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Originally published In Press as doi:10.1074/jbc.M701379200 on October 16, 2007

J. Biol. Chem., Vol. 282, Issue 52, 37471-37478, December 28, 2007
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Enhanced Excitation-coupled Calcium Entry in Myotubes Is Associated with Expression of RyR1 Malignant Hyperthermia Mutations*

Tianzhong Yang{ddagger}, Paul D. Allen{ddagger}1, Isaac N. Pessah§, and Jose R. Lopez{ddagger}

From the {ddagger}Department of Anesthesiology Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115, the §Department of Molecular Biosciences, School of Veterinary Medicine, University of California at Davis, Davis, California, 95606, and the Centro de Biofisica y Bioquimica, Instituto Venezolano de Investigaciones Cientificas, Caracas Apartado 21827, Venezuela

Received for publication, February 16, 2007 , and in revised form, September 12, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Myotubes expressing wild type RyR1 (WT) or RyR1 with one of three malignant hyperthermia mutations R615C, R2163C, and T4826I (MH) were exposed sequentially to 60 mM KCl in Ca2+-replete and Ca2+-free external buffers (Ca+ and Ca–, respectively) with 3 min of rest between exposures. Although the maximal peak amplitude of the Ca2+ transients during K+ depolarization was similar for WT and MH in both external buffers, the rate of decay of the sustained phase of the transient during K+ depolarization (decay rate) in Ca+ was 50% slower for MH. This difference was eliminated in Ca–, and the relative decay rates were faster for both genotypes than in Ca+. The integrated Ca2+ transient in Ca–compared with Ca+ was reduced by 50–60% for MH and 20% for WT. The decay rate was not affected by [K+] x [Cl–] product or NiCl2 (2 mM) supplementation of Ca–. The addition of La2+ (0.1 mM), or SKF 96365 (20 µM) to Ca+ significantly accelerated decay rates for both WT and MH, but their effect was significantly greater in MH. Nifedipine (1 µM) had no effect, suggesting that the mechanism for this difference was not a reduction in L-type Ca2+ channel Ca2+ current. These data strongly suggest: 1) the decay rate in skeletal myotubes is related in part to Ca2+ entry through the ECCE channel; 2) the MH mutations enhance ECCE compared with wild type; and 3) the increased Ca2+ entry might play a significant role in the pathophysiology of MH.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Excitation-contraction (EC) coupling2 in skeletal muscle is a cascade of events that is initiated by the depolarization of T-tubule membrane (dihydropyridine receptor (DHPR)), which is followed by activation of the intracellular Ca2+ release channels known as ryanodine receptors (RyR1) located at the terminal cisternae of the sarcoplasmic reticulum (SR). These two proteins align during development at the junction of the t-tubule with the terminal cisternae of the SR (1), a collection of structures termed the calcium release unit (2). The DHPR may serve a dual function as a slow activated voltage-dependent Ca2+ channel (3) and as a voltage sensor for EC coupling (4). The DHPR is responsible for activating RyR1 causing Ca2+ release from the SR (5–7) into the cytoplasm that in turn triggers muscle contraction. Although the exact signal transduction mechanisms between DHPR and RyR1 are still unknown, it is generally accepted that intramembrane charge movements and conformational changes in the DHPR II-III loop couple the T tubule depolarization and Ca2+ release from the SR (orthograde conformational coupling) (5, 8, 9). It is this massive release of Ca2+ from the SR into the cytosol, and not a Ca2+ influx from the extracellular space, that is conventionally thought to initiate a series of Ca2+-dependent events that results in force generation (10, 11).

Experiments in which external Ca2+ was removed (12, 13) or calcium channel blockers are added to the bathing medium around skeletal muscle fibers (14) show that EC coupling and twitch contraction persists in skeletal muscle cells under these conditions. However, the role of extracellular Ca2+ on EC coupling in mammalian skeletal muscle has been re-examined (15). Several groups have demonstrated that stimuli that deplete Ca2+ in the SR enhance Ca2+ entry through the plasma membrane by a mechanism referred as store-operated Ca2+ entry (SOCE) (16, 17). Although Ca2+ entry through SOCE is easily seen, identification of the channel(s) responsible still awaits discovery. It is highly likely that the mechanism for this entry is at least partially if not completely derived from current passing though Orai 1, 2, and/or 3, which is activated by translocation of Stim 1 or Stim 2 from the SR (18–23). In addition to SOCE, a new mechanism for Ca2+ entry has recently been described in skeletal myotubes (24, 25) that is not linked to Ca2+ depletion of the SR and is elicited by brief or sustained membrane depolarization that would block conventional SOCE current(s). Activation of this Ca2+ entry pathway depends on interaction among three different Ca2+ channels: the DHPR, RyR1, and an unidentified Ca2+ influx pathway through the plasma membrane that was termed excitation-coupled Ca2+ entry (ECCE) (24).

