Oral digoxin effects on exercise performance, K+ regulation and skeletal muscle Na+ ,K+ -ATPase in healthy humans.

We investigated whether digoxin lowered muscle Na+ ,K+ -ATPase (NKA), impaired muscle performance and exacerbated exercise K+ disturbances. Ten healthy adults ingested digoxin (0.25 mg; DIG) or placebo (CON) for 14 days and performed quadriceps strength and fatiguability, finger flexion (FF, 105%peak-workrate , 3 × 1 min, fourth bout to fatigue) and leg cycling (LC, 10 min at 33% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ and 67% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ , 90% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ to fatigue) trials using a double-blind, crossover, randomised, counter-balanced design. Arterial (a) and antecubital venous (v) blood was sampled (FF, LC) and muscle biopsied (LC, rest, 67% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ , fatigue, 3 h after exercise). In DIG, in resting muscle, [3 H]-ouabain binding site content (OB-Fab ) was unchanged; however, bound-digoxin removal with Digibind revealed total ouabain binding (OB+Fab ) increased (8.2%, P = 0.047), indicating 7.6% NKA-digoxin occupancy. Quadriceps muscle strength declined in DIG (-4.3%, P = 0.010) but fatiguability was unchanged. During LC, in DIG (main effects), time to fatigue and [K+ ]a were unchanged, whilst [K+ ]v was lower (P = 0.042) and [K+ ]a-v greater (P = 0.004) than in CON; with exercise (main effects), muscle OB-Fab was increased at 67% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ (per wet-weight, P = 0.005; per protein P = 0.001) and at fatigue (per protein, P = 0.003), whilst [K+ ]a , [K+ ]v and [K+ ]a-v were each increased at fatigue (P = 0.001). During FF, in DIG (main effects), time to fatigue, [K+ ]a , [K+ ]v and [K+ ]a-v were unchanged; with exercise (main effects), plasma [K+ ]a , [K+ ]v , [K+ ]a-v and muscle K+ efflux were all increased at fatigue (P = 0.001). Thus, muscle strength declined, but functional muscle NKA content was preserved during DIG, despite elevated plasma digoxin and muscle NKA-digoxin occupancy, with K+ disturbances and fatiguability unchanged. KEY POINTS: The Na+ ,K+ -ATPase (NKA) is vital in regulating skeletal muscle extracellular potassium concentration ([K+ ]), excitability and plasma [K+ ] and thereby also in modulating fatigue during intense contractions. NKA is inhibited by digoxin, which in cardiac patients lowers muscle functional NKA content ([3 H]-ouabain binding) and exacerbates K+ disturbances during exercise. In healthy adults, we found that digoxin at clinical levels surprisingly did not reduce functional muscle NKA content, whilst digoxin removal by Digibind antibody revealed an ∼8% increased muscle total NKA content. Accordingly, digoxin did not exacerbate arterial plasma [K+ ] disturbances or worsen fatigue during intense exercise, although quadriceps muscle strength was reduced. Thus, digoxin treatment in healthy participants elevated serum digoxin, but muscle functional NKA content was preserved, whilst K+ disturbances and fatigue with intense exercise were unchanged. This resilience to digoxin NKA inhibition is consistent with the importance of NKA in preserving K+ regulation and muscle function.

Metronomic 5-Fluorouracil Delivery Primes Skeletal Muscle for Myopathy but Does Not Cause Cachexia.

Skeletal myopathy encompasses both atrophy and dysfunction and is a prominent event in cancer and chemotherapy-induced cachexia. Here, we investigate the effects of a chemotherapeutic agent, 5-fluorouracil (5FU), on skeletal muscle mass and function, and whether small-molecule therapeutic candidate, BGP-15, could be protective against the chemotoxic challenge exerted by 5FU. Additionally, we explore the molecular signature of 5FU treatment. Male Balb/c mice received metronomic tri-weekly intraperitoneal delivery of 5FU (23 mg/kg), with and without BGP-15 (15 mg/kg), 6 times in total over a 15 day treatment period. We demonstrated that neither 5FU, nor 5FU combined with BGP-15, affected body composition indices, skeletal muscle mass or function. Adjuvant BGP-15 treatment did, however, prevent the 5FU-induced phosphorylation of p38 MAPK and p65 NF-B subunit, signalling pathways involved in cell stress and inflammatory signalling, respectively. This as associated with mitoprotection. 5FU reduced the expression of the key cytoskeletal proteins, desmin and dystrophin, which was not prevented by BGP-15. Combined, these data show that metronomic delivery of 5FU does not elicit physiological consequences to skeletal muscle mass and function but is implicit in priming skeletal muscle with a molecular signature for myopathy. BGP-15 has modest protective efficacy against the molecular changes induced by 5FU.

