Skip main navigation

Ca2+-Related Signaling and Protein Phosphorylation Abnormalities Play Central Roles in a New Experimental Model of Electrical Storm

Originally publishedhttps://doi.org/10.1161/CIRCULATIONAHA.110.016683Circulation. 2011;123:2192–2203

Abstract

Background—

Electrical storm (ES), characterized by recurrent ventricular tachycardia/fibrillation, typically occurs in implantable cardioverter-defibrillator patients and adversely affects prognosis. However, the underlying molecular basis is poorly understood. In the present study, we report a new experimental model featuring repetitive episodes of implantable cardioverter-defibrillator firing for recurrent ventricular fibrillation (VF), in which we assessed involvement of Ca2+-related protein alterations in ES.

Methods and Results—

We studied 37 rabbits with complete atrioventricular block for ≈80 days, all with implantable cardioverter-defibrillator implantation. All rabbits showed long-QT and VF episodes. Fifty-three percent of rabbits developed ES (≥3 VF episodes per 24-hour period; 103±23 VF episodes per rabbit). Expression/phosphorylation of Ca2+-handling proteins was assessed in left ventricular tissues from rabbits with the following: ES; VF episodes but not ES (non-ES); and controls. Left ventricular end-diastolic diameter increased comparably in ES and non-ES rabbits, but contractile dysfunction was significantly greater in ES than in non-ES rabbits. ES rabbits showed striking hyperphosphorylation of Ca2+/calmodulin-dependent protein kinase II, prominent phospholamban dephosphorylation, and increased protein phosphatase 1 and 2A expression versus control and non-ES rabbits. Ryanodine receptors were similarly hyperphosphorylated at Ser2815 in ES and non-ES rabbits, but ryanodine receptor Ser2809 and L-type Ca2+ channel α-subunit hyperphosphorylation were significantly greater in ES versus non-ES rabbits. To examine direct effects of repeated VF/defibrillation, VF was induced 10 times in control rabbits. Repeated VF tissues showed autophosphorylated Ca2+/calmodulin-dependent protein kinase II upregulation and phospholamban dephosphorylation like those of ES rabbit hearts. Continuous infusion of a calmodulin antagonist (W-7) to ES rabbits reduced Ca2+/calmodulin-dependent protein kinase II hyperphosphorylation, suppressed ventricular tachycardia/fibrillation, and rescued left ventricular dysfunction.

Conclusions—

ES causes Ca2+/calmodulin-dependent protein kinase II activation and phospholamban dephosphorylation, which can explain the vicious cycle of arrhythmia promotion and mechanical dysfunction that characterizes ES.

Electrical storm (ES), characterized by repetitive episodes (generally defined as ≥3 within 24 hours) of ventricular fibrillation (VF) and/or ventricular tachycardia (VT), is an increasing problem among implantable cardioverter-defibrillator (ICD) patients.1 Recent analyses of large clinical trials indicate a high risk of early mortality in patients who experience ES.2,3 Most of this excess risk is attributable to cardiac, nonsudden mechanisms, particularly progressive heart failure (HF).13 The molecular mechanisms underlying ES and associated mortality are poorly understood.

Editorial see p 2183

Clinical Perspective on p 2203

Ca2+-handling protein phosphorylation is a major control mechanism for cardiac contractility and relaxation. Increased activity of protein phosphorylation mediators like cAMP-dependent protein kinase A (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and protein kinase Cα (PKCα) and resulting hyperphosphorylation of Ca2+-handling proteins are linked to mechanical dysfunction and arrhythmias.49 CaMKII/PKA-mediated phosphorylation of ryanodine receptors (RyR2s) causes diastolic Ca2+ leak from the sarcoplasmic reticulum (SR), causing depletion of SR Ca2+ stores and arrhythmias related to delayed afterdepolarizations.47 Hyperphosphorylation of L-type Ca2+ channels (LTCCs) by CaMKII modulates Ca2+ current, predisposing to early afterdepolarizations.8 PKCα activation results in hypophosphorylation of phospholamban, enhancing phospholamban inhibition of SR Ca2+-ATPase (SERCA2a) and impairing Ca2+ uptake into the SR.9,10 These findings, together with the fact that VF is associated with increased [Ca2+]i11,12 and that ES is associated with elevated sympathetic tone,1 led us to hypothesize that Ca2+-handling alterations caused by changed expression and/or activation of Ca2+ signaling molecules may be responsible for ES-related negative outcomes. In the present study, we report a new experimental model that features repetitive episodes of ICD firing for recurrent VF, in which we tested this hypothesis.

Methods

All animal handling protocols were approved by the Animal Experimentation Ethics Committee of Nagoya University.

Surgical Technique and Experimental Protocol

Female New Zealand White rabbits (weight, 2.5 to 3.5 kg) were used. We created complete atrioventricular block and implanted ICDs (Medtronic) using methods described previously,13,14 with modifications. In brief, 2 unipolar pacing leads (6491, Medtronic) fixed to the right ventricular free wall were connected to an ICD through a Y adaptor (5866–38M, Medtronic) implanted subcutaneously in the back. A custom-made patch electrode was sutured subcutaneously in the chest wall (Figure 1A). Ventricular pacing at 150 bpm (maximum pacing rate available in the device) was instituted immediately after creating atrioventricular block to allow recovery from surgery. Seven days after surgery, the pacing rate was decreased to 90 bpm, and ICD settings were established under ketamine (25 mg/kg)/xylazine (5 mg/kg) anesthesia (day 0). To determine the defibrillation threshold, VF was induced twice by applying 50-Hz burst stimulation via the ICD. The ICD was then programmed to detect and treat VF (see below). Rabbits with complete atrioventricular block (CAVB) equipped with ICDs were subjected to sustained bradycardia with VVI pacing (90 bpm for ≈1 week, followed by 60 bpm). CAVB rabbits that survived for ≈3 months were euthanized (pentobarbital, 40 mg/kg IV). Left ventricular (LV) free-wall tissue samples were fast-frozen in liquid N2 and stored at −80°C. Echocardiograms were obtained at day 0 and end of study.

Figure 1.

Figure 1. A, Chemical atrioventricular node ablation and extracardiac implantable cardioverter-defibrillator system implantation. B, Implantable cardioverter-defibrillator parameters for rabbits. C, Mean±SEM defibrillation threshold (DFT) determined at day 0 and follow-up period in electrical storm (ES) (n=17) and non-ES (n=15) rabbits. D, Representative ventricular fibrillation (VF) episodes during 1 day in an ES rabbit. VT indicates ventricular tachycardia; EGM, electrogram.

