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Autophagy is a catabolic process that targets and degrades cytoplasmic materials. In skeletal muscle, autophagy is required for the control of mass under catabolic conditions, but is also basally active in the maintenance of myofiber homeostasis. In this study, we found that some specific autophagic markers (LC3-I, LC3-II, SQSTM1) were basally lower in glycolytic muscle compared to oxidative muscle of autophagy competent mice. In contrast, basal autophagic flux was higher in glycolytic muscle. In addition, we used several skeletal muscle-specific Atg7 transgenic mouse models to investigate the effect of acute (iAtg7-/-) and chronic (cAtg7-/-) autophagy deficiency on skeletal muscle morphology, contractility, and apoptotic signaling. While acute autophagy ablation (iAtg7-/-) resulted in increased centralized nuclei in glycolytic muscle, it did not alter contractile properties or measures of apoptosis and proteolysis. In contrast, with chronic autophagy deficiency (cAtg7-/-) there was an increased proportion of centralized nuclei, as well as reduced force and altered twitch kinetics in glycolytic muscle. Glycolytic muscle of cAtg7-/- mice also displayed an increased level of the pro-apoptotic protein BAX, as well as calpain and proteasomal enzymatic activity. Collectively, our data demonstrate cumulative damage from chronic skeletal muscle-specific autophagy deficiency with associated apoptotic and proteasomal upregulation. These findings point towards the importance of investigating different muscle/fiber types when studying skeletal muscle autophagy, and the critical role of autophagy in the maintenance of myofiber function, integrity, and cellular health.

Phospholamban (PLN) is an important Ca2+ modulator at the sarcoplasmic reticulum (SR) of striated muscles. It physically interacts and inhibits sarcoplasmic reticulum Ca2+ ATPase (SERCA2) function, whereas a protein kinase A (PKA)‐dependent phosphorylation at its serine 16 reverses the inhibition. The underlying mechanism of this post‐translational modification, however, remains not fully understood. Using publicly available databases, we identified A‐kinase anchoring protein 6 (AKAP6) as a candidate that might play some roles in PLN phosphorylation. Immunofluorescence showed colocalization between GFP‐AKAP6 and PLN in transfected HEK‐293T cells and cultured mouse neonatal cardiomyocytes (CMNCs). Co‐immunoprecipitation confirmed the functional interaction between AKAP6 and PLN in HEK‐293T and isolated adult rat cardiomyocytes in response to isoproterenol stimulation. Functionally, AKAP6 promoted Ca2+ uptake activity of SERCA1 in cotransfected HEK‐293T cells despite the presence of PLN. These results were further confirmed in adult rat cardiomyocytes. Immunofluorescence showed colocalization of both proteins around the perinuclear region, while protein–protein interaction was corroborated by immunoprecipitation of the nucleus‐enriched fraction of rat hearts. Our findings suggest AKAP6 as a novel interacting partner to PLN in HEK‐293T and murine cardiomyocytes.

This study compared both the extensor carpi radialis brevis (ECRB) and the trapezius (TRAP) muscles of women with work-related myalgia (WRM) with healthy controls (CON) to determine whether abnormalities existed in cellular energy status and the potentials of the various metabolic pathways and segments involved in energy production and substrate transport. For both the ECRB (CON, n = 6–9; WRM, n = 13) and the TRAP (CON, n = 6–7; WRM, n = 10), no differences (P > 0.05) were found for the concentrations (in millimoles per kilogram of dry mass) of ATP, PCr, lactate, and glycogen. Similarly, with one exception, the maximal activities (in moles per milligram of protein per hour) of mitochondrial enzymes representative of the citric acid cycle (CAC), the electron transport chain (ETC), and β-oxidation, as well as the cytosolic enzymes involved in high energy phosphate transfer, glycogenolysis, glycolysis, lactate oxidation, and glucose phosphorylation were not different (P > 0.05). The glucose transporters GLUT1 and GLUT4, and the monocarboxylate transporters MCT1 and MCT4, were also normal in WRM. It is concluded that, in general, abnormalities in the resting energy and substrate state, the potential of the different metabolic pathways and segments, as well as the glucose and monocarboxylate transporters do not appear to be involved in the cellular pathophysiology of WRM.

Aims Phospholamban (PLN) and sarcolipin (SLN) are small inhibitory proteins that regulate the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump. Previous work from our laboratory revealed that in the soleus and gluteus minimus muscles of mice overexpressing PLN (PlnOE), SERCA function was impaired, dynamin 2 (3–5 fold) and SLN (7–9 fold) were upregulated, and features of human centronuclear myopathy (CNM) were observed. Here, we performed structural and functional experiments to evaluate whether the diaphragm muscles of the PlnOE mouse would exhibit CNM pathology and muscle weakness. Methods Diaphragm muscles from PlnOE and WT mice were subjected to histological/histochemical/immunofluorescent staining, Ca2+-ATPase and Ca2+ uptake assays, Western blotting, and in vitro electrical stimulation. Results Our results demonstrate that PLN overexpression reduced SERCA's apparent affinity for Ca2+ but did not reduce maximal SERCA activity or rates of Ca2+ uptake. SLN was upregulated 2.5-fold, whereas no changes in dynamin 2 expression were found. With respect to CNM, we did not observe type I fiber predominance, central nuclei, or central aggregation of oxidative activity in diaphragm, although type I fiber hypotrophy was present. Furthermore, in vitro contractility assessment of PlnOE diaphragm strips revealed no reductions in force-generating capacity, maximal rates of relaxation or force development, but did indicate that ½ relaxation time was prolonged. Conclusions Therefore, the effects of PLN overexpression on skeletal muscle phenotype differ between diaphragm and the postural soleus and gluteus minimus muscles. Our findings here point to differences in SLN expression and type I fiber distribution as potential contributing factors.

X-linked muscular dystrophy of the mouse (mdx) has been reported to progressively remodel skeletal muscle to preferentially reduce fast fiber composition. Despite this, mdx muscle displays normal levels of posttetanic potentiation (PTP). Since PTP may primarily depend on phosphorylation of the myosin regulatory light chain (RLC) in fast muscle fibers, maintenance of PTP with mdx disease progression is paradoxical and may represent an adaptation of the diseased muscle. This study assesses the role of RLC phosphorylation during PTP of mdx muscle. Extensor digitorum longus muscles were isolated from mdx and from C57BL/10 (control) mice at ~50 (young) and ~300 (adult) days and stimulated in vitro (25°C) to induce PTP. During potentiation, muscles were harvested for subsequent determination of RLC phosphorylation levels. Immunofluorescence was used to assess muscle fiber type composition and no age effects were found. The magnitude of PTP was higher (P

In this study, we aimed to directly quantify the relative contribution of Ca2+cycling to resting metabolic rate in mouse fast-twitch (extensor digitorum longus, EDL) and slow-twitch (soleus) skeletal muscle. Resting oxygen consumption of isolated muscles (V̇o2, μl·g wet wt−1·s−1) measured polarographically at 30°C was ∼25% higher in soleus (0.61 ± .03) than in EDL (0.46 ± .03). To quantify the specific contribution of Ca2+cycling to resting metabolic rate, cyclopiazonic acid (CPA), a highly specific inhibitor of sarco(endo)plasmic reticulum Ca2+ATPases (SERCAs), was added to the bath at different concentrations (1, 5, 10, and 15 μM). There was a concentration-dependent effect of CPA on V̇o2, with increasing CPA concentrations up to 10 μM resulting in progressively greater reductions in muscle V̇o2. There were no differences between 10 and 15 μM CPA, indicating that 10 μM CPA induces maximal inhibition of SERCAs in isolated muscle preparations. Relative reduction in muscle V̇o2in response to CPA was nearly identical in EDL (1 μM, 10.6 ± 3.0%; 5 μM, 33.2 ± 3.4%; 10 μM, 49.2 ± 2.9%; 15 μM, 50.9 ± 2.1%) and soleus (1 μM, 11.2 ± 1.5%; 5 μM, 37.7 ± 2.4%; 10 μM, 50.0 ± 1.3%; 15 μM, 49.9 ± 1.6%). The results indicate that ATP consumption by SERCAs is responsible for ∼50% of resting metabolic rate in both mouse fast- and slow-twitch muscles at 30°C. Thus SERCA pumps in skeletal muscle could represent an important control point for energy balance regulation and a potential target for metabolic alterations to oppose obesity.

