INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

MULTIPLE EXPOSURES TO RESISTANCE exercise produce adaptive changes in skeletal muscles, including an increase in mass (hypertrophy) and contractile strength (19). Muscle adaptation involves exercise-induced changes in the expression of specific genes and an accompanying increase in protein synthesis (39). A number of cellular pathways have been reported to contribute to the adaptive response, including mechanosensitive cation-selective channels in the muscle membrane (39). Mechanosensitive or stretch-activated ion channels (SAC) may be particularly important when the exercise involves eccentric or lengthening contractions. SAC are a constituent of the cell membrane in most tissues and contribute to the regulation of a wide variety of cellular functions (23, 29). They have been identified in skeletal muscle (13), but their role in muscle function remains uncertain. SAC appear to have an important role in the remodeling of cardiac and smooth muscle cells in response to mechanical strain (17, 18, 30, 31), and they may play a similar role in exercising skeletal muscle.

Several types of SAC have been identified, and one or more of these types appears to be present in most cells (23, 29). In general, SAC provide a major pathway for the transduction of mechanical stimuli in cells, but the subsequent response to the stimulus is tissue specific. In cardiac and smooth muscles, for example, SAC have been linked to mechanically induced cell growth (17, 18), and it appears probable that SAC could contribute to modulating stretch or eccentric exercise-induced cell growth in skeletal muscle as well (37). SAC in cardiac muscle are permeant to monovalent (K⁺ and Na⁺) or divalent (Ca²⁺) cations, however, and are associated with membrane depolarization and alterations in action potential generation and conduction (14). Eccentric contraction-induced SAC activation could also contribute to membrane-voltage changes in skeletal muscle, affecting excitation-contraction coupling.

Identifying the physiological role of SAC in any tissue has been hampered by the absence of specific channel inhibitors or activators. Nonetheless, nonselective blockers, including aminoglycoside antibiotics and the trivalent cation gadolinium (Gd³⁺), have been used effectively to inhibit cation-permeable SAC in a variety of tissues, including cardiac and skeletal muscle. Sokabe et al. (33), for example, used patch-clamp methods to show that aminoglycoside antibiotics blocked SAC in cultured chick skeletal muscle cells in a voltage- and dose-dependent manner. Aminoglycosides are viewed as nonselective SAC blockers because they inhibit other types of cation channels as well, but their dissociation constants for SAC are much lower than for the other channels (25). The cation Gd³⁺ is reported to be the most potent SAC blocker available, but it is also nonselective, and its toxicity limits its use to in vitro preparations (14). Gd³⁺ blocks SAC by binding to a site in the permeation pathway for Na⁺ and Ca²⁺ (23), but it also displaces Ca²⁺ and other ions from specific and nonspecific membrane binding sites, and it can block L-type Ca²⁺ currents (14). Sadoshima et al. (32), however, compared the effect of Gd³⁺ on SAC with its effect on voltage-gated ion channels and concluded that Gd³⁺ has a higher affinity for SAC than for other cation channels. Application of 10 µM Gd³⁺ to cardiac myocytes, for example, completely inhibited SAC, whereas 50 µM Gd³⁺ produced only a 16% inhibition of voltage-gated Na⁺ channels and a 32% inhibition of L-type Ca²⁺ channels. Similarly, 10 µM Gd³⁺ produced a significant inhibition (>90%) of stretch-induced arrythmias in isolated perfused dog hearts, primarily as a consequence of its effect on SAC (14).

