PRE-CLINICAL RESEARCH
Dantrolene, a Therapeutic Agent for Malignant Hyperthermia, Markedly Improves the Function of Failing Cardiomyocytes by Stabilizing Interdomain Interactions Within the Ryanodine Receptor
Shigeki Kobayashi, MD, PhD*,
Masafumi Yano, MD, PhD*,*,
Takeshi Suetomi, MD*,
Makoto Ono, MD*,
Hiroki Tateishi, MD*,
Mamoru Mochizuki, MD, PhD*,
Xiaojuan Xu, MD*,
Hitoshi Uchinoumi, MD*,
Shinichi Okuda, MD, PhD*,
Takeshi Yamamoto, MD, PhD*,
Noritaka Koseki, BS ,
Hiroyuki Kyushiki, PhD,
Noriaki Ikemoto, PhD , and
Masunori Matsuzaki, MD, PhD*
* Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan
First Institute of New Drug Discovery, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan
Boston Biomedical Research Institute, Watertown, Massachusetts
Department of Neurology, Harvard Medical School, Boston, Massachusetts
Manuscript received June 23, 2008;
revised manuscript received January 12, 2009,
accepted January 19, 2009.
* Reprint requests and correspondence: Dr. Masafumi Yano, Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan (Email: yanoma{at}yamaguchi-u.ac.jp).
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Abstract
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Objectives: We sought to investigate the effect of dantrolene, a drug generally used to treat malignant hyperthermia, on the Ca2+ release and cardiomyocyte function in failing hearts.
Background: The N-terminal (N: 1–600) and central (C: 2000–2500) domains of the ryanodine receptor (RyR) harbor many mutations associated with malignant hyperthermia in skeletal muscle RyR (RyR1) and polymorphic ventricular tachycardia in cardiac RyR (RyR2). There is strong evidence that interdomain interaction between these regions plays an important role in the mechanism of channel regulation.
Methods: Sarcoplasmic reticulum vesicles and cardiomyocytes were isolated from the left ventricular muscles of dogs (normal or rapid ventricular pacing for 4 weeks), for Ca2+ leak, transient, and spark assays. To assess the zipped or unzipped state of the interacting domains, the RyR was labeled fluorescently with methylcoumarin acetate in a site-directed manner. We used a quartz-crystal microbalance technique to identify the dantrolene binding site within the RyR2.
Results: Dantrolene specifically bound to domain 601–620 in RyR2. In the sarcoplasmic reticulum isolated from pacing-induced failing dog hearts, the defective interdomain interaction (domain unzipping) had already occurred, causing spontaneous Ca2+ leak. Dantrolene suppressed both domain unzipping and the Ca2+ leak, demonstrating identical drug concentration-dependence (IC50 = 0.3 µmol/l). In failing cardiomyocytes, both diastolic Ca2+ sparks and delayed afterdepolarization were observed frequently, but 1 µmol/l dantrolene inhibited both events.
Conclusions: Dantrolene corrects defective interdomain interactions within RyR2 in failing hearts, inhibits spontaneous Ca2+ leak, and in turn improves cardiomyocyte function in failing hearts. Thus, dantrolene may have a potential to treat heart failure, specifically targeting the RyR2.
Key Words: heart failure • calcium • sarcoplasmic reticulum
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Abbreviations and Acronyms
| | ARVC2 = arrhythmogenic right ventricular cardiomyopathy type 2 | | CPVT = catecholaminergic polymorphic ventricular tachycardia | | DAD = delayed afterdepolarization | | LV = left ventricle/ventricular | | MH = malignant hyperthermia | | QCM = quartz crystal microbalance | | RyR = ryanodine receptor | | SR = sarcoplasmic reticulum |
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The skeletal muscle type-1 and cardiac type-2 ryanodine receptors (RyR1 and RyR2, respectively) are intracellular channels that mediate Ca2+ release from the sarcoplasmic reticulum (SR). More than 70 point mutations in RyR1 have been reported to cause malignant hyperthermia (MH) and central core disease, both of which are potentially lethal genetic disorders of skeletal muscle (1). As widely recognized, MH mutations are not randomly distributed along the RyR1 sequence. The majority of them are localized in 2 restricted regions: the N-terminal (Cys35-Arg614), and the central (Asp2129-Arg2458) domains, whereas a third, C-terminal region (Ile3916-Gly4942) contains fewer MH mutations (1). All of the RyR1 Ca2+-release channels with MH mutations are characterized by a leaky SR Ca2+ channel (1,2).
