Cardiomyocyte Adaptation to Exercise: K+ Channels, Contractility and Ischemic Injury.

Cardiovascular disease is a leading cause of morbidity and mortality, and exercise-training (TRN) is known to reduce risk factors and protect the heart from ischemia and reperfusion injury. Though the cardioprotective effects of exercise are well-documented, underlying mechanisms are not well understood. This review highlights recent findings and focuses on cardiac factors with emphasis on K+ channel control of the action potential duration (APD), ß-adrenergic and adenosine regulation of cardiomyocyte function, and mitochondrial Ca2+ regulation. TRN-induced prolongation and shortening of the APD at low and high activation rates, respectively, is discussed in the context of a reduced response of the sarcolemma delayed rectifier potassium channel (IK) and increased content and activation of the sarcolemma KATP channel. A proposed mechanism underlying the latter is presented, including the phosphatidylinositol-3kinase/protein kinase B pathway. TRN induced increases in cardiomyocyte contractility and the response to adrenergic agonists are discussed. The TRN-induced protection from reperfusion injury is highlighted by the increased content and activation of the sarcolemma KATP channel and the increased phosphorylated glycogen synthase kinase-3ß, which aid in preventing mitochondrial Ca2+ overload and mitochondria-triggered apoptosis. Finally, a brief section is presented on the increased incidences of atrial fibrillation associated with age and in life-long exercisers.

Altered contractility, Ca2+ transients, and cell morphology seen in a patient-specific iPSC-CM model of Ebstein's anomaly with left ventricular noncompaction.

Patients with two congenital heart diseases (CHDs), Ebstein's anomaly (EA) and left ventricular noncompaction (LVNC), suffer higher morbidity than either CHD alone. The genetic etiology and pathogenesis of combined EA/LVNC remain largely unknown. We investigated a familial EA/LVNC case associated with a variant (p.R237C) in the gene encoding Kelch-like protein 26 (KLHL26) by differentiating induced pluripotent stem cells (iPSCs) generated from affected and unaffected family members into cardiomyocytes (iPSC-CMs) and assessing iPSC-CM morphology, function, gene expression, and protein abundance. Compared with unaffected iPSC-CMs, CMs containing the KLHL26 (p.R237C) variant exhibited aberrant morphology including distended endo(sarco)plasmic reticulum (ER/SR) and dysmorphic mitochondria and aberrant function that included decreased contractions per minute, altered calcium transients, and increased proliferation. Pathway enrichment analyses based on RNASeq data indicated that the "structural constituent of muscle" pathway was suppressed, whereas the "ER lumen" pathway was activated. Taken together, these findings suggest that iPSC-CMs containing this KLHL26 (p.R237C) variant develop dysregulated ER/SR, calcium signaling, contractility, and proliferation.NEW & NOTEWORTHY We demonstrate here that iPSCs derived from patients with Ebstein's anomaly and left ventricular noncompaction, when differentiated into cardiomyocytes, display significant structural and functional changes that offer insight into disease pathogenesis, including altered ER/SR and mitochondrial morphology, contractility, and calcium signaling.

Ca2+ dependency of limb muscle fiber contractile mechanics in young and older adults.

Age-induced declines in skeletal muscle contractile function have been attributed to multiple cellular factors, including lower peak force (Po), decreased Ca2+ sensitivity, and reduced shortening velocity (Vo). However, changes in these cellular properties with aging remain unresolved, especially in older women, and the effect of submaximal Ca2+ on contractile function is unknown. Thus, we compared contractile properties of muscle fibers from 19 young (24 ± 3 yr; 8 women) and 21 older adults (77 ± 7 yr; 7 women) under maximal and submaximal Ca2+ and assessed the abundance of three proteins thought to influence Ca2+ sensitivity. Fast fiber cross-sectional area was ~44% larger in young (6,479 ± 2,487 µm2) compared with older adults (4,503 ± 2,071 µm2, P < 0.001), which corresponded with a greater absolute Po (young = 1.12 ± 0.43 mN; old = 0.79 ± 0.33 mN, P < 0.001). There were no differences in fast fiber size-specific Po, indicating the age-related decline in force was explained by differences in fiber size. Except for fast fiber size and absolute Po, no age or sex differences were observed in Ca2+ sensitivity, rate of force development (ktr), or Vo in either slow or fast fibers. Submaximal Ca2+ depressed ktr and Vo, but the effects were not altered by age in either sex. Contrary to rodent studies, regulatory light chain (RLC) and myosin binding protein-C abundance and RLC phosphorylation were unaltered by age or sex. These data suggest the age-associated reductions in contractile function are primarily due to the atrophy of fast fibers and that caution is warranted when extending results from rodent studies to humans.