Gd³⁺ and the aminoglycoside antibiotic streptomycin were used as SAC blockers in the present experiments to help identify conductance pathways that contribute to the significant depolarization observed in muscle cells after eccentric contractile activity. Both agents were applied to isolated muscles maintained in vitro. Rats were also treated with streptomycin before and after exposure to eccentric contractions to inhibit SAC in vivo during the contractile protocol. Gd³⁺ and streptomycin are nonselective channel blockers and may have actions in addition to their effect on SAC, but, they are, at present, the most effective SAC inhibitors available. They were used in conjunction with other Na⁺ channel blockers as one means of testing the hypothesis that eccentric contractions in skeletal muscle lead to the activation of SAC and an associated increase in Na⁺ conductance.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Female 3-mo-old Sprague-Dawley rats, with body weights ranging from 240 to 260 g, were used in the experiments. All animal care and use protocols followed approved University of California and National Institutes of Health guidelines. The animals were housed in a temperature-controlled room (19-21°C) with a 12:12-h light-dark cycle. The rats were provided unlimited access to standard rat chow and water. Rats were anesthetized with pentobarbital sodium (50 mg/kg ip) within 30 min of engaging in the exercise protocol and before the terminal experiment to evaluate muscle properties. Muscle contractile and electrophysiological properties were evaluated in one group of anesthetized rats immediately (1-2 h) after the exercise protocol. Exercised rats that were to be evaluated at later time points were placed on a warming pad and allowed to recover from the anesthesia. Those animals not immediately evaluated were returned to the animal quarters until the terminal experiment.

Exercise protocol. Anesthetized animals performed high- or low-repetition resistance exercise using a pulley device similar to that described by Wong and Booth (40). The rat was placed in the prone position on the supporting platform of the apparatus, which was designed to stabilize the lower leg and allow full ankle rotation. The right hindlimb was passed through a circular opening in the platform, and the knee was stabilized on the platform. The hind foot was attached directly to a plate connected to the lever arm of the pulley system, and the ankle was stabilized with the foot at a 90° angle with respect to the lower leg (neutral position). Two monopolar stainless steel needle electrodes were inserted percutaneously near the sciatic notch to stimulate the sciatic nerve. Supramaximal stimulation of the sciatic nerve above the branch point of the tibial and peroneal nerves caused the plantar flexors (triceps surae) to contract concentrically, stretching the dorsiflexors as they were also maximally activated. The dorsiflexors thus contract eccentrically, in opposition to the stronger ankle extensors. Data reported by Ashton-Miller et al. (3) concerning the biomechanical properties of ankle rotation in the mouse indicate that the dorsiflexors [tibialis anterior (TA) and extensor digitorum longus] and plantar flexors (soleus and plantaris) are at optimum length when the ankle is at the 90° neutral position. They also note that full plantar flexion from the neutral position produces a 10% increase in the length of TA. Supramaximal stimulation consisted of 100-Hz stimulus trains with a train duration of 2.5 s. The low-repetition exercise protocol consisted of four sets of six repetitions (24 repetitions) with a 20-s rest between repetitions and a 5-min rest between sets. Muscles contracted for 1 min over an exercise period of 30 min. The high-repetition protocol consisted of eight segments with four sets of six repetitions in each segment (192 repetitions). Sixty-second rest periods were inserted between the first 16 sets, and 30-s rest periods were inserted between the second 16 sets. A 210-s rest period was inserted between each segment. Muscles contracted for 8 min over an exercise period of 75 min. Exercise protocols were repeated after 10 days (high-repetition group) or 14 days (low-repetition group) in the groups subject to more than one exposure to the protocol.

Contractile measurements. Contractile deficits provide an indirect measure of muscle injury and recovery after eccentric exercise (5, 21). TA contractile function was determined in situ 1, 2, 5, and 14 days after exposure to the low-repetition protocol and 2 and 10 days after exposure to the high-repetition protocol. The animals were anesthetized with pentobarbital sodium (50 mg/kg ip), and the TA and peroneal nerve in each hindlimb were exposed. The rat was placed on a warming pad to maintain body temperature (38 ± 1°C), and the animal and pad were placed on a rat spinal frame. Each hindlimb was immobilized by clamping the knee and ankle joints with clamps fixed to the metal frame. The distal tendon of the TA was isolated and attached to a force transducer (Grass FT-03) with silk suture (4-0). The peroneal nerve was placed on a bipolar platinum-hook electrode and stimulated supramaximally (0.05 ms, 7-10 V). The nerve was laid over the electrode and crushed proximal to the electrode to avoid reflex or antidromic activation of the motor neurons. The TA was adjusted to optimal length by gradually increasing muscle length until maximum twitch amplitude was achieved. Maximum isometric twitch tension, time to peak twitch tension, twitch half relaxation time, rate of twitch relaxation, maximum isometric tetanic tension, and the maximum rate of force development during a tetanus at 330 Hz were recorded at 35 ± 0.5°C.

