Published online before print November 10, 2008, doi: 10.1161/CIRCULATIONAHA.108.788067
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(Circulation. 2008;118:2225-2234.)
© 2008 American Heart Association, Inc.
Arrhythmia/Electrophysiology |
Proarrhythmic Defects in Timothy Syndrome Require Calmodulin Kinase II
From Vanderbilt University, Nashville, Tenn (W.H.T.), and University of Iowa, Iowa City (W.H.T., B.C., T.J.H., O.M.K., A.P., L.-S.S., P.J.M., M.E.A.).
Correspondence to Mark E. Anderson, University of Iowa, Department of Internal Medicine, 285 Newton Rd, Iowa City, IA 52242. E-mail mark-e-anderson{at}uiowa.edu
Received April 23, 2008; accepted September 8, 2008.
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Abstract |
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Background— Timothy syndrome (TS) is a disease of excessive cellular Ca2+ entry and life-threatening arrhythmias caused by a mutation in the primary cardiac L-type Ca2+ channel (CaV1.2). The TS mutation causes loss of normal voltage-dependent inactivation of CaV1.2 current (ICa). During cellular Ca2+ overload, the calmodulin-dependent protein kinase II (CaMKII) causes arrhythmias. We hypothesized that CaMKII is a part of the proarrhythmic mechanism in TS.
Methods and Results— We developed an adult rat ventricular myocyte model of TS (G406R) by lentivirus-mediated transfer of wild-type and TS CaV1.2. The exogenous CaV1.2 contained a mutation (T1066Y) conferring dihydropyridine resistance, so we could silence endogenous CaV1.2 with nifedipine and maintain peak ICa at control levels in infected cells. TS CaV1.2–infected ventricular myocytes exhibited the signature voltage-dependent inactivation loss under Ca2+ buffering conditions, not permissive for CaMKII activation. In physiological Ca2+ solutions, TS CaV1.2–expressing ventricular myocytes exhibited increased CaMKII activity and a proarrhythmic phenotype that included action potential prolongation, increased ICa facilitation, and afterdepolarizations. Intracellular dialysis of a CaMKII inhibitory peptide, but not a control peptide, reversed increases in ICa facilitation, normalized the action potential, and prevented afterdepolarizations. We developed a revised mathematical model that accounts for CaMKII-dependent and CaMKII-independent effects of the TS mutation.
Conclusion— In TS, the loss of voltage-dependent inactivation is an upstream initiating event for arrhythmia phenotypes that are ultimately dependent on CaMKII activation.
Key Words: action potentials • calcium • ion channels 
long-QT syndrome • myocytes
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Introduction |
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Timothy syndrome (TS) is an autosomal-dominant genetic disease of the primary voltage-gated cardiac Ca2+ channel (CaV1.2) consisting of a missense mutation in the pore-forming
1c subunit protein.1 TS is associated with 3 individual mutations: G406R on exon 8a,2 G406R on exon 8,1 and G402S.1 The G406R mutation on exon 8 used for our model of TS is believed to be the most severe form of TS.1 TS patients have an average life expectancy of only 2.5 years because of severe cardiac disease. TS also is known as long-QT syndrome 8, and the prolonged QT intervals in TS patients are thought to cause cardiac arrhythmias and sudden death. TS disease phenotypes are apparently initiated by excessive Ca2+ entry due, at least in part, to impaired voltage-dependent inactivation (VDI) of CaV1.2 current (ICa).1,2 Mathematical modeling predicts that intracellular Ca2+ overload and action potential prolongation stimulate afterdepolarizations, which are the cellular mechanism for triggering ventricular arrhythmias in TS.1,2 However, these predictions have not been directly tested in ventricular myocytes.