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic syndrome in which exposure to volatile anesthetics or depolarizing neuromuscular blockers triggers a robust intracellular Ca2+ release through RyR1 from the SR, inducing a cascade of biochemical events that if untreated results in muscle rigidity, rhabdomyolysis, cardiac arrhythmia, and lethal hyperthermia (26, 27). This syndrome has been associated with a dysfunction of resting intracellular Ca2+ regulation. To date, about 112 mutations within the gene that codes for type 1 ryanodine receptor (RyR1) have been found on chromosome 19 (28), and two mutations in the {alpha}1s subunit of the DHPR found on chromosome 1 have been associated with MH (29).

To examine the possible role of abnormal sarcolemmal Ca2+ entry during MH, myotubes expressing mutations R615C, R2163C, and T48261 [GenBank] , (collectively termed MHRyR1s) were exposed to supermaximal concentrations of KCl (60 mM K+) in the presence (Ca+) and absence (Ca–) of extracellular Ca2+. In addition, we also conducted experiments in the presence or absence of Ca2+ after the addition of NiCl2, nifedipine, La3+, or SKF 96365 in an attempt to identify, or if not to identify at least to characterize, the entry pathway. The results strongly suggest that the accentuated Ca2+ transients seen in myotubes expressing MHRyR1s after KCl depolarization that we previously reported (30) were the result of a more robust sarcolemmal Ca2+ entry and not an increased SR Ca2+ release in response to depolarization. This difference potentially plays a role in the pathophysiology of the MH syndrome.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Construction and Expression of MHRyR1 cDNAs—A detailed explanation of construction and expression of the MHRyR1s (R615C, R2163C, and T48261 [GenBank] ) used in the present study has been previously described (30). All of the mutated and WTRyR1 cDNAs were packaged into HSV1 virions using a helper virus-free packaging system (31, 32) and were then used to transduce dyspedic 1B5 myotubes (32) for 2 h at an multiplicity of infection of 0.5 and then cultured for 48 h prior to imaging. 1B5 cells (RyR-1, RyR-2, and RyR-3 null) were cultured on Matrigel-coated (BD Biosciences, San Jose, CA) 96-well plates (Opticlear® COSTAR 3614) as described previously (30).

Calcium Imaging of Myotubes—Differentiated myotubes were loaded with 5 µM Fluo-4AM (Molecular Probes Inc., Eugene, OR) at 37 °C, for 20 min in imaging buffer. The cells were then washed three times with imaging buffer (IB), transferred to a Nikon TE2000 microscope. Fluo-4 was excited at 494 nm with an argon light source, and fluorescence emission was measured at 516 nm using a 40 x 1.3na objective. The data were collected with an intensified 12-bit digital intensified CCD at 30 fps (Stanford Photonics, Stanford, CA) from regions consisting of 2–12 individual cells and analyzed using QED software (QED, Pittsburgh PA). We used the average fluorescence of the calcium transient (area under the curve) to compare responses. Individual response amplitudes were calculated in the following way: the cumulative fluorescence during the 30 s challenge (Ar) minus the average base-line fluorescence for the 10 s immediately prior to the challenge (Ab) was divided by Ab and then multiplied by 100. To compare different experiments, individual response amplitudes were normalized to the peak amplitude of the transient obtained in the same cell. The peak amplitude of a transient refers to the highest fluorescence value during the stimulation time minus the average base-line fluorescence for the 10 s immediately prior to the challenge.