Implantable Cardioverter-Defibrillator Settings

The VF detection interval was set <240 ms (>250 bpm), and the VT detection algorithm was disabled. The number of intervals to detect VF (NID) and to redetect VF (NID-redetect) were programmed to maximum values available (120 of 160 and 30 of 40, respectively) to avoid inappropriate shocks for torsades de pointes (TdP)-like VTs, which occur frequently in CAVB-rabbits.13,14 Up to 6 defibrillation shocks were programmed to treat a detected VF episode. The first therapy was set to at least twice the defibrillation threshold. Figure 1B shows an example of therapy parameters. Mean defibrillation thresholds were not statistically different between groups (Figure 1C).

Implantable Cardioverter-Defibrillator Interrogation and Real-Time Electrogram Recordings

Approximately every 10 days, the ICD was interrogated under conscious conditions to assess the number of VF and VT episodes and obtain intracardiac electrograms of VF episodes and percentages of rhythm paced/sensed. Tachyarrhythmias that were ≥5 consecutive beats at >250 bpm (VF detection interval) and terminated spontaneously less than NID, were detected as non-sustained VT-episodes (Figure I in the online-only Data Supplement). ES was defined as ≥3 VF episodes per 24 hours. Real-time electrograms and electrograms between the active CAN and the patch electrode (CAN-patch electrogram) were recorded through the ICD programmer. Electrogram parameter measurements, including cycle length of escape rhythm and QT interval, were based on averages of 3 consecutive beats in the lead providing the clearest QT-interval definition. QT interval was corrected (QTc) according to the method of Carlsson et al15 for rabbits with the following formula: QTc=QT−0.175×(RR−300).

W-7 Administration

The calmodulin antagonist W-7 (BIOMOL) was infused continuously in ES rabbits via an osmotic pump (2ML1, Alzet) that delivers solutions continuously at 10 μL/h for 7 days. The pump was filled with 2 mL dimethyl sulfoxide/water (50:50 vol/vol) with or without W-7 50 mg. Under ketamine/xylazine anesthesia, 2 pumps without W-7 were implanted subcutaneously into the abdomen for 7-day baseline infusion. W-7 was then administered by 1 pump (W-7 50 mg) for 1 week, followed by 2 pumps (W-7 100 mg) for the subsequent week. During infusion, ICD interrogation was performed daily. The dosages were established on the basis of the study by Mazur et al.16

Western Blots

Protein extracts were prepared, and immunoblotting was performed as described previously.14 Primary antibodies included the following: anti-calmodulin (Fitzgerald), anti-RyR2 (Affinity BioReagents), antiphospho-Ser286-CaMKII (CaMKII-P, Santa Cruz), anti-CaMKIIδ (Santa Cruz), anti-SERCA (Affinity BioReagents), anti-Cav1.2 (Alomone), antiphospho-Ser2815-RyR2 (RyR2-P2815, a kind gift from Dr A. Marks, Columbia University), anti-Ser2809-RyR2 (RyR2-P2809, Badrilla), anti-phospholamban (Affinity BioReagents), antiphospho-Ser16-phospholamban and anti-Thr17-phospho-phospholamban (phospholamban-P16 and phospholamban-P17, both Badrilla), anti-protein phosphatase 1 (PP1) catalytic subunit and anti-protein phosphatase 2A (PP2A) catalytic subunit (both Upstate), anti-PKAα catalytic subunit (Santa Cruz), anti-PKCα (Abcam), antiphospho-Ser657-PKCα (PKCα-P, Upstate), inhibitor-1 (I-1) (Eurogentec), phospho-Thr35-I-1 (I-1 P35, Cell Signaling), phospho-Ser67-I-1 (I-1 P67, Eurogentec), troponin-I (Milipore), phosphorylated Ser23/24-troponin-I (Cell Signaling), total myosin-binding protein C and phosphorylated Ser282-myosin-binding protein C (kind gifts from Dr L. Carrier, University of Hamburg), calsequestrin (Dianova), phosphorylated myosin light chain-2a (a kind gift from Dr T. Eschenhagen, University of Hamburg), and caspase-3 (Santa Cruz). Band densities were quantified by densitometry, standardized to GAPDH (Fitzgerald), and normalized to the control sample. I-1 protein was enriched by an optimized trichloroacetic acid extraction procedure as described previously.20 Phosphorylation analysis for Cav1.2 was obtained with Pro-Q Diamond stain technology. In brief, proteins electroblotted on polyvinylidene fluoride membranes were fixed and stained with the Pro-Q Diamond Phosphoprotein Blot Stain Kit (P33356, Invitrogen). The membrane was imaged with UV transillumination and ATTO Light Capture molecular imager. Scans were quantified with CS Analyzer (ATTO, Tokyo, Japan).

Real-Time Polymerase Chain Reaction

Total RNA was extracted from 0.5- to 1.0-g LV samples with Trizol reagent (Invitrogen) according to the manufacturer's protocols. To assess mRNA transcript levels by real-time reverse transcriptase polymerase chain reaction, the first-strand cDNA was first synthesized with the High Capacity Reverse Transcriptase Kit (Applied Biosystems). TaqMan primers and probes for hypertrophy marker genes, including rabbit atrial natriuretic factor, brain natriuretic peptide (designed with Custom TaqMan Gene Expression Assay Design Tool), β-myosin heavy chain, and control 18S, were purchased from Applied Biosystems. Real-time polymerase chain reaction was performed with an ABI prism 7700 sequence detector. The fluorescent amplification curve of the product was determined, and the cycle at which the fluorescence reached a threshold was recorded (Ct) in triplicate and averaged. To control for variability in RNA quantity, the measured abundances of hypertrophic marker genes were normalized to that of 18S with the formula ΔCt=Ct (detected genes)−Ct (18S). The relative expression in ES and ES+W-7 hearts (normalized to control hearts) was then determined with the following relationship: gene expression=2−ΔΔCt, where ΔΔCt=ΔCt(ES or ES+W-7)−ΔCt (control).

Statistical Analysis

All data are expressed as mean±SEM. Western blot band intensities are expressed normalized to GAPDH signal intensity for the same sample. Two-way repeated-measures ANOVA was performed for electrogram parameters with the use of mixed model methodology with group and time as main effects. In case of a significant interaction, Tukey tests were used to compare individual means. One-way ANOVA followed by Bonferroni-corrected t-test was used to evaluate differences in echocardiographic parameters and protein and mRNA expression among groups. Unpaired t-tests were used for single comparisons between non-ES and ES group measurements, and paired t-tests were used for comparison of echocardiographic parameters before and after W-7. Two-tailed P<0.05 indicated statistical significance. The authors had full access to and take responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

Results

Model Characteristics

Thirty-two CAVB rabbits with ICDs were followed for 76±5 days. All had QTc prolongation and ICD-detected self-terminating VT before the first VF episode; 53% (17/32) subsequently developed ES. Figure 1D shows 3 VF episodes within <2 hours in an ES rabbit. This ES rabbit had 127 VF episodes and 6617 self-terminating VT episodes until euthanasia at day 90. ES appeared at day 20, occurred intermittently for 17 days, and then occurred continuously from day 78.