Editorial p 2221
Clinical Perspective p 2234
In ventricular myocytes, multiple signaling pathways are activated by increased intracellular Ca2+ entry, including the multifunctional Ca2+ and calmodulin-dependent kinase II (CaMKII),3 a procardiomyopathic and proarrhythmic signaling molecule.4 Increased CaMKII activity causes action potential prolongation and arrhythmias, similar to the observed phenotypes in TS patients, in part by increasing sarcoplasmic reticulum (SR) Ca2+ leak and ICa facilitation.5,6 On the other hand, CaMKII inhibition restores normal intracellular Ca2+ homeostasis and suppresses arrhythmias.4,5 On the basis of these concepts, we hypothesized that increased Ca2+ entry in TS ventricular myocytes enhances CaMKII actions and that activated CaMKII recruitment is important for the proarrhythmic cellular phenotype in TS. To test this hypothesis, we created an adult rat ventricular myocyte model of TS by lentiviral infection of a dihydropyridine-resistant CaV1.2 1c subunit7 harboring the TS mutation. Our studies show that TS mutation requires CaMKII activity to cause important proarrhythmic phenotypes in adult ventricular myocytes.
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Methods |
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Cloning
The plasmids pLentiNB CaV1.2 DHPR hemaglutanin wild-type (WT) TS and pLentiNB CaV1.2 DHPR hemaglutanin TS are described in the Methods section of the online-only Data Supplement.
Lentivirus
Lentivirus was prepared from the manufacturer’s (Invitrogen, Carlsbad, Calif) protocol and as described in the online-only Data Supplement Methods.
Ventricular Myocyte Isolation, Culturing, and Viral Transduction
Adult male Sprague-Dawley rat (250 to 300 g) ventricular myocytes were isolated as previously published8 and cultured as described in the online-only Data Supplement Methods. Procedures were in accordance with the Institutional Animal Care and Use Committee of the University of Iowa. Lentivirus was added to cells at a multiplicity of infection of 1 to 3, and cultures were maintained for 24 to 36 hours.
Electrophysiology
Electrophysiology for HEK293 cells and myocytes, including pipette solutions, bath solutions, voltage clamp protocols, current clamp protocols, and data analysis, is detailed in the online-only Data Supplement Methods.
Immunofluorescence
HEK293 and myocytes were fixed, permeabilized, and incubated with primary antibody Ig and a fluorescent secondary antibody Ig. Confocal images were collect on a Zeiss 510 Meta confocal microscope (Carl Zeiss, Thornwood, NY). Detailed information on cell preparation, antibodies, and acquisition of confocal images is available in the online-only Data Supplement Methods.
Calcium Imaging
Ca2+ transients, SR content, and sparks were acquired by confocal laser scanning from myocytes loaded with Fluo-3. The online-only Data Supplement Methods contain details on cell preparation and confocal Ca2+ imaging.
Mathematical Modeling
Mathematical models of the WT and TS myocytes are based on the Luo-Rudy dynamic (LRd) model of the mammalian ventricular action potential.9,10 See the online-only Data Supplement Methods for equations that differ from the published model.
