Contractile function of single muscle fibers after hindlimb suspension

The purpose of this investigation was to determine how muscle atrophy produced by the hindlimb suspension (HS) model alters the contractile function of slow- and fast-twitch single muscle fibers. After 2 wk of HS, small bundles of fibers were isolated from the soleus and the deep and superficial regions of the lateral and medial heads of the gastrocnemius, respectively. The bundles were placed in skinning solution and stored at -20 degrees C until studied. Single fibers were isolated and suspended between a motor arm and force transducer, the functional properties were studied, and subsequently the fiber type was established by myosin heavy chain (MHC) analysis on 1-D sodium dodecyl sulfate polyacrylamide gel electrophoresis. After HS, slow-twitch fibers of the soleus showed a significant reduction in fiber diameter (68 +/- 2 vs. 41 +/- 1 micron) and peak tension (1.37 +/- 0.01 vs. 0.99 +/- 0.06 kg/cm2), whereas the maximal shortening speed (Vmax) increased [1.49 +/- 0.11 vs. 1.92 +/- 0.14 fiber lengths (FL)/s]. A histogram showed two populations of fibers: one with Vmax values identical to control slow-twitch fibers and a second with significantly elevated Vmax values. This latter group frequently contained both slow and fast MHC protein isoforms. The pCa-force relation of the soleus slow-twitch fibers was shifted to the right; consequently, the free Ca2+ required for the onset of tension and for 50% of peak tension was significantly higher after HS. Slow-twitch fibers isolated from the gastrocnemius after HS showed a significant reduction in diameter (67 +/- 4 vs. 44 +/- 3 microns) and peak tension (1.2 +/- 0.06 vs. 0.96 +/- 0.07 kg/cm2), but Vmax was unaltered (1.70 +/- 0.13 vs. 1.65 +/- 0.18 FL/s). Fast-twitch fibers from the red gastrocnemius showed a significant reduction in diameter (59 +/- 2 vs. 49 +/- 3 microns) but no change in peak tension or Vmax. Fast-twitch fibers from the white superficial region of the medial head of the gastrocnemius were unaffected by HS. Collectively, these data suggest that the effects of HS on fiber function depend on the fiber type and location. Both slow-twitch type I and fast-twitch type IIa fibers atrophied; however, only slow-twitch fibers showed a decline in peak tension, and the increase in Vmax was restricted to a subpopulation of slow-twitch soleus fibers.     

Phospholamban expressed in slow-twitch and chronically stimulated fast-twitch muscles minimally affects calcium affinity of sarcoplasmic reticulum Ca(2+)-ATPase.

Chronic excitation, at 2 Hz for 6-7 weeks, of the predominantly fast-twitch canine latissimus dorsi muscle promoted the expression of phospholamban, a protein found in sarcoplasmic reticulum (SR) from slow-twitch and cardiac muscle but not in fast-twitch muscle. At the same time that phospholamban was expressed, there was a switch from the fast-twitch (SERCA1) to the slow-twitch (SERCA2a) Ca(2+)-ATPase isoform. Antibodies against Ca(2+)-ATPase (SERCA2a) and phospholamban were used to assess the relative amounts of the slow-twitch/cardiac isoform of the Ca(2+)-ATPase and phospholamban, which were found to be virtually the same in SR vesicles from the slow-twitch muscle, vastus intermedius; cardiac muscle; and the chronically stimulated fast-twitch muscle, latissimus dorsi. The phospholamban monoclonal antibody 2D12 was added to SR vesicles to evaluate the regulatory effect of phospholamban on calcium uptake. The antibody produced a strong stimulation of calcium uptake into cardiac SR vesicles, by increasing the apparent affinity of the Ca2+ pump for calcium by 2.8-fold. In the SR from the conditioned latissimus dorsi, however, the phospholamban antibody produced only a marginal effect on Ca2+ pump calcium affinity. These different effects of phospholamban on calcium uptake suggest that phospholamban is not tightly coupled to the Ca(2+)-ATPase in SR vesicles from slow-twitch muscles and that phospholamban may have some other function in slow-twitch and chronically stimulated fast-twitch muscle.     

Adenosine triphosphatases during maturation of cultured human skeletal muscle cells and in adult human muscle.