Bioenergetic basis for the increased fatigability with ageing.

KEY POINTS: The mechanisms for the age-related increase in fatigability during dynamic exercise remain elusive. We tested whether age-related impairments in muscle oxidative capacity would result in a greater accumulation of fatigue causing metabolites, inorganic phosphate (Pi ), hydrogen (H+ ) and diprotonated phosphate (H2 PO4- ), in the muscle of old compared to young adults during a dynamic knee extension exercise. The age-related increase in fatigability (reduction in mechanical power) of the knee extensors was closely associated with a greater accumulation of metabolites within the working muscle but could not be explained by age-related differences in muscle oxidative capacity. These data suggest that the increased fatigability in old adults during dynamic exercise is primarily determined by age-related impairments in skeletal muscle bioenergetics that result in a greater accumulation of metabolites. ABSTRACT: The present study aimed to determine whether the increased fatigability in old adults during dynamic exercise is associated with age-related differences in skeletal muscle bioenergetics. Phosphorus nuclear magnetic resonance spectroscopy was used to quantify concentrations of high-energy phosphates and pH in the knee extensors of seven young (22.7 ± 1.2 years; six women) and eight old adults (76.4 ± 6.0 years; seven women). Muscle oxidative capacity was measured from the phosphocreatine (PCr) recovery kinetics following a 24 s maximal voluntary isometric contraction. The fatiguing exercise consisted of 120 maximal velocity contractions (one contraction per 2 s) against a load equivalent to 20% of the maximal voluntary isometric contraction. The PCr recovery kinetics did not differ between young and old adults (0.023 ± 0.007 s-1  vs. 0.019 ± 0.004 s-1 , respectively). Fatigability (reductions in mechanical power) of the knee extensors was ∼1.8-fold greater with age and was accompanied by a greater decrease in pH (young = 6.73 ± 0.09, old = 6.61 ± 0.04) and increases in concentrations of inorganic phosphate, [Pi ], (young = 22.7 ± 4.8 mm, old = 32.3 ± 3.6 mm) and diprotonated phosphate, [H2 PO4- ], (young = 11.7 ± 3.6 mm, old = 18.6 ± 2.1 mm) at the end of the exercise in old compared to young adults. The age-related increase in power loss during the fatiguing exercise was strongly associated with intracellular pH (r = -0.837), [Pi ] (r = 0.917) and [H2 PO4- ] (r = 0.930) at the end of the exercise. These data suggest that the age-related increase in fatigability during dynamic exercise has a bioenergetic basis and is explained by an increased accumulation of metabolites within the muscle.

Effects of elevated H+ and Pi on the contractile mechanics of skeletal muscle fibres from young and old men: implications for muscle fatigue in humans.

