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MUSCLE CELL BIOLOGY AND CELL MOTILITY
1Department of Surgery, Section of Plastic Surgery and 2Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan
Submitted 8 June 2007 ; accepted in final form 27 December 2007
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ABSTRACT |
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skeletal muscle; muscle damage; dystrophin
Among the features frequently observed in dystrophic muscle is a prevalence of fibers that have "hypercontracted" to form contracture clots (4, 5, 8, 16). The presence of contracture clots suggests a history of aberrant and sustained contractile activity, which, in turn, suggests a loss of control of intracellular calcium concentration ([Ca2+]i). Elevated [Ca2+]i is associated with dystrophin deficiency (10) and is frequently attributed to an influx of extracellular Ca2+ through tears in the sarcolemma, hypothesized to occur as a result of the fragility of the membrane coupled with the high stress levels of contraction (24). However, the relationship between sarcolemmal disruptions, elevated [Ca2+]i, and muscle fiber damage and degeneration has not been clearly established. Our purpose was to investigate the mechanism by which the absence of dystrophin results in an increased susceptibility to contraction-induced damage in muscles of mdx mice.
To gain insights into the failure progression, we developed a very small whole muscle preparation using the lumbrical muscle from the foot of the mouse. The small size of the lumbrical muscle permitted individual fibers to be viewed with a compound microscope, allowing direct observation of the failure process. The short diffusion distances also facilitated the loading of fibers with a Ca2+-sensing fluorescent dye, which served as a real-time indicator of the presence of Ca2+-admitting breaches in the sarcolemma. Using this unique preparation, we show that dystrophin-null lumbrical muscle fibers do not tolerate nonuniform contractile activation caused by an influx of extracellular Ca2+ and that normal contractile activity can result in such an influx. In contrast, control lumbrical muscles demonstrated neither detectable entry of extracellular Ca2+ in response to contractile activity nor an adverse effect of nonuniform Ca2+ activation. We propose a failure mechanism for dystrophic muscle that invokes dystrophin's putative membrane stabilization and lateral mechanical coupling functions.
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MATERIALS AND METHODS |
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Histology. Freshly dissected muscles were immersed in tissue-freezing medium (Triangle Biomedical Sciences) and were frozen rapidly in isopentane cooled with dry ice. Embedded muscles were stored at –80°C for less than a week before being sectioned with a cryostat (model HM500, Microm). Cryosections of 10-µm thickness were allowed to reach room temperature and were then stained with hematoxylin (nuclear stain) and eosin-phloxine (cytoplasmic stain). Coverslips were attached to the slides with an adhesive (Permount, Fisher Scientific) and were allowed to dry overnight before being viewed with a light microscope (Olympus BX51).
Contractile properties. Whole lumbrical muscles were dissected from the medial side of digit 2 in the hind feet while immersed in cold Tyrode solution. Isolated muscles were then pinned by their tendons to the bottom of a shallow dissecting dish, also filled with cold Tyrode solution, and were gently trimmed of as much adherent fascia and epimysium as possible to enhance the visualization of fiber striations. Muscles were too small to be weighed accurately; mass was estimated to be <1 mg from approximate length, width, and depth dimensions. Trimmed muscles were transferred to a chamber designed specifically for use with small preparations. The chamber was perfused at a rate of two exchanges per minute with the following Tyrode solution (in mM): 121 NaCl, 5.0 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.4 NaH2PO4, 24 NaHCO3, 5.5 glucose, and 0.10 EDTA. For the low [Ca2+] experiments, the CaCl2 was omitted and MgCl2 increased to 2.3 mM. The solution was oxygenated and maintained at pH 7.3 by bubbling with a 95% O2-5% CO2 mix. Chamber temperature was maintained at 25.0°C, and activation was accomplished by electrical stimulation via platinum plate electrodes placed on either side of and parallel to the muscle. Muscles were mounted horizontally in the chamber with one end attached to a stationary post and the other to a force transducer (modified model 400A, Aurora Scientific). Sarcomere length was adjusted by monitoring the striation spacing microscopically while changing muscle length. Because fibers in the lumbrical are arranged parallel to the axis of the tendons, the number of sarcomeres in series within a fiber could be inferred by changing muscle length by a known amount and measuring the resulting change in sarcomere length. Fiber length was then calculated as the number of series sarcomeres multiplied by 2.5 µm. Mean fiber lengths determined with this technique were 2.16 ± 0.08 mm (mean ± SE; n = 28) and 2.20 ± 0.06 mm (n = 11) for mdx and control muscles, respectively. After the sarcomere length was adjusted to 2.5 µm, muscles from both mdx and control mice were subjected to 10 consecutive isometric (fixed-length) tetanic contractions, each lasting 1 s and separated by 60-s rest. Stimulus pulses within the 1-s tetanus were 0.4 ms in duration and were delivered at a rate of 125 s–1. Force, Ca2+-induced changes in intracellular dye fluorescence, and a video image of the muscle were recorded continuously throughout the protocol.
