Temporal changes in the muscle extracellular matrix due to volumetric muscle loss injury.

PURPOSE/AIM: Volumetric muscle loss (VML) is a devastating orthopedic injury resulting in chronic persistent functional deficits, loss of joint range of motion, pathologic fibrotic deposition and lifelong disability. However, there is only limited mechanistic understanding of VML-induced fibrosis. Herein we examined the temporal changes in the fibrotic deposition at 3, 7, 14, 28, and 48 days post-VML injury. MATERIALS AND METHODS: Adult male Lewis rats (n = 39) underwent a full thickness ~20% (~85 mg) VML injury to the tibialis anterior (TA) muscle unilaterally, the contralateral TA muscle served as the control group. All TA muscles were harvested for biochemical and histologic evaluation. RESULTS: The ratio of collagen I/III was decreased at 3, 7, and 14 days post-VML, but significantly increased at 48 days. Decorin content followed an opposite trend, significantly increasing by day 3 before dropping to below control levels by 48 days. Histological evaluation of the defect area indicates a shift from loosely packed collagen at early time points post-VML, to a densely packed fibrotic scar by 48 days. CONCLUSIONS: The shift from early wound healing efforts to a fibrotic scar with densely packed collagen within the skeletal muscle occurs around 21 days after VML injury through dogmatic synchronous reduction of collagen III and increase in collagen I. Thus, there appears to be an early window for therapeutic intervention to prevent pathologic fibrous tissue formation, potentially by targeting CCN2/CTGF or using decorin as a therapeutic.

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.

An altered response in macrophage phenotype following damage in aged human skeletal muscle: implications for skeletal muscle repair.

The purpose of this study was to test the hypothesis that macrophage polarization is altered in old compared to young skeletal muscle, possibly contributing to the poor satellite cell response observed in older muscle tissue. Muscle biopsies were collected prior to and at 3, 24, and 72 h following a muscle-damaging exercise in young and old individuals. Immunohistochemistry was used to measure i.m. macrophage content and phenotype, and cell culture experiments tested macrophage behavior and influence on primary myoblasts from older individuals. We found that macrophage infiltration was similar between groups at 24 (young: 3712 ± 2407 vs. old: 5035 ± 2978 cells/mm3) and 72 (young: 4326 ± 2622 vs. old: 5287 ± 2248 cells/mm3) hours postdamage, yet the proportion of macrophages that expressed the proinflammatory marker CD11b were markedly lower in the older subjects (young: 74.5 ± 15 vs. old: 52.6 ± 17%). This finding was coupled with a greater overall proportion of CD206+, anti-inflammatory macrophages in the old (group: P = 0.0005). We further demonstrate in vitro that proliferation, and in some cases differentiation, of old primary human myoblasts increase as much as 30% when exposed to a young macrophage-conditioned environment. Collectively, the data suggest that old macrophages appear less capable of adapting and maintaining inflammatory function, which may contribute to poor satellite cell activation and delayed recovery from muscle damage.-Sorensen, J. R., Kaluhiokalani, J. P., Hafen, P. S., Deyhle, M. R., Parcell, A. C., Hyldahl, R. D. An altered response in macrophage phenotype following damage in aged human skeletal muscle: implications for skeletal muscle repair.

α-Adrenergic receptor regulation of skeletal muscle blood flow during exercise in heart failure patients with reduced ejection fraction.

Patients suffering from heart failure with reduced ejection fraction (HFrEF) experience impaired limb blood flow during exercise, which may be due to a disease-related increase in α-adrenergic receptor vasoconstriction. Thus, in eight patients with HFrEF (63 ± 4 yr) and eight well-matched controls (63 ± 2 yr), we examined changes in leg blood flow (Doppler ultrasound) during intra-arterial infusion of phenylephrine (PE; an α1-adrenergic receptor agonist) and phentolamine (Phen; a nonspecific α-adrenergic receptor antagonist) at rest and during dynamic single-leg knee-extensor exercise (0, 5, and 10 W). At rest, the PE-induced reduction in blood flow was significantly attenuated in patients with HFrEF (-15 ± 7%) compared with controls (-36 ± 5%). During exercise, the controls exhibited a blunted reduction in blood flow induced by PE (-12 ± 4, -10 ± 4, and -9 ± 2% at 0, 5, and 10 W, respectively) compared with rest, while the PE-induced change in blood flow was unchanged compared with rest in the HFrEF group (-8 ± 5, -10 ± 3, and -14 ± 3%, respectively). Phen administration increased leg blood flow to a greater extent in the HFrEF group at rest (+178 ± 34% vs. +114 ± 28%, HFrEF vs. control) and during exercise (36 ± 6, 37 ± 7, and 39 ± 6% vs. 13 ± 3, 14 ± 1, and 8 ± 3% at 0, 5, and 10 W, respectively, in HFrEF vs. control). Together, these findings imply that a HFrEF-related increase in α-adrenergic vasoconstriction restrains exercising skeletal muscle blood flow, potentially contributing to diminished exercise capacity in this population.