Electrophysiology. Resting membrane potentials (RMPs) were obtained from control and exercised TA both in situ and in vitro. The initial observations in RMP changes in response to eccentric exercise were performed in situ at various time points after either one or two exposures to eccentric contractions. Characterization of muscle membrane conductance after eccentric contractions was performed in vitro, where the composition of the extracellular solution could be controlled and known concentrations of Na⁺ channel blockers could be applied for a given period and then washed out. The in vitro experiments were used to characterize the effect of Gd³⁺, the most potent SAC inhibitor yet described. Recordings were obtained by using standard glass microelectrode techniques (4, 26). Electrodes were filled with 3 M KCl and had tip resistances of ~20-30 M. A Grass platinum reference electrode was placed in the proximal end of the TA. RMPs were recorded from TA in situ in rats anesthetized with pentobarbital sodium (50 mg/kg ip). The animals were placed on a thermal pad to maintain body temperature (37 ± 1°C) and were arranged on the rat spinal frame as in the contractile measurements. Control and exercised TA muscles were exposed, cleared of all overlying connective tissue, and covered with warm mineral oil to maintain temperature (35 ± 1°C) and prevent drying. Intracellular recordings were obtained from exercised TA and the contralateral control TA at the end of the exercise protocol and in separate groups of animals at 24, 48, and 72 h postexercise. Each exercise group contained at least four animals, and at least 40 fibers were sampled in each muscle.

Effect of changes in [K⁺]_o or substitution for [Na⁺]_o on RMP. The influence of extracellular [K⁺] ([K⁺]_o; brackets denote concentration) on the RMP of control and exercised TA immediately after a first exposure to eccentric exercise was determined in vitro. One leg in the anesthetized rat was exercised in situ. After the exercise, the TA from both the exercised and control legs were rapidly excised and placed in a chamber containing oxygenated HEPES-buffered physiological saline (containing 5 mM [K⁺]) maintained at room temperature (21°C). [K⁺] in the bathing solution was adjusted by reducing [K⁺]_o or by substituting [K⁺]_o for extracellular [Na⁺] ([Na⁺]_o) on an equimolar basis. Muscle RMP was determined in solutions containing [K⁺]_o at concentrations of 0.5, 1.0, 5.0, 10, 50, and 100 mM. The [K⁺][Cl] product was maintained constant by replacing [Cl] with [SO²₄] when required (16, 34). Membrane potentials were first measured in muscles bathed in the control solution (5 mM [K⁺]_o). After the control measurements, the 0.5 mM [K⁺]_o solution was substituted for the control solution, and RMP was measured. The chamber was drained at the end of each recording period and filled with the solution containing the next highest [K⁺]. The muscles were allowed to equilibrate in each solution for 10 min before recordings were obtained. RMPs were obtained from at least 20 cells in each muscle at each [K⁺]_o.

Effect of amiloride, TTX, streptomycin, and Gd³⁺ on RMP. The possible role of specific Na⁺ channels in the membrane depolarization observed immediately after eccentric exercise was evaluated in vitro. The excised TA was bathed initially in the control oxygenated, HEPES-buffered physiological solution, and a series of RMP measurements was obtained. The chamber was then drained and filled with a solution containing one of the following pharmacological agents. 1) The Na⁺/H⁺ exchange inhibitor amiloride at concentrations of 0.5 and 1.0 mM was dissolved in DMSO before addition to the bathing solution. The final concentration of DMSO was <0.05% (vol/vol). Control measurements did not include the addition of DMSO to the bathing solution (11). 2) The voltage-gated Na⁺ channel blocker TTX was added at a concentration of 10⁶ M (27). 3) The SAC blockers streptomycin and Gd³⁺ were added at concentrations of 1 mM and 10 µM, respectively (7, 31). Measurements were obtained from a minimum of 20 fibers/muscle in the presence of each agent.