The Paradoxical Effect of PARP Inhibitor BGP-15 on Irinotecan-Induced Cachexia and Skeletal Muscle Dysfunction.

Chemotherapy-induced muscle wasting and dysfunction is a contributing factor to cachexia alongside cancer and increases the risk of morbidity and mortality. Here, we investigate the effects of the chemotherapeutic agent irinotecan (IRI) on skeletal muscle mass and function and whether BGP-15 (a poly-(ADP-ribose) polymerase-1 (PARP-1) inhibitor and heat shock protein co-inducer) adjuvant therapy could protect against IRI-induced skeletal myopathy. Healthy 6-week-old male Balb/C mice (n = 24; 8/group) were treated with six intraperitoneal injections of either vehicle, IRI (30 mg/kg) or BGP-15 adjuvant therapy (IRI+BGP; 15 mg/kg) over two weeks. IRI reduced lean and tibialis anterior mass, which were attenuated by IRI+BGP treatment. Remarkably, IRI reduced muscle protein synthesis, while IRI+BGP reduced protein synthesis further. These changes occurred in the absence of a change in crude markers of mammalian/mechanistic target of rapamycin (mTOR) Complex 1 (mTORC1) signaling and protein degradation. Interestingly, the cytoskeletal protein dystrophin was reduced in both IRI- and IRI+BGP-treated mice, while IRI+BGP treatment also decreased ß-dystroglycan, suggesting significant remodeling of the cytoskeleton. IRI reduced absolute force production of the soleus and extensor digitorum longus (EDL) muscles, while IRI+BGP rescued absolute force production of the soleus and strongly trended to rescue force output of the EDL (p = 0.06), which was associated with improvements in mass. During the fatiguing stimulation, IRI+BGP-treated EDL muscles were somewhat susceptible to rupture at the musculotendinous junction, likely due to BGP-15's capacity to maintain the rate of force development within a weakened environment characterized by significant structural remodeling. Our paradoxical data highlight that BGP-15 has some therapeutic advantage by attenuating IRI-induced skeletal myopathy; however, its effects on the remodeling of the cytoskeleton and extracellular matrix, which appear to make fast-twitch muscles more prone to tearing during contraction, could suggest the induction of muscular dystrophy and, thus, require further characterization.

Resistance training upregulates skeletal muscle Na+, K+-ATPase content, with elevations in both α1 and α2, but not ß isoforms.

PURPOSE: The Na+, K+-ATPase (NKA) is important in regulating trans-membrane ion gradients, cellular excitability and muscle function. We investigated the effects of resistance training in healthy young adults on the adaptability of NKA content and of the specific α and ß isoforms in human skeletal muscle. METHODS: Twenty-one healthy young males (22.9 ± 4.6 year; 1.80 ± 0.70 m, 85.1 ± 17.8 kg, mean ± SD) underwent 7 weeks of resistance training, training three times per week (RT, n = 16) or control (CON, n = 5). The training program was effective with a 39% gain in leg press muscle strength (p = 0.001). A resting vastus lateralis muscle biopsy was taken before and following RT or CON and assayed for NKA content ([3H]ouabain binding site content) and NKA isoform (α1, α2, ß1, ß2) abundances. RESULTS: After RT, each of NKA content (12%, 311 ± 76 vs 349 ± 76 pmol g wet weight-1, p = 0.01), NKA α1 (32%, p = 0.01) and α2 (10%, p < 0.01) isoforms were increased, whereas ß1 (p = 0.18) and ß2 (p = 0.22) isoforms were unchanged. NKA content and isoform abundances were unchanged during CON. CONCLUSIONS: Resistance training increased muscle NKA content through upregulation of both α1 and α2 isoforms, which were independent of ß isoform changes. In animal models, modulations in α1 and α2 isoform abundances in skeletal muscle may affect fatigue resistance during exercise, muscle hypertrophy and strength. Whether similar in-vivo functional benefits of these NKA isoform adaptations occurs in human muscle with resistance training remains to be determined.

