Department of Physiology, The University of Melbourne, Victoria 3010, Australia
Submitted 15 August 2002 ; accepted in final form 21 April 2003
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ABSTRACT |
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muscular dystrophy; depolarization-induced force; T-system; calcium release; sarcoplasmic reticulum; mdx mouse
The triadic junction is formed by the regular array of voltage sensors (dihydropyridine receptors, DHPRs) on the transverse tubular membrane and ryanodine receptors (RyRs)/Ca2+ release channels (CRCs) in the adjacent sarcoplasmic reticulum (SR) membrane. The triadic junction is involved in transferring an electrical signal in the T-system to the SR CRCs during E-C coupling. E-C coupling is defined as events that include depolarization of the T-system and activation of DHPRs, which in turn open CRCs in the adjacent SR membrane, allowing Ca2+ to enter the cytoplasm (24), and are responsible for coordinating contraction of skeletal muscles. Data on E-C coupling and SR function in dystrophic skeletal muscle are conflicting. Voltage-clamp experiments indicate that intramembrane charge movement, a step in E-C coupling, is not modified by the absence of dystrophin (13). In contrast, intact extensor digitorum longus (EDL) muscle fibers from mdx mice contracted at more negative potentials compared with age-matched controls and took longer to reach threshold for contraction- and relaxation-based calcium movements (7). These changes were attributed to the lack of dystrophin in the T-system causing a permanent alteration in E-C coupling in the muscles of mdx mice (7). Experiments on chemically skinned fibers prepared from the tibialis anterior muscles of mdx mice indicated a "more prominent" caffeine-induced contracture and a greater leakage of Ca2+ from the SR of mdx than control mice (33). In contrast, single fibers from the EDL muscles of 11-wk-old mdx mice had a decreased amplitude of caffeine-induced contraction and slower SR Ca2+ uptake but no difference in SR Ca2+ leak (8). To address these contradictory findings, we examined the contractile properties of mechanically skinned single fibers from mdx mice.
Mechanical skinning involves removal of the plasma membrane of a single muscle fiber under oil and immediate sealing of the T-system. The mechanically skinned fiber segments retain normal voltage regulation of Ca2+ release with an endogenous level of SR Ca2+, allowing for manipulation of the intracellular environment (30). These skinned fiber segments are unique in that manipulation of the bathing solution allows for examination of the individual steps in E-C coupling and SR Ca2+ handling. The purpose of this study was to characterize SR function and E-C coupling in mechanically skinned T-tubule sealed single muscle fibers from mdx mice. We tested the hypothesis that muscle fibers from dystrophic mdx mice have altered T-system function leading to impairments in E-C coupling and SR Ca2+ handling.
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METHODS |
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Solutions for Depolarization-Induced Responses of Mechanically Skinned Fibers
The composition of the solutions and the procedures used for activation of
the skinned muscle fibers have been described by us and others in detail
(19,
28). Briefly, the T-system of
the fibers was polarized by incubating the fiber in a potassium
hexamethylenediamine-tetraacetic acid solution [K-HDTA; in mM: 90 HEPES, 125
K+,36Na+, 50 HDTA, 0.1 EGTA, 8.5 Mg (total), 1
NaN3, 10 creatine phosphate, and 8 ATP] for 2 min. The fiber was
then depolarized by rapidly substituting the K-HDTA with Na-HDTA, an identical
solution but with all of the K+ replaced by equimolar
Na+. Maximum Ca2+-activated force
(Po) was determined in Ca-EGTA [in mM: 90 HEPES, 125 K+,
36 Na+, 50 Ca-EGTA, 8.12 Mg (total), 1 NaN3, 10 creatine
phosphate, and 8 ATP; pCa 4.6]. All solutions contained 1 mM free
Mg2+ (unless otherwise stated) and had an osmolality of
295 mosmol/kgH2O
(20) (see
Table 1). The pH of all
solutions was carefully adjusted to 7.10 ± 0.05 with KOH, except for
Na-HDTA, which was adjusted with NaOH. Stocks of all solutions were prepared
using ultra pure water and stored frozen in small aliquots that were defrosted
for experimentation when required. All experiments were performed at 22
± 2°C.
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Experimental Protocol
Protocol 1: Depolarization-induced force responses. After being mounted to the force transducer, the freshly skinned fiber segments were initially bathed in the K-HDTA solution for 2 min to polarize the T-system. The T-system of each fiber was depolarized by substitution of K-HDTA for Na-HDTA, causing Ca2+ release from the SR, initiating a transient depolarization-induced contractile response (DICR). Fibers then underwent a repeated depolarization (Na-HDTA) with a 30-s repriming period in K-HDTA between each DICR until they reached the point of rundown (DICR <50% of initial). The viability of SR CRC function was assessed by stimulating SR Ca2+ release with a low-[Mg2+] solution (K-HDTA with 0.1 mM free Mg2+) (29). At the conclusion of the DICR measurement, Po was determined.
