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- Publisher
- American Society for Biochemistry and Molecular Biology
- Copyright
- Copyright © 2016 Elsevier Inc.
- ISSN
- 0021-9258
- eISSN
- 1083-351X
- DOI
- 10.1074/jbc.m116.756528
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Abstract
crossmark THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 47, pp. 24528 –24537, November 18, 2016 © 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Enhanced Cytosolic Ca Activation Underlies a Common Defect of Central Domain Cardiac Ryanodine Receptor Mutations Linked to Arrhythmias Received for publication, August 31, 2016, and in revised form, October 2, 2016 Published, JBC Papers in Press, October 12, 2016, DOI 10.1074/jbc.M116.756528 ‡1 ‡2 ‡3 ‡ ‡4 ‡2 ‡ Zhichao Xiao , Wenting Guo ,BoSun , Donald J. Hunt , Jinhong Wei , Yingjie Liu , Yundi Wang , ‡ § ¶ ‡5 Ruiwu Wang , Peter P. Jones , Thomas G. Back , and S. R. Wayne Chen From the the Department of Physiology and Pharmacology, Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta T2N 4N1, Canada, the Department of Physiology, Otago School of Medical Sciences and HeartOtago, University of Otago, Dunedin 9054, New Zealand, and the Department of Chemistry, University of Calgary, Calgary, Alberta T2N 1N4, Canada Edited by Roger Colbran Recent three-dimensional structural studies reveal that the of RyR2 mutations associated with CPVT and AF, which could central domain of ryanodine receptor (RyR) serves as a trans- potentially be suppressed by carvedilol or (R)-carvedilol. ducer that converts long-range conformational changes into the gating of the channel pore. Interestingly, the central domain encompasses one of the mutation hotspots (corresponding to Ryanodine receptors are a family of intracellular Ca release amino acid residues 3778–4201) that contains a number of car- channels that are essential for excitation-contraction coupling diac RyR (RyR2) mutations associated with catecholaminergic in skeletal and cardiac muscles (1–3). They also play an impor- polymorphic ventricular tachycardia (CPVT) and atrial fibrilla- tant role in the pathogenesis of various skeletal and cardiac tion (AF). However, the functional consequences of these cen- muscle disorders (4, 5). Naturally occurring mutations in the tral domain RyR2 mutations are not well understood. To gain skeletal muscle ryanodine receptor (RyR1) are linked to malig- insights into the impact of the mutation and the role of the cen- nant hyperthermia and central core disease, whereas mutations tral domain in channel function, we generated and character- in the cardiac ryanodine receptor (RyR2) are associated with ized eight disease-associated RyR2 mutations in the central different forms of cardiac arrhythmias and cardiomyopathies, domain. We found that all eight central domain RyR2 mutations such as catecholaminergic polymorphic ventricular tachycar- 2 3 enhanced the Ca -dependent activation of [ H]ryanodine dia (CPVT), idiopathic ventricular fibrillation (IVF), atrial 2 2 binding, increased cytosolic Ca -induced fractional Ca fibrillation (AF), and dilated cardiomyopathies (4, 5). To date, release, and reduced the activation and termination thresholds more than 300 RyR1 and 150 RyR2 disease-associated muta- for spontaneous Ca release in HEK293 cells. We also showed tions have been identified. A majority of these RyR mutations that racemic carvedilol and the non-beta-blocking carvedilol are located in three regions: the N-terminal (RyR1 residues, enantiomer, (R)-carvedilol, suppressed spontaneous Ca oscil- 13–552; RyR2 residues, 44 – 466), central (RyR1 residues, lations in HEK293 cells expressing the central domain RyR2 2101–2458; RyR2 residues, 2246–2534), and C-terminal (RyR1 mutations associated with CPVT and AF. These data indicate residues, 4631–4990; RyR2 residues, 4497–4959) regions in that the central domain is an important determinant of cytosolic RyR1 and RyR2 (4, 5). In addition to these three disease muta- Ca activation of RyR2. These results also suggest that altered tion hotspots, RyR2 also contains another disease mutation cytosolic Ca activation of RyR2 represents a common defect hotspot between residues 3778–4201 (hotspot-3). Interest- ingly, mutation hotspot-3 seems to be absent in RyR1 (4, 5). This difference in the distribution of disease-associated RyR1 * This work was supported by research grants from the Canadian Institutes of Health Research, the Heart and Stroke Foundation of Canada, the Canada and RyR2 mutations raises an interesting and important ques- Foundation for Innovation, and the Heart and Stroke Foundation/Libin tion about the functional impact of RyR2 mutations in Cardiovascular Institute Professorship in Cardiovascular Research (to hotspot-3 and the role of the domain encompassing mutation S. R. W. C.). The authors declare that they have no conflicts of interest with hotspot-3 in channel function. the contents of this article. □ S This article contains supplemental Figs. S1 and S2. A near-atomic resolution (3.8 Å) three-dimensional (3D) Present address: Dept. of Cardiology of Tongji Hospital, Tongji Medical structure of RyR1 has recently been solved using cryo-electron School, Huazhong University of Science and Technology, Wuhan 430030, microscopy (6). The region corresponding to mutation hotspot-3 China. Recipients of the Alberta Innovates-Health Solutions (AIHS) Graduate Stu- dentship Award. 