Enhanced skeletal muscle contraction with myosin light chain phosphorylation by a calmodulin-sensing kinase

Repetitive low frequency stimulation results in potentiation of twitch force development in fast-twitch skeletal muscle due to myosin regulatory light chain (RLC) phosphorylation by Ca(2+)/calmodulin (CaM)-dependent skeletal muscle myosin light chain kinase (skMLCK). We generated transgenic mice that express an skMLCK CaM biosensor in skeletal muscle to determine whether skMLCK or CaM is limiting to twitch force potentiation. Three transgenic mouse lines exhibited up to 22-fold increases in skMLCK protein expression in fast-twitch extensor digitorum longus muscle containing type IIa and IIb fibers, with comparable expressions in slow-twitch soleus muscle containing type I and IIa fibers. The high expressing lines showed a more rapid RLC phosphorylation and force potentiation in extensor digitorum longus muscle with low frequency electrical stimulation. Surprisingly, overexpression of skMLCK in soleus muscle did not recapitulate the fast-twitch potentiation response despite marked enhancement of both fast-twitch and slow-twitch RLC phosphorylation. Analysis of calmodulin binding to the biosensor showed a frequency-dependent activation to a maximal extent of 60%. Because skMLCK transgene expression is 22-fold greater than the wild-type kinase, skMLCK rather than calmodulin is normally limiting for RLC phosphorylation and twitch force potentiation. The kinase activation rate (10.6 s(-1)) was only 3.6-fold slower than the contraction rate, whereas the inactivation rate (2.8 s(-1)) was 12-fold slower than relaxation. The slower rate of kinase inactivation in vivo with repetitive contractions provides a biochemical memory via RLC phosphorylation. Importantly, RLC phosphorylation plays a prominent role in skeletal muscle force potentiation of fast-twitch type IIb but not type I or IIa fibers.     

Regulation of skeletal muscle dihydropyridine receptor gene expression by biomechanical unloading.

Biomechanical unloading of the rat soleus by hindlimb unweighting is known to induce atrophy and a slow- to fast-twitch transition of skeletal muscle contractile properties, particularly in slow-twitch muscles such as the soleus. The purpose of this study was to determine whether the expression of the dihydropyridine (DHP) receptor gene is upregulated in unloaded slow-twitch soleus muscles. A rat DHP receptor cDNA was isolated by screening a random-primed cDNA lambda gt10 library from denervated rat skeletal muscle with oligonucleotide probes complementary to the coding region of the rabbit DHP receptor cDNA. Muscle mass and DHP receptor mRNA expression were assessed 1, 4, 7, 14, and 28 days after hindlimb unweighting in rats by tail suspension. Isometric twitch contraction times of soleus muscles were measured at 28 days of unweighting. Northern blot analysis showed that tissue distribution of DHP receptor mRNA was specific for skeletal muscle and expression was 200% greater in control fast-twitch extensor digitorum longus (EDL) than in control soleus muscles. A significant stimulation (80%) in receptor message of the soleus was induced as early as 24 h of unloading without changes in muscle mass. Unloading for 28 days induced marked atrophy (control = 133 +/- 3 vs. unweighted = 62.4 +/- 1.8 mg), and expression of the DHP receptor mRNA in the soleus was indistinguishable from levels normally expressed in EDL muscles. These changes in mRNA expression are in the same direction as the 37% reduction in time to peak tension and 28% decrease in half-relaxation time 28 days after unweighting. Our results suggest that muscle loading necessary for weight support modulates the expression of the DHP receptor gene in the soleus muscle.     

Differences in catecholamine-sensitive adenylate cyclase and beta-adrenergic receptor binding between fast-twitch and slow-twitch skeletal muscle membranes.

Catecholamines known to be active at the β-adrenergic receptor stimulated the adenylate cyclase in a plasmalemmal fraction of slow-twitch soleus muscle 3-fold, while they enhanced the enzyme activity in a similar fraction of fast-twitch extensor digitorum longus (EDL) muscle by only 0.6-fold. In contrast, the β-adrenergic receptors, as revealed by [³H] dihydroalprenolol binding to plasmalemmal fraction were 30–60% greater in EDL than in soleus. It is suggested that the enzyme and receptor are independent entities.     

