Author + information
- Received August 14, 2013
- Revision received October 31, 2013
- Accepted November 1, 2013
- Published online April 22, 2014.
- Simon Sedej, PhD∗,†∗ (, )
- Albrecht Schmidt, MD∗,
- Marco Denegri, PhD‡,
- Stefanie Walther, MD∗,
- Marinko Matovina∗,
- Georg Arnstein, MD∗,
- Eva-Maria Gutschi, MSc∗,†,
- Isabella Windhager, MD∗,
- Senka Ljubojević, PhD∗,†,
- Sara Negri, MSc‡,
- Frank R. Heinzel, MD, PhD∗,†,
- Egbert Bisping, MD∗,†,
- Marc A. Vos, PhD§,
- Carlo Napolitano, MD, PhD‡,‖,
- Silvia G. Priori, MD, PhD‡,‖,
- Jens Kockskämper, PhD¶ and
- Burkert Pieske, MD∗,†∗ ()
- ∗Department of Cardiology, Medical University of Graz, Graz, Austria
- †Ludwig Boltzmann Institute for Translational Heart Failure Research, Graz, Austria
- ‡IRCCS Salvatore Maugeri Foundation and Department of Molecular Medicine, University of Pavia, Pavia, Italy
- §Department of Medical Physiology, University Medical Center Utrecht, Utrecht, the Netherlands
- ‖Leon H. Charney Division of Cardiology, New York University School of Medicine, New York, New York
- ¶Institute of Pharmacology and Clinical Pharmacy, Philipps-University of Marburg, Marburg, Germany
- ↵∗Reprint requests and correspondence:
Dr. Simon Sedej and Prof. Dr. Burkert Pieske, Department of Cardiology, Medical University of Graz, Auenbruggerplatz 15, A-8036 Graz, Austria.
Objectives This study sought to explore whether subclinical alterations of sarcoplasmic reticulum (SR) Ca2+ release through cardiac ryanodine receptors (RyR2) aggravate cardiac remodeling in mice carrying a human RyR2R4496C+/– gain-of-function mutation in response to pressure overload.
Background RyR2 dysfunction causes increased diastolic SR Ca2+ release associated with arrhythmias and contractile dysfunction in inherited and acquired cardiac diseases, such as catecholaminergic polymorphic ventricular tachycardia and heart failure (HF).
Methods Functional and structural properties of wild-type and catecholaminergic polymorphic ventricular tachycardia–associated RyR2R4496C+/– hearts were characterized under conditions of pressure overload induced by transverse aortic constriction (TAC).
Results Wild-type and RyR2R4496C+/– hearts had comparable structural and functional properties at baseline. After TAC, RyR2R4496C+/– hearts responded with eccentric hypertrophy, substantial fibrosis, ventricular dilation, and reduced fractional shortening, ultimately resulting in overt HF. RyR2R4496C+/–-TAC cardiomyocytes showed increased incidence of spontaneous SR Ca2+ release events, reduced Ca2+ transient peak amplitude, and SR Ca2+ content as well as reduced SR Ca2+-ATPase 2a and increased Na+/Ca2+-exchanger protein expression. HF phenotype in RyR2R4496C+/–-TAC mice was associated with increased mortality due to pump failure but not tachyarrhythmic events. RyR2-stabilizer K201 markedly reduced Ca2+ spark frequency in RyR2R4496C+/–-TAC cardiomyocytes. Mini-osmotic pump infusion of K201 prevented deleterious remodeling and improved survival in RyR2R4496C+/–-TAC mice.
Conclusions The combination of subclinical congenital alteration of SR Ca2+ release and pressure overload promoted eccentric remodeling and HF death in RyR2R4496C+/– mice, and pharmacological RyR2 stabilization prevented this deleterious interaction. These findings suggest potential clinical relevance for patients with acquired or inherited gain-of-function of RyR2-mediated SR Ca2+ release.
