Effect of acute and chronic autophagy deficiency on skeletal muscle apoptotic signaling, morphology, and function.

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.

Effects of ovarian sex hormones and downhill running on fiber-type-specific HSP70 expression in rat soleus.

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 degrees 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.

Protective effects of Hsp70 on the structure and function of SERCA2a expressed in HEK-293 cells during heat stress.

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 (P < 0.05) in S2a/Hsp70 compared with S2a/pMT2 and approximately twofold higher (P < 0.05) following HS in all cells. Coimmunoprecipitation demonstrated that Hsp70 directly binds to SERCA2a. Following HS, maximal SERCA2a activity was reduced ( approximately 52%, P < 0.05) in S2a/pMT2 but was increased ( approximately 33%, P < 0.05) in S2a/Hsp70. Thermal inactivation of SERCA2a in S2a/pMT2 was associated with decreased ( approximately 49%, P < 0.05) binding capacity for fluorescein isothiocyanate (FITC) and increased carbonyl ( approximately 42%, P < 0.05) and nitrotyrosine ( approximately 40%, P < 0.05) levels in SERCA2a. By contrast, the HS-induced increase in maximal SERCA2a activity observed in S2a/Hsp70 corresponded with no change (P > 0.05) in FITC-binding capacity and reductions in carbonyl ( approximately 40%, P < 0.05) and nitrotyrosine ( approximately 23%, P < 0.05) 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.

Acute responses in muscle mitochondrial and cytosolic enzyme activities during heavy intermittent exercise.

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 (Vo(2 peak)) = 44.3 +/- 2.3 ml.kg(-1).min(-1)] cycled at approximately 91% Vo(2 peak) for 6 min once per hour for 16 h. Maximal enzyme activities (V(max), 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 beta-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) V(max) 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 V(max) 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 V(max).

Dissociation between changes in muscle Na+-K+-ATPase isoform abundance and activity with consecutive days of exercise and recovery.

The early plasticity of vastus lateralis Na(+)-K(+)-ATPase to the abrupt onset of prolonged submaximal cycling was studied in 12 untrained participants (Vo(2 peak) 44.8 +/- 2.0 ml x kg(-1) x 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 (beta(max)) (vanadate-facilitated [(3)H]ouabain binding), alpha- and beta-isoform concentration (quantitative immunoblotting) and maximal Na(+)-K(+)-ATPase activity (V(max)) (3-O-methylfluorescein K(+)-stimulated phosphatase assay). For beta(max) (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 alpha-isoforms alpha(1), alpha(2), and alpha(3), increases (P < 0.05) of 46, 42, and 31% were observed at R1, respectively. For the beta-isoform, beta(1) and beta(2) increased (P < 0.05) by 19 and 28% at R1, whereas beta(3) increased (P < 0.05) by 18% at R2. With the exception of alpha(2) and alpha(3), the increases in the isoforms persisted at R3. Exercise resulted in an average decrease (P < 0.05) in V(max) by 14.3%. No differences were observed in V(max) 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.

Rapid upregulation of GLUT-4 and MCT-4 expression during 16 h of heavy intermittent cycle exercise.

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 (.VO(2peak)) = 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 approximately 90% .VO(2peak), 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 approximately 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.

Muscle fiber type-specific response of Hsp70 expression in human quadriceps following acute isometric exercise.

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 approximately 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 approximately 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.

Muscle metabolic responses during 16 hours of intermittent heavy exercise.

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 (micromol.(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.

Effects of buthionine sulfoximine treatment on diaphragm contractility and SR Ca2+ pump function in rats.

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 Ca(2+)-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/mm(2)) 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 Ca(2+) uptake and maximal SERCA activity (mumol.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 Ca(2+) 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.

Muscle sarcoplasmic reticulum calcium regulation in humans during consecutive days of exercise and recovery.