Na+/K(+)-ATPase, Mg(2+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+)-ATPase are examined in cultured human skeletal muscle cells of different maturation grade and in human skeletal muscle. Na+/K(+)-ATPase is investigated by measuring ouabain binding and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-methylfluorescein phosphatase (3-O-MFPase). SR Ca(2+)-ATPase is examined by ELISA, Ca(2+)-dependent phosphorylation and its activities on ATP and 3-O-methylfluorescein phosphate. Na+/K(+)-ATPase and SR Ca(2+)-ATPase are localized by immunocytochemistry. The activities of Na+/K(+)-ATPase and SR Ca(2+)-ATPase show a good correlation with the other assayed parameters of these ion pumps. All ATPase parameters investigated increase with the maturation grade of the cultured muscle cells. The number of ouabain-binding sites and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-MFPase are significantly higher in cultured muscle cells than in muscle. The Mg(2+)-ATPase activity, the content of SR Ca(2+)-ATPase and the activities of SR Ca(2+)-ATPase and Ca(2+)-dependent 3-O-MFPase remain significantly lower in cultured cells than in muscle. The ouabain-binding constant and the molecular activities of Na+/K(+)-ATPase and SR Ca(2+)-ATPase are equal in muscle and cultured cells. During ageing of human muscle the activity as well as the concentration of SR Ca(2+)-ATPase decrease. Thus the changes of the activities of the ATPases are caused by variations of the number of their molecules. Na+/K(+)-ATPase is localized in the periphery of fast- and slow-twitch muscle fibers and at the sarcomeric I-band. SR Ca(2+)-ATPase is predominantly confined to the I-band, whereas fast-twitch fibers are much more immunoreactive than slow-twitch fibers. The presence of cross-striation for Na+/K(+)-ATPase and SR Ca(2+)-ATPase in highly matured cultured muscle cells indicate the development and subcellular organization of a transverse tubular system and SR, respectively, which resembles the in vivo situation.     

Effects of modulators of sarcoplasmic Ca2+ release on the development of skeletal muscle fatigue.

The reduced release of Ca2+ from sarcoplasmic reticulum (SR) is considered a major determinant of muscle fatigue. In the present study, we investigated whether the presence of dantrolene, an established inhibitor of SR Ca2+ release, or caffeine, a drug facilitating SR Ca2+ release, modifies muscle fatigue development. Accordingly, the effects of Ca2+ release modulators were analyzed in vitro in mouse fast-twitch [extensor digitorum longus (EDL)] and slow-twitch (soleus) muscles, fatigued by repeated short tetani (40 Hz for 300 ms, 0.5 s(-1) in soleus and 60 Hz for 300 ms, 0.3 s(-1) in EDL, for 6 min). Caffeine produced a substantial increase of tetanic tension of both EDL and soleus muscles, whereas dantrolene decreased tetanic tension only in EDL muscle. In both EDL and soleus muscles, 5 microM dantrolene did not affect fatigue development, whereas 20 microM dantrolene produced a positive staircase during the first 3 min of stimulation in EDL muscle and a slowing of fatigue development in soleus muscle. The development of the positive staircase was abolished by the addition of 15 microM ML-7, a selective inhibitor of myosin light chain kinase. On the other hand, caffeine caused a larger and faster loss of tension in both EDL and soleus muscles. The results seem to indicate that the changes in fatigue profile induced by caffeine or dantrolene are mainly due to the changes in the initial tetanic tension caused by the drugs, with the resulting changes in the level of contraction-dependent factors of fatigue, rather than to changes in the SR Ca2+ release during fatigue development.     

