Acta Pharmacologica Sinica

Invited review

Acta Pharmacologica Sinica 2006 July; 27 (7): 791-798

Ca²⁺ sparks as a plastic signal for skeletal muscle health, aging, and dystrophy

Noah Weisleder, Jian-jie Ma¹

Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA

¹Correspondence to Dr Jian-jie MA.
Phn 86-732-235-4494.
Fax 86-732-235-4483.
E-mail maj2@umdnj.edu
Received 2006-05-12
Accepted 2006-05-17
doi: 10.1111/j.1745-7254.2006.00384.x

Abstract

Ca²⁺ sparks are the elementary units of intracellular Ca²⁺ signaling in striated muscle cells revealed as localized Ca²⁺ release events from sarcoplasmic reticulum (SR) by confocal microscopy. While Ca²⁺ sparks are well defined in cardiac muscle, there has been a general belief that these localized Ca²⁺ release events are rare in intact adult mammalian skeletal muscle. Several laboratories determined that Ca²⁺ sparks in mammalian skeletal muscle could only be observed in large numbers when the sarcolemmal membranes are permeabilized or the SR Ca²⁺ content is artificially manipulated, thus the cellular and molecular mechanisms underlying the regulation of Ca²⁺ sparks in skeletal muscle remain largely unexplored. Recently, we discovered that membrane deformation generated by osmotic stress induced a robust Ca²⁺ spark response confined in close spatial proximity to the sarcolemmal membrane in intact mouse muscle fibers. In addition to Ca²⁺ sparks, prolonged Ca²⁺ transients, termed Ca²⁺ bursts, are also identified in intact skeletal muscle. These induced Ca²⁺ release events are reversible and repeatable, revealing a plastic nature in young muscle fibers. In contrast, induced Ca²⁺ sparks in aged muscle are transient and cannot be re-stimulated. Dystrophic muscle fibers display uncontrolled Ca²⁺ sparks, where osmotic stress-induced Ca²⁺ sparks are not reversible and they are no longer spatially restricted to the sarcolemmal membrane. An understanding of the mechanisms that underlie generation of osmotic stress-induced Ca²⁺ sparks in skeletal muscle, and how these mechanisms are altered in pathology, will contribute to our understanding of the regulation of Ca²⁺ homeostasis in muscle physiology and pathophysiology.

Key words

calcium signaling; calcium channel; aging; muscular dystrophies; endoplasmic reticulum

Extract

Note: Please read the complete full text with Figures and Tables at

Ca²⁺ sparks: elemental unit of intracellular Ca²⁺ release in striated muscle
Excitation-contraction coupling in skeletal and cardiac muscle requires close association between voltage-gated Ca²⁺ channels of the dihydropyridine receptor (DHPR) class in the sarcolemmal membrane and Ca²⁺ release channels of ryanodine receptor (RyR) in the sarcoplasmic reticulum (SR). In the heart, the entry of extracellular Ca²⁺ via DHPR triggers opening of RyR to amplify Ca²⁺ signaling through the Ca²⁺-induced Ca²⁺ release (CICR) mechanism^[1_3]. Membrane depolarization, rather than external Ca²⁺ entry, triggers SR Ca²⁺ release in skeletal muscle. In skeletal muscle, CICR represents an important amplification mechanism following voltage-induced Ca²⁺ release (VICR), especially under stress conditions^[4_6].
The close juxtaposition of the transverse-tubular (TT) invagination of the sarcolemma and the SR terminal cisternae allows relay of the depolarizing signal^[7_10]. These TT invaginations run in close spatial proximity to the SR terminal cisternae and establish the triad junction complex in skeletal muscle fibers^[11,12]. This membrane structure allows contact between RyR and DHPR to ensure tight control of the Ca²⁺ release machinery to limit Ca²⁺ leak from SR. A majority of RyR channels in the muscle fiber are coupled with DHPR at the triad junctional region. Under normal physiological condition, Ca²⁺ sparks termination and repression in skeletal muscle can be achieved from either deactivation of the DHPR voltage sensor or intrinsic inactivation of the RyR channel^[13].
The elementary units of Ca²⁺ release from SR in striated muscle cells are discreet events known as Ca²⁺ sparks. These events are visualized by laser confocal scanning microscopy as a localized increase of signal from a fluorescent Ca²⁺ indicator dye loaded within a muscle cell. Ca²⁺ sparks were first discovered in cardiac muscle as quantal Ca²⁺ release events that originate from paracrystalline arrays of RyR channels on the SR surface, and therefore represent the elemental units of CICR^[1,14,15]. The discovery of Ca²⁺ sparks has revolutionized understanding of the physiology and pathophysiology of Ca²⁺ signaling in cardiac and smooth muscles^[16,17]. How-ever, since the discovery of Ca²⁺ sparks in cardiac muscle, investigators have had difficulty in detecting these localized Ca²⁺ release events in intact adult mammalian skeletal muscle (Table 1).