KEY POINTS: The mechanisms responsible for the loss in muscle power and increased fatigability with ageing are unresolved. We show that the contractile mechanics of fibres from the vastus lateralis of old men were well-preserved compared to those of young men, but the selective loss of fast myosin heavy chain II muscle was strongly associated with age-related decrements in whole-muscle strength and power. We reveal that the combination of acidosis (H+ ) and inorganic phosphate (Pi ) is an important mediator of muscle fatigue in humans by inhibiting the low- to high-force state of the cross-bridge cycle and peak power, but the depressive effects of these ions on cross-bridge function were similar in fibres from young and old men. These findings suggest that the age-related loss in muscle power is primarily determined by the atrophy of fast fibres, but the age-related increased fatigability cannot be explained by an increased sensitivity of the cross-bridge to H+ and Pi . ABSTRACT: The present study aimed to identify the mechanisms responsible for the loss in muscle power and increased fatigability with ageing by integrating measures of whole-muscle function with single fibre contractile mechanics. After adjusting for the 22% smaller muscle mass in old (73-89 years, n = 6) compared to young men (20-29 years, n = 6), isometric torque and power output of the knee extensors were, respectively, 38% and 53% lower with age. Fatigability was ∼2.7-fold greater with age and strongly associated with reductions in the electrically-evoked contractile properties. To test whether cross-bridge mechanisms could explain age-related decrements in knee extensor function, we exposed myofibres (n = 254) from the vastus lateralis to conditions mimicking quiescent muscle and fatiguing levels of acidosis (H+ ) (pH 6.2) and inorganic phosphate (Pi ) (30 mm). The fatigue-mimicking condition caused marked reductions in force, shortening velocity and power and inhibited the low- to high-force state of the cross-bridge cycle, confirming findings from non-human studies that these ions act synergistically to impair cross-bridge function. Other than severe age-related atrophy of fast fibres (-55%), contractile function and the depressive effects of the fatigue-mimicking condition did not differ in fibres from young and old men. The selective loss of fast myosin heavy chain II muscle was strongly associated with the age-related decrease in isometric torque (r = 0.785) and power (r = 0.861). These data suggest that the age-related loss in muscle strength and power are primarily determined by the atrophy of fast fibres, but the age-related increased fatigability cannot be explained by an increased sensitivity of the cross-bridge to H+ and Pi .

Effects of regular exercise on ventricular myocyte biomechanics and KATP channel function.

Exercise training is known to protect the heart from ischemia and improve function during exercise by reducing cardiomyocyte action potential duration (APD) and increasing contractility. The cellular mechanisms involve ß-adrenergic regulation and the ATP-sensitive K+ (KATP) channel, but how each alters function of the left ventricle and sex specificity is unknown. To address this, female and male Sprague-Dawley rats were randomly assigned to wheel-running (TRN) or sedentary (SED) groups. After 6-8 wk of training, myocytes were isolated from the left ventricle and field stimulated at 1, 2, and 5 Hz. TRN significantly increased cardiomyocyte contractility, the kinetics of the Ca2+ transient, and responsiveness to the adrenergic receptor agonist isoproterenol (ISO), as reflected by an increased sarcomere shortening. Importantly, we demonstrated a TRN-induced upregulation of KATP channels, which was reflected by elevated content, current density, and the channel's contribution to APD shortening at high activation rates and in the presence of the activator pinacidil. TRN induced increase in KATP current occurred throughout the left ventricle, but channel subunit content showed regional specificity with increases in Kir6.2 in the apex and SUR2A in base regions. In summary, TRN elevated cardiomyocyte cross-bridge kinetics, Ca2+ sensitivity, and the responsiveness of contractile function to ß-adrenergic receptor stimulation in both sexes. Importantly, upregulation of the KATP channel accelerates repolarization and shortens APD during stress and exercise. These adaptations have clinical importance, as increased contractility and reduced APD would help protect cardiac output and reduce intracellular Ca2+ overload during stresses such as regional ischemia. NEW & NOTEWORTHY Our results demonstrate that regular exercise significantly increased ventricular myocyte shortening and relaxation velocity and the rate of rise in intracellular Ca2+ transient and enhanced the response of biomechanics and Ca2+ reuptake to ß-adrenergic stimulation. Importantly, exercise training upregulated the cardiomyocyte sarcolemma ATP-sensitive K+ channel across the left ventricle in both sexes, as reflected by elevated channel subunit content, current density, and the channel's contribution to reduced action potential duration at high activation rates.

Skeletal muscle PGC-1ß signaling is sufficient to drive an endurance exercise phenotype and to counteract components of detraining in mice.

Peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α and -1ß serve as master transcriptional regulators of muscle mitochondrial functional capacity and are capable of enhancing muscle endurance when overexpressed in mice. We sought to determine whether muscle-specific transgenic overexpression of PGC-1ß affects the detraining response following endurance training. First, we established and validated a mouse exercise-training-detraining protocol. Second, using multiple physiological and gene expression end points, we found that PGC-1ß overexpression in skeletal muscle of sedentary mice fully recapitulated the training response. Lastly, PGC-1ß overexpression during the detraining period resulted in partial prevention of the detraining response. Specifically, an increase in the plateau at which O2 uptake (V̇o2) did not change from baseline with increasing treadmill speed [peak V̇o2 (ΔV̇o2max)] was maintained in trained mice with PGC-1ß overexpression in muscle 6 wk after cessation of training. However, other detraining responses, including changes in running performance and in situ half relaxation time (a measure of contractility), were not affected by PGC-1ß overexpression. We conclude that while activation of muscle PGC-1ß is sufficient to drive the complete endurance phenotype in sedentary mice, it only partially prevents the detraining response following exercise training, suggesting that the process of endurance detraining involves mechanisms beyond the reversal of muscle autonomous mechanisms involved in endurance fitness. In addition, the protocol described here should be useful for assessing early-stage proof-of-concept interventions in preclinical models of muscle disuse atrophy.