Effect of exposure to streptomycin in vivo on the response to eccentric exercise. Rats were treated with streptomycin in vivo by adding it to the drinking water (4 g/l). Treatment began 6 days before the exercise. RMPs were recorded immediately (1-2 h) after or 1 or 2 days after exposure to eccentric exercise. Isometric contractile properties were evaluated in the low-repetition group 2 days after either the first or second exercise to assess exercise-induced muscle injury in the presence of streptomycin. The TA in the nonexercised, contralateral leg served as the control in each group.

Statistics. Results are expressed as means ± SD of the indicated number of measurements. Statistical analyses were performed by using the Statview SE software program (Abacus Concepts, Berkeley, CA). Statistical significance was accepted at P  0.05. An ANOVA was used to compare RMP in the exercise groups with the RMP in control groups. Differences indicated by the ANOVA were further analyzed by using the Fisher's t-test.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Maximum contractile force after initial exposure to eccentric exercise. A reduction in maximum contractile force has been used as an index of muscle injury after eccentric exercise (5, 21). The initial exposure to low- or high-repetition eccentric exercise was associated with a significant reduction in maximum isometric tension after the exercise (Fig. 1, A and B). The low-repetition contractile deficit persisted through 5 days, and force was restored by 14 days postexercise (Fig. 1A). High-repetition eccentric exercise produced a force deficit that was greater than the low-repetition deficit (as a percentage of control force) 2 days postexercise (Fig. 1B). Maximum force in the exercised TA was not different from the force produced by the contralateral control TA at 10 days postexercise. The maximum rate of tension development and the rate of relaxation of twitch force were also significantly lower 2 days after both the low-repetition (see Table 3) and high-repetition exercise (data not shown). The eccentric exercise protocol used in these experiments produced a significant and reproducible contractile deficit in the exercised TA, as reported previously (20).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effect of initial exposure to low-repetition (A; 24 contractions) or high-repetition (B; 192 contractions) resistance exercise protocol on maximum tetanic tension that could be generated by exercised tibialis anterior (TA) at indicated no. of days after exercise. Contractile deficit after exposure to eccentric exercise is a reflection of muscle injury produced by exercise (5, 12, 21). C, control TA. Values are means ± SD of measurements from at least 6 animals at each time point (in situ).

RMP measured in situ after a single exposure to the eccentric exercise protocol. Muscle cells in the TA showed persistent depolarization after a single exposure to either the low- or high-repetition eccentric exercise protocol. Mean RMP was significantly depolarized within 1-2 h after the exercise (Figs. 2A and 3, A and B) and remained significantly depolarized for 24 h (low repetition, Figs. 2B and 3A) or 48 h (high repetition, Fig. 3B). RMP in exercised muscle fibers returned to control values by 48 h (low repetition, Figs. 2C and 3A) or 72 h (high repetition, Fig. 3B) postexercise. Muscle fibers exposed to the high-repetition protocol were significantly more depolarized than fibers exposed to the low-repetition protocol 1-2 h after the exercise, but depolarization was similar in the two groups at 24 h postexercise. In contrast, the mean RMP of concentrically exercised TA 1-2 h after the exercise was not significantly different from the mean RMP of control TA (Fig. 2D; concentrically exercised RMP: 84.1 ± 2.5 mV, n = 4 muscles; control RMP: 83.9 ± 1.2 mV; n = 16 muscles).

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Frequency distribution of resting membrane potential (RMP) in TA fibers (recorded in situ) after single exposure to low-repetition protocol (solid bars) compared with control fibers in contralateral TA (open bars). A: same day after exercise (1-2 h); B: 1 day postexercise; C: 2 days postexercise; D: same day (1-2 h) after low-repetition concentric contractions with use of same protocol as the eccentric contractions. Each set of frequency distributions represents measurements from muscles from at least 4 animals with recordings from at least 40 fibers/muscle.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Means ± SD of RMP recorded from control and exercised TA (in situ) after single exposure to low- (A) or high-repetition (B) resistance exercise protocol. Each mean value represents measurements from at least 4 animals with recordings from at least 40 fibers/muscle. , Recordings from exercised TA pretreated with streptomycin for 6 days before exercise, with treatment continuing until recordings were obtained; , recordings from exercised, untreated TA. Time 0 represents control. Recordings made same day as exercise protocol were taken within 1-2 h after exercise. * Significantly different (P < 0.05) from mean RMP of contralateral control TA.

