO mice that likely contributes to the mitochondrial defects. PGC-1α is post-translationally regulated via phosphorylation by AMP-activated protein kinase (AMPK). Treatment with the AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) failed to rescue mitochondrial deficits in Fktn KO mice (P = 0.458) but did have beneficial (∼30% greater) effects on recovery of muscle contractility following injury in both LM and Fktn KO mice compared to saline treatment (P = 0.006). The beneficial effects of AMPK stimulation via AICAR on muscle contractile function may be partially explained by AMPK's other role of regulating skeletal muscle autophagy, a cellular process critical for clearance of damaged and/or dysfunctional organelles. Two primary conclusions can be drawn from this data: (1) fukutin deletion produces intrinsic muscular metabolic defects that likely contribute to dystroglycanopathy disease pathology, and (2) AICAR treatment accelerates recovery of muscle contractile function following injury suggesting AMPK signalling as a possible target for therapeutic strategies.

Mitochondrial-specific autophagy linked to mitochondrial dysfunction following traumatic freeze injury in mice.

The objective of this study was to interrogate the link between mitochondrial dysfunction and mitochondrial-specific autophagy in skeletal muscle. C57BL/6J mice were used to establish a time course of mitochondrial function and autophagy induction after fatigue (n = 12), eccentric contraction-induced injury (n = 20), or traumatic freeze injury (FI, n = 28); only FI resulted in a combination of mitochondrial dysfunction, i.e., decreased mitochondrial respiration, and autophagy induction. Moving forward, we tested the hypothesis that mitochondrial-specific autophagy is important for the timely recovery of mitochondrial function after FI. Following FI, there is a significant increase in several mitochondrial-specific autophagy-related protein contents including dynamin-related protein 1 (Drp1), BCL1 interacting protein (BNIP3), Pink1, and Parkin (~2-fold, P < 0.02). Also, mitochondrial-enriched fractions from FI muscles showed microtubule-associated protein light chain B1 (LC3)II colocalization suggesting autophagosome assembly around the damaged mitochondrial. Unc-51 like autophagy activating kinase (Ulk1) is considered necessary for mitochondrial-specific autophagy and herein we utilized a mouse model with Ulk1 deficiency in adult skeletal muscle (myogenin-Cre). While Ulk1 knockouts had contractile weakness compared with littermate controls (-27%, P < 0.02), the recovery of mitochondrial function was not different, and this may be due in part to a partial rescue of Ulk1 protein content within the regenerating muscle tissue of knockouts from differentiated satellite cells in which Ulk1 was not genetically altered via myogenin-Cre. Lastly, autophagy flux was significantly less in injured versus uninjured muscles (-26%, P < 0.02) despite the increase in autophagy-related protein content. This suggests autophagy flux is not upregulated to match increases in autophagy machinery after injury and represents a potential bottleneck in the clearance of damaged mitochondria by autophagy.

Five-dimensional two-photon volumetric microscopy of in-vivo dynamic activities using liquid lens remote focusing.

Multi-photon scanning microscopy provides a robust tool for optical sectioning, which can be used to capture fast biological events such as blood flow, mitochondrial activity, and neuronal action potentials. For many studies, it is important to visualize several different focal planes at a rate akin to the biological event frequency. Typically, a microscope is equipped with mechanical elements to move either the sample or the objective lens to capture volumetric information, but these strategies are limited due to their slow speeds or inertial artifacts. To overcome this problem, remote focusing methods have been developed to shift the focal plane axially without physical movement of the sample or the microscope. Among these methods is liquid lens technology, which adjusts the focus of the lens by changing the wettability of the liquid and hence its curvature. Liquid lenses are inexpensive active optical elements that have the potential for fast multi-photon volumetric imaging, hence a promising and accessible approach for the study of biological systems with complex dynamics. Although remote focusing using liquid lens technology can be used for volumetric point scanning multi-photon microscopy, optical aberrations and the effects of high energy laser pulses have been concerns in its implementation. In this paper, we characterize a liquid lens and validate its use in relevant biological applications. We measured optical aberrations that are caused by the liquid lens, and calculated its response time, defocus hysteresis, and thermal response to a pulsed laser. We applied this method of remote focusing for imaging and measurement of multiple in-vivo specimens, including mesenchymal stem cell dynamics, mouse tibialis anterior muscle mitochondrial electrical potential fluctuations, and mouse brain neural activity. Our system produces 5 dimensional (x,y,z,λ,t) data sets at the speed of 4.2 volumes per second over volumes as large as 160 x 160 x 35 µm3.

Early rehabilitation for volumetric muscle loss injury augments endogenous regenerative aspects of muscle strength and oxidative capacity.