Effect of colchicine on alkaline triglyceride lipase activity and triglyceride content in rat skeletal muscle

One purpose of this study was to determine if colchicine increased intracellular alkaline triglyceride (TG) lipase activity above control levels in rat skeletal muscle. The second aim was to determine the effects of colchicine treatment on the concentration of TG in skeletal muscle. The results show that colchicine was a potent inducer of alkaline TG lipase activity, increasing enzyme activity approximately twofold in slow-twitch red, fast-twitch red, and fast-twitch white muscle types. It was found that in slow-twitch red soleus and fast-twitch red vastus, the two muscle groups with the highest levels of enzyme activity, 76% or more of enzyme activity resides in the intracellular compartment. These results provide evidence that colchicine blocks the export of alkaline TG lipase from skeletal muscle cells similar to that seen in the heart. The finding that TG were reduced at a time when enzyme activity was elevated suggests that intracellular alkaline TG lipase may be playing a role in the hydrolysis of the intramuscular TG droplet.     

Excitation-induced Ca(2+) influx in rat soleus and EDL muscle: mechanisms and effects on cellular integrity

In rat skeletal muscle, electrical stimulation increases Ca(2+) influx leading to progressive accumulation of calcium. Excitation-induced Ca(2+) influx in extensor digitorum longus (EDL; fast-twitch fibers) and soleus muscle (slow-twitch fibers) is compared. In EDL and soleus, stimulation at 40 Hz increased (45)Ca uptake 34- and 21-fold and (22)Na uptake 17- and 7-fold, respectively. These differences may be related to the measured 70% higher concentration of Na(+) channels in EDL. Repeated stimulation at 40 Hz elicited a delayed release of lactic acid dehydrogenase (LDH) from EDL (11-fold increase) and soleus (5-fold increase). Continuous stimulation at 1 Hz increased LDH release only from EDL (18-fold). This was associated with increased Ca(2+) content and was augmented at high extracellular Ca(2+) concentration ([Ca(2+)](o)) and suppressed at low [Ca(2+)](o). The data support the hypothesis that excitation-induced Ca(2+) influx is mediated in part by Na(+) channels and that the ensuing increase in intracellular Ca(2+) induces cellular damage. This is most pronounced in EDL, which may account for the repeated observation that prolonged exercise leads to preferential damage to fast-twitch fibers.     

Phorbol esters affect skeletal muscle glucose transport in a fiber type-specific manner

Recent evidence has shown that activation of lipid-sensitive protein kinase C (PKC) isoforms leads to skeletal muscle insulin resistance. However, earlier studies demonstrated that phorbol esters increase glucose transport in skeletal muscle. The purpose of the present study was to try to resolve this discrepancy. Treatment with the phorbol ester 12-deoxyphorbol-13-phenylacetate 20-acetate (dPPA) led to an approximately 3.5-fold increase in glucose transport in isolated fast-twitch epitrochlearis and flexor digitorum brevis muscles. Phorbol ester treatment was additive to a maximally effective concentration of insulin in fast-twitch skeletal muscles. Treatment with dPPA did not affect insulin signaling in the epitrochlearis. In contrast, phorbol esters had no effect on basal glucose transport and inhibited maximally insulin-stimulated glucose transport approximately 50% in isolated slow-twitch soleus muscle. Furthermore, dPPA treatment inhibited the insulin-stimulated tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and the threonine and serine phosphorylation of PKB by approximately 50% in the soleus. dPPA treatment also caused serine phosphorylation of IRS-1 in the slow-twitch soleus muscle. In conclusion, our results show that phorbol esters stimulate glucose transport in fast-twitch skeletal muscles and inhibit insulin signaling in slow-twitch soleus muscle of rats. These findings suggest that mechanisms other than PKC activation mediate lipotoxicity-induced whole body insulin resistance.     

Adaptation of rat skeletal muscle to creatine depletion: AMP deaminase and AMP deamination.

AMP deaminase catalyzes deamination of the AMP formed in contracting muscles to inosine 5'-monophosphate (IMP). Slow-twitch muscle has only approximately 30% as high a level of AMP deaminase activity as fast-twitch muscle in the rat, and rates of IMP formation during intense contractile activity are much lower in slow-twitch muscle. We found that feeding the creatine analogue beta-guanidinopropionic acid (beta-GPA) to rats, which results in creatine depletion, causes a large decrease in muscle AMP deaminase. This adaptation was used to evaluate the role of AMP deaminase activity level in accounting for differences in IMP production in slow-twitch and fast-twitch muscles. beta-GPA feeding for 3 wk lowered AMP deaminase activity in fast-twitch epitrochlearis muscle to a level similar to that found in the normal slow-twitch soleus muscle but had no effect on the magnitude of the increase in IMP in response to intense contractile activity. Despite a similar decrease in ATP in the normal soleus and the epitrochlearis from beta-GPA-fed rats, the increase in IMP was only approximately 30% as great in the soleus in response to intense contractile activity. These results demonstrate that the accumulation of less IMP in slow- compared with fast-twitch skeletal muscle during contractile activity is not due to the lower level of AMP deaminase in slow-twitch muscle.     