Impact of age on exercise-induced ATP supply during supramaximal plantar flexion in humans.

Currently, the physiological factors responsible for exercise intolerance and bioenergetic alterations with age are poorly understood due, at least in art, to the confounding effect of reduced physical activity in the elderly. Thus, in 40 healthy young (22 ± 2 yr) and old (74 ± 8 yr) activity-matched subjects, we assessed the impact of age on: 1) the relative contribution of the three major pathways of ATP synthesis (oxidative ATP synthesis, glycolysis, and the creatine kinase reaction) and 2) the ATP cost of contraction during high-intensity exercise. Specifically, during supramaximal plantar flexion (120% of maximal aerobic power), to stress the functional limits of the skeletal muscle energy systems, we used (31)P-labeled magnetic resonance spectroscopy to assess metabolism. Although glycolytic activation was delayed in the old, ATP synthesis from the main energy pathways was not significantly different between groups. Similarly, the inferred peak rate of mitochondrial ATP synthesis was not significantly different between the young (25 ± 8 mM/min) and old (24 ± 6 mM/min). In contrast, the ATP cost of contraction was significantly elevated in the old compared with the young (5.1 ± 2.0 and 3.7 ± 1.7 mM·min(-1)·W(-1), respectively; P < 0.05). Overall, these findings suggest that, when young and old subjects are activity matched, there is no evidence of age-related mitochondrial and glycolytic dysfunction. However, this study does confirm an abnormal elevation in exercise-induced skeletal muscle metabolic demand in the old that may contribute to the decline in exercise capacity with advancing age.

In vivo evidence of an age-related increase in ATP cost of contraction in the plantar flexor muscles.

Impaired skeletal muscle efficiency potentially contributes to the age-related decline in exercise capacity and may explain the altered haemodynamic response to exercise in the elderly. Thus we examined whether (i) the ATP cost of contraction increases with age, and (ii) this results in altered convective O(2) delivery to maintain microvascular oxygenation in the calf muscle. To this aim, we used an integrative experimental approach combining (31)P-MRS (magnetic resonance spectroscopy), Doppler ultrasound imaging and NIRS (near-IR spectroscopy) during dynamic plantar flexion exercise at 40% of WR(max) (maximal power output) in 20 healthy young and 20 older subjects matched for physical activity. The ATP cost of contraction was significantly higher in the old (7.2±4.1 mM/min per W) compared with the young (2.4±1.9 mM/min per W; P<0.05) and this was only significantly correlated with the plantar flexion WR(max) value in the old subjects (r=-0.52; P<0.05). Even when differences in power output were taken into account, end-exercise blood flow (old, 259±168 ml/min per W and young, 134±40 ml/min per W; P<0.05) and convective O(2) delivery (old, 0.048±0.031 l/min per W and young, 0.026±0.008 l/min per W; P<0.05) were greater in the old in comparison with the young subjects. In contrast, the NIRS oxyhaemoglobin, deoxyhaemoglobin and microvascular oxygenation indices were not significantly different between the groups (P>0.05). Therefore the present study reveals that, although the peripheral haemodynamic responses to plantar flexion exercise appear to be appropriate, the elevated energy cost of contraction and associated reduction in the WR(max) value in this muscle group may play a role in limiting exercise capacity with age.

FOXK1 regulates Wnt signalling to promote cardiogenesis.

