Published online before print August 14, 2003, doi: 10.1161/01.RES.0000091335.07574.86
© 2003 American Heart Association, Inc.
Molecular Medicine |
Ryanodine Receptor Mutations Associated With Stress-Induced Ventricular Tachycardia Mediate Increased Calcium Release in Stimulated Cardiomyocytes
From the Wales Heart Research Institute, Department of Cardiology, University of Wales College of Medicine, Cardiff, UK.
Correspondence to Dr Christopher H. George, Wales Heart Research Institute, University of Wales College of Medicine, Heath Park, Cardiff, UK CF14 4XN. E-mail georgech{at}cf.ac.uk
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Abstract |
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Ca2+ release from the sarcoplasmic reticulum mediated by the cardiac ryanodine receptor (RyR2) is a fundamental event in cardiac muscle contraction. RyR2 mutations suggested to cause defective Ca2+ channel function have recently been identified in catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia (ARVD) affected individuals. We report expression of three CPVT-linked human RyR2 (hRyR2) mutations (S2246L, N4104K, and R4497C) in HL-1 cardiomyocytes displaying correct targeting to the endoplasmic reticulum. N4104K also localized to the Golgi apparatus. Phenotypic characteristics including intracellular Ca2+ handling, proliferation, viability, RyR2:FKBP12.6 interaction, and beat rate in resting HL-1 cells expressing mutant hRyR2 were indistinguishable from wild-type (WT) hRyR2. However, Ca2+ release was augmented in cells expressing mutant hRyR2 after RyR activation (caffeine and 4-chloro-m-cresol) or ß-adrenergic stimulation (isoproterenol). RyR2:FKBP12.6 interaction remained intact after caffeine or 4-CMC activation, but was dramatically disrupted by isoproterenol or forskolin, an activator of adenylate cyclase. Isoproterenol and forskolin elevated cyclic-AMP to similar magnitudes in all cells and were associated with equivalent hyperphosphorylation of mutant and WT hRyR2. CPVT-linked mutations in hRyR2 did not alter resting cardiomyocyte phenotype but mediated augmented Ca2+ release on RyR-agonist or ß-AR stimulation. Furthermore, equivalent interaction between mutant and WT hRyR2 and FKBP12.6 was demonstrated.
Key Words: ryanodine receptors • mutations • ventricular tachycardia • stress • cardiomyocytes
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Introduction |
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Normal cardiac E-C coupling requires the precise coordination of ionic currents and molecular defects in ion channels ("channelopathies") are causative of cardiac pathophysiologies including long-QT syndrome, ventricular fibrillation (VF), and ventricular tachycardia (VT).1–3 Ca2+ is the most important ion involved in the cardiac muscle contraction and is rapidly mobilized from intracellular stores into the sarcoplasm to trigger the activation of contractile elements. Termination of these events requires the removal of sarcoplasmic Ca2+ via sequestration into the sarcoplasmic/endoplasmic reticulum (SR/ER) through Ca2+-ATPase (SERCA) activity and extrusion of Ca2+ via the Na+-Ca2+ exchanger (NCX).1 Duration and amplitude of Ca2+ efflux from the SR is regulated by cardiac ryanodine receptors (RyR2), large homotetrameric complexes that are organized into functional Ca2+ release units via interaction with a multitude of accessory proteins.4 Recently, defective regulation of RyR2, which leads to abnormal cellular Ca2+ handling, has been implicated in heart failure, hypertrophy, and arrhythmia.5–8 To date, 21 point mutations in hRyR2 have been reported in individuals affected by catecholaminergic polymorphic VT (CPVT) and arrhythmogenic right ventricular dysplasia (ARVD), and defective RyR Ca2+ channel function is implicated in the disease phenotype.9–12 Interestingly, these hRyR2 mutations cluster in highly conserved regions corresponding to mutation-containing domains of the skeletal muscle isoform (RyR1) associated with malignant hyperthermia (MH).13 This positional clustering of mutation loci together with the requirement for a "trigger" in both CPVT (physical/emotional stress) and MH (inhalational anesthetic) raises the possibility that CPVT/ARVD is a cardiac equivalent of MH, and indeed, certain MH-linked RyR1 mutations (eg, R614C) are associated with a stress-induced phenotype. Chronic administration of ß-blockers or implantable cardiac defibrillators are currently used to control CPVT although these do not represent optimal therapeutic strategies.8 Thus, understanding the molecular events underlying the disease phenotype is critical for the development of novel therapeutic approaches.
