Although the primary deficiency in dystrophin and the concomitant reduction in surface glycoproteins are well established factors in the molecular pathogenesis of Duchenne muscular dystrophy, the pathophysiological events that render a muscle fibre more susceptible to necrosis are not well understood. One proposed mechanism involves abnormal calcium homeostasis in mechanically stressed fibres that eventually leads to skeletal muscle weakness. This chapter examines the calcium hypothesis of muscular dystrophy and outlines how unbalanced ion cycling through the sarcolemma and the sarcoplasmic reticulum may contribute to enhanced degradation of muscle proteins. Studying calcium handling in muscular dystrophy is not only important for increasing our knowledge on the multifaceted process of muscle degeneration, but might also have implications for the future design of therapeutic approaches to treating x-linked muscular dystrophy. In this respect, it is encouraging that the pharmacological elimination of Ca²⁺-dependent proteolysis counteracts dystrophic changes and that the removal of excess cytosolic Ca²⁺ appears to convey natural protection to dystrophin-deficient fibres.

Calcium Handling Proteins

Calcium ions represent an ubiquitous second messenger molecule involved in the regulation of various metabolic and physiological processes.¹ In skeletal muscle, cytosolic Ca²⁺-levels dictate the overall contractile status, and Ca²⁺-cycling through intracellular compartments is maintained under precise spatial and temporal control.² Signal transduction during excitation-contraction coupling and fibre relaxation is mediated by complex interactions between voltage sensors, Ca²⁺-channels, Ca²⁺-binding proteins, Ca²⁺-transporters, ion exchangers and ion pumps.^3-5 Table 1 lists established elements involved in ion handling that are potentially impaired in muscular dystrophy. Almost all Ca²⁺-regulatory elements exist as several isoforms⁶ reflecting the heterogeneity of skeletal muscle fibres.⁷ Due to the enormous complexity of the Ca²⁺-handling apparatus, small changes in individual steps involved in modulating Ca²⁺-signals might result in major pathophysiological consequences. Although it is not yet established how many auxiliary proteins act as intrinsic regulators of Ca²⁺-release complexes, Ca²⁺-uptake units and the luminal Ca²⁺-reservoir complex, abnormal expression patterns of ion-regulatory components are clearly involved in muscular dystrophy.^8-12 The exact degree of abnormal Ca²⁺-handling is difficult to judge,¹³ since it is not fully understood whether key Ca²⁺-handling proteins operate at their full capacity under normal conditions.^3,14-16

Table 1

Ion-regulatory proteins of the excitation-contraction-relaxation cycle.

Calcium Hypothesis of Muscular Dystrophy

Historically, the formulation of the calcium hypothesis of muscular dystrophy¹⁷ preceded the pioneering discovery of the genetic defect responsible for Duchenne muscular dystrophy.¹⁸ A decade before Kunkel and coworkers described the primary abnormality in x-linked muscular dystrophy using a positional cloning strategy,^18,19 anomalous intracellular Ca²⁺-accumulation was described in fibres from patients afflicted with x-linked muscular dystrophy.²⁰ In conjunction with the electron microscopical demonstration of membrane damage in dystrophic fibres,²¹ this finding led to the idea that chronic Ca²⁺-overload might play a central role in fibre degeneration.¹⁷ Since dystrophinopathies are primarily diseases of the membrane cytoskeleton,^22,23 it is important to consider that disruption of the cytoskeletal network has a direct effect on Ca²⁺-fluxes.^24,25 This suggests a direct involvement of the membrane cytoskeleton in the maintenance of subsarcolemmal Ca²⁺-levels and Ca²⁺-mediated signal transduction pathways.

