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Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a hereditary heart disease characterized by fatty infiltration, life-threatening arrhythmias, and increased risk of sudden cardiac death. The guideline for management of ARVC in patients is to improve quality of life by reducing arrhythmic symptoms and to prevent sudden cardiac death. However, the mechanism underlying ARVC-associated cardiac arrhythmias remains poorly understood. Methods: Using protein mass spectrometry analyses, we identified that integrin β1 is downregulated in ARVC hearts without changes to Ca 2+ -handling proteins. As adult cardiomyocytes express only the β1D isoform, we generated a cardiac specific β1D knockout mouse model and performed functional imaging and biochemical analyses to determine the consequences of integrin β1D loss on function in the heart in vivo and in vitro. Results: Integrin β1D deficiency and RyR2 Ser-2030 hyperphosphorylation were detected by Western blotting in left ventricular tissues from patients with ARVC but not in patients with ischemic or hypertrophic cardiomyopathy. Using lipid bilayer patch clamp single channel recordings, we found that purified integrin β1D protein could stabilize RyR2 function by decreasing RyR2 open probability, mean open time, and increasing mean close time. Also, β1D knockout mice exhibited normal cardiac function and morphology but presented with catecholamine-sensitive polymorphic ventricular tachycardia, consistent with increased RyR2 Ser-2030 phosphorylation and aberrant Ca 2+ handling in β1D knockout cardiomyocytes. Mechanistically, we revealed that loss of DSP (desmoplakin) induces integrin β1D deficiency in ARVC mediated through an ERK1/2 (extracellular signal–regulated kinase 1 and 2)–fibronectin–ubiquitin/lysosome pathway. Conclusions: Our data suggest that integrin β1D deficiency represents a novel mechanism underlying the increased risk of ventricular arrhythmias in patients with ARVC.

Background: Enhanced diastolic calcium (Ca 2+ ) release through ryanodine receptor type-2 (RyR2) has been implicated in atrial fibrillation (AF) promotion. Diastolic sarcoplasmic reticulum Ca 2+ leak is caused by increased RyR2 phosphorylation by PKA (protein kinase A) or CaMKII (Ca 2+ /calmodulin-dependent kinase-II) phosphorylation, or less dephosphorylation by protein phosphatases. However, considerable controversy remains regarding the molecular mechanisms underlying altered RyR2 function in AF. We thus aimed to determine the role of SPEG (striated muscle preferentially expressed protein kinase), a novel regulator of RyR2 phosphorylation, in AF pathogenesis. Methods: Western blotting was performed with right atrial biopsies from patients with paroxysmal AF. SPEG atrial knockout mice were generated using adeno-associated virus 9. In mice, AF inducibility was determined using intracardiac programmed electric stimulation, and diastolic Ca 2+ leak in atrial cardiomyocytes was assessed using confocal Ca 2+ imaging. Phosphoproteomics studies and Western blotting were used to measure RyR2 phosphorylation. To test the effects of RyR2-S2367 phosphorylation, knockin mice with an inactivated S2367 phosphorylation site (S2367A) and a constitutively activated S2367 residue (S2367D) were generated by using CRISPR-Cas9. Results: Western blotting revealed decreased SPEG protein levels in atrial biopsies from patients with paroxysmal AF in comparison with patients in sinus rhythm. SPEG atrial-specific knockout mice exhibited increased susceptibility to pacing-induced AF by programmed electric stimulation and enhanced Ca 2+ spark frequency in atrial cardiomyocytes with Ca 2+ imaging, establishing a causal role for decreased SPEG in AF pathogenesis. Phosphoproteomics in hearts from SPEG cardiomyocyte knockout mice identified RyR2-S2367 as a novel kinase substrate of SPEG. Western blotting demonstrated that RyR2-S2367 phosphorylation was also decreased in patients with paroxysmal AF. RyR2-S2367A mice exhibited an increased susceptibility to pacing-induced AF, and aberrant atrial sarcoplasmic reticulum Ca 2+ leak, as well. In contrast, RyR2-S2367D mice were resistant to pacing-induced AF. Conclusions: Unlike other kinases (PKA, CaMKII) that increase RyR2 activity, SPEG phosphorylation reduces RyR2-mediated sarcoplasmic reticulum Ca 2+ release. Reduced SPEG levels and RyR2-S2367 phosphorylation typified patients with paroxysmal AF. Studies in S2367 knockin mouse models showed a causal relationship between reduced S2367 phosphorylation and AF susceptibility. Thus, modulating SPEG activity and phosphorylation levels of the novel S2367 site on RyR2 may represent a novel target for AF treatment.

Article, see p 1595 Arrhythmogenic cardiomyopathy (AC) is a genetic disorder characterized by high risk of life-threatening ventricular arrhythmias, sudden cardiac death, and progressive heart failure. Currently, there is evidence that AC includes a spectrum of cardiomyopathy phenotypes, ranging from the classical form of arrhythmogenic right ventricular cardiomyopathy (ARVC) to more recently identified forms of arrhythmogenic left ventricular cardiomyopathy.1,2 ARVC is considered a disease of the desmosome, because in the majority of cases, it is caused by mutations in genes encoding proteins of the cardiac desmosomes.3 However, mutations in nondesmosomal genes have also been found in ARVC, such as the genes encoding the cardiac ryanodine receptor 2 ( RYR2 ), the transforming growth factor β-3 ( TGFB3 ), the nuclear transmembrane protein 43 ( TMEM43 ), and desmin ( DES ). Mutations in LMNA , encoding the nuclear envelope protein lamin A/C, known to cause dilated cardiomyopathy with an arrhythmic phenotype, was reported to cause also an ARVC phenotype,3 even though the association between LMNA and ARVC is still questioned. In the left dominant form, which clinically presents as an arrhythmogenic dilated cardiomyopathy, the same desmosomal genes causing ARVC can be found,1 as well as LMNA and the more recently identified filamin C gene ( FLNC ), encoding an actin …

Background— β-Adrenergic receptor (β-AR) activation can provoke cardiac arrhythmias mediated by cAMP-dependent alterations of Ca 2+ signaling. However, cAMP can activate both protein kinase A and an exchange protein directly activated by cAMP (Epac), but their functional interaction is unclear. In heart, selective Epac activation can induce potentially arrhythmogenic sarcoplasmic reticulum (SR) Ca 2+ release that involves Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) effects on the ryanodine receptor (RyR). Methods and Results— We tested whether physiological β-AR activation causes Epac-mediated SR Ca 2+ leak and arrhythmias and whether it requires Epac1 versus Epac2, β 1 -AR versus β 2 -AR, and CaMKIIδ-dependent phosphorylation of RyR2-S2814. We used knockout (KO) mice for Epac1, Epac2, or both. All KOs exhibited unaltered basal cardiac function, Ca 2+ handling, and hypertrophy in response to pressure overload. However, SR Ca 2+ leak induced by the specific Epac activator 8-CPT in wild-type mice was abolished in Epac2-KO and double-KO mice but was unaltered in Epac1-KO mice. β-AR–induced arrhythmias were also less inducible in Epac2-KO versus wild-type mice. β-AR activation with protein kinase A inhibition mimicked 8-CPT effects on SR Ca 2+ leak and was prevented by blockade of β 1 -AR but not β 2 -AR. CaMKII inhibition (KN93) and genetic ablation of either CaMKIIδ or CaMKII phosphorylation on RyR2-S2814 prevented 8-CPT–induced SR Ca 2+ leak. Conclusions— β 1 -AR activates Epac2 to induce SR Ca 2+ leak via CaMKIIδ-dependent phosphorylation of RyR2-S2814. This pathway contributes to β-AR–induced arrhythmias and reduced cardiac function.