Representative real-time electrograms during ICD interrogation in an ES and non-ES rabbit are shown in Figure 2A. The ES rabbit had a slow escape rhythm and QT prolongation/abnormal QTU complexes at day 71 compared with the non-ES rabbit at day 72. Figure 2B shows the daily number of VF episodes from the same rabbits. The ES rabbit had a progressive increase in VF episodes from day 67 until death at day 83. The stored electrogram of the last VF episode showed unsuccessful defibrillation with the sixth therapy after 5 programmed therapies for repeated postshock VF. In contrast, the non-ES rabbit had sporadic single VF episodes. Figure 2C summarizes time-dependent electrogram changes in all rabbits. The RR intervals of escape rhythms were longer in ES rabbits versus non-ES rabbits. In ES rabbits, QTc increased from 183±5 ms on day 10 to near steady state values (229±7 ms) by day 20. Although non-ES rabbits also showed increased QTc (from 187±3 to 215±6 ms by day 40), QTc was significantly greater in ES rabbits. The number of VF episodes per rabbit averaged 103±23 for ES rabbits and 3.2±0.9 for non-ES rabbits (P<0.001; Figure 2D) during follow-up periods of 78±8 and 70±8 days, respectively (P=0.53; Figure 1C). The number of self-terminating VT episodes was also substantially greater for ES rabbits (Figure 2D). ES development appeared rate dependent: Clustered VF episodes could be suppressed by increasing the pacing rate and reappeared on returning the rate to 60 bpm (Figure II in the online-only Data Supplement).

Figure 2.

Figure 2. A, CAN-patch electrocardiograms (ECGs) and intracardiac electrograms (EGM) recorded during implantable cardioverter-defibrillator interrogation in an electrical storm (ES) and non-ES rabbit. B, The daily number of ventricular fibrillation (VF) episodes in the same rabbits. C, Mean±SEM time-dependent changes in RR and QTc intervals at approximately days 10 (the beginning of follow-up period), 20, 40, 60, and 70 (end of follow-up) in ES (n=13) and non-ES (n=13) rabbits. Statistical analysis could not be performed for day 70 data because of animal death, which caused excessive data dropout. *P<0.05 vs day 10; †P<0.05 for ES vs non-ES rabbits. D, Mean number of VF episodes and self-terminating ventricular tachycardia (VT) episodes per rabbit in ES (n=17) and non-ES (n=15) groups. **P<0.01 vs non-ES group.

ES sometimes occurred early, and only transiently (Figure III in the online-only Data Supplement). Of 17 ES rabbits, 9 died prematurely. Six died during ES, with the last VF episode recording showing VF undersensing or defibrillation failure. Two were euthanized for severe pocket infection, and 1 died of unknown causes. The remaining 8 ES rabbits that survived for 89±7 days were used for immunoblotting studies. ES episodes in 5 rabbits and single VF episodes in 3 rabbits were documented within the 24 hours before euthanasia.

Of 15 non-ES rabbits, 7 surviving for 94±5 days were used for immunoblotting studies. All 7 rabbits had at least 50 self-terminating VT episodes within 24 hours of euthanasia. The remaining 8 died prematurely. Five died suddenly of VF with defibrillation failure, and 3 died of unknown causes.

Echocardiography

Figure 3A shows representative M-mode echocardiograms at day 0 (baseline) and at the end of follow-up. LV end-diastolic diameter increased similarly, by >20%, in non-ES and ES rabbits (Figure 3B). LV fractional shortening decreased from 38±1% at day 0 to 32±1% in non-ES rabbits and more severely to 25±1% in ES rabbits (P=0.006 versus non-ES) by end of study (Figure 3C). There were no significant differences in interventricular septum or posterior LV wall thicknesses among groups (Figure 3D). Pleural effusions were observed in 4 of 7 ES rabbits but not in non-ES rabbits.

Figure 3.

Figure 3. A, Representative M-mode echocardiograms for 1 rabbit in each group. B through D, Overall mean±SEM data at day 0 (n=10) and the end of the follow-up period in electrical storm (ES) (n=7) and non-ES (n=7) rabbits. LVEDd indicates left ventricular end-diastolic diameter; FS, fractional shortening; IVS, interventricular septum; and PW, posterior wall of left ventricle. *P<0.05 vs day 0; †P<0.05 vs non-ES group.

Protein Kinases and Phosphatases

Figure 4A shows representative immunoblots for CaMKII-P, CaMKIIδ, and calmodulin on 1 gel each. CaMKII-P expression was significantly increased by ≈500% and ≈165% in ES and non-ES rabbits, respectively, without significant change in total CaMKIIδ expression. Fractional CaMKII autophosphorylation was ≈2 times greater in ES than in non-ES rabbits, suggesting striking CaMKII activation. Calmodulin expression was unaltered. CaMKII activation was also observed in the right ventricle (Figure IV in the online-only Data Supplement) and was qualitatively similar to LV changes. Immunoblots for PKAα catalytic subunit, PKCα-P, and total PKCα are shown in Figure 4B and for phosphatase proteins in Figure 4C. PKAα catalytic subunit expression was reduced similarly by 61% and 55% in ES and non-ES rabbits, respectively, whereas total PKCα and PKCα-P were unchanged (Figure 4D). Expression of PP1 and PP2A catalytic subunits was increased by ≈85% in ES rabbits only (Figure 4E).

Figure 4.

Figure 4. A, Top, Representative immunoblots of autophosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII-P), CaMKIIδ, calmodulin (CaM), and GAPDH in left ventricular tissue samples from 4 control (CTL), 4 non–electrical storm (ES), and 4 ES rabbits. Bottom, Mean±SEM band intensities in control (n=6), non-ES (n=7), and ES (n=8) rabbits. B and C, Immunoblots of protein kinase A (PKA)α catalytic subunit, autophosphorylated protein kinase Cα (PKCα-P), total PKCα, protein phosphatases 1 and 2A (PP1 and PP2A). D and E, Mean±SEM band intensities corresponding to B and C in control (n=6 to 8), non-ES (n=5 to 7), and ES (n=6 to 8) groups. *P<0.05, **P<0.01 vs control; ††P<0.01 vs non-ES group.

Phosphorylation of Cav1.2, RyR2, and Phospholamban

Figure 5 shows immunoblots for total and phosphorylated forms of key Ca2+-handling proteins. The LTCC α-subunit Cav1.2 band was observed at the expected molecular mass of ≈205 kDa (Figure 5A, top). Phosphoprotein bands corresponding to Cav1.2 were stronger in both non-ES and ES rabbits versus controls (Figure 5B), with a larger fractional Cav1.2 phosphorylation (≈500%) in ES versus non-ES rabbits (≈130%).

Figure 5.