This study examined the influence of the ovarian sex hormones, estrogen and progesterone, on the fiber-type-specific response of the inducible 70-kDa heat shock protein (HSP70) to damaging exercise in rat soleus. Ovariectomized female rats were divided into three treatment groups ( n = 16 per group): sham (S), progesterone (P; 25 mg pellet), and estrogen (E; 0.25 mg pellet). Each treatment group was divided into control and exercised groups. After 8 days of sham or hormone treatment, animals ran downhill intermittently for 90 min (17 m/min, −13.5° grade) on a treadmill, and soleus muscles were removed 24 h postexercise. HSP70 expression was assessed in whole muscle homogenates by Western blotting and in individual muscle fiber types by immunohistochemical analysis of serial cross sections of soleus samples. Comparisons between control groups showed that HSP70 expression in soleus was increased ( P < 0.05) in E compared with both S and P. No difference ( P > 0.05) was observed between S and P. Following downhill running, HSP70 content in soleus was increased ( P < 0.05) compared with control in S and P, but not ( P > 0.05) in E. As a result, soleus HSP70 content following downhill running was not different ( P > 0.05) between any of the treatment groups. Under all conditions, HSP70 content was higher in type I vs. type II fibers, and the effects of both estrogen and exercise on HSP70 expression in soleus were also more pronounced in type I vs. type II fibers. These results demonstrate that 1) estrogen regulates HSP70 expression in skeletal muscle, increasing basal HSP70 expression and preventing further increases in HSP70 in response to exercise; 2) progesterone is not involved in the regulation of HSP70 expression in skeletal muscle; and 3) the effects of estrogen and exercise on HSP70 expression in skeletal muscle are fiber type specific.

Heat shock protein 70 (Hsp70) can physically interact with and prevent thermal inactivation of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) 1a, the SERCA isoform expressed in adult fast-twitch skeletal muscle. This study examined whether Hsp70 could physically interact with and prevent thermal inactivation of SERCA2a, the SERCA isoform expressed in heart. HEK-293 cells were cotransfected with cDNAs encoding human Hsp70 and rabbit SERCA2a (S2a/Hsp70). Cells cotransfected with SERCA2a cDNA and pMT2 (S2a/pMT2) were used as control. One-half of the cells was heat shocked at 40 degrees C for 1 h (HS), and one-half was maintained at 37 degrees C before harvesting the cells and isolating microsomes. Western blot analysis showed that Hsp70 and SERCA2a were colocalized in the microsomal fraction. The levels of Hsp70 were approximately fivefold higher (P0.05) in S2a/Hsp70 compared with S2a/pMT2 and approximately twofold higher (P0.05) following HS in all cells. Coimmunoprecipitation demonstrated that Hsp70 directly binds to SERCA2a. Following HS, maximal SERCA2a activity was reduced ( approximately 52%, P0.05) in S2a/pMT2 but was increased ( approximately 33%, P0.05) in S2a/Hsp70. Thermal inactivation of SERCA2a in S2a/pMT2 was associated with decreased ( approximately 49%, P0.05) binding capacity for fluorescein isothiocyanate (FITC) and increased carbonyl ( approximately 42%, P0.05) and nitrotyrosine ( approximately 40%, P0.05) levels in SERCA2a. By contrast, the HS-induced increase in maximal SERCA2a activity observed in S2a/Hsp70 corresponded with no change (P0.05) in FITC-binding capacity and reductions in carbonyl ( approximately 40%, P0.05) and nitrotyrosine ( approximately 23%, P0.05) levels in SERCA2a compared with S2a/pMT2. These results show that Hsp70 forms a protective interaction with SERCA2a during HS actually reducing oxidation and nitrosylation of SERCA2a thus increasing its maximal activity.

The early plasticity of vastus lateralis Na+-K+-ATPase to the abrupt onset of prolonged submaximal cycling was studied in 12 untrained participants (V̇o2 peak 44.8 ± 2.0 ml·kg−1·min−1, mean ± SE) using a 6-day protocol (3 days of exercise plus 3 days of recovery). Tissue samples were extracted prior to (Pre) and following exercise (Post) on day 1 (E1) and day 3 (E3) and on each day of recovery (R1, R2, R3) and analyzed for changes in maximal protein (βmax) (vanadate-facilitated [3H]ouabain binding), α- and β-isoform concentration (quantitative immunoblotting) and maximal Na+-K+-ATPase activity ( Vmax) (3- O-methylfluorescein K+-stimulated phosphatase assay). For βmax (pmol/g wet wt), an increase ( P < 0.05) of 11.8% was observed at R1 compared with E1-Pre (340 ± 14 vs 304 ± 17). For the α-isoforms α1, α2, and α3, increases ( P < 0.05) of 46, 42, and 31% were observed at R1, respectively. For the β-isoform, β1 and β2 increased ( P < 0.05) by 19 and 28% at R1, whereas β3 increased ( P < 0.05) by 18% at R2. With the exception of α2 and α3, the increases in the isoforms persisted at R3. Exercise resulted in an average decrease ( P < 0.05) in Vmax by 14.3%. No differences were observed in Vmax at E1 - Pre and E3 - Pre or between R1, R2, and R3. It is concluded that 3 days of prolonged exercise is a powerful stimulus for the rapid upregulation of the Na+-K+-ATPase subunit isoforms. Contrary to our hypothesis, the increase in subunit expression is not accompanied by increases in the maximal catalytic activity.

The alterations in muscle metabolism were investigated in response to repeated sessions of heavy intermittent exercise performed over 16 h. Tissue samples were extracted from the vastus lateralis muscle before (B) and after (A) 6 min of cycling at approximately 91% peak aerobic power at repetitions one (R1), two (R2), nine (R9), and sixteen (R16) in 13 untrained volunteers (peak aerobic power = 44.3 ± 0.66 mL·kg–1·min–1, mean ± SE). Metabolite content (mmol·(kg dry mass)–1) in homogenates at R1 indicated decreases (p < 0.05) in ATP (21.9 ± 0.62 vs. 17.7 ± 0.68) and phosphocreatine (80.3 ± 2.0 vs. 8.56 ± 1.5) and increases (p < 0.05) in inosine monophosphate (IMP, 0.077 ± 0.12 vs. 3.63 ± 0.85) and lactate (3.80 ± 0.57 vs. 84.6 ± 10.3). The content (µmol·(kg dry mass)–1) of calculated free ADP ([ADPf], 86.4 ± 5.5 vs. 1014 ± 237) and free AMP ([AMPf], 0.32 ± 0.03 vs. 78.4 ± 31) also increased (p < 0.05). No differences were observed between R1 and R2. By R9 and continuing to R16, pronounced reductions (p < 0.05) at A were observed in IMP (72.2%), [ADPf] (58.7%), [AMPf] (85.5%), and lactate (41.3%). The 16-hour protocol resulted in an 89.7% depletion (p < 0.05) of muscle glycogen. Repetition-dependent increases were also observed in oxygen consumption during exercise. It is concluded that repetitive heavy exercise results in less of a disturbance in phosphorylation potential, possibly as a result of increased mitochondrial respiration during the rest-to-work non-steady-state transition.