Background— Recently, we reported that sarcoplasmic reticulum Ca 2+ ATPase 2a (SERCA2a), the pump responsible for reuptake of cytosolic calcium during diastole, plays a central role in the molecular mechanism of cardiac alternans. Heart failure (HF) is associated with impaired myocardial calcium handling, deficient SERCA2a, and increased susceptibility to cardiac alternans. Therefore, we hypothesized that restoring deficient SERCA2a by gene transfer will significantly reduce arrhythmogenic cardiac alternans in the failing heart. Methods and Results— Adult guinea pigs were divided into 3 groups: control, HF, and HF+AAV9.SERCA2a gene transfer. HF resulted in a decrease in left ventricular fractional shortening compared with controls ( P 2+ release, and increased diastolic Ca 2+ ( P P m alternans and Ca alternans compared with controls ( P P P o secondary to reduction of ryanodine receptor 2–P S2814 ( P P= 0.05). Conclusions— These data show that SERCA2a gene transfer in the failing heart not only improves contractile function but also directly restores electric stability through the amelioration of key arrhythmogenic substrate (ie, cardiac alternans) and triggers (ie, sarcoplasmic reticulum Ca 2+ leak).

Background— Delayed afterdepolarizations (DADs) carried by Na + -Ca 2+ -exchange current (I NCX ) in response to sarcoplasmic reticulum (SR) Ca 2+ leak can promote atrial fibrillation (AF). The mechanisms leading to delayed afterdepolarizations in AF patients have not been defined. Methods and Results— Protein levels (Western blot), membrane currents and action potentials (patch clamp), and [Ca 2+ ] i (Fluo-3) were measured in right atrial samples from 76 sinus rhythm (control) and 72 chronic AF (cAF) patients. Diastolic [Ca 2+ ] i and SR Ca 2+ content (integrated I NCX during caffeine-induced Ca 2+ transient) were unchanged, whereas diastolic SR Ca 2+ leak, estimated by blocking ryanodine receptors (RyR2) with tetracaine, was ≈50% higher in cAF versus control. Single-channel recordings from atrial RyR2 reconstituted into lipid bilayers revealed enhanced open probability in cAF samples, providing a molecular basis for increased SR Ca 2+ leak. Calmodulin expression (60%), Ca 2+ /calmodulin-dependent protein kinase-II (CaMKII) autophosphorylation at Thr287 (87%), and RyR2 phosphorylation at Ser2808 (protein kinase A/CaMKII site, 236%) and Ser2814 (CaMKII site, 77%) were increased in cAF. The selective CaMKII blocker KN-93 decreased SR Ca 2+ leak, the frequency of spontaneous Ca 2+ release events, and RyR2 open probability in cAF, whereas protein kinase A inhibition with H-89 was ineffective. Knock-in mice with constitutively phosphorylated RyR2 at Ser2814 showed a higher incidence of Ca 2+ sparks and increased susceptibility to pacing-induced AF compared with controls. The relationship between [Ca 2+ ] i and I NCX density revealed I NCX upregulation in cAF. Spontaneous Ca 2+ release events accompanied by inward I NCX currents and delayed afterdepolarizations/triggered activity occurred more often and the sensitivity of resting membrane voltage to elevated [Ca 2+ ] i (diastolic [Ca 2+ ] i –voltage coupling gain) was higher in cAF compared with control. Conclusions— Enhanced SR Ca 2+ leak through CaMKII-hyperphosphorylated RyR2, in combination with larger I NCX for a given SR Ca 2+ release and increased diastolic [Ca 2+ ] i -voltage coupling gain, causes AF-promoting atrial delayed afterdepolarizations/triggered activity in cAF patients.

Background Approximately half of patients with heart failure die suddenly as a result of ventricular arrhythmias. Although abnormal Ca 2+ release from the sarcoplasmic reticulum through ryanodine receptors (RyR2) has been linked to arrhythmogenesis, the molecular mechanisms triggering release of arrhythmogenic Ca 2+ remain unknown. We tested the hypothesis that increased RyR2 phosphorylation by Ca 2+ /calmodulin-dependent protein kinase II is both necessary and sufficient to promote lethal ventricular arrhythmias. Methods and Results Mice in which the S2814 Ca 2+ /calmodulin-dependent protein kinase II site on RyR2 is constitutively activated (S2814D) develop pathological sarcoplasmic reticulum Ca 2+ release events, resulting in reduced sarcoplasmic reticulum Ca 2+ load on confocal microscopy. These Ca 2+ release events are associated with increased RyR2 open probability in lipid bilayer preparations. At baseline, young S2814D mice have structurally and functionally normal hearts without arrhythmias; however, they develop sustained ventricular tachycardia and sudden cardiac death on catecholaminergic provocation by caffeine/epinephrine or programmed electric stimulation. Young S2814D mice have a significant predisposition to sudden arrhythmogenic death after transverse aortic constriction surgery. Finally, genetic ablation of the Ca 2+ /calmodulin-dependent protein kinase II site on RyR2 (S2814A) protects mutant mice from pacing-induced arrhythmias versus wild-type mice after transverse aortic constriction surgery. Conclusions Our results suggest that Ca 2+ /calmodulin-dependent protein kinase II phosphorylation of RyR2 Ca 2+ release channels at S2814 plays an important role in arrhythmogenesis and sudden cardiac death in mice with heart failure.

Background— Catecholaminergic polymorphic ventricular tachycardia is a disease characterized by ventricular arrhythmias elicited exclusively under adrenergic stress. Additional features include baseline bradycardia and, in some patients, right ventricular fatty displacement. The clinical spectrum is expanded by the 2 families described here. Methods and Results— Sixteen members from 2 separate families have been clinically evaluated and followed over the last 15 years. In addition to exercise-related ventricular arrhythmias, they showed abnormalities in sinoatrial node function, as well as atrioventricular nodal function, atrial fibrillation, and atrial standstill. Left ventricular dysfunction and dilatation was present in several affected individuals. Linkage analysis mapped the disease phenotype to a 4-cM region on chromosome 1q42-q43. Conventional polymerase chain reaction–based screening did not reveal a mutation in either the Ryanodine receptor 2 gene ( RYR2 ) or ACTN2 , the most plausible candidate genes in the region of interest. Multiplex ligation-dependent probe amplification and long-range polymerase chain reaction identified a genomic deletion that involved RYR2 exon-3, segregated in all the affected family members (n=16) in these 2 unlinked families. Further investigation revealed that the genomic deletion occurred in both families as a result of Alu repeat–mediated polymerase slippage. Conclusions— This is the first report on a large genomic deletion in RYR2 , which leads to extended clinical phenotypes (eg, sinoatrial node and atrioventricular node dysfunction, atrial fibrillation, atrial standstill, and dilated cardiomyopathy). These features have not previously been linked to RYR2 .