AIMS: Congenital heart disease (CHD) is the most common genetic birth defect, which has considerable morbidity and mortality. We focused on deciphering key regulators that govern cardiac progenitors and cardiogenesis. FOXK1 is a forkhead/winged helix transcription factor known to regulate cell cycle kinetics and is restricted to mesodermal progenitors, somites, and heart. In the present study, we define an essential role for FOXK1 during cardiovascular development. METHODS AND RESULTS: We used the mouse embryoid body system to differentiate control and Foxk1 KO embryonic stem cells into mesodermal, cardiac progenitor cells and mature cardiac cells. Using flow cytometry, immunohistochemistry, cardiac beating, transcriptional and chromatin immunoprecipitation quantitative polymerase chain reaction assays, bulk RNA sequencing (RNAseq) and assay for transposase-accessible chromatin using sequencing (ATACseq) analyses, FOXK1 was observed to be an important regulator of cardiogenesis. Flow cytometry analyses revealed perturbed cardiogenesis in Foxk1 KO embryoid bodies (EBs). Bulk RNAseq analysis at two developmental stages showed a significant reduction of the cardiac molecular program in Foxk1 KO EBs compared to the control EBs. ATACseq analysis during EB differentiation demonstrated that the chromatin landscape nearby known important regulators of cardiogenesis was significantly relaxed in control EBs compared to Foxk1 KO EBs. Furthermore, we demonstrated that in the absence of FOXK1, cardiac differentiation was markedly impaired by assaying for cardiac Troponin T expression and cardiac contractility. We demonstrate that FOXK1 is an important regulator of cardiogenesis by repressing the Wnt/ß-catenin signalling pathway and thereby promoting differentiation. CONCLUSION: These results identify FOXK1 as an essential transcriptional and epigenetic regulator of cardiovascular development. Mechanistically, FOXK1 represses Wnt signalling to promote the development of cardiac progenitor cells.

Response of terminal Schwann cells following volumetric muscle loss injury.

An often-overlooked component of traumatic skeletal muscle injuries is the impact on the nervous system and resultant innervation of the affected muscles. Recent work in a rodent model of volumetric muscle loss (VML) injury demonstrated a progressive, secondary loss of neuromuscular junction (NMJ) innervation, supporting a role of NMJ dysregulation in chronic functional deficits. Terminal Schwann cells (tSCs) are known to be vital for the maintenance of NMJ structure and function, in addition to guiding repair and regeneration after injury. However, the tSC response to a traumatic muscle injury such as VML is not known. Thus, a study was conducted to investigate the effect of VML on tSC morphological characteristics and neurotrophic signaling proteins in adult male Lewis rats that underwent VML injury to the tibialis anterior muscle using a temporal design with outcome assessments at 3, 7, 14, 21, and 48 days post-injury. The following salient observations were made; first, although there is a loss of innervation over time, the number of tSCs per NMJ increases, significantly so at 48 days post-injury compared to control. The degree of NMJ fragmentation was positively correlated with tSC number after injury. Moreover, neurotrophic factors such as NRG1 and BDNF are elevated after injury through at least 48 days. These results were unanticipated and in contrast to neurodegenerative disease models, in which there is a reduction in tSC number that precedes denervation. However, we found that while there are more tSCs per NMJ after injury, they cover a significantly smaller percent of the post-synaptic endplate area compared to control. These findings support a sustained increase in neurotrophic activity and tSC number after VML, which is a maladaptive response occurring in parallel to other aspects of the VML injury, such as over-accumulation of collagen and aberrant inflammatory signaling.

Secondary denervation is a chronic pathophysiologic sequela of volumetric muscle loss.