Influence of [K⁺]_o on RMP of control and eccentrically exercised TA. The low-repetition protocol was used to investigate the possible cellular basis for membrane depolarization after eccentric exercise. Measurements were made on the contralateral control and exercised TA in vitro immediately after the initial exposure to the exercise protocol. The P_Na⁺/P_K⁺ in muscle cells was evaluated by measuring the response of the RMP to changes in [K⁺]_o while holding [K⁺]_o + [Na⁺]_o constant. The results are shown in Fig. 4. RMPs were recorded from at least 20 fibers in each muscle (n = 6). Figure 4A shows three theoretical curves calculated from the Goldman constant-field equation for RMP as a function of [K⁺]_o. The curves assume P_Na⁺/P_K⁺ values of 0.02, 0.03, and 0.05, respectively. The best-fit relationship between the theoretical curves and the measured values from control TA is provided by the curve where P_Na⁺/P_K⁺ = 0.03. The best-fit relationship for exercised TA is provided by the curve where P_Na⁺/P_K⁺ = 0.05. A linear graph of the data with the use of the exponential form of the Goldman equation (Fig. 4B) provided P_Na⁺/P_K⁺ values of 0.031 for control TA and 0.053 for the eccentrically exercised TA. The difference in P_Na⁺/P_K⁺ in control and exercised TA suggests that the Na⁺ permeability in exercised muscles increases by 70% within 1-2 h after an initial exposure to the low-repetition protocol.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Mean RMP of muscle fibers (in vitro) exposed to different concentrations of intracellular K+ concentrations ([K+]o). , Mean values for control TA; , mean values for exercised TA 1-2 h after single exposure to low-repetition eccentric exercise protocol. A: curves represent calculated values of RMP at various [K+]o as determined from Goldman-Hodgkin-Katz equation. Curves are given for permeability ratios of Na+ to K+ (PNa+/PK+) of 0.02, 0.03, and 0.05 as indicated. Mean values of recorded RMP for exercised and control TA are superimposed on theoretical curves to estimate PNa+/PK+ for the two groups. Estimated PNa+/PK+ for control TA was 0.03, and estimated value for exercised TA was 0.05. B: mean RMP values from A were graphed as an exponential function of the constant field equation. The y-intercept is an estimate of PNa+/PK+ (1, 10). e, Exponential form of equation; Vm, membrane potential; F, Faraday constant; R, gas constant; T, absolute temperature.

Effect of replacement of [Na⁺]_o with an impermeant cation on muscle membrane depolarization after eccentric exercise. The impermeant organic cation NMDG was used to replace [Na⁺] in the bathing solution to determine whether Na⁺ influx contributed to the membrane depolarization observed after eccentric exercise. RMP of fibers in the eccentrically exercised TA, measured in vitro, depolarized from 81.9 ± 1.8 to 72.7 ± 0.5 mV within 1-2 h after an initial exposure to the low-repetition protocol (Table 1). Replacement of [Na⁺]_o with equimolar NMDG produced a repolarization of exercised fibers to 79.1 ± 2.0 mV within 10 min after the change in solutions (Table 1). The NMDG solution was removed and replaced by the control solution, and the exercised muscle cells again depolarized to 71.5 ± 0.7 mV. Control muscles maintained normal RMP in the presence of NMDG (Table 1). These data support the hypothesis that an increase in Na⁺ permeability, and an accompanying increase in Na⁺ influx, is primarily responsible for postexercise depolarization of eccentrically exercised muscle cells.