Molecular Defects in Calcium Homeostasis

The current mechanical calcium hypothesis of muscular dystrophy assumes that a causal connection exists between the disintegration of sarcolemmal integrity and Ca²⁺-dependent proteolysis of muscle proteins,^8-12 as outlined in the flow chart of Figure 1. Various aspects of individual steps involved in this destructive process have been discussed in recent reviews.^8-12,26,27 Deficiency in the dystrophin isoform Dp427¹⁹ results in a drastic reduction in various sarcolemma-associated proteins,²⁸ such as the sarcoglycans²⁹ and dystroglycans.³⁰ The resulting destabilisation of the linkage between the actin cytoskeleton and the extracellular matrix component laminin renders the muscle periphery more susceptible to micro-rupturing.³¹ This in turn, triggers a Ca²⁺-dependent membrane resealing process at the sites of sarcolemmal disintegration. The influx of extracellular Ca²⁺-ions initiates an exocytotic event that adds plasma membrane patches to seal leaky micro domains of the disrupted muscle periphery.⁸ Ca²⁺-leak channels present in the newly introduced sarcolemmal patches cause an increased influx of Ca²⁺-ions into the cytoplasm of dystrophic fibres. The exact extent of perturbation of Ca²⁺-handling in dystrophin-deficient fibres is still controversial.^8-12 Contradictory findings may be partially due to the usage of different physiological techniques and standardisation in the measurement of intracellular Ca²⁺-levels. However, it is generally accepted that the Ca²⁺-overload is not global but restricted to cytosolic domains adjacent to the sarcolemma.^32,33 The recent characterisation of dysferlin-null mice has highlighted the importance of proper membrane resealing in muscular disorders. Although dysferlin-deficient fibres maintain a functional dystrophin-glycoprotein complex, they develop a progressive muscular dystrophy.³⁴ In skeletal muscle, surface membrane repair is an active process involving dysferlin whereby disruption of the sarcolemma repair apparatus triggers muscle degeneration.³⁴ This clearly demonstrates the importance of a fast Ca²⁺-dependent sarcolemma resealing process to efficiently counter-act injuries to the muscle plasma membrane.

Figure 1

Flow chart of the mechanical calcium hypothesis of muscular dystrophy. Outlined are two major factors involved in the elevated cytosolic Ca²⁺-levels observed in muscular dystrophy. Deficiency in the membrane cytoskeletal component dystrophin causes a (more...)

The pathological consequence of increased cytosolic Ca²⁺-levels is an activation of Ca²⁺-dependent proteases, such as skeletal muscle-specific calpains, resulting in the net degradation of muscle proteins in dystrophic muscle cells.^35-38 Localised Ca²⁺-elevations are believed to contribute to a destructive cycle of enhanced protease activity and Ca²⁺-leak channel activation. ³⁶ Because increased proteolytic activity renders Ca²⁺-leak channels constitutively active, insertion of membrane vesicles for resealing, following exercise-induced membrane rupturing, results in persistent Ca²⁺ influx at affected sarcolemma domains. Hence, more Ca²⁺ influx causes increased proteolysis, and more proteolysis causes increased Ca²⁺ influx, giving rise to a pathophysiological cycle.⁸ The theory of the initiating role of mechano-sensitive Ca²⁺-channels in triggering fibre degradation has recently been challenged by the study of transgenic mdx muscle that over-expresses utrophin.³⁹ It therefore can not be excluded that the Ca²⁺-leak channel activation in mechanically stressed fibres is preceded by modifications in other Ca²⁺-handling proteins. For example, surface membrane leakage of Ca²⁺ into the cytoplasm may also be due to abnormalities in the nicotinic acetylcholine receptor.^40,41 This fact, however, does not challenge the overall concept of deranged intracellular Ca²⁺-handling being involved in muscular dystrophy. Both, Ca²⁺-permeable growth factor-regulated channels42 (GRC) and transient receptor potential channels³³ (TRPC) may facilitate abnormal Ca²⁺-influxes through the dystrophin-deficient muscle periphery. The expression of Ca²⁺-permeable nonselective cation channels of the GRC type were shown to be elevated in the sarcolemma of mdx fibres⁴² and isoforms TRPC-1, 4 and 6 of the transient receptor potential channels are present in mdx membrane.³³ In contrast, surface L-type Ca²⁺-channels of the dihydropyridine receptor class do not appear to contribute to elevated cytosolic Ca²⁺-levels in muscular dystrophy.⁴³

