Sarcoplasmic Reticulum Calcium Overloading in Junctin Deficiency Enhances Cardiac Contractility but Increases Ventricular Automaticity
Background— Abnormal sarcoplasmic reticulum calcium (Ca) cycling is increasingly recognized as an important mechanism for increased ventricular automaticity that leads to lethal ventricular arrhythmias. Previous studies have linked lethal familial arrhythmogenic disorders to mutations in the ryanodine receptor and calsequestrin genes, which interact with junctin and triadin to form a macromolecular Ca-signaling complex. The essential physiological effects of junctin and its potential regulatory roles in sarcoplasmic reticulum Ca cycling and Ca-dependent cardiac functions, such as myocyte contractility and automaticity, are unknown.
Methods and Results— The junctin gene was targeted in embryonic stem cells, and a junctin-deficient mouse was generated. Ablation of junctin was associated with enhanced cardiac function in vivo, and junctin-deficient cardiomyocytes exhibited increased contractile and Ca-cycling parameters. Short-term isoproterenol stimulation elicited arrhythmias, including premature ventricular contractions, atrioventricular heart block, and ventricular tachycardia. Long-term isoproterenol infusion also induced premature ventricular contractions and atrioventricular heart block in junctin-null mice. Further examination of the electrical activity revealed a significant increase in the occurrence of delayed afterdepolarizations. Consistently, 25% of the junctin-null mice died by 3 months of age with structurally normal hearts.
Conclusions— Junctin is an essential regulator of sarcoplasmic reticulum Ca release and contractility in normal hearts. Ablation of junctin is associated with aberrant Ca homeostasis, which leads to fatal arrhythmias. Thus, normal intracellular Ca cycling relies on maintenance of junctin levels and an intricate balance among the components in the sarcoplasmic reticulum quaternary Ca-signaling complex.
Received July 28, 2006; accepted November 10, 2006.
One of the most prevalent causes for premature death in Western countries is lethal ventricular arrhythmias. The proximate cause of death in approximately half of all heart failure patients is ventricular tachycardia or fibrillation,1 and polymorphic ventricular tachycardia is responsible for unexpected exercise-induced sudden death in children and adolescents with no overt evidence of heart disease.2 It has been shown that abnormal automaticity plays a great role in the induction and maintenance of different types of ventricular arrhythmias in patients with either structurally normal hearts or severe structural diseases.3–5 One type of the abnormal automaticity, known as “triggered activity,” induces ventricular arrhythmias through activation of delayed afterdepolarizations (DADs),6 the spontaneously occurring secondary depolarizations sometimes observed in abnormal cardiomyocytes after complete repolarization.7 Although the precise biochemical mechanisms that lead to propagation of arrhythmias by DADs are not entirely known, altered ryanodine receptor (RyR)–mediated calcium release has been implicated as a potentially important pathological mediator.8
Clinical Perspective p 309
Ca release by RyRs occurs subsequent to their activation by the L-type Ca channel–mediated inward Ca current. It has been proposed that RyRs are not only activated by cytosolic Ca during excitation-contraction coupling but also by increases in sarcoplasmic reticulum (SR) luminal Ca,9 which is itself dependent on proteins, including calsequestrin (CSQ), triadin, and junctin,10 that together with RyR constitute the macromolecular SR Ca-regulating complex.11 Within this complex, CSQ can act as a Ca sensor for RyRs as follows: Decreased SR luminal [Ca] (and therefore Ca-bound CSQ) during Ca-induced Ca release permits the association of junctin and triadin with CSQ, which inhibits RyR channel activity.8,10 Thus, CSQ, triadin, and junctin appear to be critical for normal regulation of RyR-mediated Ca release. Consistent with this notion, mutations in CSQ are associated with the development of DAD-triggered ventricular tachycardia and sudden death, and forced expression of triadin in cardiomyocytes decreases the threshold for DAD-related arrhythmias.12,13
Less is known about junctin, another component of the RyR protein complex. Junctin and triadin exhibit 60% to 70% amino acid homology in their transmembrane domains, including repeated KEKE motifs important for macromolecular protein–protein interactions within their SR luminal tails.14 The effects on cardiac function of both junctin and triadin have been examined with transgenic overexpression strategies in mouse hearts, which exhibit varying phenotypes ranging from mild SR structural alterations, prolongation of Ca transient decay, impaired relaxation, and cardiac hypertrophy and/or heart failure.15–18 Although demonstrating that these components of the RyR complex can modify SR Ca cycling and Ca-dependent cardiac function, the potential for promiscuous or nonspecific activity with protein overexpression precludes a clear assessment of the physiological/pathophysiological role of junctin in normal SR Ca cycling. Therefore, the present study generated junctin-deficient mice to elucidate its essential functional roles in the in vivo heart. We find that junctin gene ablation enhances SR Ca cycling and contractility but is associated with DAD-induced arrhythmias and premature mortality under conditions of physiological stress.