3 6 Recipient of the Heart and Stroke Foundation of Canada Junior Fellowship The abbreviations used are: RyR, ryanodine receptor; RyR1, skeletal ryano- Award and the AIHS Fellowship Award. dine receptor; RyR2, cardiac ryanodine receptor; CPVT, catecholaminergic A recipient of the Libin Cardiovascular Institute of Alberta and Cumming polymorphic ventricular tachycardia; AF, atrial fibrillation; ER, endoplasmic 2 2 School of Medicine Postdoctoral Fellowship Award. reticulum; SR, sarcoplasmic reticulum; CICR, Ca -induced Ca release; 5 2 An AIHS Scientist. To whom correspondence should be addressed: 3330 SOICR, store overload-induced Ca release; EC, excitation-contraction; Hospital Dr. N.W., Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-4235; ICM, intracellular-like medium; ex, excitation; em, emission; KRH buffer, E-mail: [email protected]. Krebs-Ringer-Hepes buffer. 24528 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 47 • NOVEMBER 18, 2016 This is an Open Access article under the CC BY license. 2 Enhanced RyR2 Ca Activation and Cardiac Arrhythmias FIGURE 1. Location of the RyR2 mutation hotspot-3 region in the central domain of the RyR channel. A, the open bar represents the5,000-amino acid (5000 aa) sequence of RyR. Mutations in RyR1 associated with malignant hyperthermia (MH) and central core disease (CCD) are largely clustered in three hotspot regions (red bars): MH/CCD I, II, and III. Mutations in RyR2 associated with CPVT are mainly clustered in four hotspot regions (red bars): CPVT I, II, III, and IV. The FKBP12.6 binding sites, PKA phosphorylation sites, calmodulin (CaM) binding domain, Ca activation sites, and pore-forming segment are depicted as circles. The divergent and transmem- brane regions are also indicated. B, three-dimensional structure of two RyR monomers (6). The mutation hotspot-3 (CPVT III), the S6 inner helix (S6), and the C-terminal domain (CTD) are highlighted.C, a close-up view showing the locations of eight disease-associated RyR2 mutations in the mutation hotspot-3 region within the central domain (green). The location of the Glu-3987 residue (9) critical for cytosolic Ca activation of RyR2 is also shown. is located within the central domain that directly interacts with spontaneous activities of CPVT- and AF-associated RyR2 the C-terminal domain, which controls the gate of the RyR mutants. These observations suggest that the central domain is channel (6). Comparison of the closed and open states of the an important determinant of cytosolic Ca activation of RyR2, RyR1 channel reveals that the central domain acts as a trans- and that altered cytosolic Ca activation of RyR2 may enhance ducer that couples the conformational changes in the cytosolic the susceptibility to both ventricular tachycardias and AF. assembly to the gating of the central pore of the channel (7). Carvedilol may be beneficial for patients with RyR2 mutation- Furthermore, we showed previously that a point mutation associated CPVT and AF. E4032A in RyR1, E3987A in RyR2, and E3885A in RyR3, located Results in the central domain, dramatically diminished cytosolic Ca activation of the channel (8–10). These observations suggest Effect of Disease-associated RyR2 Mutations Located in 2 2 3 that the central domain may mediate the Ca -dependent gat- the Central Domain on Ca Activation of [ H]Ryanodine ing of the RyR channel, and that disease-associated RyR2 muta- Binding—To assess the functional impact of disease-associated tions in mutation hotspot-3 located within the central domain RyR2 mutations located in mutation hotspot-3 (residues 3778– may alter the cytosolic Ca activation of RyR2. To test this 4201) within the central domain of the 3D structure of RyR (6) hypothesis, in the present study, we generated a number of (Fig. 1), we generated a number of disease-associated RyR2 disease-associated RyR2 mutations in hotspot-3 within the cen- mutations in this region, including G3946A, G3946S, M3978I, tral domain and determined their functional impact. Consist- H4108N, H4108Q, S4124T, T4158P, and Q4159P (16–23). We ent with this hypothesis, we found that central domain RyR2 then determined the effect of these mutations on the Ca -de- 2 3 mutations enhanced the cytosolic Ca -dependent activation pendent activation of [ H]ryanodine binding. Because ryano- of RyR2. Interestingly, the central domain contains RyR2 muta- dine only binds to the open conformation of RyRs, [ H]ryano- tions that are associated with CPVT and AF. We showed pre- dine binding has widely been used for monitoring the opening viously that carvedilol, a clinically used beta-blocker, sup- of the RyR channel. As shown in Fig. 2, [ H]ryanodine binding pressed spontaneous activities of CPVT-linked RyR2 mutants to RyR2 WT was activated by submicromolar Ca with an (11). Here we found that carvedilol and the