Volumetric muscle loss (VML) is the traumatic loss of muscle tissue that results in long-term functional impairments. Despite the loss of myofibers, there remains an unexplained significant decline in muscle function. VML injury likely extends beyond the defect area, causing negative secondary outcomes to the neuromuscular system, including the neuromuscular junctions (NMJs), yet the extent to which VML induces denervation is unclear. This study systematically examined NMJs surrounding the VML injury, hypothesizing that the sequela of VML includes denervation. The VML injury removed ∼20% of the tibialis anterior (TA) muscle in adult male inbred Lewis rats (n = 43), the noninjured leg served as an intra-animal control. Muscles were harvested up to 48 days post-VML. Synaptic terminals were identified immunohistochemically, and quantitative confocal microscopy evaluated 2,613 individual NMJ. Significant denervation was apparent by 21 and 48 days post-VML. Initially, denervation increased ∼10% within 3 days of injury; with time, denervation further increased to ∼22% and 32% by 21 and 48 days post-VML, respectively, suggesting significant secondary denervation. The appearance of terminal axon sprouting and polyinnervation were observed as early as 7 days post-VML, increasing in number and complexity throughout 48 days. There was no evidence of VML-induced NMJ size alteration, which may be beneficial for interventions aimed at restoring muscle function. This work recognizes VML-induced secondary denervation and poor remodeling of the NMJ as part of the sequela of VML injury; moreover, secondary denervation is a possible contributing factor to the chronic functional impairments and potentially an overlooked treatment target.NEW & NOTEWORTHY This work advances our understanding of the pathophysiologic complexity of volumetric muscle loss injury. Specifically, we identified secondary denervation in the muscle remaining after volumetric muscle loss injuries as a novel aspect of the injury sequela. Denervation increased chronically, in parallel with the appearance of irregular morphological characteristics and destabilization of the neuromuscular junction, which is expected to further confound chronic functional impairments.

Humanized skeletal muscle in MYF5/MYOD/MYF6-null pig embryos.

Because post-mortem human skeletal muscle is not viable, autologous muscle grafts are typically required in tissue reconstruction after muscle loss due to disease or injury. However, the use of autologous tissue often leads to donor-site morbidity. Here, we show that intraspecies and interspecies chimaeric pig embryos lacking native skeletal muscle can be produced by deleting the MYF5, MYOD and MYF6 genes in the embryos via CRISPR, followed by somatic-cell nuclear transfer and the delivery of exogenous cells (porcine blastomeres or human induced pluripotent stem cells) via blastocyst complementation. The generated intraspecies chimaeras were viable and displayed normal histology, morphology and function. Human:pig chimaeras generated with TP53-null human induced pluripotent stem cells led to higher chimaerism efficiency, with embryos collected at embryonic days 20 and 27 containing humanized muscle, as confirmed by immunohistochemical and molecular analyses. Human:pig chimaeras may facilitate the production of exogenic organs for research and xenotransplantation.

Accumulation of Skeletal Muscle T Cells and the Repeated Bout Effect in Rats.

PURPOSE: The purpose of this investigation was to characterize skeletal muscle T-cell accumulation after contraction-induced muscle damage and test the hypothesis that T cells contribute to postdamage muscle protection (i.e., the repeated bout effect) in a way reminiscent of their role in adaptive immunity. METHODS: In vivo lengthening contractions were used to model the repeated bout effect and contralateral repeated bout effect in rats. Intramuscular T-cell subsets were characterized by flow cytometry after single and repeated bouts of lengthening contractions, and an adoptive T-cell transfer experiment was done to test whether T cells from muscle damage-experienced rats can confer protection from injury to damage-naive rats. RESULTS: Electrically stimulated lengthening contractions elicited the repeated bout effect, but not the contralateral repeated bout effect. Although leukocytes (CD45+) were scarce in undamaged muscle (2.1% of all cells), substantially more (63% of all cells) were observed after a single bout of lengthening contractions. Within the leukocyte population were several subsets of T cells, including conventional CD4+, CD8+, memory, and regulatory T cells. In contrast, a minimal increase in T cells was observed after a second bout of lengthening contractions. Conventional CD4+ T cells (FoxP3-) were the most abundant subset in muscle after lengthening contractions. Adoptive T-cell transfer from damage-experienced rats did not confer protection to damage-naive recipient rats. CONCLUSIONS: The robust T-cell accumulation, particularly the CD4 subset, after contraction-induced damage suggests a role for these cells in muscle repair and adaptation to muscle damaging contractions. Moreover, T cells are unlikely to mediate the protective adaptations of the repeated bout effect in a manner similar to their role in adaptive immunity.

Passive leg movement in chronic obstructive pulmonary disease: evidence of locomotor muscle vascular dysfunction.