Hypertension is the most prevalent cardiovascular risk factor for congestive heart failure (HF) (1). High blood pressure induces left ventricular (LV) hypertrophy as an adaptive compensatory mechanism to reduce LV wall stress in response to hemodynamic overload. Sustained overload induces maladaptive cardiac remodeling, LV dilation, and contractile dysfunction (2), resulting in arrhythmias and HF, a major cause for mortality in the Western world (3).
Pressure overload–induced changes that contribute to cardiac remodeling include an increase in the size of cardiomyocytes, alterations in the extracellular matrix with increased fibrosis (4), and abnormalities of the coronary vasculature (5). Development of hypertrophy and its progression to HF varies considerably between hypertensive individuals, both in the magnitude of LV mass increase and its geometric pattern, such as chamber dilation and wall thickening (6,7). However, the mechanisms that modulate afterload-dependent remodeling processes responsible for these interindividual differences remain incompletely understood (8).
Altered cardiomyocyte Ca2+ homeostasis is a central pathophysiological mechanism in hypertrophy and HF (9). Disturbed intracellular Ca2+ handling accounts for abnormalities in Ca2+ signaling during excitation-contraction coupling (10) and also for disturbances in maladaptive gene expression and activation of many Ca2+-dependent hypertrophic signaling pathways (9). Dysfunction of the cardiac ryanodine receptor (RyR2), the intracellular sarcoplasmic reticulum (SR) Ca2+ release channel, is associated with increased spontaneous diastolic SR Ca2+ release, delayed afterdepolarizations (DADs), and triggered activity (11). Consequently, contractile dysfunction and arrhythmias occur in patients with hypertrophy and HF (12), and many of these patients have hypertension as an underlying risk factor or co-morbidity. Increased SR Ca2+ leak also occurs in congenital arrhythmia syndromes associated with RyR2 or calsequestrin mutations, such as catecholaminergic polymorphic ventricular tachycardia (CPVT) (13). CPVT is an inherited arrhythmic disorder characterized by sudden cardiac death during physical or emotional stress in the structurally normal heart (13,14). However, the central role of RyR2 in intracellular Ca2+ homeostasis makes it plausible that even subtle abnormalities in RyR2 function may facilitate adverse cardiac remodeling. Therefore, we hypothesized that dysfunctional SR Ca2+ release through mutated RyR2 accelerates maladaptive LV remodeling under conditions of pressure overload. We used a knock-in mouse model carrying the RyR2R4496C+/– mutation associated with increased diastolic SR Ca2+ leak and CPVT in humans to test this hypothesis.
In the present study, we show that constitutively abnormal RyR2-mediated SR Ca2+ leak promotes myocardial remodeling, including eccentric hypertrophy, dilation, and contractile dysfunction, and increases mortality due to pump failure in RyR2R4496C+/– mice under pressure overload. Conversely, pharmacological stabilization of SR Ca2+ release by K201 ameliorates the development of HF and preserves systolic function.
An expanded version of the Methods section can be found in the Online Appendix.
Heterozygous RyR2R4496C+/– knock-in mice (R4496C) and their wild-type (WT) littermates underwent surgery without (sham) or with transverse aortic constriction (TAC). Dimensions and function of the left ventricle were assessed by using transthoracic echocardiography (microimaging system Vevo770, 30-MHz transducer, VisualSonics, Inc., Toronto, Ontario, Canada). Arrhythmias were continuously monitored by using electrocardiogram (ECG) radio telemetry (DSI, St. Paul, Minnesota). The RyR2-stabilizing agent K201 was administered via mini-osmotic pumps (Alzet, DURECT Corporation, Cupertino, California). Cardiomyocytes were isolated 1 week post-surgery as described previously (15). [Ca2+]i measurements were acquired in line-scan mode by using a confocal microscope (LSM 510 Meta, Carl Zeiss Inc., Jena, Germany) using the solutions, recording protocols, and analysis as described previously (16). Data are shown as mean ± SEM. Statistical differences between groups were considered significant when p < 0.05.
Additional figures of the Results section can be found in the Online Appendix.
