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ENVIRONMENTAL, EXERCISE AND RESPIRATORY PHYSIOLOGY
1Departments of Applied Physics and Chemistry, University of Electro-Communications, Chofu, Tokyo, Japan; 2Departments of Anatomy, Physiology and Kinesiology, Kansas State University, Manhattan, Kansas; and 3School of Sports and Health Sciences, University of Exeter, Exeter, Devon, United Kingdom
Submitted 9 November 2007 ; accepted in final form 9 January 2008
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ABSTRACT |
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spinotrapezius; muscle damage; streptomycin; gadolinium
0.1 µmol (2). When myocytes contract, there is a transient elevation of [Ca2+]i that helps trigger muscle contraction. In nonfatigued and nondamaged myocytes, as the muscle relaxes after contraction, [Ca2+]i decreases immediately. However, long repeated contractions induce myocyte fatigue (reduction of tetanic force), myocyte damage, and prolonged elevation of [Ca2+]i after contraction(s) (15, 43). It is well known that eccentric (ECC) contractions induce muscle damage (8, 26, 44), and it has been suggested that one principal cause of that damage is high [Ca2+]i (1, 17). Moreover, if ECC does cause increased [Ca2+]i, it is possible that stretch-activated ion channels (SAC) are involved in this response (1, 54–56) and the associated muscle damage. Franco and Lansman (11, 12) initially reported SAC function in skeletal muscle and noted that the channels were blocked by streptomycin and gadolinium (Gd3+) (13, 41). Recently, Yeung and colleagues (54–56) demonstrated that SAC blocker treatment prevented an increase in the resting [Ca2+]i in isolated single fibers from the mdx mouse, a model of human Duchenne muscular dystrophy, following ECC contractions. Furthermore, it has been suggested that inhibition of SAC during ECC contraction attenuates activation of muscle growth-related signaling pathways (42). These phenomena may be associated with perturbations of the intracellular ionic environment; for example, [Ca2+]i levels may rise through ECC contraction-induced SAC activation.
Because of the difficulty in measuring [Ca2+]i under in vivo conditions, almost all investigations of [Ca2+]i have been performed under in vitro conditions. However, such in vitro conditions are likely to perturb [Ca2+]i regulation, and, therefore, development of an in vivo preparation, capable of resolving [Ca2+]i in single fibers, might provide a unique and valuable opportunity to better understand the role of [Ca2+]i in muscle function and dysfunction. Bioimaging techniques can visualize intracellular ions directly (9, 39, 50, 51), and, whereas measurements of [Ca2+]i are potentially feasible under intravital conditions routinely used for microcirculation studies, to date, because skeletal muscle was considered to be too thick for microscopy, most studies of [Ca2+]i have used isolated or cultured single myocytes. Unfortunately, such isolated or cultured cells have quite a different environment than in vivo skeletal muscle with respect to their absence of a microcirculation, different oxygen and substrate availabilities, and metabolism, among other considerations (43, 47).
Since the development of the spinotrapezius intravital microscopy preparation by Gray in 1973 (18), this muscle has served as a keystone for the understanding of muscle microvascular control. The spinotrapezius is sufficiently thin to permit transmission light microscopy, is composed of all three major mammalian muscle fiber types (10), and has an oxidative capacity similar to that of the human quadriceps (30). To date, there are a few reports of [Ca2+]i measured using bioimaging techniques in the spinotrapezius muscle (23, 48), but the effects of repeated isometric (ISO) and ECC contractions on [Ca2+]i have not been investigated.
The purpose of the present investigation was to test the following original hypotheses in the spinotrapezius muscle of healthy rats. 1) Compared with the surgically excised in vitro spinotrapezius, the in vivo preparation (i.e., exteriorized, as for intravital miscroscopy; Refs. 18, 38, 45, 46) prolonged (90 min) observation at rest would not elevate [Ca2+]i. 2) An extended series of ECC would elevate [Ca2+]i to a greater extent than ISO contractions. 3) The elevated [Ca2+]i accompanying ECC contractions would be prevented or substantially reduced by the SAC blockers.
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METHODS |
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Male Wistar rats (n = 36, Japan SLC), 8–12 wk of age, were used in this study. Rats were maintained on a 12:12-h light-dark cycle and received food and water ad libitum. All experiments were conducted under the guidelines established by the Physiological Society of Japan and were approved by University of Electro-Communications Institutional Animal Care and Use Committee. The rats were anesthetized with intraperitoneal injection of pentobarbital sodium (70 mg/kg ip), and supplemental doses of anesthesia were administered, as needed.