Effects of testosterone suppression, hindlimb immobilization, and recovery on [3H]ouabain binding site content and Na+, K+-ATPase isoforms in rat soleus muscle.

We investigated the effects of testosterone suppression, hindlimb immobilization, and recovery on skeletal muscle Na+,K+-ATPase (NKA), measured via [3H]ouabain binding site content (OB) and NKA isoform abundances (α1-3, ß1-2). Male rats underwent castration or sham surgery plus 7 days of rest, 10 days of unilateral immobilization (cast), and 14 days of recovery, with soleus muscles obtained at each time from cast and noncast legs. Testosterone reduction did not modify OB or NKA isoforms in nonimmobilized control muscles. With sham surgery, OB was lower after immobilization in the cast leg than in both the noncast leg (-26%, P = 0.023) and the nonimmobilized control (-34%, P = 0.001), but OB subsequently recovered. With castration, OB was lower after immobilization in the cast leg than in the nonimmobilized control (-34%, P = 0.001), and remained depressed at recovery (-34%, P = 0.001). NKA isoforms did not differ after immobilization or recovery in the sham group. After castration, α2 in the cast leg was ~60% lower than in the noncast leg (P = 0.004) and nonimmobilized control (P = 0.004) and after recovery remained lower than the nonimmobilized control (-42%, P = 0.039). After immobilization, ß1 was lower in the cast than the noncast leg (-26%, P = 0.018), with ß2 lower in the cast leg than in the noncast leg (-71%, P = 0.004) and nonimmobilized control (-65%, P = 0.012). No differences existed for α1 or α3. Thus, both OB and α2 decreased after immobilization and recovery in the castration group, with α2, ß1, and ß2 isoform abundances decreased with immobilization compared with the sham group. Therefore, testosterone suppression in rats impaired restoration of immobilization-induced lowered number of functional NKA and α2 isoforms in soleus muscle.NEW & NOTEWORTHY: The Na+,K+-ATPase (NKA) is vital in muscle excitability and function. In rats, immobilization depressed soleus muscle NKA, with declines in [3H]ouabain binding, which was restored after 14 days recovery. After testosterone suppression by castration, immobilization depressed [3H]ouabain binding, depressed α2, ß1, and ß2 isoforms, and abolished subsequent recovery in [3H]ouabain binding and α2 isoforms. This may have implications for functional recovery for inactive men with lowered testosterone levels, such as in prostate cancer or aging.

Co-treatment With BGP-15 Exacerbates 5-Fluorouracil-Induced Gastrointestinal Dysfunction.

Gastrointestinal (GI) side-effects of chemotherapy present a constant impediment to efficient and tolerable treatment of cancer. GI symptoms often lead to dose reduction, delays and cessation of treatment. Chemotherapy-induced nausea, bloating, vomiting, constipation, and/or diarrhea can persist up to 10 years post-treatment. We have previously reported that long-term 5-fluorouracil (5-FU) administration results in enteric neuronal loss, acute inflammation and intestinal dysfunction. In this study, we investigated whether the cytoprotectant, BGP-15, has a neuroprotective effect during 5-FU treatment. Balb/c mice received tri-weekly intraperitoneal 5-FU (23 mg/kg/d) administration with and without BGP-15 (15 mg/kg/d) for up to 14 days. GI transit was analyzed via in vivo serial X-ray imaging prior to and following 3, 7, and 14 days of treatment. On day 14, colons were collected for assessment of ex vivo colonic motility, neuronal mitochondrial superoxide, and cytochrome c levels as well as immunohistochemical analysis of myenteric neurons. BGP-15 did not inhibit 5-FU-induced neuronal loss, but significantly increased the number and proportion of choline acetyltransferase (ChAT)-immunoreactive (IR) and neuronal nitric oxide synthase (nNOS)-IR neurons in the myenteric plexus. BGP-15 co-administration significantly increased mitochondrial superoxide production, mitochondrial depolarization and cytochrome c release in myenteric plexus and exacerbated 5-FU-induced colonic inflammation. BGP-15 exacerbated 5-FU-induced colonic dysmotility by reducing the number and proportion of colonic migrating motor complexes and increasing the number and proportion of fragmented contractions and increased fecal water content indicative of diarrhea. Taken together, BGP-15 co-treatment aggravates 5-FU-induced GI side-effects, in contrast with our previous findings that BGP-15 alleviates GI side-effects of oxaliplatin.