Myocardial remodeling and fibrosis
Increased afterload on the left ventricle was induced by TAC (Fig. 1A). In WT and R4496C mice, 1 week of TAC induced similar degrees of cardiac hypertrophy as defined by an increased heart weight–to-tibia length (Fig. 1B). Three weeks' post-TAC, however, relative heart weight had increased significantly more in R4496C-TAC hearts compared with WT-TAC hearts (11.3 ± 0.7 mg/mm vs. 9.4 ± 0.3 mg/mm; p < 0.05). R4496C-TAC mice had elevated lung weight–to-tibia length ratio (5.7 ± 0.6 mg/mm; p < 0.05) compared with WT-TAC mice (3.9 ± 0.3 mg/mm) (Fig. 1C). Increased relative heart weight correlated with induction of the hypertrophic gene program (Figs. 1E and 1F), and activation of hypertrophic genes was more prominent in R4496C-TAC hearts. Accelerated remodeling of R4496C-TAC hearts was associated with more fibrosis (compared with WT-TAC, p < 0.05) (Figs. 1A and 1D) and increased myocyte length/width ratio in isolated R4496C-TAC cardiomyocytes (Fig. 1G).
Accelerated progression from hypertrophy to HF
R4496C-TAC mice underwent progressive LV chamber dilation that started within the first week after TAC (Figs. 2A and 2B). In contrast, WT-TAC mice effectively compensated for pressure overload at 1 week after TAC with moderate increases in LV end-diastolic diameter 3 weeks' post-TAC. Increased dilation in conjunction with smaller LV wall thickness suggest that R4496C-TAC hearts underwent accelerated transition from load-induced hypertrophy to HF (Fig. 2B). Fractional shortening declined more in R4496C-TAC hearts at 1 week (WT-TAC: –6.2 ± 2.4%, R4496C-TAC: –18.1 ± 2.3%; p < 0.05) and 3 weeks post-TAC (WT-TAC: –14.3 ± 1.9%, R4496C-TAC: –23.9 ± 1.7; p < 0.05) (Fig. 2C). WT hearts responded with a significant increase in the relative wall thickness to pressure overload. This physiological response was absent in R4496C-TAC mice (compared with WT-TAC, p < 0.05) (Fig. 2D). Eccentric LV remodeling in R4496C-TAC mice was associated with premature death: only 36% of animals were alive after 6 weeks of TAC compared with a 74% survival rate in WT-TAC mice (p < 0.05) (Fig. 2E).
Impaired survival due to pump failure death in R4496C-TAC mice
Susceptibility to catecholamine-induced tachyarrhythmias and sudden cardiac death (17) could explain the increased mortality in R4496C-TAC mice. ECG transmitters were implanted in a subset of R4496C-TAC mice (n = 11) to allow continuous recording of ambulatory ECG waveforms. All of these 11 R4496C-TAC mice, however, had sinus rhythm followed by progressing sinus bradycardia and atrioventricular conduction block III, and they ultimately died of pump failure (Figs. 3A and 3B1-3). In another set of experiments, we examined whether R4496C-TAC mice were vulnerable to CPVT under conditions that resemble those eliciting tachyarrhythmias in CPVT patients (14). Epinephrine and RyR2-agonist caffeine administration increased heart rate and induced sustained bidirectional ventricular tachycardia in 100% (6 of 6) of the R4496C-TAC survivors 1 week after TAC (Fig. 3C). Persistent bidirectional ventricular tachycardia induced by epinephrine/caffeine did not deteriorate into bradycardia followed by asystole, but it subsided within 30 min and failed to induce sudden cardiac death in 100% (6 of 6) of the R4496C-TAC mice. These mice developed progressive sinus bradycardia a few days later and died thereafter of pump failure.
Increased SR Ca2+ leak in R4496C-TAC mice
We investigated whether pressure overload increases RyR2-mediated SR Ca2+ leak measured as Ca2+ sparks (Fig. 4A). Ca2+ spark frequency at matched SR Ca2+ load (Fig. 4B) revealed a pronounced increase in SR Ca2+ leak in R4496C-sham compared with WT-sham (173.6 ± 20.9 pL–1s–1 and 102.2 ± 9.7 pL–1s–1, respectively; p < 0.05) (Fig. 4C). Ca2+ spark frequency at matched SR Ca2+ load was largely increased in R4496C-TAC cardiomyocytes (332.7 ± 42.9 pL–1s–1, p < 0.05) versus WT-TAC (190.1 ± 27 pL–1s–1).