Intracellular Ca2+. The floor of the experimental chamber was made of polished quartz, allowing viewing of the muscle and excitation of fluorescent dyes with ultraviolet light when the chamber was placed on the stage of an inverted microscope (Zeiss Axiovert 100). Intracellular Ca2+ levels were monitored using the Ca2+-sensitive fluorescent dye fura-2 (11). The dye was loaded into fibers by incubating muscles in Tyrode solution containing 0.01% Pluronic F-127 and 15 µM fura-2 (Molecular Probes) in the membrane-permeant acetoxymethyl ester form for 30 min at 25°C. Records of the variations in [Ca2+]i with time were obtained using an illumination and photometer system (model Deltascan 4000, Photon Technology International) and were reported as the response to excitation at 340 nm divided by the response at 375 nm (ratio units). Fluorescence responses were collected from an area 0.23 mm by 0.23 mm in the distal half of the muscle. Other details of the intracellular Ca2+-monitoring technique have been reported previously (19).
Regional activation.
Nonuniform activation was induced by topical application of the Ca2+-ionophore ionomycin. This was accomplished by pressure ejection of an ionomycin-Tyrode solution (20 µM) from glass micropipettes made with a Flaming-Brown puller (model P-87, Sutter Instruments) and having tip diameters of 
40 µm. For these experiments, the chamber perfusion was stopped and muscles remained submerged in normal Tyrode solution. The micropipette holder was maneuvered with the aid of x-y-z manipulators such that the micropipette tip was placed 50–100 µm above the midline of the muscle before application. The duration of the application was 60 s.
Image acquisition.
All images were obtained with transmitted, brightfield illumination. The images shown in Fig. 1 were obtained with a digital still camera (Olympus DP70) mounted on an upright microscope (Olympus BX51) with a x20 objective (Olympus UPlan Apo, numerical aperture 0.7). All others were obtained with an inverted microscope (Zeiss Axiovert 100) with a x10 objective (Zeiss Fluar, numerical aperture 0.5). Images shown in Fig. 2 and in movies 1 and 2 (the online version of this article contains supplemental data) were captured with a charge-coupled device video camera (model HC 7001, Elmo), and those in 
Fig. 5 were captured using a digital still camera (Nikon, model D1). Images shown in Fig. 1 were adjusted by using the "auto levels" function followed by brightness and contrast modifications using image editing software (Adobe Photoshop CS2). All others were converted to grayscale, and then contrast and brightness were adjusted with image editing software: Adobe Premiere Elements 2.0 for video images and Adobe Photoshop CS2 for still images.
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RESULTS |
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Isometric contractions. During the first and second tetanic contractions, the contractile behavior and microscopic appearance of the mdx and control muscles were indistinguishable. However, during the rest period between the second and third tetanic contractions of mdx muscles, multiple individual fibers were observed to be undergoing sustained contractile activity (contractures), and the myoplasm of those fibers was separating longitudinally. Myoplasmic separation was followed by the retraction of myoplasm from the site of the separation and then formation of contracture clots, all occurring during the rest period (see movie 1). Subsequent tetanic stimulations of the mdx muscles produced force responses that reached steady levels and were in all respects normal in appearance except that each force level was progressively lower than that of the preceding contraction because of the declining number of functional fibers. Each tetanus was followed by a 60-s rest period that featured additional fibers in which contracture activity was observed to result in separation and retraction of the myoplasm. Retraction of myoplasm was rarely observed in mdx muscles during the tetanic stimulation periods, even when contraction duration was increased to as much as 5 s. A sequence illustrating a typical progression of both the microscopic appearance of and force generated by an mdx muscle is shown on the right in Fig. 2. Mean force responses from 12 experiments on mdx muscles are shown in Fig. 3 (filled squares); by the tenth tetanic contraction, force production had declined to <50% of that of the first.