Introduction: We have been previously reported that calmodulin (CaM) binding affinity to RyR2 decreases upon beta stimulation in catecholaminergic polymorphic ventricular tachycardia (CPVT) type knock-in mice with mutation at central domain (R2474S-KI). Meanwhile, amino acid residues from 4026 to 4172 of RyR2 have EF hand motifs and are called CaM-like domain (CaMLD). Some of the human CPVT mutations were reported in this CaMLD. In the present study we investigated the molecular mechanism of arrhythmogenesis of CPVT with mutation at CaMLD using N4103K KI mice. Methods and Results: VT and/or Vf were frequently observed after 120 mg/kg caffeine plus 1 mg/kg epinephrine infusion in KI mice, but not in WT mice (WT: 0%, KI: 43%). CaM binding affinity to RyR2 was assessed by incorporation of exogenous CaM labeled with HiLyte Fluor® in isolated saponin-permeabilized cardiomyocytes. The binding affinity of CaM decreased in KI cells (Kd: 161 nM) compared with WT cells (Kd: 89 nM). Addition of 1μM cAMP did not change the affinity of CaM to RyR2 in both KI and WT cells, while it decreased the affinity in R2474S-KI. Frequency of the Ca 2+ sparks was measured in isolated saponin-permeabilized cells. The Ca 2+ spark frequency (SpF: s -1 100μm -1 ) was much higher in KI than in WT cells {KI (n=79): 21.5±6.3; WT (n=34): 10.6±4.1; p2+ transient (SCaT) was measured during sequential pacing from 1 to 5 Hz in isolated intact cardiomyocytes. The SCaT was rarely observed in WT cells even with 10 nM isoproterenol and 5 Hz pacing, whereas that was observed in 33% of KI cells. Incorporation of CaM to intact KI cells using protein delivery kit (BioPORTER®) did not decrease incidence of SCaT (34% of KI cells), however, significantly decreased by HA-CaM incorporation. Conclusions: Different from R2474S-KI mice, CaM binding affinity to RyR2 is decreased without beta stimulation in KI mice with CaMLD mutation. Conformational change of CaMLD might affect CaM-binding domain (CaMBD) and decrease the affinity of CaM to CaMBD. The CaMLD-CaMBD interaction may be a novel therapeutic target of lethal arrhythmias.

Background: We have been reported that dantrolene (DAN) binds to N-terminal domain of the cardiac ryanodine receptor (RyR2) and corrects the aberrant Ca 2+ leak. We have been also reported that DAN enhances binding affinity of Calmodulin (CaM) to RyR2 in failing cardiomyocytes. In this study, we investigated whether CaM is essential for the anti-arrhythmogenic effect of DAN using pressure-overload heart failure induced by transverse aortic constriction (TAC). Methods and results: Pressure overload to 12 weeks old mice was induced by TAC using a 27-gauge needle. Cardiomyocytes isolated from mice 8 weeks after TAC operation were used in the present study. The survival rate of our TAC mice at 8 weeks after TAC operation was 32 %. LV end-diastolic dimension was significantly enlarged and LV ejection fraction was significantly decreased. Lethal arrhythmias were frequently observed after 2 mg epinephrine infusion in TAC mice, but not in Sham mice. All following experiments were done under 1 μM of KN93 to avoid the effect of CaMKII. The Ca 2+ spark frequency (SpF: s -1 ·100 μm -1 ; by fluo3) was much higher in TAC than in Sham cardiomyocytes {TAC (n=10): 7.6±4.7; Sham (n=5): 5.7±1.9; p2+ spark frequency {(n=5): 5.6±1.4}. This effect of DAN disappeared after suramin, CaM remover from RyR2, treatment {(n=4): 7.2±3.2}. Spontaneous Ca 2+ transients (SCaT) were measured during sequential pacing of intact cardiomyocytes from 1 Hz to 5 Hz. SCaT was rarely observed in Sham cells even at 5 Hz pacing, whereas it was observed in 20 % of TAC cells. Pre-incubation with DAN to intact TAC cells normalize inceidence of SCaT (11 %). After suramin incorporation, effect of DAN was abolished (19 %). Conclusions: In conclusion, CaM is essential for the channel stabilization effect of DAN. DAN corrects the aberrant domain-domain interaction, and as the result, CaM binding affinity to the RyR2 improves. These are the possible mechanism underlying anti-arrhythmogenic effect of DAN.

Introduction: Long-QT-syndrome type 2 (LQT2) has been linked to heterogeneities on a whole-heart scale. Here, we systematically investigated regional electromechanical function in wild-type (WT) and transgenic LQT2 rabbits. Methods: Rabbits (n=7 WT, 6 LQT2) were examined by phase-contrast magnetic resonance imaging (MRI) to assess cardiac wall movement velocities. A novel data-processing algorithm was developed to calculate regional contraction-like profiles. Using patch clamping, action potentials (AP) were recorded from cardiomyocytes isolated from the left ventricular (LV) apex and LV, septum, and right ventricular base. Cav1.2, KCNQ1, Kv1.4, Kir2.1, Nav1.5, SERCA, RyR2, Phospholamban (PLB) and NCX expression were quantified by western blot. Results: AP was longer in WT LV apex than in all three base regions (APD90 median 291 vs. 202, 209, 196ms; p Conclusions: Electromechanical heterogeneity is not exclusive to the arrhythmogenic heart: we demonstrate the presence of an electromechanical, in silico reproducible apico-basal gradient in healthy rabbit hearts with an associated regionally heterogeneous expression of 5 key proteins. This pattern can be explained logically as apical mechanical dominance reduces outflow resistance and increases efficiency. LQT2 rabbits show increased intra-cardiac heterogeneity with diastolic asynchrony that can be determined non-invasively and might have prognostic value.

Background— K201 (JTV519) is a newly developed 1,4-benzothiazepine drug with antiarrhythmic and cardioprotective properties. It functions via stabilization of the ryanodine receptor–calcium release channel in the heart (RyR2). This receptor has been identified in the kidney, and in vitro studies suggest a role in the control of renal hemodynamics. To date, the in vivo function of this receptor is undefined. We hypothesized that this new drug, which is being developed for the treatment of heart failure for its myocardial actions, also possesses renal hemodynamic enhancing and excretory properties. We also used immunohistochemistry to identify RyR2 in the normal canine kidney. Methods and Results— We investigated the renal actions of K201 during intrarenal infusion in normal anesthetized dogs. K201 was infused after baseline measurements at 2 doses (0.1 and 0.5 mg · kg −1 · min −1 ). Immunohistochemistry was used to identify RyR2 presence in the kidney not exposed to K201. K201 was potently natriuretic and diuretic, with glomerular filtration rate– and renal blood flow–enhancing actions. The excretory responses to K201 administration were associated with decreases in distal tubular reabsorption of sodium despite a mild decrease in mean arterial pressure, which returned to baseline levels after K201 discontinuation. Immunohistochemistry of the normal canine kidney revealed the presence of RyR2 in the medullary collecting duct cells. Conclusions— We report for the first time that the newly developed cardioprotective drug K201 possesses natriuretic, diuretic, glomerular filtration rate–enhancing, and vasodilating properties that go beyond myocardial actions and may support its therapeutic role in treatment of heart failure.

