Marissa K. Caldow

Repeated Sprints Alter Signaling Related to Mitochondrial Biogenesis in Humans

PURPOSE We investigated the effects of acute and chronic repeated-sprint exercise (RSE) on the skeletal muscle messenger RNA (mRNA) expression and protein abundance/phosphorylation associated with mitochondrial biogenesis. METHODS Ten healthy young adults (seven males, three females) performed the RSE trial, comprising three sets of 5 × 4-s maximal sprints on a nonmotorized treadmill, with a 20-s recovery between sprints and 4.5 min between sets. After 4 wk of repeated-sprint training, three times per week, participants repeated the RSE. A vastus lateralis muscle biopsy was obtained at rest, immediately after, and 1 and 4 h after RSE, before and after training. Venous blood lactate and glucose were measured at rest and during recovery. Real-time reverse transcriptase polymerase chain reaction and Western blot techniques were used to measure mRNA expression and protein abundance, respectively. RESULTS Acute RSE increased the phosphorylation of acetyl-CoA carboxylase (86%, effect size (ES) = 1.4 ± 0.8, P < 0.001) and Ca calmodulin-dependent protein kinase II (69%, ES = 0.7 ± 0.6). Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α; 208%, ES = 1.5 ± 0.7, P < 0.001) and nuclear respiratory factor 1 (92%, ES = 0.7 ± 0.8) mRNA expression was increased after RSE. Four weeks of training increased the abundance of PGC-1α protein at rest (33%, ES = 0.9 ± 0.7). CONCLUSIONS Both acute and chronic RSE, despite only 60 s and 12 min of exercise, respectively, altered the molecular signaling associated with mitochondrial adaptations and PGC-1α mRNA expression in skeletal muscle. However, the small-to-moderate changes in resting PGC-1α protein abundance after training, together with the absence of changes in aerobic fitness, require further research to understand the functional significance of PGC-1α in response to RSE.

Ribosome biogenesis adaptation in resistance training-induced human skeletal muscle hypertrophy

Resistance training (RT) has the capacity to increase skeletal muscle mass, which is due in part to transient increases in the rate of muscle protein synthesis during postexercise recovery. The role of ribosome biogenesis in supporting the increased muscle protein synthetic demands is not known. This study examined the effect of both a single acute bout of resistance exercise (RE) and a chronic RT program on the muscle ribosome biogenesis response. Fourteen healthy young men performed a single bout of RE both before and after 8 wk of chronic RT. Muscle cross-sectional area was increased by 6 ± 4.5% in response to 8 wk of RT. Acute RE-induced activation of the ERK and mTOR pathways were similar before and after RT, as assessed by phosphorylation of ERK, MNK1, p70S6K, and S6 ribosomal protein 1 h postexercise. Phosphorylation of TIF-IA was also similarly elevated following both RE sessions. Cyclin D1 protein levels, which appeared to be regulated at the translational rather than transcriptional level, were acutely increased after RE. UBF was the only protein found to be highly phosphorylated at rest after 8 wk of training. Also, muscle levels of the rRNAs, including the precursor 45S and the mature transcripts (28S, 18S, and 5.8S), were increased in response to RT. We propose that ribosome biogenesis is an important yet overlooked event in RE-induced muscle hypertrophy that warrants further investigation.