Impaired global Ca2+ homeostasis
At baseline, sham-operated WT and R4496C cardiomyocytes displayed similar Ca2+ transients (Online Figs. 1A through 1D). However, the peak of the caffeine-induced Ca2+ transient was diminished in R4496C-sham cardiomyocytes versus WT-sham cardiomyocytes (p < 0.05) (Online Fig. 1E). One week after TAC, R4496C-TAC myocytes displayed significantly reduced peak amplitudes of Ca2+ transients, prolonged [Ca2+]i transient decline, and further reduced SR Ca2+ content (all p < 0.05 vs. WT-TAC) (Online Figs. 1B, 1C, and 1E, respectively).
Altered expression and phosphorylation of Ca2+ regulatory proteins
Phosphorylation of RyR2 at Ser2808 and at Ser2814 and also at Ser16 of PLB was unaltered in all groups (Online Figs. 2A and 2B). However, phosphorylation of PLB at Thr17 was significantly decreased in R4496C (sham and TAC) mice (Online Fig. 2B). SERCA 2a levels were reduced in both WT-TAC and R4496C-TAC hearts compared with WT-sham hearts (p < 0.05) (Online Fig. 2C). In contrast, Na+/Ca2+ exchanger (NCX) expression was significantly increased in R4496C-TAC mice only but unchanged in WT-sham hearts (Online Fig. 2C).
Altered differential expression and phosphorylation of CaMKII, calcineurin, and protein phosphatase 1
Under baseline conditions, overall CaMKII expression and phosphorylation was similar, but the expression of CaMKIIγ was significantly higher in R4496C compared with WT (Online Fig. 2D). After 1 week of TAC, CaMKII expression and phosphorylation were significantly increased in both WT and R4496C hearts. However, this increase was significantly higher in R4496C-TAC hearts (compared with WT-TAC hearts, p < 0.05). Interestingly, the overall increase in CaMKII after TAC was related to differential regulation of its isoforms γ and δ. Whereas CaMKIIγ was largely increased after TAC in WT, CaMKIIδ remained unchanged; in contrast, the smaller increase in CaMKIIγ in R4496C was associated with a parallel increase in CaMKIIδ. Calcineurin protein levels significantly increased with TAC in WT hearts but were already elevated in R4496C hearts at baseline, with no further change after TAC (Online Fig. 2E). Protein phosphatase 1 levels were not affected by TAC but were up-regulated in both R4496C groups (Online Fig. 2E).
K201 ameliorated the progression of HF and improved survival in R4496C-TAC mice
Infusion of K201 resulted in a mean K201 plasma concentration of 69.1 ± 10.8 ng/ml (n = 6). After TAC, K201 prevented LV dilation (LV end-systolic diameter) (Fig. 5A), prevented the decline in fractional shortening (1 week 6.6 ± 2.6%; 3 weeks 7.9 ± 2.6%; both p < 0.05 vs. control) (Fig. 5B), restored an increase in the relative wall thickness (Fig. 5C), and largely prevented the accelerated transition from concentric to eccentric remodeling and LV dilation in R4496C-TAC mice (LV end-diastolic diameter) (Fig. 5D). K201 treatment normalized survival over 6 weeks after TAC (Fig. 5E). At 6 weeks, 83% of K201-treated R4496C-TAC mice were alive compared with 31% of vehicle-treated mice (p < 0.05).
