Quantitative morphology of stimulation-induced damage in rabbit fast-twitch skeletal muscles

Summary The purpose of this study was to examine the contention that stimulation-induced damage, resulting in degeneration with subsequent regeneration, plays a major role in the transformation of fibre type brought about by chronic electrical stimulation. Data from histological and histochemical sections of 9-day-stimulated rabbit fast-twitch muscles were analysed with multivariate statistical techniques. Fibre degeneration and regeneration varied non-systematically between sample areas at any given cross-sectional level. In the extensor digitorum longus muscle, but not in the tibialis anterior, there was more degeneration in proximal than in distal portions of the muscle. The extensor digitorum longus muscle consistently showed more degeneration than the tibialis anterior muscle. Degeneration was less extensive for an intermittent pattern of stimulation that delivered half the aggregate number of impulses of continuous stimulation. Degeneration and regeneration varied markedly between individual rabbits in each of the groups. Sections that revealed the most degeneration and regeneration also had more fibres that reacted positively with an anti-neonatal antibody. Rigorous analysis of different sources of variation has helped to explain apparent conflicts in the literature. The incidence of muscle fibre damage in the stimulated tibialis anterior muscle is low, showing that the contribution of degenerative-regenerative phenomena to fibre type conversion in this muscle is insignificant.     

Estimation of instantaneous moment arms of lower-leg muscles

Muscle moment arms at the human knee and ankle were estimated from muscle length changes measured as a function of joint flexion angle in cadaver specimens. Nearly all lower-leg muscles were studied: extensor digitorum longus, extensor hallucis longus, flexor digitorum longus, flexor hallucis longus, gastrocnemius lateralis, gastrocnemius medialis, peroneus brevis, peroneus longus, peroneus tertius, plantaris, soleus, tibialis anterior, and tibialis posterior. Noise in measured muscle length was filtered by means of quintic splines. Moment arms of the mm. gastrocnemii appear to be much more dependent on joint flexion angles than was generally assumed by other investigators. Some consequences for earlier analyses are mentioned.     

CYP4A metabolites of arachidonic acid and VEGF are mediators of skeletal muscle angiogenesis

Vascular endothelial growth factor (VEGF) has been implicated in angiogenesis induced by electrical stimulation in skeletal muscle. Less is known about the role of arachidonic acid metabolites in the control of growth of blood vessels in vivo. The present study examined the role of 20-hydroxyeicosatetraenoic acid (20-HETE) on the angiogenesis induced by electrical stimulation in skeletal muscle. The tibialis anterior and extensor digitorum longus muscles of rats were stimulated for 7 days. Electrical stimulation significantly increased the 20-HETE formation and angiogenesis in the muscles, which was blocked by chronic treatment with N-hydroxy-N'-(4-butyl-2-methylphenol)formamidine (HET0016) or 1-aminobenzotriazole (ABT). Chronic treatment with either HET0016 or ABT did not block the increases in VEGF protein expression in both muscles. To analyze the role of VEGF on 20-HETE formation, additional rats were treated with VEGF-neutralizing antibody (VEGF Ab). VEGF Ab blocked the increases of 20-HETE formation induced by stimulation. These results place 20-HETE in the downstream signaling pathway for angiogenesis and show that both VEGF and 20-HETE are involved in the angiogenesis induced by electrical stimulation in skeletal muscle.     

Muscle damage and repair in voluntarily running mice: strain and muscle differences

Summary Soleus, extensor digitorum longus and tibialis anterior muscles of mice voluntarily running in wheels for periods of 5 to 120 days were studied in spaced serial and serial cross-sections. Shortly after the onset of running and during the next 2 weeks, degeneration, necrosis, phagocytosis and regeneration of muscle fibers, satellite cell proliferation and cellular infiltration were found in soleus muscles of mice from all strains investigated (CBA/J, NMRI, C57b, NIH, SWS and Balb/c). Tibialis anterior but not extensor digitorum longus muscles were also damaged. Predominantly high-oxidative fibers were affected (both slow-oxidative and fast oxidative glycolytic in soleus, fast-oxidative glycolytic in tibialis anterior). Denervated soleus muscles that had been passively stretched during running were not damaged. Evidence was found that, during the early period of running, split fibers form by myogenesis within (regeneration) or outside (satellite cell proliferation) necrotic muscle fiber segments. Split fibers persisted in solei of long-term (2 to 3 months) exercised CBA/J but not NMRI mice. In 6 out of 20 solei of CBA/J runners exercised for 2 months or longer, fiber-type grouping was observed in the areas where extensive damage usually occurred in the early periods. The results show that different muscles are damaged and repaired to varying degrees and that marked interstrain and inter-individual differences are present. It appears that acute muscle injury occurring upon onset of voluntary running is a usual event in the adaptation of muscles to altered use.     