Treatment with ActRIIB-mFc Produces Myofiber Growth and Improves Lifespan in the Acta1 H40Y Murine Model of Nemaline Myopathy.

Nemaline myopathies (NMs) are a group of congenital muscle diseases caused by mutations in at least 10 genes and associated with a range of clinical symptoms. NM is defined on muscle biopsy by the presence of cytoplasmic rod-like structures (nemaline rods) composed of cytoskeletal material. Myofiber smallness is also found in many cases of NM and may represent a cause of weakness that can be counteracted by treatment. We have used i.p. injection of activin type IIB receptor (ActRIIB)-mFc (an inhibitor of myostatin signaling) to promote hypertrophy and increase strength in our prior murine work; we therefore tested whether ActRIIB-mFc could improve weakness in NM mice through myofiber hypertrophy. We report a study of ActRIIB-mFc treatment in the Acta1 H40Y mouse model of NM. Treatment of Acta1 H40Y mice produced significant increases in body mass, muscle mass, quadriceps myofiber size, and survival, but other measurements of strength (forelimb grip strength, ex vivo measurements of contractile function) did not improve. Our studies also identified that the complications of urethral obstruction are associated with mortality in male hemizygote Acta1 H40Y mice. The incidence of urethral obstruction and histologic evidence of chronic obstruction (inflammation) were significantly lower in Acta1 H40Y mice that had been treated with ActRIIB-mFc. ActRIIB-mFc treatment produces a mild benefit to the disease phenotype in Acta1 H40Y mice.

Effects of low cell pH and elevated inorganic phosphate on the pCa-force relationship in single muscle fibers at near-physiological temperatures.

Intense muscle contraction induces high rates of ATP hydrolysis with resulting increases in Pi, H(+), and ADP, factors thought to induce fatigue by interfering with steps in the cross-bridge cycle. Force inhibition is less at physiological temperatures; thus the role of low pH in fatigue has been questioned. Effects of pH 6.2 and collective effects with 30 mM Pi on the pCa-force relationship were assessed in skinned fast and slow rat skeletal muscle fibers at 15 and 30°C. At 30°C, pH 6.2 + 30 mM Pi significantly depressed peak force in all fiber types, with the greatest effect in type IIx fibers. Across fiber types, Ca(2+) sensitivity was depressed by low pH and low pH + high Pi, with the greater effect at 30°C. For type IIx fibers at 30°C, half-maximal activation (pCa50) was 5.36 at pH 6.2 (no added Pi) and 4.98 at pH 6.2 + 30 mM Pi compared with 6.58 in the control condition (pH 7, no added Pi). At 30°C, n2, reflective of thick filament cooperativity, was unchanged by low cell pH but was depressed from 5.02 to 2.46 in type IIx fibers with pH 6.2 + 30 mM Pi. With acidosis, activation thresholds of all fiber types required higher free Ca(2+) at 15 and 30°C. With the exception of type IIx fibers, the Ca(2+) required to reach activation threshold increased further with added Pi. In conclusion, it is clear that fatigue-inducing effects of low cell pH and elevated Pi at near-physiological temperatures are substantial.

Phosphate and acidosis act synergistically to depress peak power in rat muscle fibers.