甲状腺功能亢进大鼠比目鱼肌肌浆网Ca2+-ATP酶活性增高可加速强直收缩疲劳

甲状腺功能亢进(甲亢)时甲状腺素分泌增加,不仅使具有神经支配的慢缩型肌纤维向快缩型转化,而且改变骨骼肌的强直收缩功能.因此,甲亢性肌病的肌肉乏力可能与骨骼肌强直收缩易发生疲劳有关.本实验在离体条件下,观测甲亢4周引起的大鼠慢缩肌--比目鱼肌(soleus, SOL)单收缩与间断强直收缩功能的变化.结果显示,甲亢4周大鼠体重明显低于同步对照组[(292±13)g vs (354±10)g],但SOL湿重没有明显改变[(107.3±8.6)mg vs (115.1±6.9)mg].甲亢大鼠SOL单收缩张力达到峰值的时间(time to peak tension, TPT)、从峰值降至75%舒张时间(time from peak tension to 75% relaxation, TR75)均明显缩短;强直收缩的TR75也明显缩短[(102.8±4.1)ms vs (178.8±15.8)ms];强直收缩的最适频率从对照组的100Hz增加到140Hz;间断强直收缩期间容易发生疲劳.甲亢大鼠SOL肌浆网Ca2 -ATP酶(sarcoplasmic-reticulum Ca2 -ATPase, SERCA)活性增高.采用SERCA特异性抑制剂CPA (1.0μmol/L)处理后,对照组与甲亢大鼠SOL间断强直收缩的TR75均延长,同时不易出现疲劳.5.0μmol/L CPA灌流虽可进一步抵抗甲亢大鼠SOL间断强直收缩引起的疲劳,但强直收缩期间的静息张力却明显升高.将CPA浓度增至10.0μmol/L,甲亢大鼠SOL间断强直收缩又趋向易发生疲劳.这些结果提示,与心肌相同,骨骼肌肌纤维SERCA活性亦可影响单收缩与强直收缩的舒张时间,SERCA活性升高可加速间断强直收缩发生疲劳.     

Effects of high myoplasmic L-lactate concentration on E-C coupling in mammalian skeletal muscle.

The effects of high myoplasmic L-lactate concentrations (20-40 mM) at constant pH (7.1) were investigated on contractile protein function, voltage-dependent Ca(2+) release, and passive Ca(2+) leak from the sarcoplasmic reticulum (SR) in mechanically skinned fast-twitch (extensor digitorum longus; EDL) and slow-twitch (soleus) fibers of the rat. L-Lactate (20 mM) significantly reduced maximum Ca(2+)-activated force by 4 +/- 0.5% (n = 5, P < 0.05) and 5 +/- 0.4% (n = 6, P < 0.05) for EDL and soleus, respectively. The Ca(2+) sensitivity was also significantly decreased by 0.06 +/- 0. 002 (n = 5, P < 0.05) and 0.13 +/- 0.01 (n = 6, P < 0.001) pCa units, respectively. Exposure to L-lactate (20 mM) for 30 s reduced depolarization-induced force responses by ChCl substitution by 7 +/- 3% (n = 17, P < 0.05). This inhibition was not obviously affected by the presence of the lactate transport blocker quercetin (10 microM), or the chloride channel blocker anthracene-9-carboxylic acid (100 microM). L-Lactate (20 mM) increased passive Ca(2+) leak from the SR in EDL fibers (the integral of the response to caffeine was reduced by 16 +/- 5%, n = 9, P < 0.05) with no apparent effect in soleus fibers (100 +/- 2%, n = 3). These results indicate that the L-lactate ion per se has negligible effects on either voltage-dependent Ca(2+) release or SR Ca(2+) handling and exerts only a modest inhibitory effect on muscle contractility at the level of the contractile proteins.     

Distribution of calcium ATPase in the sarcoplasmic reticulum of fast- and slow-twitch muscles determined with monoclonal antibodies

Summary Four monoclonal antibodies against the calcium ATPase in sarcoplasmic reticulum (SR) of rabbit fast-twitch skeletal muscle were characterized using SDS-PAGE, Western blots and immunofluorescence. The ultrastructural distribution of the antigens was determined using post-embedding immunolabeling. The antibodies recognized the calcium ATPase in the SR but not in transverse (T-) tubule or plasma membranes. The antibody, D12, had the same binding affinity for the calcium ATPase from fast-twitch (rabbit sternomastoid) and slow-twitch (rabbit soleus) fibers and the affinity fell by 30% after fixation for electron microscopy in both types of muscle fiber. Ultrastructural studies revealed that the density of D12 antibody binding to the terminal cisternae membrane of extensor digitorum longus (edl) and sternomastoid fibers was on average seven times greater than in the slow-twitch soleus and semimembranosus fibers. Since the affinity of the ATPase for the antibody was the same in SR from fast- and slow-twitch muscles, the concentration of calcium ATPase in the terminal cisternae membrane of fast-twitch fibers was seven times greater than in slow-twitch fibers. This conclusion was supported by the fact that the concentration of calcium ATPase in light SR membranes was six times greater in SR from fast-twitch fibers than in SR from slow-twitch fibers. The results provide strong evidence that the different calcium accumulation rates in mammalian fast- and slow-twitch muscles are due to different concentrations of calcium ATPase molecules in the SR membrane.     