Chronic obstructive pulmonary disease (COPD), characterized by pulmonary dysfunction, is now also recognized to be associated with free radical-mediated vascular dysfunction. However, as previous investigations have utilized the brachial artery flow-mediated dilation technique, whether such vascular dysfunction exists in the locomotor muscle of patients with COPD remains unclear. Therefore, in patients with COPD (n = 13, 66 ± 6 yr) and healthy age- and sex-matched control subjects (n = 12, 68 ± 6 yr), second-by-second measurements of leg blood flow (LBF) (ultrasound Doppler), mean arterial pressure (MAP) (Finapres), and leg vascular conductance (LVC) were recorded before and during both 2 min of continuous upright seated continuous-movement passive leg movement (PLM) and a single-movement PLM (sPLM). In response to PLM, both peak change in LBF (COPD 321 ± 54, Control 470 ± 55 ∆mL/min) and LVC (COPD 3.0 ± 0.5, Control 5.4 ± 0.5 ∆mL·min-1·mmHg-1) were significantly attenuated in patients with COPD compared with control subjects (P < 0.05). This attenuation in the patients with COPD was also evident in response to sPLM, with peak change in LBF tending to be lower (COPD 142 ± 26, Control 169 ± 14 ∆mL/min) and LVC being significantly lower (P < 0.05) in the patients than the control subjects (COPD 1.6 ± 0.4, Control 2.5 ± 0.3 ∆mL·min-1·mmHg-1). Therefore, utilizing both PLM and sPLM, this study provides evidence of locomotor muscle vascular dysfunction in patients with COPD, perhaps due to redox imbalance and reduced nitric oxide bioavailability, which is in agreement with an increased cardiovascular disease risk in this population. This locomotor muscle vascular dysfunction, in combination with the clearly dysfunctional lungs, may contribute to the exercise intolerance associated with COPD.NEW & NOTEWORTHY Utilizing both the single and continuous passive leg movement (PLM) models, which induce nitric oxide (NO)-dependent hyperemia, this study provides evidence of vascular dysfunction in the locomotor muscle of patients with chronic obstructive pulmonary disease (COPD), independent of central hemodynamics. This impaired hyperemia may be the result of an oxidant-mediated attenuation in NO bioavailability. In addition to clearly dysfunctional lungs, vascular dysfunction in locomotor muscle may contribute to the exercise intolerance associated with COPD and increased cardiovascular disease risk.

Acute extracellular matrix, inflammatory and MAPK response to lengthening contractions in elderly human skeletal muscle.

To uncover potential factors that may be involved in the impaired regenerative capacity of aged skeletal muscle, we comprehensively assessed the molecular stress response following muscle damage in old and young individuals. 10 young (22.7 ±â€¯2.25 yrs) and 8 physically active old (70.9 ±â€¯7.5 yrs) subjects completed a bout of 300 lengthening contractions (LC), and muscle biopsies were taken pre-exercise and at 3, 24, and 72 h post-LC. Both age groups performed the same amount of work during LC, with the old group displaying a resistance to LC-induced fatigue during the exercise. Muscle damage was evident by soreness and losses in isokinetic force and power production, though older subjects experienced reduced force and power losses relative to the young group. The acute extracellular matrix (ECM) response was characterized by substantial increases in the glycoproteins tenascin C and fibronectin in the young, which were blunted in the old muscle following damage. Old muscle displayed a generally heightened and asynchronous inflammatory response compared to young muscle, with higher expression of MCP-1 that appeared at later time points, and increased NF-κb activity. Expression of the stress-related MAPKs P38 and JNK increased only in the old groups following muscle damage. In summary, aberrations appear in the inflammatory, ECM and MAPK responses of aged skeletal muscle following damaging LC, each of which may individually or collectively contribute to the deterioration of muscle repair mechanisms that accompanies aging.

Repeated exposure to heat stress induces mitochondrial adaptation in human skeletal muscle.

