Junctophilin damage contributes to early strength deficits and EC coupling failure after eccentric contractions.

Junctophilins (JP1 and JP2) are expressed in skeletal muscle and are the primary proteins involved in transverse (T)-tubule and sarcoplasmic reticulum (SR) membrane apposition. During the performance of eccentric contractions, the apposition of T-tubule and SR membranes may be disrupted, resulting in excitation-contraction (EC) coupling failure and thus reduced force-producing capacity. In this study, we made three primary observations: 1) through the first 3 days after the performance of 50 eccentric contractions in vivo by the left hindlimb anterior crural muscles of female mice, both JP1 and JP2 were significantly reduced by approximately 50% and 35%, respectively, while no reductions were observed after the performance of nonfatiguing concentric contractions; 2) following the performance of a repeated bout of 50 eccentric contractions in vivo, only JP1 was immediately reduced ( approximately 30%) but recovered by 3-day postinjury in tandem with the recovery of strength and EC coupling; and 3) following the performance of 10 eccentric contractions at either 15 degrees or 35 degrees C by isolated mouse extensor digitorum longus (EDL) muscle, isometric force, EC coupling, and JP1 and JP2 were only reduced after the eccentric contractions performed at 35 degrees C. Regression analysis of JP1 and JP2 content in tibialis anterior and EDL muscles from each set of experiments indicated that JP damage is significantly associated with early (0-3 days) strength deficits after performance of eccentric contractions (R = 0.49; P < 0.001). As a whole, the results of this study indicate that JP damage plays a role in early force deficits due to EC coupling failure following the performance of eccentric contractions.

Effects of eccentric exercise training on cortical bone and muscle strength in the estrogen-deficient mouse.

The purpose of this study was to determine whether eccentrically biased exercise training could attenuate changes in muscle and bone function associated with estrogen deficiency in the mouse model. Four groups of ICR mice were used: control (Con), sham ovariectomized (Sham), ovariectomized (OVX), and ovariectomized + high-force resistance training (OVX+Train). All groups except Con were implanted with a nerve cuff surrounding the peroneal nerve to stimulate the left ankle dorsiflexors. Training consisted of 30 stimulated eccentric contractions of the left ankle dorsiflexors at approximately 150% of peak isometric torque every third day for 8 wk. After the training period, groups were not significantly different with regard to peak torque or muscle size. However, the tibial midshaft of the trained leg in the OVX+Train mice exhibited greater stiffness (+15%) than that in the untrained OVX mice, which could not be explained by changes in cross-sectional geometry of the tibia. Scaling of bone mechanical properties to muscle strength were not altered by ovariectomy or training. These data indicate that eccentric exercise training in adult mice can significantly increase bone stiffness, despite the absence of ovarian hormones.

Enantiopure 2,3-dihydro-4-pyridones as synthetic intermediates: a concise asymmetric synthesis of (+)-allopumiliotoxin 267A.

[figure: see text] A concise asymmetric synthesis of (+)-allopumillotoxin 267A has been accomplished using an enantiopure dihydropyridone building block. The synthesis is highly stereoselective and requires 10 steps from readily available material.

Excitation-contraction uncoupling: major role in contraction-induced muscle injury.

The mechanisms that account for the strength loss after contraction-induced muscle injury remain controversial. We present data showing that (1) most of the early strength loss results from a failure of excitation-contraction coupling and (2) a slow loss of contractile protein in the days after injury prolongs the recovery time.

Time course changes in [Ca2+]i, force, and protein content in hindlimb-suspended mouse soleus muscles.

BACKGROUND: Exposure to reduced gravitational forces during spaceflight is associated with significant reductions in skeletal muscle mass and strength. The purpose of this study was to test the hypothesis that increases in resting cytosolic free calcium concentration ([Ca2+]i) would precede reductions in protein content and maximal isometric tetanic force (Po) in mouse soleus muscle after initiation of hindlimb suspension. METHODS: Female ICR mice (n = 42) were hindlimb suspended for 1, 2, 3, 5, or 7 d; weight-matched mice were used as controls. Following the hindlimb suspension, the left soleus muscle was used to determine Po in vitro and the right soleus muscle was used to determine protein content and [Ca2+]i via confocal laser scanning microscopy. RESULTS: Compared with controls, [Ca2+]i was elevated by 38% at 2 d, and 117% at 7 d. Compared with controls, soleus muscle total and myofibrillar protein contents were reduced 27-29% and 30-34%, respectively, at 5-7 d after initiation of hindlimb suspension. Compared with controls, soleus muscle Po was decreased by 24% at 3 d, and 38% at 7 d. CONCLUSION: It appears that resting cytosolic Ca2+ homeostasis is disturbed soon after the initiation of hindlimb suspension, and these elevations in [Ca2+]i may play a role in initiating soleus muscle atrophy.