Oxaliplatin-induced enteric neuronal loss and intestinal dysfunction is prevented by co-treatment with BGP-15.

BACKGROUND AND PURPOSE: Gastrointestinal side effects of chemotherapy are an under-recognized clinical problem, leading to dose reduction, delays and cessation of treatment, presenting a constant challenge for efficient and tolerated anti-cancer treatment. We have found that oxaliplatin treatment results in intestinal dysfunction, oxidative stress and loss of enteric neurons. BGP-15 is a novel cytoprotective compound with potential HSP72 co-inducing and PARP inhibiting properties. In this study, we investigated the potential of BGP-15 to alleviate oxaliplatin-induced enteric neuropathy and intestinal dysfunction. EXPERIMENTAL APPROACH: Balb/c mice received oxaliplatin (3 mg·kg-1 ·day-1 ) with and without BGP-15 (15 mg·kg-1 ·day-1 : i.p.) tri-weekly for 14 days. Gastrointestinal transit was analysed via in vivo X-ray imaging, before and after treatment. Colons were collected to assess ex vivo motility, neuronal mitochondrial superoxide and cytochrome c levels and for immunohistochemical analysis of myenteric neurons. KEY RESULTS: Oxaliplatin-induced neuronal loss increased the proportion of neuronal NO synthase-immunoreactive neurons and increased levels of mitochondrial superoxide and cytochrome c in the myenteric plexus. These changes were correlated with an increase in PARP-2 immunoreactivity in the colonic mucosa and were attenuated by BGP-15 co-treatment. Significant delays in gastrointestinal transit, intestinal emptying and pellet formation, impaired colonic motor activity, reduced faecal water content and lack of weight gain associated with oxaliplatin treatment were restored to sham levels in mice co-treated with BGP-15. CONCLUSION AND IMPLICATIONS: Our results showed that BGP-15 ameliorated oxidative stress, increased enteric neuronal survival and alleviated oxaliplatin-induced intestinal dysfunction, suggesting that BGP-15 may relieve the gastrointestinal side effects of chemotherapy.

Intense interval training in healthy older adults increases skeletal muscle [3H]ouabain-binding site content and elevates Na+,K+-ATPase α2 isoform abundance in Type II fibers.

Young adults typically adapt to intense exercise training with an increased skeletal muscle Na+,K+-ATPase (NKA) content, concomitant with reduced extracellular potassium concentration [K+] during exercise and enhanced exercise performance. Whether these changes with longitudinal training occur in older adults is unknown and was investigated here. Fifteen older adults (69.4 ± 3.5 years, mean ± SD) were randomized to either 12 weeks of intense interval training (4 × 4 min at 90-95% peak heart rate), 3 days/week (IIT, n = 8); or no exercise controls (n = 7). Before and after training, participants completed an incremental cycle ergometer exercise test until a rating of perceived exertion of 17 (very hard) on a 20-point scale was attained, with measures of antecubital venous [K+]v Participants underwent a resting muscle biopsy prior to and at 48-72 h following the final training session. After IIT, the peak exercise work rate (25%), oxygen uptake (16%) and heart rate (6%) were increased (P < 0.05). After IIT, the peak exercise plasma [K+]v tended to rise (P = 0.07), while the rise in plasma [K+]v relative to work performed (nmol.L-1J-1) was unchanged. Muscle NKA content increased by 11% after IIT (P < 0.05). Single fiber measurements, increased in NKA α2 isoform in Type II fibers after IIT (30%, P < 0.05), with no changes to the other isoforms in single fibers or homogenate. Thus, intense exercise training in older adults induced an upregulation of muscle NKA, with a fiber-specific increase in NKA α2 abundance in Type II fibers, coincident with increased muscle NKA content and enhanced exercise performance.