BACKGROUND: Volumetric muscle loss (VML) injuries occur due to orthopaedic trauma or the surgical removal of skeletal muscle and result in debilitating long-term functional deficits. Current treatment strategies do not promote significant restoration of function; additionally appropriate evidenced-based practice physical therapy paradigms have yet to be established. The objective of this study was to develop and evaluate early rehabilitation paradigms of passive range of motion and electrical stimulation in isolation or combination to understand the genetic and functional response in the tissue remaining after a multi-muscle VML injury. METHODS: Adult male mice underwent an ~ 20% multi-muscle VML injury to the posterior compartment (gastrocnemius, soleus, and plantaris muscle) unilaterally and were randomized to rehabilitation paradigm twice per week beginning 2 days post-injury or no treatment. RESULTS: The most salient findings of this work are: 1) that the remaining muscle tissue after VML injury was adaptable in terms of improved muscle strength and mitigation of stiffness; but 2) not adaptable to improvements in metabolic capacity. Furthermore, biochemical (i.e., collagen content) and gene (i.e., gene arrays) assays suggest that functional adaptations may reflect changes in the biomechanical properties of the remaining tissue due to the cellular deposition of non-contractile tissue in the void left by the VML injury and/or differentiation of gene expression with early rehabilitation. CONCLUSIONS: Collectively this work provides evidence of genetic and functional plasticity in the remaining skeletal muscle with early rehabilitation approaches, which may facilitate future evidenced-based practice of early rehabilitation at the clinical level.

Mitochondria-specific antioxidant supplementation does not influence endurance exercise training-induced adaptations in circulating angiogenic cells, skeletal muscle oxidative capacity or maximal oxygen uptake.

KEY POINTS: Reducing excessive oxidative stress, through chronic exercise or antioxidants, can decrease the negative effects induced by excessive amounts of oxidative stress. Transient increases in oxidative stress produced during acute exercise facilitate beneficial vascular training adaptations, but the effects of non-specific antioxidants on exercise training-induced vascular adaptations remain elusive. Circulating angiogenic cells (CACs) are an exercise-inducible subset of white blood cells that maintain vascular integrity. We investigated whether mitochondria-specific antioxidant (MitoQ) supplementation would affect the response to 3 weeks of endurance exercise training in CACs, muscle mitochondrial capacity and maximal oxygen uptake in young healthy men. We show that endurance exercise training increases multiple CAC types, an adaptation that is not altered by MitoQ supplementation. Additionally, MitoQ does not affect skeletal muscle or whole-body aerobic adaptations to exercise training. These results indicate that MitoQ supplementation neither enhances nor attenuates endurance training adaptations in young healthy men. ABSTRACT: Antioxidants have been shown to improve endothelial function and cardiovascular outcomes. However, the effects of antioxidants on exercise training-induced vascular adaptations remain elusive. General acting antioxidants combined with exercise have not impacted circulating angiogenic cells (CACs). We investigated whether mitochondria-specific antioxidant (MitoQ) supplementation would affect the response to 3 weeks of endurance exercise training on CD3+ , CD3+ /CD31+ , CD14+ /CD31+ , CD31+ , CD34+ /VEGFR2+ and CD62E+ peripheral blood mononuclear cells (PBMCs), muscle mitochondrial capacity, and maximal oxygen uptake (VO2 max ) in healthy men aged 22.1 ± 0.7 years, with a body mass index of 26.9 ± 0.9 kg m-2 , and 24.8 ± 1.3% body fat. Analysis of main effects revealed that training induced 33, 105 and 285% increases in CD14+ /CD31+ , CD62E+ and CD34+ /VEGFR2+ CACs, respectively, and reduced CD3+ /CD31- PBMCs by 14%. There was no effect of MitoQ on CAC levels. Also independent of MitoQ supplementation, exercise training significantly increased quadriceps muscle mitochondrial capacity by 24% and VO2 max by roughly 7%. In conclusion, endurance exercise training induced increases in multiple CAC types, and this adaptation is not modified by MitoQ supplementation. Furthermore, we demonstrate that a mitochondrial-targeted antioxidant does not influence skeletal muscle or whole-body aerobic adaptations to exercise training.

Mitochondrial maintenance via autophagy contributes to functional skeletal muscle regeneration and remodeling.