Decreased EMG median frequency during a second bout of eccentric contractions.

PURPOSE: Others have reported preferential recruitment of fast motor units in muscles during performance of eccentric contractions and there is evidence that fast muscle fibers are more susceptible to eccentric contraction-induced injury. We tested the hypothesis that during a second bout of maximal eccentric contractions 1 wk after the first, there would be a reduction in the electromyographic (EMG) median frequency (MF) with minimal change in the EMG root-mean-square (RMS), indicating greater reliance on slower motor units. This could provide an explanation for the enhanced resistance to eccentric contraction-induced injury after a single bout of eccentric exercise. METHODS: Human subjects performed 50 maximal voluntary eccentric (N = 10) or concentric (N = 10) contractions of the anterior crural muscles on two occasions separated by 1 wk. To determine whether MF changes during the second bout could be a consequence of injury to fibers in fast motor units, the anterior crural muscles of mice were electrically stimulated to perform 50 maximal eccentric (N = 10) or concentric (N = 9) contractions on two occasions separated by 1 wk. In both the humans and mice, torque production and tibialis anterior muscle RMS and MF were measured during the two exercise bouts. RESULTS: In human tibialis anterior muscle, MF was 30% lower (P < 0.01) during the second eccentric bout although RMS was the same. In the mice, RMS and MF were unchanged at any time after the first eccentric bout despite torque deficits similar to those observed in the humans. CONCLUSIONS: The data indicate that with repetition of maximal voluntary eccentric contractions, there is an increased activation of slow motor units and a concomitant decrease in activation of fast units.

Intracellular Ca2+ transients in mouse soleus muscle after hindlimb unloading and reloading.

The objective of this study was to determine whether altered intracellular Ca(2+) handling contributes to the specific force loss in the soleus muscle after unloading and/or subsequent reloading of mouse hindlimbs. Three groups of female ICR mice were studied: 1) unloaded mice (n = 11) that were hindlimb suspended for 14 days, 2) reloaded mice (n = 10) that were returned to their cages for 1 day after 14 days of hindlimb suspension, and 3) control mice (n = 10) that had normal cage activity. Maximum isometric tetanic force (P(o)) was determined in the soleus muscle from the left hindlimb, and resting free cytosolic Ca(2+) concentration ([Ca(2+)](i)), tetanic [Ca(2+)](i), and 4-chloro-m-cresol-induced [Ca(2+)](i) were measured in the contralateral soleus muscle by confocal laser scanning microscopy. Unloading and reloading increased resting [Ca(2+)](i) above control by 36% and 24%, respectively. Although unloading reduced P(o) and specific force by 58% and 24%, respectively, compared with control mice, there was no difference in tetanic [Ca(2+)](i). P(o), specific force, and tetanic [Ca(2+)](i) were reduced by 58%, 23%, and 23%, respectively, in the reloaded animals compared with control mice; however, tetanic [Ca(2+)](i) was not different between unloaded and reloaded mice. These data indicate that although hindlimb suspension results in disturbed intracellular Ca(2+) homeostasis, changes in tetanic [Ca(2+)](i) do not contribute to force deficits. Compared with unloading, 24 h of physiological reloading in the mouse do not result in further changes in maximal strength or tetanic [Ca(2+)](i).

Uncoupling of in vivo torque production from EMG in mouse muscles injured by eccentric contractions.