A separate group of fibers was used to assess the repriming rate of DICR in fibers from control and mdx mice. As mentioned previously, a 30-s repriming period in a high-K+ solution between successive DICRs is required to repolarize the T-system. However, with shorter repriming periods (5, 10, and 20 s) between DICRs, submaximal force responses are observed. EDL muscle fibers from control and mdx mice were initially depolarized and then allowed a 5-, 10-, or 20-s repriming period in K-HDTA before being depolarized. The DICR at each repriming period was normalized to the maximum DICR (30-s repriming period) for each fiber.
Protocol 2: SR function. Another group of fibers was used to examine the ability of the Ca2+-depleted SR to load Ca2+. The SR of each fiber was first completely depleted of Ca2+ by incubation in a SR Ca2+ release solution (low-[Mg2+] K-HDTA containing 0.1 mM free Mg2+, 30 mM caffeine, and 0.5 mM EGTA) for 2 min. Each fiber was then incubated in a Ca2+-loading solution (Table 1) for a set period (10, 20, 30, and 60 s) to partially replenish SR Ca2+. The SR was depleted of Ca2+ after each loading period, and the integration of the tension curve for each load duration was compared with maximum (60 s of Ca2+ loading) to establish a load duration-SR Ca2+ content relationship, a measure of SR Ca2+-ATPase activity (25).
Additional muscle fibers were used to examine Ca2+ leakage from the SR. As described previously, fibers were prepared by first depleting the SR of Ca2+ (2-min incubation in the release solution). The SR was then reloaded with Ca2+ (30 s) and equilibrated for 2 min in a solution in which Ca2+ uptake and leak were prevented (equilibration solution: K-HDTA containing 10 mM free Mg2+ and 0.5 mM EGTA), and then the SR was allowed to "leak" Ca2+ for 30 s (K-HDTA with 1 mM total EGTA) before all Ca2+ remaining in the SR was released. The integration of the tension curve after the leak period was compared with the integration of the tension curve after normal (30 s) SR Ca2+ loading (no leak period) to estimate the amount of Ca2+ leaked from the SR.
The sensitivity of SR Ca2+ release was determined by contraction in a low caffeine concentration. Again, each fiber was prepared by completely depleting the SR of Ca2+ and then partially reloading the SR with Ca2+ (30-s load duration). Peak force of contraction was determined in a series of K-HDTA solutions containing 2, 3, 5 and 7 mM caffeine, with complete SR Ca2+ depletion and then a partial reload of the SR with Ca2+ (30-s load duration) between each caffeine contraction. At the conclusion of testing, Po was determined, and the peak height of caffeine-induced contraction was expressed as a percentage of Po to estimate the relative activity of the SR CRC.
Statistical Analysis
Values in the text are presented as means ± SE. Fibers from mdx and control mice were compared using either analysis of variance with Newman-Keuls post hoc analysis or by Student's paired and unpaired t-tests, where appropriate. Results were considered significant when P < 0.05.
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RESULTS |
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Substituting K-HDTA for the release solution caused sustained tension, and the area under the tension curve was used as an approximate indicator of SR Ca2+ content (29). After 2 min of equilibration in the release solution and complete SR Ca2+ depletion, the SR was partially reloaded with Ca2+ by incubation in the load solution for defined intervals (10, 20, 30, and 60 s). The load duration-SR Ca2+ content relationship was established by comparing the area under the tension curve after each "load duration," to maximum (60 s of Ca2+ loading; see Fig. 3). There was no difference in the load duration-SR Ca2+ content relationship in single muscle fibers from mdx and control mice.
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A protocol similar to the Ca2+-reloading experiments (above) was used to measure SR Ca2+ leak. After partial SR Ca2+ reloading to the Ca2+-depleted SR, SR Ca2+ leak was also measured. SR Ca2+ content (integration of the tension curve) after the leak period was compared with normal loading (30-s load duration) as an estimate of the amount of Ca2+ leaked from the SR. No difference in SR Ca2+ leak was observed in fibers from control and mdx mice (Fig. 4). When fibers from control mice were exposed to K-HDTA solutions containing caffeine, submaximal force responses were observed, and when normalized to Po for each fiber, force increased with increasing concentrations of caffeine (Fig. 5, A and C). However, submaximal force responses to caffeine were lower in fibers from mdx than control mice at all concentrations of caffeine tested (P < 0.05; Fig. 5, B and C).