Solutions—Ionic compositions of Ca2+-replete imaging buffer (Ca+) with and without K+ (40 or 60 mM) are shown in Table 1. In KCl solutions, NaCl concentrations were adjusted to maintain a total ionic strength (Na+ concentration ([Na+]) + K+ concentration ([K+]) at 130 mM, but the product of [K+] x [Cl–] changes with different concentrations of KCl. In K2SO4 solutions, K2SO4 was used to increase [K+], and Na2SO4 was used to replace NaCl to maintain the [K+] x [Cl–] product at 670 and [Na+] + [K+] at 130 (the same values as Ca+). All nominally Ca2+-free buffers (Ca–) were prepared using the same protocol as the corresponding regular solutions but without CaCl2 added. Caffeine, NiCl2, nifedipine, LaCl3, and SKF96365 solutions were prepared by adding these compounds directly into either of the external buffers as well as the corresponding KCl solutions. Because of frequent trace Ca2+ contamination in other chemicals, residual free Ca2+ concentrations in Ca–(same composition as Ca+, but no Ca2+ added) and 60 mM Ca2+-free KCl solution (60KCl–Ca) were measured using Ca2+-selective microelectrodes and were ~0.8 and ~1.0 µM, respectively.


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  		TABLE 1
Composition of buffer solutions

 
Determination of Threshold for Maximum Ca2+ Transient in Response to KCl—From our previous studies we had determined that 50 mM [K+]o was sufficient to produce the maximum peak Ca2+ transient in myotubes (30). However, to make certain that our previous observations did not diverge from the measurements performed in the present study, we measured the magnitude of Ca2+ transients in myotubes expressing WT RyR1 produced by challenges with 40, 60, 80, and 100 mM [K+]o applied in a random order.

Statistics—All of the values are expressed as the means ± S.E. Prism software (version 4.0b) was used for statistical analysis (GraphPad Software, San Diego, CA; www.graphpad.com). Student's t tests, one-way analysis of variance, and Tukey's multiple comparison tests were used to compare the response sizes and peak amplitudes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Extracellular Ca2+ on KCl-induced Ca2+ Transients—A significant observation in our previous study (30) was that dyspedic 1B5 myotubes expressing MHRyR1s consistently exhibited Ca2+ transients (area under the curve) in response to 60 mM KCl (60KCl) that were greater than those in myotubes expressing WTRyR1. The selection of 60 mM [K+]o to induce Ca2+ transients in WTRyR1 and MHRyR1 myotubes for this study was based both on our results obtained from the dose-response relationship measured in WT myotubes (Fig. 1) and previously published data (33, 34). The collective results from these studies show that in muscle fibers the amount of membrane depolarization caused by >50 mM [K+]o will elicit an elevation of myoplasmic [Ca2+] above the level required to saturate the contractile apparatus.

Closer examination of data from our previous study revealed differences in the shapes of the Ca2+ transients induced by K+ between WTRyR1 and each of the seven MHRyR1s. Although there was no difference in the peak amplitude between the two groups, all MHRyR1 Ca2+ transients had a slower rate of decay than WTRyR1 Ca2+ transients (Fig. 1A, inset) and accounted for the greater areas under the Ca2+ transients elicited by 60KCl in myotubes expressing MHRyR1s.


Figure 1
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  		FIGURE 1.
The effects of increasing KCl concentration on the depolarization-induced Ca2+ transient. Shown is the average calcium fluorescence response in n = 7 1B5 cells expressing WTRyR1 after being stimulated with 40, 60, 80, and 100 mM KCl applied in random order for individual cells. afu, arbitrary fluorescence units. Note that in 1B5 cells expressing WTRyR1, as KCl concentration increases inactivation becomes faster to the point where at 100 mM KCl inactivation begins before the peak Ca2+ response is attained.

 


Figure 2
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  		FIGURE 2.
The effects of nominally Ca2+-free imaging buffer on the size of the Ca2+ transient in WT and MH myotubes. A, top panel, comparison of KCl Ca2+ transients between myotubes expressing WTRyR1 and seven common MHRyR1s (from Ref. 30). Bottom panels, responses to 60 mM KCl in Ca2+ replete (60KCl) and nominally Ca2+-free (60KCl–Ca) IB. The responses are superimposed on the right. B, total response size to 60 mM KCl in WT and MH myotubes in Ca2+ replete and nominally Ca2+-free IB. WT = 100%. C, the ratio of the response to 60 mM KCl between Ca2+ replete and nominally Ca2+-free IB.