Is creatine kinase responsible for fatigue? Studies of isolated skeletal muscle deficient in creatine kinase.

Creatine kinase (CK) is a key enzyme for maintaining a constant ATP/ADP ratio during rapid energy turnover. To investigate the role of CK in skeletal muscle fatigue, we used isolated whole muscles and intact single fibers from CK-deficient mice (CK(-/-)). With high-intensity electrical stimulation, single fibers from CK(-/-) mice displayed a transient decrease in both tetanic free myoplasmic [Ca(2+)] ([Ca(2+)](i), measured with the fluorescent dye indo-1) and force that was not observed in wild-type fibers. With less intense, repeated tetanic stimulation single fibers and EDL muscles, both of which are fast-twitch, fatigued more slowly in CK(-/-) than in wild-type mice; on the other hand, the slow-twitch soleus muscle fatigued more rapidly in CK(-/-) mice. In single wild-type fibers, tetanic force decreased and [Ca(2+)](i) increased during the first 10 fatiguing tetani, but this was not observed in CK(-/-) fibers. Fatigue was not accompanied by phosphocreatine breakdown and accumulation of inorganic phosphate in CK(-/-) muscles. In conclusion, CK is important for avoiding fatigue at the onset of high-intensity stimulation. However, during more prolonged stimulation, CK may contribute to the fatigue process by increasing the myoplasmic concentration of inorganic phosphate.     

Enhanced technique to measure proteins in single segments of human skeletal muscle fibers: fiber-type dependence of AMPK-alpha1 and -beta1

Human physiological studies typically use skeletal muscle biopsies from the heterogeneous vastus lateralis muscle comprised of both fast-twitch and slow-twitch fiber types. It is likely that potential changes of physiological importance are overlooked because fiber-type specific responses may not be apparent in the whole muscle preparation. A technological advance in Western blotting is presented where proteins are analyzed in just one small segment (<2 mm) of individual fibers dissected from freeze-dried muscle samples using standard laboratory equipment. A significant advance is being able to classify every fiber at the level of both contractile (myosin heavy chain and tropomyosin) and sarcoplasmic reticulum [sarco(endo)plasmic reticulum Ca(2+)-ATPase type 1] properties and then being able to measure specific proteins in the very same segments. This removes the need to fiber type segments before further analyses and, as such, dramatically reduces the time required for sample collection. Compared with slow-twitch fibers, there was less AMP-activated protein kinase (AMPK)-α(1) (～25%) and AMPK-β(1) (～60%) in fast-twitch fibers from human skeletal muscle biopsies.     

Relaxation of diaphragm muscle.

Relaxation is the process by which, after contraction, the muscle actively returns to its initial conditions of length and load. In rhythmically active muscles such as diaphragm, relaxation is of physiological importance because diaphragm must return to a relatively constant resting position at the end of each contraction-relaxation cycle. Rapid and complete relaxation of the diaphragm is likely to play an important role in adaptation to changes in respiratory load and breathing frequency. Regulation of diaphragm relaxation at the molecular and cellular levels involves Ca(2+) removal from the myofilaments, active Ca(2+) pumping by the sarcoplasmic reticulum (SR), and decrease in the number of working cross bridges. The relative contribution of these mechanisms mainly depends on sarcomere length, muscle tension, and the intrinsic contractile function. Increased capacity of SR to take up Ca(2+) can arise from increased density of active SR pumping sites or in slow-twitch fibers from phosphorylation of phospholamban, whereas impaired coupling between ATP hydrolysis and Ca(2+) transport into the SR or intracellular acidosis reduces SR Ca(2+) pump activity. In experimental conditions of decreased contractile performance, slowed, enhanced, or unchanged relaxation rates have been reported in vitro. In vivo, a slowing in the rate of decline of the respiratory pressure is generally considered an early reliable index of respiratory muscle fatigue. Impaired relaxation rate may, in turn, favor mismatch between blood flow and metabolic demand, especially at high breathing frequencies.     