Which Na⁺ channels are responsible for the increase in Na⁺ permeability after eccentric exercise? Amiloride in high concentrations is reported to block the Na⁺/H⁺ antiporter in many tissues, including skeletal muscle (11). If eccentrically exercised TA depolarized as a consequence of intracellular acidification and a resulting increase in Na⁺/H⁺ exchange, amiloride should block the pathway and produce membrane repolarization. The addition of amiloride to the bathing solution at concentrations of 0.5 and 1.0 mM did not reverse the depolarization observed in exercised muscle cells (Table 1). Addition of amiloride to the bathing solution also had no significant effect on the RMP of control muscles (82.2 ± 1.2 mV in control fibers compared with 82.5 ± 1.8 mV in 1 mM amiloride). This observation suggests that Na⁺/H⁺ exchange in resting mammalian muscles is less active than in resting frog muscles in which the addition of 0.4 mM amiloride produces a 12-mV hyperpolarization of the muscle (11).

6

6

5

Effect of pretreatment with streptomycin in vivo on TA RMP after low-repetition eccentric exercise. One group of rats received continuous oral administration of streptomycin from 6 days before the first exposure to low-repetition eccentric exercise until the muscle RMP was evaluated at various times after the exercise. Control streptomycin-treated TA had a mean RMP not significantly different from that of control untreated TA (Fig. 3A). The mean RMP of exercised TA from the treated group was significantly less depolarized immediately after the first exercise (79.6 ± 1.3 mV; n = 5 muscles) than was the mean RMP of exercised untreated TA (70.7 ± 1.4 mV; n = 12). The streptomycin-treated exercised muscles, however, were still significantly depolarized compared with their controls. The mean RMP of treated exercised TA was not significantly different from that of control, nonexercised TA 1 day after the first exercise, whereas the mean RMP of exercised TA from untreated animals remained significantly depolarized (Fig. 3A). Exercised muscles in both treated and untreated groups had a mean RMP similar to that of control TA at 2 days after the exercise.

Effect of pretreatment with streptomycin in vivo on the TA response to multiple bouts of resistance exercise. The contractile properties of control, nonexercised muscles were not significantly different between TA treated with streptomycin for 6 days and untreated TA (Tables 3 and 4). The contractile deficit 2 days after the first exposure to the low-repetition exercise was unaffected by treatment with streptomycin (Table 3), despite a significant reduction in the magnitude and duration of the exercise-induced membrane depolarization produced by streptomycin treatment (Fig. 3A). A second exposure to the low-repetition protocol, 14 days after the first, did not produce a significant contractile deficit 2 days after the exercise (Table 4). In contrast, TA muscles exposed to a second low-repetition exercise were still significantly depolarized immediately after the exercise (Figs. 5A and 6). Pretreatment with streptomycin for 6 days before the first exercise increased the magnitude and duration of the exercise-induced depolarization after the second exercise (Figs. 5B and 6). The membrane voltage changes in the pretreated TA after the second exercise resembled the changes seen in the untreated TA after the first exposure to the exercise (Fig. 2, A and B).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Frequency distribution of RMP in TA (recorded in situ) 1 day after second exposure to low-repetition protocol. One group of rats was treated with streptomycin starting 6 days before first exercise. Treatment was discontinued between first and second exposure to contraction protocol. A: distribution of RMP in exercised (open bars) and contralateral control (solid bars) TA from untreated animals 1 day after exercise. There was no significant difference between the 2 populations. B: distribution of RMP in exercised and contralateral control TA from streptomycin-treated animals 1 day after exercise. Mean RMP of treated, exercised fibers (76.2 ± 0.7 mV) was significantly different (P < 0.05) from mean RMP of treated control fibers (82.7 ± 0.4 mV). Each set of frequency distributions represents measurements from at least 4 animals with recordings from at least 40 fibers/muscle.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Means ± SD of RMP (in situ) recorded from control and exercised TA after second exposure to low-repetition resistance exercise protocol 14 days after first exposure. Each mean value represents measurements from at least 4 animals with recordings from at least 40 fibers/muscle. , Recordings from exercised TA pretreated with streptomycin for 6 days before and continuing through the first exercise; , recordings from untreated, exercised TA. Treatment was discontinued after the first exercise. Time 0 represents control. Recordings made same day as exercise protocol were taken within 1-2 h after exercise. Significant difference (P < 0.05) from * control TA, and # untreated, exercised TA.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   RMP means ± SD (in situ) of control and exercised TA at indicated times after second (A) and third (B) exposure to high-repetition protocol. Each exercise exposure occurred 10 days after previous exposure. , Measurements from untreated, exercised TA; , measurements from TA in rats treated with streptomycin beginning 6 days before indicated exercise and continuing until recordings were obtained. Time 0 indicates mean of control measurements. Recordings indicated as made same day of exercise were made 1-2 h post-exercise. Values are means of RMP from at least 4 animals per time point, with recordings obtained from at least 40 fibers per muscle. * Significantly different (P < 0.05) from control and treated TA.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Eccentric contractions in the predominately fast-twitch rat TA induced a significant, prolonged depolarization of the muscle. The magnitude and duration of the depolarization were related to the number of contractions during resistance exercise and to the number of previous exposures to the exercise protocol. Depolarization was greatest and most prolonged after the first exposure to the exercise, but subsequent exposures also produced significant, prolonged depolarization. Exposure to the same number of concentric contractions did not produce a significant change in muscle RMP. In vitro measurements showed that the membrane depolarization was due, for the most part, to an increase in Na⁺ influx triggered by the eccentric contractions. Experiments comparing the ability of specific Na⁺ blockers to reverse the postexercise depolarization showed that the Na⁺/H⁺ antiport inhibitor amiloride and the voltage-gated Na⁺ channel inhibitor TTX did not affect the depolarization. In contrast, the addition of either of two nonspecific SAC inhibitors, streptomycin or Gd³⁺, led to a significant repolarization of the membrane. The two agents are not selective for specific mechanosensitive ion channels, but they have been shown to inhibit ion conductance through SAC in several different systems (29, 33). The effect of the two agents given together at supramaximal concentrations was not additive, which also suggests that they were acting on the same conductance pathway. The results are consistent with a prolonged, exercise-induced activation of mechanosensitive cation channels (SAC) in eccentrically contracting skeletal muscle.