BGP-15 Protects against Oxaliplatin-Induced Skeletal Myopathy and Mitochondrial Reactive Oxygen Species Production in Mice.

Chemotherapy is a leading intervention against cancer. Albeit highly effective, chemotherapy has a multitude of deleterious side-effects including skeletal muscle wasting and fatigue, which considerably reduces patient quality of life and survivability. As such, a defense against chemotherapy-induced skeletal muscle dysfunction is required. Here we investigate the effects of oxaliplatin (OXA) treatment in mice on the skeletal muscle and mitochondria, and the capacity for the Poly ADP-ribose polymerase (PARP) inhibitor, BGP-15, to ameliorate any pathological side-effects induced by OXA. To do so, we investigated the effects of 2 weeks of OXA (3 mg/kg) treatment with and without BGP-15 (15 mg/kg). OXA induced a 15% (p < 0.05) reduction in lean tissue mass without significant changes in food consumption or energy expenditure. OXA treatment also altered the muscle architecture, increasing collagen deposition, neutral lipid and Ca2+ accumulation; all of which were ameliorated with BGP-15 adjunct therapy. Here, we are the first to show that OXA penetrates the mitochondria, and, as a possible consequence of this, increases mtROS production. These data correspond with reduced diameter of isolated FDB fibers and shift in the fiber size distribution frequency of TA to the left. There was a tendency for reduction in intramuscular protein content, albeit apparently not via Murf1 (atrophy)- or p62 (autophagy)- dependent pathways. BGP-15 adjunct therapy protected against increased ROS production and improved mitochondrial viability 4-fold and preserved fiber diameter and number. Our study highlights BGP-15 as a potential adjunct therapy to address chemotherapy-induced skeletal muscle and mitochondrial pathology.

Dissociation between short-term unloading and resistance training effects on skeletal muscle Na+,K+-ATPase, muscle function, and fatigue in humans.

Physical training increases skeletal muscle Na+,K+-ATPase content (NKA) and improves exercise performance, but the effects of inactivity per se on NKA content and isoform abundance in human muscle are unknown. We investigated the effects of 23-day unilateral lower limb suspension (ULLS) and subsequent 4-wk resistance training (RT) on muscle function and NKA in 6 healthy adults, measuring quadriceps muscle peak torque; fatigue and venous [K+] during intense one-legged cycling exercise; and skeletal muscle NKA content ([3H]ouabain binding) and NKA isoform abundances (immunoblotting) in muscle homogenates (α1-3, ß1-2) and in single fibers (α1-3, ß1). In the unloaded leg after ULLS, quadriceps peak torque and cycling time to fatigue declined by 22 and 23%, respectively, which were restored with RT. Whole muscle NKA content and homogenate NKA α1-3 and ß1-2 isoform abundances were unchanged with ULLS or RT. However, in single muscle fibers, NKA α3 in type I (-66%, P = 0.006) and ß1 in type II fibers (-40%, P = 0.016) decreased after ULLS, with other NKA isoforms unchanged. After RT, NKA α1 (79%, P = 0.004) and ß1 (35%, P = 0.01) increased in type II fibers, while α2 (76%, P = 0.028) and α3 (142%, P = 0.004) increased in type I fibers compared with post-ULLS. Despite considerably impaired muscle function and earlier fatigue onset, muscle NKA content and homogenate α1 and α2 abundances were unchanged, thus being resilient to inactivity induced by ULLS. Nonetheless, fiber type-specific downregulation with inactivity and upregulation with RT of several NKA isoforms indicate complex regulation of muscle NKA expression in humans.

Cell specific differences in the protein abundances of GAPDH and Na(+),K(+)-ATPase in skeletal muscle from aged individuals.