Phosphorylation of anchoring protein by calmodulin protein kinase associated to the sarcoplasmic reticulum of rabbit fast-twitch muscle

Regulatory phosphorylation of phospholamban and of SR Ca(2+)-ATPase SERCA2a isoform by endogenous CaM-K II in slow-twitch skeletal and cardiac sarcoplasmic reticulum (SR) is well documented, but much less is known of the exact functional role of CaM K II in fast-twitch muscle SR. Recently, it was shown that RNA splicing of brain-specific alpha CaM K II, gives rise to a truncated protein (alpha KAP), consisting mainly of the association domain, serving to anchor CaM K II to SR membrane in rat skeletal muscle [Bayer, K.-U., et al. (1998) EMBO J. 19, 5598-5605]. In the present study, we searched for the presence of alpha KAP in sucrose-density purified SR membrane fractions from representative fast-twitch and slow-twitch limb muscles, both of the rabbit and the rat, using immunoblot techniques and antibody directed against the association domain of alpha CaM K II. Putative alpha KAP was immunodetected as a 23-kDa electrophoretic component on SDS-PAGE of the isolated SR from fast-twitch but not from slow-twitch muscle, and was further identified as a specific substrate of endogenous CaM K II, in the rabbit. Immunodetected, (32)P-labeled, non-calmodulin binding protein, behaved as a single 23-kDa protein species under several electrophoretic conditions. The 23-kDa protein, with defined properties, was isolated as a complex with 60-kDa delta CaM K II isoform, by sucrose-density sedimentation analysis. Moreover, we show here that putative alphaKAP, in spite of its inability to bind CaM in ligand blot overlay, co-eluted with delta CaM K II from CaM-affinity columns. That raises the question of whether CaM K II-mediated phosphorylation of alpha KAP and triadin together might be involved in a molecular signaling pathway important for SR Ca(2+)-release in fast-twitch muscle SR.     

IP(3)-induced tension and IP(3)-receptor expression in rat soleus muscle during postnatal development

The present study was designed to examine whether changes in Ca(2+) release by inositol-1,4,5-trisphosphate (IP(3)) in 8-, 15-, and 30-day-old rat skeletal muscles could be associated with the expression of IP(3) receptors. Experiments were conducted in slow-twitch muscle in which both IP(3)-induced Ca(2+) release and IP(3)-receptor (IP(3)R) expression have been shown to be larger than in fast-twitch muscle. In saponin-skinned fibers, IP(3) induced transient contractile responses in which the amplitude was dependent on the Ca(2+)-loading period with the maximal IP(3) contracture being at 20 min of loading. The IP(3) tension decreased during postnatal development, was partially inhibited by ryanodine (100 microM), and was blocked by heparin (20-400 microg/ml). Amplification of the DNA sequence encoding for IP(3)R isoforms (using the RT-PCR technique) showed that in slow-twitch muscle, the type 2 isoform is mainly expressed, and its level decreases during postnatal development in parallel with changes in IP(3) responses in immature fibers. IP(3)-induced Ca(2+) release would then have greater participation in excitation-contraction coupling in developing fibers than in mature muscle.     

Histochemical properties of skeletal muscle fibers in streptozotocin-diabetic rats

Summary The response of rat gastrocnemius muscle fibers to chronic streptozotocin-diabetes was studied. Transverse sections of this muscle from normal and diabetic rats were histochemically assayed for reduced diphosphopyridine nucleotide-diaphorase, myofibrillar adenosine triphosphatase, mitochondrial alpha-glycerophosphate dehydrogenase, beta-hydroxybutyrate dehydrogenase, and alkaline phosphatase activities. Cross-sectional areas of the fiber types were measured, and fiber capillarization and populations estimated. Chemically-induced diabetes appeared to have little effect on the metabolic or morphological properties of slow-twitch fibers. However, a general dedifferentiation occurred in the 2 fast-twitch fiber populations. There was a loss of oxidative potential in the fast-twitch-oxidative-glycolytic fibers, and a significant decrease in size in the fast-twitch-glycolytic fibers. No change in the proportions of slow- and fast-twitch fibers in the muscles of diabetic rats occurred. It is concluded that hypoinsulinism has differential effects on the 3 fiber types in heterogeneous rat skeletal muscle, and that slow-twitch fibers are least affected by the diabetic condition.     