Statistics
Data are presented as means with SEM. Sigma Stat was used to compare 2 groups with Student t test and multiple groups with ANOVA. Significance was set at a value of P<0.05. Categorical data between 2 groups were compared by use of a 2-tailed Fisher exact test with significance set at P<0.05.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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An Adult Ventricular Myocyte TS Model
We marked exogenous CaV1.2 by adding an extracellular hemaglutanin epitope11 (Figure 1A, green circle) and introduced a validated dihydropyridine-insensitivity mutation7 (Figure 1A, black circle). The dihydropyridine-insensitivity mutation allows the virally introduced CaV1.2 to remain functional while using nifedipine to inhibit endogenous CaV1.2.7 Exogenous CaV1.2 expression was confirmed by immunoblot (Figure 1B) and immunofluorescence (Figure 1C) in transduced HEK293T cells. The functions of CaV1.2 WT and TS (G406R exon 8) were confirmed by recording ICa using whole-cell voltage clamp in HEK293T cells. ICa recorded from TS-expressing HEK293T cells exhibited a significant loss of VDI (Figure 1D), as previously published.1,2,12
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Overexpression of CaV1.2 in ventricular myocytes yielded a 33.7% increase in peak ICa (Figure 1F) and an average 31.9% increase in total CaV1.2 protein (online-only Data Supplement Figure I). Because of the dihydropyridine-resistance mutation,7 peak ICa in CaV1.2-infected ventricular myocytes was significantly resistant to nifedipine compared with uninfected cells (Figure 1E and 1F). In TS and WT infected ventricular myocytes, 10 nmol/L nifedipine resulted in a peak ICa (WT, 6.6±0.7 pA/pF [n=5]; TS, 6.9±0.7 pA/pF [n=6]) that was similar to the peak ICa (6.7±1.0 pA/pF; n=8) measured in noninfected myocytes recorded without nifedipine (Figure 1E and 1F). This nifedipine-engineered balance of endogenous and exogenous CaV1.2 allowed us to determine the effects of the TS mutation on cardiac electrophysiology independently of overexpression-induced changes in peak ICa.
TS Ventricular Myocytes Exhibit Increased CaMKII Autophosphorylation
We confirmed expression of exogenous CaV1.2 in cultured adult ventricular myocytes by immunostaining for the hemaglutanin epitope (Figure 2D and 2G). Virally introduced CaV1.2 was properly targeted to the transverse-tubule (T-tubule) network on the basis of the punctate appearance and 1.8-µm spacing of the hemaglutanin immunofluorescence that is consistent with known distances between T tubules in a resting sarcomere.13 No hemaglutanin immunostaining was detected in uninfected ventricular myocytes (Figure 2A).
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We immunostained for the CaMKII autophosphorylation site, Thr 286, which is a marker of CaMKII activation.14 TS ventricular myocytes (Figure 2H and 2I) exhibited greater levels of CaMKII autophosphorylation compared with both WT (Figure 2E and 2F) and uninfected (Figure 2B and 2C) ventricular myocytes. Total CaMKII immunostaining revealed no changes in CaMKII protein levels between WT, TS, and uninfected ventricular myocytes (online-only Data Supplement Figure II). These data show that activated CaMKII is recruited in TS CaV1.2–expressing ventricular myocytes and suggest that CaMKII activity may contribute to the cellular arrhythmia phenotypes in TS.
Action Potential Prolongation in TS Ventricular Myocytes Is Reversed by CaMKII Inhibition
Stimulated action potentials (arrowhead in Figure 3A) were recorded in nifedipine-treated (10 nmol/L) WT and TS ventricular myocytes. Compared with WT, the TS mutation significantly prolonged the action potential duration (Figure 3A and 3B) as determined by the time to 90% repolarization. Excessive action potential prolongation favors the generation of afterdepolarizations.15 We observed afterdepolarizations from TS ventricular myocytes (5 of 10 cells; Figure 3A and 3C), whereas none were observed in any of the WT cells (0 of 10 cells; Figure 3A and 3C). Most afterdepolarizations were delayed afterdepolarizations, but early afterdepolarizations also were recorded from TS ventricular myocytes. Delayed afterdepolarizations are favored by increased diastolic Ca2+ leak from the SR;16,17 early afterdepolarizations are caused by increased ICa facilitation.18 The action potential prolongation and the tendency for afterdepolarizations in TS ventricular myocytes are consistent with predictions from computational modeling.1,2
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Action potential durations from WT and TS ventricular myocytes in 1 µmol/L nifedipine were reduced to equivalent times, and neither WT nor TS ventricular myocytes exhibited afterdepolarizations under these conditions (Figure 3B and 3C). The 1-µmol/L nifedipine bath solution overcomes the dihydropyridine resistance mutation and inhibited the total peak ICa by >50% (Figure 1F, double arrows). These findings indicate that the observed TS phenotypes were initiated by increased ICa.