On the basis of the distribution of these mutations, Yamamoto et al. (3,4) proposed the hypothesis that the interactions between the N-terminal domain and the central domain of RyR1 are involved in Ca2+ channel regulation as an intrinsic regulator of the Ca2+ channel and that these interacting domains comprise a "domain switch." According to this hypothesis, in the resting or nonactivated state, the N-terminal and central domains make close contact at several subdomains (domain zipping). The conformational constraints imparted by the "zipped" configuration of these 2 domains stabilize and maintain the closed state of Ca2+ channel. Stimulation via excitation-contraction coupling or pharmacological agents weakens these critical interdomain contacts, resulting in a reduction of conformational constraints (domain unzipping), thereby lowering the energy barrier for Ca2+ channel opening. Weakening of these interdomain interactions may also occur via mutation or with the use of synthetic domain peptides.
To date, more than 60 RyR2 missense mutations have been found to be linked with 2 inherited forms of sudden cardiac death, that is, catecholaminergic polymorphic ventricular tachycardia (CPVT), and arrhythmogenic right ventricular cardiomyopathy type 2 (ARVC2) (1). All RyR2 mutations cluster into 3 regions of the channel that correspond to the 3 MH/central core disease mutation regions (N-terminal domain, central domain, and channel-forming domain) of RyR1.
Particularly, some mutations of RyR2, which have been reported in cardiac disease patients, are located in the regions corresponding to the skeletal N-terminal and central domains harboring most of the MH mutations that cause an increased Ca2+ leak (5). This finding suggests that RyR2 shares a common domain-mediated channel regulation mechanism with RyR1 and that the increased Ca2+ leak of diseased RyR2 channels may be attributable to defective modes of interdomain interaction. We recently reported that the defective interdomain interaction within the RyR2 (namely, domain unzipping) destabilizes the channel gating of the RyR2, associated with FK506 binding protein 12.6 dissociation from RyR2 and Ca2+ leak, causing contractile dysfunction of cardiomyocyte (6). On the basis of these findings, we have proposed that the weakened interdomain interaction is one of the key mechanisms underlying the pathogenesis of ARVC2, CPVT, and heart failure and that normal operation of the domain switch is essential for proper function of both RyR1 and RyR2 channels.
Paul-Pletzer et al. (7) have identified the dantrolene-binding site within RyR1 as Leu590-Cys609 (named DP1) by using [3H]azidodantrolene, a pharmacologically active photoaffinity analog of dantrolene. Recently, we demonstrated a novel mechanism by which dantrolene improves the hypersensitization of channel gating to Ca2+ observed in MH channels (8). Upon adding dantrolene to defective RyR1 Ca2+ release channel mimicking MH, we found dantrolene to produce a local conformational change, resulting in reinforcement of the interdomain interactions (8).
Because the amino acid sequence of the DP1 region is exactly identical between RyR1 and RyR2, we hypothesized that dantrolene would be equally effective in correcting the defective interdomain interaction in failing hearts. Here we report that dantrolene in fact corrects defective interdomain interaction within RyR2, inhibits Ca2+ leak, and improves cardiomyocyte function in failing hearts.
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Methods
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Materials.
Dantrolene was purchased from Sigma Chemical Co. (St. Louis, Missouri).
Animal disease model.
In beagles, heart failure was induced by chronic rapid right ventricular pacing (Online Appendix).
Preparation of SR vesicles.
We prepared SR vesicles from normal or 4-week paced failing left ventricles (LVs) obtained from dogs, as described previously (9).
Peptides used and peptide synthesis.
Domain peptides (DPc10), corresponding to amino acids Gly2460-Pro2495 of RyR2, were synthesized and used for analysis of domain–domain interaction within RyR2 (Online Appendix).
Construction and expression of RyR2 fragments.
The peptides corresponding to various regions of RyR2 (1–610, 494–1000, 741–1260, 1245–1768, 1741–2270, 1981–2520, and 2234–2750) were amplified via polymerase chain reaction (Online Appendix).