Leucine as a treatment for muscle wasting: A critical review

Amino acids are potent modulators of protein turnover and skeletal muscle cells are highly sensitive to changes in amino acid availability. During amino acid abundance increased activity of mTORC1 drives protein synthesis and growth. In skeletal muscle, it has been clearly demonstrated that of all the amino acids, leucine is the most potent stimulator of mTORC1 and protein synthesis in vitro and in vivo. As such, leucine has received considerable attention as a potential pharmaconutrient for the treatment of numerous muscle wasting conditions. However, despite a multitude of studies showing enhanced acute protein synthesis with leucine or leucine-rich supplements in healthy individuals, additional leucine intake does not necessarily enhance protein synthesis during muscle wasting conditions. In addition, long-term, placebo controlled, iso-caloric studies in humans consistently show no beneficial effect of leucine supplementation on skeletal muscle mass or function. This review, critically evaluates the therapeutic potential of leucine to attenuate the skeletal muscle wasting associated with ageing, cancer and immobilization/bed rest. It also highlights the impact of inflammation on amino acid sensing, mTORC1 activation and stimulation of protein synthesis and challenges the underlying hypothesis that the acute activation of mTORC1 and stimulation of protein synthesis by leucine increases in muscle mass over time. We conclude that leucine, as a standalone nutritional intervention, is not effective in the prevention of muscle wasting. Future work should focus on identifying and utilizing other nutrients or treatments that sensitize skeletal muscle to leucine, thereby enhancing its therapeutic potential for muscle wasting conditions.

Resistance exercise increases NF-κB activity in human skeletal muscle

Intense resistance exercise causes a significant inflammatory response. NF-κB has been identified as a prospective key transcription factor mediating the postexercise inflammatory response. The purpose of this study was to determine whether a single bout of intense resistance exercise regulates NF-κB signaling in human skeletal muscle. Muscle biopsy samples were obtained from the vastus lateralis of five recreationally active, but not strength-trained, males (21.9 ± 1.3 yr) prior to, and at 2 and 4 h following, a single bout of intense resistance exercise. A further five subjects (4 males, 1 female) (23 ± 0.89 yr) were recruited as a nonexercise control group to examine the effect of the muscle biopsy protocol on key markers of skeletal muscle inflammation. Protein levels of IκBα and phosphorylated NF-κB (p65), as well as the mRNA expression of inflammatory myokines monocyte chemoattractant protein 1 (MCP-1), IL-6, and IL-8 were measured. Additionally, NF-κB (p65) DNA binding to the promoter regions of MCP-1, IL-6, and IL-8 was investigated. IκBα protein levels decreased, while p-NF-κB (p65) protein levels increased 2 h postexercise and returned to near-baseline levels by 4-h postexercise. Immunohistochemical data verified these findings, illustrating an increase in p-NF-κB (p65) protein levels, and nuclear localization at 2 h postexercise. Furthermore, NF-κB DNA binding to MCP-1, IL-6, and IL-8 promoter regions increased significantly 2 h postexercise as did mRNA levels of these myokines. No significant change was observed in the nonexercise control group. These novel data provide evidence that intense resistance exercise transiently activates NF-κB signaling in human skeletal muscle during the first few hours postexercise. These findings implicate NF-κB in the transcriptional control of myokines known to be central to the postexercise inflammatory response.

Association between skeletal muscle inflammatory markers and walking pattern in people with knee osteoarthritis.

Patients with knee osteoarthritis (OA) are characterized by increased muscle inflammation and altered gait. We investigated the association between proinflammatory mediators in the vastus lateralis and physical function and gait in patients with knee OA.

Impact of SOCS3 overexpression on human skeletal muscle development in vitro.

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling cascade has been identified as a crucial factor for myogenesis. The STAT3 isoform is essential for satellite cell migration and myogenic differentiation as it mediates the expression of muscle specific myogenic factors. The SOCS (suppressors of cytokine signaling) family of proteins down-regulates STAT activation. Primary human skeletal muscle cells were isolated and cultured to investigate the effect of SOCS3 adenoviral overexpression on myotube maturation. It was demonstrated that STAT3 inhibition did not influence myotube development or survival. Moreover, SOCS3 overexpression enhances the mRNA expression of downstream targets of STAT3, c-FOS and VEGF. These increases were correlated with enhanced mRNA expression of genes associated with muscle maturation and hypertrophy. Thus SOCS3 influences myoblast differentiation and SOCS3 may be significant in regulating the activity of genes previously identified as transcriptionally regulated by STAT3.