 
To determine whether Ca2+ entry contributed to the slower decline of the Ca2+ transient in myotubes expressing MHRyR1s during KCl depolarization, we increased the duration of depolarization from 10 s as it was in the previous study (30) to 30 s to record their rates of decay of the sustained phase of the transient and increased the interval between two 60KCl challenges from 50 s (30) to 3 min to permit the myotubes to return to their original resting intracellular free Ca2+ concentration between responses. As shown in Fig. 2A, the 1B5 myotubes expressing WTRyR1 or each of three common MHRyR1s (R615C, R2163C, and T4826I from RyR1 gene "hot spots" I, II, and III, respectively) were sequentially exposed to 60KCl, 60KCl–Ca, and 60KCl using the protocol described above. Consistent with our previous observation (2), all three types of MH myotubes demonstrated transients with much slower decay rates of the sustained phase of the transient during 60KCl depolarization compared with those expressing WTRyR1. Prior to exposure to 60KCl, the Ca+ replete buffer was exchanged with nominally Ca2+-free (Ca–) buffer for 5 s. The decay rate of the sustained phase of the Ca2+ transient during exposure to 60KCl–Ca was accelerated for all four types (one WT and three MH) of myotubes and was more rapid in all groups than the rate obtained from WTRyR1 depolarized in Ca+ buffer. On the other hand, the peak amplitude of the response was not significantly different for all three KCl exposures. In addition the ratio of the peak amplitude to the amplitude at 30 s in Ca2+-free medium (pure RyR1 Ca2+ release) was not different between cells expressing WTRyR1 compared with those expressing any of the three MHRyR1s. After 3 min of rest, the rate of decay of the sustained phase of the Ca2+ transient after the third 60KCl exposure was essentially the same as the first 60KCl response in both WTRyR1 and MHRyR1 myotubes. Therefore, the increased rate of decay seen during the 60KCl–Ca response was not the result of progressive store depletion in either group. Superimposing the first 60KCl response with the 60KCl–Ca response in the same myotube clearly showed the pronounced difference in their decay rate in the presence and the absence of external Ca2+. Importantly the relative increase in the decay rate in Ca–was much greater for all MHRyR1s than for WTRyR1. Comparison of the response sizes (Fig. 2B) demonstrated that 1) all three groups of MH myotubes had significantly larger total Ca2+ responses (p < 0.01) to 60KCl compared with WTRyR1, 2) the response sizes to 60KCl–Ca were significantly smaller than those to 60KCl in both WT and MH myotubes, and 3) the peak and total response sizes to 60KCl–Ca for WTRyR1 and MHRyR1 myotubes was the same. The ratio of the 60KCl–Ca response size to the 60KCl (Fig. 2C) response size for each of the three MHRyR1s was significantly (p < 0.01) less than that for WTRyR1 (42.6 ± 3.9, 48.6 ± 1.6, and 52.0 ± 3.3% for R615CRyR1, R2163CRyR1, and T4826IRyR1, respectively, versus 80.5 ± 4.6%, for WTRyR1), reflecting the difference in the rate of decay during depolarization for MHRyR1s (for superimposed normalized responses, see Fig. 2A). Because of the similarity in the ratios of these three MH mutations in their slower rate of decay compared with WTRyR1, we randomly picked T4826I for further investigation for the possible mechanisms for this difference.

It is well known that extracellular [Cl–] also contributes to the membrane potential of frog muscle fibers when extracellular [K+] and [Cl–] were changed reciprocally to keep a constant product (35). In our 60 mM KCl solution both [K+] and [Cl–] were increased, substantially increasing the product of [K+] x [Cl–], compared with Ca+ buffers without KCl (Table 1). Thus it was theoretically possible that the responses to 60KCl we observed could have been affected by a Cl– overload. To rule this in or out, we exposed WTRyR1 and T4826IRyR1 myotubes to 60 mM KCl, whose [K+] x [Cl–] product in the buffer was the same as Ca+ buffer. Comparison of the shape and response sizes after depolarization with these two K+ solutions ([K+] x [Cl–] not constant versus [K+] x [Cl–] constant) showed that they were not significantly different from one another (Fig. 3A).