Intracellular Na+ and K+ shifts induced by contractile activities of rat skeletal muscles

The effects of direct and indirect electrical stimulation on intracellular potassium and sodium contents ([K]_i and [Na]_i, respectively) in rat soleus muscle (SOL) and extensor digitorum longus muscle (EDL) were investigated under in vivo conditions. The changes of [K]_i and [Na]_i contents in both muscles which were stimulated indirectly reached respective values at 30 min or 1 hr after the beginning of stimulation, whereas those of EDL stimulated with 60 Hz changed gradually through 2 hr stimulation. The shifts of [K]_i and [Na]_i in EDL occurred during the twitch contraction at considerably lower frequency stimulation (0.5–10 Hz), whereas those in SOL were observed during the tetanus contraction at high frequency stimulation (10–40 Hz). The difference of change in cationic shifts between EDL and SOL under low frequency stimulation was reduced by ouabain treatment, though the difference was still significant. When the muscles were indirectly stimulated 6000 times at 1,5,10 and 20 Hz, the cationic shifts in EDL were greater than those in SOL, extending over all frequencies. It was concluded that such a difference in ionic shift between contracting EDL and SOL may be primarily due to the difference in unidirectional ionic fluxes per stimulation and, secondly, to the difference in Na⁺-K⁺ pump activity.     

Discovery of TBC1D1 as an insulin-, AICAR-, and contraction-stimulated signaling nexus in mouse skeletal muscle

The Akt substrate of 160 kDa (AS160) is phosphorylated on Akt substrate (PAS) motifs in response to insulin and contraction in skeletal muscle, regulating glucose uptake. Here we discovered a dissociation between AS160 protein expression and apparent AS160 PAS phosphorylation among soleus, tibialis anterior, and extensor digitorum longus muscles. Immunodepletion of AS160 in tibialis anterior muscle lysates resulted in minimal depletion of the PAS band at 160 kDa, suggesting the presence of an additional PAS immunoreactive protein. By immunoprecipitation and mass spectrometry, we identified this protein as the AS160 paralog TBC1D1, an obesity candidate gene regulating GLUT4 translocation in adipocytes. TBC1D1 expression was severalfold higher in skeletal muscles compared with all other tissues and was the dominant protein detected by the anti-PAS antibody at 160 kDa in tibialis anterior and extensor digitorum longus but not soleus muscles. In vivo stimulation by insulin, contraction, and the AMP-activated protein kinase (AMPK) activator AICAR increased TBC1D1 PAS phosphorylation. Using mass spectrometry on TBC1D1 from mouse skeletal muscle, we identified several novel phosphorylation sites on TBC1D1 and found the majority were consensus or near consensus sites for AMPK. Semiquantitative analysis of spectra suggested that AICAR caused greater overall phosphorylation of TBC1D1 sites compared with insulin. Purified Akt and AMPK phosphorylated TBC1D1 in vitro, and AMPK, but not Akt, reduced TBC1D1 electrophoretic mobility. TBC1D1 is a major PAS immunoreactive protein in skeletal muscle that is phosphorylated in vivo by insulin, AICAR, and contraction. Both Akt and AMPK phosphorylate TBC1D1, but AMPK may be the more robust regulator.     

