Robert D. Hyldahl

Lengthening our perspective: Morphological, cellular, and molecular responses to eccentric exercise

The response of skeletal muscle to unaccustomed eccentric exercise has been studied widely, yet it is incompletely understood. This review is intended to provide an up‐to‐date overview of our understanding of how skeletal muscle responds to eccentric actions, with particular emphasis on the underlying molecular and cellular mechanisms of damage and recovery. This review begins by addressing the question of whether eccentric actions result in physical damage to muscle fibers and/or connective tissue. We next review the symptomatic manifestations of eccentric exercise (i.e., indirect damage markers, such as delayed onset muscle soreness), with emphasis on their relatively poorly understood molecular underpinnings. We then highlight factors that potentially modify the muscle damage response following eccentric exercise. Finally, we explore the utility of using eccentric training to improve muscle function in populations of healthy and aging individuals, as well as those living with neuromuscular disorders. Muscle Nerve 49: 155–170, 2014

Activation of nuclear factor-κB following muscle eccentric contractions in humans is localized primarily to skeletal muscle-residing pericytes

Limited data exist on the molecular mechanisms that govern skeletal muscle regeneration in humans. This study characterized the early molecular alterations in humans to eccentric contractions (ECs), a stimulus known to induce a muscle regenerative response. Thirty‐five subjects completed 100 ECs of the knee extensors with 1 leg, and muscle biopsies were taken from both legs 3 h post‐EC. The sample from the non‐EC leg served as the control. We first conducted a well‐powered transcriptomic screen and network analysis. Our screen identified significant changes in several transcripts with functions relating to inflammation, cell growth, and proliferation. Network analysis then identified the transcription factor NF‐κPB as a key molecular element affected by ECs. A transcription factor ELISA, using nuclear extracts from EC and control muscle samples, showed a 1.6‐fold increase in NF‐κPB DNA binding activity following ECs. Immunohistochemical experiments localized the majority of NF‐κPB‐positive nuclei to cells in the interstitium, which stained positive for the pericyte markers NG2 proteoglycan and alkaline phosphatase. Our results provide the first evidence of NF‐κPB activation in human muscle following ECs and suggest a novel role for muscle residing pericytes in the early adaptive response to ECs.—Hyldahl, R. D., Xin, L., Hubal, M. J., Moeckel‐Cole, S., Chipkin, S., Clarkson, P. M. Activation of nuclear factor‐κPB following muscle eccentric contractions in humans is localized primarily to skeletal muscle‐residing pericytes. FASEB J. 25, 2956–2966 (2011). www.fasebj.org

Satellite cell activity is differentially affected by contraction mode in human muscle following a work-matched bout of exercise.

Optimal repair and adaptation of skeletal muscle is facilitated by resident stem cells (satellite cells). To understand how different exercise modes influence satellite cell dynamics, we measured satellite cell activity in conjunction with markers of muscle damage and inflammation in human skeletal muscle following a single work- and intensity-matched bout of eccentric (ECC) or concentric contractions (CON). Participants completed a single bout of ECC (n = 7) or CON (n = 7) of the knee extensors. A muscle biopsy was obtained before and 24 h after exercise. Functional measures and immunohistochemical analyses were used to determine the extent of muscle damage and indices of satellite cell activity. Cytokine concentrations were measured using a multiplexed magnetic bead assay. Isokinetic peak torque decreased following ECC (p < 0.05) but not CON. Greater histological staining of the damage marker Xin was observed in muscle samples of ECC vs. CON. Tenasin C immunoreactivity increased 15 fold (p < 0.01) following ECC and was unchanged following CON. The inflammatory cytokines interferon gamma-induced protein 10 (IP-10) and monocyte chemotactic protein 1 (MCP-1) increased pre- to post-ECC (4.26 ± 1.4 vs. 10.49 ± 5.8 pg/ml, and 3.06 ± 0.7 vs. 6.25 ± 4.6 pg/ml, respectively; p < 0.05). There was no change in any cytokine post-CON. Satellite cell content increased 27% pre- to post-ECC (0.10 ± 0.031 vs. 0.127 ± 0.041, respectively; p < 0.05). There was no change in satellite cell number in CON (0.099 ± 0.027 vs. 0.102 ± 0.029, respectively). There was no fiber type-specific satellite cell response following either exercise mode. ECC but not CON resulted in an increase in MyoD positive nuclei per myofiber pre- to post-exercise (p < 0.05), but there was no change in MyoD DNA binding activity in either condition. In conclusion, ECC but not CON results in functional and histological evidence of muscle damage that is accompanied by increased satellite cell activity 24 h post-exercise.