We tested the role of CaMKII activity in the observed proarrhythmic cellular phenotypes observed from TS ventricular myocytes by dialysis of AC3-I, a selective CaMKII inhibitory peptide.4,19 AC3-I normalized the action potential duration in TS to WT levels (P=0.40; Figure 3D and 3E). The inactive control peptide, AC3-C,4,19 had no effect, suggesting that CaMKII-dependent increases in ICa contributed to action potential prolongation in TS. The CaMKII inhibitory peptide also eliminated afterdepolarizations in TS ventricular myocytes (P=1.0; Figure 3D and 3F), whereas AC3-C did not (P=0.04; Figure 3D and 3F). These data support the concept that CaMKII activity is required for the proarrhythmic electrophysiological phenotypes in TS ventricular myocytes.
In WT ventricular myocytes, the CaMKII inhibitory peptide, AC3-I, resulted in a nonsignificant (P=0.28) shortening of the action potential duration (Figure 3E) compared with WT ventricular myocytes dialyzed with the control peptide, AC3-C. WT ventricular myocytes did not exhibit afterdepolarizations after dialysis with AC3-I or AC3-C (Figure 3F). We assessed additional action potential parameters, including resting cell membrane potential and peak cell membrane depolarization amplitude. Both TS and WT ventricular myocytes exhibited equivalent resting membrane potentials and peak action potential amplitudes (online-only Data Supplement Table I). Action potential parameters from WT ventricular myocytes in the presence of 10 nmol/L nifedipine were similar to uninfected ventricular myocytes cultured for the same time period (24 to 36 hours) and recorded without nifedipine (online-only Data Supplement Table I). These controls suggest that viral expression of CaV1.2 does not alter the action potential when peak ICa is adjusted to normal levels (by 10 nmol/L nifedipine) and that the proarrhythmic phenotype observed in TS ventricular myocytes was due to the TS mutation.
Taken together, these findings are the first to demonstrate experimentally that the action potential phenotypes observed in TS ventricular myocytes were dependent on increased Ca2+ entry through CaV1.2. Our findings suggest that the TS VDI defect is insufficient, in the absence of increased CaMKII activity, to cause significant action potential prolongation in ventricular myocytes.
TS Reduces VDI in Ventricular Myocytes Independently of CaMKII Activity
Expression of TS CaV1.2 in Xenopus oocytes1,2 and heterologous cells1,2,12 (Figure 1D) showed a loss of CaV1.2 VDI. The Xenopus ooctye experiments1,2 included Ca2+-independent conditions that would not favor CaMKII activation because Ba2+ replaced Ca2+ as the charge carrier. To test the effect of the TS mutation on VDI in ventricular myocytes under conditions not permissive to CaMKII activation, we recorded ICa from TS and WT ventricular myocytes (10 nmol/L nifedipine) using Ba2+ (1.8 mmol/L) as the charge carrier and under high intracellular Ca2+ buffering (20 mmol/L BAPTA). TS ventricular myocytes exhibited a loss of VDI as a significant (P=0.008; Figure 4A) rightward shift compared with WT. The TS V1/2 (–30.75 mV) shifted to more positive potentials compared with WT V1/2 (–35.89 mV). In contrast, the peak ICa elicited by the conditioning pulses showed no difference between WT and TS (Figure 4B), confirming equivalent expression of exogenous WT and TS CaV1.2. No differences were observed in peak ICa or VDI recorded from adult ventricular myocytes expressing WT dihydropyridine-resistant CaV1.2 with 10 nmol/L nifedipine compared with uninfected adult ventricular myocytes without nifedipine (online-only Data Supplement Table II). These findings show that TS causes a loss of CaV1.2 VDI in ventricular myocytes, establishing the initial requirement for increased cellular Ca2+ entry necessary to recruit CaMKII.