1. The main objective of this study was to determine whether eccentric contraction-induced muscle injury causes impaired plasmalemmal action potential conduction, which could explain the injury-induced excitation-contraction coupling failure. Mice were chronically implanted with stimulating electrodes on the left common peroneal nerve and with electromyographic (EMG) electrodes on the left tibialis anterior (TA) muscle. The left anterior crural muscles of anaesthetized mice were stimulated to perform 150 eccentric (ECC) (n = 12 mice) or 150 concentric (CON) (n = 11 mice) contractions. Isometric torque, EMG root mean square (RMS) and M-wave mean and median frequencies were measured before, immediately after, and at 1, 3, 5 and 14 days after the protocols. In parallel experiments, nicotinic acetylcholine receptor (AChR) concentration was measured in TA muscles to determine whether the excitation failure elicited a denervation-like response. 2. Immediately after the ECC protocol, torque was reduced by 47-89 %, while RMS was reduced by 9-21 %; the RMS decrement was not different from that observed for the CON protocol, which did not elicit large torque deficits. One day later, both ECC and CON RMS had returned to baseline values and did not change over the next 2 weeks. However, torque production by the ECC group showed a slow recovery over that time and was still depressed by 12-30 % after 2 weeks. M-wave mean and median frequencies were not affected by performance of either protocol. 3. AChR concentration was elevated by 79 and 368 % at 3 and 5 days, respectively, after the ECC protocol; AChR concentration had returned to control levels 2 weeks after the protocol. At the time of peak AChR concentration in the ECC protocol muscles (i.e. 5 days), AChR concentration in CON protocol muscles was not different from the control level. 4. In conclusion, these data demonstrate no major role for impaired plasmalemmal action potential conduction in the excitation-contraction coupling failure induced by eccentric contractions. Additionally, a muscle injured by eccentric contractions shows a response in AChR concentration similar to a transiently denervated muscle.

E-C coupling failure in mouse EDL muscle after in vivo eccentric contractions.

The objectives of this research were to determine the contribution of excitation-contraction (E-C) coupling failure to the decrement in maximal isometric tetanic force (Po) in mouse extensor digitorum longus (EDL) muscles after eccentric contractions and to elucidate possible mechanisms. The left anterior crural muscles of female ICR mice (n = 164) were injured in vivo with 150 eccentric contractions. Po, caffeine-, 4-chloro-m-cresol-, and K+-induced contracture forces, sarcoplasmic reticulum (SR) Ca2+ release and uptake rates, and intracellular Ca2+ concentration ([Ca2+]i) were then measured in vitro in injured and contralateral control EDL muscles at various times after injury up to 14 days. On the basis of the disproportional reduction in Po (approximately 51%) compared with caffeine-induced force (approximately 11-21%), we estimate that E-C coupling failure can explain 57-75% of the Po decrement from 0 to 5 days postinjury. Comparable reductions in Po and K+-induced force (51%), and minor reductions (0-6%) in the maximal SR Ca2+ release rate, suggest that the E-C coupling defect site is located at the t tubule-SR interface immediately after injury. Confocal laser scanning microscopy indicated that resting [Ca2+]i was elevated and peak tetanic [Ca2+]i was reduced, whereas peak 4-chloro-m-cresol-induced [Ca2+]i was unchanged immediately after injury. By 3 days postinjury, 4-chloro-m-cresol-induced [Ca2+]i became depressed, probably because of decreased SR Ca2+ release and uptake rates (17-31%). These data indicate that the decrease in Po during the first several days after injury primarily stems from a failure in the E-C coupling process.

A stimulating nerve cuff for chronic in vivo measurements of torque produced about the ankle in the mouse.

Specific muscle training and chronic contractile measurements are difficult in rodents, especially in the mouse. The primary reason for this is the lack of a means for stimulating the motor nerve that does not damage the nerve and that permits reproducible measurements of contractility. In this paper, we describe procedures for the construction and implantation of a stimulating nerve cuff for use on the mouse common peroneal nerve. We demonstrate that nerve cuff implantation success rates can be high (i.e., 75-93%), as determined from measurements of maximal isometric torque produced by the anterior crural muscles. Isometric torque production is not adversely affected by the nerve cuff because the torque produced matches that observed in our established percutaneous stimulation model. We also demonstrate that use of the nerve cuff for stimulation is compatible with electromyographic measurements made on the tibialis anterior muscle, with no sign of stimulation artifact in the electromyographic signal.

Dissociation of force production from MHC and actin contents in muscles injured by eccentric contractions.