In Vivo Muscle Preparation
The spinotrapezius was exteriorized, as described previously (3, 25, 27, 28, 38). Briefly, the right spinotrapezius muscle was carefully accessed through an 4-cm-long midline incision through the skin, starting at the lower cervical level and extending caudally to the upper lumbar vertebral level. Exteriorization was performed with as little disruption as possible to minimize tissue damage. With the exception of the distal feed artery, all of the vascular and nervous connections remained intact as the caudal perimeter of the muscle was dissected free of connective tissue attachments. The exposed spinotrapezius muscle was attached to a thin-wire horseshoe around the caudal periphery by five to six equidistant sutures placed around the caudal perimeter. For the contraction protocols, electrodes were placed on the dorsal spinotrapezius surface proximal to the motor point and along the caudal periphery, facilitating indirect whole muscle contractions. The muscle surface was kept moist by superfusing with warmed Krebs-Henseleit buffer solution (KHB; 132 NaCl, 4.7 KCl, 21.8 NaHCO3, 2 MgSO4, 2 CaCl2 mM), equilibrated with 95% N2–5% CO2 and adjusted to pH 7.4, at 37°C. All other drugs were dissolved into KHB solution. In the experiments examining the effects of SAC blockade, streptomycin and Gd3+ were used. Aqueous stock solutions of streptomycin and GdCl3 were diluted in normal KHB to final concentrations of 200 and 20 µM, respectively (52, 55, 56). Fluorescence Ca2+ indicator fura 2-AM (5 mM, Dojindo Laboratories) was dissolved in dimethyl sulfoxide and Pluronic F-127 (final < 0.1%) and dispersed into the KHB solution at a final concentration of 20 µM. Muscles were incubated in fura 2-AM/KHB solution for 30 min on a 37°C hotplate to facilitate AM esterase activity. After incubation, muscles were rinsed with dye-free KHB solution to remove nonloaded fura 2-AM, and the microscopy protocol was initiated within 15 min.
In Vitro Muscle Preparation
The preparation was similar to the in vivo condition, except that the rostral (scapular) perimeter of the muscle was also dissected free from connective tissue connections. Subsequently, the detached muscle was fixed in an immoveable clamp at its rostral extremity, while the caudal boundary was sutured to the horseshoe, as described for the in vivo preparation above.
Microscopy and Fluorescence Measurement
The fura 2-loaded spinotrapezius muscles were mounted on the 37°C glass hotplate (Kitazato Supply), which reduced any movements due to breathing and cardiac contractions, and observed by fluorescence microscopy using a x10 objective lens (Nikon). After ensuring that the muscle was not grossly damaged and supported blood flow, a sampling area (880 x 663 µm) was selected using branching vessels from the main feed artery as landmarks, and bright-field images were captured (Fig. 1). Thereafter, 340-nm and 380-nm wavelength excitation light was delivered using a xenon lamp equipped with appropriate fluorescent filters, and pairs of fluorescence images were captured for ratiometry at 1.6 pixels/µm monitor resolution.
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When the spinotrapezius muscles were observed in bright-field illumination, muscle fibers and microvessels, including capillaries, were clearly visualized. Whereas there was no arteriolar or capillary red blood cell (RBC) flow in the in vitro preparation, these microvessels maintained good blood flow until the end of the measurement period in the in vivo preparation. In any preparations in which the arteriolar blood flow ceased during the observation period, the results from that muscle were not analyzed. After selecting an appropriate region of interest, the spinotrapezius was observed fluorescently, and 340- and 380-nm excitation wavelength fluorescent filters were switched manually. Images were converted to 340/380 ratio image by ImageJ software (National Institutes of Health), and the 340/380 ratio image data were averaged over the whole area sampled. The 340/380 images were assumed to indicate [Ca2+]i (Fig. 2).
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Experimental Protocols
Animals were divided into six groups: 1) in vitro control (n = 5); 2) in vivo control (n = 8); 3) in vivo ISO contractions (n = 7); 4) in vivo ECC contractions (n = 7); 5) in vivo ECC contractions with streptomycin (n = 5); and 6) in vivo ECC contractions with Gd3+ (n = 4). Groups 1 and 2 addressed hypothesis 1 by comparing in vitro with in vivo muscle. Groups 3 and 4 addressed hypothesis 2 by comparing [Ca2+]i changes across different contraction types (i.e., ISO and ECC). Finally, groups 5 and 6 addressed hypothesis 3 by investigating the involvement of SAC in [Ca2+]i accumulation.