Induction of neuronal type nitric oxide synthase in skeletal muscle by chronic electrical stimulation in vivo

Reiser, Peter J., William O. Kline, and Pal L. Vaghy.Induction of neuronal type nitric oxide synthase in skeletal muscle by chronic electrical stimulation in vivo. J. Appl. Physiol. 82(4): 1250-1255, 1997.Fast-twitch skeletal muscles contain more neuronal-type nitricoxide synthase (nNOS) than slow-twitch muscles because nNOS is presentonly in fast (type II) muscle fibers. Chronic in vivo electricalstimulation of tibialis anterior and extensor digitorum longus musclesof rabbits was used as a method of inducing fast-to-slow fiber typetransformation. We have studied whether an increase in musclecontractile activity induced by electrical stimulation alters nNOSexpression, and if so, whether the nNOS expression decreases to thelevels present in slow muscles. Changes in the expression of myosinheavy chain isoforms and maximum velocity of shortening of skinnedfibers indicated characteristic fast-to-slow fiber type transformationafter 3 wk of stimulation. At the same time, activity of NOS doubled inthe stimulated muscles, and this correlated with an increase in theexpression of nNOS shown by immunoblot analysis. These data suggestthat nNOS expression in skeletal muscle is regulated by muscle activityand that this regulation does not necessarily follow the fast-twitchand slow-twitch pattern during the dynamic phase of phenotypetransformation.

     

Dihydropyridine and ryanodine receptor binding after eccentric contractions in mouse skeletal muscle.

The purpose of this study was to determine whether there are alterations in the dihydropyridine and/or ryanodine receptors that might explain the excitation-contraction uncoupling associated with eccentric contraction-induced skeletal muscle injury. The left anterior crural muscles (i.e., tibialis anterior, extensor digitorum longus, and extensor hallucis longus) of mice were injured in vivo by 150 eccentric contractions. Peak isometric tetanic torque of the anterior crural muscles was reduced approximately 45% immediately and 3 days after the eccentric contractions. Partial restoration of peak isometric tetanic and subtetanic forces of injured extensor digitorum longus muscles by 10 mM caffeine indicated the presence of excitation-contraction uncoupling. Scatchard analysis of [3H]ryanodine binding indicated that the number of ryanodine receptor binding sites was not altered immediately postinjury but decreased 16% 3 days later. Dihydropyridine receptor binding sites increased approximately 20% immediately after and were elevated to the same extent 3 days after the injury protocol. Muscle injury did not alter the sensitivity of either receptor. These data suggest that a loss or altered sensitivity of the dihydropyridine and ryanodine receptors does not contribute to the excitation-contraction uncoupling immediately after contraction-induced muscle injury. We also concluded that the loss in ryanodine receptors 3 days after injury is not the primary cause of excitation-contraction uncoupling at that time.     

Nerve stimulation with a multi-contact cuff electrode: validation of model predictions

The recruitment characteristics of muscle selective nerve stimulation by a multi-contact nerve cuff electrode, as predicted by computer modeling, have been investigated in acute experiments on rabbits. A nerve cuff containing five or six dot electrodes was placed around the sciatic nerve in five rabbits. M-waves were recorded with wire electrodes from the lateral gastrocnemius, soleus, tibialis anterior, and extensor digitorum longus muscles. The muscle recruitment performances of three contact configurations (monopole, transverse bipole, transverse tripole) were compared. The selectivity was quantified by the recruitment of two muscles (one extensor and one flexor) in response to a particular stimulus. The results showed that only in a few cases, transverse bi- and tripolar stimulation provided a better selectivity than monopolar stimulation. Neither of the two extensors, nor of the two flexors could be stimulated separately. In accordance with the results of the modeling studies, bi- and tripolar stimulation required higher stimulus currents than monopolar stimulation, whereas maximum recruitment and slopes of recruitment curves were lower. The rabbit sciatic nerve appears to be a less suitable preparation for reproducible selectivity experiments, due to the variability in the number and size of the fascicles and their position in this nerve.     

Unlike effects of denervation on the rate of entry of inorganic phosphate into rat slow and fast muscles.

The increased inorganic phosphate flow, characteristic of denervated gastrocnemius muscle is shown to be present in additional denervated fast muscles, i.e. the plantaris, tibialis anterior and extensor digitorum longus muscles. The response of the soleus, a slow muscle, to denervation is biphasic. After an initial decrease of the phosphate flow at the end of the first postoperative day, there is a secondary rise which has the same general characteristics as the rise observed in fast muscles i.e. an exponential or hyperbolic increase to an asymptotic value reached after thirty days. The denervated fast and slow muscles are not converging to an intermediate metabolic pattern. The changes in phosphate flow induced by denervation are reversible in the soleus as well as in the gastrocnemius muscles.     