The activation of SAC produced by eccentric contractions could contribute to the muscle response to eccentric exercise in several ways. Na⁺ and/or Ca²⁺ influx through SAC may initiate a cascade of events leading to the delayed, or secondary, contraction-induced injury and reduction in contractile capacity observed several days after eccentric exercise (12). Increases in [Na⁺]_i and intracellular [Ca²⁺] ([Ca²⁺]_i), for example, are associated with the contractile and metabolic deficits observed after prolonged ischemia and reperfusion of skeletal muscle (38). It has been difficult, however, to demonstrate a clear association between an increase in [Ca²⁺]_i and contraction-induced muscle injury (2). Moreover, the present experiments suggest that SAC-associated ion currents were not associated with postexercise muscle deficits. Treatment of the rats with streptomycin in vivo to block SAC before and during the first exposure to the low-repetition protocol prevented membrane depolarization after the eccentric contractions but did not prevent the reduction in maximum isometric contractile force observed 2 days after the exercise (Table 3). Thus SAC-associated ion currents do not appear to contribute to muscle injury or force deficits after eccentric contractions.

Ionic permeability increased after the first exposure to eccentric exercise, but the increase was not due entirely to the activation of SAC. Treatment with streptomycin before the first low-repetition or the second high-repetition exercise did not prevent a significant depolarization 1-2 h after the exercise, suggesting that pathways other than SAC also contributed to the increase in membrane permeability. The other pathways have yet to be identified, although Na⁺/H⁺ exchange and voltage-dependent fast Na⁺ channels may presumably be ruled out, because neither amiloride nor TTX had any significant effect on the postexercise depolarization. Eccentric exercise is associated with sarcolemmal injury, which is most prominent after the first exposure to the exercise (8, 22). Sarcolemmal injury is characterized by the leak of large molecules, including creatine kinase and lactic dehydrogenase, out of the muscle (2, 8) and the movement of serum albumin into the muscle (22). Membrane damage could also produce nonspecific increases in membrane permeability to all small ions, including Na⁺ and Ca²⁺, and contribute to membrane depolarization, contractile deficits, and muscle injury. We speculate that the initial depolarization measured after a novel exposure to an eccentric exercise protocol is due to a combination of both membrane damage and the opening of SAC.