The primary objective of this study was to determine whether alterations in mitochondria affect recovery of skeletal muscle strength and mitochondrial enzyme activity following myotoxic injury. 3-Methyladenine (3-MA) was administered daily (15 mg/kg) to blunt autophagy, and the creatine analog guanidionpropionic acid (ß-GPA) was administered daily (1% in chow) to enhance oxidative capacity. Male C57BL/6 mice were randomly assigned to nontreatment (Con, n = 6), 3-MA-treated (n = 6), and ß-GPA-treated (n = 8) groups for 10 wk. Mice were euthanized at 14 days after myotoxic injury for assessment of mitochondrial remodeling during regeneration and its association with the recovery of muscle strength. Expression of several autophagy-related proteins, e.g., phosphorylated Ulk1 (∼2- to 4-fold, P < 0.049) was greater in injured than uninjured muscles, indicating a relationship between muscle regeneration/remodeling and autophagy. By 14 days postinjury, recovery of muscle strength (18% less, P = 0.03) and mitochondrial enzyme (e.g., citrate synthase) activity (22% less, P = 0.049) were significantly lower in 3-MA-treated than Con mice, suggesting that the autophagy process plays an important role during muscle regeneration. In contrast, muscle regeneration was nearly complete in ß-GPA-treated mice, i.e., muscle strength recovered to 93% of baseline vs. 78% for Con mice. Remarkably, 14 days allowed sufficient time for a near-complete recovery of mitochondrial function in ß-GPA-treated mice (e.g., no difference in citrate synthase activity between injured and uninjured, P = 0.49), indicating a robust mitochondrial remodeling process during muscle regeneration. In conclusion, autophagy is likely activated following muscle injury and appears to play an important role in functional muscle regeneration.

Effect of acute exercise on circulating angiogenic cell and microparticle populations.

Subpopulations of peripheral blood mononuclear cells (PMBCs), known as circulating angiogenic cells (CACs), have been implicated in endothelial repair, angiogenesis and vascular homeostasis. Conversely, microparticles released from endothelial cells, platelets and leucocytes in response to injury or apoptosis are elevated in chronic diseases. We investigated the effect of acute exercise on CAC subpopulations, specifically CD34(+)/VEGFR2(+), CD3(+)/CD31(+), CD14(+)/CD31(+) and CD62E(+) PBMCs and CD62E(+), CD31(+)/CD42b(-) and CD34(+) MPs in men and women. Additionally, we examined angiogenesis-related gene expression in CD34(+), CD31(+) and CD62E(+) PBMCs at baseline and after exercise. Finally, we examined whether acute exercise modulates CD62E(+) PBMC paracrine actions on cultured endothelial cells. Blood samples for CAC and MP analyses were obtained before and after cycling exercise at 70% peak oxygen uptake that elicited an energy expenditure of 600 kcal. Exercise produced a decrease in CD14(+)/CD31(+) PBMCs, whereas CD62E expression on PBMCs increased with exercise. CD34(+)/VEGFR2(+) and CD3(+)/CD31(+) PBMC levels were not altered with exercise. Gene expression analysis revealed a more proangiogenic phenotype in CD62E(+) cells at baseline compared with CD31(+) and CD34(+) cells. Conditioned media from CD62E(+) PBMCs obtained after exercise exerted a proangiogenic influence on human umbilical vein endothelial cells, with increases in genes encoding receptors for growth factors (KDR, FGFR1 and EGFR) and inflammatory mediators (TLR4 and TNFR1). Finally, exercise increased CD62E(+) endothelial MPs in men and increased CD34(+) MPs in women. Our work highlights the potential role of CD62E(+) cells as a novel, exercise-responsive proangiogenic cell population and demonstrates sex-specific exercise-induced changes in circulating MPs.

A cross-validation of near-infrared spectroscopy measurements of skeletal muscle oxidative capacity with phosphorus magnetic resonance spectroscopy.

The purpose of this study was to cross-validate measurements of skeletal muscle oxidative capacity made with near-infrared spectroscopy (NIRS) measurements to those made with phosphorus magnetic resonance spectroscopy ((31)P-MRS). Sixteen young (age = 22.5 ± 3.0 yr), healthy individuals were tested with both (31)P-MRS and NIRS during a single testing session. The recovery rate of phosphocreatine was measured inside the bore of a 3-Tesla MRI scanner, after short-duration (∼10 s) plantar flexion exercise as an index of skeletal muscle oxidative capacity. Using NIRS, the recovery rate of muscle oxygen consumption was also measured using repeated, transient arterial occlusions outside the MRI scanner, after short-duration (∼10 s) plantar flexion exercise as another index of skeletal muscle oxidative capacity. The average recovery time constant was 31.5 ± 8.5 s for phosphocreatine and 31.5 ± 8.9 s for muscle oxygen consumption for all participants (P = 0.709). (31)P-MRS time constants correlated well with NIRS time constants for both channel 1 (Pearson's r = 0.88, P < 0.0001) and channel 2 (Pearson's r = 0.95, P < 0.0001). Furthermore, both (31)P-MRS and NIRS exhibit good repeatability between trials (coefficient of variation = 8.1, 6.9, and 7.9% for NIRS channel 1, NIRS channel 2, and (31)P-MRS, respectively). The good agreement between NIRS and (31)P-MRS indexes of skeletal muscle oxidative capacity suggest that NIRS is a valid method for assessing mitochondrial function, and that direct comparisons between NIRS and (31)P-MRS measurements may be possible.