Impaired Excitation-Contraction Coupling

In addition to abnormal sarcolemma physiology, changes in the Ca²⁺-cycling patterns through the lumen of the sarcoplasmic reticulum⁴⁴ and mitochondria⁴⁵ have also been shown to occur in dystrophin-deficient skeletal muscle. The excitation-contraction-relaxation cycle is regulated by the physiological interplay between the voltage-sensing dihydropyridine receptor of the transverse tubules, the ryanodine receptor Ca²⁺-release channel of the junctional sarcoplasmic reticulum and the Ca²⁺-ATPases of the longitudinal tubules.^3-5 It would not be surprising if these ion-regulatory elements were secondarily involved in muscular dystrophy, since numerous inherited muscle diseases exhibit defects in excitation-contraction coupling or muscle relaxation. This includes malignant hyperthermia, central core disease, hypokalemic periodic paralysis and Brody's disease.⁴⁶ In normal muscle fibres, the release of intracellular Ca²⁺ in response to sarcolemmal depolarization is one of the key steps of excitation-contraction coupling. ³ Following voltage-sensing by the α1S-subunit of the dihydropyridine receptor, physical coupling between the II-III loop domain of the transverse-tubular tetrad complex and the cytosolic foot region of the junctional ryanodine receptor tetramer triggers transient opening of the Ca²⁺-release channel.^5,16 Thus, at least initially, a direct physical receptor interaction process couples membrane depolarization to the release of Ca²⁺-ions into the cytosol. In a subsequent step, comparable to the cardiac Ca²⁺-induced Ca²⁺ -release mechanism, sarcoplasmic reticulum Ca²⁺-channels are also locally activated by excess Ca²⁺ that greatly amplifies the overall Ca²⁺-signal.^3-5,16 Fibre relaxation is initiated by the energy-dependent reversal of the cytosolic Ca²⁺-signal. Reuptake of Ca²⁺-ions in the lumen of the sarcoplasmic reticulum is achieved by the SERCA type Ca²⁺-pumps.¹⁴ In contrast to heart muscle, Ca²⁺-removal via the surface Na⁺/Ca²⁺-exchanger does not play a major role in skeletal muscle fibres. An important physiological regulator that mediates between Ca²⁺-release and Ca²⁺-uptake is the luminal Ca²⁺-reservoir complex of the sarcoplasmic reticulum.¹⁵ Besides the histidine-rich Ca²⁺-binding protein, calreticulin, sarcalumenin and calsequestrin-like proteins, the most abundant Ca²⁺-binding element of the terminal cisternae are clusters of 63 kDa calsequestrin molecules.⁴

It has not been determined how many auxiliary proteins are involved in the fine regulation of receptor coupling, Ca²⁺-release, Ca²⁺-buffering and Ca²⁺-uptake and remains to be elucidated which triad-associated components prevent passive disintegration of supramolecular membrane assemblies. Recently identified auxiliary proteins such as triadin, junctin, JP-45 and JP-90 only represent part of the total protein complement of the junctional couplings involved in excitation-contraction coupling.^47-50 Current muscle proteome projects are addressing this question and aim at the identification of all sarcoplamic reticulum proteins involved in Ca²⁺-cycling. Until these results become available, we lack an overall understanding of the complexity of the Ca²⁺-handling apparatus. Nevertheless, the initial analysis of the main players involved in excitation-contraction coupling revealed that calsequestrin-like proteins (CLP) are greatly reduced in muscular dystrophy,⁴⁴ as illustrated in the immunoblot analysis shown in Figure 2. While the expression of the junctional ryanodine receptor Ca²⁺-release channel, the voltage-sensing dihydropyridine receptor, the Ca²⁺-ATPase and calsequestrin was not affected, a drastic decline in CLP-150, CLP-170 and CLP-220 was observed in dystrophic microsomes.⁴⁴ Although immunoblotting could clearly demonstrate the reduction in these terminal cisternae markers, this technique could not address the question of whether the reduction in high-molecular-mass isoforms of calsequestrin is due to decreased expression levels or because of an impaired oligomersiation pattern.