Challenges in observation of Ca²⁺ sparks in skeletal muscle fibers
A major obstacle facing research on Ca²⁺ spark in skeletal muscles is the intrinsic difficulty in measuring spontaneous Ca²⁺ sparks in intact adult mammalian skeletal muscle fibers^[18], as most available studies were conducted with amphibian muscle or permeabilized mammalian muscle^[19,20]. Ca²⁺ sparks were also soon detected in neonatal mammalian skeletal muscle^[21] where they were attributed to the activity of the type 3 RyR^[22], which is preferentially expressed in mammalian skeletal muscle during fetal development and present at low expression levels in a minority of adult skeletal muscles^[23]. While rare observations of Ca²⁺ sparks have been made in resting intact adult mammalian fibers^[18,21], significant numbers of events in mammalian skeletal fibers were only observed in cells where the sarcolemmal integrity was disrupted by various physical or chemical skinning methods^[20,24]. Since the sarcolemmal membrane is a major regulator of Ca²⁺ release at the triad junction, disruption of membrane integrity of skeletal muscle will likely alter the intracellular Ca²⁺ release machinery. Thus, most of the biophysical studies with Ca²⁺ sparks in skeletal muscle were carried out under non-physiological conditions. This disadvantage, coupled with the intrinsic difficulties with monitoring Ca²⁺ spark activity in intact mammalian muscle fibers, has limited our understanding of the cellular and molecular mechanisms underlying the regulation of Ca²⁺ spark in skeletal muscle and the adaptive changes of Ca²⁺ spark in muscle aging and disease.

Osmotic stress-induced Ca²⁺ sparks in skeletal muscle
Recently, our laboratory discovered that transient osmotically-induced membrane deformation resulted in a fluttered Ca²⁺ spark response adjacent to the sarcolemmal membrane in intact mouse muscle fibers^[25,26](Figure 1). In a series of serendipitous experiments with mouse skeletal muscle fibers, we discovered that subtle alterations of membrane structure produced drastic elevation of intracellular Ca²⁺ spark activity. When bathed in isotonic solution, isolated intact muscle fibers from non-exercised mice do not reveal any spontaneous Ca²⁺ spark activity, confirming that Ca²⁺ sparks are suppressed under resting conditions. After equilibration in isotonic solution (290 mOsm), fibers were perfused with either a hypotonic (170 mOsm) or hypertonic (450 mOsm) solution. Cell volume was markedly altered by exposure to solutions of varying osmolarity, swelling in a hypotonic environment and shrinking upon exposure to hypertonic conditions. These changes in cell volume result in alterations to the fluorescent Ca²⁺ dye signal observed, which may reflect dilution due to water entry or a decrease in intracellular Ca²⁺ that could influence Ca²⁺ spark induction. Shrinkage of the fiber, resulting from either hypertonic solution or a return to isotonic solution after exposure to hypotonic solution, induces a remarkable elevation of Ca²⁺ spark activity (Figure 1A). Kinetic analysis reveals the presence of two modes of Ca²⁺ spark signaling, the first being short release events similar to those seen in cardiac muscle and permeabilized skeletal muscle. A second group contains events with extended openings of the Ca²⁺ release machinery, known as Ca²⁺ bursts (Figure 1B). These Ca²⁺ burst events uncovered in our intact muscle preparation may have a significant physiological function, as they are not observed in permeabilized skeletal muscle fibers.
The sudden increase in Ca²⁺ spark activity following cell shrinkage is reversible in young, healthy muscle fibers as it returns back to baseline levels in 10 to 15 min after return to normal cell volume. These Ca²⁺ sparks are absent in the presence of high levels of ryanodine, indicating the spark activity is dependent on RyR channel activity. Short term removal of extracellular Ca²⁺ does not have a major effect on osmotic shock-induced Ca²⁺ spark activity, suggesting that osmotic stress-induced sparks are not induced by Ca²⁺ entry from outside the fiber. Our discovery of legitimate manifestations of Ca²⁺ sparks in skeletal muscle enables us to address some fundamental questions in skeletal muscle physiology.