Adaptive strength gains in dystrophic muscle exposed to repeated bouts of eccentric contraction

The objective of this study was to determine the functional recovery and adaptation of dystrophic muscle to multiple bouts of contraction-induced injury. Because lengthening (i.e., eccentric) contractions are extremely injurious for dystrophic muscle, it was considered that repeated bouts of such contractions would exacerbate the disease phenotype in mdx mice. Anterior crural muscles (tibialis anterior and extensor digitorum longus) and posterior crural muscles (gastrocnemius, soleus, and plantaris) from mdx mice performed one or five repeated bouts of 100 electrically stimulated eccentric contractions in vivo, and each bout was separated by 10-18 days. Functional recovery from one bout was achieved 7 days after injury, which was in contrast to a group of wild-type mice, which still showed a 25% decrement in electrically stimulated isometric torque at that time point. Across bouts there was no difference in the immediate loss of strength after repeated bouts of eccentric contractions for mdx mice (-70%, P = 0.68). However, after recovery from each bout, dystrophic muscle had greater torque-generating capacity such that isometric torque was increased ～38% for both anterior and posterior crural muscles at bout 5 compared with bout 1 (P < 0.001). Moreover, isolated extensor digitorum longus muscles excised from in vivo-tested hindlimbs 14-18 days after bout 5 had greater specific force than contralateral control muscles (12.2 vs. 10.4 N/cm(2), P = 0.005) and a 20% greater maximal relaxation rate (P = 0.049). Additional adaptations due to the multiple bouts of eccentric contractions included rapid recovery and/or sparing of contractile proteins, enhanced parvalbumin expression, and a decrease in fiber size variability. In conclusion, eccentric contractions are injurious to dystrophic skeletal muscle; however, the muscle recovers function rapidly and adapts to repeated bouts of eccentric contractions by improving strength.     

Differential adaptation to weightlessness of functional and structural characteristics of rat hindlimb muscles

Soleus, vastus intermedius, tibialis anterior, and extensor digitorum longus muscles were removed from rats following space flight onboard the SLS-2 mission and from control animals. Muscle tissues were studied for their calcium and strontium activated tension characteristics and for structural changes. Muscles were also examined for myosin composition using electrophoresis. Results indicate that changes occurred in structural and functional muscle characteristics in both slow and fast muscle fiber types. These results are detailed and discussed.     

The isometric length-force models of nine different skeletal muscles.

The length-force relations of nine different skeletal muscles in the hindlimb of the cat were determined experimentally, with electrical stimulation of the sciatic nerve as the activation mode. It was shown that the active-, passive-, and total-force patterns varied widely among the muscles. The tibialis posterior (TP), medial and lateral gastrocnemius (MG, LG) and flexor digitorum longus (FDL) had a symmetric active-force curve, whereas the tibialis anterior (TA), peroneus brevis (PB), peroneus longus (PL), extensor digitorum longus (EDL), and soleus (SOL) had an asymmetric curve which exhibits about 25% of the maximal isometric force at extreme lengths. The SOL, EDL, and LG had a low-level passive force which appeared at short muscle length, whereas all other muscles exhibited initial passive force just before the optimal length. The total force was rising quasi-linearly for the SOL, whereas the other muscles exhibited an intermediate plateau about the optimal length. The LG and FDL had a substantial but temporary intermediate dip in the total force as the muscle was elongated past the optimal length. The elongation range of the various muscles also varied, ranging from +/- 15 to +/- 30% of the optimal length. The elongation range was symmetric for the FDL, LG, MG, TP, SOL, and EDL, and asymmetric for the PL, PB, and TA, being -12 to + 17%, -12 to + 17%, and -35 to + 12%, respectively. Two different models which incorporate muscle architecture were successfully fitted to the experimental data of the muscles except for the MG and TA. The architecture of these two muscles is highly nonhomogeneous and contains compartments with two pennation patterns or two different optimal lengths. New models, which add spatially and temporally the individual characteristics of each compartment of the muscles, were constructed for these two muscles. The new models demonstrated high correlation to the experimental data obtained from the MG and TA. It was concluded that the length-force relation varies widely among various skeletal muscles and is probably dependent on the primary function of the muscle in the context of integrated movement; this is a manifestation of architectural factors such as fiber pennation pattern and angle, cross-sectional area, ratio of muscle to tendon length, distribution of the fiber length within the muscle and compartmental pennation.     