K201 prevented the spontaneous SR Ca2+ release in R4496C-TAC mice
To test whether the beneficial effect of K201 is related to a reduction in SR Ca2+ leak in R4496C-TAC cardiomyocytes, we assessed the effect of K201 300 nmol/L and dantrolene 1 μmol/L (RyR2 stabilizers) and KN-93 1 μmol/L (CaMKII inhibitor) on Ca2+ spark frequency and global Ca2+ homeostasis. K201 and dantrolene markedly reduced the increase in Ca2+ spark frequency at matched SR Ca2+ load in R4496C-TAC and WT-TAC cells as well as in R4496C-sham cells (Figs. 6A1-2 and 6B). The RyR2-stabilizing effect was more pronounced in the presence of K201 than dantrolene. None of the RyR2-channel stabilizers changed the peak Ca2+ transient amplitude (Fig. 6C) and kinetics (data not shown) in TAC groups. However, the peak of the caffeine-induced Ca2+ transient was increased with K201 and dantrolene treatment in WT-TAC and R4496C-TAC cells (Fig. 6D). In contrast to K201 and dantrolene administration, KN-93 treatment did not significantly alter the Ca2+ spark frequency and global Ca2+ homeostasis in any of the groups studied (Figs. 6A3 to 6D).
In the present study, we demonstrated that the CPVT-associated RyR2R4496C+/– gain-of-function mutation promotes adverse structural and functional myocardial remodeling, HF, and pump failure death in response to pressure overload. RyR2 stabilization by K201 attenuated the SR Ca2+ leak, ameliorated the TAC-induced transition from hypertrophy to HF, and improved survival of RyR2R4496C+/– mice.
Ca2+ dysregulation and adverse remodeling
Increased SR Ca2+ leak via RyR2 channels and reduced Ca2+ uptake by SERCA 2a contribute to reduced SR Ca2+ content and increased diastolic Ca2+ concentration in hypertrophy and HF (18). RyR2R4496C+/– knock-in mice display increased SR Ca2+ release (15) in structurally normal hearts, resulting in increased diastolic Ca2+ concentration and catecholamine-induced bidirectional tachycardias and ventricular fibrillation (14) similar to humans (13). Our results show that the combination of pressure overload and dysregulated SR Ca2+ release through mutated RyR2R4496C+/– channel promotes rapid cardiac remodeling. A remarkable shift in the pattern of hypertrophic remodeling related to increased SR Ca2+ leak was reflected in altered gene expression, increased fibrosis and relative heart weight, eccentric hypertrophy, and LV dilation and dysfunction, which progressed into overt HF. The involvement of RyR2-mediated SR Ca2+ release for the development of pressure overload–induced hypertrophy has been recently demonstrated in RyR2R176Q+/– knock-in mice (19) and RyR2+/–-deficient mice (20). While reduced SR Ca2+ release was associated with less concentric hypertrophy and fibrosis after 3 weeks of TAC (20), elevated SR Ca2+ release via mutated channels of RyR2R176Q+/– enhanced the hypertrophic response accompanied by impaired diastolic and systolic function and LV dilation by activation of the calcineurin/NFAT-signaling pathway in response to aortic constriction (19). Our study extends these findings and provides evidence that the congenital RyR2 dysfunction promotes adverse myocardial remodeling, including aggravation of RyR2-mediated SR Ca2+ leak and altered intracellular Ca2+ handling, both of which may contribute to the pronounced shift in phenotypic remodeling during pressure overload.
During the development of myocardial hypertrophy and before the transition to HF, the contribution of SERCA 2a to Ca2+ removal is reduced and already shifted in favor of the NCX, and defective Ca2+ uptake into the SR is partially compensated for by increased NCX expression (21). In R4496C-TAC mice, this shift was reflected in reduced SR Ca2+ content, reduced peak amplitude of the Ca2+ transient, and the prolonged [Ca2+]i transient decline, indicating decreased contractility and impaired relaxation, respectively. Reduced SR Ca2+ content, a major cause for contractile dysfunction in R4496C-TAC hearts, was associated with the reduced SERCA 2a–dependent Ca2+ removal, increased expression of NCX, and increased Ca2+ spark frequency. These findings provide compelling evidence for the pathophysiological relevance of dysfunctional SR Ca2+ release in the dysregulation of SR Ca2+ handling, which, in turn, provides the basis for the cardiomyocyte malfunctioning associated with the development of pressure overload–induced hypertrophy.