Skeletal muscle fatigue is characterized by the buildup of H(+) and inorganic phosphate (Pi), metabolites that are thought to cause fatigue by inhibiting muscle force, velocity, and power. While the individual effects of elevated H(+) or Pi have been well characterized, the effects of simultaneously elevating the ions, as occurs during fatigue in vivo, are still poorly understood. To address this, we exposed slow and fast rat skinned muscle fibers to fatiguing levels of H(+) (pH 6.2) and Pi (30 mM) and determined the effects on contractile properties. At 30°C, elevated Pi and low pH depressed maximal shortening velocity (Vmax) by 15% (4.23 to 3.58 fl/s) in slow and 31% (6.24 vs. 4.55 fl/s) in fast fibers, values similar to depressions from low pH alone. Maximal isometric force dropped by 36% in slow (148 to 94 kN/m(2)) and 46% in fast fibers (148 to 80 kN/m(2)), declines substantially larger than what either ion exerted individually. The strong effect on force combined with the significant effect on velocity caused peak power to decline by over 60% in both fiber types. Force-stiffness ratios significantly decreased with pH 6.2 + 30 mM Pi in both fiber types, suggesting these ions reduced force by decreasing the force per bridge and/or increasing the number of low-force bridges. The data indicate the collective effects of elevating H(+) and Pi on maximal isometric force and peak power are stronger than what either ion exerts individually and suggest the ions act synergistically to reduce muscle function during fatigue.

Significance of α-Myosin Heavy Chain (MYH6) Variants in Hypoplastic Left Heart Syndrome and Related Cardiovascular Diseases.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) with complex genetic inheritance. HLHS segregates with other left ventricular outflow tract (LVOT) malformations in families, and can present as either an isolated phenotype or as a feature of a larger genetic disorder. The multifactorial etiology of HLHS makes it difficult to interpret the clinical significance of genetic variants. Specific genes have been implicated in HLHS, including rare, predicted damaging MYH6 variants that are present in >10% of HLHS patients, and which have been shown to be associated with decreased transplant-free survival in our previous studies. MYH6 (α-myosin heavy chain, α-MHC) variants have been reported in HLHS and numerous other CHDs, including LVOT malformations, and may provide a genetic link to these disorders. In this paper, we outline the MYH6 variants that have been identified, discuss how bioinformatic and functional studies can inform clinical decision making, and highlight the importance of genetic testing in HLHS.

Effect of recombinant growth hormone replacement in a growth hormone deficient subject recovering from mild traumatic brain injury: A case report.

OBJECTIVE: To assess the effects of growth hormone (GH) replacement in an individual who sustained mild traumatic brain injury (mTBI) as an adult and was found to have GH deficiency by glucagon stimulation testing. PARTICIPANT: A 43-year old woman who sustained a mild TBI at age 37 years. She was 6.8 years post-injury when she began supplementation. INTERVENTION: Recombinant human GH (rhGH) subcutaneously per day for 1 year. MAIN OUTCOME MEASURES: Single fibre muscle function was evaluated from muscle biopsies. Body composition, muscle strength and peak aerobic capacity were also measured. In addition, neuropsychological tests of memory, processing speed and motor dexterity and speed, as well as a self-report depression inventory were administered. All assessments were performed at baseline and after 6 and 12 months of rhGH replacement therapy. RESULTS: Single muscle fibre changes were greatest at 6 months. Body composition showed continuous improvement. Muscle strength improved for knee extension. Peak oxygen consumption increased at 6 months and total work and ventilatory equivalents continued to improve at 12 months. Significant improvements in neuropsychological test performance were not found, with the exception of performance on a test of motor dexterity and speed. CONCLUSION: rhGH replacement in a subject with GH deficiency after mild TBI improves muscle force production, body composition and aerobic capacity. Reliable improvements on tests of cognition were not found in this subject.

Cardiomyocyte slowly activating delayed rectifier potassium channel: regulation by exercise and ß-adrenergic signaling.