Figure 2

Drastic reduction in calsequestrin-like proteins in dystrophin-deficient skeletal muscle. Shown is an immunoblot of microsomal proteins from normal (lane 1) and dystrophic mdx (lane 2) muscle fibres labelled with monoclonal antibody VIIID1₂ to the fast (more...)

Comparative chemical crosslinking analysis of normal and dystrophic microsomal membranes revealed a crosslinker-induced reappearance of CLP species in dystrophin-deficient preparations. ⁵¹ Hence, CLP-150, CLP-170 and CLP-220 appear to represent clusters of the calsequestrin monomers and not distinct high-molecular-mass isoforms. Since the introduction of short crosslinker probes could restore the appearance of CLPs in dystrophic membranes, their drastically reduced expression in muscular dystrophy is due to impaired oligomerisation of calsequestrin monomers, and not based on a loss in individual isoforms of this Ca²⁺-binding protein. Equilibrium dialysis experiments have shown that the overall Ca²⁺-binding capacity was reduced by approximately 20% in dystrophic sarcoplasmic reticulum. ⁴⁴ Thus, impaired protein coupling between calsequestrin units in the terminal cisternae region might be directly involved in triggering impaired Ca²⁺-sequestration within the lumen of the sarcoplasmic reticulum. In muscular dystrophy, disturbed sarcolemmal Ca²⁺-fluxes appear to influence the complex Ca²⁺-cycling apparatus causing distinct changes in the oligomerisation pattern of a subset of Ca²⁺-handling proteins (fig. 3).

Figure 3

Diagrammatic presentation of the pathophysiological role of abnormal Ca²⁺-handling in muscular dystrophy. In adult skeletal muscle, excitation-contraction (EC) coupling is mediated by direct physical interactions between the voltage-sensing α (more...)

With respect to cytosolic Ca²⁺-binding elements, parvalbumin plays an ion-buffering role in fast muscle fibres.⁵² Chronic low-frequency stimulation suppresses parvalbumin expression in fast muscles demonstrating that a slow motoneuron-like impulse pattern rapidly silences the parvalbumin gene.⁵³ An increased turnover of parvalbumin has been described in dystrophic fast-twitching mdx fibres.⁵⁴ Elevated serum parvalbumin levels appear to be indicative of the severity of the disease status in dystrophic mouse muscle.⁵⁴ Compared to the severe dystrophic phenotype of Duchenne patients, the mdx mouse model exhibits a much milder dystrophy. Since parvalbumin is expressed at only very low levels in human fibres, but is abundant in rodent IIB fibres,⁵⁵ the presence of parvalbumin might be responsible for this difference in the severity of the dystrophinopathy. This relatively species-specific Ca²⁺-binding element could buffer the increased Ca²⁺-influx through the mdx sarcolemma thereby maintaining a lower cytosolic Ca²⁺-level as compared to dystrophic human muscle fibres. However, double mutant mice that are deficient in both dystrophin and parvalbumin exhibit a cytosolic Ca²⁺-content similar to that of mdx fibres.⁵⁶ Thus, parvalbumin does not appear to play a central role in eliminating abnormal Ca²⁺-cycling through the cytosol of mdx muscle.