Evolution of pyruvate carboxylase and other biotin containing enzymes in developing rat liver and kidney

During contractions, when the rate of ATP hydrolysis exceeds that of ADP phosphorylation, inosine 5'-monophosphate (IMP) accumulates in skeletal muscle. If the cellular energy balance is not promptly restored, subsequent purine degradation to inosine via 5'-nucleotidase can occur, a process that is most robust in the slow-twitch red, as compared to fast-twitch, skeletal muscle. We measured the distribution of 5'-nucleotidase activity among membrane-bound and soluble fractions of fiber specific skeletal muscle sections and found most (80-90%) of the total 5'-nucleotidase activity to be membrane-bound. The 5' IMP nucleotidase activity present in the soluble fraction of muscle extracts differs among fiber types with slow-twitch red > fast-twitch red > mixed fibered > fast-twitch white. Experiments testing the substrate dependence of IMP and AMP dephosphorylation by the soluble fraction of muscle extracts revealed a lower Km toward IMP (approximately 0.7-1.5 mM) than AMP (1.9-2.8 mM). Among skeletal muscle fiber sections, the soluble 5'-nucleotidase activity present in slow-twitch red muscle extracts had the highest substrate affinity, the highest activity with IMP as substrate, and an estimated catalytic efficiency (Vmax/Km) that was > 3-fold higher than calculated for fast-twitch muscle extracts. This is likely due to the Mg2+ dependent cytosolic 5' IMP nucleotidase isoform, since immunoprecipitation experiments revealed 3-4 times more activity in slow-twitch red than in fast-twitch red or fast-twitch white fibers, respectively. These finding are consistent with the previously recognized in vivo pattern of nucleoside formation by muscle where the soleus demonstrated extensive inosine formation at a much lower IMP content than fast-twitch red or fast-twitch white muscle fiber sections.     

Immunochemical quantification of sarcoplasmic reticulum Ca-ATPase, of calsequestrin and of parvalbumin in rabbit skeletal muscles of defined fiber composition

Antibodies directed against purified Ca-ATPase from sarcoplasmic reticulum, calsequestrin and parvalbumin from rabbit fast-twitch muscle were raised in sheep. The specificity of the antibodies was shown by immunoblot analysis and by enzyme-linked immunoadsorbent assays (ELISAs). IgG against the sarcoplasmic reticulum Ca-ATPase inhibited the catalytic activities of Ca-ATPase from fast-twitch (psoas, tibialis anterior) and slow-twitch (soleus) muscles to the same degree. In non-equilibrium competitive ELISAs the anti(Ca-ATPase) IgG displayed a slightly higher affinity for the Ca-ATPase from fast-twitch muscle than for that from slow-twitch muscle. This suggests a fiber-type-specific polymorphism of the sarcoplasmic reticulum Ca-ATPase. Quantification of Ca-ATPase, calsequestrin and parvalbumin in various rabbit skeletal muscles of histochemically determined fiber composition was achieved by sandwich ELISA. Ca-ATPase was found to be 6-7 times higher in fast than in slow-twitch muscles. A slightly higher concentration was found in fast-twitch muscles with a higher percentage of IIb fibers when compared with fast-twitch muscles with a higher percentage of IIa fibers. Thus Ca-ATPase is distributed as follows, IIb greater than or equal to IIa much greater than I. Calsequestrin was uniformly distributed in fast-twitch muscles independently of their IIa/IIb fiber ratio and displayed 50% lower concentrations in slow than in fast-twitch muscles (IIb = IIa greater than I). Parvalbumin contents were 200-300-fold higher in fast than in slow-twitch muscles. Significantly lower parvalbumin concentrations were found in fast-twitch muscles with a higher percentage of IIa fibers than in fast-twitch muscles with a higher percentage of IIb fibers (IIb greater than IIa much greater than I).     

Effects of body suspension on soleus muscle fibres and spinal motoneurones in the rat.