We determined the functional impact of CPVT-linked mutations in the human recombinant RyR2 (hRyR2) on intracellular Ca2+ handling in HL-1 cardiomyocytes. RyR2 mutations (S2246L, N4104K, and R4497C) were expressed in HL-1 cells and we demonstrate that the resting phenotype (resting cytoplasmic [Ca2+] ([Ca2+]c), ER Ca2+ load, beat rate, cell size, and proliferation) were indistinguishable from cells expressing nonmutant WT hRyR2. The interaction between RyR2 and FKBP12.6 was intact in resting cells expressing WT or mutant hRyR2. However, activation of Ca2+ release using mechanistically different RyR agonists [caffeine and 4-chloro-m-cresol (4-CMC)] or ß-adrenergic (ß-AR) stimulation (isoproterenol) was associated with significantly augmented Ca2+ release through mutant hRyR2. The RyR2:FKBP12.6 complex remained intact after caffeine or 4-CMC addition but was severely disrupted after ß-AR stimulation, which was associated with equivalent hyperphosphorylation of WT and mutant hRyR2. Importantly, we determined equivalent dissociation of FKBP12.6 from both WT and mutant hRyR2 after ß-AR stimulation. Our data provide direct evidence that mutations in hRyR2 associated with CPVT mediates augmented Ca2+ release in stimulated HL-1 cardiomyocytes, which may underlie the disease phenotype.
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Materials and Methods |
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Construction of eGFP-Tagged WT and Mutant hRyR2
A fragment of peGFP-C3 (Clontech) (35 to 1330 bp) was amplified by PCR, digested withMluI/SpeI, and inserted intoMluI/SpeI digested pcDNA3/hRyR2.14 This strategy placed eGFP at the N-terminus of hRyR2 separated by a four amino acid spacer (Thr-Ser-Gly-Ser). To generate the S2246L point mutation, anEcoRV cassette from hRyR2 (4015 to 7013 bp) was transferred into pSL1180 (Amersham) and the TCG (Ser) codon was replaced with TTG (Leu) using oligonucleotide directed mutagenesis (Quikchange;Stratagene). A similar strategy was used to generate N4104K [AAC (Asn) to AAG (Lys)] and R4497C [CGC (Arg) toTGC (Cys)] using anNheI/XhoI (10015 to 15335 bp) fragment of hRyR2. Full-length hRyR2 mutants were generated by the ligation of mutated cassettes into pcDNA3/hRyR2 usingEcoRV (S2246L) orFseI/XhoI (N4104K, R4497C) strategies. All constructs were verified by automated sequencing (ABI 3700,Applied Biosystems).
Analysis of RyR2 and FKBP Expression in HL-1 Cardiomyocytes
HL-1 cardiomyocytes were cultured on a gelatin (0.02% [wt/vol])/fibronectin (10 µg/mL) matrix and were maintained in Claycomb media (JRH Biosciences) supplemented with fetal calf serum (10% [vol/vol]), glutamine (2 mmol/L), norepinephrine (0.1 mmol/L), penicillin (100 u/mL), and streptomycin (100 µg/mL).15 Cells were transiently transfected with plasmid DNA using Lipofectamine2000 (Invitrogen) as described previously.14
The expression levels of endogenous and recombinant RyR2 were determined after immunoblot analysis of cellular fractions using anti-RyR2 (pAb129), and recombinant GFP-tagged hRyR2 was detected using anti-GFP (Clontech) as described.14 Antiphosphoserine (clone 16B4,Calbiochem) was used to determine the phosphorylation status of RyR2. FKBP12 and 12.6 were detected using rabbit polyclonal antiserum, which cross-reacts with both FKBP12 and 12.6 isoforms.16 Densitometric analysis was performed using a GS700 scanner and Quantity One software (Biorad) as described in the expanded Materials and Methods section in the online data supplement available at http://www.circresaha.org. For direct analysis of FKBP12.6:hRyR2 interaction, coimmunoprecipitation assays were performed on HL-1 cell lysates using pAb129 and anti-FKBP to immunoprecipitate and immunoblot, respectively (see online data supplement).