Exercise training is known to prolong the ventricular cardiomyocyte action potential duration (APD), increasing Ca2+ influx and contractility. The prolonged APD is caused, in part, by a decreased responsiveness to ß-adrenergic agonists. The study's aims were to elucidate the mechanisms by which exercise training alters ß-adrenergic regulation and to determine the involvement of delayed rectifier potassium channels (IKr and IKs) in the response. Rats were randomly assigned to wheel running-trained (TRN) or sedentary (SED) groups. After 6-8 wk of training, myocytes were isolated from the apex and base regions of the left ventricle, and current-voltage relationships of IKr and IKs were measured. Myocytes from SED and TRN rats exhibit lower IKr current compared with IKs, and a regional difference in IKs was observed, with higher current in apex compared with base myocytes. Wheel running decreased IKs at positive voltages and reduced IKs responsiveness to ß-agonist. IKs channel subunit KCNQ1 content was higher in apex compared with base, and exercise training decreased KCNQ1 and KCNE1 subunit content in both regions. Exercise training had no effect on ß1-adrenergic receptor content but reduced the kinase anchoring protein yotiao and ß-adrenergic receptor kinase GRK2 compared with SED rats. The reduced KCNQ1, KCNE1, and yotiao provide a mechanism underlying the training-induced decrease in IKs current, while downregulation of GRK2 would reduce inactivation of the ß-AR, maintaining adrenergic stimulation of contractility. Collectively, these membrane protein changes in response to TRN provide a mechanism for prolonging the APD, increasing myocyte efficiency in low stress conditions, while increasing contractility.NEW & NOTEWORTHY Results demonstrate that exercise training (TRN) downregulates ventricular IKs channel current and the channel's responsiveness to ß-agonist factors mediated by TRN-induced decline in channel subunits KCNQ1 and KCNE1 and the A-kinase anchoring protein yotiao. The reduced IKs current helps explain the TRN-induced prolongation of the action potential in basal conditions and, coupled with previously reported upregulation of the KATP channel, results in a more efficient heart that is better able to respond to beat-by-beat changes in metabolism.

Contractility of Induced Pluripotent Stem Cell-Cardiomyocytes With an MYH6 Head Domain Variant Associated With Hypoplastic Left Heart Syndrome.

Hypoplastic left heart syndrome (HLHS) is a clinically and anatomically severe form of congenital heart disease; however, its etiology remains largely unknown. We previously demonstrated that genetic variants in the MYH6 gene are significantly associated with HLHS. Additionally, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from an HLHS-affected family trio (affected parent, unaffected parent, affected proband) carrying an MYH6-R443P head domain variant demonstrated dysmorphic sarcomere structure and increased compensatory MYH7 expression. Analysis of iPSC-CMs derived from the HLHS trio revealed that only beta myosin heavy chain expression was observed in CMs carrying the MYH6-R443P variant after differentiation day 15 (D15). Functional assessments performed between D20-D23 revealed that MYH6-R443P variant CMs contracted more slowly (40 ± 2 vs. 47 ± 2 contractions/min, P < 0.05), shortened less (5.6 ± 0.5 vs. 8.1 ± 0.7% of cell length, P < 0.05), and exhibited slower shortening rates (19.9 ± 1.7 vs. 28.1 ± 2.5 µm/s, P < 0.05) and relaxation rates (11.0 ± 0.9 vs. 19.7 ± 2.0 µm/s, P < 0.05). Treatment with isoproterenol had no effect on iPSC-CM mechanics. Using CRISPR/Cas9 gene editing technology, introduction of the R443P variant into the unaffected parent's iPSCs recapitulated the phenotype of the proband's iPSC-CMs, and conversely, correction of the R443P variant in the proband's iPSCs rescued the cardiomyogenic differentiation, sarcomere organization, slower contraction (P < 0.05) and decreased velocity phenotypes (P < 0.0001). This is the first report to identify that cardiac tissues from HLHS patients with MYH6 variants can exhibit sarcomere disorganization in atrial but not ventricular tissues. This new discovery was not unexpected, since MYH6 is expressed predominantly in the postnatal atria in humans. These findings demonstrate the feasibility of employing patient-derived iPSC-CMs, in combination with patient cardiac tissues, to gain mechanistic insight into how genetic variants can lead to HLHS. Results from this study suggest that decreased contractility of CMs due to sarcomere disorganization in the atria may effect hemodynamic changes preventing development of a normal left ventricle.

Exercise in space: human skeletal muscle after 6 months aboard the International Space Station.