Quartz crystal microbalance (QCM) measurements.
Binding of dantrolene to RyR2 fragments was detected with the use of a 27-MHz QCM (Initium, Inc., Tokyo, Japan) (Online Appendix).
Ca2+ uptake and leak assays.
Ca2+ uptake and the following Ca2+ leak assays were conducted as described previously (9).
Site-directed fluorescent labeling of the RyR2.
Specific fluorescent labeling of the RyR2 moiety of the SR membrane was performed with the cleavable hetero-bifunctional cross-linking reagent sulfosuccinimidyl 3-([2-(7-azido-4-methylcoumarin-3-acetamido) ethyl] dithio) propionate from Pierce (now Thermo Scientific, Rockford, Illinois), with DPc10 as a site-specific carrier following the site-directed fluorescent labeling method (6).
Fluorescence quenching of the methylcoumarin acetate fluorescence attached to the DPc10 binding site.
The zipped and unzipped states of RyR were evaluated by chemical quencher analysis, as described previously (Online Appendix) (4,6,8).
Isolation of cardiac cardiomyocytes.
Cardiomyocytes were isolated from the LV free wall of normal or 4-week paced failing dogs, as described previously (Online Appendix) (6).
Cell shortening and Ca2+ transient measurement.
Measurements of cardiomyocyte cell shortening and intracellular Ca2+ were performed with fura-2 acetoxymethyl ester, as described previously (Online Appendix) (6).
Analysis of Ca2+ sparks with laser scanning confocal microscopy.
Ca2+ sparks were measured on a laser scanning confocal microscope (Online Appendix).
Measurement of membrane potential with laser scanning confocal microscopy.
Measurements of membrane potential were performed on a laser scanning confocal microscope following the same protocol for the measurement of Ca2+ sparks (Online Appendix).
Statistics.
The unpaired t test was used for statistical comparison of data between the 2 states. We also used analysis of variance for repeated measures with a post-hoc Scheffe test for statistical comparison of concentration-dependent data. Data are expressed as mean ± SD. We accepted a value of p < 0.05 as statistically significant.
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Results
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Effect of dantrolene on in vivo cardiac function.
In the dantrolene-treated dogs with long-term right ventricular pacing, both LV end-diastolic and -systolic diameters were smaller than in dantrolene-untreated dogs, and fractional shortening was significantly improved (Table 1, Fig. 1). This result indicates that the development of heart failure by pacing was attenuated by the treatment with dantrolene.
Specific binding of dantrolene to domain 601–620 of RyR2.
Dantrolene was previously reported to bind to the domain Leu590-Cys609 in the case of RyR1 (7). We investigated whether dantrolene also binds to the corresponding domain 601–620 of RyR2 by using a quartz crystal microbalance technique (a highly sensitive mass-measuring technique). To identify the dantrolene-binding region, we screened several recombinant fragments corresponding to various regions covering the area from the N-terminus to residue 2750, which include both of the aforementioned critical regulatory domains (N-terminal [1–600] and central [2000–2500] domains). Figure 2A shows the gel image of these recombinant fragments. As shown in Figure 2B, of all recombinant fragments we tested, only the fragment494–1000 showed the signal of dantrolene binding.
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Figure 2 Identification of the Dantrolene Binding Site Within RyR2
(A) Gel image of the recombinant fragments corresponding to various regions covering the area from the N-terminus to residue 2750. (B to D). Screening of the dantrolene binding site within the ryanodine receptor (RyR)2 with recombinant RyR2 fragments and synthetic peptides with the use of quartz-crystal microbalance. Dantrolene was immobilized on the sensor chip surface, as described in the Methods section. The traces represent the time course of the binding of various peptides or RyR2 fragments to dantrolene fixed on the sensor chip. Note that DP601–620 and RyR2 fragment 494–1000 specifically bound to the dantrolene (B and C) but not in the presence of excess added dantrolene (D).