Immunofluorescent localization of endogenous RyR2 and FKBP was performed using antibodies pAb129 and N-19 (Santa-Cruz Biotechnology), respectively, followed by detection using donkey anti-rabbit Alexa488 and donkey anti-goat Alexa546 conjugated antibodies (Molecular Probes), respectively.14 Recombinant GFP-tagged WT and mutant hRyR2 were directly localized using eGFP fluorescence (Ex 488 nm, Em 511 nm) and in these cells, FKBP was detected using an Alexa546-linked secondary antibody as above.
Phenotypic Characterization of HL-1 Cardiomyocytes
The Ca2+-dependent fluorescence of calcium orange (10 µmol/L) in HL-1 cardiomyocytes was determined using a confocal microscope (SP2,Leica).16 ER Ca2+ load was estimated from peak Ca2+ release after the addition of thapsigargin (5 µmol/L) to cells.14 Caffeine (0.01 to 40 mmol/L), 4-CMC (0.01 µmol/L to 10 mmol/L), and isoproterenol (0.1 nmol/L to 10 µmol/L) were used to mobilize intracellular Ca2+ in cells maintained in KRH buffer containing 1.3 mmol/L CaCl2.14 RyR2 and ß-AR were inhibited by the addition of ryanodine (1 mmol/L) and nadolol (100 µmol/L), respectively. Data were acquired in bidirectional scan mode (130 ms/frame) at 512x512 pixel resolution.
Cell surface area and beating rate (see online data supplement) were measured using confocal microscopy (Leica). Cell viability and proliferation were determined as described previously.16 In phenotypic analysis of transfected cells, data were collected from "GFP-positive" cells.
Measurement of Intracellular [cAMP]
Intracellular [cAMP] was measured using an enzyme-linked immunoassay (EIA) (Amersham) after stimulation of cells with caffeine (10 mmol/L), 4-CMC (1 mmol/L), isoproterenol (1 µmol/L), or forskolin (5 µmol/L). Reactions were terminated by the addition of dodecyltrimethylammonium bromide (2.5% [wt/vol]) and intracellular [cAMP] was calculated using a calibration curve constructed from known [cAMP] (12.5 to 3200 fmol).
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Results |
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Immunoblot analysis of microsomal fractions demonstrates that transfected HL-1 cardiomyocytes express significantly higher levels of full-length RyR2 (
560 kDa) than untransfected cells (Figure 1Aa), and that expression of recombinant WT and mutant hRyR2 is essentially equivalent (hRyR2 242±24%; S2246L 221±29%; N4104K 259±36%; R4497C 270±37%; HL-1 100%). RyR2 is the only endogenously expressed RyR isoform in HL-1 cells, as RyR1 and RyR3 were undetectable using isoform-specific antibodies (data not shown). An anti-GFP antibody enabled monitoring of recombinant WT and mutant hRyR2 expression distinct from endogenous RyR2, confirming high-level, equivalent expression of recombinant protein (Figure 1Ab). Immunoblots of total cell lysates using antiserum recognizing both FKBP12 and FKBP12.6,16 revealed FKBP12 expression of 4-fold higher level than FKBP12.6, and that both their cellular levels remained unchanged after expression of recombinant hRyR2 (Figure 1Ac). However, only FKBP12.6 was associated with microsomal fractions obtained from cells expressing WT and mutant hRyR2 (Figure 1Ad) and this association was completely abolished after rapamycin treatment (5 µmol/L) (Figure 1Ae), consistent with specific protein:protein interaction occurring between FKBP12.6 and WT and mutant RyR2. The elevation of hRyR2 in transfected cells (Figure 1Aa) accompanied the parallel increase in FKBP12.6 association with microsomes, a phenomenon observed with cells expressing recombinant WT or mutant hRyR2 (Figure 1Ad). In contrast, we did not detect FKBP12 in microsomal fractions, although it was abundant in postmicrosomal supernatants (Figure 1Af). Coimmunoprecipitation studies using pAb129 confirmed that a specific, comparable interaction occurred between FKBP12.6 and hRyR2 (0.88±0.18), S2246L (1.06±0.14), N4104K (0.91±0.16), and R4497C (0.87±0.12) when compared with FKBP12.6 association with endogenous RyR2 in resting HL-1 cardiomyocytes (assigned 1.0) (Figure 1Ag), which was ablated after incubation of immune complexes with rapamycin (Figure 1Ah). The coimmunoprecipitation studies demonstrate that interaction between FKBP12.6 and CPVT-linked hRyR2 mutations is indistinguishable from that determined with WT hRyR2 in resting HL-1 cardiomyocytes and agrees with data obtained using the microsomal association assays (Figures 1Ad and 1Ae).