Concurrent with the separation and clotting of fiber myoplasm that occurred between tetanic contractions, the mdx muscles exhibited elevations in resting [Ca2+]i that were accompanied by increases in resting tension (Fig. 4, A and B). This finding, taken together with the stable force responses (Fig. 2) and absence of myoplasmic retraction during the tetanic contractions, suggests strongly that the processes responsible for the decline in tetanic force production in the mdx muscles took place during the rest periods between contractions rather than during the contractions themselves. When subjected to the same contraction protocol, muscles from control mice exhibited no fiber clotting (Fig. 2, left, and movie 2), no decline in maximum isometric force (Fig. 3, filled circles), and only small elevations in resting tension and resting [Ca2+]i during the 60-s periods between tetanic contractions (Fig. 4, A and B).
To determine whether the source of excess Ca2+ in the fibers of mdx muscles was intracellular or extracellular, the 10-contraction protocol was repeated with muscles bathed in a physiological solution in which Ca2+ was replaced by magnesium (Mg2+). The response of the mdx muscles in reduced extracellular Ca2+ was similar to that of control muscles (Fig. 3, filled triangles); there was no decline in maximum isometric force and no separation and clotting of fiber myoplasm. This result established the extracellular compartment as the source of activating Ca2+ responsible for the increases in intercontraction fluorescence and tension.
Response to regional activation. The finding that fibers lacking dystrophin undergo myoplasmic separation and retraction during quiescent periods when overall [Ca2+]i and force remain quite low, but not during tetanic activation periods when [Ca2+]i and force are both very high, presented a paradox. It suggested that nonuniform Ca2+ activation, expected if Ca2+ enters the myoplasm via nonuniformly distributed leaks rather than by uniformly distributed intracellular release, may be responsible for the observed fiber failures. This idea was tested directly in a series of experiments in which lumbrical muscles from both mdx and control mice were exposed to localized applications of ionomycin, a Ca2+ ionophore. All muscles treated in this way responded with an immediate contracture-like force production that was clearly nonuniform, with pronounced shortening of some regions of the muscles and corresponding lengthening of the remaining regions. In the presence of ionomycin, the force produced by the mdx muscles reached a maximum early in the observation period and then began to decline (Fig. 5, A and B). The force decline was accompanied by numerous occurrences of myoplasmic separation and retraction identical in appearance to those observed during the rest periods between tetanic contractions shown in movie 1. Four of the 10 ionomycin experiments on mdx muscles were performed while monitoring fura-2 fluorescence. These experiments confirmed that [Ca2+]i began to increase coincident with the introduction of ionomycin and generally continued to rise throughout an 800 s observation period. By the end of the 800 s period, fibers in the mdx muscles exhibited extensive clotting (Fig. 5C, bottom right image) similar to that observed in the mdx muscles subjected to tetanic contractions (Fig. 2, bottom right image).
In contrast, control muscles maintained force production throughout the observation period of nearly 15 min with no decrement in magnitude (Fig. 5, A and B) and no evidence of separation or clotting of myoplasm (Fig. 5C, bottom left image). These experiments confirmed that membrane breaches and the resultant nonuniform activation are tolerated well in control muscle fibers but have catastrophic consequences in fibers lacking dystrophin. The results indicate that, in addition to rendering the sarcolemma vulnerable to Ca2+ breaches, the absence of dystrophin has further adverse functional consequences that are ultimately responsible for separation, retraction, and clotting of myoplasm.
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DISCUSSION |
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In addition to providing confirming and direct evidence of the importance of dystrophin in preventing entry of extracellular calcium, we present evidence supporting an essential role for dystrophin in stabilizing the underlying sarcomeric structure. The observations that control muscle fibers tolerate ionophore-induced entry of extracellular Ca2+ and the resultant localized activation of contractile activity without apparent negative consequences indicate that, in the presence of an intact dystrophin complex, significant disruptions of the plasma membrane do not result acutely in damage to the fibers. We conclude that it is the combined loss of both the membrane and sarcomeric stabilization functions of dystrophin that causes such catastrophic consequences in dystrophic skeletal muscle fibers. The observation that localized application of ionophore causes myoplasmic separation and clotting in mdx muscle fibers shows that Ca2+ entry can initiate these events rather than simply being one of their consequences.