We investigated the hypothesis that muscles of different oxidative potential would display differences in sarcoplasmic reticulum (SR) Ca2+handling responses to repetitive contractile activity and recovery. Repetitive activity was induced in two muscles of high oxidative potential, namely, soleus (SOL) and red gastrocnemius (RG), and in white gastrocnemius (WG), a muscle of low oxidative potential, by stimulation in adult male rats. Measurements of SR properties, performed in crude homogenates, were made on control and stimulated muscles at the start of recovery (R0) and at 25 min of recovery (R25). Maximal Ca2+-ATPase activity ( Vmax, μmol·g protein−1·min−1) at R0 was lower in stimulated SOL (105 ± 9 vs. 135 ± 7) and RG (269 ± 22 vs. 317 ± 26) and higher ( P < 0.05) in WG (795 ± 32 vs. 708 ± 34). At R25, Vmaxremained lower ( P < 0.05) in SOL and RG but recovered in WG. Ca2+uptake, measured at 2,000 nM, was depressed ( P < 0.05) in SOL and RG by 34 and 13%, respectively, in stimulated muscles at R0 and remained depressed ( P < 0.05) at R25. In contrast, Ca2+uptake was elevated ( P < 0.05) in stimulated WG at R0 by 9% and remained elevated ( P < 0.05) at R25. Ca2+release, unaltered in SOL and RG at both R0 and R25, was increased ( P < 0.05) in stimulated WG at both R0 and R25. We conclude that SR Ca2+-handling responses to repetitive contractile activity and recovery are related to the oxidative potential of muscle.

To examine the effects of exercise and exercise plus active and passive recovery on sarcoplasmic reticulum (SR) Ca2+-handling properties.Crude muscle homogenates were prepared from adult rat gastrocnemius muscle from two experiments. In one experiment, the muscle was extracted immediately after prolonged treadmill running (RUN), after a 45 min period of reduced exercise intensity (RUN+) following RUN and compared with controls (CON). In the second experiment, muscle was extracted during passive recovery following the same run protocol at 10 min (REC10), 25 min (REC25) and 45 min (REC45) and compared with CON.Sarcoplasmic reticulum Ca2+-uptake was 31% higher (P0.05) in RUN+ compared with CON and RUN. Higher values (P0.05) were also found in REC25 (48%) and REC45 (50%) compared with CON. Maximal Ca2+-ATPase was increased by 23% (P0.05) in RUN+ compared with CON and RUN and by 65-68% (P0.05) in REC25 and REC45 compared with CON. A higher (P0.05) Hill coefficient for Ca2+-ATPase activity was observed in RUN+ (2.3 +/- 0.2) compared with CON (1.7 +/- 0.2) or RUN (1.6 +/- 0.2), but not for any REC conditions. In addition, the coupling ratio (Ca2+-uptake/Ca2+-ATPase activity) was higher (P0.05) in RUN+ (2.2 +/- 0.10) compared with CON (1.9 +/- 0.05) and RUN (1.9 +/- 0.08).It is concluded that in crude homogenates, SR Ca2+-uptake and Ca2+-ATPase activity are elevated in recovery following prolonged running and that the elevation in these properties is more pronounced during passive compared with active recovery.

To investigate the effects of intermittent heavy exercise (HE) on sarcoplasmic reticulum (SR) maximal Ca2+-ATPase activity ( Vmax) and Ca2+uptake, a continuous two-stage standardized cycling test was performed before and after HE by untrained men [peak aerobic power (V̇o2 peak) = 42.9 ± 2.7 ml · kg-1· min-1]. The HE consisted of 16 bouts of cycling performed for 6 min each hour at 90% V̇o2 peak. Tissue was obtained from the vastus lateralis by needle biopsy before and during each cycle test. Before HE, reductions ( P < 0.05; μmol · g protein-1· min-1) of 16 and 31% were observed in Vmaxand Ca2+uptake, respectively, after 40 min of the standardized test. Resting Vmaxand Ca2+uptake were depressed ( P < 0.05) by 19 and 30%, respectively, when measured 36–48 h after HE. During the standardized test, after HE, Vmaxincreased ( P < 0.05) by 20%, whereas no change was observed in Ca2+uptake. The HE protocol resulted in small increases ( P < 0.05) and decreases ( P < 0.05) in sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 2a and SERCA1 expression, respectively, as determined by Western blotting techniques. These results indicate that SR Ca2+-sequestering function in response to a prolonged exercise test depends on prior activity status, such that rested muscles exhibit a decrease and prior exercised muscles, an increase in Ca2+-ATPase activity. Moreover, it appears that changes in SERCA content can occur in response to a sustained session of intermittent exercise.

Silver nitrate (AgNO3) is a sulfhydryl oxidizing agent that induces a biphasic Ca2+release from isolated sarcoplasmic reticulum (SR) vesicles by presumably oxidizing critical sulfhydryl groups in the Ca2+release channel (CRC), causing the channel to open. To further examine the effects of AgNO3on the CRC and the Ca2+-ATPase, Ca2+release was measured in muscle homogenates prepared from rat hindlimb muscle using indo 1. Cyclopiazonic acid (CPA) and ruthenium red (RR) were used to inhibit the Ca2+-ATPase and block the CRC, respectively, before inducing Ca2+release with both AgNO3and 4-chloro- m-cresol (4-CMC), a releasing agent specific for the CRC. With AgNO3and CPA, the early rapid rate of release (phase 1) was increased ( P < 0.05) by 42% (314 ± 5 vs. 446 ± 39 μmol · g protein−1· min−1), whereas the slower, more prolonged rate of release (phase 2) was decreased ( P < 0.05) by 72% (267 ± 39 vs. 74 ± 7.7 μmol · g protein−1· min−1). RR, in combination with AgNO3, had no effect on phase 1 ( P > 0.05) (314 ± 51 vs. 334 ± 43 μmol · g protein−1· min−1) and decreased phase 2 ( P < 0.05) by 65% (245 ± 34 vs. 105 ± 8.2 μmol · g protein−1· min−1). With 4-CMC, CPA had no effect ( P > 0.05) on either phase 1 or 2. With addition of RR, phase 1 was reduced ( P < 0.05) by 59% (2,468 ± 279 vs. 1,004 ± 87 μmol · g protein−1· min−1), and RR completely blocked phase 2. Both AgNO3and 4-CMC fully inhibited Ca2+-ATPase activity measured in homogenates. These findings indicate that AgNO3, but not 4-CMC, induces Ca2+release by acting on both the CRC and the Ca2+-ATPase.

We investigated the potential role of selected excitation–contraction coupling processes in females with work-related myalgia (WRM) by comparing WRM with healthy controls (CON) using tissue from extensor carpi radialis brevis (ECRB) and trapezius (TRAP) muscles. For the ECRB, age (mean ± SE) was 29.6 ± 3.5 years for CON (n = 9) and 39.2 ± 2.8 years for WRM (n = 13), while for the TRAP, the values were 26.0 ± 2.1 years for CON (n = 7) and 44.6 ± 2.9 years for WRM (n = 11). For the sarcoplasmic reticulum (SR) of the ECRB, WRM displayed concentrations (nmol·(mg protein)−1·min−1) that were lower (P < 0.05) for Total (202 ± 4.4 vs 178 ± 7.1), Basal (34 ± 1.6 vs 30.1 ± 1.3), and maximal Ca2+-ATPase activity (Vmax, 168 ± 4.9 vs 149 ± 6.3), and Ca2+-uptake (5.06 ± 0.31 vs 4.13 ± 0.29), but not SERCA1a and SERCA2a isoforms, by comparison with CON. When age was incorporated as a co-variant, Total, Basal, and Ca2+-uptake remained different from CON (P < 0.05), but not Vmax(P = 0.13). For TRAP, none of the ATPase properties differed between groups (P > 0.05) either before or following adjustment for age. No differences (P > 0.05) were observed between the groups for Ca2+-release in the SR for either TRAP or ECRB. Similarly, no deficiencies, regardless of muscle, were noted for either the Na+–K+-ATPase content or the α and β subunit isoform distribution in WRM. This preliminary study provides a basis for further research, with expanded numbers, investigating the hypothesis that abnormalities in SR Ca2+-regulation are involved in the cellular etiology of WRM.