non-beta-blocking EC of 0.21 0.01 M. All eight central domain RyR2 muta- carvedilol enantiomer, (R)-carvedilol (12–15), also suppressed tions tested significantly reduced the EC of Ca -dependent NOVEMBER 18, 2016 • VOLUME 291 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 24529 2 Enhanced RyR2 Ca Activation and Cardiac Arrhythmias the regulation of RyR2 by cytosolic Ca in a cellular environ- ment. HEK293 cells expressing the RyR2 WT and eight central domain mutants were permeabilized to allow access to cytoso- lic Ca . Permeabilized cells were then perfused with various 2 2 cytosolic Ca concentrations (0.1–10 M) to induce ER Ca release. The fractional Ca release induced by elevating cyto- solic Ca concentrations was monitored by measuring the steady state ER Ca level using a FRET-based ER luminal Ca -sensing protein D1ER (24–26). As shown in Fig. 3, increasing cytosolic Ca concentrations (0.1–10 M) reduced the steady state ER Ca level in permeabilized HEK293 cells expressing RyR2 WT (Fig. 3, A and F). This reduction in the 2 2 steady state ER Ca level likely reflects cytosolic Ca -induced 2 2 fractional Ca release from the ER Ca store. The steady state ER Ca levels in permeabilized HEK293 cells expressing central domain RyR2 mutants at various cytosolic Ca con- centrations are shown in Figs. 3 and 4. All eight RyR2 mutations located in the central domain significantly reduced the steady state ER Ca levels in the presence of 0.1–1.0 M cytosolic Ca concentrations as compared with those in WT cells. These reduced steady state ER Ca levels or enhanced levels of 2 2 cytosolic Ca -induced fractional Ca release suggest that central domain RyR2 mutations increase the cytosolic Ca -de- pendent activation of Ca release. This is consistent with the observation that central domain RyR2 mutations increase the 2 3 Ca -dependent activation of [ H]ryanodine binding (Fig. 2 and supplemental Fig. S1). Interestingly, the steady state ER Ca level in HEK293 cells expressing central domain RyR2 mutants at 10 M cytosolic Ca concentration was signifi- cantly higher than that in WT cells (Figs. 3 and 4). In other words, central domain RyR2 mutant cells displayed reduced 2 2 fractional Ca release at 10 M cytosolic Ca concentra- tion as compared with WT cells. This suggests that RyR2 mutations located in the central domain of the channel may FIGURE 2. Central domain RyR2 mutations increase the Ca -dependent 2 2 also alter the cytosolic Ca -dependent inhibition of Ca 3 3 activation of [ H]ryanodine binding. A and B,[ H]ryanodine binding to cell release. lysate prepared from HEK293 cells expressing the RyR2 WT, G3946A, G3946S, M3978I, or H4108N mutant (A) or the H4108Q, S4124T, T4158P, or Q4159P Effect of Central Domain RyR2 Mutations on the Activation mutant (B) was carried out at various Ca concentrations (0.2 nM to 0.1 mM) 3 3 and Termination Thresholds for Spontaneous Ca Release— with 150 mM KCl and 5 nM [ H]ryanodine. The amounts of [ H]ryanodine bind- ing at various Ca concentrations were normalized to the maximal binding Disease-associated RyR2 mutations have been shown to (100%). C, the EC of WT and central domain mutants. Data points shown are 2 increase the propensity for arrhythmogenic spontaneous Ca mean S.E. from 3–9 separate experiments (*, p 0.05 versus WT). release during store Ca overload, a process also known as [ H]ryanodine binding (Fig. 2 and supplemental Fig. S1). For store overload-induced Ca release (SOICR) (27, 28). It is of example, the H4108N and H4108Q mutations markedly interest and importance to assess whether RyR2 mutations decreased the EC of Ca -dependent activation to 0.086 located in the central domain of the channel also alter the prop- 0.004 and 0.106 0.003 M (p 0.05), respectively. The erties of SOICR. To this end, we monitored the ER luminal Ca expression levels of the RyR2 WT and central domain mutants dynamics in HEK293 cells using D1ER (24, 25). As shown are shown in supplemental Fig. S2. Except for the H4108Q in Fig. 5, elevating extracellular Ca from0to2mM induced mutant, all central domain mutants tested were expressed at spontaneous ER Ca oscillations in HEK293 cells expressing a level comparable with that of the WT. Because the Ca RyR2 WT (depicted as downward deflections of the FRET sig- dependence of [ H]ryanodine binding reflects the depen- nal). SOICR occurred when the ER luminal Ca content dence of single RyR2 channels to activation by cytosolic increased to a threshold level (F ), and terminated when SOICR 2 3 2 Ca (9), our [ H]ryanodine binding data suggest that dis- the ER luminal Ca content fell to another threshold level ease-associated RyR2 mutations located in the central (F ) (Fig. 5A). The ER luminal Ca dynamics during SOICR termi domain of the channel enhance the sensitivity of RyR2 to in HEK293 cells expressing eight RyR2 mutants located in the cytosolic Ca activation. central domain is shown in Fig. 5, B–I. All eight central domain Effect of Central Domain RyR2 Mutations on Cytosolic Ca RyR2 mutations significantly reduced both the activation (Fig. Regulation of Ca Release in HEK293 Cells—We next assessed 6A) and