Group 1: In vitro control group. The resting (nonstimulated) control experiment was performed in vitro by surgically isolating and loading the spinotrapezius muscle with fura 2 before microscopy. Sequential fluorescence images were captured every 5 min for 90 min. Each pair of 340- and 380-nm images was analyzed and quantified. Quantified data were graphed as changes from precontraction (baseline) levels.
Group 2: In vivo control group. The protocol was the same as for group 1, except that the muscle was exteriorized with the principal vascular and neural pathways maintained intact.
Group 3: In vivo ISO contractions group. The time course of [Ca2+]i change was observed after each of 10 discrete sets of ISO muscle stimulation, in a similar fashion to that described previously (46). Specifically, each set consisted of the muscle being stimulated tetanically at resting spinotrapezius sarcomere length (100 Hz, 5–8 V, stimulus duration 700 ms, 2.6- to 2.8-µm sarcomere length) every 3 s for 2.5 min (i.e., 50 contractions). Pairs of fluorescence images were captured precontraction and after each set of contractions, as well as at the end of the 5-min between-set recovery (immediately before initiation of the subsequent set of contractions).
Group 4: In vivo ECC contractions group.
Muscle lengthening ECC contractions in in vivo spinotrapezius muscle were compared with ISO contractions in group 3. The ECC contraction protocol was modified slightly from that reported previously (26). The motor device coupled with electro-stimulator (model RU-72, NEC Medical Systems) evoked strain via the caudal edge of the muscle, which was attached to the wire horseshoe and delivered a stretch (lengthening) of 10% of resting sarcomere length (i.e., to 3.0 µm). Muscle lengthening was started 0.2 s after initiation of electrical stimulation and immediately returned to resting length at the end of electrical stimulation. Other settings and measurements were the same as for protocol 3 above (ISO contractions).
Groups 5 and 6: Streptomycin and Gd3+ groups. Protocol was the same as for group 4 above, except that the KHB superfusate contained either streptomycin or Gd3+.
Force Measurement
The wire horseshoe attached to the spinotrapezius muscle was connected by fine wire to a strain gauge. Torque (0–10 mN·m, scale) was monitored by computer using Mac Lab/8s (A/D Instruments Pty.) via strain-gauge-linked motor device during all contraction protocols. The first and last five contractions of sets 1, 5, and 10 were averaged and plotted graphically as the index of fatigue.
Statistical Analysis
All statistical analyses were performed in Prism version 4.0 (GraphPad Software). A two-way repeated-measures ANOVA and Bonferroni post hoc test was used for in vitro vs. in vivo, ISO vs. ECC, and ECC vs. streptomycin or Gd3+ comparisons. A one-way repeated-measures ANOVA and Bonferroni post hoc test was used for relative force comparison. Measured values are presented as means ± SE. Significance was established at P < 0.05.
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RESULTS |
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2030% within a set, and at the final 10th set it was significantly decreased to 41.9 ± 7.5% of prefatigue conditions. While ECC also indicates decreasing tetanic force within a set, there was not any significant attenuation of eccentric force production throughout 10 sets of contractions. SAC blockers streptomycin and Gd3+ with ECC contractions did not affect eccentric force.
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DISCUSSION |
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Methodological Considerations
The spinotrapezius muscle has formed a cornerstone of our understanding of muscle microcirculatory control (18, 21, 24, 29, 34). As well as possessing optical properties requisite for intravital microscopy, it is composed of all three major muscle fiber types (10) and has an oxidative capacity similar to that found in the human quadriceps (30). As detailed in METHODS, the spinotrapezius can be exteriorized for microscopy while preserving vascular and neural pathways, and sarcomere length can be set with precision, if required (38). These considerations are paramount if in vivo microcirculatory structure and function and, therefore, muscle viability are to be maintained. In the present investigation, it was crucial that each muscle fiber could be discerned clearly by bright-field microscopy, and it was ensured that arterioles and capillaries sustained RBC flow throughout the experimental period.
For the present investigation, we specifically chose fura 2 because, using a ratiometry technique, this indicator facilitates the determination of [Ca2+]i without problems associated with variations of tissue and liquid thicknesses, amount of intracellular indicator, and/or the specific power of the luminous source (51). We set the image exposure time to 5 s, which facilitated capture of the fluorescence images required. Comparison was made between fura 2-loaded and unloaded muscles, and it was determined that there was no significant background fluorescence at 340- and 380-nm wavelengths. In addition, because fura 2 quenching Mn2+ solution (19) decreased fluorescence intensity, it was believed that the fluorescence signal in this study could be attributed exclusively to fura 2 binding with [Ca2+]i.