To investigate the hypothesis that prolonged partial ischemia would result in a depression in homogenate sarcoplasmic reticulum (SR) Ca2+-sequestering and mechanical properties in muscle, a cuff was placed around the hindlimb of 8 adult Sprague–Dawley rats (267 ± 5.8 g; × ± S.E.) and partially inflated (315 mm Hg) for 2 h. Following occlusion, the EDL was sampled both from the ischemic (I) and contralateral control (C) leg and SR properties compared with the EDL muscles extracted from rats (n = 8) immediately following anaesthetization (CC). Ischemia was indicated by a lower (p < 0.05) concentration (mmol.kg dry wt–1) of ATP (19.0 ± 0.7 vs. 16.7 ± 0.7) and phosphocreatine (58.1 ± 5.7 vs. 35.0 ± 4.6) in I compared to C. Although Ca2+-ATPase activity (μmol·g protein–1.sec–1 ), both maximal and submaximal, was not different between C and I (19.7 ± 0.4 vs. 18.5 ± 1.3), reductions (p < 0.05) in Ca2+-uptake (mmol·g protein–1.sec–1 ) of between 18.2 and 24.7% across a range of submaximal free Ca2+-levels were observed in I compared to C. Lower submaximal Ca2+-ATPase activity and Ca2+-uptake were also observed in the EDL in C compared to CC animals. Time dependent reductions (p < 0.05) were found in peak twitch and maximal tetanic tension in EDL from I but not C. It is concluded that partial ischemia, resulting in modest reductions in energy state in EDL, induces a reduction in Ca2+-uptake independent of changes in Ca2+-ATPase activity. These changes reduce the coupling ratio and the efficiency of Ca2+-transport by SR.

We investigated the effects of a 21-day expedition to the summit of Mount Denali, Alaska (6,194 m) on selected Ca2+ sequestration properties of sarcoplasmic reticulum (SR) calcium pump in vastus lateralis muscle. Muscle samples were obtained by biopsy from 5 male climbers (peak oxygen consumption, VO2peak = 52.3 +/- 2.1 mL.kg(-1).min(-1)) approximately 7 days prior to (PRE) and 4 days following (POST) the expedition. A comparison of PRE versus POST measures of maximal Ca2+-ATPase activities (117 +/- 8.5 vs. 97.6 +/- 5.6 nmol.mg protein(-1).min(-1)) and Ca2+-uptake (204 +/- 15 vs. 161 +/- 11 nmol.mg protein(-1).min(-1)) measured in crude homogenates obtained from pre-exercised muscle, indicated only an effect (p0.05) of the expedition on Ca2+-uptake. The reduction in Ca2+-ATPase activity, representing 16.6%, was not significant (p = 0.089). The sarco endoplasmic reticulum calcium (SERCA)-ATPase isoforms, measured using Western blotting techniques, revealed a small reduction (p0.05) in SERCA 1 (-4.6 +/- 1.9%), but not in SERCA 2a (+2.0 +/- 1.4%). Prior to the expedition, both Ca2+-ATPase activity and Ca2+-uptake were reduced (p0.05) by approximately 34 and 18%, respectively, following 40 min of a two-step continuous cycling task (20 min at 59% VO2peak and 20 min at 74% VO2peak). The exercise-induced reduction in Ca2+-ATPase activity was independent of fiber type. Only in the case of Ca2+-uptake was a lower exercise response (p0.05) observed following the expedition, an effect that was due to the lower resting value. It is concluded that acclimatization as experienced during a mountaineering expedition induces changes in the properties of the SR Ca2+-pump, and particularly to Ca2+-sequestering function.

To investigate the hypothesis that acclimatization to altitude would result in a downregulation in muscle Na+-K+-ATPase pump concentration, tissue samples were obtained from the vastus lateralis muscle of six volunteers (5 males and 1 female), ranging in age from 24 to 35 yr, both before and within 3 days after a 21-day expedition to the summit of Mount Denali, Alaska (6,194 m). Na+-K+-ATPase, measured by the [3H]ouabain-binding technique, decreased by 13.8% [348 ± 12 vs. 300 ± 7.6 (SE) pmol/g wet wt; P< 0.05]. No changes were found in the maximal activities (mol ⋅ kg protein− 1 ⋅ h− 1) of the mitochondrial enzymes, succinic dehydrogenase (3.63 ± 0.20 vs. 3.25 ± 0.23), citrate synthase (4.76 ± 0.44 vs. 4.94 ± 0.44), and malate dehydrogenase (12.6 ± 1.8 vs. 12.7 ± 1.2). Similarly, the expedition had no effect on any of the histochemical properties examined, namely fiber-type distribution (types I, IIA, IIB, IC, IIC, IIAB), area, capillarization, and succinic dehydrogenase activity. Peak aerobic power (52.3 ± 2.1 vs. 50.6 ± 1.9 ml ⋅ kg− 1 ⋅ min− 1) and body mass (76.9 ± 3.7 vs. 75.5 ± 2.9 kg) were also unaffected. We concluded that acclimatization to altitude results in a downregulation in muscle Na+-K+-ATPase pump concentration, which occurs without changes in oxidative potential and other fiber-type histochemical properties.

This study examined the alterations in sarcoplasmic reticulum (SR) Ca2+ sequestration function in homogenates during eccentric exercise and recovery and following additional eccentric exercise, and correlated these alterations with changes in force output. Eight healthy, untrained females, aged 20-25 years, cycled for a total of 60 min on an eccentric cycle ergometer (30 min at 66 ± 3% O2 peak and 30 min at 76 ± 3% O2 peak, determined during concentric exercise). Biopsies (extracted from the vastus lateralis) were taken before and after the exercise as well as on days 2, 6 and prior to and following identical exercise on day 14. Ca2+-uptake (nmol/min/mg protein) was unaffected (p > 0.05) following the first session of eccentric exercise; however, by day 2 a depression in uptake (p < 0.05) was observed which persisted throughout the remainder of the experiment. Maximal Ca2+-ATPase activity (nmol/min/mg protein) was elevated (p < 0.05) immediately following the first exercise session, remained elevated through day 2 and returned to pre-exercise levels by day 6 of recovery and increased again by day 14. No changes in either Ca2+-ATPase activity or Ca2+-uptake were observed with exercise on day 14. Both eccentric sessions, performed on days 0 and 14, resulted in similar depressions in force (p < 0.05) immediately following exercise. By day 2 force had recovered to pre-exercise levels. The results demonstrate that a prolonged alteration in SR Ca2+-uptake occurs following eccentric work that is unaccompanied by parallel changes in either SR Ca2+-ATPase activity or mechanical performance.