Figure 3
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  		FIGURE 3.
The effects of [K+] x [Cl–] product and moderate membrane depolarization on the Ca2+ transient in response to KCl depolarization. A, Ca2+ transients in response to 40 and 60 mM KCl is unchanged in response to changes in the [K+] x [Cl–] product (responses directly overlaid at right and shown in the bar graph). B, relieving the partial depolarization caused by removing Ca2+ in nominally Ca2+-free IB does not change the rate of decay of the sustained phase of the Ca2+ transient in response to 60 mM KCl (responses directly overlaid at right and shown in the bar graph).

 
It has also been demonstrated in single frog muscle fibers that removing extracellular Ca2+ to levels identical with that used for our Ca–experiments resulted in a slight membrane depolarization (9 ± 2 mV) and potentiation of twitch responses (12). Substitution of Ni2+ for missing Ca2+ in extracellular solutions resolved this problem (36). To examine this as a possible mechanism for the differences we observed between WTRyR1 and MHRyR1, we prepared 60KClNi solution by adding 2 mM NiCl2 to the 60KCl–Ca solution. Myotubes expressing T4826IRyR1 were exposed to 60KCl, 60KCl–Ca, 60KClNi and 60KCl sequentially (Fig. 3B). As observed before, the rate of decay of the Ca2+ transient in response to 60KCl–Ca depolarization was accelerated compared with the 60KCl response. Addition of 2 mM NiCl2 (60KCl–Ca+Ni) did not slow the accelerated rate of decay. However, the slow rate of decay of the sustained phase of KCl responses in T4826IRyR1 myotubes was restored in Ca+ buffer (Fig. 3B).

Effects of Ca2+ Entry Blockers on Depolarization-induced Ca2+ Entry—To better define the mechanism for the enhanced Ca2+ entry caused by depolarization of cells with MHRyR1 mutations, we used a pharmacologic library to attempt to block the response. To do this, we chose (0.1 mM) lanthanum chloride (La3+; a nonspecific Ca2+ entry blocker (37)), 20 µM SKF 96365 (a SOCE and TRP channel blocker (38) that has also been shown to block cation influx attributable to ECCE (24)), and 1 µM nifedipine (the selective L-type Ca2+ channel blocker (39). As shown in Fig. 4A, La3+ (0.1 mM) added 10 s after 60KCl exposure immediately accelerated the decay rate of the sustained phase of the Ca2+ transients for both WTRyR1 and T4826IRyR1 myotubes (responses labeled #2 for WTRyR1 and T4826IRyR1). The 60KCl responses (responses labeled #3) with La3+ (0.1 mM) added 5 s before KCl exposure demonstrated a more significantly accelerated decay rate of the sustained phase for both WT and T4826IRyR1 myotubes, which is better illustrated by superimposed normalized traces on the right side of Fig. 4A. The last 60KCl responses (responses labeled #4) in the absence of extracellular La3+ partially restored the slower decay rate of the sustained phase in both WTRyR1 and T4826IRyR1 myotubes, although the recovery was not complete compared with the #1 responses. The ratio of response sizes of 60KCl + La3+ (responses labeled #3) to 60KCl (responses labeled #1) (Fig. 4B) is significantly smaller (p < 0.01) for T4826I (64.7 ± 3.1%) compared with WT (85.3 ± 3.6%), indicating that a significantly larger portion of the 60KCl response in T4826IRyR1 myotubes was diminished by La3+ compared with WTRyR1. Of note, no change in the peak amplitude of the KCl responses was observed with the sequential KCl stimulations.

The decay rate of the sustained phase of the Ca2+ transient during KCl depolarization was drastically accelerated in both WT and T4826I myotubes by 20 µM SKF 96365 (responses labeled #2 in Fig. 5A). Removing SKF 96365 prior to the next 60KCl stimulation restored the slower decay of sustained phase to pre-exposure rates in T4826I myotubes. 20 µM of SKF 96365 appears to accelerate the decay rate instantaneously (Fig. 5A, inset), and the ratios of response #2 to response #1 sizes for WT and T4826I are 72.8 ± 3.2 and 49.0 ± 3.9%, respectively, which suggests a more significant effect (p < 0.01) of SKF 96365 on T4826I myotubes compared with WT myotubes (Fig. 5). Interestingly, the response #2 to response #1 ratios for the SKF 96365 effect tended to be smaller than even those for 60KCl–Ca shown in Fig. 2 (80.5 ± 4.6 and 52.0 ± 3.3% for WT and T4826I, respectively).