Peripheral confined nature of osmotic stress-induced Ca²⁺ sparks
The numerous Ca²⁺ sparks induced upon shrinkage of the fiber appear in the periphery of the cell (Figure 2A). The osmotic stress-induced Ca²⁺ spark response is localized near the sarcolemma surfaces despite global swelling of the TT system by osmotic shock. Electron microscopy studies from Chawla et al have shown that hypertonic treatment induces subtle changes of triad junction structure in skeletal muscle^[27]. We have replicated these experiments and found similar swelling of triad structures throughout the muscle fiber, without significant disruption of sarcolemmal or myofibrillar integrity. It is well known that RyR channels and other associated Ca²⁺ release machinery are found throughout the TT system, and since osmotic shock results in global swelling of TT, the spatial restriction of Ca²⁺ sparks suggests two criteria for the regulation of Ca²⁺ sparks. First, a signal for the initiation of Ca²⁺ sparks must be spatially confined, eg, either membrane-delimited signaling cascades or localized cytosolic factors should be involved in triggering the SR Ca²⁺ release machinery. Second, a coordinated mechanism must be present to prevent propagation of the Ca²⁺ spark signaling from the periphery toward the central region of a healthy muscle fiber.
Spatial inhomogeneities of SR Ca²⁺ release are not unique to skeletal muscle, as cardiac and smooth muscles also display preferential SR Ca²⁺ release at the periphery of the muscle fiber, at least at the initial stages of cell depolarization^[28_32]. Indeed, Ca²⁺ release following membrane deformation is a common phenomena observed in many cell types^[33]. While in many of these cases subsarcolemmal Ca²⁺ sparks or waves have been attributed to the limited TT system within these cell types^[34], other cell types do display well developed TT structures^[35]. In addition, a previous study also identified osmotic-shock induced Ca²⁺ sparks and Ca²⁺ waves in amphibian skeletal muscle^[36].

Role of ryanodine receptor isoforms in Ca²⁺ spark induction
While Ca²⁺ sparks are mediated by opening of RyR, it is not clear how the expression of different RyR isoforms contributes to Ca²⁺ spark formation, particularly within adult mammalian skeletal muscle fibers. Various studies have determined that two RyR isoforms are expressed in mammalian skeletal muscle, RyR1 and RyR3^[37_39]. While most adult skeletal muscle primarily expresses RyR1, the RyR3 protein is expressed mainly in neonatal muscle fibers and at a low level in a minority of adult skeletal muscles^[40,41]. The function of RyR1 is tightly controlled by DHPR while RyR3 acts as an secondary component that amplifies RyR1-mediated Ca²⁺ release in neonatal skeletal muscle^[42]. Overexpression of RyR3 in cultured myotubes^[22,43] and non-excitable cells^[44] has been shown to produce Ca²⁺ sparks. Homozygous ablation of RyR1 in mice results in post-natal lethality due to defective excitation-contraction coupling^[45], while ablation of RyR3 produces viable animals^[46_48]. The viability of the ryr3(-/-) mouse provides an opportunity to use osmotic shock to determine the contribution of RyR3 to the generation of Ca²⁺ sparks (Table 1).
Osmotic shock-induced Ca²⁺ sparks also occur in ryr3(-/-) muscle with a spatial distribution similar to that seen in wild type (wt) muscle, suggesting that RyR1 alone is sufficient to produce the dynamic Ca²⁺ spark signal in skeletal muscle fibers. Although RyR3 is not essential for induction of Ca²⁺ sparks in skeletal muscle, kinetic analysis reveals that the absence of RyR3 in the adult muscle fiber leads to significant changes in the elemental properties of Ca²⁺ spark signaling. Our results show that the spatial restriction, initiation rate and amplitude of individual Ca²⁺ release events are altered in the ryr3(-/-) muscle, whereas the peripheral localization of active Ca²⁺ release sites and their cross-talk remain unchanged. These changes could reflect the contribution of residual RyR3 function in adult skeletal muscle fibers or adaptations that take place in the developing skeletal muscle to compensate for the loss of RyR3 expression.