Generation of Targeted Mice
The murine junctin gene was isolated from the PAC (P1-derived Artificial Chromosome) genomic library by screening it with mouse junctin cDNA. The targeting strategy was designed to replace exons 1 and 2 with a neomyocin (neo)-resistance gene. The generation of the targeting construct, targeting of embryonic stem cells, and generation of junctin homozygous mice were performed in Gene-Targeting Core at the University of Cincinnati, Cincinnati, Ohio, as described previously.19 Animals used in the present study were littermates at 12 to 14 weeks of age, unless specified otherwise. There were no differences between males and females in any of the parameters examined; thus, both sexes were used in the present study. All the animals were housed at the University of Cincinnati and handled and maintained according to protocols by the ethics committee of the University of Cincinnati. The investigation conformed to the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health.
Quantitative Immunoblotting Analysis of Cardiac Ca-Handling Protein Levels
Mice were anesthetized, and hearts were excised, washed with ice-cold PBS, and then quickly frozen in liquid nitrogen before homogenization. To assess the levels of Ca-cycling proteins in junctin-knockout (KO) hearts, quantitative immunoblotting was performed.20,21 The antibody to histidine-rich Ca-binding protein was a generous gift from Dr Woo Jin Park (Gwangju Institute of Science and Technology, Gwangju, Korea); the junctin antibody was homemade and specific to the last 15 C-terminal residues of mouse junctin; the polyclonal antibody to SR Ca-ATPase was homemade; the polyclonal antibody to phosphorylated RyR at serine 2809 was a generous gift from Dr Andrew R. Marks (Columbia University, New York, NY); and the polyclonal antibodies to CaMKII and CaMKII-δ were generous gifts from Dr Harold A. Singer (Albany Medical College, Albany, NY). All the other antibodies were purchased from Affinity Bioreagents (Golden, Colo). For immunoblotting of triadin, a specific polyclonal antibody to residues 146 to 160 of mouse triadin 1 was used, and immunoblotting was performed as described previously.18 Protein levels were determined with AlphaEaseFC software (Alpha Innotech, San Leandro, Calif).
Isolated Myocyte Mechanics and Ca Kinetics
Isolation of mouse ventricular myocytes and measurements of mechanics and Ca kinetics were performed in the absence or presence of isoproterenol 100 nmol/L at 0.5 Hz.20
Electrophysiology of Isolated Left Ventricular Myocytes
The L-type Ca current and the Na-Ca exchanger (NCX) current were recorded from isolated ventricular myocytes with the whole-cell patch-clamp technique with an Axopatch-200B amplifier (Axon Instruments, Foster City, Calif), as described previously.20,22
SR Ca Uptake
SR Ca uptake in whole-heart homogenates was measured by a modification of the Millipore (Billerica, Mass) filtration technique.19
Induction of Aftercontractions and DADs in Isolated Cardiomyocytes
Rod-shaped ventricular myocytes, which exhibited no spontaneous activity at rest, were paced at 5 Hz in the presence of 1 μmol/L isoproterenol in 1.8 mmol/L Ca-Tyrode’s solution at room temperature. After 2 to 3 trains of stimulations, pacing was stopped to allow the recording of spontaneous aftercontractions within 2 to 5 seconds. Ca transients were examined with the same protocol. Ryanodine (10−7 or 10−6 mol/L) was applied to myocytes once aftercontractions were observed. Action potentials were recorded under current-clamp mode and triggered by 2-ms just-threshold current steps at a frequency of 5 Hz in the presence of isoproterenol 1 μmol/L at 32°C. Cells were then rested to determine the presence of DADs.
Measurement of Ca Sparks
To assess the effects of junctin ablation on RyR activity, Ca sparks were measured in permeabilized cardiomyocytes as described previously,23,24 except that the free Ca was 50 nmol/L.
Surface ECG Recordings After Injection of Isoproterenol
Mice were anesthetized with pentobarbital 90 mg/kg, and the ECG was monitored on an ECG recording system (PowerLab, ADInstruments, Colorado Springs, Colo) after the mice were injected with isoproterenol 0.25 μg/g IP (Sigma, St. Louis, Mo).