Mechanisms and Mediators of the Skeletal Muscle Repeated Bout Effect

Skeletal muscle adapts to exercise-induced damage by orchestrating several but still poorly understood mechanisms that endow protection from subsequent damage. Known widely as the repeated bout effect, we propose that neural adaptations, alterations to muscle mechanical properties, structural remodeling of the extracellular matrix, and biochemical signaling work in concert to coordinate the protective adaptation.

A contralateral repeated bout effect attenuates induction of NF-κB DNA binding following eccentric exercise

We investigated the existence of contralateral repeated bout effect and tested if the attenuation of nuclear factor-kappa B (NF-κB; an important regulator of muscle inflammation) induction following eccentric exercise is a potential mechanism. Thirty-one healthy men performed two bouts of knee extension eccentric exercise, initially with one leg and then with the opposite leg 4 wk later. Vastus lateralis muscle biopsies of both exercised and control legs were taken 3 h postexercise. Knee extension isometric and isokinetic strength (60°/sec and 180°/sec) were measured at baseline, pre-exercise, immediately postexercise, and 1/day for 5 days postexercise. Serum creatine kinase (CK) activity and muscle soreness were assessed at baseline and 1/day for 5 days postexercise. NF-κB (p65) DNA-binding activity was measured in the muscle biopsies. Isometric strength loss was lower in bout 2 than in bout 1 at 24, 72, and 96 h postexercise (P < 0.05). Isokinetic strength (60°/s and 180°/s) was reduced less in bout 2 than in bout 1 at 72 h postexercise (P < 0.01). There were no significant differences between bouts for postexercise CK activity or muscle soreness. p65 DNA-binding activity was increased following eccentric exercise (compared with the control leg) in bout 1 (122.9% ± 2.6%; P < 0.001) and bout 2 (109.1% ± 3.0%; P < 0.05). Compared with bout 1, the increase in NF-κB DNA-binding activity postexercise was attenuated after bout 2 (P = 0.0008). Repeated eccentric exercise results in a contralateral repeated bout effect, which could be due to the attenuated increase in NF-κB activity postexercise.

Skeletal Muscle Inflammation Following Repeated Bouts of Lengthening Contractions in Humans.

Skeletal muscle responds to exercise-induced damage by orchestrating an adaptive process that protects the muscle from damage by subsequent bouts of exercise, a phenomenon called the repeated bout effect (RBE). The mechanisms underlying the RBE are not understood. We hypothesized that an attenuated inflammation response following a repeated bout of lengthening contractions (LC) would be coincidental with a RBE, suggesting a potential relationship. Fourteen men (n = 7) and women (n = 7) completed two bouts of lengthening contractions (LC) separated by 28 days. Muscle biopsies were taken before the first bout (B1) from the non-exercised leg, and from the exercised leg 2- and 27-d post-B1 and 2-d following the second bout (B2). A 29-plex cytokine array identified alterations in inflammatory cytokines. Immunohistochemistry quantified inflammatory cell infiltration and major histocompatibility complex class 1 (MHC-1). Muscle soreness was attenuated in the days following B2 relative to B1, indicating a RBE. Intramuscular monocyte chemoattractant protein (MCP1) and interferon gamma-induced protein 10 (IP10) increased following B2 relative to the pre-exercise sample (7–52 and 11–36 pg/ml, respectively p < 0.05). Interleukin 4 (IL4) decreased (26–13 pg/ml, p < 0.05) following B2 relative to the pre-exercise sample. Infiltration of CD68+ macrophages and CD8+ T-cells were evident following B2, but not B1. Moreover, CD8+ T-cells were observed infiltrating apparently necrotic muscle fibers. No changes in MHC-1 were found. We conclude that inflammation is not attenuated following a repeated bout of LC and that CD8+ T-cells may play a role in muscle adaptation following LC. Moreover, it appears that the muscle or the immune system becomes sensitized to an initial bout of damaging exercise such that inflammatory cell infiltration into the muscle is enhanced upon a repeated bout of damaging exercise.