Figure 5. A, Immunoblots for Cav1.2-P and Cav1.2 (top) and ryanodine receptor (RyR)2, RyR2–P2815, RyR2–P2809, and calsequestrin (CSQ) (bottom). B and C, Mean±SEM protein expression and phosphorylation fraction in control (CTL) (n=8), non–electrical storm (ES) (n=7), and ES (n=8) rabbits. D, Immunoblots for sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), phospholamban (PLB), phospholamban-P16 (PLB-P16), and phospholamban-P17 (PLB-P17). E, Corresponding mean±SEM protein expression and phosphorylation fraction in control (n=8), non-ES (n=7), and ES (n=8) rabbits. *P<0.05, **P<0.01 vs control; †P<0.01 vs non-ES group.

RyR2 has multiple phosphorylation sites including RyR2-Ser2815, phosphorylated by CaMKII,17 and RyR2-Ser2809, which can be phosphorylated by CaMKII and/or PKA.5,18Figure 5A (bottom) shows representative immunoblots of total RyR2, CaMKII-phosphorylated RyR2-P2815, and PKA/CaMKII-phosphorylated RyR2-P2809. Fractional RyR2-P2815 phosphorylation was increased significantly in both ES and non-ES rabbits, but that of RyR2-P2809 was significantly increased, by ≈70%, in ES rabbits only (Figure 5C). Total RyR2 and calsequestrin expression were unaltered in either group.

Phospholamban is an endogenous inhibitor of SERCA2a function. Phospholamban phosphorylation causes phospholamban dissociation from SERCA2a and removes inhibition. SERCA2a activity and SR Ca2+ uptake are enhanced on phospholamban phosphorylation. Phospholamban is phosphorylated at Ser16 by PKA and at Thr17 by CaMKII.19Figure 5D shows representative immunoblots for SERCA2a, phospholamban-P16, phospholamban-P17, and total phospholamban. Fractional phospholamban phosphorylation was greatly reduced in ES rabbits, by 96% at Ser16 and 89% at Thr17 (Figure 5E), whereas there was no statistically significant change in phospholamban phosphorylation in non-ES rabbits. SERCA2a and total phospholamban expression were unchanged (Figure 5E).

Data for expression/phosphorylation of myofibrillar proteins are shown in Figure V in the online-only Data Supplement. PKA-phosphorylated myosin-binding protein C and troponin-I were reduced by ≈75% and ≈50% in ES and by ≈66% and ≈47% in non-ES groups, respectively, which was compatible with similar reductions in PKAα catalytic subunit expression in both groups.

Protein Phosphatase I-1 Phosphorylation

PP1 activity is modulated by protein phosphatase I-1, which is phosphorylated at Thr35 by PKA.9,10 PKA-phosphorylated I-1 inhibits PP1, augmenting the phospholamban phosphorylation resulting from direct PKA action. PKCα phosphorylates I-1 at a different site, Ser67, suppressing I-1 inhibition of PP1 and thereby reducing phospholamban phosphorylation.9,10 Figure VI in the online-only Data Supplement shows representative blots for total I-1, PKA-phosphorylated I-1 (I-1-P35), and PKCα-phosphorylated I-1 (I-1-P67). Bands were observed at the expected molecular mass.20 There were no significant differences among groups, suggesting that phospholamban dephosphorylation in ES rabbits is due to enhancement of protein phosphatase activity unrelated to changes in PKA or PKCα phosphorylation of I-1.

Direct Effects of VF/Defibrillation on CaMKII and Phospholamban Phosphorylation

CaMKII hyperphosphorylation is arrhythmogenic, and phospholamban dephosphorylation negatively affects SR Ca2+ stores and contractility. We considered the possibility that repeated VF and defibrillation can cause these changes, initiating a vicious cycle of self-reinforcing contractile failure and arrhythmogenesis. To examine the effect of repeated VF/defibrillation on protein phosphorylation, we obtained LV tissue samples from day 0 rabbits subjected to repeated VF induction and termination. Under ketamine/xylazine anesthesia, repetitive VF induction by 50-Hz burst stimulation was performed 10 times over 1 hour (each VF episode was ICD terminated like spontaneous VF), and then rabbits were euthanized with pentobarbital (40 mg/kg IV). LV free-wall tissue was removed, fast-frozen in liquid N2, and stored at −80°C. Figure 6A and 6B show representative immunoblots for CaMKII and phospholamban phosphorylation and corresponding mean data. CaMKII-P expression and fractional CaMKII phosphorylation were increased by ≈400% and ≈700%, respectively. Phospholamban phosphorylation was reduced at Ser16 and Thr17 by ≈80% and ≈50%, respectively, which was qualitatively similar to results in ES rabbit hearts.

Figure 6.

Figure 6. A and B, Direct effects of ventricular fibrillation (VF)/defibrillation on protein phosphorylation. Left ventricular tissue samples were prepared from day 0 rabbits subjected to 10 times VF induction to mimic electrical storm (ES). Immunoblots for Ca2+/calmodulin-dependent protein kinase II (CaMKII) and phospholamban (PLB) in control (CTL) and acute ES tissue samples and corresponding mean data in control (n=5) and acute ES tissue (n=5) are shown. C and D, Direct effects of electrical shocks. Left ventricular tissue samples were prepared from control rabbits subjected to 10 times shock pulse. Immunoblots for CaMKII and phospholamban and mean data in control (n=5) and shock tissue (n=5) are shown. *P<0.05, **P<0.01 vs control.

We then tested whether repeated electrical shocks per se contribute to altered protein phosphorylation, using LV tissue samples from additional control rabbits that received ten 5-J shock pulses without VF induction over 1 hour. Unlike repeated VF/defibrillation cycles, electrical shocks alone did not reproduce the ES-associated Ca2+-handling protein changes (compare Figure 6C and 6D with Figures 4A, 5D, 6A, and 6B). These findings suggest that VF/defibrillation and electrical shocks differentially modulate Ca2+ handling and that alterations associated with ES derive from repeated VF/defibrillation cycles rather than electrical shocks alone.

Responses of ES Rabbits to Calmodulin Inhibition

CaMKII activation results from Ca2+/calmodulin binding. Anderson and colleagues16,21 have demonstrated in rabbit models of drug-induced long-QT syndrome that the CaMKII inhibitor KN-93 suppresses early afterdepolarizations and that the calmodulin antagonist W-7 prevents torsades de pointes, suggesting that CaMKII is a proarrhythmic signal. In the present study, to assess the potential central role of Ca2+/calmodulin/CaMKII signaling in ES, we administered W-7 to ES rabbits. Figure 7A illustrates the response in 1 ES rabbit. Multiple VF and VT episodes were documented almost every day during baseline infusion of dimethyl sulfoxide. Both VF and VT episodes decreased transiently after initiating 50-mg W-7 infusion and disappeared completely with 100-mg W-7 infusion. After discontinuation of W-7, arrhythmias eventually recurred. One-week infusion of W-7 to 5 rabbits caused dose-dependent reductions in VF episodes (from 25±7 during dimethyl sulfoxide infusion to 13±4 with the lower dose and 2.0±0.9 with the higher dose), self-terminating VT episodes (from 2022±579 to 711±178 and 160±25, respectively), and ES days (from 3.8±0.7 to 1.5±0.6 and 0.3±0.2, respectively) over 1-week observation periods (Figure 7B). W-7 did not significantly alter RR interval or QTc (Figure VII in the online-only Data Supplement).