1. After 14 days of body suspension, fibre type composition and fibre cross-sectional area in the soleus muscle of 17-week-old male Sprague-Dawley rats were investigated. Oxidative enzyme activity of soleus motoneurones in the spinal cord was also examined. 2. After suspension, soleus muscle weight decreased by 44.2%, the cross-sectional area of SO and FOG fibres decreased by 60.4% and 58.6%, respectively. 3. The percentage of fibre types was not changed by suspension. However, ATPase activity after alkaline preincubation was markedly inhibited in FOG fibres. 4. Oxidative enzyme activity of soleus motoneurones was not changed by suspension. 5. This study demonstrates that using mature animals body suspension induces atrophy of fast- and slow-twitch fibres accompanied with the selective inhibition in ATPase activity of fast-twitch fibres, and without changes in histochemical profiles of the corresponding motoneurones.     

S100a0 (αα) Protein, a Calcium-Binding Protein, Is Localized in the Slow-Twitch Muscle Fiber

We previously showed that, in contrast to the distribution of S100b (beta beta), S100a0 (alpha alpha) is mainly present in human skeletal and heart muscles at the level of 1-2 micrograms/mg of soluble protein and is universally distributed at high levels in skeletal and heart muscles of various mammals. To elucidate cellular and ultrastructural localizations of the alpha subunit of S100 protein (S100-alpha) in skeletal muscle, we used immunohistochemical and enzyme immunoassay methods. The immunohistochemical study revealed that S100-alpha is mainly localized in slow-twitch muscle fibers, whereas the beta subunit of S100 protein (S100-beta) was not detected in both types of muscle fibers, an observation indicating that the predominant form of S100 protein in the slow-twitch muscle fiber is not S100a or S100b, but S100a0. The quantitative analysis using enzyme immunoassay corroborates the immunohistochemical finding: The S100-alpha concentration of mouse soleus muscle (mainly composed of slow-twitch muscle fibers) is about threefold higher than that of mouse rectus femoris muscle (mainly composed of fast-twitch muscle fibers). At the ultrastructural level, S100-alpha is associated with polysomes, sarcoplasmic reticulum, the plasma membrane, the pellicle around lipid droplets, the outer membrane of mitochondria, and thin and thick filaments, by immunoelectron microscopy.     

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.     

Angiotensin-converting enzyme inhibition attenuates myonuclear addition in overloaded slow-twitch skeletal muscle

Because optimal overload-induced skeletal muscle hypertrophy requires ANG II, we aimed to determine the effects of blocking ANG II production [via angiotensin-converting enzyme (ACE) inhibition] on potential mediators of hypertrophy in overloaded skeletal muscle, namely, myonuclear addition and fibroblast content. In a 2 x 2 design, adult (200-225 g) female Sprague-Dawley rats were placed into one of four groups (n = 8/group): 7-day skeletal muscle overload, sham operation, 7-day skeletal muscle overload with ACE inhibition, or sham operation with ACE inhibition. Functional overloads of the plantaris and soleus muscles were produced via bilateral surgical ablation of the synergistic gastrocnemius muscle, and ACE inhibition was accomplished by the addition of the ACE inhibitor enalapril maleate to the animals' daily drinking water (0.3 mg/ml). Myonuclear addition and extrasarcolemmal nuclear proliferation, as measured by in vivo 5-bromo-2'-deoxyuridine labeling, were significantly (P < or = 0.05) increased by overload in both the slow-twitch soleus and fast-twitch plantaris muscles. Furthermore, ACE inhibition attenuated these overload-induced increases in the soleus muscle but not in the plantaris muscle. However, the effect of ACE inhibition on soleus extrasarcolemmal nuclei was not likely due to differences in fibroblast content because overload elicited significant increases in vimentin-positive areas in soleus and plantaris muscles, and these areas were unaffected by ACE inhibition in either muscle. There was no effect of ACE inhibition on any measure in sham-operated muscles. Collectively, these data indicate that ANG II may mediate the satellite cell response to overload in slow-twitch soleus but not in fast-twitch plantaris muscles and that this effect may occur independently of changes in fibroblast content.     