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To test the hypothesis that RyR2 and RyR1 share the identical dantrolene binding sequence (as discussed in the Introduction section), DP601–620 (a synthetic peptide, described previously, corresponding to DP1) was subjected to the QCM binding assay. As shown in the QCM recording of Figure 2C, there was a rapid binding of dantrolene to DP601–620. There was no detectable drug binding to the central domain peptide: DPc10 (2460–2495) and to the N-terminal domain peptide: DP163–195. As shown in Figure 2D, the binding of dantrolene to fragment494–1000 or DP601–620 was abolished in the copresence of excess dantrolene (30 µmol/l).
Dantrolene inhibited both DPc10-induced Ca2+ leak from the normal SR and spontaneous Ca2+ leak from the failing SR.
The addition of 0.3 µmol/l thapsigargin to normal SR vesicles at a steady-state of adenosine triphosphate-dependent Ca2+ uptake produced little Ca2+ leak, whereas the addition of 100 µmol/l DPc10 together with 0.3 µmol/l thapsigargin produced a pronounced leak. However, the Ca2+ leak was almost completely suppressed by 1 µmol/l dantrolene (Fig. 3A). In failing SR, the addition of 0.3 µmol/l thapsigargin produced spontaneous Ca2+ leak. Dantrolene (1 µmol/l) abolished the spontaneous Ca2+ leak that otherwise would have occurred in the failing SR (Fig. 3A).
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Figure 3 Effect of Dantrolene on SR Ca2+ Leak
(A) Representative time courses of Ca2+ uptake and Ca2+ leak in normal and failing sarcoplasmic reticulum (SR) vesicles in the presence or absence of dantrolene. (B) Concentration dependence of dantrolene inhibition of the DPc10 (100 µmol/l)-induced Ca2+ leak in normal SR vesicles. (C) Concentration dependence of dantrolene inhibition on spontaneous Ca2+ leak in failing SR vesicles. Each data point is a mean of 4 to 6 measurements ± SD. (D) Effect of dantrolene on cyclic adenosine monophosphate (cAMP)-induced Ca2+ leak in normal SR vesicles. Thapsigargin (0.3 µmol/l) was added after adenosine triphosphate-dependent Ca2+ uptake after the pre-incubation of the SR vesicles with 30 µmol/l cAMP plus 1 µmol/l okadaic acid.
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As shown in Figure 3B, the concentration dependence of dantrolene inhibition of DPc10-induced Ca2+ leak in normal SR occurs in the concentration range of 0.1 to 1.0 µmol/l, with the concentration of a half-maximal inhibition (IC50) of 0.3 µmol/l. The concentration dependence and IC50 (0.3 µmol/l) of dantrolene inhibition of the spontaneous Ca2+ leak in failing SR (Fig. 3C) are identical to those of DPc10-induced Ca2+ leak in the normal SR. We also evaluated the effect of dantrolene on Ca2+ leak induced by protein kinase A phosphorylation of RyR2 (by cyclic adenosine monophosphate [cAMP]) (Fig. 3D). In the presence of 1 µmol/l dantrolene, cAMP-induced Ca2+ leak was completely inhibited. Dantolene (0.1 to 1 µmol/l) had no effect on the Ca2+ uptake time course (data not shown).
Dantrolene reversed an abnormal domain unzipping of the domain switch.
To investigate the effect of dantrolene on the mode of interdomain interaction between the N-terminal domain and the central domain (2 regulatory domains of the domain switch; for more details, see the Introduction section), we used the methylcoumarin acetate fluorescence quenching technique we developed to determine the extent of unzipping of the domain switch (see the Methods section). The slope of the plot, which is equal to the Stern-Volmer quenching constant (KQ), is a measure of the degree of domain unzipping.
As shown in our previous report (6), KQ increased with increasing concentrations of DPc10 and leveled off at approximately 100 µmol/l peptide. In the experiment shown in Figure 4A, we investigated the effect of increased concentrations of dantrolene on the KQ that had reached a maximal level by 100 µmol/l peptide. As shown in Figure 4B, the K'Q/KQ value (where K'Q is the quenching constant in the DPc10-treated SR and KQ is the quenching constant in the untreated control SR) was reduced with an increase of dantrolene concentration, and at concentrations >1 µmol/l it reached the control level (K'Q/KQ = 1).