Figure 7.

Figure 7. A, The daily number of ventricular fibrillation (VF) and ventricular tachycardia (Vt)episodes in 1 electrical storm (ES) rabbit during continuous infusion of dimethyl sulfoxide (DMSO) solution without and with W-7. B, Dose-dependent effects on VF and VT episodes and ES days in 5 ES rabbits. C, Representative M-mode echocardiograms before and after W-7 in 1 ES rabbit. D, Summarized echocardiographic parameters in 5 ES rabbits. LVEDd indicates left ventricular end-diastolic diameter; LVESd, left ventricular end-systolic diameter; FS, fractional shortening; IVS, interventricular septum; and PW, posterior wall of left ventricle. *P<0.05, **P<0.01.

Five ES rabbits were added to study whether W-7 rescues LV dysfunction. We obtained LV tissue samples from ES rabbits at the end of W-7 infusion for molecular studies. Figure 7C shows representative M-mode echocardiograms in 1 ES rabbit before and after 2-week W-7 infusion. Mean echocardiographic parameters in 5 ES rabbits are summarized in Figure 7D. LV end-diastolic and end-systolic diameters were decreased significantly, by 11% and 19%, respectively, and LV fractional shortening was increased to 30±2% from 23±2% before infusion (P=0.047). Representative immunoblots show that CaMKII-P bands were attenuated by W-7 in ES rabbits (Figure 8A). Overall, W-7 significantly reduced CaMKII-P expression and fractional CaMKII phosphorylation (Figure 8B). Because CaMKII promotes apoptosis and stimulates hypertrophic transcriptional programs, we examined whether the cardiomyopathic effects of CaMKII were inhibited by W-7. Compared with controls, ES rabbits showed significant increases in caspase-3 subunits (Figure 8C and 8D) and atrial natriuretic factor and brain natriuretic peptide mRNA expression (Figure 8E), which were not suppressed by W-7 treatment. These findings indicate that the beneficial effects of W-7 are not associated with suppression of biomarkers of apoptosis or cardiac hypertrophy.

Figure 8.

Figure 8. A, Immunoblots of autophosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII-P) and CaMKIIδ in left ventricular tissue samples from 1 control (CTL), 3 electrical storm (ES) rabbits, and 5 ES rabbits treated with W-7 (ES+W-7). B, Mean±SEM band intensities in control (n=6), ES (n=6), and ES+W-7 (n=5) rabbits. C and D, Immunoblots of procaspase-3 and caspase-3 subunits (activated forms) and mean data in control (n=6), ES (n=6), and ES+W-7 (n=5) rabbits. E, Mean±SEM mRNA expression of atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), and β-myosin heavy chain (β-MHC) in control (n=5), ES (n=5), and ES+W-7 (n=5) rabbits. *P<0.05, **P<0.01 vs control; ††P<0.01 vs ES rabbits.

Discussion

ES is a significant clinical problem with a substantial mortality rate, particularly in the ICD population.1 In this article, we report findings in an animal model of ES based on an arrhythmic substrate induced by severe bradycardia due to CAVB.13,14 ES, manifested as frequently recurrent VT/VF episodes, was observed in approximately half of CAVB rabbits equipped with ICDs and allowed us to assess associated changes in Ca2+-handling protein expression/phosphorylation. ES rabbits showed LV functional deterioration along with prominent alterations in phosphorylation of multiple proteins including CaMKII, Cav1.2, RyR2, and phospholamban, similar to findings in some human and animal models of HF.22 Repeated VF/defibrillation cycles reproduced ES-related changes in phosphorylation of CaMKII and phospholamban, suggesting that they may result from ES rather than simply being an associated finding. Calmodulin inhibition with W-7 suppressed VT/VF episodes and rescued LV dysfunction in association with a significant reduction in CaMKII hyperphosphorylation.

Relationship to Previous Studies in Related Arrhythmogenic Animal Models

The cellular and molecular bases of arrhythmogenic ventricular remodeling in CAVB rabbits have been characterized previously.13,14,23 CAVB rabbit cardiomyocytes show action potential duration (APD) prolongation and increases in cell shortening, systolic [Ca2+]i transients, and SR Ca2+ content.23 Downregulation of subunits encoding rapid and slow delayed-rectifier K+ current components underlies APD prolongation.13,14 CaMKII activation and Ca2+-handling alterations enhance contractile function but at the same time promote arrhythmogenic afterdepolarizations.23 SR Ca2+ load is increased by APD prolongation and enhanced SR Ca2+ uptake due to phospholamban hyperphosphorylation, leading to increased systolic Ca2+ release.

The findings in the present study of ES rabbits show some similarities and differences relative to previous work in shorter-term CAVB rabbits. QT prolongation and CaMKII hyperphosphorylation are prominent in both. However, phospholamban was dephosphorylated in ES rabbits, in contrast to phospholamban hyperphosphorylation in rabbits with 2-week CAVB. Perhaps as a result, contractility was reduced in ES rabbits, contrasting with hypercontractility in shorter-term CAVB. The discrepancies may be partly due to the effects of repeated VF/defibrillation, which directly induces phospholamban dephosphorylation (Figure 6B).

The striking CaMKII hyperphosphorylation (indicating CaMKII autophosphorylation and activation) and phospholamban dephosphorylation observed in the present study are likely key findings. CaMKII-P expression was increased 5.7-fold in ES rabbits, a level exceeding changes seen previously in other arrhythmogenic animal models, including CaMKIIδ-overexpressing mice (≈3-fold wild-type values),7 CaMKIV-overexpressing mice (CaMKII-P data not shown, but CaMKII activity increased ≈1.5-fold),8 rabbits with HF after aortic banding/regurgitation (≈1.5-fold),24 and 2-week CAVB rabbits (≈2.5-fold),23 whereas changes in non-ES rabbits remained comparable to previous reports. CaMKII activation causes RyR2 hyperphosphorylation, producing RyR diastolic Ca2+ leak that promotes both HF (because of loss of SR Ca2+) and arrhythmogenesis (via arrhythmogenic afterdepolarizations).22,25,26 CaMKII activation also increases LTCC phosphorylation, as observed in the present study, leading to arrhythmogenic increases in Ca2+ window current.23,25 Phospholamban phosphorylation removes phospholamban-induced SERCA inhibition, enhancing SR Ca2+ uptake and improving cardiac relaxation and contraction, whereas phospholamban dephosphorylation has opposite effects, impairing diastolic and systolic function.10,22 Thus, both CaMKII hyperphosphorylation and phospholamban hypophosporylation are likely to be important contributors to the contractile failure we observed in ES rabbits.