Na(+), K(+)-ATPase (NKA) isoforms (α1,α2,α3,ß1,ß2,ß3) are involved in the maintenance of membrane potential and hence are important regulators of cellular homeostasis. Given the age-related decline in skeletal muscle function, we investigated whether the natural physiological process of aging is associated with altered abundance of NKA isoforms (α1,α2,α3,ß1,ß2,ß3) or of the commonly used control protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Importantly, measurements were made in both whole muscle or specific fiber types obtained from skeletal muscle biopsies. Seventeen healthy older (AGED, 69.4 ± 3.5 years, mean ± SD) and 14 younger (YOUNG, 25.5 ± 2.8 years) adults underwent a muscle biopsy for biochemical analyses. Comparing homogenates from AGED and YOUNG individuals revealed higher ß3 isoform (p<0.05) and lower GAPDH (p<0.05). Analysis of individual fibers in muscle from YOUNG individuals, showed greater α3 and ß2 isoforms, and more GAPDH in Type II compared with Type I fibers (p<0.05). In the AGED, GAPDH was higher in Type II compared with Type I fibers (p<0.05), there were no fiber type differences in the NKA isoforms (p>0.05). Compared with the same fiber type in YOUNG, α1 was greater (Type I) and α3 lower (Type II), while in both fiber types, ß2 was lower, ß3 greater and GAPDH lower, in muscle from AGED individuals (all p<0.05). Overall, we demonstrate that (i) GAPDH is an inappropriate choice of protein for normalization in all skeletal muscle research and (ii) full understanding of the role of NKA isoforms in human skeletal muscle requires consideration of age and muscle fiber type.

The effects of knee injury on skeletal muscle function, Na+, K+-ATPase content, and isoform abundance.

While training upregulates skeletal muscle Na(+), K(+)-ATPase (NKA), the effects of knee injury and associated disuse on muscle NKA remain unknown. This was therefore investigated in six healthy young adults with a torn anterior cruciate ligament, (KI; four females, two males; age 25.0 ± 4.9 years; injury duration 15 ± 17 weeks; mean ± SD) and seven age- and BMI-matched asymptomatic controls (CON; five females, two males). Each participant underwent a vastus lateralis muscle biopsy, on both legs in KI and one leg in CON. Muscle was analyzed for muscle fiber type and cross-sectional area (CSA), NKA content ([(3)H]ouabain binding), and α1-3 and ß1-2 isoform abundance. Participants also completed physical activity and knee function questionnaires (KI only); and underwent quadriceps peak isometric strength, thigh CSA and postural sway assessments in both injured and noninjured legs. NKA content was 20.1% lower in the knee-injured leg than the noninjured leg and 22.5% lower than CON. NKA α2 abundance was 63.0% lower in the knee-injured leg than the noninjured leg, with no differences in other NKA isoforms. Isometric strength and thigh CSA were 21.7% and 7.1% lower in the injured leg than the noninjured leg, respectively. In KI, postural sway did not differ between legs, but for two-legged standing was 43% higher than CON. Hence, muscle NKA content and α2 abundance were reduced in severe knee injury, which may contribute to impaired muscle function. Restoration of muscle NKA may be important in rehabilitation of muscle function after knee and other lower limb injury.

Dose-dependent increases in p70S6K phosphorylation and intramuscular branched-chain amino acids in older men following resistance exercise and protein intake.

Resistance exercise and whey protein supplementation are effective strategies to activate muscle cell anabolic signaling and ultimately promote increases in muscle mass and strength. In the current study, 46 healthy older men aged 60-75 (69.0 ± 0.55 years, 85.9 ± 1.8 kg, 176.8 ± 1.0 cm) performed a single bout of unaccustomed lower body resistance exercise immediately followed by ingestion of a noncaloric placebo beverage or supplement containing 10, 20, 30, or 40 g of whey protein concentrate (WPC). Intramuscular amino acid levels in muscle biopsy samples were measured by Gas Chromatography-Mass Spectrometry (GC-MS) at baseline (before exercise and WPC supplementation) plus at 2 h and 4 h post exercise. Additionally, the extent of p70S6K phosphorylation at Thr389 in muscle biopsy homogenates was assessed by western blot. Resistance exercise alone reduced intramuscular branch chain amino acid (BCAA; leucine, isoleucine, and valine) content. Supplementation with increasing doses of whey protein prevented this fall in muscle BCAAs during postexercise recovery and larger doses (30 g and 40 g) significantly augmented postexercise muscle BCAA content above that observed following placebo ingestion. Additionally, the fold change in the phosphorylation of p70S6K (Thr389) at 2 h post exercise was correlated with the dose of whey protein consumed (r = 0.51, P < 001) and was found to be significantly correlated with intramuscular leucine content (r = 0.32, P = 0.026). Intramuscular BCAAs, and leucine in particular, appear to be important regulators of anabolic signaling in aged human muscle during postexercise recovery via reversal of exercise-induced declines in intramuscular BCAAs.