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Endogenous RyR2 or recombinant hRyR2, S2246L, N4104K, and R4497C (Figure 1B) were all correctly targeted to the ER as determined by the localization of calreticulin, an ER resident protein (see online data supplement). However, N4104K was also localized to a perinuclear concentration (Figure 1B), which was identified as the Golgi apparatus using an antibody to Golgi 58K protein (see online data supplement). Image analysis demonstrated a significant interaction (Merge) between membrane localized WT/mutant hRyR2 (green) and FKBP (red) (Figures 1B and 1C). However, much FKBP, presumably FKBP12 (Figures 1Ad and 1Af), remained in the cytoplasm. Taken together, these data demonstrate that in resting HL-1 cells, a specific interaction occurs between FKBP12.6 and mutants S2246L, N4104K, and R4497C that is indistinguishable from that which occurs with WT hRyR2. The validity of our confocal imaging approach to accurately determine the in situ association of the RyR2:FKP12.6 complex was confirmed by the separation of fluorescent signals from RyR2 and FKBP after treatment of the cells with rapamycin (Figures 1B and 1C). These findings are entirely complementary with data obtained using immunoblotting of microsomal fractions or coimmunoprecipitation assays (Figure 1A).
Cell size, viability, and proliferation remained unaltered after expression of recombinant WT or mutant RyR2s in resting HL-1 cells, and we determined an equivalent increase in ER Ca2+ storage capacity in cells expressing WT or mutant RyR2s when compared with untransfected HL-1 cells (Table). However, mutant RyR2s exhibited enhanced sensitivity to caffeine activation (Figure 2A), and we measured a significant increase in the magnitude and duration of Ca2+ release after addition of caffeine (40 mmol/L) to cells expressing S2246L, N4104K, and R4497C when compared with untransfected cells or those expressing WT hRyR2 (Figures 2B through 2D). The caffeine-induced Ca2+ release in cells expressing either WT hRyR2 or mutant hRyR2 was increased when compared with untransfected HL-1 cells due to the augmentation of the ER Ca2+ store (Table).14 Ryanodine (1 mmol/L) completely abolished caffeine-induced Ca2+ release demonstrating that channels assembled from S2246L, N4104K or R4497C were sensitive to ryanodine modification (Figure 2B, lower traces). Importantly, the augmented Ca2+ release determined in cells expressing mutant RyR2s in response to caffeine stimulation was not underscored by dissociation of the RyR:FKBP12.6 complex, which remained intact (Figures 2E and 2F).
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Mutant RyR2s also displayed an increased sensitivity to activation by 4-CMC (Figure 3A), and 4-CMC caused a sustained elevation in [Ca2+]c, the magnitude of which was significantly augmented in cells expressing mutant hRyR2 (Figures 3B and 3C). The rate of Ca2+ mobilization after 4-CMC addition was similar in all cells (Figure 3D) and Ca2+ release could be inhibited by preincubation with ryanodine (Figure 3B, lower traces). RyR2:FKBP12.6 association remained intact after stimulation of cells with 4-CMC (Figures 3E and 3F). The Golgi localization of N4104K persisted after caffeine and 4-CMC stimulation of cells (Figs. 2E and 3
E, respectively).