Background— Defective interdomain interaction within the ryanodine receptor (RyR2) seems to play a key role in the pathogenesis of heart failure, as shown in recent studies. In the present study we investigated the effect of oxidative stress on the interdomain interaction, its outcome in the cardiac function in heart failure, and the possibility of preventing the problem with antioxidants. Methods and Results— Sarcoplasmic reticulum (SR) vesicles were isolated from dog left ventricular (LV) muscle (normal or rapid ventricular pacing for 4 weeks with or without the antioxidant edaravone). In the edaravone-treated paced dogs (EV+), but not in the untreated paced dogs (EV−), normal cardiac function was restored almost completely. In the SR vesicles isolated from the EV−, oxidative stress of the RyR2 (reduction in the number of free thiols) was severe, but it was negligible in EV+. The oxidative stress of the RyR2 destabilized interdomain interactions within the RyR2 (EV−), but its effect was reversed in EV+. Abnormal Ca 2+ leak through the RyR2 was found in EV− but not in EV+. The amount of the RyR2-bound FKBP12.6 was less in EV− than in normal dogs, whereas it was restored almost to a normal amount in EV+. The NO donor 3-morpholinosydnonimine (SIN-1) reproduced, in normal SR, several abnormal features seen in failing SR, such as defective interdomain interaction and abnormal Ca 2+ leak. Both cell shortening and Ca 2+ transients were impaired by SIN-1 in isolated normal myocytes, mimicking the pathophysiological conditions in failing myocytes. Incubation of failing myocytes with edaravone restored the normal properties. Conclusions— During the development of heart failure, edaravone ameliorated the defective interdomain interaction of the RyR2. This prevented Ca 2+ leak and LV remodeling, leading to an improvement of cardiac function and an attenuation of LV remodeling.

Background— Mutations in the ryanodine 2 receptor (RyR2) gene have been identified in patients with catecholaminergic polymorphic ventricular tachycardia. We examined the cellular basis for the ECG features and arrhythmia mechanisms using low-dose caffeine to mimic the defective calcium homeostasis encountered under these conditions. Methods and Results— A transmural ECG and action potentials were recorded simultaneously from epicardial, M, and endocardial cells in arterially perfused canine ventricular wedge preparations. Caffeine alone produced no change (10 to 100 μmol/L) or a slight abbreviation (300 μmol/L) of the QT interval and no change in transmural dispersion of repolarization. Isoproterenol (100 nmol/L) alone induced sustained monomorphic ventricular tachycardia (VT) that originated in the epicardium in 3 of 14 wedge preparations. Isoproterenol in the presence of caffeine (100 to 300 μmol/L) induced epicardial VT in 9 of 16 wedge preparations. Delayed afterdepolarization–induced triggered beats that originated in the epicardium were associated with an increased T peak -T end interval and transmural dispersion of repolarization. Bidirectional VT developed in 11 of 16 wedge preparations as a consequence of alternation in the origin of ectopic activity between endocardial, M, and epicardial regions. Single extrastimuli delivered during sustained epicardial VT induced a rapid polymorphic VT/ventricular fibrillation (VF) in 3 of 9 wedges. Spontaneous polymorphic VT was observed in 3 of 16 preparations. Propranolol (1.0 μmol/L) or verapamil (1.0 μmol/L) completely suppressed ectopic activity that arose from the epicardium and prevented induction of polymorphic VT. Conclusions— Our data suggest delayed afterdepolarization–induced extrasystolic activity serves to trigger catecholamine-induced VT/VF under conditions of defective calcium handling. Epicardial origin of the ectopic beats increases transmural dispersion of repolarization, thus providing the substrate for the development of reentrant tachyarrhythmias that underlie rapid polymorphic VT/VF.

The cardiac ryanodine receptor (RyR2)/Ca2+ release channel on the sarcoplasmic reticulum (SR) is regulated by evolutionarily highly conserved signaling pathways that control excitation-contraction (EC) coupling in the heart. Phosphorylation of RyR2 by cAMP-dependent protein kinase (PKA) plays a key role in regulating the channel in response to stress via activation of the sympathetic nervous system (the “fight-or-flight response”).1 Maladaptive PKA hyperphosphorylation of RyR2 in failing hearts alters channel function, which may cause depletion of SR Ca2+ and diastolic release of SR Ca2+. This can initiate delayed afterdepolarizations that trigger ventricular arrhythmias.1 Mutations in RyR2 recently have been identified in patients with catecholaminergic induced sudden cardiac death (SCD).2–4⇓⇓ There may be a direct link between the PKA hyperphosphorylation of RyR2 that occurs during the progression of heart failure and fatal cardiac arrhythmias. Regulation of cardiac EC coupling by the release of Ca2+ from the SR via RyR2 in cardiomyocytes, known as Ca2+-induced Ca2+ release (CICR), has been appreciated for more than a decade.5,6⇓ Furthermore, it is well known that the amplitude of the Ca2+ transient generated by SR Ca2+ release determines contractile force in cardiomyocytes. The systems that regulate SR Ca2+ release include: (1) the triggers (predominantly Ca2+ influx through the voltage-gated Ca2+ channel on the plasma membrane); (2) the SR Ca2+ release channel or type 2 RyR2; and (3) the SR Ca2+ reuptake pump (SERCA2a) and its regulator phospholamban. These systems (trigger, release, and reuptake) are modulated by signaling pathways, including the β-adrenergic receptor (β-AR) signaling pathway (ie, phosphorylation by PKA). Activation of the sympathetic nervous system in response to stress results in elevation of cAMP levels and activation of PKA. Phosphorylation of RyR2 may not correlate directly …

Understanding the pathogenetic chain of events between genotype and phenotype is critical to the appropriate diagnosis and treatment of heritable diseases. Although our knowledge of the molecular substrate of many diseases continues to increase, identifying the mechanistic links between a susceptibility variant and disease expression remains a major challenge. A minority of mendelian diseases display complete clinical penetrance in individuals harboring known disease-causing mutations, with rare examples such as achondroplasia and Huntington disease. Even in the most well-characterized hereditary arrhythmia syndrome, the long-QT syndrome (LQTS), in which genotype-phenotype relationships and gene-specific management strategies1 exist for the most common molecular subtypes (LQTS 1 to 3), the reasons for incomplete penetrance and variable expression in this disease remain largely unknown. In fact, LQTS has relatively low penetrance; however, in those LQTS genotype-positive individuals who do exhibit symptoms, the initial presentation can be catastrophic, and all too often, families come to clinical attention as a result of aborted cardiac arrest or sudden cardiac death. Therefore, identification of disease-associated mutations in individuals at risk for heritable arrhythmogenic disorders is imperative for subsequent risk reduction in their family members and them. Article see p 1001 Over the past 10 to 15 years, it has been shown that mutations in SCN5A , the gene encoding the α subunit of the human cardiac sodium channel (Na v 1.5), have pleiotropic effects, including risk for type 3 LQTS, Brugada syndrome, progressive cardiac conduction disease, atrial fibrillation, sick sinus syndrome, sudden infant death syndrome, and dilated cardiomyopathy. This phenotypic plasticity is not unique to SCN5A , but is also observed with other cardiovascular disease–associated genes. For example, mutations in the cardiac ryanodine receptor gene, RYR2 , are associated with catecholaminergic polymorphic ventricular tachycardia and arrhythmogenic right ventricular dysplasia/cardiomyopathy. In the current issue of Circulation , Watanabe et al …