Telemetry Recordings After Implantation of Isoproterenol Minipumps
A telemetry transmitter (EA-F20, Data Science International, St Paul, Minn) and an Alzet 2002 pump (Braintree Scientific, Inc, Braintree, Mass) that can release isoproterenol at a rate of 15 mg · kg−1 · d−1 for 2 weeks were implanted. Subsequently, ECG recordings were monitored for 2 weeks. Values from ECG (R-Rint, P-wave duration, PRint, QRS, and QTint) were analyzed with Dataquest A.R.T. software (EA-F20, Data Science International).
Assessment of In Vivo Cardiac Function With Echocardiography
Echocardiography was performed to examine cardiac contractile function in a noninvasive manner.20
All data are expressed as mean±SEM. Comparison between groups was evaluated with the Student t test. Protein levels in wild-type (WT) and KO hearts were also compared with 1-way ANOVA. For the studies that used isolated myocytes, 5 to 10 cells per heart were studied; n indicated the number of hearts. Aftercontractions and DADs were analyzed with χ2 test. Probability values of <0.05 were considered significant.
The authors had full access to the data and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
Ablation of Junctin In Vivo by Gene Targeting
The physiological role of junctin in vivo was investigated with gene targeting to ablate its expression in the intact mouse (Figure 1A, 1B, and 1C). First, the PAC genomic library was screened with mouse junctin cDNA. One of 5 positive clones, which contained all 5 exons of the junctin gene, was sequenced, characterized (Figure 1A), and used to generate the targeting construct, which was designed to delete exons 1 and 2 of the junctin gene (Figure 1B and 1C). Heterozygous mice were interbred to produce the homozygous deficient mice, which were screened by polymerase chain reaction (online-only Data Supplement Figure IB and IC). Offspring exhibited the expected mendelian ratios, and the homozygous junctin-KO mice were viable, without any overt abnormalities. Quantitative immunoblotting revealed that there was no junctin protein detected in homozygous hearts (Figure 1D).
Because junctin interacts with CSQ, triadin, and the RyR, ablation of junctin may result in compensation from 1 or more of its partners in the SR Ca-cycling complex.11 Quantitative immunoblotting, however, did not reveal any significant changes in the levels of RyR, phosphorylation of RyR at serine 2809, triadin, or calsequestrin in junctin-KO hearts. Likewise, there were no alterations in the histidine-rich Ca-binding protein, FKBP12.6, SR Ca-ATPase, or phospholamban levels (Figure 1D and 1E). Because junctin ablation may alter SR Ca homeostasis, we also examined SR Ca-transport activity in KO and WT hearts. There were no significant changes in Vmax (58.6±5.6 versus 60.6±8.7 nmol Ca · mg−1 · min−1 in WT versus junctin-KO hearts; n=6) or EC50 (279±15 versus 274±12 nmol/L in WT versus KO hearts, respectively; n=6) of the SR Ca transport for Ca (online Data Supplement, Figure II). Further assessment of the major sarcolemmal Ca-cycling proteins indicated no alterations in L-type Ca-channel protein level on junctin ablation (Figure 1D and 1E). Moreover, the L-type Ca currents were recorded in isolated WT and junctin-KO ventricular myocytes by whole-cell voltage clamp. There were no differences in membrane capacitance (WT: 174.4±11.2 pF, n=23; KO: 174±7.5 pF, n=27 myocytes) or average peak current density (−9.4±0.6 and −9.2±0.4 pA/pF, WT versus KO; Figure 2A and 2B). There were no differences in the average current-voltage relationships of the L-type Ca currents (Figure 2B) and kinetic properties of the Ca current (data not shown) between WT and KO cells; however, the current levels of NCX in junctin-KO myocytes were significantly enhanced. The average NCX density at 80 mV was increased by 67% in junctin-KO cells (1.06±0.09 pA/pF) compared with WT cells (0.64±0.07 pA/pF; Figure 2E). Consistent with these findings, there was a 70% increase in NCX protein levels in junctin-KO hearts (Figure 2F and 2G). Thus, ablation of junctin was not associated with alterations in any of the major Ca-cycling proteins except the NCX.