Comparison of the effects of inorganic phosphate on caffeine-induced Ca2+ release in fast- and slow-twitch mammalian skeletal muscle

We compared the effects of 50 mM P(i) on caffeine-induced Ca(2+) release in mechanically skinned fast-twitch (FT) and slow-twitch (ST) skeletal muscle fibers of the rat. The time integral (area) of the caffeine response was reduced by approximately 57% (FT) and approximately 27% (ST) after 30 s of exposure to 50 mM P(i) in either the presence or absence of creatine phosphate (to buffer ADP). Differences in the sarcoplasmic reticulum (SR) Ca(2+) content between FT and ST fibers [ approximately 40% vs. 100% SR Ca(2+) content (pCa 6.7), respectively] did not contribute to the different effects of P(i) observed; underloading the SR of ST fibers so that the SR Ca(2+) content approximated that of FT fibers resulted in an even smaller ( approximately 21%), but not significant, reduction in caffeine-induced Ca(2+) release by P(i). These observed differences between FT and ST fibers could arise from fiber-type differences in the ability of the SR to accumulate Ca(2+)-P(i) precipitate. To test this, fibers were Ca(2+) loaded in the presence of 50 mM P(i). In FT fibers, the maximum SR Ca(2+) content (pCa 6.7) was subsequently increased by up to 13 times of that achieved when loading for 2 min in the absence of P(i). In ST fibers, the SR Ca(2+) content was only doubled. These data show that Ca(2+) release in ST fibers was less affected by P(i) than FT fibers, and this may be due to a reduced capacity of ST SR to accumulate Ca(2+)-P(i) precipitate. This may account, in part, for the fatigue-resistant nature of ST fibers.     

Intracellular calcium movements during excitation-contraction coupling in mammalian slow-twitch and fast-twitch muscle fibers

In skeletal muscle fibers, action potentials elicit contractions by releasing calcium ions (Ca(2+)) from the sarcoplasmic reticulum. Experiments on individual mouse muscle fibers micro-injected with a rapidly responding fluorescent Ca(2+) indicator dye reveal that the amount of Ca(2+) released is three- to fourfold larger in fast-twitch fibers than in slow-twitch fibers, and the proportion of the released Ca(2+) that binds to troponin to activate contraction is substantially smaller.     

Nonuniform changes in arteriolar myogenic tone within skeletal muscle following hindlimb unweighting.

Hindlimb unweighting (HLU) has been shown to alter myogenic tone distinctly in arterioles isolated from skeletal muscles composed predominantly of fast-twitch (white gastrocnemius) compared with slow-twitch (soleus) fibers. Based on these findings, we hypothesized that HLU would alter myogenic tone differently in arterioles isolated from distinct fiber-type regions within a single skeletal muscle. We further hypothesized that alterations in myogenic tone would be associated with alterations in voltage-gated Ca(2+) channel current (VGCC) density of arteriolar smooth muscle. After 14 days of HLU or weight bearing (control), first-order arterioles were isolated from both fast-twitch and mixed fiber-type regions of the gastrocnemius muscle, cannulated, and pressurized at 90 cmH(2)O. Mixed gastrocnemius arterioles of HLU rats demonstrated increased spontaneous tone [43 +/- 5% (HLU) vs. 27 +/- 4% (control) of possible constriction] and an approximately twofold enhanced myogenic response when exposed to step changes in intraluminal pressure (10-130 cmH(2)O) compared with control rats. In contrast, fast-twitch gastrocnemius arterioles of HLU rats demonstrated similar levels of spontaneous tone [6 +/- 2% (HLU) vs. 6 +/- 2% (control)] and myogenic reactivity to control rats. Neither KCl-induced contractile responses (10-50 mM KCl) nor VGCC density was significantly different between mixed gastrocnemius arterioles of HLU and control rats. These results suggest that HLU produces diverse adaptations in myogenic reactivity of arterioles isolated from different fiber-type regions of a single skeletal muscle. Furthermore, alterations in myogenic responses were not attributable to altered VGCC density.     

Simulation of Ca2+ movements within the sarcomere of fast-twitch mouse fibers stimulated by action potentials