The aim of this investigation was to document the exercise program used by crewmembers (n = 9; 45 +/- 2 yr) while aboard the International Space Station (ISS) for 6 mo and examine its effectiveness for preserving calf muscle characteristics. Before and after spaceflight, we assessed calf muscle volume (MRI), static and dynamic calf muscle performance, and muscle fiber types (gastrocnemius and soleus). While on the ISS, crewmembers had access to a running treadmill, cycle ergometer, and resistance exercise device. The exercise regimen varied among the crewmembers with aerobic exercise performed approximately 5 h/wk at a moderate intensity and resistance exercise performed 3-6 days/wk incorporating multiple lower leg exercises. Calf muscle volume decreased (P < 0.05) 13 +/- 2% with greater (P < 0.05) atrophy of the soleus (-15 +/- 2%) compared with the gastrocnemius (-10 +/- 2%). Peak power was 32% lower (P < 0.05) after spaceflight. Force-velocity characteristics were reduced (P < 0.05) -20 to -29% across the velocity spectrum. There was a 12-17% shift in myosin heavy chain (MHC) phenotype of the gastrocnemius and soleus with a decrease (P < 0.05) in MHC I fibers and a redistribution among the faster phenotypes. These data show a reduction in calf muscle mass and performance along with a slow-to-fast fiber type transition in the gastrocnemius and soleus muscles, which are all qualities associated with unloading in humans. Future long-duration space missions should modify the current ISS exercise prescription and/or hardware to better preserve human skeletal muscle mass and function, thereby reducing the risk imposed to crewmembers.

Bioenergetic basis of skeletal muscle fatigue.

Energetic demand from high-intensity exercise can easily exceed ATP synthesis rates of mitochondria leading to a reliance on anaerobic metabolism. The reliance on anaerobic metabolism results in the accumulation of intracellular metabolites, namely inorganic phosphate (Pi) and hydrogen (H+), that are closely associated with exercise-induced reductions in power. Cellular and molecular studies have revealed several steps where these metabolites impair contractile function demonstrating a causal role in fatigue. Elevated Pi or H+ directly inhibits force and power of the cross-bridge and decreases myofibrillar Ca2+ sensitivity, whereas Pi also inhibits Ca2+ release from the sarcoplasmic reticulum (SR). When both metabolites are elevated, they act synergistically to cause marked reductions in power, indicating that fatigue during high-intensity exercise has a bioenergetic basis.

The cross-bridge cycle and skeletal muscle fatigue.

The functional correlates of fatigue observed in both animals and humans during exercise include a decline in peak force (P0), maximal velocity, and peak power. Establishing the extent to which these deleterious functional changes result from direct effects on the myofilaments is facilitated through understanding the molecular mechanisms of the cross-bridge cycle. With actin-myosin binding, the cross-bridge transitions from a weakly bound low-force state to a strongly bound high-force state. Low pH reduces the number of high-force cross bridges in fast fibers, and the force per cross bridge in both fast and slow fibers. The former is thought to involve a direct inhibition of the forward rate constant for transition to the strong cross-bridge state. In contrast, inorganic phosphate (Pi) is thought to reduce P0 by accelerating the reversal of this step. Both H+ and Pi decrease myofibrillar Ca2+ sensitivity. This effect is particularly important as the amplitude of the Ca2+ transient falls with fatigue. The inhibitory effects of low pH and high Pi on P0 are reduced as temperature increases from 10 to 30 degrees C. However, the H+-induced depression of peak power in the slow fiber type, and Pi inhibition of myofibrillar Ca2+ sensitivity in slow and fast fibers, are greater at high compared with low temperature. Thus the depressive effects of H+ and Pi at in vivo temperatures cannot easily be predicted from data collected below 25 degrees C. In vitro, reactive oxygen species reduce myofibrillar Ca2+ sensitivity; however, the importance of this mechanism during in vivo exercise is unknown.

Ventricular action potential adaptation to regular exercise: role of ß-adrenergic and KATP channel function.