The study investigated the hypothesis that three consecutive days of prolonged cycle exercise would result in a sustained reduction in the Ca(2+)-cycling properties of the vastus lateralis in the absence of changes in the sarcoplasmic (endoplasmic) reticulum Ca(2+)-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 Ca(2+)-ATPase activity (V(max)), Ca(2+) uptake and Ca(2+) release (phase 1 and phase 2), and SERCA isoform expression (SERCA1a and SERCA2a). At E1, reductions (P < 0.05) from Pre to Post in V(max) (150 +/- 7 vs. 121 +/- 7), Ca(2+) uptake (7.79 +/- 0.28 vs. 5.71 +/- 0.33), and both phases of Ca(2+) 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 V(max), which recovered at Pre E3 and then remained stable at Post E3 and throughout recovery, Ca(2+) uptake remained depressed (P < 0.05) at E3 Pre and Post and at R1 as did phase 2 of Ca(2+) 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 V(max) following the onset of regular exercise but not Ca(2+) uptake and Ca(2+) release.

Muscle Na+-K+-ATPase response during 16 h of heavy intermittent cycle exercise.

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 approximately 91% peak aerobic power (Vo(2 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 Vo(2 peak) of 44.3 +/- 2.3 ml x kg(-1) x min(-1) participated in the study. Maximal Na(+)-K(+)-ATPase activity (V(max), in nmol x mg protein(-1) x h(-1)) as measured by the 3-O-methylfluorescein K(+)-stimulated phosphatase assay was reduced (P < 0.05) by approximately 15% with exercise regardless of the number of repetitions performed. In addition, V(max) at R9 and R16 was lower (P < 0.05) than at R1 and R2. Vanadate-facilitated [(3)H]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 alpha(2)-isoform by R2 and a 29% increase in alpha(3) by R9. At R16, beta(3) was lower (P < 0.05) than at R2 and R9. No changes were observed in alpha(1), beta(1), or beta(2). It is concluded that repeated sessions of heavy exercise, although resulting in increases in the alpha(2)- and alpha(3)-isoforms and decreases in beta(3)-isoform, also result in depression in maximal catalytic activity.

Differential effects of repetitive activity on sarcoplasmic reticulum responses in rat muscles of different oxidative potential.

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, micromol x g protein(-1) x 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, Vmax remained 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.

Muscle sarcoplasmic reticulum Ca2+ cycling adaptations during 16 h of heavy intermittent cycle exercise.

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 (VO2(peak)) = 44.3 +/- 0.66 ml x kg(-1) x min(-1)]. The protocol involved 6 min of cycle exercise performed at approximately 91% VO2(peak) once 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. Vmax remained 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.

Reversal of muscle fatigue during 16 h of heavy intermittent cycle 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 approximately 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 approximately 53% at 10 Hz (113 +/- 12 vs. 52.6 +/- 7.4 N) and approximately 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.

Effects of prolonged exercise and recovery on sarcoplasmic reticulum Ca2+ cycling properties in rat muscle homogenates.

AIM: To examine the effects of exercise and exercise plus active and passive recovery on sarcoplasmic reticulum (SR) Ca2+-handling properties. METHODS: 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. RESULTS: Sarcoplasmic reticulum Ca2+-uptake was 31% higher (P < 0.05) in RUN+ compared with CON and RUN. Higher values (P < 0.05) were also found in REC25 (48%) and REC45 (50%) compared with CON. Maximal Ca2+-ATPase was increased by 23% (P < 0.05) in RUN+ compared with CON and RUN and by 65-68% (P < 0.05) in REC25 and REC45 compared with CON. A higher (P < 0.05) 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 (P < 0.05) in RUN+ (2.2 +/- 0.10) compared with CON (1.9 +/- 0.05) and RUN (1.9 +/- 0.08). CONCLUSIONS: 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.

Paradoxical effects of prior activity on human sarcoplasmic reticulum Ca2+-ATPase response to exercise.

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 (Vo -->Vo2 peak) = 42.9 +/- 2.7 ml. kg-1 x min-1]. The HE consisted of 16 bouts of cycling performed for 6 min each hour at 90% Vo2 peak. Tissue was obtained from the vastus lateralis by needle biopsy before and during each cycle test. Before HE, reductions (P < 0.05 micromol. g protein-1x min-1) of 16 and 31% were observed in Vmax and Ca2+ uptake, respectively, after 40 min of the standardized test. Resting Vmax and 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, Vmax increased (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.