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F.U. s-1) relative to that measured with untransfected HL-1 cells) (D) after 4-CMC addition (1 mmol/L) were determined. Data are plotted as mean±SEM (n=5) and **P<0.01 when compared with control HL-1 cells and cells expressing mutant hRyR2 and *P<0.01 when compared with control cells and those expressing WT hRyR2. E, Colocalization (yellow) between endogenous RyR2 (HL-1) or recombinant hRyR2, S2246L, N4104K, and R4497C (green) and FKBP (red) after 4-CMC addition. Bar=15 µm. F, Association of FKBP12.6 with microsomes (200 µg) obtained from untreated cells (-, open bars) or from cells after 4-CMC addition (1 mmol/L) (+, filled bars). Densitometric values were obtained as optical density (OD; signal intensity per mm2) and are plotted as mean±SEM from three separate experiments.ß-Adrenergic status is implicated in the pathogenesis of the CPVT phenotype,8 and we investigated the effect of ß-AR activation of cells expressing mutant hRyR2. Mutant RyR2s exhibited a left-shifted dose response after addition of isoproterenol, a ß-AR agonist that induces VT in individuals carrying hRyR2 mutations10 (Figure 4A). The maximal Ca2+ release elicited by isoproterenol (1 µmol/L) was equivalently augmented in cells expressing WT or mutant RyR2s (Figure 4C), but we determined a significant increase in the duration of the Ca2+ transient in cells expressing S2246L, N4104K, or R4497C (Figure 4D). Ryanodine (1 mmol/L) and nadolol (100 µmol/L), a ß-AR antagonist,10 prevented isoproterenol induced Ca2+ release (Figure 4B, middle and lower traces, respectively), suggesting that ß-AR stimulation triggered the activation of hRyR2 Ca2+ release. Furthermore, the finding that isoproterenol-mediated activation of the ß-AR cascade markedly augmented the duration of elevated [Ca2+]c release in cells expressing mutant RyR2s (>60s) (Figures 4B and 4D) is in concert with the characteristic Ca2+ overload associated with the CPVT disease phenotype.10,12 In situ localization and microsomal association assays demonstrated that isoproterenol addition disrupted RyR2:FKBP12.6 interaction in HL-1 cardiomyocytes, in agreement with the finding that ß-AR stimulation causes the dissociation of the RyR2:FKBP12.6 complex,5 but importantly, we determined equivalent dissociation between FKBP12.6 and WT and mutant RyR2s (Figures 4E and 4F).
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We used forskolin, a potent activator of adenylate cyclase, to determine the effect of direct stimulation of downstream effectors of the ß-adrenergic signaling cascade on RyR2:FKBP12.6 interaction. In concert with our data from receptor-mediated activation of the ß-adrenergic cascade (Figure 4), forskolin dramatically reduced RyR2:FKBP12.6 interaction but importantly, the dissociation was equivalent in WT and mutant RyR2 expressing cells (Figures 5A and 5B). The dramatic disruption of the RyR:FKBP12.6 complex induced by isoproterenol and forskolin persisted after ryanodine-mediated inhibition of RyR indicating its ablation of RyR2:FKBP12.6 interaction was independent of the open/closed status of RyR (data not shown).