Myofascial force transmission between antagonistic rat lower limb muscles: Effects of single muscle or muscle group lengthening

Effects of lengthening of the whole group of anterior crural muscles (tibialis anterior and extensor hallucis longus muscles (TA + EHL) and extensor digitorum longus (EDL)) on myofascial interaction between synergistic EDL and TA + EHL muscles, and on myofascial force transmission between anterior crural and antagonistic peroneal muscles, were investigated. All muscles were either passive or maximally active. Peroneal muscles were kept at a constant muscle tendon complex length. Either EDL or all anterior crural muscles were lengthened so that effects of lengthening of TA + EHL could be analyzed. For both lengthening conditions, a significant difference in proximally and distally measured EDL passive and active forces, indicative of epimuscular myofascial force transmission, was present. However, added lengthening of TA + EHL significantly affected the magnitude of the active and passive load exerted on EDL. For the active condition, the direction of the epimuscular load on EDL was affected; at all muscle lengths a proximally directed load was exerted on EDL, which decreased at higher muscle lengths. Lengthening of anterior crural muscles caused a 26% decrease in peroneal active force.

Extramuscular myofascial connections are thought to be the major contributor to the EDL proximo-distal active force difference. For antagonistic peroneal complex, the added distal lengthening of a synergistic muscle increases the effects of extramuscular myofascial force transmission.     

Restoration of fast muscle characteristics following cessation of chronic stimulation: physiological, histochemical and metabolic changes during slow-to-fast transformation

Implantable electronic stimulators were used to subject fast-twitch tibialis anterior and extensor digitorum longus muscles of adult rabbits to a chronically increased level of use. Stimulation was discontinued after 6 weeks and physiological, histochemical and biochemical properties of the muscles were examined at intervals over the ensuing 20 weeks. Previous work had shown that 6 weeks of stimulation was sufficient to bring about a substantial transformation of type in fast-twitch muscles, which then exhibited much of the character of muscles of the slow-twitch type. The present experiments showed that these stimulation-induced changes were completely reversible. The time-course of reversion was such that the muscles had recovered their original fast properties by about 12 weeks after the cessation of stimulation. The contractile characteristics and post-tetanic potentiation typical of fast muscle returned rapidly, in only 3-4 weeks, and over the same period the proportion of histochemical type 1 fibres declined from about 70% to control levels. Changes in fatigue-resistance, capillary density and enzyme activity followed a more prolonged time-course; in particular, the decline in the activity of enzymes of oxidative metabolism corresponded closely to that already established for the mitochondrial volume fraction. Reacquisition of fast properties was not accompanied by any changes in specific force-generating capacity. Observations from these experiments and from a related morphological study fit into a 'first-in, last-out' pattern for the response to stimulation and recovery. The slow-to-fast reversion that takes place during the recovery period provides a further opportunity for testing causal associations within the events underlying type transformation. It has important consequences for therapeutic applications that make use of the fatigue-resistant character of chronically stimulated muscle.     

Effect of prior chronic contractile activity on mitochondrial function and apoptotic protein expression in denervated muscle.