Figure 4
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  		FIGURE 4.
The effects lanthanum (La3+) on the Ca2+ transient in response to KCl depolarization. A, La3+ immediately increases the decay rate of the sustained phase of the Ca2+ transient in response to 60 mM KCl. Responses with and without La3+ are compared at right. B, the increase in the decay rate in response to La3+ is greater in MH than in WT myotubes.

 


Figure 5
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  		FIGURE 5.
The effects of SKF96365 on the Ca2+ transient in response to KCl depolarization. A, SKF 96365 immediately increases the decay rate of the sustained phase of the Ca2+ transient in response to 60 mM KCl. Responses with and without SKF 96365 are compared at right. B, the increase in the decay rate in response to SKF 96365 is greater in MH than in WT myotubes.

 


Figure 6
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  		FIGURE 6.
The effects of Nifedipine on the Ca2+ transient in response to KCl depolarization. 1 µM nifedipine has no effect on the rate of decay of the sustained phase of the Ca2+ transient in response to 60 mM KCl depolarization. Responses with and without nifedipine are compared directly at right (A). There is also no difference in the effect of nifedipine between WT and MH myotubes (B).

 
Nifedipine administered 5 s before 60KClNif (60KCl containing 1 µM nifedipine) stimulation (#2 responses in Fig. 6A) had no significant effect on the sustained phase in either WT or MH myotubes compared with the corresponding control #1 responses.

Caffeine-induced Ca2+ Transient Is Not Mediated by Sarcolemmal Ca2+ Entry—To investigate whether the presence of extracellular Ca2+ has any effect on the Ca2+ transient elicited by direct RyR1 activators, 20 mM caffeine was used to stimulate Ca2+ release from SR from myotubes expressing WTRyR1 and T4826IRyR1, using the same experimental protocol (Fig. 7A). In contrast to the KCl responses shown before, the responses to regular 20 mM caffeine (20caff) and Ca2+-free 20 mM caffeine (20caff–Ca) were identical in both WTRyR1 and T4826IRyR1 myotubes. These similarities were clearly illustrated when the normalized data were superimposed. Interestingly, the peak amplitude of the caffeine responses decreased significantly with sequential caffeine successive caffeine challenges and was independent of external Ca2+ (Fig. 7B). As a result, the response sizes showed a similar decrease in both WTRyR1 and T4826IRyR1 myotubes (Fig. 7C). When the peak amplitudes and response sizes for the first 20caff response and the 20caff–Ca response were superimposed (Fig. 7D), the decreases in peak amplitude were coincident with the decrease in response size (area under the curve) for both genotypes, suggesting that the decreased response size to 20caff–Ca resulted from decreased peak amplitude rather than a change in the sustained phase. These observations are also born out when the cells are stimulated with caffeine in the presence of SKF 96365. (Fig. 7E). Neither WTRyR1 nor T4826IRyR1 expressing cells showed any decrease in the amplitude or area under the curve of their 20caffeine Ca2+ transient in the presence of 20 µM SKF 96365 compared with the control 20caff response.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The purpose of this study was to determine whether sarcolemmal Ca2+ entry was responsible for maintaining and prolonging the Ca2+ transient in MH skeletal muscle myotubes in response to depolarization and, if so, to attempt to use pharmacological means to better define the pathway for this entry. Our study demonstrates that in myotubes expressing WTRyR1 or MHRyR1, there is a significant amount of Ca2+ entry in response to K+-induced depolarization. This Ca2+ entry is most easily detectable during the sustained phase of a long depolarization that follows the peak Ca2+ transient and is enhanced in cells expressing any of three widely spaced MHRyR1s. Removing extracellular Ca2+ significantly reduced the area under the Ca2+ transient in both WTRyR1- and MHRyR1-expressing myotubes and was attributed to an accelerated decay of the sustained phase without affecting the peak amplitude. Interestingly, the rate of decay of the Ca2+ signal during the sustained phase was significantly greater in myotubes expressing any of the MHRyR1s tested. Sarcolemmal channel blockers La3+ or SKF 96365 both instantaneously accelerated the rate of decay in both WTRyR1 and MHRyR1 myotubes, and similar to the responses seen with low extracellular Ca2+ their effect is larger in myotubes expressing MHRyR1s than in WTRyR1. Although these two Ca2+ entry blockers like almost all pharmacologic agents have off site actions and thus are never specific for a single channel type, we can be certain their ability to reduce Ca2+ entry was not due to their ability to block L-type Ca2+ current, because nifedipine had no significant effect on the Ca2+ entry in either WTRyR1 or MHRyR1s myotubes. The importance of extracellular Ca2+ during K+-induced contracture has been previously suggested in early studies of frog muscle fibers (12, 40, 41). In these studies it was found that Ca2+ influx, measured by Ca45 entry, was almost proportional to the extracellular Ca2+ concentration during the K+ contracture (41). The same study also showed that the difference between the height of contractures evoked by high K+ solutions (95 mM) with or without Ca2+ was very small or absent, whereas the duration time of the contracture is directly proportional to the extracellular Ca2+ concentration. Similar results were obtained in a later study on effects of external calcium deprivation on single frog muscle fibers (12), which demonstrated that Ca2+ removal immediately increased the rate of relaxation of K+ contractures, whereas a longer time (3 min) of Ca2+ withdrawal was needed to decrease the action potential amplitude. On the other hand, in experiments in which muscle fibers were subjected to brief electrical stimulation, removal of extracellular Ca2+ did not appear to impair EC coupling and force production in amphibian skeletal muscle cells (12, 13). More recently a Ca2+ entry that is not linked to Ca2+ depletion of the SR, named ECCE has been shown to be elicited by K+ depolarization and trains of electrical pulses (24). Activation of ECCE depends on interaction among three different Ca2+ channels: the DHPR, RyR1, and an as of yet unidentified Ca2+ channel at the plasma membrane (24). Its distinctive properties are: 1) it is initiated by depolarization; 2) it is dependent on the presence of both RyR1 and DHPR; 3) it is independent of the DHPR L-type Ca2+ current; 4) it does not depend on SR Ca2+ release; and 5) it can be effectively inhibited by La3+ or SKF96365 (24).