Therapeutic Implications

In addition to helping in understanding the molecular pathways underlying muscular dystrophy, studying Ca²⁺-regulatory proteins might also lead to the discovery of new therapeutic targets. One potential way forward is the analysis of naturally protected muscle phenotypes that lack dystrophin but do not exhibit severe symptoms. The sparing of dystrophin-deficient extraocular fibres is attributed to the special protective properties of small-diameter fibres.⁵⁷ Recently, it was shown that in contrast to leg muscles, toe fibres from the dystrophic animal model mdx also show a very low degree of muscle degeneration.^58,59 This is histochemically evident by a low percentage of centrally located nuclei. Toe fibres from the mdx mouse exhibit a preserved expression of the critical trans-sarcolemmal linker α-dystroglycan⁵⁸ and an up-regulation and extra-junctional localization of the autosomal dystrophin homologue utrophin.^58,59 Possibly utrophin acts as a substitute for the deficient Dp427 isoform and thereby prevents the disintegration of the surface-associated glycoprotein complex.⁶⁰ Interestingly, an increase in both the expression of the fast SERCA1 isoform of the sarcoplasmic reticulum Ca²⁺-pump and the total Ca²⁺-ATPase activity was described in protected mdx toe fibres.⁵⁸ Possibly, the up-regulation of Ca²⁺-ATPase units causes an increased removal of cytosolic Ca²⁺-ions from mdx toe fibres thereby significantly reducing the degree of Ca²⁺-induced myonecrosis in dystrophin-deficient fibres.⁵⁸ That a decrease in cytosolic Ca²⁺-levels has a protective effect was shown by experiments with carnitine-linked leupeptin.^61,62 The targeted introduction of protease inhibitors appears to specifically inactivate Ca²⁺-dependent calpain activity. ⁶² Calpain inhibition could be correlated with rentention of myofibre size.⁶¹ Overexpression of the calpain inhibitor calpastatin in dystrophin-deficient fibres reduced the dystrophic phenotype⁶³ suggesting that specific calpains are primarily involved in muscle necrosis. Based on the pathophysiological role of abnormal Ca²⁺-cycling in dystrophic fibres, potential sites of pharmacological interventions present themselves at various Ca²⁺ regulatory processes including the sarcolemma and sarcoplasmic reticulum.^64-66 Especially calpain inhibition promises to be an excellent therapeutic target to prevent muscular degeneration in Duchennne muscular dystrophy.^61-63 Hence, besides myoblast transfer,⁶⁷ stem cell treatment⁶⁸ or gene therapy⁶⁹ for the treatment of x-linked muscular dystrophy (see Chapters 17 and 18), a more traditional pharmacological approach might also be a promising option.⁷⁰

Conclusions

In conclusion, abnormal Ca²⁺-handling might be an important factor in the progressive functional decline of dystrophic muscle fibres. Although the total extent and exact micro-domain localisation of the initial Ca²⁺-disturbance has not yet been fully established, it is understood that even small changes in Ca²⁺-cycling trigger a cascade of modifications in ion-regulatory muscle proteins. The deficiency in dystrophin leads primarily to a reduction in dystrophin-associated proteins which in turn destroys sarcolemmal integrity by weakening the linkage between the extracellular matrix and the membrane cytoskeleton. The introduction of Ca²⁺-leak channels during membrane resealing appears to be the central pathophysiological incident that causes an increase in cytosolic Ca²⁺-levels and consequently Ca²⁺-induced myonecrosis. In addition, impaired calsequestrin oligomerisation causes decreased Ca²⁺-sequestration within the lumen of the sarcoplasmic reticulum. Thus, the disintegration of the surface dystrophin-glycoprotein complex triggers disturbed sarcolemmal Ca²⁺-fluxes which in turn impairs other Ca²⁺-handling steps thereby resulting in distinct changes in the oligomerisation pattern of Ca²⁺-binding elements. This pathophysiological scenario might explain one of the key routes leading to cellular degeneration in muscular dystrophy. Most importantly, a more detailed knowledge of impaired Ca²⁺ handling in muscular dystrophy might lead to the design of novel approaches to counter-act fibre degeneration in genetic muscle diseases.

Acknowledgments

Research in the author's laboratory on calcium handling in neuromuscular disorders was supported by project grants from the Irish Health Research Board (HRB-95/95, HRB-01/98, HRB-01/99, HRB-01/01), Enterprise Ireland (SC/2000/386) and Muscular Dystrophy Ireland (MDI-95, MDI-02), as well as network grants from the European Commission (FMRX-CT960032, QLRT-1999-02034, RTN2-2001-00337). I would like to thank Dr. Kevin Culligan for the immunoblot analysis of dystrophic fibres.

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