The effects of osteoarthritis and age on skeletal muscle strength, Na+-K+-ATPase content, gene and isoform expression

Knee osteoarthritis (OA) is a debilitating disorder prevalent in older populations that is accompanied by declines in muscle mass, strength, and physical activity. In skeletal muscle, the Na(+)-K(+) pump (NKA) is pivotal in ion homeostasis and excitability and is modulated by disuse and exercise training. This study examined the effects of OA and aging on muscle NKA in 36 older adults (range 55-81 yr), including 19 with OA (69.9 ± 6.5 yr, mean ± SD) and 17 asymptomatic controls (CON, 66.8 ± 6.4 yr). Participants completed knee extensor strength testing and a physical activity questionnaire. A vastus lateralis muscle biopsy was analyzed for NKA content ([(3)H]ouabain binding sites), α1-3- and β1-3-isoform protein abundance (immunoblotting), and mRNA (real-time RT-PCR). The association between age and NKA content was investigated within the OA and CON groups and in pooled data. The NKA content was also contrasted between subgroups below and above the median age of 68.5 yr. OA had lower strength (-40.8%, P = 0.005), but higher NKA α2- (∼34%, P = 0.006) and α3-protein (100%, P = 0.016) abundance than CON and performed more incidental physical activity (P = 0.035). No differences were found between groups for NKA content, abundance of other NKA isoforms, or gene expression. There was a negative correlation between age and NKA content within OA (r = -0.63, P = 0.03) and with both groups combined (r = -0.47, P = 0.038). The NKA content was 25.5% lower in the older (69-81 yr) than in the younger (55-68 yr) subgroup. Hence older age, but not knee OA, was related to lowered muscle NKA content in older adults.

Unchanged [3H]ouabain binding site content but reduced Na +-K+ pump α2-protein abundance in skeletal muscle in older adults

Aging is associated with reduced muscle mass, weakness, and increased fatigability. In skeletal muscle, the Na(+)-K(+) pump (NKA) is important in regulating Na(+)-K(+) gradients, membrane excitability, and thus contractility, but the effects of aging on muscle NKA are unclear. We investigated whether aging is linked with reduced muscle NKA by contrasting muscle NKA isoform gene expression and protein abundance, and NKA total content in 17 Elderly (66.8 ± 6.4 yr, mean ± SD) and 16 Young adults (23.9 ± 2.2 yr). Participants underwent peak oxygen consumption assessment and a vastus lateralis muscle biopsy, which was analyzed for NKA α(1)-, α(2)-, α(3)-, β(1)-, β(2)-, and β(3)-isoform gene expression (real-time RT-PCR), protein abundance (immunoblotting), and NKA total content ([(3)H]ouabain binding sites). The Elderly had lower peak oxygen consumption (-36.7%, P = 0.000), strength (-36.3%, P = 0.001), NKA α(2)- (-24.4%, 11.9 ± 4.4 vs. 9.0 ± 2.7 arbitrary units, P = 0.049), and NKA β(3)-protein abundance (-23.0%, P = 0.041) than Young. The β(3)-mRNA was higher in Elderly compared with Young (P = 0.011). No differences were observed between groups for other NKA isoform mRNA or protein abundance, or for [(3)H]ouabain binding site content. Thus skeletal muscle in elderly individuals was characterized by decreased NKA α(2)- and β(3)-protein abundance, but unchanged α(1) abundance and [(3)H]ouabain binding. The latter was likely caused by reduced α(2) abundance with aging, preventing an otherwise higher [(3)H]ouabain binding that might occur with a greater membrane density in smaller muscle fibers. Further study is required to verify reduced muscle NKA α(2) with aging and possible contributions to impaired exercise capability and daily living activities.

L-Citrulline Protects Skeletal Muscle Cells from Cachectic Stimuli through an iNOS-Dependent Mechanism.