Mechanisms underlying increases in SR Ca2+-ATPase activity after exercise in rat skeletal muscle.

Prolonged exercise followed by a brief period of reduced activity has been shown to result in an overshoot in maximal sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity [maximal velocity (V(max))] in rat locomoter muscles (Ferrington DA, Reijneveld JC, Bär PR, and Bigelow DJ. Biochim Biophys Acta 1279: 203-213, 1996). To investigate the functional significance and underlying mechanisms for the increase in V(max), we analyzed Ca(2+)-ATPase activity and Ca(2+) uptake in SR vesicles from the fast rat gastrocnemius muscles after prolonged running (RUN) and after prolonged running plus 45 min of low-intensity activity (RUN+) or no activity (REC45) and compared them with controls (Con). Although no differences were observed between RUN and Con, both V(max) and Ca(2+) uptake were higher (P < 0.05) by 43 and 63%, respectively, in RUN+ and by 35 and 34%, respectively, in REC45. The increase in V(max) was accompanied by increases (P < 0.05) in the phosphorylated enzyme intermediate measured by [gamma-(32)P]ATP. No differences between groups for each condition were found for the fluorescent probes FITC and (N-cyclohexyl-N(1)-dimethylamino-alpha-naphthyl)carbodiimide, competitive inhibitors of the nucleotide-binding and Ca(2+)-binding sites on the enzyme, respectively. Similarly, no differences for the Ca(2+)-ATPase were observed between groups in nitrotyrosine and phosphoserine residues, a measure of nitrosylation and phosphorylation states, respectively. Western blots indicated no changes in relative isoform content of sarcoendoplasmic reticulum (SERCA)1 and SERCA2a. It is concluded that the increase in V(max) of the Ca(2+)-ATPase observed in recovery is not the result of changes in enzyme nitroslyation or phosphorylation, changes in ATP and Ca(2+)-binding affinity, or changes in protein content of the Ca(2+)-ATPase.

Reduced activity of muscle Na(+)-K(+)-ATPase after prolonged running in rats.

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.

Thermal instability of rat muscle sarcoplasmic reticulum Ca(2+)-ATPase function.

To examine the thermal instability and the role of sulfhydryl (SH) oxidation on sarcoplasmic reticulum (SR) Ca(2+)-ATPase function, crude homogenates were prepared from the white portion of the gastrocnemius (WG) adult rat muscles (n = 9) and incubated in vitro for < or =60 min either at a normal resting body temperature (37 degrees C) or at a temperature indicative of exercise-induced hyperthermia (41 degrees C) with DTT and without DTT (CON). In general, treatment with DTT resulted in higher Ca(2+)-ATPase and Ca(2+) uptake values (nmol. mg protein(-1). min(-1), P < 0.05), an effect that was not specific to time of incubation. Incubations at 41 degrees C resulted in lower (P < 0.05) Ca(2+) uptake rates (156 +/- 18 and 35.9 +/- 3.3) compared with 37 degrees C (570 +/- 54 and 364 +/- 26) at 30 and 60 min, respectively. At 37 degrees C, ryanodine (300 microM), which was used to block Ca(2+) release from the calcium release channel, prevented the time-dependent decrease in Ca(2+) uptake. A general inactivation (P < 0.05) of maximal Ca(2+)-ATPase activity (V(max)) in CON was observed with incubation time (0 > 30 > 60 min), with the effect being more pronounced (P < 0.05) at 41 degrees C compared with 37 degrees C. The Hill slope, a measure of co-operativity, and the pCa(50), the cytosolic Ca(2+) concentration required for half-maximal activation of Ca(2+)-ATPase activity, decreased (P < 0.05) at 41 degrees C only. Treatment with DTT attenuated the alterations in enzyme kinetics. The increase in V(max) with the Ca(2+) ionophore A-23187 was less pronounced at 41 degrees C compared with 37 degrees 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 Ca(2+) uptake and occurs as a result of increases in membrane permeability to Ca(2+). Moreover, the decreases in Ca(2+)-ATPase kinetics in WG with sustained heat stress result from SH oxidation.