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CaMKII Is Required for TS Effects on ICa
To test the importance of CaMKII for ICa changes other than VDI in our TS model, we measured CaMKII-dependent ICa facilitation.18,20 ICa facilitation consists of dynamic increases in peak ICa and slowing of inactivation with repetitive depolarizations.21,22 TS ventricular myocytes exhibited maximal peak ICa during the first depolarization, whereas WT attained peak ICa after the initial depolarization (Figure 5A and online-only Data Supplement Figure III). Subsequent depolarizations showed no difference in peak ICa between TS and WT (Figure 5A and online-only Data Supplement Figure III). To measure the effects of ICa facilitation on cellular Ca2+ entry, we integrated total ICa during the voltage clamp command step. Integrated ICa was significantly greater in TS compared with WT during all depolarization steps (first step, P=0.029; remaining steps, P<0.001; Figure 5B). We found that the fast component of ICa inactivation (
fast) was slower in TS compared with WT (first step, P=0.006; remaining steps, P<0.001; Figure 5C), consistent with increased ICa facilitation and augmented cellular Ca2+ entry in TS ventricular myocytes.
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AC3-I restored the dynamic response characteristics of integrated ICa and fast in TS to levels recorded from WT cells (integrated ICa, P=0.522; fast, P=0.294; Figure 5E and 5F). In contrast, dialysis of AC3-C had no effect on fast or integrated ICa. Dialysis of the CaMKII inhibitory peptide prevented ICa facilitation in WT ventricular myocytes (online-only Data Supplement Figure III), whereas the control peptide had no effect on WT ventricular myocyte ICa facilitation. These measurements show that CaMKII is a significant determinant of ICa from TS mutant channels and that CaMKII actions are distinct from the previously reported shift in VDI.
TS Augments Intracellular Ca2+
Mathematical modeling studies predicted alterations in intracellular Ca2+ handling in TS, including increased Ca2+ transient amplitude and increased SR Ca2+ content.1 We recorded Ca2+ transients (Figure 6A) from WT and TS ventricular myocytes loaded with Fluo-3 AM and field stimulated at 1 Hz.23 TS caused a significant increase in the peak Ca2+ transient compared with WT (P=0.04; Figure 6B), which is consistent with computer models.1,23,24 Interestingly, the 50% decay time for Ca2+ transients in TS was significantly shortened compared with WT (P=0.02; Figure 6C). A faster decay time implicates increased SR/endoplasmic reticulum calcium ATPase activity,25 which was not predicted by modeling studies but is associated with CaMKII signaling.26–29 These experimental data reveal that TS alters intracellular Ca2+ handling by increasing the peak Ca2+ transient amplitude and enhancing the decay of the intracellular Ca2+ transient.
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Mathematical modeling also predicted increased SR Ca2+ content with TS as a result of enhanced ICa from TS CaV1.2. Surprisingly, we found that TS SR Ca2+ content was not different from WT (P=0.55; Figure 6D). We considered that increased SR Ca2+ leak in TS balances faster SR Ca2+ uptake,25 thereby preventing a net increase in SR Ca2+ content compared with WT. Increased SR Ca2+ leak is implicated in CaMKII signaling6,30,31 and in triggering delayed afterdepolarizations,16,17 a prominent feature of the TS ventricular myocytes (Figure 3A and 3C). We assessed diastolic SR Ca2+ leak by measuring spontaneous Ca2+ sparks from TS and WT ventricular myocytes (Figure 6E).32 The SR Ca2+ sparks were significantly increased in TS compared with WT (P=0.001; Figure 6E and 6F), indicating increased SR Ca2+ leak in TS. The spark amplitude for TS was significantly greater than WT (P=0.002; online-only Data Supplement Table III), consistent with the increase in peak Ca2+ transient observed with TS. The effects of TS on intracellular Ca2+ handling had 2 unexpected results: the faster decay time and the increase in spark frequency. Taken together, these data suggest that SR Ca2+ cycling is enhanced in TS ventricular myocytes, resulting in significantly increased SR Ca2+ uptake and diastolic Ca2+ leak but without a change in SR Ca2+ content.