Ca(2+) release from the sarcoplasmic reticulum (SR) of skeletal muscle takes place at the triadic junctions; following release, Ca(2+) spreads within the sarcomere by diffusion. Here, we report multicompartment simulations of changes in sarcomeric Ca(2+) evoked by action potentials (APs) in fast-twitch fibers of adult mice. The simulations include Ca(2+) complexation reactions with ATP, troponin, parvalbumin, and the SR Ca(2+) pump, as well as Ca(2+) transport by the pump. Results are compared with spatially averaged Ca(2+) transients measured in mouse fibers with furaptra, a low-affinity, rapidly responding Ca(2+) indicator. The furaptra Deltaf(CaD) signal (change in the fraction of the indicator in the Ca(2+)-bound form) evoked by one AP is well simulated under the assumption that SR Ca(2+) release has a peak of 200-225 microM/ms and a FDHM of approximately 1.6 ms (16 degrees C). Deltaf(CaD) elicited by a five-shock, 67-Hz train of APs is well simulated under the assumption that in response to APs 2-5, Ca(2+) release decreases progressively from 0.25 to 0.15 times that elicited by the first AP, a reduction likely due to Ca(2+) inactivation of Ca(2+) release. Recovery from inactivation was studied with a two-AP protocol; the amplitude of the second release recovered to >0.9 times that of the first with a rate constant of 7 s(-1). An obvious feature of Deltaf(CaD) during a five-shock train is a progressive decline in the rate of decay from the individual peaks of Deltaf(CaD). According to the simulations, this decline is due to a reduction in available Ca(2+) binding sites on troponin and parvalbumin. The effects of sarcomere length, the location of the triadic junctions, resting [Ca(2+)], the parvalbumin concentration, and possible uptake of Ca(2+) by mitochondria were also investigated. Overall, the simulations indicate that this reaction-diffusion model, which was originally developed for Ca(2+) sparks in frog fibers, works well when adapted to mouse fast-twitch fibers stimulated by APs.     

Interactions between intracellular calcium and phosphate in intact mouse muscle during fatigue

Fatigue was studied in intact tibialis anterior muscle of anesthetized mice. The distal tendon was detached and connected to a force transducer while blood flow continued normally. The muscle was stimulated with electrodes applied directly to the muscle surface and fatigued by repeated (1 per 4 s), brief (0.4 s), maximal (100-Hz stimulation frequency) tetani. Force declined monotonically to 49 ± 5% of the initial value with a half time of 36 ± 5 s and recovered to 86 ± 4% after 4 min. Intracellular phosphate concentration ([P(i)]) was measured by (31)P-NMR on perchloric acid extracts of muscles. [P(i)] increased during fatigue from 7.6 ± 1.7 to 16.0 ± 1.6 mmol/kg muscle wet wt and returned to control during recovery. Intracellular Ca(2+) was measured with cameleons whose plasmids had been transfected in the muscle 2 wk before the experiment. Yellow cameleon 2 was used to measure myoplasmic Ca(2+), and D1ER was used to measure sarcoplasmic reticulum (SR) Ca(2+). The myoplasmic Ca(2+) during tetani declined steadily during the period of fatigue and showed complete recovery over 4 min. The SR Ca(2+) also declined monotonically during fatigue and showed a partial recovery with rest. These results show that the initial phase of force decline is accompanied by a rise in [P(i)] and a reduction in the tetanic myoplasmic Ca(2+). We suggest that both changes contribute to the fatigue. A likely cause of the decline in tetanic myoplasmic Ca(2+) is precipitation of CaP(i) in the SR.     

Effects of fatigue and training on sarcoplasmic reticulum Ca(2+) regulation in human skeletal muscle.

Little is known about fatigue and training effects on sarcoplasmic reticulum (SR) function in human muscle, and we therefore investigated this in eight untrained controls (UT), eight endurance-trained (ET), and eight resistance-trained athletes (RT). Muscle biopsies (vastus lateralis) taken at rest and after 50 maximal quadriceps contractions (180 degrees/s, 0.5 Hz) were analyzed for fiber composition, metabolites and maximal SR Ca(2+) release, Ca(2+) uptake, and Ca(2+)-ATPase activity. Fatigue reduced (P < 0.05) Ca(2+) release (42.1 +/- 3.8%, 43.4 +/- 3.9%, 31.3 +/- 6.1%), Ca(2+) uptake (43.0 +/- 5.2%, 34.1 +/- 4.6%, 28.4 +/- 2.8%), and Ca(2+)-ATPase activity (38.6 +/- 4.2%, 48.5 +/- 5.7%, 29.6 +/- 5.0%), in UT, RT, and ET, respectively. These decreases were correlated with fatigability and with type II fiber proportion (P < 0.05). Resting SR measures were correlated with type II proportion (r > or = 0.51, P < 0.05). ET had lower resting Ca(2+) release, Ca(2+) uptake, and Ca(2+)-ATPase (P < 0.05) than UT and RT (P < 0.05), probably because of their lower type II proportion; only minor effects were found in RT. Thus SR function is markedly depressed with fatigue in controls and in athletes, is dependent on fiber type, and appears to be minimally affected by chronic training status.