Increased Contractility in Junctin-Deficient Mice
Because we proposed that junctin is important for the regulation of SR Ca cycling, it was important to determine whether junctin deficiency had any effects on overall cardiac contractility. Cardiac echocardiography of intact mice indicated that ejection time was significantly abbreviated (47.2±1.91 versus 40.4±0.62 seconds, P<0.05), whereas the fractional shortening (35.7±1.93 versus 42.5±2.54, P<0.05) and the rate-corrected velocity of circumferential fiber shortening (8.1±0.59 versus 10.5±0.58 circumference/s, P<0.05) were markedly increased by 19% and 30%, respectively, in junctin-KO mice compared with WTs (mean±SEM; WT, n=9; KO, n=11). Consistent with these findings, assessment of the mechanical parameters and Ca transients in isolated cardiomyocytes, which represent a mechanically unloaded preparation, revealed that junctin ablation resulted in significant increases in the fractional shortening (51%), +dL/dt (66%), and −dL/dt (89%; Figure 3A, 3B, 3C, and 3D). Analysis of the Ca transients showed that there were no significant changes in diastolic Ca concentration; however, the Ca peak was increased by 54% in junctin-KO myocytes (Figure 3E and 3F), and the time constant of Ca transient decay (τ) was significantly shorter than in WTs (Figure 3G). The amplitude of caffeine-induced Ca release was also increased by 30% (Figure 3H and 3I), which indicates a higher SR Ca content in junctin-deficient cells. Furthermore, the τ of caffeine-induced Ca release was significantly abbreviated in junctin-KO cells compared with WTs (Figure 3J), consistent with the increased NCX activity in these cardiomyocytes. The present data are in agreement with those obtained from transgenic mice overexpressing junctin in cardiac muscle, which exhibited opposite phenotypic alterations: depressed contractile function, Ca kinetics, and SR Ca content.16,25
To examine the effects of β-adrenergic agonists, isolated cardiomyocytes were subjected to maximal isoproterenol (100 nmol/L) stimulation, and the contractile parameters and Ca kinetics were evaluated. Isoproterenol stimulation was associated with significant increases in contractile parameters, including ±dL/dt and fractional shortening in both WT and junctin-deficient myocytes. Although the relative increases were smaller in the KO cells, the maximally stimulated parameters were similar between WTs and KOs (Figure 3B, 3C, and 3D). Similarly, Ca transient amplitude and τ were significantly stimulated in both groups, and the maximally stimulated parameters were not different between WT and KO cells (Figure 3F and 3G). The apparently blunted responses of the KO cardiomyocytes to isoproterenol stimulation may be due to their highly elevated basal levels, which allows smaller further increases by β-adrenergic stimulus, similar to the phospholamban KO models.26
Ca Sparks on Ablation of Junctin
The increases in SR Ca load and luminal [Ca] have been shown to stimulate the open probability of the RyR channel, which results in increased spontaneous SR Ca release.27 Ca sparks, the local and temporally restricted fluorescence Ca signals that represent the coordinated openings of RyRs gating in situ, were examined. Experiments in permeabilized cells, although not entirely physiological, resolve more directly the effect of junctin ablation on RyR gating. In the absence of external Ca fluxes (no L-type Ca channel or NCX contribution), in permeabilized cells, the excitation-contraction coupling machinery is practically reduced to SR Ca2+ release and uptake, with the greatest regulation of these 2 events dictated by RyR/luminal proteins and SR Ca2+ ATPase/phospholamban, respectively. Cell permeabilization also allows for controlled buffering of the internal medium, which dictates the level of SR Ca2+ load. Because, as we show in the present study, WT and junctin-KO cardiomyocytes exhibit dissimilar NCX activity (Figure 2E) but similar Ca uptake rates (online Data Supplement, Figure II), experiments in permeabilized cells resolve directly the effect of junctin ablation on RyR gating without the compound effects of unequal Ca removal processes. Thus, we examined Ca sparks in saponin-permeabilized myocytes. Representative line-scan images of Ca sparks acquired at a constant cytosolic [Ca] of ≈50 nmol/L and their surface plots, obtained by averaging multiple individual events, are illustrated in Figure 4A and 4B for WT and junctin-KO cells. In WT myocytes, spontaneous Ca sparks occurred with an average frequency of 0.187±0.018 sparks/μm · s. In myocytes from junctin-null hearts, spark frequency was increased to 0.266±0.010 sparks/μm · s (Figure 4C). Furthermore, the event amplitude was significantly increased in junctin-KO cells with respect to WTs (2.19±0.01 and 1.76±0.01; Figure 4D), but duration was decreased (35.452±0.543 and 33.433±0.304 ms; Figure 4E). Ca spark width was unchanged (Figure 4F). The decreased duration of Ca sparks in junctin-KO cells likely indicates faster termination of the Ca release event once [Ca] underneath the active RyRs drops to subthreshold levels. The increased spark frequency and amplitude strongly suggest that SR Ca load is increased in junctin-KO cells, in agreement with our findings in cardiomyocytes (Figure 3H and 3I).