Figure 7
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  		FIGURE 7.
The effects of nominally free Ca2+ medium and SKF 96365 on 20 mM caffeine induced Ca2+ transients. A shows responses in Ca2+ replete or nominally Ca2+-free IB with normalized responses shown at the right. Note that there is a steady decline in the total response on successive caffeine exposure, presumably because of reduction in SR stores. The peak amplitude (B) and the total response size (area under the curve of the Ca2+ transient) (C) are significantly smaller in MH myotubes (p < 0.01) than the comparable responses in WT myotubes. Both the total response (RS) and peak amplitude (PA) decline proportionally in MH and WT myotubes (D), and in E it can be seen that SKF 96365 has no effect on the peak amplitude or total response of the Ca2+ transient in response to 20 mM Caffeine. Responses with and without SKF 96365 compared at the right (E).

 
Previous studies of KCl responses in MH muscle cells showed significantly larger muscle contractures and lower KCl contracture thresholds in muscle fibers dissected from MH susceptible swine compared with WT (42–44). In addition, it has been reported that the total calcium transient in MH susceptible muscle fibers was larger than control fibers when exposed to subcontracture and contracture concentrations of KCl (30, 43, 45, 46).

This study is the first detailed investigation of the effects of external Ca2+ on the kinetics of the Ca2+ transient in response to K+ depolarization in myotubes expressing WTRyR1 and MHRyR1s. Our results demonstrate that a significant portion of the observed KCl depolarization-induced Ca2+ transient is the result of sarcolemmal Ca2+ entry in both groups of myotubes. This is most easily detectable during the sustained phase that follows the peak of the Ca2+ transient. The data from our previous study on seven common MHRyR1s expressed in dyspedic myotubes gave us the first clue that the kinetics of MH-associated Ca2+ transients, especially the sustained phase, is significantly different compared with WTRyR1 myotubes (30). The experimental protocol adopted in this study was specifically designed to facilitate a more detailed characterization of the sustained phase of the KCl response, i.e. the 3-min interval between KCl stimulations allowed the myotubes to restore the SR calcium pools so the peak amplitudes of repeated KCl responses remained the same in any individual cell. We have characterized the class of channels responsible for the increased Ca2+ entry seen in cells expressing MHRyR1s during KCl-induced Ca2+ transients in myotubes by exposing them to several different experimental conditions as follows.