termination (Fig. 6B) thresholds for SOICR. However, these mutations altered the activation and termination thresh- the effect of RyR2 mutations located in the central domain on 24530 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 47 • NOVEMBER 18, 2016 2 Enhanced RyR2 Ca Activation and Cardiac Arrhythmias 2 2 FIGURE 3. RyR2 mutations G3946A, G3946S, M3978I, and H4108N increase cytosolic Ca -induced fractional Ca release in HEK293 cells. A–E, stable, inducible HEK293 cell lines expressing RyR2 WT (A), G3946A (B), G3946S (C), M3978I (D), or H4108N (E) were transfected with the FRET-based ER luminal Ca -sensing protein D1ER and induced using tetracycline. The transfected and induced cells were permeabilized with saponin, washed, and perfused with intracellular-like medium plus increasing levels of free Ca (0.1, 0.2, 0.4, 1, and 10 M). FRET recordings from representative cells (total 45–59 cells each) are shown. F, to minimize the influence by CFP/YFP cross-talk, we used relative FRET measurements for calculating the steady state ER Ca level, which was determined by the equation ((F F )/(F F )) 100%. The dashed lines (F to F ) indicate the steady state FRET levels after perfusion with each cyto min max min 0.1 10 Ca concentration (0.1, 0.2, 0.4, 1, or 10 M). The maximum FRET signal F is defined as the FRET level after tetracaine treatment. The minimum FRET signal max F is defined as the FRET level after caffeine treatment. Data shown are mean S.E. (n 3) (*, p 0.05 versus WT). min olds to different extents. As a result, they differentially affected Racemic Carvedilol and the (R)-Carvedilol Enantiomer Sup- the fractional Ca release (activation threshold termination press SOICR in HEK293 Cells Expressing Central Domain RyR2 threshold) during SOICR. Mutations H4108Q, T4158P, and Mutations Associated with Atrial Fibrillation—We have previ- Q4159P significantly reduced the fractional Ca release (p ously shown that racemic carvedilol, a clinically used beta- 0.05), whereas mutations H4108N and S4124T slightly blocker, and the non-beta-blocking (R)-carvedilol enantiomer increased fractional release as compared with that in RyR2 WT- suppress spontaneous Ca oscillations (SOICR) in HEK293 expressing cells (p 0.05). On the other hand, mutations cells expressing a RyR2 mutation R4496C that is associated G3946A, G3946S, and M3978I exerted no significant impact with CPVT (11, 15). Interestingly, some of the RyR2 mutations on the fractional Ca release (Fig. 6C). It should be noted located in the central domain, G3946A, M3978I, and Q4159P, that there was no significant difference in the store capacity have been associated with AF (16, 19, 23). It is unknown (F F ) between RyR2 WT and eight central domain whether racemic carvedilol or (R)-carvedilol is able to suppress max min RyR2 mutant cells (Fig. 6D). Furthermore, SOICR did not SOICR mediated by CPVT- and AF-associated RyR2 mutants. occur in control HEK293 cells expressing no RyR2, and To this end, we perfused HEK293 cells expressing the CPVT- SOICR was not affected by the inositol trisphosphate recep- and AF-associated RyR2 mutants (G3946A, M3978I, or tor (IP3R) inhibitor, xestospongin C (29), indicating that Q4159P) with elevated extracellular Ca from0to2mM to SOICR is mediated by RyR2. Collectively, these data indicate induce spontaneous Ca oscillations in these cells. The frac- that disease-associated RyR2 mutations located in the cen- tions of HEK293 cells displaying Ca oscillations were then tral domain reduce both the SOICR activation and termina- determined before and after application of increasing concen- tion thresholds. trations (0, 1, 3, 10, and 30 M) of racemic carvedilol or the NOVEMBER 18, 2016 • VOLUME 291 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 24531 2 Enhanced RyR2 Ca Activation and Cardiac Arrhythmias 2 2 FIGURE 4. RyR2 mutations H4108Q, S4124T, T4158P, and Q4159P increase cytosolic Ca -induced fractional Ca release in HEK293 cells. A–E, stable, inducible HEK293 cell lines expressing RyR2 WT (A), H4108Q (B), S4124T (C), T4158P (D), or Q4159P (E) were transfected with the FRET-based ER luminal Ca -sensing protein D1ER and induced using tetracycline. The transfected and induced cells were permeabilized with saponin, washed, and perfused with intracellular-like medium plus increasing levels of free Ca (0.1, 0.2, 0.4, 1, and 10 M). FRET recordings from representative cells (total 20 –59 cells each) are shown. F, to minimize the influence by CFP/YFP cross-talk, we used relative FRET measurements for calculating the steady state ER Ca level, which was determined by the equation ((F F )/(F F )) 100%. The dashed lines (F to F ) indicate the steady state FRET levels after perfusion with each cyto min max min 0.1 10 Ca concentration (0.1, 0.2, 0.4, 1, or 10 M). The maximum FRET signal F is defined as the FRET level after tetracaine treatment. The minimum FRET signal max F is defined as the FRET level after caffeine treatment. Data shown are mean S.E. (n 3) (*, p 0.05 versus WT). min non-beta-blocking (R)-carvedilol. As shown in Fig. 7, racemic understood. Recent structural analysis of the