In Vitro vs. In Vivo
Historically, it has been common practice to superfuse in vitro muscle preparations with very high (nonphysiological) partial pressures of O2 (PO2) between 100 and 600 Torr. Specifically, microvascular PO2 is 30 Torr in the resting in vivo spinotrapezius muscle (5). Whereas, in the absence of an intact microcirculation, PO2 values of 100–600 Torr are necessary to provide O2 for mitochondrial oxidative phosphorylation, high PO2 values, in and of themselves, can be injurious to the tissue. Hence, the in vitro preparation is far from ideal in terms of hyperoxic/hypoxic damage, as well as provision of substrates such as free fatty acids and glucose. In the present investigation, we chose to compare the in vivo preparation to an in vitro one that was not damaged by nonphysiologically high PO2, recognizing that it would become overtly hypoxic as resting metabolism utilized the small available O2 reserves in RBCs, dissolved in plasma and extra- and intracellular fluids and on myoglobin. Consequently, the [Ca2+]i elevation observed herein likely resulted from the process of hypoxia/anoxia-induced muscle cell degradation (33, 36, 49). This phenomenon was also reported by Terada et al. (47), who measured [Ca2+]i in the thin (similar to the spinotrapezius) epitrochlearis muscle. Despite provision of a glucose and O2-supplemented buffer, [Ca2+]i was significantly elevated after 70 min of observation. Collectively, these results indicate that survivability of muscle tissue depends strongly on the maintenance of resting blood flow. Indeed, under such in vitro conditions, whole muscle tissue may be more damaged than single-muscle fibers (57). In contrast, in the present investigation, there were no significant changes in resting muscle [Ca2+]i under the circulation-intact in vivo conditions. Our microscopic observation area in the present investigation included at least the main feed artery and/or the first- and second-order branching arteriolar vessels (1A and 2A). It was ensured that, for the in vivo condition, these vessels maintained blood flow during the experiment. We consider that this is the most likely explanation for the muscle fibers in the in vivo (in contrast to the in vitro) preparation maintaining cellular function, such that [Ca2+]i accumulation was prevented and [Ca2+]i, therefore, remained at resting levels (Fig. 3).
ISO vs. ECC Contractions
Although some reports indicate that [Ca2+]i accumulation may not occur following shorter bouts of ISO contractions (4, 22), longer lasting bouts of muscle contractions that cause fatigue do elicit muscle [Ca2+]i accumulation (7, 15, 16). It is possible that, initially, during ISO contractions, Ca2+ originates exclusively from the sarcoplasmic reticulum (SR; i.e., intracellularly) with quantitatively little Ca2+ migrating across the sarcolemma. However, more chronic stimulation protocols, such as the 10 bouts used herein, may cause a gradual [Ca2+]i accumulation that becomes evident after five sets of contractions and increased progressively thereafter. In agreement with McBride et al. (32), who have shown no SAC activation with concentric contractions, we speculate that the most likely source of the elevated [Ca2+]i was SR-released Ca2+, and its accumulation reflects a progressive inability for the SR to recover during each resting period.
On the other hand, [Ca2+]i elevation of ECC contracted muscle was larger and faster compared with the ISO condition. As shown in many previous studies (1, 2, 4), our results support that ECC contraction induces high [Ca2+]i accumulation. There are two major potential sources of this Ca2+: either SR (i.e., intracellular), or the extracellular space where [Ca2+] is 
10,000-fold greater than resting [Ca2+]i. It has been recognized that ECC contraction can damage the sarcolemmal microstructure, and that SAC are present in the muscle cell membrane (12). In consideration of the above, we sought to block the sarcolemmal SAC to test the novel hypothesis that these channels were responsible for the [Ca2+]i accumulation caused by ECC contractions.