Increased expression and activity of CaMKII in R4496C hearts at 1 week after aortic constriction suggest that an early development of HF phenotype in R4496C-TAC mice is, at least partially, attributed to the activation of CaMKII-dependent hypertrophy pathways. This contrasts with the study by van Oort et al. (19), in which the authors ruled out the possible role of elevated SR Ca2+ leak in the activation of CaMKII-dependent hypertrophy signaling. Recent studies demonstrated that CaMKIIδ is required for alterations in the expression of Ca2+ regulatory proteins during the development of pressure overload–induced HF (22). Conversely, CaMKIIδ deletion protects from adverse remodeling after pressure overload (23) and progression from pressure overload hypertrophy to HF (22). A disparity in the expression between CaMKIIγ and CaMKIIδ isoforms in R4496C-TAC hearts support the hypothesis that increased expression of CaMKIIδ provoked early onset of adverse myocardial remodeling. Up-regulation of CaMKIIδc also contributes to abnormal Ca2+ handling and increased mortality in R4496C mice (24). Notably, increased levels of CaMKII did not affect the phosphorylation of RyR2 and PLB in R4496C mice, likely due to increased PP1 levels. Hence, our findings suggest that maladaptive CaMKII signaling was affecting structural myocardial remodeling rather than functionally interfering with Ca2+ cycling. In support of this hypothesis, we found that CaMKII inhibition with KN-93 did not attenuate Ca2+ spark frequency and interfere with excitation-contraction coupling in the absence of catecholaminergic stimulation in R4496C-TAC cardiomyocytes, as described previously (17). Previous studies emphasize the importance of CaMKII-mediated phosphorylation of RyR2 in the progression of the nonischemic form of HF in both humans (12) and TAC mice (25,26). However, our results suggest a minor role of the CaMKII-mediated phosphorylation of RyR2 at Ser2814 on aberrant SR Ca2+ release via RyR2 and in vivo cardiac dysfunction. This discrepancy may be related to different time points at which the phosphorylation of RyR2 at Ser2814 was assessed or the severity of cardiac decompensation. In agreement with the study by van Oort et al. (19), we found increased expression levels of calcineurin in pressure overload–imposed R4496C hearts (but also in WT), indicating the activation of the Ca2+-dependent calcineurin/NFAT hypertrophic signaling pathway.
Increased mortality and mode of death
Our study shows, for the first time, that pressure overload impairs the survival of R4496C mice due to pump failure rather than ventricular arrhythmias. Given that acute beta-adrenergic stimulation predisposes R4496C mice to CPVT and ventricular fibrillation (27), we anticipated that increased propensity for ventricular arrhythmia was a major cause for increased mortality in R4496C mice imposed to pressure overload. However, even though R4496C-TAC cardiomyocytes exhibited an increased arrhythmogenic potential in vitro, including increased SR Ca2+ leak, reduced SERCA 2a–dependent Ca2+ removal, and increased expression of NCX, ventricular arrhythmias and sudden cardiac death were not observed in vivo in R4496C mice under pressure overload. Several reasons may account for this phenomenon, including reduced SR Ca2+ content and beta-adrenergic responsiveness and the absence of CaMKII-dependent phosphorylation of RyR2, which has been shown to be required for arrhythmogenic events leading to sudden cardiac death (24,25). Triggered activity was effectively compensated for by reduced SR Ca2+ load in the R4496C-TAC myocytes, such that the diastolic SR Ca2+ leak was insufficient to produce triggered activity unless the SR Ca2+ content was increased (28). In response to beta-adrenergic stimulation, however, the R4496C mutation results in largely increased SR Ca2+ leak and SR Ca2+ content due to increased CaMKII site-dependent RyR2 phosphorylation and increased SR Ca2+ re-uptake, respectively (17). This has been related to an increased propensity to DAD-mediated triggered activity underlying ventricular tachyarrhythmia (27), which could also be elicited in our R4496C-TAC hearts upon catecholamine stimulation. Faster heart rate and SR Ca2+ uptake during adrenergic stimulation increases SR Ca2+ load and induces subsequent Ca2+ waves followed by DAD-mediated triggered beats in ventricular cardiomyocytes harboring RyR2 mutations (27,29). Notably, triggered activity in R4496C cardiomyocytes also occurs with increasing SR Ca2+ content in the absence of adrenergic stimulation (15). These findings jointly highlight the importance of elevated SR Ca2+ content as a principal trigger in the CPVT arrhythmogenesis.