After exposure to nominally Ca2+-free imaging buffer, the cells were exposed to Ca–shortly (5 s) before the exposure to 60K–Ca to ensure the KCl response occurred in a calcium-free solution while minimizing the possible reduction of SR calcium stores caused by Ca2+ leakage out of the cell. EGTA was not used in the Ca2+-free solutions because 1) high concentrations of EGTA result in a blockade of excitation-contraction coupling (46) and 2) adding EGTA (1 or 2 mM) to extracellular solutions can significantly increase the spontaneous Ca2+ release activities in primary myotubes.3 We believe that the Ca2+-free buffers used in this study did not have an observable effect on the membrane potential and the voltage sensitivity of the myotubes because 1) the peak amplitudes of KCl-induced Ca2+ transients in the presence or absence of extracellular Ca2+ are the same and 2) supplementation of the Ca2+-free KCl solution with 2 mM NiCl2, which has been shown to prevent the partial depolarization observed in Ca2+-free medium (12), did not change the amplitude or rate of decay of the sustained phase of KCl-induced Ca2+ transients in myotubes.

In the presence of sarcolemmal Ca2+ entry channel blockers, La3+, a nonspecific blocker of membrane Ca2+ permeability and SKF 96365 that has been widely used as a SOC channel blocker (38), accelerated the decay of the sustained phase of the Ca2+ transients in both WTRyR1 and T4826IRyR1 myotubes, proving that the sustained phase of the KCl responses is largely due to Ca2+ entry. Their pharmacological effects were more evident in myotubes expressing T4826IRyR1 than WTRyR1 expressing myotubes. Both compounds have been demonstrated to effectively block ECCE (24), which suggests the possibility that ECCE is enhanced in myotubes carrying MH mutations.

Any conventional contribution of the slow inward Ca2+ current through the DHPR to the Ca2+ entry observed during sustained depolarization in WTRyR1 and MHRyR1 myotubes was ruled out by the fact that neither group was affected by 1 µM nifedipine.

Another interesting finding in support of a role for Ca2+ entry during KCl depolarization being the cause of the difference between WTRyR1 and MHRyR1 was the result seen when 20 mM caffeine was used to stimulate Ca2+ release in myotubes expressing WTRyR1 and T4826IRyR1. The fact that the responses to 20 mM caffeine were almost identical in Ca2+ replete, Ca2+-free, and Ca2+ replete buffer containing SKF 96365 in both WTRyR1 and T4826IRyR1 myotubes rules out the possible contribution of an increased SR Ca2+ release being responsible for the larger KCl-induced Ca2+ transients seen in cells expressing MHRyRs. In addition, the fact that both the peak amplitude and response size of the caffeine responses for myotubes expressing T4826IRyR1 were smaller and decreased more significantly with sequential caffeine stimulations compared with those for WTRyR1 suggests that our previous conclusion that the SR Ca2+ load is not diminished in MH myotubes (30) was incorrect and gives support to hypothesis that there are a greater number of MHRyR1 channels in leak state in the SR than are present in cells expressing WTRyR1 (47).

In summary, these results demonstrate that RyR1 MH mutations are associated with an enhanced Ca2+ entry through the sarcolemma during depolarization. They further suggest that Ca2+ entry may contribute to maintaining Ca2+ homeostasis in mammalian skeletal EC coupling and may play an important role in the pathophysiology of malignant hyperthermia.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Anesthesiology Perioperative and Pain Medicine, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. E-mail: pdallen{at}partners.org.

2 The abbreviations used are: EC, excitation-contraction; WT, wild type; MH, malignant hyperthermia; DHPR, dihydropyridine receptor; SR, sarcoplasmic reticulum; SOCE, store-operated Ca2+ entry; ECCE, excitation-coupled Ca2+ entry; IB, imaging buffer. Back

3 T. Yang, P. D. Allen, I. N. Pessah, and J. R. Lopez, unpublished observations. Back



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 ABSTRACT
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 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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