Extensive work implicates CaMKII hyperphosphorylation of RyR2 in arrhythmogenesis and defective Ca2+ handling.6,7,17,24 In a rabbit HF model, Ai et al24 recently showed CaMKII activation and RyR hyperphosphorylation at both Ser2809 (PKA/CaMKII) and Ser2815 (CaMKII) sites. HF cardiomyocytes had enhanced SR Ca2+ leak and reduced SR Ca2+ load, which improved with CaMKII inhibition. CaMKIIδ overexpression in rabbit cardiomyocytes also causes RyR2 hyperphosphorylation at both sites, as well as increased diastolic Ca2+ spark frequency.27 There have been disagreements about which RyR2 phosphorylation site(s) (eg, Ser2809, Ser2815, and/or others) induces diastolic SR Ca2+ leak. In the present study, ES rabbits showed RyR2 hyperphosphorylation at both sites, whereas non-ES rabbits demonstrated Ser2815 hyperphosphorylation alone, potentially contributing to the arrhythmic phenotype and LV dysfunction in ES rabbits.

The effect of CaMKII on LTCCs has also been implicated in arrhythmogenesis. Recently, Koval et al28 provided direct evidence that CaV1.2 β-subunits Thr498 and Leu493 are responsible for CaMKII actions to promote a high-activity Ca2+ channel gating mode (mode 2), CaV1.2 current facilitation, and early afterdepolarizations and that CaMKII actions at the β-subunit are sufficient to induce early afterdepolarizations in the absence of SR Ca2+ release. These findings, together with our results showing CaMKII activation and CaV1.2 hyperphosphorylation, suggest that LTCC reactivation might be a more central mechanism than RyR2 Ca2+ leak for ES in this model.

Mechanism of Postshock VF Reinitiation

Few studies have addressed mechanisms of postshock VF reinitiation. Zaugg et al12 showed in Langendorff-perfused rat hearts that VF-induced [Ca2+]i overload causes failure of electric defibrillation and postshock VF reinitiation. Successful defibrillation led to a reduction in [Ca2+]i load. Incomplete reversal of [Ca2+]i overload after defibrillation was followed by spontaneous [Ca2+]i oscillations and VF reinitiation. Impaired SERCA function caused by phospholamban dephosphorylation impairs diastolic Ca2+ clearance from the cytosol and, along with diastolic Ca2+ releases from hyperphosphorylated RyRs, promotes diastolic [Ca2+]i overload. Another mechanism proposed by Ogawa et al29 is that postshock APD abbreviation associated with unaltered [Ca2+]i transient duration enhances Na+-Ca2+ exchanger currents, increasing the likelihood of afterdepolarizations that reinduce VF. Recently, Wagner et al30 demonstrated that CaMKII differentially modulates K+ currents. Chronic CaMKIIδ overexpression in a transgenic mouse model prolonged APD by downregulating Kv4.2/KChIP2 and Kir2.1 responsible for Ito,fast and IK1, whereas acute CaMKIIδ overexpression shortened APD by enhancing IK1 and Ito,slow and accelerating Ito recovery in rabbit cardiomyocytes.

Novel Findings and Potential Significance

To our knowledge, this study is the first to report the molecular basis of ES in a clinically relevant chronic animal model. Many studies have described CaMKII activation as a signal for proarrhythmia and myocardial dysfunction,16,21,25,26 but ours is the first to implicate Ca2+/calmodulin/CaMKII signaling in ES pathophysiology. Not only was CaMKII hyperphosphorylation observed at functionally important CaMKII autophosphorylation, RyR2, and LTCC sites, but a therapeutic intervention targeting Ca2+/calmodulin interaction demonstrated ES suppression and LV dysfunction improvement along with a reduction in CaMKII hyperphosphorylation. However, the phosphorylation abnormalities associated with ES were more complex than would be expected from CaMKII activation alone. The catalytic subunit of PKA was downregulated, and protein phosphatases (PP1 and PP2A) were upregulated, accounting for profound hypophosphorylation of phospholamban. The phosphorylation state of phospholamban determines its association with and inhibition of SERCA2a. Phospholamban dephosphorylation reduces SERCA2a function and, in combination with diastolic SR Ca2+ leak due to RyR2 hyperphosphorylation, would be expected to reduce SR Ca2+ stores and impair contractility, potentially accounting for the LV function impairment that we observed in ES rabbits. These observations may account for the tendency of ES to induce myocardial pump failure, which is the principal cause of excess mortality in ES patients.1

Our results provide potential insights into the pathophysiology of ES. The initiating factors for ES are often cryptic, although psychological stress, acute myocardial ischemia, and electrolyte imbalances have all been implicated.1 We noted that repeated cycles of VF/defibrillation by themselves cause CaMKII hyperphosphorylation, a central component of CaMKII overactivity that favors arrhythmia occurrence and myocardial dysfunction.6,8 It is very possible that repeated VF/defibrillation cycles induce a positive-feedback cycle that enhances the probability of recurrent VF episodes and exacerbates contractile failure. This notion is supported by successful therapeutic effects of W-7.

Our findings also have potential implications for the clinical management of ES. Drug therapy of ES is problematic, with conventional antiarrhythmic agents providing little benefit and β-blockers and amiodarone seeming to be of the greatest value.1 Adrenergic receptor stimulation activates CaMKII68; thus, antagonism of adrenergic activation of CaMKII may account for the beneficial actions of β-blockers and amiodarone. Our results also point to potential new approaches to treating and/or preventing ES. A Ca2+/calmodulin interaction inhibitor (W-7) was highly effective in suppressing ES in our study. Interventions targeting the Ca2+/calmodulin/CaMKII system may prove highly beneficial in preventing both further arrhythmic episodes and hemodynamic deterioration in ES patients.

Limitations

We focused on molecular changes underlying ES, performing detailed immunoblotting studies of Ca2+-handling proteins in ES and non-ES rabbits. Because of the long preparation time and high mortality rates in this model, the number of available ES and non-ES rabbits was limited, and therefore we were unable to perform extensive functional studies of the cellular mechanisms predicted to result from protein phosphorylation disturbances.

The precise mechanisms underlying the prominent phospholamban dephosphorylation observed in ES rabbits remain to be determined. Further studies on subcellular distribution of PP1 are needed to understand why phospholamban was dephosphorylated, a change opposite that seen for RyR2 and LTCC, which were hyperphosphorylated. Specific macromolecular compartmentalization of kinases, phosphatases, and target proteins is likely involved.