Effects of pharmacological RyR2 stabilization on adverse remodeling
Prevention of excessive diastolic RyR2-mediated SR Ca2+ release with K201 suppresses SR Ca2+ leak by stabilizing defective RyR2 gating (30) and improves cardiac and skeletal muscle function in HF (31). Thus, use of RyR2 stabilizers has been proposed as a therapeutic strategy in patients with HF (31,32). We found that the correction of dysfunctional Ca2+ release by chronic administration of K201 (plasma concentration ∼160 nmol/l) ameliorated the development of eccentric dilation, improved LV systolic function, and, most importantly, improved survival of R4496C-TAC mice. The evidence that K201 decelerates the progression of HF in CPVT mice after pressure overload extends previous findings, which demonstrated that K201 improves contractile function in tissue from end-stage human failing hearts (33) and delays the progression of ventricular remodeling and dysfunction by inhibiting the SR Ca2+ leak (31). Our in vitro experiments demonstrated that K201 (at 300 nmol/L) and dantrolene abolished acquired as well as congenital RyR2-mediated SR Ca2+ leak to almost normal levels. Three findings confirm that K201 reduces RyR2 open probability in WT-TAC and R4496C cells. First, K201 attenuated Ca2+ spark frequency in all “diseased” groups. Second, K201 decreased systolic [Ca2+]i in R4496C-sham cells. Third, in WT- and R4496C-TAC cardiomyocytes, K201 (and dantrolene) increased the SR Ca2+ content and reduced fractional release. This finding is consistent with our recent study (15), in which we demonstrated that the dominant effect of K201 is RyR2 stabilization associated with reduced SR Ca2+ leak in R4496C cells under conditions of similar and/or increased SR Ca2+ content. Interestingly, K201 prevented SR Ca2+ leak more effectively than dantrolene, implying that differences in the mode of interdomain interaction in those different regions, to which K201 and dantrolene bind, may result in the observed differences in their RyR2-stabilizing efficacy (34). Notably, K201 and dantrolene treatment did not alter Ca2+ spark frequency and intracellular Ca2+ handling in WT-sham cells, suggesting that both RyR2 stabilizers may be effective only in repairing defective interdomain interactions of the RyR2. Our study showed that the R4496C-TAC model has a much more complex pathophysiology than the non-TAC CPVT model. Therefore, the finding that K201 does not seem to prevent arrhythmias in R4496C mice without TAC (27) may simply mean that K201 acts on different pathways to eliminate store overload–induced Ca2+ release (which is probably the reason for arrhythmias in CPVT). Taken together, we found that K201 and dantrolene suppressed SR Ca2+ leak by stabilizing defective RyR2 gating, confirming that destabilization of RyR2 and consequent SR Ca2+ leak plays a pivotal role in TAC-induced remodeling.
Potential clinical relevance
Our finding that increased diastolic SR Ca2+ release is causally involved in the accelerated pathogenesis of pressure overload–induced HF has important clinical implications. First, our study suggests that patients with CPVT (with RyR2 mutations) that eventually develop hypertension may be particularly vulnerable to the development of eccentric hypertrophy and HF. Thus, it is possible that CPVT patients present a “latent” substrate for mechanical dysfunction that becomes overt with appropriate triggers. This concept is corroborated by evidence for an atypical form of cardiomyopathy reported in association with some RyR2 mutations (35,36). Interestingly, genome-wide association studies demonstrate that some RyR2 variants (single nucleotide polymorphisms) increase susceptibility to hypertension (37), indicating that aberrant RyR2 function could represent an underlying pathogenic risk factor in heart disease. Second, acquired imbalances of SR Ca2+ release are common findings in patients with hypertrophy and HF. Many of these patients have hypertension as an underlying risk factor or co-morbidity. This unfavorable combination may underlie rapid progression of myocardial remodeling in some of these patients and may partially explain interindividual differences in the pattern of remodeling (eccentric vs. concentric hypertrophy).