In contrast to TS, WT exhibited no difference in the Ca2+ transient peak amplitude or decay time compared with uninfected ventricular myocytes (online-only Data Supplement Table III). No significant changes were observed in the width or duration of the Ca2+ sparks (online-only Data Supplement Table III) between WT, TS, and uninfected cells. The spark frequency and profile of individual sparks showed no difference between WT and uninfected ventricular myocytes (online-only Data Supplement Table III).33
Revised TS Mathematical Modeling
Several studies have modeled the impact of TS on myocardial electrophysiology by using a shift in CaV1.2 VDI estimated from measurements in nonmyocytes.1,2,24 Using data from our TS ventricular myocyte model, we developed a new mathematical model of TS incorporating CaMKII signaling (Figure 7A). As the basis for our new model of TS, we used the LRd model9,10 because of its established utility in studying cardiac arrhythmia mechanisms.
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Our model of TS incorporated 3 modifications to match our experimental observations. First, we shifted the CaV1.2 steady-state VDI in the LRd model to simulate the measured TS defect on channel gating. Second, we simulated the downstream CaMKII effect on CaV1.2 ICa facilitation associated with TS by slowing ICa inactivation to increase integrated ICa as measured experimentally (online-only Data Supplement Figure IV). Third, we simulated the CaMKII actions on intracellular Ca2+ handling associated with TS by increasing the mean open time of the ryanodine receptor SR Ca2+ release channels, decreasing the threshold for spontaneous SR Ca2+ release, and increasing SR Ca2+ release. Consistent with our experimental measurements, the new model of TS predicted an increase in the intracellular Ca2+ transient amplitude without any change in SR Ca2+ load compared with WT (online-only Data Supplement Figure IV). The model also predicted an increase in action potential duration (Figure 7B) and afterdepolarizations (Figure 7C) during a pause after pacing. We also simulated CaMKII inhibition using the TS LRd model by reversing the simulated downstream CaMKII effects but leaving in place the shift in CaV1.2 VDI that we measured under conditions not permissive for CaMKII activity (Figure 4A). The resulting TS LRd model with "CaMKII inhibition" prevented action potential prolongation and afterdepolarizations (Figure 7C). Our mathematical models of TS, with and without CaMKII inhibition, are consistent with our experimental data from our TS ventricular myocyte model.
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Discussion |
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TS is the first arrhythmia syndrome (long-QT syndrome 8) resulting from a genetic mutation in the CaV1.2 pore-forming
subunit.1,2 Compared with cardiac Na+ and K+ channels, CaV1.2 has proved to be remarkably resistant to genetic disease. One key difference between Ca2+, Na+, and K+ is the prominent role that Ca2+ plays as a second messenger. TS patients have not only extremely profound QT interval prolongation but also structural cardiac abnormalities that are not typical of Na+ or K+ channel gene-related long-QT syndrome patients. QT interval prolongation reflects increased duration of the ventricular action potential. The action potential duration prolongation in TS was attributed entirely to the defect in VDI,1 but this defect in TS VDI was ascertained in heterologous (nonmyocardial) cells in which action potentials could not be directly measured. Furthermore, heterologous cells lack the highly ordered ultrastructure that is present in ventricular myocytes for Ca2+ homeostasis and excitation-contraction coupling. The ventricular myocyte TS model allowed us to measure electrophysiological, intracellular Ca2+ handling and Ca2+-mediated signaling changes that occur downstream of the loss of VDI.