Bcl-2/Bax protein expression in heart,slow-twitch and fast-twitch muscles in young rats growing under chronic hypoxia conditions

We have studied the magnitude of apoptosis in heart, slow-twitch skeletal muscle (soleus) and fast-twitch skeletal muscle (gastrocnemius) of rats exposed to 3 weeks in vivo chronic hypoxia. Apoptosis was evaluated biochemically by DNA laddering and by TUNEL and annexin V-staining. The expression of Bax and Bcl-2 proteins was determined by immunohistochemistry and Western blotting.Western blot analysis revealed only a slight difference in Bax expression among the different tissues under normoxic and hypoxic conditions; therefore we can consider that Bax protein is constitutively expressed in muscle tissues. However a singular pattern of Bcl-2 expression was observed among the different tissues under normoxic conditions. Bcl-2 protein was more expressed in fast-twitch glycolytic muscles than in slow-twitch or oxidative muscles with a highest value found in gastrocnemius (4926 ± 280 AU), followed by soleus (2138 ± 200 AU) and a very low expression was displayed in the heart muscle (543 ± 50 AU). After exposure to hypoxia for 21 days (10% O2), Bcl-2 protein expression markedly increased, (44%) in gastrocnemius, (323%) in soleus and (1178%) in heart, with significant differences (p < 0.05 student t-test), reaching a similar threshold of expression in both types of muscles. Furthermore, no sign of apoptosis was detected by TUNEL assay, annexin V-binding assay or DNA electrophoresis analysis. The latter suggested some indiscriminate fragmentations of DNA without apoptosis. In conclusion, we postulate that these protein modifications could represent a adaptative mechanism allowing a better protection against the lack of oxygen in oxidative muscles by preventing apoptosis.     

Fasting-related autophagic response in slow- and fast-twitch skeletal muscle

This study investigated regulation of autophagy in slow-twitch soleus and fast-twitch plantaris muscles in fasting-related atrophy. Male Fischer-344 rats were subjected to fasting for 1, 2, or 3 days. Greater weight loss was observed in plantaris muscle than in soleus muscle in response to fasting. Western blot analysis demonstrated that LC3-II, a marker protein for macroautophagy, was expressed at a notably higher level in plantaris than in soleus muscle, and that the expression level was fasting duration-dependent. To identify factors related to LC3-II enhancement, autophagy-related signals were examined in both types of muscle. Phosphorylated mTOR was reduced in plantaris but not in soleus muscle. FOXO3a and ER stress signals were unchanged in both muscle types during fasting. These findings suggest that preferential atrophy of fast-twitch muscle is associated with induction of autophagy during fasting and that differences in autophagy regulation are attributable to differential signal regulation in soleus and plantaris muscle.     

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.     

Diazepam reveals different rate-limiting processes in rat skeletal muscle contraction

The action of the tranquilizer diazepam on rat skeletal muscle showed that relaxation of isometric twitches is controlled by different processes in extensor digitorum longus (fast-twitch) and soleus (slow-twitch) muscles. Diazepam caused an increase in the amplitude of twitches in fibres from both muscles but increased the twitch duration only in soleus. The amplitude of fused tetani were reduced in both muscles and the rate of relaxation after the tetanus slowed by as much as 34% when the amplitude of the tetanus was reduced by only 11%. The slower tetanic relaxation indicated that calcium uptake by the sarcoplasmic reticulum was slower than normal in slow- and fast-twitch fibres. We conclude therefore that calcium uptake by the sarcoplasmic reticulum is rate limiting for twitch relaxation in slow-twitch but not fast-twitch fibres and suggest that calcium binding to parvalbumin controls relaxation in the fast fibres.     

Fiber type changes in rat skeletal muscle after intense interval training

Female Sprague-Dawley rats were subjected to a ten week training program to determine the influence of intense interval running on the fiber type composition of selected hindlimb muscles; soleus (S), plantaris (P), deep vastus lateralis (DVL), and superficial vastus lateralis (SVL). The muscles of one hindlimb were used for histochemical ATPase analysis to determine the distribution of fiber types and those of the contralateral hindlimb were assayed biochemically for citrate synthase activity (an aerobic marker). Training induced a significant increase in citrate synthase activity in each muscle section. The largest absolute increase occurred in the DVL and the largest relative increase occurred in the SVL. The distribution of fiber types within the S (85% slow-twitch) and SVL (100% fast-twitch) remained unchanged with training. However, significant increases in the percentage of type I (slow-twitch) fibers in both the P (2-fold) and DVL (3-fold) were observed with concomitant decreases in the type II (fast-twitch) population. In addition, training induced significant changes in the fast-twitch subtype populations of the DVL (IIB----IIA). These data suggest exercise-induced fiber type transformations occurring both within the fast-twitch population and between fast-twitch and slow-twitch fibers in certain hindlimb muscles of the rat following a high intensity interval training program.