To examine the effects of repetitive bouts of heavy exercise on the maximal activities of enzymes representative of the major metabolic pathways and segments, 13 untrained volunteers [peak aerobic power (V̇o2 peak) = 44.3 ± 2.3 ml·kg−1·min−1] cycled at ∼91% V̇o2 peak for 6 min once per hour for 16 h. Maximal enzyme activities ( Vmax, mol·kg−1·protein·h−1) were measured in homogenates from tissue extracted from the vastus lateralis before and after exercise at repetitions 1 (R1), 2 (R2), 9 (R9), and 16 (R16). For the mitochondrial enzymes, exercise resulted in reductions ( P < 0.05) in cytochrome- c oxidase (COX, 14.6%), near significant reductions in malate dehydrogenase (4.06%; P = 0.06) and succinic dehydrogenase (4.82%; P = 0.09), near significant increases in β-hydroxyacyl-CoA dehydrogenase (4.94%; P = 0.08), and no change in citrate synthase (CS, 2.88%; P = 0.37). For the cytosolic enzymes, exercise reduced ( P < 0.05) Vmax in hexokinase (Hex, 4.4%), creatine phosphokinase (9.0%), total phosphorylase (13.5%), phosphofructokinase (16.6%), pyruvate kinase (PK, 14.1%) and lactate dehydrogenase (10.7%). Repetition-dependent reductions ( P < 0.05) in Vmax were observed for CS (R1, R2 > R16), COX (R1, R2 > R16), Hex (1R, 2R > R16), and PK (R9 > R16). It is concluded that heavy exercise results in transient reductions in a wide range of enzymes involved in different metabolic functions and that in the case of selected enzymes, multiple repetitions of the exercise reduce average Vmax.

In this study, we have investigated the hypothesis that an exercise protocol designed to repeatedly induce a large dependence on carbohydrate and large increases in glycolytic flux rate would result in rapid increases in the principal glucose and lactate transporters in working muscle, glucose transporter (GLUT)-4 and monocarboxylate transporter (MCT)4, respectively, and in activity of hexokinase (Hex), the enzyme used to phosphorylate glucose. Transporter abundance and Hex activity were assessed in homogenates by Western blotting and quantitative chemiluminescence and fluorometric techniques, respectively, in samples of tissue obtained from the vastus lateralis in 12 untrained volunteers [peak aerobic power (V̇o2peak) = 44.3 ± 2.3 ml·kg−1·min−1] before cycle exercise at repetitions 1 (R1), 2 (R2), 9 (R9), and 16 (R16). The 16 repetitions of the exercise were performed for 6 min at ∼90% V̇o2peak, once per hour. Compared with R1, GLUT-4 increased ( P < 0.05) by 28% at R2 and remained elevated ( P < 0.05) at R9 and R16. For MCT-4, increases ( P < 0.05) of 24% were first observed at R9 and persisted at R16. No changes were observed in GLUT-1 and MCT-1 or in Hex activity. The ∼17- to 24-fold increase ( P < 0.05) in muscle lactate observed at R1 and R2 was reduced ( P < 0.05) to an 11-fold increase at R9 and R16. It is concluded that an exercise protocol designed to strain muscle carbohydrate reserves and to result in large increases in lactic acid results in a rapid upregulation of both GLUT-4 and MCT-4.

To investigate the time course of fiber type-specific heat shock protein 70 (Hsp70) expression in human skeletal muscle after acute exercise, 10 untrained male volunteers performed single-legged isometric knee extensor exercise at 60% of their maximal voluntary contraction (MVC) with a 50% duty cycle (5-s contraction and 5-s relaxation) for 30 min. Muscle biopsies were collected from the vastus lateralis before (Pre) exercise in the rested control leg (C) and immediately after exercise (Post) in the exercised leg (E) only and on recovery days 1 (R1), 2 (R2), 3 (R3), and 6 (R6) from both legs. As demonstrated by Western blot analysis, whole muscle Hsp70 content was unchanged ( P > 0.05) immediately after exercise (Pre vs. Post), was increased ( P < 0.05) by ∼43% at R1, and remained elevated throughout the entire recovery period in E only. Hsp70 expression was also assessed in individual muscle fiber types I, IIA, and IIAX/IIX by immunohistochemistry. There were no fiber type differences ( P > 0.05) in basal Hsp70 expression. Immediately after exercise, Hsp70 expression was increased ( P < 0.05) in type I fibers by ∼87% but was unchanged ( P > 0.05) in type II fibers (Pre vs. Post). At R1 and throughout recovery, Hsp70 content in E was increased above basal levels ( P < 0.05) in all fiber types, but Hsp70 expression was always highest ( P < 0.05) in type I fibers. Hsp70 content in C was not different from Pre at any time throughout recovery. Glycogen depletion was observed at Post in all type II, but not type I, fibers, suggesting that the fiber type differences in exercise-induced Hsp70 expression were not related to glycogen availability. These results demonstrate that the time course of exercise-induced Hsp70 expression in human skeletal muscle is fiber type specific.

The purpose of this study was to examine the effects of glutathione (GSH) depletion and cellular oxidation on rat diaphragm contractility and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) function in vitro under basal conditions and following fatiguing stimulation. Buthionine sulfoximine (BSO) treatment ( n = 10) for 10 days (20 mM in drinking water) reduced ( P < 0.05) diaphragm GSH content (nmol/mg protein) and the ratio of GSH to glutathione disulfide (GSH/GSSG) by 91% and 71%, respectively, compared with controls (CTL) ( n = 10). Western blotting showed that Hsp70 expression in diaphragm was not increased ( P > 0.05) with BSO treatment. As hypothesized, basal peak twitch force (g/mm2) was increased ( P < 0.05), and fatigability in response to repetitive stimulation (350-ms trains at 100 Hz once every 1 s for 5 min) was also increased ( P < 0.05) in BSO compared with CTL. Both Ca2+uptake and maximal SERCA activity (μmol·g protein−1·min−1) measured in diaphragm homogenates that were prepared at rest were increased ( P < 0.05) with BSO treatment, an effect that could be partly explained by a twofold increase ( P < 0.05) in SERCA2a expression with BSO. In response to the 5-min stimulation protocol, both Ca2+uptake and maximal SERCA activity were increased ( P < 0.05) in CTL but not ( P > 0.05) in BSO diaphragm. We conclude that 1) cellular redox state is more optimal for contractile function and fatigability is increased in rat diaphragm following BSO treatment, 2) SERCA2a expression is modulated by redox signaling, and 3) regulation of SERCA function in working diaphragm is altered following BSO treatment.

The study investigated the hypothesis that three consecutive days of prolonged cycle exercise would result in a sustained reduction in the Ca2+-cycling properties of the vastus lateralis in the absence of changes in the sarcoplasmic (endoplasmic) reticulum Ca2+-ATPase (SERCA) protein. Tissue samples were obtained at preexercise (Pre) and postexercise (Post) on day 1 (E1) and day 3 (E3) and during recovery day 1 (R1), day 2 (R2), and day 3 (R3) in 12 active but untrained volunteers (age 19.2 ± 0.27 yr; mean ± SE) and analyzed for changes (nmol·mg protein−1·min−1) in maximal Ca2+-ATPase activity ( Vmax), Ca2+uptake and Ca2+release (phase 1 and phase 2), and SERCA isoform expression (SERCA1a and SERCA2a). At E1, reductions ( P < 0.05) from Pre to Post in Vmax(150 ± 7 vs. 121 ± 7), Ca2+uptake (7.79 ± 0.28 vs. 5.71 ± 0.33), and both phases of Ca2+release (phase 1, 20.3 ± 1.3 vs. 15.2 ± 1.1; phase 2, 7.70 ± 0.60 vs. 4.99 ± 0.48) were found. In contrast to Vmax, which recovered at Pre E3 and then remained stable at Post E3 and throughout recovery, Ca2+uptake remained depressed ( P < 0.05) at E3 Pre and Post and at R1 as did phase 2 of Ca2+release. Exercise resulted in an increase ( P < 0.05) in SERCA1a (14% at R2) but not SERCA2a. It is concluded that rapidly adapting mechanisms protect Vmaxfollowing the onset of regular exercise but not Ca2+uptake and Ca2+release.