Our findings are consistent with the working model of the failure mechanism in dystrophic fibers illustrated in Fig. 6. Three major parallel pathways, referred to in the present study as the "cross bridge," "titin," and "costamere" pathways, contribute to longitudinal force transmission in skeletal muscle fibers (22). The cross bridge pathway, which passes from Z disc to thin filaments, through cross bridges to thick filaments, and again through cross bridges to thin filaments within a sarcomere, has primary responsibility for force transmission in an activated fiber. This pathway is present and fully functional in both control and dystrophin-deficient fibers during normal activation due to a uniformly distributed high [Ca2+]i that allows myosin cross bridges from the thick filament to form strong attachments to actin in the thin filament. Under these circumstances of uniformly high [Ca2+]i and, therefore, uniform activation, both the titin (Z disc, titin, thick filament, titin) (14) and costamere (Z disc, costamere, sarcolemma + basement membrane) (21) pathways are of secondary importance.
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The findings that fibers in lumbrical muscles from mdx mice develop contracture-induced myoplasmic separations and hypercontraction clots associated with localized increases in [Ca2+]i while "at rest," and that control fibers do not, support two functional consequences of the absence of dystrophin: 1) a sarcolemma that is vulnerable to stress, making it more likely that a given mechanical event will result in entry of extracellular Ca2+, either through small stress-induced tears (17) or stretch-activated Ca2+ channels (30), and 2) reduced shunting of the forces developed within the contractile core of a fiber to its neighboring extracellular matrix. The long-recognized sarcolemmal vulnerability in dystrophic muscles is alone insufficient to cause fiber failure as evidenced by the observation that fibers in muscles from control mice tolerate ionomycin-induced Ca2+ breaches and subsequent nonuniform activation without damage. Similarly, the ability of dystrophin-deficient fibers to tolerate high levels of force during prolonged periods of uniform activation shows that a reduced ability to shunt forces is not, in isolation, sufficient to cause failure. It is during "quiescent" periods between contractions, after the uniform distribution of Ca2+ afforded by its depolarization-induced release from intracellular stores has subsided, that the myoplasm of dystrophin-deficient fibers undergoes separation, retraction, and clotting. In addition to identifying the source of excess intracellular Ca2+, our finding that muscle structure and force-generating capability are maintained in the absence of extracellular Ca2+ suggests that breaches in the membrane permeability barrier do not, alone, damage dystrophin-deficient muscle fibers. Finally, while we demonstrated that extracellular Ca2+ was responsible for the nonuniform activation and subsequent damage to dystrophin-deficient fibers, the route by which the Ca2+ enters the cells was not identified and remains an active area of investigation. Both stretch-activated cation channels (30) and transient tears in the sarcolemma (2, 17) are consistent with our present findings.
The extent of damage and magnitude of the reduction in force reported in the present study for lumbrical muscles of mdx mice following 10 isometric contractions were surprising. Such deficits are more commonly associated with lengthening-contraction protocols (1, 3, 6, 15, 18, 24). Relatively few studies have characterized the response of dystrophin-deficient muscles to isometric contractions. Petrof et al. (24) showed increased uptake of a membrane-impermeant dye in response to five isometric contractions in strips of diaphragm muscle fibers from mdx mice but did not report the duration of the contractions or whether they resulted in force deficits. Moens et al. (18) found no force deficit after six isometric tetanic contractions in extensor digitorum longus muscles from mdx mice, but tetanus duration was 0.35 s rather than the 1 s used in the present study, which suggests that contraction duration may be a factor. Another likely factor is the large variation in susceptibility to contraction-induced injury observed among muscles within mdx mice (3, 15, 18, 24). The lumbrical muscles used in the present study are intrinsic muscles of the foot and appear to be highly susceptible to damage induced by contractions in the absence of dystrophin and its associated proteins.
Sarcolemma stabilization (24) and lateral mechanical coupling (25) are among the most frequently suggested functions for dystrophin. The mechanism proposed in the present study invokes both and asserts that, while the absence of either function alone is not sufficient to cause fiber failure, their combined absence leads to a catastrophic cascade that results in fiber damage and loss in dystrophin-deficient muscle.
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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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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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