Introduction: Targeting leaky cardiac ryanodine receptors (RyR2) to prevent diastolic Ca2+ release from the sarcoplasmic reticulum (SR) is a promising pharmacological approach, to rescue the impaired cardiac contraction and prevent Ca2+-dependent arrhythmias in heart failure (HF) and disease. Hypothesis: Based on prior work from the Marks group, the Rycal S44121 (also known as ARM036) is an experimental small molecule stabilizer of RyR. We investigated the effects of S44121 in a post-myocardial infarction (PMI) mouse model of HF. Methods and results: Mice were randomly assigned to 3 groups: Sham, PMI (subjected to left coronary artery ligation), and PMI-S (treated for 3 weeks with S44121 by subcutaneous osmotic pumps on day 7 post-MI, 10 mg/kg/day). Intracellular Ca2+ was measured on single left ventricular myocytes. PMI mice exhibited a 4-fold increase in the frequency of spontaneous Ca2+ release events, Ca2+ sparks, as measured in quiescent cells using the fluorescent Ca2+ indicator Fluo-4. PMI mice also exhibited higher global diastolic Ca2+, measured with the ratiometric fluorescent probe, Indo-1 AM, and increased the occurrence of ectopic diastolic Ca2+ waves. Acute application of S44121 (10 μM for 15 min) reduced Ca2+ sparks frequency. Chronic treatment of mice with S44121 also normalized the frequency of Ca2+ sparks and of ectopic Ca2+ waves, and corrected diastolic cellular Ca2+ overload. Effects were maximal at 20 mg/kg/day. Furthermore, treatment with S44121 abolished Ca2+ waves promoted by β-adrenergic challenge (acute application of isoproterenol, 10 nM). The potential anti-arrhythmic benefit of S44121 was assessed in vivo using telemetric surface electrocardiograms. S44121 had no effect on ECG intervals and did not alter the heart rate. However, anti-arrhythmic effects were confirmed by observation of a dose-dependent reduction of spontaneous ventricular extrasystoles and ventricular tachycardia. Near maximum benefits were observed at 10 mg/kg/day, both in basal conditions or following a challenge with acute treatment of isoproterenol (0.5 mg/kg, dosed ip). Conclusion: In mice with post-ischemic HF, treatment with S44121 prevented the abnormal diastolic SR Ca2+ leak and ectopic Ca2+ waves, and reduced ventricular arrhythmias.

Background: Mutations in the gene encoding the cardiac sodium channel (SCN5A) have been implicated in arrhythmia syndromes including Brugada (BrS) and long-QT type 3 syndromes. Also, wide spectrums of mixed phenotypes, known as “overlap syndromes”, have been associated to SCN5A mutations. The contributions of single nucleotide polymorphisms (SNPs) add more complexity to the disease phenotype. Here we report a missense mutation in SCN5A (T1708N) in three Hispanic families with history of sudden cardiac death (SCD) and extensive variability and severity of symptoms, including syncope, atrial and ventricular tachycardia, and conduction abnormalities. Methods and Results: We used the Ion Torrent platform and AmpliSeq panel to sequence 19 genes associated with arrhythmia syndromes. In addition to SCN5A-T1708N mutation, the patients carry two common SNPs, SCN5A-H558R and the cardiac ryanodine receptor RYR2-Q2958R, which we previously reported to be highly prevalent (~30%) in Hispanics and to increase the risk of SCD in homozygous individuals. To study the functional properties of SCN5A-T1708N, the mutation was engineered by site-directed mutagenesis into SCN5A with or without the SCN5A-H558R and expressed in HEK cells for voltage clamp experiments. SCN5A-T1708N showed a significant increase of late INa compared to WT. Also, the kinetic properties of SCN5A-T1708N showed slower time constants of recovery and increase in slow inactivation, which would tend to reduce INa; these effects were not modified by the presence of SCN5A-H558R. On the other hand, INa density for SCN5A-T1708N was reduced compared to WT (289± 63 and 387± 62 pA/pF, respectively), yet, for the double mutant H558R-T1708N, INa was more severely depressed (145± 32 pA/pF). Conclusion: The profound biophysical phenotype of T1708N with loss and gain of function could account for the variability and the severity of the clinical phenotype in these families. The RYR2-Q2958R polymorphism may function as a pro-arrhythmic genetic modifier and it is conceivable that it may predispose to SCD in some members of these families. The study of these polymorphisms in large cohorts may identify common haplotypes associated to cardiac arrhythmias and facilitate identification of patients at high risk for SCD.

Background— Ca 2+ -dependent signaling through CaM Kinase II (CaMKII) and calcineurin was suggested to contribute to adverse cardiac remodeling. However, the relative importance of CaMKII versus calcineurin for adverse cardiac remodeling remained unclear. Methods and Results— We generated double-knockout mice (DKO) lacking the 2 cardiac CaMKII genes δ and γ specifically in cardiomyocytes. We show that both CaMKII isoforms contribute redundantly to phosphorylation not only of phospholamban, ryanodine receptor 2, and histone deacetylase 4, but also calcineurin. Under baseline conditions, DKO mice are viable and display neither abnormal Ca 2+ handling nor functional and structural changes. On pathological pressure overload and β-adrenergic stimulation, DKO mice are protected against cardiac dysfunction and interstitial fibrosis. But surprisingly and paradoxically, DKO mice develop cardiac hypertrophy driven by excessive activation of endogenous calcineurin, which is associated with a lack of phosphorylation at the auto-inhibitory calcineurin A site Ser411. Likewise, calcineurin inhibition prevents cardiac hypertrophy in DKO. On exercise performance, DKO mice show an exaggeration of cardiac hypertrophy with increased expression of the calcineurin target gene RCAN1-4 but no signs of adverse cardiac remodeling. Conclusions— We established a mouse model in which CaMKII’s activity is specifically and completely abolished. By the use of this model we show that CaMKII induces maladaptive cardiac remodeling while it inhibits calcineurin-dependent hypertrophy. These data suggest inhibition of CaMKII but not calcineurin as a promising approach to attenuate the progression of heart failure.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmic syndrome characterized by exercise- or stress-induced polymorphic ventricular tachyarrhythmias and sudden cardiac death in the context of a structurally normal heart.1,2 It is estimated to occur in ≈1 in 10 000 of the population and has a poor prognosis, with up to 50% mortality by 20 years of age.3 The pathophysiology has been studied extensively since the first genetic mutation was first identified by Priori and colleagues in 2002.1,2 Abnormal calcium storage and release from the sarcoplasmic reticulum (SR) within the cardiomyocytes underpin the disease, with causative mutations in 2 critical proteins in the SR complex: the cardiac SR calcium release channel known as the ryanodine receptor (RyR2) and the calcium buffering and RyR2 regulatory protein calsequestrin (CASQ2). Mutations have been identified in the RyR2 or CASQ2 genes in ≈60% to 70% of individuals with the clinical syndrome. Article see p 2673 Diastolic SR calcium release via RyR2 is the molecular basis for delayed afterdepolarizations (DADs), triggered activity, and ventricular ectopy and, if oscillatory, can be sustained ventricular tachyarrhythmias, leading to cardiac arrest and sudden death. Increased sympathetic activity activates the cardiac β-adrenoceptor (βAR) pathways, leading to phosphorylation of RyR2 and increasing the RyR2 channel opening probability.4 βAR activation also increases sarcoplasmic endoplasmic reticular calcium ATPase2 (SERCA2a) activity via phosphorylation of the inhibitory protein phospholamban, increasing SR calcium loading. Both of these effects are important for the normal increase in cardiac inotropy during exercise or stress as part of the “flight or fight” response. Individuals with CPVT harbor mutations in RyR2 or CASQ2, lowering the threshold for abnormal SR calcium release during diastole, when the RyR2 channel should be closed.1 Hence, individuals with CPVT develop complex ventricular tachyarrhythmias secondary to maladaptive handling of …