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PKA-mediated hyperphosphorylation of RyR2 has been detected in failing hearts,5,17 although the functional consequences of this remain highly controversial.18,19 We determined whether (1) ß-AR stimulation resulted in differential phosphorylation of mutant and WT hRyR2 and (2) intracellular elevations in [cAMP] after ß-AR activation were equivalent in all cells. Microsomal proteins (>80 kDa) obtained from HL-1 cells expressing WT hRyR2 exhibited a broad ladder of bands followingSDS-PAGE separation (Figure 6A, lane 1) and RyR2 was identified using pAb129 (Figure 6A, lane 2). An anti-phosphoserine antibody (16B4) confirmed that RyR2 was phosphorylated in nonstimulated cells (Figure 6A, lane 3). Endogenous RyR2, recombinant WT, and mutant RyR2s were phosphorylated to equivalent levels in untreated cells (endogenous RyR2, 1.0; hRyR2, 0.88±0.11; S2246L, 0.81±0.09; N4104K, 0.75±0.12; R4497C, 0.97±0.14) (Figure 6Ba), and the phosphorylation status was unaltered after stimulation of cells with caffeine or 4-CMC (Figure 6Bb and 6Bc, respectively). The increased phosphorylation signals from transfected cells (Figure 6B) proportionally reflect the increased expression levels of both WT and mutant RyR2s (Figure 1A). ß-AR stimulation using isoproterenol significantly but equivalently increased the phosphorylation of WT hRyR2 (1.91±0.23) and mutants S2246L (2.28±0.31), N4104K (2.32±0.29), and R4497C (1.98±0.23) (Figure 6Bd) relative to the phosphorylation status of WT and mutant hRyR2 determined in untreated cells (Figure 6Ba and data above). Similarly, forskolin addition also resulted in hyperphosphorylation of mutant hRyR2 (S2246L, 3.11±0.31; N4104K, 3.65±0.4; R4497C, 3.20±0.35), which was indistinguishable from that of WT (2.89±0.39) (Figure 6Be). Staurosporine, a broad-spectrum inhibitor of protein kinases, completely inhibited RyR2 phosphorylation (Figure 6Bf). Thus, ß-adrenergic stimulation resulted in hyperphosphorylation of hRyR2, but our data clearly demonstrate that the extent of phosphorylation of CPVT-linked hRyR2 mutants S2246L, N4104K and R4497C was indistinguishable from that of WT hRyR2 in HL-1 cardiomyocytes.
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Isoproterenol and forskolin treatment was associated with significant elevations in intracellular [cAMP], which was similar in untransfected HL-1 cells or those expressing WT or mutant RyR2s (Figure 6C). However, the extent of equivalent hyperphosphorylation of WT and mutant RyR2s after stimulation of the ß-AR cascade was proportional to the increased cellular levels of cAMP (forskolin>isoproterenol) (Figure 6C). The smaller caffeine-induced increase in [cAMP] (Figure 6C) did not result in hRyR2 hyperphosphorylation (Figure 6Bb) or dissociation of the RyR2:FKBP12.6 complex (Figures 2E and 2F), indicating that in our experiments, increases in cellular [cAMP] greater than 5-fold over basal levels are associated with disruption of the RyR2:FKBP12.6 complex.
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Discussion |
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We expressed recombinant GFP-tagged hRyR2 channels (
560 kDa), which correctly localized to the ER and formed functional Ca2+ release channels in HL-1 cardiomyocytes. Resting cell phenotype was unaltered after expression of hRyR2 containing CPVT-linked mutations (S2246L, N4104K, and R4497C), and thus our data obtained in cardiomyocytes strongly suggest that there is normal and appropriate regulation of CPVT-linked mutant hRyR2 channels in nonstimulated cells. In our experiments, we did not observe the increased basal "activity" previously associated with expression of a CPVT-linked RyR2 mutation (mouse R4496C) in HEK cells.20 However, it is likely that appropriate regulation of RyR2 requires the precise interaction of a multitude of accessory proteins, some of which may be absent in the RyR-deficient cells used by Jiang and colleagues.20 Intracellular Ca2+ release was significantly augmented in cells expressing mutant hRyR2 after addition of RyR agonists (caffeine and 4-CMC) or ß-AR stimulation (isoproterenol), providing strong evidence that abnormal Ca2+ release during periods of emotional or physical stress may underlie the delayed afterdepolarizations and Ca2+ overload phenomena associated with CPVT.10 Defective regulation of RyR2 via PKA mediated hyperphosphorylation and dissociation of FKBP12.6 is implicated in heart failure.5,21 Recently, the selective dissociation of FKBP12.6 from ARVD-linked RyR2 mutants has also been suggested,22 although these data were not