The heat stress response is associated with several beneficial adaptations that promote cell health and survival. Specifically, in vitro and animal investigations suggest that repeated exposures to a mild heat stress (~40°C) elicit positive mitochondrial adaptations in skeletal muscle comparable to those observed with exercise. To assess whether such adaptations translate to human skeletal muscle, we produced local, deep tissue heating of the vastus lateralis via pulsed shortwave diathermy in 20 men and women ( n = 10 men; n = 10 women). Diathermy increased muscle temperature by 3.9°C within 30 min of application. Immediately following a single 2-h heating session, we observed increased phosphorylation of AMP-activated protein kinase and ERK1/2 but not of p38 MAPK or JNK. Following repeated heat exposures (2 h daily for 6 consecutive days), we observed a significant cellular heat stress response, as heat shock protein 70 and 90 increased 45% and 38%, respectively. In addition, peroxisome proliferator-activated receptor gamma, coactivator-1 alpha and mitochondrial electron transport protein complexes I and V expression were increased after heating. These increases were accompanied by augmentation of maximal coupled and uncoupled respiratory capacity, measured via high-resolution respirometry. Our data provide the first evidence that mitochondrial adaptation can be elicited in human skeletal muscle in response to repeated exposures to mild heat stress. NEW & NOTEWORTHY Heat stress has been shown to elicit mitochondrial adaptations in cell culture and animal research. We used pulsed shortwave diathermy to produce deep tissue heating and explore whether beneficial mitochondrial adaptations would translate to human skeletal muscle in vivo. We report, for the first time, positive mitochondrial adaptations in human skeletal muscle following recurrent heat stress. The results of this study have clinical implications for many conditions characterized by diminished skeletal muscle mitochondrial function.

Preclinical characterization of the JAK/STAT inhibitor SGI-1252 on skeletal muscle function, morphology, and satellite cell content.

BACKGROUND: Recent studies have highlighted the JAK/STAT signaling pathway in the regulation of muscle satellite cell behavior. Herein we report preclinical studies designed to characterize the effects of a novel JAK/STAT inhibitor on plantar flexor skeletal muscle function, morphology, and satellite cell content. METHODS: The compound, SGI-1252, was administered orally (400mg/kg) in a 10% dextrose solution to wild type mice (n = 6) 3 times per week for 8 weeks. A control group (n = 6) received only the dextrose solution. RESULTS: SGI-1252 was well tolerated, as animals displayed similar weight gain over the 8-week treatment period. Following treatment, fatigue in the gastrocnemius-soleus-plantaris complex was greater in the SGI-1252 mice during a 300 second tetanic contraction bout (p = 0.035), though both the rate of fatigue and maximal force production were similar. SGI-1252 treated mice had increased type II myofiber cross-sectional area (1434.8 ± 225.4 vs 1754.7 ± 138.5 µm2), along with an increase in wet muscle mass (125.45 ± 5.46 vs 139.6 ± 12.34 mg, p = 0.032) of the gastrocnemius relative to vehicle treated mice. SGI-1252 treatment reduced gastrocnemius STAT3 phosphorylation 53% (94.79 ± 45.9 vs 44.5 ± 6.1 MFI) and significantly increased the concentration of Pax7+ satellite cells (2589.2 ± 105.5 vs 2859.4 ± 177.5 SC/mm3) in the gastrocnemius. SGI-1252 treatment suppressed MyoD (p = 0.013) and Myogenin (p<0.0001) expression in human primary myoblasts, resulting in reduced myogenic differentiation (p = 0.039). CONCLUSIONS: Orally delivered SGI-1252 was well tolerated, attenuates skeletal muscle STAT3 activity, and increases satellite cell content in mouse gastrocnemius muscle, likely by inhibiting myogenic progression.

CXCL10 increases in human skeletal muscle following damage but is not necessary for muscle regeneration.

CXCL10 is a chemokine for activated and memory T cells with many important immunological functions. We recently found that CXCL10 is upregulated in human muscle following contraction-induced damage. No information is available on the role of CXCL10 in the context of muscle damage or repair. In this study, we confirm that CXCL10 is elevated in human muscle at 2 and 3 days following damage and perform cell culture and animal studies to examine the role of CXCL10 in muscle repair. CXCL10 did not impact proliferation rates of human primary myoblasts but it did promote myogenic differentiation in vitro, suggesting a possible direct impact on muscle regeneration. To test if CXCL10 was dispensable for effective muscle regeneration in vivo, we measured functional and histological markers of muscle repair out to 14 days postmuscle injury caused by a myotoxin in wild-type (WT) mice and CXCL10 knockout (KO) mice. Between genotypes, no significant differences were found in loss or restoration of in situ muscle force, cross-sectional area of newly formed myofibers, or the number of embryonic myosin heavy chain-positive myofibers. In addition, KO animals were not deficient in T-cell accumulation in the damaged muscle following injury. Gene expression of the other two ligands (CXCL9 and 11) that bind to the same receptor as CXCL10 were also elevated in the damaged muscle of KO mice. Thus, other ligands may have compensated for the lack of CXCL10 in the KO mice. We conclude that CXCL10 is not necessary for effective muscle regeneration.