Skeletal muscle is highly adaptable in response to increases and decreases in contractile activity. The purpose of this study was to determine whether the preconditioning of skeletal muscle has a protective effect against subsequent denervation-induced apoptotic protein expression. To investigate this, we chronically stimulated the tibialis anterior and extensor digitorum longus muscles for 7 days (10 Hz, 3 h/day) before 7 days of denervation. Denervation reduced total cytochrome-c oxidase activity by 39%, which was likely a consequence of a decrease in subsarcolemmal (SS) mitochondria. This decrease in the SS subfraction was prevented by prior chronic stimulation and, as a result, maintained total mitochondrial content at control levels. The expression of Bax was elevated 2.2-fold by denervation, and prior chronic stimulation did not attenuate this increase. This produced a increase in the Bax-to-Bcl-2 ratio, indicating greater muscle apoptotic susceptibility. Denervation also decreased state 3 respiration in SS and intermyofibrillar mitochondria and elevated state 4 reactive oxygen species production within both mitochondrial subfractions. These changes were not prevented by prior chronic stimulation. Furthermore, the antioxidant protein MnSOD was also reduced by denervation, whereas Beclin-1 was markedly elevated. This suggests that autophagic cell death could also play a significant part in denervation-induced muscle atrophy. Thus, despite prior chronic stimulation, denervation increases the apoptotic susceptibility of skeletal muscle by altering the Bax-to-Bcl-2 ratio, by increasing reactive oxygen species production, and by reducing the expression of MnSOD. Whether a more extensive stimulation paradigm would be more effective in attenuating apoptosis before muscle disuse remains to be determined.     

Muscle plasticity: comparison of a 30-Hz burst with 10-Hz continuous stimulation

The changes in the contractile properties induced by a 30-Hz phasic stimulation paradigm were measured and compared with the changes induced by a 10-Hz continuous stimulation paradigm. The study was performed on the tibialis anterior muscles of cats with one paradigm applied to one hindlimb muscle and the other to the contralateral limb. Both hindlimb muscles received the same number of stimuli in a day, making the average stimulation frequency 10 Hz. Two periods of daily stimulation were studied, 8 and 24 h/day. Muscles stimulated at 30 Hz produced greater overall tetanic tension and, during a prolonged stimulation test, exerted a greater mean tension than muscles stimulated at 10 Hz (50 and 32% increase for animals stimulated for 8 and 24 h/day, respectively). Muscle mass was least reduced and fewer pathological abnormalities were observed in the muscles stimulated at 30 Hz. There were no apparent differences in the histochemistry or biochemistry between muscles stimulated at 10 and 30 Hz, which could account for these differences in muscle properties. These results indicate the 30-Hz paradigm may be better suited than 10 Hz continuous stimulation for applications requiring sustained muscle tension such as correction of scoliosis or muscle conditioning for motor prostheses.     

Estrogen and gender do not affect fatigue resistance of extensor digitorum longus muscle in rats.

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.     

Effects of re-feeding after prolonged starvation on pyruvate dehydrogenase activities in heart, diaphragm and selected skeletal muscles of the rat.

We investigated the capacity for pyruvate oxidation in skeletal muscle, diaphragm and heart after starvation and re-feeding. Starvation for 48 h decreased pyruvate dehydrogenase (PDH) activity in soleus (by 47%), extensor digitorum longus (64%), gastrocnemius (86%), diaphragm (87%), adductor longus (90%), tibialis anterior (92%) and heart (99%). Chow re-feeding increased PDH activity in all muscles to 43-78% of the fed value within 2 h. However, complete re-activation was not observed for at least 4-6 h, during which time hepatic glycogen was replenished. We discuss the importance of muscle PDH activity in relation to sparing carbohydrate for hepatic glycogen synthesis.     

Effect of electrical stimulation on intracellular triacylglycerol in isolated skeletal muscle

The contribution of intracellular triacylglycerol (TG) as a substrate for skeletal muscle during electrical stimulation is equivocal. Therefore, the purpose of this study was to investigate the effect of electrical stimulation on the TG content in the isolated intact rat flexor digitorum brevis skeletal muscle preparation by use of two different stimulation protocols. Muscles were electrically stimulated for 1 h either continuously at 1 Hz or intermittently (30 s on, 60 s off) at 5 Hz while incubated in 21 degrees C Krebs bicarbonate buffer (pH 7.4) that contained 11 mM glucose. Control muscles were either frozen immediately after excision or incubated for 1 h. TG content was significantly decreased (P less than 0.05) compared with control concentrations in both stimulated muscle groups, with the greatest reduction (60%) occurring after 5-Hz intermittent stimulation. These data indicate that intramuscular TG is hydrolyzed in response to electrical stimulation in the isolated flexor digitorum brevis muscle preparation. In addition, the type of stimulation (higher frequency intermittent vs. lower frequency continuous) employed influences the amount of intracellular TG hydrolyzed.