Despite the relatively modest reduction in CaV1.2 VDI measured in our TS model, we found action potential prolongation and spontaneous afterdepolarizations that were due to secondary activation of CaMKII. We conclude that the shift in VDI provides the initial stimulus to trigger intracellular Ca2+ signaling that includes CaMKII activation. Increased CaMKII activity appears to be necessary for the cellular phenotype of prolonged action potentials and afterdepolarizations insofar as CaMKII inhibition prevents these phenotypes. CaMKII inhibition may be a viable alternative therapeutic approach for TS patients treated with the ICa antagonist verapamil.34 Our results showed that CaMKII amplifies Ca2+ entry through CaV1.2 in TS by slowing fast and shifting the V1/2 of ICa inactivation. Our studies do not exclude the possibility that CaMKII inhibition also could affect other depolarizing or repolarizing currents such as Na+ current35 or K+ current.36 Our finding that SR Ca2+ leak is increased in TS is consistent with other reports that show that proarrhythmic actions of CaMKII are due to increasing SR Ca2+ leak,37 thereby enabling a transient inward current16 (INCX) that triggers delayed afterdepolarizations. Thus, our data support the concept that the ryanodine receptor is a secondary proarrhythmic target for excessive CaMKII activity in TS. Our data highlight how small changes in cellular Ca2+ entry through CaV1.2 can lead to unanticipated, maladaptive, and far-reaching changes in Ca2+ activated signaling.
Interestingly a connection between CaMKII and a TS mutation was suggested on the basis of single-channel recordings from heterologous expression of TS CaV1.2 in baby hamster kidney 6 cells.38 These experiments found that TS CaV1.2 was more likely than WT to exhibit frequent long openings, so-called mode 2 gating, that are the single-channel mechanisms underlying CaMKII-mediated ICa facilitation.20 Our new studies add to evidence supporting a connection between TS and CaMKII by showing that CaMKII is critical for increased ICa facilitation action potential prolongation and afterdepolarizations in our TS ventricular myocyte model. Enhanced CaMKII activity increases ICa facilitation,17 which may cause generation of early afterdepolarizations.5
Although major Ca2+ homeostatic proteins are conserved in ventricular myocytes across mammalian species, differences exist between species as to the quantitative contribution of these components to the action potential.39 Thus, one goal of future studies should be to determine whether CaMKII or other Ca2+-activated signaling molecules contribute to TS phenotypes in ventricular myocytes from other species. However, the use of our TS adult ventricular myocyte model has contributed new insights into arrhythmia mechanisms in TS by illustrating how a concise defect in CaV1.2 gating can initiate downstream recruitment of CaMKII that ultimately enables the electrophysiological cellular disease phenotype in TS. The central nervous system defects of TS patients also may be due to secondary recruitment of Ca2+-activated signaling molecules, including CaMKII. Overexpression of CaMKII is known to interfere with neuronal growth and differentiation,40 and a constitutively active CaMKII within the mouse brain causes significantly impaired spatial memory.41 CaMKII recruitment in TS ventricular myocytes also suggests the possibility that other disease phenotypes in TS patients (eg, structural heart disease or mental retardation) may be initiated by defects in VDI but carried forward indirectly by recruitment of Ca2+-dependent signaling molecules.
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Acknowledgments |
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We thank the University of Iowa Gene Transfer Vector Core (a National Institutes of Health [NIH]–funded resource) for help in preparing the lentivirus, particularly Maria Scheel and Dalyz Ochoa for their assistance and expertise.
Sources of Funding
This work was funded by NIH grants R01 HL 079031, R01 HL 62494, and R01 HL 70250 (Dr Anderson); NIH grants R01 HL084583 and R01 HL083422 and Pew Scholars Trust (Dr Mohler); the University of Iowa Cardiovascular Center Interdisciplinary Research Fellowship (Dr Hund); and the University of Iowa Research Foundation. W.H. Thiel was supported in part by an American Heart Association predoctoral fellowship award.
Disclosures
None.
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The online-only Data Supplement can be found with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.788067/DC1.
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