closed and open carvedilol or (R)-carvedilol at 10 M markedly suppressed states of RyR1 identified the central domain as the transducer SOICR in HEK293 cells expressing the CPVT- and AF-associ- that couples the long-range conformational changes between ated mutants (G3946A, M3978I, and Q4159P), and completely the cytosolic peripheral domains and the channel domain (7). abolished SOICR in these cells at 30 M. Because both the beta- Interestingly, the central domain of RyR2 encompasses one of blocking carvedilol and non-beta-blocking (R)-carvedilol sup- the disease-associated mutation hotspots (hotspot-3), but the press SOICR, their SOICR-inhibiting effect is independent of functional impact of hotpsot-3 RyR2 mutations is not well beta-blockade. Thus, racemic carvedilol and the (R)-carvedilol defined. We have previously shown that a point mutation enantiomer are also able to suppress SOICR mediated by E3987A located in the central domain of RyR2 dramatically CPVT- and AF-linked RyR2 mutants. reduced the sensitivity of the channel to activation by cytosolic Ca (9). This suggests that the central domain may mediate Discussion the cytosolic Ca activation of RyR2. Consistent with this The three-dimensional structure of RyR consists of a number view, here we show that disease-associated RyR2 mutations of major domains, including the N-terminal domain, SPRY located in hotspot-3 within the central domain enhance the cytosolic Ca activation of the channel and the propensity for domains, phosphorylation domains, handle domain, helical domain, central domain, and channel domain (6). The chan- spontaneous Ca release. Hence, our data provide novel nel domain, which possesses a structural fold similar to that of insights into the molecular mechanisms of action of hotspot-3 the superfamily of voltage-gated ion channels, is involved in the RyR2 mutations and the functional role of the central domain. gating and ion conduction of RyR (6). However, the roles of Comparison of the distribution patterns of disease-associ- other major domains in RyR function and regulation are poorly ated RyR1 and RyR2 mutations reveals that mutation hotspot-3 24532 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 47 • NOVEMBER 18, 2016 2 Enhanced RyR2 Ca Activation and Cardiac Arrhythmias FIGURE 5. Effect of central domain RyR2 mutations on SOICR activation and termination. A–I, stable, inducible HEK293 cell lines expressing RyR2 WT (A), G3946A (B), G3946S (C), M3978I (D), H4108N (E), H4108Q (F), S4124T (G), T4158P (H), or Q4159P (I) were transfected with the FRET-based ER luminal Ca - sensing protein D1ER for 48 h. The expression of RyR2 WT and central domain mutants was induced by tetracycline 24 h before imaging. The cells were perfused with KRH buffer containing increasing levels of extracellular Ca (0 –2 mM) to induce SOICR. This was followed by the addition of 10 mM tetracaine to inhibit SOICR, and then 20 mM caffeine to deplete the ER Ca store. FRET recordings from representative RyR2 WT (A) and central domain mutant (B–I) cells (a total of 33–97 cells each) are shown. appears to be absent in RyR1 (4, 5). The reason for the lack of mechanism known as voltage-induced Ca release. In this disease-causing RyR1 mutations in the hotspot-3 region within process, membrane depolarization causes a conformational the central domain is unknown. It is well established that the change in the voltage-gated Ca channel, the voltage sensor mechanism of excitation-contraction (EC) coupling in skeletal (Ca 1.1), which in turn activates the RyR1 channel through a muscle differs from that in cardiac muscle. In skeletal muscle, direct physical interaction between Ca 1.1 and RyR1. On the contraction is triggered by the release of Ca from the sarco- other hand, EC coupling in cardiac muscle is mediated by a 2 2 2 plasmic reticulum through the RyR1 Ca release channel via a mechanism known as Ca -induced Ca release (CICR), in NOVEMBER 18, 2016 • VOLUME 291 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 24533 2 Enhanced RyR2 Ca Activation and Cardiac Arrhythmias Consistent with this view, we found that central domain RyR2 mutations also reduce the threshold for activation and termi- nation of spontaneous Ca release. Therefore, it is possible that, by increasing the cytosolic Ca activation of RyR2, the central domain RyR2 mutations may reduce the threshold for CICR and enhance the propensity for the initiation and propa- gation of spontaneous Ca waves in atrial and Purkinje myo- cytes. It will be of interest to generate mouse models harboring central domain RyR2 mutations and determine whether central domain RyR2 mutations alter Ca signaling in atrial and Pur- kinje cells and increase the susceptibility to CPVT and AF in mice. Spontaneous Ca release is believed to be the trigger for FIGURE 6. Central domain RyR2 mutations reduce the thresholds for SOICR activation and termination. A and B, to minimize the influence by CPVT (5). Hence, suppressing spontaneous Ca release rep- CFP/YFP cross-talk, we used relative FRET measurements for calculating the resents a promising strategy for the treatment of CPVT. We