Plasma K+ dynamics and implications during and following intense rowing exercise.

We investigated whether potassium (K(+)) disturbances during and following intense exercise may be pronounced when utilizing a large contracting muscle mass, examining maximal 2,000-m rowing exercise effects on radial arterial plasma K(+) concentration ([K(+)]a) in 11 healthy adults. Blood was sampled at baseline, preexercise, each 30 s during rowing, and for 30 min postexercise. Time to complete 2,000 m was 7.26 ± 0.59 min; power output at 30 s was 326 ± 81 W (mean ± SD). With exercise time expressed in deciles, power output fell 16.5% from the first to fourth decile (P < 0.05) and 19.9% at the ninth decile (P < 0.05); EMG median frequency declined 4.6% by the third decile and 5.5% by the eighth decile (P < 0.05). Plasma [K(+)]a increased from 3.89 ± 0.13 mM at rest to 6.13 ± 0.46 mM by 90 s rowing (P < 0.001) and was then sustained until end exercise (P < 0.001). In recovery, [K(+)]a decreased abruptly, reaching 3.33 ± 0.22 mM at 5 min postexercise (P < 0.001) and remaining below preexercise after 30 min (P < 0.005). At end exercise, blood [lactate]a (preexercise 0.64 ± 0.18 mM) reached 10.87 ± 1.33 mM, plasma volume decreased 9.7 ± 2.3% from preexercise, and pHa decreased to 7.10 ± 0.07 units (P < 0.001). In conclusion, arterial hyperkalemia was sustained during exhaustive rowing reflecting a balance between K(+) release and reuptake in contracting muscles and K(+) uptake by inactive muscles. While high, the [K(+)]a was lower than anticipated compared with maximal cycling or sprinting, possibly reflecting greater adrenergic response and Na(+),K(+)-ATPase activity in contracting muscles; fatigue was evidenced by reduced power output and EMG median frequency. A prolonged hypokalemia after rowing likely reflected continuing muscular Na(+),K(+)-ATPase activity.

N-acetylcysteine alters substrate metabolism during high-intensity cycle exercise in well-trained humans.

We investigated the effects of N-acetylcysteine (NAC) on metabolism during fixed work rate high-intensity interval exercise (HIIE) and self-paced 10-min time-trial (TT10) performance. Nine well-trained male cyclists (V̇O2peak, 69.4 ± 5.8 mL · kg(-1) · min(-1); peak power output (PPO), 385 ± 43 W; mean ± SD) participated in a double-blind, repeated-measures, randomised crossover trial. Two trials (NAC supplementation and placebo) were performed 7 days apart consisting of 6 × 5 min HIIE bouts at 82% PPO (316 ± 40 W) separated by 1 min at 100 W, and then after 2 min of recovery at 100 W, TT10 was performed. Expired gases, venous blood, and electromyographic (EMG) data were collected. NAC did not influence blood glutathione but decreased lipid peroxidation compared with the placebo (P < 0.05). Fat oxidation was elevated with NAC compared with the placebo during HIIE bouts 5 and 6 (9.9 ± 8.9 vs. 3.9 ± 4.8 µmol · kg(-1) · min(-1); P < 0.05), as was blood glucose throughout HIIE (4.3 ± 0.6 vs. 3.8 ± 0.6 mmol · L(-1); P < 0.05). Blood lactate was lower with NAC after TT10 (3.3 ± 1.3 vs. 4.2 ± 1.3 mmol · L(-1); P < 0.05). Median EMG frequency of the vastus lateralis was lower with NAC during HIIE (79 ± 10 vs. 85 ± 10 Hz; P < 0.05), but not TT10 (82 ± 11 Hz). Finally, NAC decreased mean power output 4.9% ± 6.6% (effect size = -0.3 ± 0.4, mean ± 90% CI) during TT10 (305 ± 57 W vs. 319 ± 45 W). These data suggest that NAC alters substrate metabolism and muscle fibre type recruitment during HIIE, which is detrimental to time-trial performance.