Premature Death in Junctin-KO Mice
It was unexpected that ≈25% of the junctin-KO mice died by 3 months of age, and >50% of them died before 12 months of age, which is the mid point of the average mouse life span (24 to 30 months; Figure 5); there were no deaths among WT control mice up to 12 months of age, however. Histological examination of cardiac muscle did not reveal any cardiac morphological alterations or structural abnormalities in junctin-null hearts at 3 months of age (online Data Supplement, Figure IV). Furthermore, the heart weight/body weight ratio was not different between WT and KO mice (online Data Supplement, Table II), and there were no increases in hypertrophic signaling cascades, including p38, ERK, calcineurin, CaMKII, and Akt (data not shown) in the KO hearts.
In Vivo Cardiac Arrhythmias Induced by Isoproterenol Stimulation
The sporadic premature death without notable cardiac pathology suggested that junctin-deficient mice may be predisposed to arrhythmias. Thus, catecholaminergic-induced stress, such as isoproterenol stimulation, would be expected to trigger arrhythmias in junctin-KO mice. To test this hypothesis, the surface ECG was monitored on short-term intraperitoneal injection of isoproterenol or after implantation of an isoproterenol minipump with a telemetry system. Short-term isoproterenol administration (0.25 μg/g IP) was associated with ventricular tachycardia in junctin-null mice (Figure 6A). Frequent premature ventricular contractions and atrioventricular heart block were also observed (data not shown). However, WT mice showed only benign and expected arrhythmias, such as sinus tachycardia and infrequent atrial premature contractions. Similarly, telemetry studies under long-term isoproterenol stimulation revealed cardiac arrhythmias that included premature ventricular contractions (Figure 6B), sinus asystole, and atrioventricular heart block (online Data Supplement, Figure V). The predominant arrhythmias were premature ventricular contractions, which occurred in isolation and as couplets over 2 weeks.
Stress-Induced Aftercontractions and DADs in Junctin-Null Cardiomyocytes
We postulated that in vivo catecholaminergic-elicited arrhythmias might be the consequence of increased RyR spontaneous Ca release in the junctin-null mice. If this was the case, arrhythmias could also be observed in isolated myocytes under stress, and inhibition or stabilization of the RyR activity could block these events. To test our hypothesis, junctin-null and WT cardiomyocytes were subjected to 2 to 3 trains of 5-Hz field stimulation in the absence or presence of isoproterenol. Increased stimulation did not cause any disturbance in contractile cycles in WT cardiac myocytes, whereas there were aftercontractions in ≈16±6% of junctin-deficient cells (Figure 7B). Inclusion of isoproterenol resulted in aftercontractions in 54±13% of the junctin-null cells, compared with 8±3% of WT cells (Figure 7A and 7B). Importantly, incubation of WT cells with 10−7 mol/L ryanodine or junctin-KO cells with 10−6 mol/L ryanodine completely blocked the aftercontractions within 9 to 15 minutes, which suggests that the aftercontractions were associated with aberrant discharge of SR Ca through the RyR channel. Consistent with the increase in aftercontractions, further investigation of the Ca transients at 5 Hz in the presence of isoproterenol 1 μmol/L showed that 60±14% of the junctin-KO cells exhibited Ca aftertransients, whereas only 9±3% of the WT cells developed abnormal Ca transients after the electrical stimulation was stopped (Figure 7C).
Previous studies indicated that aberrant RyR Ca release may induce arrhythmias by activation of DADs.8 To determine whether the mechanism for the arrhythmias elicited by junctin ablation is related to DADs, electrical activity was examined under increased frequency of stimulation (5 Hz) in the absence or presence of isoproterenol, as well as at 2 to 5 seconds after termination of the stimulation in separate experiments. In the absence of isoproterenol, 32±7% of the junctin-KO cells exhibited DADs compared with 6±4% of the WT cells (Figure 7E). In the presence of isoproterenol, DADs were observed in 90±5% of the junctin-deficient cells, and among those, 72% were suprathreshold. However, only 21±2% of WT cells developed DADs (Figure 7D and 7E), and 43% of them were suprathreshold, which suggests that the increased frequency of DADs may underlie the mechanisms for the elicited arrhythmias in the junctin-null hearts.