activation threshold (A) and termination threshold (B). The activation thresh- old was determined by the equation ((F F )/(F F )) 100%, have previously shown that carvedilol, a beta-blocker, is able to SOICR min max min and the termination threshold was determined by the equation ((F termi reduce the duration of openings of single RyR2 channels and F )/(F F )) 100%. F indicates the FRET level at which SOICR min max min SOICR 2 suppress RyR2-mediated spontaneous Ca release and stress- occurs, whereas F represents the FRET level at which SOICR terminates. C, termi the fractional Ca release was calculated by subtracting the termination induced ventricular tachycardias in a mouse model harboring a threshold from the activation threshold. The maximum FRET signal F is max CPVT-causing RyR2 mutation (11). We also showed that the defined as the FRET level after tetracaine treatment. The minimum FRET sig- non-beta-blocking carvedilol enantiomer, (R)-carvedilol, is also nal F is defined as the FRET level after caffeine treatment. D, the store min capacity was calculated by subtracting F from F . Data shown are min max able to suppress spontaneous Ca waves and wave-evoked mean S.E. (n 3–5) (*, p 0.05 versus WT; NS, not significant). CPVT without the bradycardia associated with racemic carve- dilol (15). Here we demonstrated that racemic carvedilol and which membrane depolarization opens the voltage-dependent the non-beta-blocking (R)-carvedilol enantiomer also sup- 2 2 2 L-type Ca channel (Ca 1.2), leading to an influx of Ca . This pressed spontaneous Ca oscillations in HEK293 cells 2 2 small Ca entry then activates the RyR2 Ca release channel, expressing the CPVT- and AF-associated RyR2 mutations. resulting in a large Ca release from the sarcoplasmic reticu- These observations suggest that carvedilol or (R)-carvedilol lum and subsequent muscle contraction. Hence, activation of may provide some benefits for patients with CPVT and AF RyR by cytosolic Ca is absolutely required for EC coupling in associated with RyR2 mutations. cardiac muscle, but not in skeletal muscle (1–3). Therefore, In summary, our present study demonstrates that CPVT- mutations that alter the cytosolic Ca activation of RyR2 and AF-associated RyR2 mutations located in the central would be expected to result in cardiac dysfunction and diseases. domain enhance cytosolic Ca activation of RyR2 and sponta- 2 2 However, because cytosolic Ca activation of RyR1 is not neous Ca release, which can be limited by racemic carvedilol absolutely required for EC coupling, mutations that alter the or the (R)-carvedilol enantiomer. Our data suggest that the cen- cytosolic Ca activation of RyR1 may be tolerable. Thus, tral domain of RyR2 is an important determinant of cytosolic 2 2 mutations in the central domain that affect the cytosolic Ca Ca activation of the channel, and that enhanced cytosolic activation of RyR1 may not be highly pathogenic. This may Ca activation represents a common defect of RyR2 mutations account, in part, for the lack of disease-associated RyR1 muta- associated with CPVT and AF. tions in the central domain. Future studies on the impact of Experimental Procedures central domain RyR1 mutations on skeletal muscle function and disease will be required to test this hypothesis. Construction of Disease-associated RyR2 Mutations Located To date, a large number of RyR2 mutations have been iden- in the Central Domain of RyR2—The central domain RyR2 tified, most of which are associated with CPVT. RyR2 muta- mutations (G3946A, G3946S, M3978I, H4108N, H4108Q, tions have also been associated with AF (5). Interestingly, the S4124T, T4158P, and Q4159P) were generated by the overlap central domain appears to be a hotspot for CPVT- and AF- extension method using PCR (32, 33). Briefly, The SalI and associated RyR2 mutations, such as G3946A (16), M3978I (19), MluI restriction sites were introduced into the full-length Q4159P (23), and S4153R (30). The exact mechanisms by which mouse RyR2 cDNA at positions 11816 and 12340, respectively, mutations in the central domain of RyR2 enhance the propen- without altering the amino acid sequence. The SalI-MluI sity for both CPVT and AF are unknown. In the present study, fragment containing G3946A, G3946S, M3978I, H4108N, or we found that RyR2 mutations located in the central domain H4108Q was obtained by overlapping PCR and was used to enhance the cytosolic Ca activation of RyR2. Because cytoso- replace the corresponding WT fragment in the full-length lic Ca activation of RyR2 is a central step in CICR, enhanced mouse RyR2 cDNA in pBluescript, which was then subcloned cytosolic Ca activation of RyR2 would be expected to result in into pcDNA5. The MluI-Bsu36I (at position 13237) fragment a reduced threshold for CICR. A reduced CICR threshold is containing S4124T, T4158P, or Q4159P was obtained by over- thought to increase the propensity for and propagation of spon- lapping PCR and was used to replace the corresponding WT taneous pro-arrhythmic Ca waves especially in non-tubu- fragment in the BsiwI (at position 8864)-NotI (in the vector) lated cardiac cells, such as atrial