This study investigated the effects of a 16-h protocol of heavy intermittent exercise on the intrinsic activity and protein and isoform content of skeletal muscle Na+-K+-ATPase. The protocol consisted of 6 min of exercise performed once per hour at ∼91% peak aerobic power (V̇o2 peak) with tissue sampling from vastus lateralis before (B) and immediately after repetitions 1 (R1), 2 (R2), 9 (R9), and 16 (R16). Eleven untrained volunteers with a V̇o2 peakof 44.3 ± 2.3 ml·kg−1·min−1participated in the study. Maximal Na+-K+-ATPase activity ( Vmax, in nmol·mg protein−1·h−1) as measured by the 3- O-methylfluorescein K+-stimulated phosphatase assay was reduced ( P < 0.05) by ∼15% with exercise regardless of the number of repetitions performed. In addition, Vmaxat R9 and R16 was lower ( P < 0.05) than at R1 and R2. Vanadate-facilitated [3H]ouabain determination of Na+-K+-ATPase content (maximum binding capacity, pmol/g wet wt), although unaltered by exercise, increased ( P < 0.05) 8.3% by R9 with no further increase observed at R16. Assessment of relative changes in isoform abundance measured at B as determined by quantitative immunoblotting showed a 26% increase ( P < 0.05) in the α2-isoform by R2 and a 29% increase in α3by R9. At R16, β3was lower ( P < 0.05) than at R2 and R9. No changes were observed in α1, β1, or β2. It is concluded that repeated sessions of heavy exercise, although resulting in increases in the α2- and α3-isoforms and decreases in β3-isoform, also result in depression in maximal catalytic activity.

The repetition-dependent effects of a repetitive heavy exercise protocol previously shown to alter muscle mechanic behavior (Green HJ, Duhamel TA, Ferth S, Holloway GP, Thomas MM, Tupling AR, Rich SM, and Yau JE. J Appl Physiol 97: 2166–2175, 2004) on muscle sarcoplasmic reticulum (SR) Ca2+-transport properties, measured in vitro, were examined in 12 untrained volunteers [peak aerobic power (V̇o2 peak) = 44.3 ± 0.66 ml·kg−1·min−1]. The protocol involved 6 min of cycle exercise performed at ∼91% V̇o2 peakonce per hour for 16 h. Tissue samples were obtained from the vastus lateralis before (B) and after (A) exercise at repetitions 1 (R1), 2 (R2), 9 (R9), and 16 (R16). Reductions ( P < 0.05) in maximal Ca2+-ATPase activity ( Vmax) of 26 and 12% with exercise were only observed at R1 and R16, respectively. Vmaxremained depressed ( P < 0.05) at R2 (B) but not at R9 (B) and R16 (B). No changes were observed in two other kinetic properties of the enzyme, namely the Hill coefficient (defined as the slope of the relationship between Ca2+-ATPase activity and free Ca2+concentration) and the Ca50(defined as the free Ca2+concentration needed to elicit 50% Vmax). Changes in Ca2+uptake (measured at 2,000 nM) with exercise and recovery generally paralleled Vmax. The apparent coupling ratio, defined as the ratio between Ca2+uptake and Vmax, was unaffected by the intermittent protocol. Reductions ( P < 0.05) in phase 1 Ca2+release (32%) were only observed at R1. No differences were observed between B and A for R2, R9, and R16 or between B and B for R1, R2, R9, and R16. The changes in phase 2 Ca2+release were as observed for phase 1 Ca2+release. It is concluded that the SR Ca2+-handling properties, in general, display rapid adaptations to repetitive exercise.

This study examined the effects of extended sessions of heavy intermittent exercise on quadriceps muscle fatigue and weakness. Twelve untrained volunteers (10 men and 2 women), with a peak oxygen consumption of 44.3 ± 2.3 ml·kg−1·min−1, exercised at ∼91% peak oxygen consumption for 6 min once per hour for 16 h. Muscle isometric properties assessed before and after selected repetitions (R1, R2, R4, R7, R12, and R15) were used to quantitate fatigue (before vs. after repetitions) and weakness (before vs. before repetitions). Muscle fatigue at R1 was indicated by reductions ( P < 0.05) in peak twitch force (135 ± 13 vs. 106 ± 11 N) and by a reduction ( P < 0.05) in the force-frequency response, which ranged between ∼53% at 10 Hz (113 ± 12 vs. 52.6 ± 7.4 N) and ∼17% at 50 Hz (324 ± 27 vs. 270 ± 30 N). No recovery of force, regardless of stimulation frequency, was observed during the 54 min between R1 and R2. At R2 and for all subsequent repetitions, no reduction in force, regardless of stimulation frequency, was generally found after the exercise. The only exception was for R2, where, at 20 Hz, force was reduced ( P < 0.05) by 18%. At R15, force before repetitions for high frequencies (i.e., 100 Hz) returned to R1 (333 ± 29 vs. 324 ± 27 N), whereas force at low frequency (i.e., 10 Hz) was only partially ( P < 0.05) recovered (113 ± 12 vs. 70 ± 6.6 N). It is concluded that multiple sessions of heavy exercise can reverse the fatigue noted early and reduce or eliminate weakness depending on the frequency of stimulation.

The purpose of this study was to investigate the hypothesis that Na+-K+-ATPase activity is reduced in muscle of different fiber composition after a single session of aerobic exercise in rats. In one experiment, untrained female Sprague-Dawley rats (weight 275 ± 21 g; means ± SE; n = 30) were run (Run) on a treadmill at 21 m/min and 8% grade until fatigue, or to a maximum of 2 h, which served as control (Con), or performed an additional 45 min of low-intensity exercise at 10 m/min (Run+). In a second experiment, utilizing rats of similar characteristics (weight 258 ± 18 g; n = 32), Run was followed by passive recovery (Rec). Directly after exercise, rats were anesthetized, and tissue was extracted from Soleus (Sol), red vastus lateralis (RV), white vastus lateralis (WV), and extensor digitorum longus (EDL) and frozen for later analysis. 3- O-methylfluorescein phosphatase activity (3- O-MFPase) was determined as an indicator of Na+-K+-ATPase activity, and glycogen depletion identified recruitment of each muscle during exercise. 3- O-MFPase was decreased ( P < 0.05) at Run+ by an average of 12% from Con in all muscles ( P < 0.05). No difference was found between Con and Run. Glycogen was lower ( P < 0.05) by 65, 57, 44, and 33% (Sol, EDL, RV, and WV, respectively) at Run, and there was no further depletion during the continued low-intensity exercise period. No differences in Na+-K+-ATPase activity was observed between Con and Rec. The results of this study indicate that inactivation of Na+-K+-ATPase can be induced by aerobic exercise in a volume-dependent manner and that the inactivation that occurs is not specific to muscles of different fiber-type composition. Inactivation of Na+-K+-ATPase suggests intrinsic structural modifications by mechanisms that are unclear.