Uncontrolled Ca²⁺ sparks as a dystrophic signal in skeletal muscle
In young, healthy muscle fibers, osmotic shock-induced Ca²⁺ spark activity is transient and eventually returns to a silent mode several minutes after the initial shock^[25]. This response is plastic in nature as typical muscle fiber can receive up to three osmotic shocks and still maintain a reproducible response. Thus, membrane deformation can induce spontaneous Ca²⁺ spark activity in intact mammalian skeletal muscle in a reversible and repeatable manner.
Duchenne and Becker muscular dystrophy results from mutations within the dystrophin gene. Dystrophin is a protein that links actin in the muscle cytoskeleton to laminin in the extracellular matrix through the dystroglycan complex. It is likely that the dystrophic phenotype is not a direct result of alterations to the myofibrillar structures, rather it is a dis ruption of sarcolemmal membrane integrity that normally confers control of intracellular Ca²⁺ homeostasis that leads to muscle degeneration^[49]. To determine if Ca²⁺ spark signaling was altered in dystrophic skeletal muscle, we used a mouse model that lacks dystrophin, the muscular dystrophic (mdx) mouse^[50]. One hallmark of the mdx muscle is its increased fragility during endurance training and hypotonic shock^[51]. Although Ca²⁺ sparks do not appear in mdx muscle fibers at a resting state, there is a striking difference in the manifestation of Ca²⁺ sparks following osmotic shock. Similar to wt muscle fibers, exposure of the mdx muscle to either hypotonic or hypertonic solution also converts a resting and apparently silent muscle fiber into a highly active Ca²⁺ signaling state. In contrast to the transient Ca²⁺ spark activity seen in wt fibers, either a hypotonic or hypertonic shock results in sustained Ca²⁺ spark activity that is irreversible in the time period of observation^[25](Table 2).
Differences in the membrane-deformation-induced Ca²⁺ spark response in mdx muscle are not limited to its irreversible nature. As with wt fibers, Ca²⁺ sparks in mdx muscle are usually localized in the peripheral region at the initial stage. Surprisingly, Ca²⁺ sparks progressively penetrate into the center of the mdx muscle fiber at later stages following osmotic shock (Figure 2B). Furthermore, the peak amplitude of Ca²⁺ bursts in mdx muscle appears to decline with time after the osmotic shock, possibly due to reduced Ca²⁺ content in the SR. Resting cytosolic Ca²⁺ concentrations are elevated in both wt and mdx fibers following hypotonic shock, however cytosolic Ca²⁺ levels in mdx fibers are consistently increased over wt. These results suggest that membrane deformation results in a leaky SR Ca²⁺ release machinery in mdx muscle. Although our results do not exclude potential contribution of other factors, it suggests that a leaky intracellular Ca²⁺ release pathway can function as a primary trigger for the dystrophic signal cascade in mammalian skeletal muscle.