The primary purpose of this study was to determine the relationship between myosin heavy chain (MHC) and actin contents and maximum isometric tetanic force (Po) in mouse extensor digitorum longus (EDL) muscles following eccentric contraction-induced injury. Po and protein contents were measured in injured (n = 80) and contralateral control (n = 80) EDL muscles at the following time points after in vivo injury: sham, 0, 0.25, 1, 3, 5, 14, and 28 days. Po was reduced by 37 +/- 2.3% to 49 +/- 3.8% (p < or = 0.05), while MHC and actin contents were unaltered from 0 to 3 days after injury. Whereas Po partially recovered between 3 and 5 days (from -49 +/- 3.8% to -35 +/- 3.6%), MHC and actin contents in the injured muscles declined by 19 +/- 4.9% and 20 +/- 5.3%, respectively, by 5 days compared with control muscles. Decrements in Po were similar to the reductions in MHC and actin contents at 14 (approximately 24%) and 28 (approximately 11%) days. Evaluation of myofibrillar and soluble protein fractions indicated significant reductions in the content of major proteins at 5 and 14 days. Immunoblots of heat shock protein 72 revealed elevations starting at 0.25 days, peaking during 1-3 days, and declining after 5 days. These findings indicate that decreased contractile protein content is not related to the initial decrease in Po. However, decreased MHC and actin contents could account for 58% of the Po reduction at 5 days, and for nearly all the decrements in Po from 14 to 28 days.

Decreased contraction economy in mouse EDL muscle injured by eccentric contractions.

The objective of this study was to find out whether basal and/or active energy metabolism are altered in isolated mouse extensor digitorum longus muscle injured by eccentric (Ecc) contractions. Measurements of basal O2 consumption and isometric tetanus O2 recovery cost were made at 25 degrees C on muscles that had done either 10 Ecc, 10 isometric (Iso), or no contractions (No). In parallel experiments, rates of lactate and pyruvate production were measured to estimate the anaerobic contribution. Basal O2 consumption was unaffected by the type of protocol performed (P = 0.07). However, the tetanus O2 cost per force-time integral was elevated by 30-36% for the Ecc protocol muscles over that for the Iso and No protocol muscles. When including the increased lactate production by the Ecc protocol muscles, the total energetic cost per force-time integral was 53% higher than that for the Iso protocol muscles [2.35 +/- 0.17 vs. 1.54 +/- 0.18 mumol O2/(N.m.s)]. The decreased economy was attributed to two factors. First, in skinned fibers isolated from the injured muscles, the ratio of maximal actomyosin adenosinetriphosphatase activity to force production was up by 37.5%, suggesting uncoupling of ATP hydrolysis from force production. Second, increased reliance on anaerobic metabolism along with the fluorescent microscopic study of mitochondrial membrane potential and histochemical study of ATP synthase suggested an uncoupling of oxidative phosphorylation in the injured muscles.

Interaction between clenbuterol and run training: effects on exercise performance and MLC isoform content.

The purpose of this study was to determine the separate and combined effects of clenbuterol (CB) administration and interval training on running performance and myosin light-chain (MLC) isoform expression in mouse skeletal muscle. Mice were randomly assigned to one of four treatment groups: 1) control (Con), 2) exercise (Ex), 3) drug (CB), or 4) exercise + drug (Ex + CB). CB and Ex + CB mice were given CB (1.6 mg/kg) orally 4 days/wk. Ex and Ex + CB mice were trained 4 days/wk on a motorized treadmill (3 sets of 3 min, 36-40 m/min, 10-17% grade, 30-s recovery). After 8 wk of treatment, exercise conditioning increased total work performed 58% in the Ex group during a run-to-exhaustion treadmill test, whereas CB decreased total work by 25% in the CB group; in combination with exercise training, CB treatment eliminated the Ex-induced increase in work. Polyacrylamide gel electrophoresis indicated that run training, CB treatment, or a combination did not (P > 0.01) promote changes in fast and slow MLC isoforms in the soleus, gastrocnemius, or tibialis anterior muscles. Although not different from each other after 8 wk, CB and Ex + CB treatments produced significantly greater values than Con and Ex for the following variables: muscle mass (17-46%), total protein (22-50%), and myofibrillar protein (19-53%). It was concluded that CB decreases exercise performance and that the combination of Ex and CB have antagonistic effects on running performance; the two treatments do not interact to diminish the anabolic effects of CB on skeletal muscle and do not alter MLC isoform profiles.