To examine the thermal instability and the role of sulfhydryl (SH) oxidation on sarcoplasmic reticulum (SR) Ca2+-ATPase function, crude homogenates were prepared from the white portion of the gastrocnemius (WG) adult rat muscles ( n = 9) and incubated in vitro for ≤60 min either at a normal resting body temperature (37°C) or at a temperature indicative of exercise-induced hyperthermia (41°C) with DTT and without DTT (CON). In general, treatment with DTT resulted in higher Ca2+-ATPase and Ca2+uptake values (nmol · mg protein−1· min−1, P < 0.05), an effect that was not specific to time of incubation. Incubations at 41°C resulted in lower ( P< 0.05) Ca2+uptake rates (156 ± 18 and 35.9 ± 3.3) compared with 37°C (570 ± 54 and 364 ± 26) at 30 and 60 min, respectively. At 37°C, ryanodine (300 μM), which was used to block Ca2+release from the calcium release channel, prevented the time-dependent decrease in Ca2+uptake. A general inactivation ( P < 0.05) of maximal Ca2+-ATPase activity ( Vmax) in CON was observed with incubation time (0 > 30 > 60 min), with the effect being more pronounced ( P < 0.05) at 41°C compared with 37°C. The Hill slope, a measure of co-operativity, and the pCa50, the cytosolic Ca2+concentration required for half-maximal activation of Ca2+-ATPase activity, decreased ( P < 0.05) at 41°C only. Treatment with DTT attenuated the alterations in enzyme kinetics. The increase in Vmaxwith the Ca2+ionophore A-23187 was less pronounced at 41°C compared with 37°C. It is concluded that exposure of homogenates to a temperature typically experienced in exercise results in a reduction in the coupling ratio, which is mediated primarily by lower Ca2+uptake and occurs as a result of increases in membrane permeability to Ca2+. Moreover, the decreases in Ca2+-ATPase kinetics in WG with sustained heat stress result from SH oxidation.

The purpose of this study was to investigate the hypothesis that reductions in Na+-K+- ATPase activity are associated with neuromuscular fatigue following isometric exercise. In control (Con) and exercised (Ex) legs, force and electromyogram were measured in 14 volunteers [age, 23.4 ± 0.7 (SE) yr] before and immediately after (PST0), 1 h after (PST1), and 4 h after (PST4) isometric, single-leg extension exercise at ∼60% of maximal voluntary contraction for 30 min using a 0.5 duty cycle (5-s contraction, 5-s rest). Tissue was obtained from vastus lateralis muscle before exercise in Con and after exercise in both the Con (PST0) and Ex legs (PST0, PST1, PST4), for the measurements of Na+-K+-ATPase activity, as determined by the 3- O-methylfluorescein phosphatase (3- O-MFPase) assay. Voluntary (maximal voluntary contraction) and elicited (10, 20, 50, 100 Hz) force was reduced 30–55% ( P < 0.05) at PST0 and did not recover by PST4. Muscle action potential (M-wave) amplitude and area (measured in the vastus medialis) and 3- O-MFPase activity at PST0-Ex were less than that at PST0-Con ( P < 0.05) by 37, 25, and 38%, respectively. M-wave area at PST1-Ex was also less than that at PST1-Con ( P < 0.05). Changes in 3- O-MFPase activity correlated to changes in M-wave area across all time points ( r = 0.38, P < 0.05, n= 45). These results demonstrate that Na+-K+- ATPase activity is reduced by sustained isometric exercise in humans from that in a matched Con leg and that this reduction in Na+-K+-ATPase activity is associated with loss of excitability as indicated by M-wave alterations.

To investigate the hypothesis that prolonged partial ischemia would result in a depression in homogenate sarcoplasmic reticulum (SR) Ca2+-sequestering and mechanical properties in muscle, a cuff was placed around the hindlimb of 8 adult Sprague-Dawley rats (267+/-5.8 g; x +/- S.E.) and partially inflated (315 mm Hg) for 2 h. Following occlusion, the EDL was sampled both from the ischemic (I) and contralateral control (C) leg and SR properties compared with the EDL muscles extracted from rats (n = 8) immediately following anaesthetization (CC). Ischemia was indicated by a lower (p0.05) concentration (mmol.kg dry wt(-1)) of ATP (19.0+/-0.7 vs. 16.7+/-0.7) and phosphocreatine (58.1+/-5.7 vs. 35.0+/-4.6) in I compared to C. Although Ca2+-ATPase activity (micromol x g protein(-1) x sec(-1)), both maximal and submaximal, was not different between C and I (19.7+/-0.4 vs. 18.5+/-1.3), reductions (p0.05) in Ca2+-uptake (mmol x g protein(-1) x sec(-1)) of between 18.2 and 24.7% across a range of submaximal free Ca2+-levels were observed in I compared to C. Lower submaximal Ca2+-ATPase activity and Ca2+-uptake were also observed in the EDL in C compared to CC animals. Time dependent reductions (p0.05) were found in peak twitch and maximal tetanic tension in EDL from I but not C. It is concluded that partial ischemia, resulting in modest reductions in energy state in EDL, induces a reduction in Ca2+-uptake independent of changes in Ca2+-ATPase activity. These changes reduce the coupling ratio and the efficiency of Ca2+-transport by SR.

In this study, we employed an in vivo model of prolonged ischemia in rat skeletal muscle to investigate the hypothesis that structural modifications to the sarcoplasmic reticulum (SR) Ca2+-ATPase can explain the alterations in Ca2+-ATPase activity that occur with ischemia. To induce total ischemia, a tourniquet was placed around the upper hindlimb in 27 female Sprague-Dawley rats weighing 256 ± 6.7 g (mean ± SE) and was inflated to 350 mmHg for 4 h. The contralateral limb served as control (C) to the ischemic limb (I), and the limbs of animals killed immediately after anesthetization served as a double control (CC). Mixed gastrocnemius and tibialis anterior muscles were sampled and used for SR vesicle preparation. Maximal Ca2+-ATPase activity (μmol · g protein−1· min−1) of C (15,802 ± 1,246) and I (11,609 ± 1,029) was 90 and 73% ( P < 0.05) of CC (17,562 ± 1,682), respectively. No differences were found between groups in either the Hill coefficient or the free Ca2+at half-maximal activity. The fluorescent probes, FITC and N-cyclohexyl- N′-(dimethylamino-α-naphthyl) carbodiimide, used to assess structural alterations in the regions of the ATP binding site and the Ca2+binding sites of the Ca2+-ATPase, respectively, indicated a 26% reduction ( P < 0.05) in FITC binding capacity (absolute units) in I (0.22 ± 0.01) compared with CC (0.29 ± 0.02) and C (0.29 ± 0.03). Our results suggest that the reduction in maximal SR Ca2+-ATPase activity in SR vesicles with ischemia is related to structural modification in the region of the nucleotide binding domain by mechanisms that are as yet unclear.

To investigate the hypothesis that ischemia and reperfusion would impair sarcoplasmic reticulum (SR) Ca2+regulation in skeletal muscle, Sprague-Dawley rats ( n = 20) weighing 290 ± 3.5 g were randomly assigned to either a control control (CC) group, in which only the effects of anesthetization were studied, or to a group in which the muscles in one hindlimb were made ischemic for 4 h and allowed to recover for 1 h (I). The nonischemic, contralateral muscles served as control (C). Measurements of Ca2+-ATPase properties in homogenates and SR vesicles, in mixed gastrocnemius and tibialis anterior muscles, indicated no differences between groups on maximal activity, the Hill coefficient, and Ca50, defined as the Ca2+concentration needed to elicit 50% of maximal activity. In homogenates, Ca2+uptake was lower ( P < 0.05) by 20–25%, measured at 0.5 and 1.0 μM of free Ca2+([Ca2+]f) in C compared with CC. In SR vesicles, Ca2+uptake was lower ( P < 0.05) by 30–38% in I compared with CC at [Ca2+]fbetween 0.5 and 1.5 μM. Silver nitrate induced Ca2+release, assessed during both the initial, early rapid ( phase 1), and slower, prolonged late ( phase 2) phases, in homogenates and SR vesicles, indicated a higher ( P < 0.05) release only in phase 1in SR vesicles in I compared with CC. These results indicate that the alterations in SR Ca2+regulation, previously observed after prolonged ischemia by our group, are reversed within 1 h of reperfusion. However, the lower Ca2+uptake observed in long-term, nonischemic homogenates suggests that altered regulation may occur in the absence of ischemia.