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DISCUSSION |
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The failure of mechanically skinned muscle fibers to maintain E-C coupling and force production during repeated depolarization has been attributed to Ca2+-induced damage (E-C uncoupling), because exposure to elevated intracellular Ca2+ levels abolishes the DICR (18). Thus the more rapid rundown observed in fibers from mdx mice could be associated with a greater fiber susceptibility to E-C uncoupling. The Ca2+-dependent loss of DICR has been proposed to play an important feedback role in muscle by stopping Ca2+ release in localized areas where Ca2+ levels are elevated and may contribute to muscle weakness in the various dystrophies (18). The rapid rundown in fibers from mdx mice was also associated with a decreased repriming rate of the DICR. Both E-C uncoupling and the decreased repriming rate might account for the observation that initial DICR of fibers from mdx mice was equivalent to the initial DICR of fibers from control mice but that rundown was reached more rapidly.
The SR Ca2+-handling properties of EDL muscle fibers from mdx mice were generally similar to those from control mice, because there were no differences in the rate of SR Ca2+ reloading or SR Ca2+ leak. Our finding of normal Ca2+-ATPase activity in mechanically skinned T-tubule sealed fibers is in agreement with a previous report of SR Ca2+-handling in cooling contracture experiments on bundles of diaphragm muscle fibers from mdx mice (16). These results differ from another study by Kargacin and Kargacin (15), who reported decreased SR Ca2+-ATPase activity in reconstituted SR membrane vesicles prepared from muscles of mdx compared with control mice. In contrast, we have measured SR Ca2+ reuptake in single fibers with an intact SR membrane and functionally preserved T-system, and this may account for the differences between the studies. Our finding of normal SR Ca2+ leak in fibers from mdx mice also confirms previous observations from saponin-skinned single muscle fibers (8). The results support the notion that although dystrophin-deficient fibers have an increased sarcolemmal membrane permeability to divalent ions (35), SR membrane permeability is not affected.
Our finding of a decreased sensitivity of the Ca2+ release channel (when stimulated by caffeine) is in contrast to previous reports of normal electrically elicited Ca2+ transients in muscles from mdx mice (4, 14, 35). Data on caffeine-induced contraction in saponin-skinned single fibers from mdx mice are conflicting, with either an increase or decrease in the amplitude of caffeine-induced contraction being reported (8, 33). It should be noted that a consequence of using saponin to permeabilize the sarcolemmal membrane is an increase in SR permeability to Ca2+ and reduced SR Ca2+ loading, due to the activation of Ca2+ release channels (21). Therefore, measurements from fibers prepared with saponin may not be truly representative of physiological responses because of the effect of saponin on the SR Ca2+ release channel. In contrast, the preparation of mechanically skinned fibers (as employed in our experiments) does not affect the SR Ca2+ release channel. Our findings demonstrate that the amplitude of caffeine-induced contraction is lower in fast muscle fibers from mdx than in fibers from control mice.
The decrease in caffeine-induced contraction is not likely to be mediated by an effect on the myofilaments because caffeine has similar effects in permeabilized fibers from mdx and control mice, i.e., decreased maximum Ca2+-activated tension and increased Ca2+ sensitivity of the myofilaments (8). Rather, the effect could be attributed to differences in the saturation of SR Ca2+ loading in fibers from mdx mice. Previous studies have determined that the sensitivity to caffeine is greater in slow than fast muscle fibers, most likely due to the level of saturation of SR Ca2+ loading (9). In our experiments we found no differences in SR Ca2+ loading or Ca2+ leak in EDL muscle fibers from mdx and control mice and assumed an identical SR Ca2+ content in the fibers (16). Further experiments comparing the saturation of SR Ca2+ loading between fibers from mdx and control mice are warranted. It is interesting to note that the lower amplitude of caffeine-induced contraction we observed in EDL muscle fibers from mdx mice was similar to that we have reported for mechanically skinned fibers from EDL muscles of aged (28 mo old) C57BL/10 mice (28).
In this study we have successfully characterized the events of E-C coupling and SR function in fast muscle fibers from mdx mice. We found that mechanically skinned T-tubule sealed fibers from mdx mice have normal voltage activation of contraction and release of Ca2+ from the SR, with peak depolarization-induced force being similar to that of muscle fibers from control mice. Repriming of E-C coupling in mechanically skinned muscle fibers from mdx mice was disrupted, because during repeated depolarizations they reached the point of rundown more rapidly than fibers from control mice. SR Ca2+ reuptake and leak of SR Ca2+ were equivalent in fibers from mdx and control mice, but peak caffeine-induced Ca2+ release was decreased in fibers from mdx mice. We conclude that although E-C coupling and SR function in mechanically skinned fibers are similar in mdx and control mice, SR CRC activity is depressed and the ability to perform repeated depolarizations is impaired, causing E-C uncoupling. The differences in E-C coupling and SR function between fibers from mdx and control mice provide further support for the usefulness of the mdx mouse not only for studying of the pathogenesis of dystrophinopathies but also for developing potential therapies for successful treatment of DMD.
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DISCLOSURES |
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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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