Induction and Assessment of Exertional Skeletal Muscle Damage in Humans.

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery in humans. Herein we discuss the design of an eccentric exercise protocol to induce damage in the quadriceps muscles, marked by changes in strength, soreness, and plasma creatine kinase levels. This method is simple, ethical, and widely applicable since it is performed in human participants and eliminates the interspecies translation of the results. Subjects perform 300 maximal eccentric contractions of the knee extensor muscles at a speed of 120°/sec on an isokinetic dynamometer. The extent of the damage is measurable using relatively non-invasive isokinetic and isometric measures of strength loss, soreness, and plasma creatine kinase levels over several days following the exercise. Therefore, its application can be directed to specific populations in an attempt to identify mechanisms for muscle adaptation and regeneration.

Evidence of a metabolic reserve in the skeletal muscle of elderly people.

The purpose of the present study was to determine whether mitochondrial function is limited by O2 availability or the intrinsic capacity of mitochondria to synthesize ATP in elderly individuals. To this aim, we examined, in comparison to free-flow conditions (FF), the effect of superimposing reactive hyperemia (RH), induced by a period of brief ischemia during the last min of exercise, on O2 availability and mitochondrial function in the calf muscle. 12 healthy, untrained, elderly subjects performed dynamic plantar flexion exercise and phosphorus magnetic resonance spectroscopy (31P-MRS), near-infrared spectroscopy (NIRS), and Doppler ultrasound were used to assess muscle metabolism and peripheral hemodynamics. Limb blood flow [area under the curve (AUC), FF: 1.5±0.5L; RH: 3.2±1.1L, P<0.01] and convective O2 delivery (AUC, FF: 0.30±0.13L; RH: 0.64±0.29L, P<0.01) were significantly increased in RH in comparison to FF. RH was also associated with significantly higher capillary blood flow (P<0.05) and this resulted in a 33% increase in estimated peak mitochondrial ATP synthesis rate (FF: 24±11 mM.min-1; RH: 31±7 mM.min-1, P<0.05). These results document a hemodynamic reserve in the contracting calf muscle of the elderly accessible by superimposing reactive hyperemia. Furthermore, this increase in O2 availability enhanced mitochondrial function thus indicating a skeletal muscle metabolic reserve despite advancing age and low level of physical activity.

Evidence of Preserved Oxidative Capacity and Oxygen Delivery in the Plantar Flexor Muscles With Age.

Studies examining the effect of aging on skeletal muscle oxidative capacity have yielded equivocal results; however, these investigations may have been confounded by differences in oxygen (O(2)) delivery, physical activity, and small numbers of participants. Therefore, we evaluated skeletal muscle oxidative capacity and O(2) delivery in a relatively large group (N = 40) of young (22 ± 2 years) and old (73 ± 7 years) participants matched for physical activity. After submaximal dynamic plantar flexion exercise, phosphocreatine (PCr) resynthesis ((31)P magnetic resonance spectroscopy), muscle reoxygenation (near-infrared spectroscopy), and popliteal artery blood flow (Doppler ultrasound) were measured. The phosphocreatine recovery time constant (Tau) (young: 33 ± 16; old: 30 ± 11 seconds), maximal rate of adenosine triphosphate (ATP) synthesis (young: 25 ± 9; old: 27 ± 8 mM/min), and muscle reoxygenation rates determined by the deoxyhemoglobin/myoglobin recovery Tau (young: 48 ± 5; old: 47 ± 9 seconds) were similar between groups. Similarly, although tending to be higher in the old, there were no significant age-related differences in postexercise popliteal blood flow (area under the curve: young: 1,665 ± 227 vs old: 2,404 ± 357 mL, p = .06) and convective O(2) delivery (young: 293 ± 146 vs old: 404 ± 191 mL, p = .07). In conclusion, when physical activity and O(2) delivery are similar, oxidative capacity in the plantar flexors is not affected by aging. These findings reveal that diminished skeletal muscle oxidative capacity is not an obligatory accompaniment to the aging process.