Nonspecific permeability changes produced by membrane damage are clearly reduced with continuing exposure to a resistance exercise protocol (8), but SAC activation appears to accompany each exposure to the protocol. Addition of streptomycin before the third exposure to the high-repetition protocol completely blocked the postexercise membrane depolarization (Fig. 7), suggesting that depolarization after this exercise was due entirely to SAC activation. The SAC-induced depolarization was also prolonged, indicating that the increase in Na⁺ influx was maintained well past the period of active contraction. In cardiac muscle, SAC are directly gated by mechanical stimuli and are slow to desensitize or adapt (14). SAC identified in cardiac myocytes, for example, can maintain their open state for long periods of stretch without adaptation (28). The prolonged SAC-mediated depolarization in eccentrically exercised TA suggests that mechanical strain at the molecular level may persist well beyond the period of exercise, possibly as a consequence of cellular edema or cytoskeletal changes related to the lengthening contractions. Sustained SAC activation could mediate the long-term changes in muscle associated with repeated exposure to resistance exercise (19).

SAC contribute to a wide array of cellular activities, including volume regulation, electrolyte homeostasis, sensory transduction, and cell growth (29). SAC in cardiac and smooth muscle cells modulate electrical and mechanical activity (14, 31, 36, 41) and appear to contribute to the cellular hypertrophy produced by volume overload or mechanical stretch (17, 18, 31). The role of SAC in skeletal muscle has yet to be determined, but there are considerable similarities in the response of cardiac and skeletal muscle cells to sustained mechanical stretch. Stretch stimulates an increase in gene expression, an increase in protein synthesis, and the development of cellular hypertrophy in both types of muscle (15, 17, 31, 39). Sudden or sustained stretch in both muscle types also leads to membrane depolarization linked to the activation of SAC (41). Na⁺ influx through SAC is reported to stimulate an increase in protein synthesis and cell growth in the mammalian myocardium (17), although this relationship has not been established in skeletal muscle. Growth in both cardiac and skeletal muscles has also been linked to activation of the Na⁺ pump (17, 36), which could be driven by a SAC-mediated increase in Na⁺ influx. The magnitude and duration of a change in pump activity, however, will vary, depending on the magnitude and duration of the increase in Na⁺ influx. The muscle may adapt to continuing stimulation by increasing the number of pump sites in the membrane (9), which might affect the contribution of either increased Na⁺ influx or increased Na⁺ pump activity to muscle growth.

Summary. The experiments reported in the present study were designed to test the hypothesis that the mechanical strain produced by lengthening or eccentric contractions in skeletal muscle fibers activates SAC in the fibers. The experiments characterized the SAC contribution to the magnitude and duration of changes in RMP after one or more exposures to low- or high-repetition eccentric contractions. The results indicated that skeletal muscle cells were significantly depolarized soon after (1-2 h) and remained significantly depolarized for at least 24 h after exposure to even low-repetition contractions. The magnitude and duration of the postexercise depolarization, however, were related to the intensity (number of eccentric contractions) and to the number of previous exposures to the exercise. The results also suggest that postexercise depolarization occurred as a consequence of an increase in Na⁺ permeability in the muscle. The increase in permeability observed after the initial exposure to a series of eccentric contractions may involve both a nonspecific increase associated with damage to the sarcolemma and an increase due to the activation of SAC. Repeated exposures appear to activate only the SAC pathway. SAC, however, did not appear to contribute to the force deficit after the first exposure, because blocking SAC with streptomycin did not reduce the deficit. Nonetheless, SAC activation in skeletal muscle may contribute to the stimulation of specific gene expression and protein synthesis (35), as is the case in cardiac and smooth muscle cells exposed to prolonged mechanical strain (17, 18, 31). We speculate that SAC activation may serve as an initial step in muscle adaptation and in the development of muscle hypertrophy during training involving eccentric contractions.