Impaired exercise performance and muscle Na(+),K(+)-pump activity in renal transplantation and haemodialysis patients.

BACKGROUND: We examined whether abnormal skeletal muscle Na(+),K(+)-pumps underlie impaired exercise performance in haemodialysis patients (HDP) and whether these are improved in renal transplant recipients (RTx). METHODS: Peak oxygen consumption ( O(2peak)) and plasma [K(+)] were measured during incremental exercise in 9RTx, 10 HDP and 10 healthy controls (CON). Quadriceps peak torque (PT), fatigability (decline in strength during thirty contractions), thigh muscle cross-sectional area (TMCSA) and vastus lateralis Na(+),K(+)-pump maximal activity, content and isoform (α(1)-α(3), ß(1)-ß(3)) abundance were measured. RESULTS: O(2peak) was 32 and 35% lower in RTx and HDP than CON, respectively (P < 0.05). PT was less in RTx and HDP than CON (P < 0.05) but did not differ when expressed relative to TMCSA. Fatigability was ∼1.6-fold higher in RTx (24 ± 11%) and HDP (25 ± 4%) than CON (15 ± 5%, P < 0.05). Na(+),K(+)-pump activity was 28 and 31% lower in RTx and HDP, respectively than CON (P < 0.02), whereas content and isoform abundance did not differ. Pooled (n = 28) O(2peak) correlated with Na(+),K(+)-pump activity (r = 0.45, P = 0.02). CONCLUSIONS: O(2peak) and muscle Na(+),K(+)-pump activity were depressed and muscle fatigability increased in HDP, with no difference observed in RTx. These findings are consistent with the possibility that impaired exercise performance in HDP and RTx may be partially due to depressed muscle Na(+),K(+)-pump activity and relative TMCSA.

N-acetylcysteine attenuates the decline in muscle Na+,K+-pump activity and delays fatigue during prolonged exercise in humans.

Reactive oxygen species (ROS) have been linked with both depressed Na(+),K(+)-pump activity and skeletal muscle fatigue. This study investigated N-acetylcysteine (NAC) effects on muscle Na(+),K(+)-pump activity and potassium (K(+)) regulation during prolonged, submaximal endurance exercise. Eight well-trained subjects participated in a double-blind, randomised, crossover design, receiving either NAC or saline (CON) intravenous infusion at 125 mg kg(-1) h(-1) for 15 min, then 25 mg kg(-1) h(-1) for 20 min prior to and throughout exercise. Subjects cycled for 45 min at 71% , then continued at 92% until fatigue. Vastus lateralis muscle biopsies were taken before exercise, at 45 min and fatigue and analysed for maximal in vitro Na(+),K(+)-pump activity (K(+)-stimulated 3-O-methyfluorescein phosphatase; 3-O-MFPase). Arterialized venous blood was sampled throughout exercise and analysed for plasma K(+) and other electrolytes. Time to fatigue at 92% was reproducible in preliminary trials (c.v. 5.6 +/- 0.6%) and was prolonged with NAC by 23.8 +/- 8.3% (NAC 6.3 +/- 0.5 versus CON 5.2 +/- 0.6 min, P < 0.05). Maximal 3-O-MFPase activity decreased from rest by 21.6 +/- 2.8% at 45 min and by 23.9 +/- 2.3% at fatigue (P < 0.05). NAC attenuated the percentage decline in maximal 3-O-MFPase activity (%Deltaactivity) at 45 min (P < 0.05) but not at fatigue. When expressed relative to work done, the %Deltaactivity-to-work ratio was attenuated by NAC at 45 min and fatigue (P < 0.005). The rise in plasma [K(+)] during exercise and the Delta[K(+)]-to-work ratio at fatigue were attenuated by NAC (P < 0.05). These results confirm that the antioxidant NAC attenuates muscle fatigue, in part via improved K(+) regulation, and point to a role for ROS in muscle fatigue.