Herein, we used in vivo genetic techniques to establish that junctin is essential for normal cardiomyocyte SR Ca cycling, for Ca-dependent cardiac inotropy, and to suppress DAD-triggered arrhythmias that can lead to sudden death. Ablation of junctin was associated with enhanced SR Ca cycling in cardiomyocytes and increased cardiac function in vivo, which is expected with maneuvers that increase SR Ca load. Unexpectedly, however, when the KO mice were subjected to forms of physiological cardiac stress, malignant ventricular arrhythmias developed that were associated with DAD-related aftercontractions in junctin-deficient cardiac myocytes. These results not only demonstrate a necessary role for junctin in cardiomyocyte SR Ca homeostasis but also reveal a novel aspect of the previously postulated relationship between RyR function and electrical stability of cardiomyocytes, the critical regulatory role of junctin.7
By what mechanisms did ablation of junctin result in increased SR Ca load? It is known that CSQ-binding capacity, SR Ca uptake, and NCX activity are the key determinants of SR Ca load.19,28–30 We did not find significant changes, however, in the SR Ca-ATPase Ca uptake activity or its affinity for Ca, and the CSQ protein levels were not altered. By contrast, the expression level and the activity of the NCX were enhanced, which would be expected to result in diminished SR Ca load. Thus, we hypothesize that the increased SR Ca load in the absence of junctin reflected increases in the CSQ Ca-binding capacity. It is well known that the repeated aspartate-rich region at the C-terminus of CSQ is the Ca-binding domain and acts as a Ca reservoir.31 Importantly, this is also the domain that interacts with the KEKE motifs of junctin/triadin.31 Intuitively then, ablation of junctin could elicit availability of additional Ca-binding sites in the aspartate-rich region of CSQ, leading to an increase in the CSQ Ca-binding capacity.
The increase in SR Ca load was likely the primary factor responsible for the augmented Ca transient and hypercontractility in junctin-deficient cardiomyocytes. In this regard, the increase in contractile function was consistent with the enhanced Ca transients and kinetics. The gain of function of NCX appeared to also be an important compensatory mechanism along these lines, because the forward mode of the NCX favors Ca extrusion to maintain a normal diastolic [Ca] in the face of increased SR Ca load.32
Importantly, ablation of junctin was associated with DAD-induced arrhythmias under stress. Indeed, genetic defects of 2 other proteins in the SR Ca release channel complex, RyR and CSQ, have been also linked to catecholaminergic polymorphic ventricular tachycardia in human patients through activation of DADs.2 The cellular basis for DADs is associated with Ca aftertransients, caused by RyR aberrant Ca release.8,33 The molecular mechanisms associated with RyR inappropriate channel opening during diastole may include increased sensitivity to SR luminal Ca; impaired RyR domain–domain interaction, which may alter RyR gating properties; and dissociation of FKBP12.6, which destabilizes RyR and increases spontaneous Ca release. Two of the CSQ mutations were shown to mediate these effects through their defective interaction with junctin/triadin in the RyR complex, resulting in enhanced sensitivity to SR luminal Ca and increased RyR spontaneous Ca release.8,34 Thus, we hypothesize that junctin ablation may act in a similar manner on RyR activity, leading to lethal cardiac arrhythmias.
What are the mechanisms underlying the increased likelihood of DAD-associated arrhythmias in junctin-deficient mice? Junctin ablation may be associated with defective regulation of RyR activity by SR luminal Ca. Similar to the scheme in catecholaminergic polymorphic ventricular tachycardia–related CSQ mutations, junctin ablation may directly prevent the interaction between CSQ and the RyR complex, impairing the sensitivity of RyR to SR Ca, which may lead to aberrant RyR openings. Potentially, ablation of junctin may directly enhance RyR activity. It was shown that Ca negatively regulates the interaction between CSQ and junctin/triadin.11 Thus, when SR [Ca] increases during diastole, junctin and triadin may be free to interact and modulate RyR activity. On consequent decrease in [Ca] during Ca-induced Ca release, the interaction between CSQ and junctin/triadin may increase, and the effects of junctin/triadin on RyRs may be weakened by CSQ. Alternatively, the effects of junctin/triadin on regulation of RyR activity may be dominant during diastole. Indeed, the present Ca spark data from permeabilized myocytes clearly showed that spontaneous RyR Ca release was increased, which constituted an important molecular mechanism for the induction of DAD-associated arrhythmias, which suggests that junctin may represent a brake that inhibits RyR activity. Although it has been widely accepted that junctin and triadin mediate the interaction between RyR and CSQ,10,11,15,31,35 studies in skeletal muscle have indicated a direct interaction between RyR and CSQ, with CSQ enhancing RyR activity.35,36 Because it was suggested that junctin/triadin may also prevent this direct interaction,35,37 deletion of junctin might favor the positive regulation of CSQ on RyR activity and contribute to the increase in RyR spontaneous Ca release. In addition, it is known that catecholamines can induce spontaneous SR Ca release by increasing SR Ca load.38 Thus, isoproterenol stimulation most likely exacerbated the high SR Ca load of junctin-null cells. The suprathreshold levels of SR Ca load coupled with decreased FKBP12.6 binding to RyR enhanced the spontaneous RyR openings. The larger amount of Ca released during each RyR opening increased the likelihood of DAD-associated arrhythmias. However, DADs and aftercontractions were also observed in the absence of isoproterenol, which indicates that dissociation of FKBP12.6 may not be required for the generation of arrhythmias in this model. Finally, because DAD is activated by an inward current via the NCX,39 the enhanced NCX activity in junctin-deficient cardiomyocytes could be an additional contributor to the generation of DADs, which further induce arrhythmias.