Long-term CAVB rabbits studied here had lower LV fractional shortening than at baseline, which is in contrast to a dog model of CAVB in which contractile function remains preserved.31 The ventricular rate decreases comparably, from ≈280 bpm in sinus rhythm to 90 to 115 bpm in escape rhythm in rabbits versus from ≈115 to 40 to 50 bpm in dogs. The discrepancy in cardiac function response may be due to different species-dependent adaptation mechanisms or to greater spontaneous VT/VF occurrence in rabbits.

The detailed electrophysiological mechanisms underlying VF storm, particularly related to the relative roles of afterdepolarization-related triggered activity versus reentry, remain to be defined. These issues go beyond the scope of the present study. We corrected QT intervals by the Carlsson formula for rabbits, the validity of which was not tested under the bradycardic conditions of this study. We primarily examined LV tissue and function because LV function appears to be the principal factor associated with the occurrence and consequences of ES. We found that CaMKII activation, the central change involved in ES pathophysiology, occurs in the right ventricle as well as the LV, but we did not otherwise examine the right ventricle in detail.

Acknowledgments

The authors thank Medtronic Japan for providing ICDs.

Sources of Funding

This work was supported by funds from Japan Society for the Promotion of Science (18890081, 22590777), Suzuken Memorial Foundation, Takeda Science Foundation, and Mitsubishi Pharma Research Foundation (to Dr Tsuji); by the Canadian Institutes of Health Research (grant MOP 68929 to Dr Nattel); and by a Leducq Foundation Network Grant (07/CVD/03 to Drs Dobrev and Nattel).

Disclosures

None.

Footnotes

The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.110.016683/DC1.

Correspondence to Yukiomi Tsuji, MD,
Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan
. E-mail

References

  • 1. Huang DT, Traub D. Recurrent ventricular arrhythmia storms in the age of implantable cardioverter defibrillator therapy: a comprehensive review. Prog Cardiovasc Dis. 2008; 51: 229–236.CrossrefMedlineGoogle Scholar
  • 2. Exner DV, Pinski SL, Wyse DG, Renfroe EG, Follmann D, Gold M, Beckman KJ, Coromilas J, Lancaster S, Hallstrom AP. Electrical storm presages nonsudden death : the Antiarrhythmics Versus Implantable Defibrillators (AVID) trial. Circulation. 2001; 103: 2066–2071.LinkGoogle Scholar
  • 3. Sesselberg HW, Moss AJ, McNitt S, Zareba W, Daubert JP, Andrews ML, Hall WJ, McClinitic B, Huang DT. Ventricular arrhythmia storms in postinfarction patients with implantable defibrillators for primary prevention indications: a MADIT-II substudy. Heart Rhythm. 2007; 4: 1395–1402.CrossrefMedlineGoogle Scholar
  • 4. Antos CL, Frey N, Marx SO, Reiken S, Gaburjakova M, Richardson JA, Marks AR, Olson EN. Dilated cardiomyopathy and sudden death resulting from constitutive activation of protein kinase A. Circ Res. 2001; 89: 997–1004.LinkGoogle Scholar
  • 5. Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N, Marks AR. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell. 2000; 101: 365–376.CrossrefMedlineGoogle Scholar
  • 6. Maier LS, Zhang T, Chen L, DeSantiago J, Brown JH, Bers DM. Transgenic CaMKIIδC overexpression uniquely alters cardiac myocyte Ca2+ handling: reduced SR Ca2+ load and activated SR Ca2+ release. Circ Res. 2003; 92: 904–911.LinkGoogle Scholar
  • 7. Zhang T, Maier LS, Dalton ND, Miyamoto S, Ross J, Bers DM, Brown JH. The δC isoform of CaMKII is activated in cardiac hypertrophy and induces dilated cardiomyopathy and heart failure. Circ Res. 2003; 92: 912–919.LinkGoogle Scholar
  • 8. Wu Y, Temple J, Zhang R, Dzhura I, Zhang W, Trimble R, Roden DM, Passier R, Olson EN, Colbran RJ, Anderson ME. Calmodulin kinase II and arrhythmias in a mouse model of cardiac hypertrophy. Circulation. 2002; 106: 1288–1293.LinkGoogle Scholar
  • 9. Braz JC, Gregory K, Pathak A, Zhao W, Sahin B, Klevitsky R, Kimball TF, Lorenz JN, Nairn AC, Liggett SB, Bodi I, Wang S, Schwartz A, Lakatta EG, Paoli-Roach AA, Robbins J, Hewett TE, Bibb JA, Westfall MV, Kranias EG, Molkentin JD. PKC-alpha regulates cardiac contractility and propensity toward heart failure. Nat Med. 2004; 10: 248–254.CrossrefMedlineGoogle Scholar
  • 10. Champion HC, Kass DA. Calcium handler mishandles heart. Nat Med. 2004; 10: 239–240.CrossrefMedlineGoogle Scholar
  • 11. Koretsune Y, Marban E. Cell calcium in the pathophysiology of ventricular fibrillation and in the pathogenesis of postarrhythmic contractile dysfunction. Circulation. 1989; 80: 369–379.LinkGoogle Scholar
  • 12. Zaugg CE, Wu ST, Barbosa V, Buser PT, Wikman-Coffelt J, Parmley WW, Lee RJ. Ventricular fibrillation-induced intracellular Ca2+ overload causes failed electrical defibrillation and post-shock reinitiation of fibrillation. J Mol Cell Cardiol. 1998; 30: 2183–2192.CrossrefMedlineGoogle Scholar
  • 13. Tsuji Y, Opthof T, Yasui K, Inden Y, Takemura H, Niwa N, Lu Z, Lee JK, Honjo H, Kamiya K, Kodama I. Ionic mechanisms of acquired QT prolongation and torsades de pointes in rabbits with chronic complete atrioventricular block. Circulation. 2002; 106: 2012–2018.LinkGoogle Scholar
  • 14. Tsuji Y, Zicha S, Qi XY, Kodama I, Nattel S. Potassium channel subunit remodeling in rabbits exposed to long-term bradycardia or tachycardia: discrete arrhythmogenic consequences related to differential delayed-rectifier changes. Circulation. 2006; 113: 345–355.LinkGoogle Scholar
  • 15. Carlsson L, Abrahamsson C, Andersson B, Duker G, Schiller-Linhardt G. Proarrhythmic effects of the class III agent almokalant: importance of infusion rate, QT dispersion, and early afterdepolarisations. Cardiovasc Res. 1993; 27: 2186–2193.CrossrefMedlineGoogle Scholar
  • 16. Mazur A, Roden DM, Anderson ME. Systemic administration of calmodulin antagonist W-7 or protein kinase A inhibitor H-8 prevents torsade de pointes in rabbits. Circulation. 1999; 100: 2437–2442.LinkGoogle Scholar
  • 17. Wehrens XHT, Lehnart SE, Reiken SR, Marks AR. Ca2+/calmodulin-dependent protein kinase II phosphorylation regulates the cardiac ryanodine receptor. Circ Res. 2004; 94: e61–e70.LinkGoogle Scholar
  • 18. Witcher DR, Kovacs RJ, Schulman H, Cefali DC, Jones LR. Unique phosphorylation site on the cardiac ryanodine receptor regulates calcium channel activity. J Biol Chem. 1991; 266: 11144–11152.CrossrefMedlineGoogle Scholar
  • 19. Hagemann D, Xiao RP. Dual site phospholamban phosphorylation and its physiological relevance in the heart. Trends Cardiovasc Med. 2002; 12: 51–56.CrossrefMedlineGoogle Scholar
  • 20. El-Armouche A, Pamminger T, Ditz D, Zolk O, Eschenhagen T. Decreased protein and phosphorylation level of the protein phosphatase inhibitor-1 in failing human hearts. Cardiovasc Res. 2004; 61: 87–93.CrossrefMedlineGoogle Scholar
  • 21. Anderson ME, Braun AP, Wu Y, Lu T, Wu Y, Schulman H, Sung RJ. KN-93, an inhibitor of multifunctional Ca++/calmodulin-dependent protein kinase, decreases early afterdepolarizations in rabbit heart. J Pharmacol Exp Ther. 1998; 287: 996–1006.MedlineGoogle Scholar
  • 22. Nattel S, Maguy A, Le Bouter S, Yeh YH. Arrhythmogenic ion-channel remodeling in the heart: heart failure, myocardial infarction, and atrial fibrillation. Physiol Rev. 2007; 87: 425–456.CrossrefMedlineGoogle Scholar
  • 23. Qi X, Yeh YH, Chartier D, Xiao L, Tsuji Y, Brundel BJ, Kodama I, Nattel S. The calcium/calmodulin/kinase system and arrhythmogenic afterdepolarizations in bradycardia-related acquired long-QT syndrome. Circ Arrhythm Electrophysiol. 2009; 2: 295–304.LinkGoogle Scholar
  • 24. Ai X, Curran JW, Shannon TR, Bers DM, Pogwizd SM. Ca2+/calmodulin-dependent protein kinase modulates cardiac ryanodine receptor phosphorylation and sarcoplasmic reticulum Ca2+ leak in heart failure. Circ Res. 2005; 97: 1314–1322.LinkGoogle Scholar
  • 25. Anderson ME. Multiple downstream proarrhythmic targets for calmodulin kinase II: moving beyond an ion channel-centric focus. Cardiovasc Res. 2007; 73: 657–666.CrossrefMedlineGoogle Scholar
  • 26. Maier LS, Bers DM. Role of Ca2+/calmodulin-dependent protein kinase (CaMK) in excitation-contraction coupling in the heart. Cardiovasc Res. 2007; 73: 631–640.CrossrefMedlineGoogle Scholar
  • 27. Kohlhaas M, Zhang T, Seidler T, Zibrova D, Dybkova N, Steen A, Wagner S, Chen L, Heller Brown J, Bers DM, Maier LS. Increased sarcoplasmic reticulum calcium leak but unaltered contractility by acute CaMKII overexpression in isolated rabbit cardiac myocytes. Circ Res. 2006; 98: 235–244.LinkGoogle Scholar
  • 28. Koval OM, Guan X, Wu Y, Joiner ML, Gao Z, Chen B, Grumbach IM, Luczak ED, Colbran RJ, Song LS, Hund TJ, Mohler PJ, Anderson ME. CaV1.2 beta-subunit coordinates CaMKII-triggered cardiomyocyte death and afterdepolarizations. Proc Natl Acad Sci U S A. 2010; 107: 4996–5000.CrossrefMedlineGoogle Scholar
  • 29. Ogawa M, Morita N, Tang L, Karagueuzian HS, Weiss JN, Lin SF, Chen PS. Mechanisms of recurrent ventricular fibrillation in a rabbit model of pacing-induced heart failure. Heart Rhythm. 2009; 6: 784–792.CrossrefMedlineGoogle Scholar
  • 30. Wagner S, Hacker E, Grandi E, Weber SL, Dybkova N, Sossalla S, Sowa T, Fabritz L, Kirchhof P, Bers DM, Maier LS. Ca/calmodulin kinase II differentially modulates potassium currents. Circ Arrhythm Electrophysiol. 2009; 2: 285–294.LinkGoogle Scholar
  • 31. Oros A, Beekman JD, Vos MA. The canine model with chronic, complete atrio-ventricular block. Pharmacol Ther. 2008; 119: 168–178.CrossrefMedlineGoogle Scholar