and Purkinje myocytes (31). construct of mouse RyR2 in pBluescript. This construct was 24534 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 47 • NOVEMBER 18, 2016 2 Enhanced RyR2 Ca Activation and Cardiac Arrhythmias FIGURE 7. Racemic carvedilol and the (R)-carvedilol enantiomer suppress SOICR in HEK293 cells expressing CPVT- and AF-associated RyR2 mutations. A, B, C, E, F, and G, inhibition of spontaneous Ca oscillations by carvedilol (left panels)or(R)-carvedilol (right panels) in HEK293 cells expressing the CPVT- and AF-associated RyR2 mutations G3946A (A and E), M3978I (B and F), or Q4159P (C and G). The cells were loaded with Fura-2 AM and perfused continuously with 2mM Ca in KRH buffer containing increasing concentrations of carvedilol or (R)-carvedilol (0, 1, 3, 10, and 30 M). D and H, the fractions of cells that displayed Ca oscillations after perfusion of different concentrations of carvedilol (D)or(R)-carvedilol (H) were determined using a total of 152–320 cells each. Data shown are mean S.E. (n 3). then subcloned into the full-length mouse RyR2 cDNA in the Western Blotting—HEK293 cells grown for 24 h after trans- pcDNA5. All mutations were confirmed by DNA sequencing. fection with RyR2 WT and central domain mutant cDNAs were Generation of Stable, Inducible Cell Lines Expressing WT and M EDTA and harvested in the same washed with PBS plus 2.5 m Central Domain Mutants of RyR2—Stable, inducible HEK293 solution by centrifugation for 8 min at 700 g in an IEC Cen- tra-CL2 centrifuge. The cells were then washed with PBS with- cell lines expressing RyR2 WT and central domain mutants were generated using the Flp-In T-REx Core Kit from Invitro- out EDTA and centrifuged again at 700 g for 8 min. The gen. Briefly, Flp-In T-REx HEK293 cells were co-transfected PBS-washed cells were solubilized in a lysis buffer containing 25 M Tris, 50 mM Hepes (pH 7.4), 137 mM NaCl, 1% CHAPS, with the inducible expression vector pcDNA5/FRT/TO con- m 0.5% soy bean phosphatidylcholine, 2.5 mM DTT, and a prote- taining the mutant cDNAs and the pOG44 vector encoding the phosphate precip- ase inhibitor mix (1 mM benzamidine, 2 g/ml leupeptin, 2 Flp recombinase in 1:5 ratios using the Ca itation method. The transfected cells were washed with PBS g/ml pepstatin A, 2 g/ml aprotinin, and 0.5 mM PMSF). This M NaCl, 8 mM Na HPO , 1.5 mM KH PO , and 2.7 mM mixture was incubated on ice for 1 h. Cell lysate was obtained by (137 m 2 4 2 4 KCl, pH 7.4) 24 h after transfection followed by a change into centrifuging twice at 16,000 g in a microcentrifuge at 4 °C for fresh medium for 24 h. The cells were then washed again with 30 min to remove unsolubilized materials. The RyR2 WT and PBS, harvested, and plated onto new dishes. After the cells had mutant proteins were subjected to SDS-PAGE (6% gel) (34) and attached (4 h), the growth medium was replaced with a selec- transferred onto nitrocellulose membranes at 90 V for 1.5 h at g/ml hygromycin (Invitrogen). tion medium containing 200 4 °C in the presence of 0.01% SDS (35). The nitrocellulose mem- The selection medium was changed every 3–4 days until the branes containing the transferred proteins were blocked for 30 desired number of cells was grown. The hygromycin-resistant min with PBS containing 0.5% Tween 20 and 5% (w/v) nonfat cells were pooled, aliquoted (1 ml), and stored at80 °C. These dried skimmed milk powder. The blocked membrane was incu- positive cells are believed to be isogenic, because the integration bated with the anti-RyR antibody (34C) (Thermo Scientific, of RyR2 cDNA is mediated by the Flp recombinase at a single MA3-925, lot number PG200294) (1:1000 dilution) and then FRT (flippase recognition target) site. incubated with the secondary anti-(mouse IgG (heavy and NOVEMBER 18, 2016 • VOLUME 291 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 24535 2 Enhanced RyR2 Ca Activation and Cardiac Arrhythmias light)) antibodies conjugated to horseradish peroxidase NaCl, and 10 mM HEPES, pH 7.4, with KOH). The cells were (1:20,000 dilution). After washing for 5 min three times, the then switched to a complete ICM (incomplete ICM plus 2 mM bound antibodies were detected using an enhanced chemilumi- ATP, 2 mM MgCl , 0.05 mM EGTA, and 100 nM free Ca ,pH nescence kit from Pierce. The intensity of each band was deter- 7.4, with KOH) for 5–6 min to remove saponin. The permea- mined from its intensity profile obtained by ImageQuant LAS bilized cells were then perfused with various concentrations of 4000 (GE Healthcare Life Sciences) and analyzed by using the Ca (0.1, 0.2, 0.4, 1, and 10 M) followed by tetracaine (1 mM) ImageJ software (36). for estimating the store capacity and caffeine (10 mM) for esti- [ H]Ryanodine Binding—HEK293 cells were grown to 95% mating the minimum store level by depleting the ER Ca confluence in a 75-cm flask, dissociated with PBS, and plated stores. Images were captured with Compix SimplePCI 6 soft- in 100-mm tissue culture dishes at10% confluence for 18–20 ware every 2 s using an inverted microscope (Nikon TE2000-S) h before transfection with RyR2 WT and central domain equipped with an S-Fluor 