In conclusion, the present findings show that cardiomyocyte SR Ca content and contractile function are inversely related to the level of cardiac junctin expression under normal conditions, which establishes that junctin can be an important modulator of SR Ca cycling. Surprisingly, junctin deficiency also decreased the threshold for malignant ventricular arrhythmias by increasing the incidence of DADs, which may have been related to our observations of sudden death in junctin-KO mice. Thus, maintenance of junctin expression levels appears necessary for proper SR Ca handling, cardiac function, and resistance to abnormal ventricular automaticity.
Sources of Funding
This study was supported by grants HL26057, HL64018 (Dr Kranias), and HL 77101; by the Leducq Foundation (Drs Kranias and Dorn); and by grant HL-55438 (Dr Valdivia).
Packer M. Sudden unexpected death in patients with congestive heart failure: a second frontier. Circulation. 1985; 72: 681–685.
Pogwizd SM, Hoyt RH, Saffitz JE, Corr PB, Cox JL, Cain ME. Reentrant and focal mechanisms underlying ventricular tachycardia in the human heart. Circulation. 1992; 86: 1872–1887.
Pogwizd SM. Nonreentrant mechanisms underlying spontaneous ventricular arrhythmias in a model of nonischemic heart failure in rabbits. Circulation. 1995; 92: 1034–1048.
Pogwizd SM. Focal mechanisms underlying ventricular tachycardia during prolonged ischemic cardiomyopathy. Circulation. 1994; 90: 1441–1458.
Nam GB, Burashnikov A, Antzelevitch C. Cellular mechanisms underlying the development of catecholaminergic ventricular tachycardia. Circulation. 2005; 111: 2727–2733.
Viatchenko-Karpinski S, Terentyev D, Gyorke I, Terentyeva R, Volpe P, Priori SG, Napolitano C, Nori A, Williams SC, Gyorke S. Abnormal calcium signaling and sudden cardiac death associated with mutation of calsequestrin. Circ Res. 2004; 94: 471–477.
Zhang L, Kelley J, Schmeisser G, Kobayashi YM, Jones LR. Complex formation between junctin, triadin, calsequestrin, and the ryanodine receptor: proteins of the cardiac junctional sarcoplasmic reticulum membrane. J Biol Chem. 1997; 272: 23389–23397.
Terentyev D, Cala SE, Houle TD, Viatchenko-Karpinski S, Gyorke I, Terentyeva R, Williams SC, Gyorke S. Triadin overexpression stimulates excitation-contraction coupling and increases predisposition to cellular arrhythmia in cardiac myocytes. Circ Res. 2005; 96: 651–658.
Postma AV, Denjoy I, Hoorntje TM, Lupoglazoff JM, Da Costa A, Sebillon P, Mannens MM, Wilde AA, Guicheney P. Absence of calsequestrin 2 causes severe forms of catecholaminergic polymorphic ventricular tachycardia. Circ Res. 2002; 91: e21–e26.
Jones LR, Zhang L, Sanborn K, Jorgensen AO, Kelley J. Purification, primary structure, and immunological characterization of the 26-kDa calsequestrin binding protein (junctin) from cardiac junctional sarcoplasmic reticulum. J Biol Chem. 1995; 270: 30787–30796.
Kirchhefer U, Neumann J, Bers DM, Buchwalow IB, Fabritz L, Hanske G, Justus I, Riemann B, Schmitz W, Jones LR. Impaired relaxation in transgenic mice overexpressing junctin. Cardiovasc Res. 2003; 59: 369–379.
Hong CS, Cho MC, Kwak YG, Song CH, Lee YH, Lim JS, Kwon YK, Chae SW, Kim DH. Cardiac remodeling and atrial fibrillation in transgenic mice overexpressing junctin. FASEB J. 2002; 16: 1310–1312.
Kirchhefer U, Neumann J, Baba HA, Begrow F, Kobayashi YM, Reinke U, Schmitz W, Jones LR. Cardiac hypertrophy and impaired relaxation in transgenic mice overexpressing triadin 1. J Biol Chem. 2001; 276: 4142–4149.