Pericyte NF-κB Activation Enhances Endothelial Cell Proliferation and Proangiogenic Cytokine Secretion in Vitro

Pericytes are skeletal muscle resident, multipotent stem cells that are localized to the microvasculature. In vivo, studies have shown that they respond to damage through activation of nuclear‐factor kappa‐B (NF‐κB), but the downstream effects of NF‐κB activation on endothelial cell proliferation and cell–cell signaling during repair remain unknown. The purpose of this study was to examine pericyte NF‐κB activation in a model of skeletal muscle damage; and use genetic manipulation to study the effects of changes in pericyte NF‐κB activation on endothelial cell proliferation and cytokine secretion. We utilized scratch injury to C2C12 cells in coculture with human primary pericytes to assess NF‐κB activation and monocyte chemoattractant protein‐1 (MCP‐1) secretion from pericytes and C2C12 cells. We also cocultured endothelial cells with pericytes that expressed genetically altered NF‐κB activation levels, and then quantified endothelial cell proliferation and screened the conditioned media for secreted cytokines. Pericytes trended toward greater NF‐κB activation in injured compared to control cocultures (P = 0.085) and in comparison to C2C12 cells (P = 0.079). Second, increased NF‐κB activation in pericytes enhanced the proliferation of cocultured endothelial cells (1.3‐fold, P = 0.002). Finally, we identified inflammatory signaling molecules, including MCP‐1 and interleukin 8 (IL‐8) that may mediate the crosstalk between pericytes and endothelial cells. The results of this study show that pericyte NF‐κB activation may be an important mechanism in skeletal muscle repair with implications for the development of therapies for musculoskeletal and vascular diseases, including peripheral artery disease.

Effects of Ibuprofen Topical Gel on Muscle Soreness

PURPOSE Muscle soreness is a common symptom after novel exercise and may influence exercise adherence. This study examined the effect of an ibuprofen topical gel and the effect of age and sex on muscle soreness after a gym exercise. METHODS One hundred and six participants completed six sets of 10 repetitions of the elbow and knee flexor muscles. Thirty-six hours after exercise, participants were randomized to apply an ibuprofen topical gel or placebo treatment to the affected muscle groups. Soreness evaluations were taken each hour for the first 6 h (36-42 h), then at 48, 60, 66, 72, 84, 90, 96, and 108 h after exercise. Subjects then returned to the laboratory after 3 wk and repeated the same study protocol with the opposite arm/leg and treatment. RESULTS We found no significant differences in soreness between the active ibuprofen gel and the placebo treatment and no difference in effectiveness between men and women or between older and younger subjects. For the placebo groups, there was no sex differences in muscle soreness; however, when the data were analyzed by dividing participants into young (18-29 yr) and old (40-65 yr) cohort, the old cohort reported significantly less soreness in response to the elbow flexion exercise than the young cohort (P < 0.01). CONCLUSION The results of this study suggest that the topical application of ibuprofen is not an effective treatment for muscle soreness after an unaccustomed gym exercise. Furthermore, our results show that there is no sex difference in the soreness response and that older subjects have less soreness in response to a similar exercise stimulus as young subjects.

NF-KB activity functions in primary pericytes in a cell- and non-cell-autonomous manner to affect myotube formation.

Introduction: Skeletal muscle regeneration following damage relies on proliferation and differentiation of muscle precursor cells (MPCs). We recently observed increased NF‐kB activity in vascular‐associated muscle resident pericytes following muscle damage in humans. We determined how altered NF‐kB activity in human primary pericytes (HPPs) affects their myogenic differentiation (cell‐autonomous effects), as well as proliferation and differentiation of co‐cultured MPCs (non–cell‐autonomous effects). Methods: HPPs were transfected with vectors that increased or decreased NF‐kB activity. Transfected HPPs were co‐cultured with C2C12 myoblasts under differentiation conditions, and HPP fusion to myotubes was measured. We also co‐cultured HPPs with C2C12 myoblasts and measured proliferation and myotube formation. Results: Inhibition of NF‐kB activity increased HPP fusion to C2C12 myotubes. Moreover, enhanced NF‐kB activity in HPPs suppressed differentiation and enhanced proliferation of co‐cultured myoblasts. Conclusions: NF‐kB activity acts cell‐autonomously to inhibit HPP myogenic differentiation and non–cell‐autonomously to promote MPC proliferation and suppress MPC differentiation in vitro. Muscle Nerve, 2013

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.