Regular exercise training is known to affect the action potential duration (APD) and improve heart function, but involvement of ß-adrenergic receptor (ß-AR) subtypes and/or the ATP-sensitive K+ (KATP) channel is unknown. To address this, female and male Sprague-Dawley rats were randomly assigned to voluntary wheel-running or control groups; they were anesthetized after 6-8 wk of training, and myocytes were isolated. Exercise training significantly increased APD of apex and base myocytes at 1 Hz and decreased APD at 10 Hz. Ca2+ transient durations reflected the changes in APD, while Ca2+ transient amplitudes were unaffected by wheel running. The nonselective ß-AR agonist isoproterenol shortened the myocyte APD, an effect reduced by wheel running. The isoproterenol-induced shortening of APD was largely reversed by the selective ß1-AR blocker atenolol, but not the ß2-AR blocker ICI 118,551, providing evidence that wheel running reduced the sensitivity of the ß1-AR. At 10 Hz, the KATP channel inhibitor glibenclamide prolonged the myocyte APD more in exercise-trained than control rats, implicating a role for this channel in the exercise-induced APD shortening at 10 Hz. A novel finding of this work was the dual importance of altered ß1-AR responsiveness and KATP channel function in the training-induced regulation of APD. Of physiological importance to the beating heart, the reduced response to adrenergic agonists would enhance cardiac contractility at resting rates, where sympathetic drive is low, by prolonging APD and Ca2+ influx; during exercise, an increase in KATP channel activity would shorten APD and, thus, protect the heart against Ca2+ overload or inadequate filling.NEW & NOTEWORTHY Our data demonstrated that regular exercise prolonged the action potential and Ca2+ transient durations in myocytes isolated from apex and base regions at 1-Hz and shortened both at 10-Hz stimulation. Novel findings were that wheel running shifted the ß-adrenergic receptor agonist dose-response curve rightward compared with controls by reducing ß1-adrenergic receptor responsiveness and that, at the high activation rate, myocytes from trained animals showed higher KATP channel function.

Muscle Fatigue from the Perspective of a Single Crossbridge.

The repeated intense stimulation of skeletal muscle rapidly decreases its force- and motion-generating capacity. This type of fatigue can be temporally correlated with the accumulation of metabolic by-products, including phosphate (Pi) and protons (H). Experiments on skinned single muscle fibers demonstrate that elevated concentrations of these ions can reduce maximal isometric force, unloaded shortening velocity, and peak power, providing strong evidence for a causative role in the fatigue process. This seems to be due, in part, to their direct effect on muscle's molecular motor, myosin, because in assays using isolated proteins, these ions directly inhibit myosin's ability to move actin. Indeed, recent work using a single molecule laser trap assay has revealed the specific steps in the crossbridge cycle affected by these ions. In addition to their direct effects, these ions also indirectly affect myosin by decreasing the sensitivity of the myofilaments to calcium, primarily by altering the ability of the muscle regulatory proteins, troponin and tropomyosin, to govern myosin binding to actin. This effect seems to be partially due to fatigue-dependent alterations in the structure and function of specific subunits of troponin. Parallel efforts to understand the molecular basis of muscle contraction are providing new technological approaches that will allow us to gain unprecedented molecular detail of the fatigue process. This will be crucial to fully understand this ubiquitous phenomenon and develop appropriately targeted therapies to attenuate the debilitating effects of fatigue in clinical populations.

Weekly versus monthly testosterone administration on fast and slow skeletal muscle fibers in older adult males.

CONTEXT: In older adults, loss of mobility due to sarcopenia is exacerbated in men with low serum T. T replacement therapy is known to increase muscle mass and strength, but the effect of weekly (WK) vs monthly (MO) administration on specific fiber types is unknown. OBJECTIVE: To determine the efficacy of WK vs MO T replacement on the size and functional capacity of individual fast and slow skeletal muscle fiber types. DESIGN, SETTING, AND PATIENTS: Subjects were randomized into a 5-month, double-blind, placebo-controlled trial. All subjects (ages, 61-71 y) were community-dwelling men who had T levels < 500 ng/dL. INTERVENTION: Subjects were dosed weekly for 5 months, receiving continuous T (WK, n = 5; 100 mg T enanthate, im injection), monthly cycled T (MO, n = 7; alternating months of T and placebo), or placebo (n = 7). Muscle biopsies of the vastus lateralis were obtained before and after treatment. MAIN OUTCOME MEASURES: Main outcomes for individual slow and fast fibers included fiber diameter, peak force (P0), rate of tension development, maximal shortening velocity, peak power, and Ca(2+) sensitivity. RESULTS: Both treatments increased fiber diameter and peak power, with WK treatment 5-fold more effective than MO in increasing type I fiber P0. WK effects on fiber diameter and force were 1.5-fold higher in slow fibers compared to fast fibers. In fast type II fibers, diameter and P0 increased similarly between treatments. The increased power was entirely due to increased fiber size and force. CONCLUSIONS: In conclusion, T replacement effects were fiber-type dependent, restricted to increases in cell size, P0, and peak power, and dependent on the paradigm selected (WK vs MO).