Background —Familial polymorphic ventricular tachycardia is an autosomal-dominant, inherited disease with a relatively early onset and a mortality rate of ≈30% by the age of 30 years. Phenotypically, it is characterized by salvoes of bidirectional and polymorphic ventricular tachycardias in response to vigorous exercise, with no structural evidence of myocardial disease. We previously mapped the causative gene to chromosome 1q42-q43. In the present study, we demonstrate that patients with familial polymorphic ventricular tachycardia have missense mutations in the cardiac sarcoplasmic reticulum calcium release channel (ryanodine receptor type 2 [RyR2]). Methods and Results —In 3 large families studied, 3 different RyR2 mutations (P2328S, Q4201R, V4653F) were detected and shown to fully cosegregate with the characteristic arrhythmic phenotype. These mutations were absent in the nonaffected family members and in 100 healthy controls. In addition to identifying 3 causative mutations, we identified a number of single nucleotide polymorphisms that span the genomic structure of RyR2 and will be useful for candidate-based association studies for other arrhythmic disorders. Conclusions —Our data illustrate that mutations of the RyR2 gene cause at least one variety of inherited polymorphic tachycardia. These findings define a new entity of disorders of myocardial calcium signaling.

Background— Sarcoplasmic reticulum (SR) Ca 2+ leak through ryanodine receptor type 2 (RyR2) dysfunction is of major pathophysiological relevance in human heart failure (HF); however, mechanisms underlying progressive RyR2 dysregulation from cardiac hypertrophy to HF are still controversial. Methods and Results— We investigated healthy control myocardium (n=5) and myocardium from patients with compensated hypertrophy (n=25) and HF (n=32). In hypertrophy, Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) and protein kinase A (PKA) both phosphorylated RyR2 at levels that were not different from healthy myocardium. Accordingly, inhibitors of these kinases reduced the SR Ca 2+ leak. In HF, however, the SR Ca 2+ leak was nearly doubled compared with hypertrophy, which led to reduced systolic Ca 2+ transients, a depletion of SR Ca 2+ storage and elevated diastolic Ca 2+ levels. This was accompanied by a significantly increased CaMKII-dependent phosphorylation of RyR2. In contrast, PKA-dependent RyR2 phosphorylation was not increased in HF and was independent of previous β-blocker treatment. In HF, CaMKII inhibition but not inhibition of PKA yielded a reduction of the SR Ca 2+ leak. Moreover, PKA inhibition further reduced SR Ca 2+ load and systolic Ca 2+ transients. Conclusions— In human hypertrophy, both CaMKII and PKA functionally regulate RyR2 and may induce SR Ca 2+ leak. In the transition from hypertrophy to HF, the diastolic Ca 2+ leak increases and disturbed Ca 2+ cycling occurs. This is associated with an increase in CaMKII- but not PKA-dependent RyR2 phosphorylation. CaMKII inhibition may thus reflect a promising therapeutic target for the treatment of arrhythmias and contractile dysfunction.

The key event in cardiac excitation-contraction coupling is calcium-induced calcium release from the sarcoplasmic reticulum (SR) after activation of cardiac ryanodine receptors (RyRs).1 Two distinct genes encode the cardiac (RyR2) and skeletal muscle (RyR1) specific receptors.2,3 A third type of ryanodine receptor (RyR3) exhibits functional properties distinct from those of RyR1 and RyR2.4 The RyR channels form a tetrameric structure composed of 4 monomeric subunits, each of about 565 000 Daltons. These channels are about 10 times larger than the voltage-gated Ca2+ and Na+ channels.5 RyR2 opening increases cytosolic Ca2+ from approximately 100 nmol/L to 1 μmol/L with consecutive activation of contractile proteins. The RyR was recently shown to be a macromolecular complex including protein kinase A (PKA) and its anchoring protein (mAKAP), the protein phosphatases PP1 and PP2A, sorcin, calmodulin, FK506 binding protein (FKBP), and other proteins.5 FKBP12.0 and its isoform FKBP12.6 seem to play an important role for the regulation of the RyR. The immunophilin FKBP12 was originally identified as the cytosolic receptor for the immunosuppressant FK506 in T-cells. FKBP12 is a cis/trans peptidyl-prolyl isomerase. However, it is unlikely that this enzymatic activity is directly involved in RyR regulation. FKBP12 has been postulated to bind to the RyR with a stoichiometry of 4 or less FKBP12 molecules per single functional channel.6 FKBP12.0 is tightly bound to and regulates the function of the skeletal RyR1, whereas RyR2 has been found to co-purify selectively with FKBP12.6 despite a higher expression level of FKBP12.0 in cardiac myocytes.7 However, considerable species differences in binding preferences of FKBPs to RyR isoforms have been observed.8 See p 477 Marx et al9 recently suggested that phosphorylation of RyR2 dissociates the regulatory protein FKBP12.6 from the channel, resulting in pronounced altered channel function. First, …