Dietary L-citrulline is thought to modulate muscle protein turnover by increasing L-arginine availability. To date, the direct effects of increased L-citrulline concentrations in muscle have been completely neglected. Therefore, we determined the role of L-citrulline in regulating cell size during catabolic conditions by depriving mature C2C12 myotubes of growth factors (serum free; SF) or growth factors and nutrients (HEPES buffered saline; HBS). Cells were treated with L-citrulline or equimolar concentrations of L-arginine (positive control) or L-alanine (negative control) and changes in cell size and protein turnover were assessed. In myotubes incubated in HBS or SF media, L-citrulline improved rates of protein synthesis (HBS: +63%, SF: +37%) and myotube diameter (HBS: +18%, SF: +29%). L-citrulline treatment substantially increased iNOS mRNA expression (SF: 350%, HBS: 750%). The general NOS inhibitor L-NAME and the iNOS specific inhibitor aminoguanidine prevented these effects in both models. Depriving myotubes in SF media of L-arginine or L-leucine, exacerbated wasting which was not attenuated by L-citrulline. The increased iNOS mRNA expression was temporally associated with increases in mRNA of the endogenous antioxidants SOD1, SOD3 and catalase. Furthermore, L-citrulline prevented inflammation (LPS) and oxidative stress (H2O2) induced muscle cell wasting. In conclusion, we demonstrate a novel direct protective effect of L-citrulline on skeletal muscle cell size independent of L-arginine that is mediated through induction of the inducible NOS (iNOS) isoform. This discovery of a nutritional modulator of iNOS mRNA expression in skeletal muscle cells could have substantial implications for the treatment of muscle wasting conditions.

Citrulline Does Not Prevent Skeletal Muscle Wasting or Weakness in Limb-Casted Mice

BACKGROUND Increasing arginine (Arg) availability reduces atrophy in cultured skeletal muscle cells. Supplementation with its metabolic precursor citrulline (Cit) is more effective at improving skeletal muscle Arg concentrations. OBJECTIVE We tested the hypothesis that Cit supplementation would attenuate skeletal muscle atrophy and loss of function during hindlimb immobilization in mice. METHODS Male C57BL/6JArc mice underwent 14 d of unilateral hindlimb immobilization/plaster casting and were supplemented with ~0.81 g Cit · kg⁻¹ · d⁻¹ (CIT group) or Ala (ALA group) mixed into their food. The uncasted contralateral limb (internal control) and an uncasted group (CON) served as controls. Muscle atrophy was evaluated with mass, fiber area, and in situ muscle function. RESULTS Tibialis anterior (TA) muscle mass [ALA: 37.6 ± 0.92 mg; CIT: 38.3 ± 1.25 mg] and peak tetanic force (ALA: 1150 ± 38.5 mN; CIT: 1150 ± 52.0 mN) were lower (P < 0.001) in the ALA (53.9 ± 0.42 mg) and CIT (1760 ± 28.5 mN) groups than in the CON group. No difference was found between ALA and CIT groups for TA mass, fiber area, or peak force. The mRNA expression of the nitric oxide synthase 2, inducible (Nos2; ~15-fold) and B-cell chronic lymphoid leukemia/lymphoma 2/adenovirus E1B 19 kDa interacting protein 3 (Bnip3; ~17-fold) genes and the ratio of microtubule-associated protein light chain 3BII to 3BI (LC3BII:LC3BI) (50.5% ± 17.7%) were higher (P < 0.05) in the ALA group than in the CON group, suggesting increased autophagy. In the CIT group, Bnip3 mRNA was lower (-70%; P < 0.05) and Nos2 mRNA tended to be lower (-45%; P = 0.05) than in the ALA group, whereas LC3BII:LC3BI was not different from the CON group. CONCLUSIONS Cit treatment of male mice did not affect therapeutically relevant outcome measures such as skeletal muscle mass and peak muscle force after 14 d of hindlimb immobilization.