Luo W, Grupp IL, Harrer J, Ponniah S, Grupp G, Duffy JJ, Doetschman T, Kranias EG. Targeted ablation of the phospholamban gene is associated with markedly enhanced myocardial contractility and loss of beta-agonist stimulation. Circ Res. 1994; 75: 401–409.
Zhao W, Yuan Q, Qian J, Waggoner JR, Pathak A, Chu G, Mitton B, Sun X, Jin J, Braz JC, Hahn HS, Marreez Y, Syed F, Pollesello P, Annila A, Wang HS, Schultz Jel J, Molkentin JD, Liggett SB, Dorn GW II, Kranias EG. The presence of Lys27 instead of Asn27 in human phospholamban promotes sarcoplasmic reticulum Ca2+-ATPase superinhibition and cardiac remodeling. Circulation. 2006; 113: 995–1004.
Wang Z, Nolan B, Kutschke W, Hill JA. Na+-Ca2+ exchanger remodeling in pressure overload cardiac hypertrophy. J Biol Chem. 2001; 276: 17706–17711.
Farrell EF, Antaramian A, Rueda A, Gomez AM, Valdivia HH. Sorcin inhibits calcium release and modulates excitation-contraction coupling in the heart. J Biol Chem. 2003; 278: 34660–34666.
Mitchell RD, Simmerman HK, Jones LR. Ca2+ binding effects on protein conformation and protein interactions of canine cardiac calsequestrin. J Biol Chem. 1988; 263: 1376–1381.
Ginsburg KS, Weber CR, Bers DM. Control of maximum sarcoplasmic reticulum Ca load in intact ferret ventricular myocytes: effects of thapsigargin and isoproterenol. J Gen Physiol. 1998; 111: 491–504.
Philipson KD, Nishimoto AY. Na+-Ca2+ exchange is affected by membrane potential in cardiac sarcolemmal vesicles. J Biol Chem. 1980; 255: 6880–6882.
Liu N, Colombi B, Memmi M, Zissimopoulos S, Rizzi N, Negri S, Imbriani M, Napolitano C, Lai FA, Priori SG. Arrhythmogenesis in catecholaminergic polymorphic ventricular tachycardia: insights from a RyR2 R4496C knock-in mouse model. Circ Res. 2006; 99: 292–298.
Terentyev D, Nori A, Santoro M, Viatchenko-Karpinski S, Kubalova Z, Gyorke I, Terentyeva R, Vedamoorthyrao S, Blom NA, Valle G, Napolitano C, Williams SC, Volpe P, Priori SG, Gyorke S. Abnormal interactions of calsequestrin with the ryanodine receptor calcium release channel complex linked to exercise-induced sudden cardiac death. Circ Res. 2006; 98: 1151–1158.
Pogwizd SM, Schlotthauer K, Li L, Yuan W, Bers DM. Arrhythmogenesis and contractile dysfunction in heart failure: roles of sodium-calcium exchange, inward rectifier potassium current, and residual beta-adrenergic responsiveness. Circ Res. 2001; 88: 1159–1167.
Sudden cardiac death, largely attributable to malignant ventricular arrhythmia, is responsible for 300 000 to 400 000 annual deaths in the United States. Although sudden cardiac death is a frequent end point for patients with organic heart diseases, such as chronic or acute ischemia and dilated or hypertrophic cardiomyopathies, it often occurs in individuals with no detectable cardiac pathology, as in catecholaminergic polymorphic ventricular tachycardia. Recently, an abnormal sarcoplasmic reticulum Ca leak has been correlated with increased ventricular arrhythmias in patients with heart failure or catecholaminergic polymorphic ventricular tachycardia, caused by aberrant ryanodine receptor (RyR) Ca release. Although the underlying mechanisms for this leak have not been completely elucidated, human mutations in the RyR and calsequestrin genes, which alter RyR gating and calcium release, have been associated with lethal cardiac arrhythmias. Junctin is another member of the sarcoplasmic reticulum Ca-handling apparatus, and it complexes with RyR to affect Ca release, but its relationship to cardiac automaticity has not been examined. Here, through genetic ablation of junctin in the mouse, we reveal that junctin is essential for maintenance of normal RyR activity and Ca release: Junctin ablation was not only associated with enhanced sarcoplasmic reticulum Ca cycling and cardiac performance in vivo but also increased Ca leak, which further triggered arrhythmias and predisposed to sudden cardiac death. Thus, it may be valuable to identify polymorphic variants in the human junctin gene and determine their association with cardiac arrhythmias in subjects with and without known cardiac disease. Identification of such variants may suggest more aggressive targeted use of primary preventative measures for sudden cardiac death.
The online-only Data Supplement, consisting of an expanded Method section, tables, and figures, is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.106.654699/DC1.