In conditions of pressure overload, the gain-of-function R4496C mutation in RyR2 accelerated the development of congestive HF and increased mortality due to pump failure rather than tachyarrhythmias. K201 attenuated the development of HF and improved survival in these R4496C-TAC mice. We suggest that RyR2-mediated SR Ca2+ leak contributes to the myocardial remodeling under pressure overload. Our study proposes that RyR2 stabilizers may provide a novel and rational approach to effectively combat hypertrophy and development of HF in patients with hypertension and acquired or congenital increased RyR2-mediated Ca2+ leak.
The authors acknowledge Snježana Radulović, Karl-Patrik Kresoja, and the staff of the Institute for Biomedical Research (Arno Absenger, Ulrike Fackelmann), the Center for Medical Research (ZMF), and the Core Facility Molecular Biology for excellent technical assistance. The authors thank Dr. Gudrun Antoons for critical reading of an earlier version of the manuscript.
This work was supported by Start Funding Program of the Medical University of Graz (Dr. Sedej) and EU FP6 grant LSHM-CT-2005-018802/CONTICA (Drs. Pieske, Priori, and Vos). Drs. Priori and Napolitano are supported by Telethon grants GGP06007 and GGP11141, CARIPLO pr.2008.2275, and the Fondazione Veronesi Award on inherited arrhythmogenic diseases and the Fondation Leducq Award to the Alliance for Calmodulin Kinase Signaling in Heart Disease (08CVD01). The authors have reported that they have no relationships relevant to the contents of this paper to disclose.
- Abbreviations and Acronyms
- catecholaminergic polymorphic ventricular tachycardia
- Ca2+/calmodulin-dependent kinase II
- delayed afterdepolarizations
- heart failure
- left ventricular
- Na+/Ca2+ exchanger
- cardiac ryanodine receptor (type 2)
- SERCA 2a
- sarcoplasmic reticulum Ca2+-ATPase 2a
- sarcoplasmic reticulum
- transverse aortic constriction
- Received August 14, 2013.
- Revision received October 31, 2013.
- Accepted November 1, 2013.
- American College of Cardiology Foundation
- Ganau A.,
- Devereux R.B.,
- Roman M.J.,
- et al.
- Drazner M.H.
- Pieske B.,
- Maier L.S.,
- Bers D.M.,
- Hasenfuss G.
- Fischer T.H.,
- Herting J.,
- Tirilomis T.,
- et al.
- Priori S.G.,
- Napolitano C.,
- Memmi M.,
- et al.
- Cerrone M.,
- Colombi B.,
- Santoro M.,
- et al.
- Sedej S.,
- Heinzel F.R.,
- Walther S.,
- et al.
- Bers D.M.,
- Eisner D.A.,
- Valdivia H.H.
- Zou Y.,
- Liang Y.,
- Gong H.,
- et al.
- Hasenfuss G.
- Backs J.,
- Backs T.,
- Neef S.,
- et al.
- Dybkova N.,
- Sedej S.,
- Napolitano C.,
- et al.
- van Oort R.J.,
- McCauley M.D.,
- Dixit S.S.,
- et al.
- Respress J.L.,
- van Oort R.J.,
- Li N.,
- et al.
- Liu N.,
- Colombi B.,
- Memmi M.,
- et al.
- Kashimura T.,
- Briston S.J.,
- Trafford A.W.,
- et al.
- Kannankeril P.J.,
- Mitchell B.M.,
- Goonasekera S.A.,
- et al.
- Tateishi H.,
- Yano M.,
- Mochizuki M.,
- et al.
- Wehrens X.H.,
- Lehnart S.E.,
- Reiken S.,
- et al.
- Yano M.,
- Kobayashi S.,
- Kohno M.,
- et al.
- Suetomi T.,
- Yano M.,
- Uchinoumi H.,
- et al.
- Milting H.,
- Lukas N.,
- Klauke B.,
- et al.