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Figure 4 Stern-Volmer Plots of the Fluorescence Quenching Data
(A) The Stern-Volmer plots of fluorescence quenching in the presence of various concentrations of dantrolene. Domain unzipping was produced by adding 100 µmol/l DPc10 to normal sarcoplasmic reticulum (SR). (B) Concentration dependence of dantrolene inhibition of domain unzipping (K'Q/KQ) in DPc10-added and untreated normal SR. Note that dantrolene reduced the K'Q/KQ value (where K'Q is the quenching constant in DPc10-added normal SR and KQ is the quenching constant in normal SR) in DPc10-added normal SR in a concentration-dependent manner (half-maximal inhibition [IC50] = 0.3 µmol/l), whereas dantrolene had no appreciable effect in untreated normal SR. (C) The slope of Stern-Volmer plots of fluorescence quenching as a function of various concentrations of dantrolene added to failing SR. (D) Concentration dependence of dantrolene inhibition of K''Q/KQ (where K''Q is the quenching constant in failing SR and KQ is the quenching constant in normal SR) in failing SR. Note that dantrolene reduced on the K''Q/KQ in failing SR in a concentration-dependent manner (IC50 = 0.3 µmol/l). Each data point is a mean of 4 to 6 measurements ± SD. BSA = bovine serum albumin.
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Importantly, the IC50 (0.3 µmol/l) of dantrolene inhibition of domain unzipping shown here is identical to that of dantrolene inhibition of DPc10-induced Ca2+ leak shown in Figure 3B. Dantrolene had no appreciable effect on KQ unless DPc10 was added (Fig. 4B). This finding suggests that in the normal SR the domain switch is in a fully zipped state. In contrast, the KQ value of failing SR was comparable to the value of normal SR that had been treated with 100 µmol/l DPc10, suggesting that in the failing RyR2 the domain switch had already been unzipped. Dantrolene reduced KQ with a concentration dependence identical to what we have observed in the DPc10-added normal SR (Figs. 4C and 4D). Here again, the IC50 value (0.3 µmol/l) of dantrolene inhibition of domain unzipping is identical with that of spontaneous Ca2+ leak in failing SR. These results indicate that dantrolene reverses domain unzipping and restabilizes the zipped configuration of the domain switch, thereby inhibiting Ca2+ leak.
Effects of dantrolene on Ca2+ transient, cell shortening, Ca2+ spark, and membrane potential of normal and failing cardiomyocytes.
To investigate the effect of dantrolene on the isolated normal and failing cardiomyocytes, we measured both Ca2+ transient and cell shortening simultaneously in these cardiomyocytes. As shown in Figure 5A and as summarized in Table 2, Ca2+ transient and cell shortening in the cardiomyocytes isolated from the pacing-induced failing dog hearts were deteriorated compared with those in normal cardiomyocytes. Both Ca2+ transient and cell shortening were partially restored toward normal by the addition of dantrolene (1.0 µmol/l). In normal cardiomyocytes, there was no detectable change in Ca2+ transient and cell shortening after addition of dantrolene.
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Figure 5 Effect of Dantrolene on Ca2+ Transient and Cell Shortening
(A) Ca2+ transient and cell shortening in normal and failing cardiomyocytes in the presence or the absence of dantrolene (1.0 µmol/l). (B) Ca2+ transients before and after the addition of caffeine in normal and failing cardiomyocytes. The caffeine-induced increase in Ca2+ transients reflecting sarcoplasmic reticulum (SR) Ca2+ load was measured after a train of stimuli at 0.5 Hz by rapidly switching the perfusing solution to the one containing 20 mmol/l caffeine for 5 to 6 s. Note that the peak Ca2+ transient after addition of caffeine (indicative of SR Ca2+ load) was greater in normal cardiomyocyte than in failing cardiomyocyte and that dantrolene treatment ameliorated the SR load by inhibiting SR Ca2+ leak only in failing cardiomyocyte.
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Because intracellular peak Ca2+ transient strongly depends on the SR Ca2+ loading, we assessed SR Ca2+ content by measuring peak Ca2+ transient after the addition of caffeine. In normal cardiomyocytes, dantrolene was without effect on peak Ca2+ transient after the addition of caffeine (Fig. 5B). In failing cardiomyocytes, peak Ca2+ transient after the addition of caffeine was reduced, whereas it was restored toward normal in the presence of dantrolene (1 µmol/l) (Fig. 5C). These findings suggest that prevention of Ca2+ leak by dantrolene is effective to increase cardiomyocyte contractility by increasing SR Ca2+ loading.