The cardiac ryanodine receptor (RyR2) is the major calcium (Ca2+) release channel on the sarcoplasmic reticulum (SR) in cardiomyocytes. During excitation-contraction, coupling intracellular Ca2+ stored in the SR is released via RyR2 to activate muscle contraction. In the heart, excitation-contraction coupling is activated by Ca2+ influx via the L-type Ca2+ channel that activates RyR2, a process referred to as Ca2+-induced Ca2+ release.1,2⇓ The cardiac muscle RyR2 and its homologue, the skeletal muscle RyR1, are macromolecular complexes that include four ≈565-kDa RyR1 or RyR2, four FKBP12 or FKBP12.6 (12-kDa peptidyl-prolyl isomerases that are required for normal gating of the channels), as well as cAMP-dependent kinase (PKA), phosphatases, and their targeting proteins.3–5⇓⇓ One key role for the macromolecular signaling complex is to modulate channel function in response to activation of the sympathetic nervous system (ie, the classic “fight-or-flight” stress response).5,6⇓ See p 69 In the past year, three groups have independently discovered at least 21 mutations in RyR2 (Figure) that are linked to stress-induced sudden cardiac death.7–9⇓⇓ To date, RyR2 mutations have been associated with 2 forms of sudden cardiac death (SCD): (1) catecholaminergic polymorphic ventricular tachycardia (CPVT) or familial polymorphic ventricular tachycardia (FPVT), and (2) arrhythmogenic right ventricular dysplasia type 2 (ARVD2). Exercise-induced SCD-linked RyR2 mutations. Locations of SCD mutations in human RyR2 compared with MH/CCD regions of RyR1 and known regulatory domains of the channel. Eleven reported SCD-linked RyR2 mutations7–9⇓⇓ cluster in three regions homologous to three MH/CCD regions.30,31⇓ Common polymorphisms are indicated by italics. The location of 3 RyR2 leucine/isoleucine zippers (LZ) that target PP1, PP2A, and PKA to RyR2 are shown,5,6⇓ as is the FKBP12.6 binding region,32 and the CaM binding site.33 …

We recently identified the K201(JTV519)-binding site as domain2114–2149 of the ryanodine receptor (RyR2), and the binding of K201 to this domain corrects the defective inter-domain interaction between N-terminal (0–600) and central regions (2000–2500) of RyR2 in pacing-induced failing hearts. Here, we further investigated the role of the domain peptide2114–2149 (DP 2114–2149 ) on the Ca 2+ release in human CPVT-associated RyR2 R2474S/+ knock-in (KI) mice model. The KI mice revealed no structural or histological abnormality in hearts, and also had no contractile or relaxation dysfunction at rest. In all KI mice (n= 6), however, bi-directional ventricular tachycardia (VT) was observed after exercise with treadmill (Ex:6/6), but not observed in wild-type (WT) mice (Ex:0/7 ). In saponin-permeabilized cardiomyocytes from WT and KI mice, line scan images in cardiomyocytes were obtained to measure local Ca 2+ release events using a confocal microscopy with Rhod-2; [Ca 2+ ] was buffered at 30 nM by 0.5 mM EGTA. In KI, the frequency of Ca 2+ sparks (SpF: s −1 ·100μm −1 ) was much more increased in response to cAMP (0.1–1 μM) than in WT (KI:19.2±0.4 vs WT:14.9±0.4; at [cAMP]= 0.3 μM , p2114–2149 abolished the cAMP-induced increase in SpF in KI. Sarcoplasmic reticulum (SR) vesicles were isolated from WT and KI hearts (n=4), then RyR2 was fluorescently labeled with methylcoumarin acetamido (MCA) using DP 2460–2495 (DPc10), which harbors the same mutation site in CPVT as KI mice;R2474S, as a site-directing carrier. Addition of cAMP (1 μM) to the SR from KI (but not WT) mice reduced stabilizing interactions between the N-terminal 1–600 and central domains 2000–2500 (i.e. domain unzipping), as assessed by the quenching of the MCA fluorescence by a large-size fluorescence quencher (Kq: 1.16 in KI: 1.10 in WT). Interestingly, DP 2114–2149 corrected this domain unzipping in KI (Kq:1.13), although DP 2114–2149 had no effect on the cAMP-induced increase in Ser2808 phosphorylation. In conclusion, RyR2 mutation seen in human CPVT may causatively induce hyper-activated channel gating in response to PKA phosphorylation, via defective inter-domain interaction. Correction of the defective inter-domain interaction may be a new therapeutic strategy against CPVT.

We previously reported that dantrolene, a therapeutic agent for malignant hyperthermia (MH), prevented abnormal Ca 2+ leak by correction of the defective inter-domain interaction between N-terminal (1– 600) and central (2000 –2500) domains in MH-type ryanodine receptor (RyR). These N-terminal and central domains of the RyR, harbor many mutations associated with MH in RyR1 and CPVT in RyR2. Here, we examined the effect of dantrolene on the Ca 2+ release in CPVT-associated RyR2 R2474S/ + knock-in (KI) mice model. In KI (but not in wild-type:WT) mice (n=6), ventricular tachycardia was observed during or after exercise with treadmill (6/6), which was prevented by pretreatment of dantrolene (20 mg −1 kg −1 day −1 , for 7 days). Cardiac sarcoplasmic reticulum (SR) vesicles were isolated (n=4), then RyR2 was fluorescently labeled with methylcoumarin acetamido (MCA) using DP 2460 – 2495 (DPc10), harboring the CPVT mutation site; R2474S, as a site-directing carrier. Only in KI (but not WT) SR, cAMP (1 μM) reduced stabilizing interactions between N-terminal and central domains (viz. domain unzipping), as assessed by the quenching of the MCA fluorescence by a large-size fluorescence quencher. Dantrolene (1 μM) inhibited the cAMP-induced domain unzipping (in KI) without effect on the cAMP-induced increase in Ser2808 phosphorylation that showed no difference between WT and KI. Using a quartz crystal microbalance technique (a highly sensitive mass-measuring technique), dantrolene was found to specifically bind to the domain 601– 620 of RyR2. In isolated, saponin-permeabilized cardiomyocytes, local Ca 2+ release events were measured using a confocal microscopy with Rhod-2; [Ca 2+ ] was buffered at 30 nM by 0.5 mM EGTA. In KI, the frequency of Ca 2+ sparks (SpF: s −1 · 100 μm −1 ) was much more increased in response to 1μM cAMP (9.7±0.4 in WT; 14.5±0.4 in KI, p2+ release leading to lethal arrhythmia.

Background— Ca 2+ release from the sarcoplasmic reticulum via the ryanodine receptor (RyR2) activates cardiac myocyte contraction. An important regulator of RyR2 function is FKBP12.6, which stabilizes RyR2 in the closed state during diastole. β-Adrenergic stimulation has been suggested to dissociate FKBP12.6 from RyR2, leading to diastolic sarcoplasmic reticulum Ca 2+ leakage and ventricular tachycardia (VT). We tested the hypothesis that FKBP12.6 overexpression in cardiac myocytes can reduce susceptibility to VT in stress conditions. Methods and Results— We developed a mouse model with conditional cardiac-specific overexpression of FKBP12.6. Transgenic mouse hearts showed a marked increase in FKBP12.6 binding to RyR2 compared with controls both at baseline and on isoproterenol stimulation (0.2 mg/kg IP). After pretreatment with isoproterenol, burst pacing induced VT in 10 of 23 control mice but in only 1 of 14 transgenic mice ( P 2+ spark frequency was reduced by 50% ( P 2+ load remained unchanged. In parallel, peak I Ca,L density decreased by 15% ( P 2+ transient peak amplitude decreased by 30% ( P 2+ transient decay was associated with an 18% reduction in the Na + -Ca 2+ exchanger protein level ( P Conclusions— Increased FKBP12.6 binding to RyR2 prevents triggered VT in normal hearts in stress conditions, probably by reducing diastolic sarcoplasmic reticulum Ca 2+ leak. This indicates that the FKBP12.6-RyR2 complex is an important candidate target for pharmacological prevention of VT.