20/0.75 objective. The filters used mutant cDNAs. After transfection for 24 h, the cells were har- for D1ER imaging were 436 20 nm for CFP and ex ex vested and lysed in lysis buffer containing 25 mM Tris, 50 mM 500 20 nm for YFP, and 465 30 nm for CFP and em em HEPES, pH 7.4, 137 mM NaCl, 1% CHAPS, 0.5% egg phosphati- 535 30 nm for YFP with a dichroic mirror (500 nm). The dylcholine, 2.5 mM DTT, and a protease inhibitor mix (1 mM amount of FRET was determined from the ratio of the light benzamidine, 2 g/ml leupeptin, 2 g/ml pepstatin A, 2 g/ml emission at 535 and 465 nm. 2 2 aprotinin, and 0.5 mM PMSF) on ice for 60 min. Cell lysate was Single Cell Cytosolic Ca Imaging—Cytosolic Ca levels in obtained after removing the unsolubilized materials by centrif- stable, inducible HEK293 cells expressing RyR2 WT or central ugation twice in a microcentrifuge at 4 °C for 30 min each. Equi- domain mutants were monitored using single-cell Ca imag- librium [ H]ryanodine binding to cell lysates was performed as ing and the fluorescent Ca indicator dye Fura-2 AM as described previously (9) with some modifications. [ H]Ryano- described previously (27, 28). Briefly, cells grown on glass cov- lof dine binding was carried out in a total volume of 300 erslips for 8–18 h after induction (as indicated) by 1 g/ml binding solution containing 30 l of cell lysate, 150 mM KCl, 25 tetracycline (Sigma) were loaded with 5 M Fura-2 AM in KRH mM Tris, 50 mM Hepes (pH 7.4), and 5 nM [ H]ryanodine and buffer plus 0.02% Pluronic F-127 and 0.1 mg/ml BSA for 20 CaCl to set free [Ca ] from pCa 9.89 to pCa 4 and a protease min at room temperature (23 °C). The coverslips were then inhibitor mix at 37 °C for 20 min. The Ca /EGTA ratio was mounted in a perfusion chamber (Warner Instruments) on an calculated using the computer program of Fabiato and Fabiato inverted microscope (Nikon TE2000-S). The cells were per- (37). The binding mix was diluted with 5 ml of ice-cold washing fused continuously with KRH buffer containing increasing buffer containing 25 mM Tris, pH 8.0, and 250 mM KCl and extracellular Ca concentrations (0, 1.0, and 2.0 mM). The cells immediately filtered through Whatman GF/B filters presoaked 2 were then perfused continuously with 2 mM Ca in KRH with 1% polyethylenimine. The filters were washed three times, buffer containing increasing concentrations of carvedilol or and the radioactivity associated with the filters was determined (R)-carvedilol (0, 1, 3, 10, and 30 M). Caffeine (10 mM) was by liquid scintillation counting. Nonspecific binding was deter- applied at the end of each experiment to confirm the expression mined by measuring [ H]ryanodine binding in the presence of of active RyR2 channels. Time-lapse images (0.25 frame/s) were 50 M unlabeled ryanodine. All binding assays were done in captured and analyzed with Compix SimplePCI 6 software. Flu- duplicate. orescence intensities were measured from regions of interest 2 2 Single Cell Luminal Ca Imaging—Luminal Ca levels in centered on individual cells. Only cells that responded to caf- HEK293 cells expressing RyR2 WT or central domain mutants feine were analyzed. The filters used for Fura-2 imaging were were measured using single-cell Ca imaging and the FRET 340 26 nm and 387 11 nm, and 510 84 nm ex em (fluorescence resonance energy transfer)-based ER luminal with a dichroic mirror (410 nm). Ca -sensitive Cameleon protein D1ER as described previ- Statistical Analysis—All values shown are mean S.E. unless ously (24, 25). The cells were grown to 95% confluence in a indicated otherwise. To test for differences between groups, we 75-cm flask, passaged with PBS, and plated in 100-mm-diam- used Student’s t test (two-tailed) or one-way analysis of vari- eter tissue culture dishes at 10% confluence 18–20 h before ance with post hoc test. A p value 0.05 was considered to be transfection with D1ER cDNA using the Ca phosphate pre- statistically significant. cipitation method. After transfection for 24 h, the growth medium was then changed to an induction medium containing Author Contributions—Z. X., W. G., B. S., R. W., P. P. J., T. G. B., 1 g/ml tetracycline. In intact cell studies, after induction for and S. R. W. C. designed research; Z. X., W. G., B. S., D. J. H., J. W., 22 h, the cells were perfused continuously with KRH buffer Y. L., Y. W., and R. W. performed research; Z. X., W. G., B. S., (125 mM NaCl, 5 mM KCl, 6 mM glucose, 1.2 mM MgCl , and 25 D. J. H., J. W., Y. L., Y. W., and S. R. W. C. analyzed data; and Z. X., mM Hepes, pH 7.4) containing various concentrations of CaCl W. G., B. S., R. W., P. P. J., T. G. B., and S. R. W. C. wrote the paper. 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Journal
Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology
Published: Nov 1, 2016
Keywords: calcium; calcium channel; calcium imaging; calcium intracellular release; endoplasmic reticulum (ER); ryanodine receptor; sarcoplasmic reticulum (SR); ventricular tachyarrhythmia; atrial fibrillation; cardiac ryanodine receptor; disease mutations; Ca2+-dependent activation; spontaneous Ca2+ release; carvedilol; carvedilol enantiomer