To further explore the effects of dantrolene on the SR Ca2+ release mechanism, we investigated elementary Ca2+ release events (Ca2+ sparks) in normal and failing cardiomyocytes (Figs. 6A and 6B). In normal cardiomyocytes, spontaneous Ca2+ sparks occurred at a very low frequency. In DPc10-introduced cardiomyocytes, however, the frequency of spontaneous Ca2+ sparks significantly increased, but it was reduced to a normal level in the presence of dantrolene (1 µmol/l). In cardiomyocytes from failing hearts, the spark frequency was markedly increased as in the DPc10-introduced normal cardiomyocyte, whereas it decreased to a normal level in the presence of dantrolene (1 µmol/l).
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Figure 6 Effect of Dantrolene on Diastolic Ca2+ Sparks in DPc10-Incorporated Normal Cardiomyocytes and in Failing Cardiomyocytes
(A) Representative line-scan images of Ca2+ sparks. (B) Bar graphs summarize the various values on Ca2+ spark: frequency, amplitude, full width at half maximum (FWHM), full duration at half maximum (FDHM), for the same groups of cells. Each data point represents the mean ± SD. The number of cells for each group is 20 to 30.
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We further investigated the effect of dantrolene on the membrane potential of isolated cardiomyocytes. In normal cardiomyocytes, no fluctuation of membrane potential was observed, even in the presence of 100 nmol/l forskolin after 3Hz pacing (Fig. 7A), whereas the fluctuations of the membrane potential, which seem to represent delayed afterdepolarization, were observed in the DPc10-incorporated cardiomyocytes (Fig. 7A). However, the fluctuations of membrane potential observed in the DPc10-incorporated cardiomyocytes disappeared after the addition of dantrolene (Fig. 7A). As shown in Figure 7B, the spontaneous fluctuations of membrane potential were observed in failing cardiomyocytes, whereas they were prevented from occurring in the presence of dantrolene (1 µmol/l). The spontaneous fluctuations of membrane potential observed in DPc10-incorporated or failing cardiomyocytes were not induced by lower pacing rate (i.e., 1 Hz; not shown) but induced only after the application of a greater pacing rate (i.e., 3 Hz).
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Figure 7 Effect of Dantrolene on the Post-Pacing Fluctuation of Membrane Potential DAD in DPc10-Incorporated Normal Cardiomyocytes and in Failing Cardiomyocytes
Pacing was performed at a greater pacing rate (3 Hz) in the presence of forskolin (100 nmol/l) for a few seconds and stopped. Post-pacing fluctuation of the membrane potential were observed in DPc10-induced or failing cardiomyocytes but not in normal cardiomyocytes. Representative line-scan plots of membrane potential recorded in normal cardiomyocytes (A) and in failing cardiomyocytes (B). DAD = delayed afterdepolarization.
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Discussion
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Defective interdomain interactions within RyR2 play a key role in the abnormal channel gating of RyR2 in failing hearts (6). Therefore, correction of the defective interdomain interaction seems to be a new therapeutic strategy against heart failure and possibly cardiac arrhythmia. In our recent reports (10,11), we have shown that both K201 and the antioxidant edaravone prevent Ca2+ leak through RyR2 by correcting the defective interdomain interaction within RyR2, in turn improving intracellular Ca2+ handling and cardiac function and attenuating LV remodeling during development of pacing-induced heart failure.