Compromised Ca²⁺ spark signaling in aged skeletal muscle
Aging effects on muscle function have been associated with muscle fiber denervation, loss of motor units, and motor unit remodeling. Since functional alterations occur before significant muscle wasting becomes evident, changes in the excitation-contraction coupling machinery and intracellular Ca²⁺ homeostasis may act as causative factors for, or adaptive responses to, muscle aging. Altered function of several triad junction proteins, including DHPR^[52,53], calsequestrin^[54,55] and SERCA^[56,57], have been shown to contribute to disrupted Ca²⁺ homeostasis in aged skeletal muscle. It has been suggested that cumulative uncoupling of the VICR process may be part of the causative and/or adaptive changes during muscle aging^[58,59]. However, limitations in resolution of Ca²⁺ sparks in intact muscle have prevented the detailed examination of the mechanisms that underlie changes in Ca²⁺ homeostasis during muscle aging.
Extending our initial discovery of Ca²⁺ sparks in healthy young muscle, we have identified a phenotypic change of Ca²⁺ spark signaling in aged skeletal muscle. Although this Ca²⁺ spark response is located in the periphery of both young and aged muscle fibers, it appears that the plastic nature of Ca²⁺ sparks in young muscle is compromised in aged skeletal muscle where the duration of the Ca²⁺ spark response is diminished and cannot be restimulated by additional rounds of osmotic shock (Table 2). Using biochemical assays, we found that the expression of MG29 was significantly altered in aged skeletal muscle. MG29 is a synaptophysin-family protein that is essential for maintenance of membrane structure in skeletal muscle^[60_62]. One can expect that compromised Ca²⁺ spark signaling in aged muscle may be linked to the changes in membrane coupling that result from altered MG29 expression.
Our studies identified a loss of plastic Ca²⁺ spark signaling in young mg29(-/-) muscles, in a fashion very similar to that seen in aged skeletal muscle. As with aged wt muscle, there is an initial Ca²⁺ spark response to the first osmotic shock and subsequent osmotic shocks produce little to no Ca²⁺ spark response in young mg29(-/-) muscle fibers. In addition, mg29(-/-) mice display muscle weakness at age 6 months or younger, which resembles the atrophic phenotype of aged wt mice. Relative to young wt muscle, aged wt muscle appears to contain a diminished Ca²⁺ reservoir responsible for Ca²⁺ spark generation that rapidly depletes, since repeated osmotic shocks do not elicit additional Ca²⁺ spark activity. This may result from the presence of a smaller SR Ca²⁺ pool in aged wt and young mg29(-/-) skeletal muscle, relative to young wt skeletal muscle fibers, or segregation of the intracellular Ca²⁺ release machinery. These and other phenotypic similarities between young mg29(-/-) and aged wt skeletal muscle suggest the possibility that mg29(-/-) mice could mimic some Ca²⁺ related aspects of skeletal muscle aging and may serve as a model for muscle aging under certain conditions.

Conclusions
Since the maintenance of proper Ca²⁺ homeostasis is essential for normal muscle contractile function and survival of muscle fibers, it is not surprising that aberrant Ca²⁺ spark activity in skeletal muscle appears in aged and dystrophic muscle fibers. The function of Ca²⁺ spark activity is well defined in cardiac muscle^[63], however there has been relatively little investigation of Ca²⁺ sparks in skeletal muscle. Although the mechanisms underlying the membrane-deformation responses in skeletal muscle may involve changes in multiple cellular factors, our ability to resolve these elemental SR Ca²⁺ release events in intact muscle fibers provides a useful tool to address some of the fundamental questions relating to the nature of SR Ca²⁺ release in skeletal muscle health and disease. The osmotic shock-induced Ca²⁺ sparks in young, healthy skeletal muscle are plastic in nature, a characteristic that is lost during muscular dystrophy and aging. While dystrophic muscle displays uncontrolled Ca²⁺ spark activity, osmotic stress-induced Ca²⁺ sparks in aged skeletal muscle appear to be static.
Our discovery of osmotic stress-induced Ca²⁺ sparks not only opens the way for us to monitor the in vivo function of Ca²⁺ sparks, but also raises several important questions: (1) What are the factors limiting the osmotic shock-induced Ca²⁺ sparks to the periphery of muscle fibers? (2) What is the physiological role of peripherally confined Ca²⁺ sparks in mammalian muscle cells? (3) What are the consequences of loss of spatial confinement or mechanisms regulating either the frequency, magnitude or propagation of Ca²⁺ sparks in skeletal muscle?
While it is unlikely that these spatially confined Ca²⁺ spark signals can directly contribute to myofibril contraction, there are many potential cell biological functions for these peripherally localized Ca²⁺ signals. For example, localized Ca²⁺ sparks may participate in the regulation of cytoskeletal structure or may reflect changes in cytoskeletal organization. The defects observed in dystrophic mouse muscle suggest that this is certainly a possibility. Another potential physiological function of Ca²⁺ sparks could be in volume regulation. Ca²⁺-dependent K⁺ channels located at the sarcolemmal membrane may respond to a local increase of Ca²⁺ sparks and therefore participate in maintenance of cell volume^[17,64_66]. Indeed, Ca²⁺-activated K⁺ channels have been shown to be down-regulated in aged smooth muscle cells, leading to impaired arterial tone^[67,68]. We have also found in recent experiments that these peripheral Ca²⁺ sparks may be linked to Ca²⁺ entry into the myofiber. These studies and future experiments will lead to a better understanding of the regulatory role of induced Ca²⁺ sparks in the physiology of healthy muscles and the pathophysiology of muscle disease and aging.

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