corroborated using mutations in full-length RyR2 sequence and intriguingly, a robust interaction was demonstrated between FKBP12.6 and RyR1, an interaction that does not occur in vivo.6 Importantly, although our data indicate that ß-AR–induced RyR2 hyperphosphorylation is associated with disruption of the FKBP12.6:RyR2 complex, in situ localization studies and direct measurement of FKBP12.6:hRyR2 protein:protein interaction revealed that the extent of dissociation was equivalent in cells expressing WT and mutant hRyR2 (Figure 6). Thus, it is unlikely that differential phosphorylation of mutant hRyR2 or selective dissociation of FKBP12.6 from CPVT-linked hRyR2 mutants is causative of the disease phenotype. However, our results do not exclude the possibility that in stimulated cells, abnormal Ca2+ release through mutant hRyR2 is underscored by differential sensitivity to equivalent phosphorylation. Also, although our results strongly suggest that dissociation of the RyR2:FKBP12.6 complex is not causative of CPVT phenotype, it has recently been reported that genetic ablation of FKBP12.6 is associated with exercise-induced fatal arrhythmia in mice expressing wild-type RyR2.23 Hyperphosphorylation of RyR2 and RyR2:FKBP12.6 dissociation was proportional to the elevation in [cAMP]. However, elevated [cAMP] also modulates phospholamban and Na+ channels,18,24 which would directly alter intracellular Ca2+ homeostasis. Furthermore, although our results strongly support a causative role for aberrant regulation of Ca2+ release via RyR2 in CPVT, a CPVT-linked mutation in calsequestrin has been identified25 and defective ß-AR signaling via RyR2-independent mechanisms is implicated in other tachycardias.26 Therefore, although we found elevated ER Ca2+ release on ß-AR stimulation occurred specifically via mutant RyR2 (ie, was ryanodine sensitive) (Figure 4), one must consider the role of accessory cellular factors in coordinating intracellular Ca2+ release.
RyR agonists (caffeine and 4-CMC) significantly augmented the magnitude of Ca2+ release through mutant hRyR2, yet the RyR2:FKBP12.6 complex remained intact (Figures 2 and 3
, respectively). This finding strongly indicates that there are defects in FKBP12.6-independent regulation of hRyR2 mutants in HL-1 cardiomyocytes that leads to increased Ca2+ release on cell stimulation. The precise nature of these cellular regulatory components remains to be defined. Currently, chronic administration of ß-blockers are used in the management of CPVT.8,10 However, due to the many intracellular targets of the ß-AR signaling cascade, our results indicate that the precise in situ regulation of mutant RyR2 channel function represents an attractive and feasible therapeutic strategy.17 The precise molecular mechanisms by which mutations in hRyR2 mediate augmented Ca2+ release in stimulated cells are currently unknown, and clearly a more detailed understanding of structure-function aspects of hRyR2 will be necessary to develop novel therapeutic strategies in the management of CPVT/ARVD.
Cardiac arrhythmia susceptibility genes so far identified predominantly encode ion channels2,3 and our study suggests that mutations in RyR2 are bona fide additions to the list of channelopathies. An intriguing aspect of the hRyR2 mutations is that they are presented in a cardiac specific context but it is unlikely that these mutations are imprinted in the heart. RyR2 is expressed in other tissues (eg, brain and kidney) and CPVT-linked hRyR2 mutations may in the future be linked with noncardiac pathologies as is the case with KVLQT1 mutations associated with long-QT syndrome, which are also linked with epilepsy and congenital neural deafness.3 It is also likely that CPVT-linked hRyR2 mutations may be associated with other cardiac abnormalities, as is observed with SCN5A mutants that have been identified in several arrhythmogenic phenotypes including long-QT syndrome and VF.3
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Acknowledgments |
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This work was supported by a British Heart Foundation Research Fellowship (FS2000020) to C.H.G. and British Heart Foundation Grants (PG98169 and PG99087) to F.A.L. We thank W. Claycomb for HL-1 cardiomyocytes, J. Mackrill for anti-FKBP12/12.6 antibody, and N.L. Thomas for helpful discussions.
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Footnotes |
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Original received April 10, 2003; revision received August 4, 2003; accepted August 6, 2003.
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