The most important aspect of the present study is the finding that dantrolene, which might have been thought to be a skeletal muscle-specific muscle relaxant, works on the cardiac muscle as well. It is used to treat MH by correcting defective interdomain interaction within RyR1. Dantrolene is now found to bind to domain 601–620 of RyR2 and correct defective interdomain interaction within RyR2 in failing hearts. This action in turn inhibits spontaneous Ca2+ leak/Ca2+ sparks and improves cardiomyocyte function in failing hearts. This conclusion is based on the following 6 findings. First, DPc10, which is the synthetic peptide used as a domain switch unzipping agent, induced domain unzipping (Figs. 4A and 4B). Both DPc10-induced domain unzipping and the subsequent Ca2+ leak were inhibited by dantrolene. Dantrolene inhibition of domain unzipping and that of Ca2+ leak showed an identical concentration dependence (IC50 = 0.3 µmol/l) (Figs. 3 and 4). Second, in failing SR, the RyR is already in an unzipped state, as it occurs in normal SR after treatment with DPc10. Dantrolene restored the zipped state in a concentration-dependent manner (IC50 = 0.3 µmol/l) (Fig. 4D).
Similarly, dantrolene inhibited spontaneous Ca2+ leak from failing SR with the same concentration dependence (IC50 = 0.3 µmol/l) (Fig. 3C). Dantrolene also inhibited the cAMP-induced Ca2+ leak in normal SR. Third, the incorporation of DPc10 produced diastolic Ca2+ sparks in normal cardiomyocytes, whereas these sparks were inhibited by dantrolene. In failing cardiomyocytes, the magnitude of the intracellular Ca2+ transient decreased by 52% and its decay time was prolonged by 51%, but these abnormalities were normalized by 1 µmol/l dantrolene.
Fourth, the SR Ca2+ content, assessed by rapid application of caffeine, was reduced in failing cardiomyocytes, but it was restored by dantrolene, suggesting that the inhibition of SR Ca2+ leak by dantrolene increased SR Ca2+ content and thereby improved Ca2+ transient and cell shortening (Figs. 5A and 5B). Fifth, in the failing cardiomyocytes, diastolic Ca2+ sparks were observed, but they were eliminated almost completely by 1 µmol/l dantrolene.
Sixth, importantly, <1 µmol/l dantrolene had no appreciable effect on normal SR and cardiomyocyte functions in all experiments. It is also reported that dantrolene restores normal interdomain interactions within RyR1 (namely, zipped state) in DP4-treated RyR1 channel, which is analogous to MH channel (8). Taken together, all of these data strongly suggest that the stable interdomain interaction of regulatory domains within RyR1 or RyR2 is an essential requirement for normal channel function. Defective interdomain interaction (namely, domain unzipping) causes channel dysfunction in RyR1 of MH and in RyR2 of failing heart; thus, correction of the defective interdomain interaction restores normal channel gating and hence prevents diseased conditions.
Recently, Yang et al. (12) reported that DPc10 lowered the [Ca2+] threshold for spontaneous Ca2+ release in permeabilized cardiomyocytes. That is, DPc10 mimicked the abnormal channel gating seen in CPVT/ARVC2. Consistent with these data, abnormal diastolic Ca2+ sparks were observed in DPc10-incorporated cardiomyocytes, while dantrolene suppressed the diastolic Ca2+ sparks (Figs. 6A and 6B). Moreover, in DPc10-incorporated cardiomyocytes, the fluctuation of membrane potential, which represents delayed afterdepolarization (DAD), was observed, and dantrolene prevented it from occurring (Fig. 7A). Thus, the restoration of the domain interaction from defective unzipped state to normal zipped state by dantrolene may be a new strategy for the treatment of polymorphic VT and post-translationally developed heart failure as well.
Although it is widely recognized that dantrolene has an appreciable effect on RyR1, there is a controversy as to whether dantrolene modulates channel gating of RyR2. Zhao et al. (13) did not find any dantrolene inhibition of Ca2+ release in the RyR2 expressed in Chinese hamster ovary cells. In contrast, it is reported that dantrolene improved the inotropic response to norepinephrine in human failing heart (14) and reduced Ca2+ oscillations, thereby decreasing the magnitude of diastolic intracellular [Ca2+] in post-infarcted rat myocardium (15).