Effect of SAC Blocker
It has been determined that streptomycin and Gd3+ function as SAC blockers in working skeletal muscle from healthy rat (31, 32, 42, 53) or mdx mouse (52, 55, 56). However, the specificity of these agents is often brought into question. While it has been suggested that aminoglycosides and Gd3+ can block other cation channels, for example L-type Ca2+ channels, the inhibiting effect of these agents for other channels seems lower than for SAC (35, 40). Previous studies also indicate that 200 µM streptomycin or 20 µM Gd3+ have no effect on muscle contraction (20, 42, 52, 55). In our protocols, tetanic force measurements in the ECC protocol revealed that the spinotrapezius muscle maintained such good contractility in the presence of streptomycin and Gd3+ that, if streptomycin or Gd3+ does affect L-type Ca2+ channels, this effect is not so great that it impairs contractility to a noticeable degree (52). Moreover, before initiating pharmacological experiments, neither streptomycin nor Gd3+ affected [Ca2+]i in resting spinotrapezius over at least 90-min duration (T. Sonobe, T. Inagaki, Y. Kano, unpublished observations). As seen clearly in Figure 5, streptomycin and Gd3+ significantly reduced [Ca2+]i accumulation following ECC contractions, such that [Ca2+]i elevation was similar, or perhaps even less than that seen following ISO contractions. In addition, bright-field scrutiny demonstrated that, with the streptomycin and Gd3+ condition, almost all muscle fibers were spared from the hypercontractured condition commonly observed after the control ECC contraction condition. Moreover, it demonstrates, at least in this muscle preparation, that SAC are not crucial for contractions. In summary, these results suggest that critical [Ca2+]i accumulation following ECC contraction occurs across the SAC and, furthermore, that SAC blockade has the ability to reduce ECC contraction-induced [Ca2+]i accumulation and, therefore, any associated muscle fiber fatigue and/or damage.
Tetanic Force and Contraction Protocols
It has been established that voluntary concentric contractions induced a greater loss of force than voluntary ECC contractions (37). Similarly, ECC contractions have a greater resistance to fatigue than ISO and concentric contractions compared at the same electrically stimulated contraction intensity (6). We found that, while repeated ECC contractions tend to decrease tetanic force, there was not a significant attenuation of force production, at least over the number of contractions evaluated herein (Fig. 6). Also, because no difference in fatigability occurs following ECC, with or without blockers, we presume that tetanic force during repeated ECC contractions was not dependent on resting Ca2+ level.
Ca2+ Propagation in Muscle Fibers
One advantage of using bio-imaging is the ability to observe [Ca2+]i changes in real time by image analysis for each muscle fiber in whole tissue. To evaluate the time course of [Ca2+]i changes, digitizing was performed on ratio images that included 10–20 muscle fibers. This digitizing area (880 x 663 µm, 0.6 mm2) was considerably larger than a previous study in single fibers (50 x 50 µm; Ref. 56) and in in vivo skeletal muscle (100 x 100 µm; Ref. 48). In the latter study, [Ca2+]i changes were averaged over multiple muscle fibers. Using ratio-image analysis, a longitudinal progression of [Ca2+]i accumulation resembling a wave could be identified in single muscle fibers from the equated-digitized graph. These [Ca2+]i accumulations were quite different from the acute [Ca2+]i transients associated directly with each contraction. We speculated that the source of this Ca2+ was influx from an effective increase of sarcolemmal permeability rather than SR damage and the absence of such behavior under the SAC inhibiting conditions.
Heterogeneous [Ca2+]i accumulation patterns among fibers were observed under our experimental conditions. Because the tetanic contractions were elicited from direct supramaximal electrical stimulation, this heterogeneity of [Ca2+]i could not be explained by selective motor unit recruitment during contraction. It is likely that different muscle fiber types are more susceptible to such damage than others. For example, Ivanics and colleagues (23) have described a selective damage (and [Ca2+]i accumulation) in oxidative type I fibers following an ischemia-reperfusion protocol in the spinotrapezius muscle. In contrast, Suzuki and colleagues (46) found that an ischemia-reperfusion protocol selectively damaged those fibers with a low oxidative capacity (most likely type IIb fibers, as visualized using rhodamine-123 to identify relative mitochondrial content). When muscle is injured consequent to ECC contractions, muscle fibers may be selectively damaged, and, as these fibers subsequently produce less force, the remaining healthy fibers may be subjected to greater stresses and are likely to suffer progressively more injury (14). In the present investigation, those fibers with the greatest [Ca2+]i tended to be the larger diameter fibers, consistent with (but not proof of) selective damage to type II fibers.
Perspectives and Significance
This investigation has demonstrated that, under in vivo conditions, ECC contractions induce a more rapid and far greater [Ca2+]i accumulation than ISO contractions. Moreover, it appears that SAC are mechanistically involved in this ECC contraction-induced [Ca2+]i accumulation and, therefore, any damage resulting from the high [Ca2+]i. Future experiments designed specifically to determine the fiber-type specificity of this effect would prove valuable.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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