Differential effects of anesthetics on in vivo skeletal muscle contractile function in the mouse.

The purpose of this study was to evaluate the effects of four anesthetic regimens on in vivo contractile function of mouse ankle dorsiflexor muscles. The torque-frequency and torque-velocity relationships were determined for the following anesthetics: fentanyl-droperidol and diazepam (F-d/d); ketamine and xylazine (K/x); pentobarbital sodium (Ps); and methoxyflurane (Mf). Mf, Ps, and F-d/d regimens resulted in comparable contractile responses at low doses, whereas K/x produced a relative depression in isometric contractile function as shown by a decrease in the torque-time integral at the 300-Hz stimulation frequency (-13.9%; P < 0.05). Moreover, K/x caused a shift to the left in the torque-frequency curve as indicated by increases in torque-time integrals at 25 and 50 Hz. Both Ps and F-d/d regimens exhibited dose-dependent effects during the isovelocity contractions. Ps significantly reduced work (-28.7%) and average power (-28.9%) at 800 degrees/s at the high dose. In contrast, F-d/d anesthesia resulted in increases in peak torque (16-20%) and work (15-18%) output at all eccentric contraction velocities at the high dose, whereas average power was increased only at -800 (17%) and -1,000 degrees/s (17%). In conclusion, commonly used anesthetic regimens can affect the contractile response in vivo; K/x and Ps yield smaller torque outputs, whereas Mf and F-d/d consistently produce larger contractile responses. Mf and F-d/d are recommended for use in studying skeletal muscle function in mice in vivo.

Muscle function and protein metabolism after initiation of eccentric contraction-induced injury.

This study was designed to determine the relationship between skeletal muscle function and protein metabolism after initiation of eccentric contraction-induced injury. Mouse anterior crural muscles were injured in vivo, and then either immediately or 3, 6, 24, 48, 72, 120, or 336 h after injury muscles were isolated and studied for indexes of muscle function, injury, phagocyte infiltration, and protein metabolism. A group of mice were administered anti-polymorphonuclear cell and anti-macrophage antisera in an attempt to reduce phagocytic infiltration into injured muscle. Force production in extensor digitorum longus muscles was reduced 55% immediately after injury induction and did not recover significantly until 120 h postinjury (28% below baseline). However, rates of protein degradation were not elevated until 48 h postinjury (60% above normal) and were not correlated with the changes in force production (r = -0.37; P = 0.24). Phagocytic infiltration was evident 24-120 h postinjury and was correlated with the elevated protein degradation rates (r = 0.75; P < 0.01). Protein synthesis rates began to increase approximately 48 h after injury was induced and were elevated by 83% 5 days postinjury. Fourteen days after injury, muscle protein degradation and synthesis rates had returned to normal, as well as specific force production, and phagocytic infiltration was not detected. However, muscle mass, protein content, and absolute force production were lower than normal. Antisera-treated mice were rendered neutropenic, but there was no difference in any variable measured between muscles from these mice and muscles from normal mice.

The effects of active and passive recovery on short-term, high intensity power output.

The advantage of active over passive recovery from long and intermediate duration exercise is well documented. Success has been attributed to metabolite washout and/or lactate (La) utilization by the active musculature. This study was designed to determine whether active recovery was superior to passive rest during short duration, high intensity performance. On 4 separate days, six athletes performed a set of eight 6-s power tests separated by 30-s recovery intervals under two recovery conditions. Recovery conditions involved either sitting passively on the bike (P) or actively pedaling (A) at 60 rpm using 1 kg resistance. A MANOVA on peak power (PP), fatigue rate (F), and total work (TW) showed a significant difference due to recovery condition, F(3, 169); p < .0001. Separate ANOVAs revealed that PP (A = 1192.85 watts, P = 1134.57 watts; p < .0001) and TW (A = 6.59 kJ, P = 6.23 kJ; p < .0001) differed significantly between conditions. No difference was found for F (A = 80.12 watts.sec-1, P = 79.80 watts.sec-1). Results indicate that active recovery provides superior performance to passive rest in repeated short-term, high intensity power activities.