The present finding that dantrolene inhibited cAMP-induced Ca2+ leak may partly explain the improvement of the beta-adrenergic responsiveness by dantrolene. Taken together, dantrolene appears to have much more appreciable effect on Ca2+ handling in failing (Ca2+ overloaded) hearts than in normal hearts because of its inhibition of diastolic Ca2+ release that can be seen only in failing hearts. Dantrolene was found to enhance the Ca2+ uptake rate in rat cardiac SR but also showed mild negative inotropic effect in parallel with a decrease in peak Ca2+ transient (16). In that study, however, the concentration of dantrolene was 50 µmol/l, which is 50 times greater than that used in the present study. In the present study, dantrolene (0.1 to 1 µmol/l) had no effect on the SR Ca2+ uptake time course, Ca2+ transient, and cell shortening in normal hearts. Low concentrations of dantrolene (0.1 to 1 µmol/l) used in this study seem to have no significant effect on SR Ca2+ ATPase and Ca2+-induced Ca2+ release in normal hearts.
The present data also showed that dantrolene inhibited DPc10-induced Ca2+ leak in normal RyR2 and spontaneous Ca2+ leak in failing RyR2, whereas it had no appreciable effect on normal RyR2, in which interdomain interactions between N-terminal and central regulatory domains are still in the zipped state. These findings suggest that the effect of dantrolene on channel gating depends on the conformational state of the regulatory domains. That is, dantrolene may be effective for stabilizing RyR2 only in the unzipped state but not in the zipped state. The report that [3H]dantrolene-binding to the rabbit RyR2 could be seen only in particular conditions (in the presence of a very high concentration of ethylene glycol tetraacetic acid) (17) supports the idea that dantrolene binding to RyR2 is indeed dependent on a particular conformational state of RyR2 that takes place in disease conditions.
To identify the dantrolene binding site within the RyR2, we screened recombinant constructs corresponding to the region from the N-terminus to the residue 2750 by using the QCM assay and found that only the recombinant construct corresponding to aa494–1000 and the synthetic peptide corresponding to aa601–620 bound with dantrolene. This finding indicates that the aa601–620 region of RyR2 represents the site of dantrolene binding. However, because our screening did not extend beyond the residue 2750, we cannot exclude the possibility that there might be another drug-binding region in the carboxyl half of the RyR2. We also cannot exclude the possibility that there might be additional drug-binding sites, with a low affinity of binding, even in the N-terminal half, because some of the recombinant constructs may have failed to retain the native tertiary structure required for high-affinity drug binding. Altered intracellular Ca2+ handling is one of the causative mechanisms in the generation of lethal arrhythmia in the patients with heart failure and CPVT. Diastolic Ca2+ leak from SR via RyR2 will initiate DAD and triggered activity, leading arrhythmia. Therefore, stabilization of RyR2 may be a novel therapeutic strategy against heart failure and CPVT (10). The proposed molecular mechanism by which dantrolene corrects the defective channel gating in diseased RyR2 is summarized in the Figures 8A and 8B.
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Figure 8 Schematic Illustration of the Effects of Dantrolene on Abnormal Intracellular Ca2+ Handling and Membrane Potential in Cardiomyocytes
(A) In normal cardiomyocytes, domain unzipping between the N-terminal and central domains occurs upon incorporation of DPc10, thereby causing aberrant diastolic Ca2+ sparks. These Ca2+ sparks subsequently induce the DAD caused by inward Na+ current via Na+-Ca2+ exchange. Dantrolene inhibits all of these Ca2+ handling abnormalities. (B) In failing cardiomyocytes, domain unzipping has already taken place, and it induces aberrant diastolic Ca2+ sparks and DAD. Dantrolene inhibits all of the Ca2+ handling abnormalities. NCX = Na+/Ca2+ exchanger; other abbreviations as in Figures 3 and 7.
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Conclusions
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Defective interactions of the regulatory domains in RyR2 seem to play a key role in the abnormal Ca2+ channel functions of RyR2 found in the failing cardiomyocytes, such as increased Ca2+ spark frequency and DAD. The present finding that the defective domain interaction is corrected by dantrolene provides one with a new clue for the development of therapeutic strategy against heart failure and possibly lethal arrhythmia.
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Appendix
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For a supplemental Methods section, please see the online version of this article.
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Footnotes
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This work was supported by grants-in-aid for scientific research from The Ministry of Education in Japan (grant nos. 18390234 and 18659228 to Dr. Yano; 18590777 to Dr. Yamamoto; and 18591706 to Dr. Kobayashi) and the National Heart, Lung, and Blood Institute (HL072841 to Dr. Ikemoto).
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References
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