In this study, we investigated the hypothesis that prolonged ischemia would impair both sarcoplasmic reticulum (SR) Ca2+uptake and Ca2+release in skeletal muscle. To induce total ischemia (I), a tourniquet was placed around the upper hindlimb in 30 female Sprague-Dawley rats [wt = 256 ± 6.7 (SE) g] and inflated to 350 mmHg for 4 h. The contralateral limb served as control (C). Immediately after the 4 h of ischemia, mixed gastrocnemius and tibialis anterior muscle was sampled from both limbs, and both crude muscle homogenates and SR vesicles were prepared. In another 10 control animals (CC), muscles were sampled and prepared exactly the same way, but immediately after anesthetization. Ca2+uptake and Ca2+release were measured in vitro with Indo-I on both homogenates and SR vesicles. As hypothesized, submaximal Ca2+uptake was lower ( P < 0.05) in I compared with CC and C, by 25 and 45% in homogenates and SR vesicles, respectively. Silver nitrate (AgNO3)-induced Ca2+release, which occurred in two phases ( phase 1 and phase 2), was also altered in I compared with CC and C, in both muscle homogenates and SR vesicles. With ischemia, phase 1 peak Ca2+release was 26% lower ( P < 0.05) in SR vesicles only. For phase 2, peak Ca2+release was 54 and 24% lower ( P < 0.05) in SR vesicles and homogenates, respectively. These results demonstrate that prolonged skeletal muscle ischemia leads to a reduced SR Ca2+uptake in both homogenates and SR vesicles. The effects of ischemia on SR Ca2+release, however, depend on both the phase examined and the type of tissue preparation.

This study examined the alterations in sarcoplasmic reticulum (SR) Ca2+ sequestration function in homogenates during eccentric exercise and recovery and following additional eccentric exercise, and correlated these alterations with changes in force output. Eight healthy, untrained females, aged 20-25 years, cycled for a total of 60 min on an eccentric cycle ergometer (30 min at 66+/-3% VO2 peak and 30 min at 76+/-3% VO2 peak, determined during concentric exercise). Biopsies (extracted from the vastus lateralis) were taken before and after the exercise as well as on days 2, 6 and prior to and following identical exercise on day 14. Ca2+-uptake (nmol/min/mg protein) was unaffected (p0.05) following the first session of eccentric exercise; however, by day 2 a depression in uptake (p0.05) was observed which persisted throughout the remainder of the experiment. Maximal Ca2+-ATPase activity (nmol/min/mg protein) was elevated (p0.05) immediately following the first exercise session, remained elevated through day 2 and returned to pre-exercise levels by day 6 of recovery and increased again by day 14. No changes in either Ca2+-ATPase activity or Ca2+-uptake were observed with exercise on day 14. Both eccentric sessions, performed on days 0 and 14, resulted in similar depressions in force (p0.05) immediately following exercise. By day 2 force had recovered to pre-exercise levels. The results demonstrate that a prolonged alteration in SR Ca2+-uptake occurs following eccentric work that is unaccompanied by parallel changes in either SR Ca2+-ATPase activity or mechanical performance.

To investigate the hypothesis that intrinsic changes in sarcoplasmic reticulum (SR) Ca2+-sequestration function can be implicated in postcontractile depression (PCD) of force in humans, muscle tissue was obtained from the vastus lateralis and determinations of maximal Ca2+uptake and maximal Ca2+-ATPase activity were made on homogenates obtained before and after the induction of PCD. Eight untrained females, age 20.6 ± 0.75 yr (mean ± SE), performed a protocol consisting of 30 min of isometric exercise at 60% maximal voluntary contraction and at 50% duty cycle (5-s contraction and 5-s relaxation) to induce PCD. Muscle mechanical performance determined by evoked activation was measured before (0 min), during (15 and 30 min), and after (60 min) exercise. The fatiguing protocol resulted in a progressive reduction ( P < 0.05) in evoked force, which by 30 min amounted to 52% for low frequency (10 Hz) and 20% for high frequency (100 Hz). No force restoration occurred at either 10 or 100 Hz during a 60-min recovery period. Maximal SR Ca2+-ATPase activity (nmol ⋅ mg protein−1⋅ min−1) and maximal SR Ca2+uptake (nmol ⋅ mg protein−1⋅ min−1) were depressed ( P < 0.05) by 15 min of exercise [192 ± 45 vs. 114 ± 8.7 and 310 ± 59 vs. 205 ± 47, respectively; mean ± SE] and remained depressed at 30 min of exercise. No recovery in either measure was observed during the 60-min recovery period. The coupling ratio between Ca2+-ATPase and Ca2+uptake was preserved throughout exercise and during recovery. These results illustrate that during PCD, Ca2+uptake is depressed and that the reduction in Ca2+uptake is due to intrinsic alterations in the Ca2+pump. The role of altered Ca2+sequestration in Ca2release, cytosolic-free calcium, and PCD remains to be determined.

The effects of estrogen on skeletal muscle fatigue are controversial. To determine the effects of estrogen and gender on rat extensor digitorum longus (EDL) muscle, we either injected 40 microg beta-estradiol 3/benzoate.kg BW(-1) to female rats or sham injected male or female rats for 14 days. Subsequently a 90 min fatigue protocol consisting of electrical stimulation at 10 Hz delivered in 500 ms trains was administered. Force was recorded for a 5 s period at the start of the protocol (0 min) and at 5 min intervals until completion following 90 min of stimulation. After 90 min, EDL force generation at 10 Hz stimulation declined in all groups to between 50-60 % of initial values. However, no significant difference in fatigue rate or final 10 Hz stimulated force was seen between females administered estrogen, sham injected females or males. Hence, estrogen administration and gender did not significantly affect EDL muscle fatigue in this model.

To examine the hypothesis that foot-strike hemolysis alters vascular volumes and selected hematological properties is trained athletes, we have measured total blood volume (TBV), red cell volume (RCV) and plasma volume (PV) in cyclists (n = 21) and runners (n = 17) and compared them to those of untrained controls (n = 20). TBV (ml x kg(-1)) was calculated as the sum of RCV (ml x kg(-1)) and PV (ml x kg(-1)) obtained using 51Cr and 125I-labelled albumin, respectively. Hematological assessment was carried out using a Coulter counter. Peak aerobic power (VO2peak) was measured during progressive exercise to fatigue using both cycle and treadmill ergometry. RCV was 15% higher (P < 0.05) in male cyclists [35.4 (1.0), mean (SE); n = 12] and runners [35.3 (0.98); n = 9] compared to the controls [30.7 (0.92); n = 12]. Similar differences existed between the female cyclists [28.2 (2.1); n = 9] and runners [28.4 (1.0); n = 8] compared to the untrained controls [24.9 (1.4); n = 8]. For the male athletes, PV was between 19% (cyclists) and 28% (runners) higher (P < 0.05) in the trained athletes compared to the untrained controls. The differences in PV between the female groups were not significant. Although the males had a higher (P < 0.05) TBV, RCV and PV than the females, no differences between cyclists and runners were found for either gender. Mean cell volume was not different between the athletic groups. VO2peak (ml x kg(-1) x min(-1)) was higher (P < 0.05) in both male [68.4 (1.5)] and female [54.8 (2.1)] runners when compared to the untrained males [47.1 (1.0)] and females [40.5 (2.1)]. Although differences existed between the genders in VO2peak for both cyclists and runners, no differences were found between the athletic groups within a gender. Since the vascular volumes were not different between cyclists and runners for either the males or females, foot-strike hemolysis would not appear to have an effect on that parameter. The significant correlations (P < 0.05) found between VO2peak and RCV (r = 0.64 and 0.64) and TBV (r = 0.82 and 0.63) for the males and females, respectively, suggests a role for the vascular system in realizing a high aerobic power.