Clinical Perspective

Electrical storm (ES), characterized by recurrent ventricular tachycardia/fibrillation, is an increasing problem among implantable cardioverter-defibrillator patients. Despite acute cessation of ES with medical therapy and/or catheter ablation, the early mortality (within a few months of ES) is high and often nonsudden, involving progressive heart failure in particular. Underlying mechanisms are unknown. Defective Ca2+ handling is central to the pathogenesis of heart failure. Changes in phosphorylation of L-type Ca2+ channels, sarcoplasmic reticulum Ca2+-release channel ryanodine receptors, and the sarcoplasmic reticulum Ca2+ uptake regulator protein phospholamban are linked to heart failure–related mechanical dysfunction and arrhythmogenic afterdepolarizations. To study the molecular basis of ES-related cardiac deterioration, we studied rabbits with chronic complete atrioventricular block equipped with implantable cardioverter-defibrillators. They developed QT prolongation and implantable cardioverter-defibrillator–detected ventricular fibrillation episodes, with ES (defined as clustered, frequently recurrent ventricular fibrillation episodes) occurring in ≈50%. ES rabbits showed left ventricular function deterioration, along with striking activation of the Ca2+-sensitive phosphorylating enzyme Ca2+/calmodulin-dependent protein kinase II (CaMKII) and enhanced protein phosphatase expression. These alterations produced important changes in phosphorylation patterns, notably hyperphosphorylation of L-type Ca2+ channels and ryanodine receptors and dephosphorylation of phospholamban, which could explain arrhythmias and impaired contractility. Repeated ventricular fibrillation induction/defibrillation with implantable cardioverter-defibrillator shocks in control rabbits reproduced ES-related changes in CaMKII and phospholamban phosphorylation. Infusion of the calmodulin antagonist W-7 suppressed ventricular tachycardia/ventricular fibrillation episodes and rescued left ventricular dysfunction in ES rabbits, indicating a central pathophysiological role of CaMKII activation. These results strongly support the notion that CaMKII activation and Ca2+-handling abnormalities resulting from ES events might be responsible for negative outcomes and suggest that interventions targeting the Ca2+/calmodulin/CaMKII system might provide benefits in ES patients.

eLetters(0)

eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate.

Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page.