Background— We previously demonstrated that defective interdomain interaction between N-terminal (0 to 600) and central regions (2000 to 2500) of ryanodine receptor 2 (RyR2) induces Ca 2+ leak in failing hearts and that K201 (JTV519) inhibits the Ca 2+ leak by correcting the defective interdomain interaction. In the present report, we identified the K201-binding domain and characterized the role of this novel domain in the regulation of the RyR2 channel. Methods and Results— An assay using a quartz-crystal microbalance technique (a very sensitive mass-measuring technique) revealed that K201 specifically bound to recombinant RyR2 fragments 1741 to 2270 and 1981 to 2520 but not to other RyR2 fragments from the 1 to 2750 region (1 to 610, 494 to 1000, 741 to 1260, 985 to 1503, 1245 to 1768, 2234 to 2750). By further analysis of the fragment 1741–2270 , K201 was found to specifically bind to its subfragment 2114–2149 . With the use of the peptide matching this subfragment (DP 2114–2149 ) as a carrier, the RyR2 was fluorescently labeled with methylcoumarin acetate (MCA) in a site-directed manner. After tryptic digestion, the major MCA-labeled fragment of RyR2 (155 kDa) was detected by an antibody raised against the central region (Ab 2132 ). Moreover, of several recombinant RyR2 fragments, only fragment 2234–2750 was specifically MCA labeled; this suggests that the K201-binding domain 2114–2149 binds with domain 2234–2750 . Addition of DP 2114–2149 to the MCA-labeled sarcoplasmic reticulum interfered with the interaction between domain 2114–2149 and domain 2234–2750 , causing domain unzipping, as evidenced by an increased accessibility of the bound MCA to a large-size fluorescence quencher. In failing cardiomyocytes, the frequency of spontaneous Ca 2+ spark was markedly increased compared with normal cardiomyocytes, whereas incorporation of DP 2114–2149 markedly decreased the frequency of spontaneous Ca 2+ spark. Conclusions— We first identified the K201-binding site as domain 2114–2149 of RyR2. Interruption of the interdomain interaction between the domain 2114–2149 and central domain 2234–2750 seems to mediate stabilization of RyR2 in failing hearts, which may lead to a novel therapeutic strategy against heart failure and perhaps lethal arrhythmia.

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.

Background— Ca 2+ leak from the sarcoplasmic reticulum (SR) may play an important role in triggering and/or maintaining atrial arrhythmias, including atrial fibrillation (AF). Protein kinase A (PKA) hyperphosphorylation of the cardiac ryanodine receptor (RyR2) resulting in dissociation of the channel-stabilizing subunit calstabin2 (FK506-binding protein or FKBP12.6) causes SR Ca 2+ leak in failing hearts and can trigger fatal ventricular arrhythmias. Little is known about the role of RyR2 dysfunction in AF, however. Methods and Results— Left and right atrial tissue was obtained from dogs with AF induced by rapid right atrial pacing (n=6 for left atrial, n=4 for right atrial) and sham instrumented controls (n=6 for left atrial, n=4 for right atrial). Right atrial tissue was also collected from humans with AF (n=10) and sinus rhythm (n=10) and normal cardiac function. PKA phosphorylation of immunoprecipitated RyR2 was determined by back-phosphorylation and by immunoblotting with a phosphospecific antibody. The amount of calstabin2 bound to RyR2 was determined by coimmunoprecipitation. RyR2 channel currents were measured in planar lipid bilayers. Atrial tissue from both the AF dogs and humans with chronic AF showed a significant increase in PKA phosphorylation of RyR2, with a corresponding decrease in calstabin2 binding to the channel. Channels isolated from dogs with AF exhibited increased open probability under conditions simulating diastole compared with channels from control hearts, suggesting that these AF channels could predispose to a diastolic SR Ca 2+ leak. Conclusions— SR Ca 2+ leak due to RyR2 PKA hyperphosphorylation may play a role in initiation and/or maintenance of AF.

Background— Swimming is a relatively genotype-specific arrhythmogenic trigger for type 1 long-QT syndrome (LQT1). We hypothesize that mimickers of concealed LQT1, namely catecholaminergic polymorphic ventricular tachycardia (CPVT), may also underlie swimming-triggered cardiac events. Methods and Results— Between August 1997 and May 2003, 388 consecutive, unrelated patients were referred specifically for LQTS genetic testing. The presence of a personal and/or family history of a near-drowning or drowning was determined by review of the medical records and/or phone interviews and was blinded to genetic test results. Comprehensive mutational analysis of the 5 LQTS-causing channel genes, KCNQ1 (LQT1), KCNH2 (LQT2), SCN5A (LQT3), KCNE1 (LQT5), and KCNE2 (LQT6), along with KCNJ2 (Andersen-Tawil syndrome) and targeted analysis of 18 CPVT1-associated exons in RyR2 , was performed with the use of denaturing high-performance liquid chromatography and direct DNA sequencing. Approximately 11% (43 of 388) of the index cases had a positive swimming phenotype. Thirty-three of these 43 index cases had a “Schwartz” score (≥4) suggesting high clinical probability of LQTS. Among this subset, 28 patients (85%) were LQT1, 2 patients (6%) were LQT2, and 3 were genotype negative. Among the 10 cases with low clinical probability for LQTS, 9 had novel, putative CPVT1-causing RyR2 mutations. Conclusions— In contrast to previous studies that suggested universal LQT1 specificity, genetic heterogeneity underlies channelopathies that are suspected chiefly because of a near-drowning or drowning. CPVT1 and strategic genotyping of RyR2 should be considered when LQT1 is excluded in the pathogenesis of a swimming-triggered arrhythmia syndrome.

Background— Familial polymorphic ventricular tachycardia (FPVT) is characterized by exercise-induced arrhythmias and sudden cardiac death due to missense mutations in the cardiac ryanodine receptor (RyR2), an intracellular Ca 2+ release channel required for excitation-contraction coupling in the heart. Methods and Results— Three RyR2 missense mutations, P2328S, Q4201R, and V4653F, which occur in Finnish families, result in similar mortality rates of ≈33% by age 35 years and a threshold heart rate of 130 bpm, above which exercise induces ventricular arrhythmias. Exercise activates the sympathetic nervous system, increasing cardiac performance as part of the fight-or-flight stress response. We simulated the effects of exercise on mutant RyR2 channels using protein kinase A (PKA) phosphorylation. All 3 RyR2 mutations exhibited decreased binding of calstabin2 (FKBP12.6), a subunit that stabilizes the closed state of the channel. After PKA phosphorylation, FPVT-mutant RyR2 channels showed a significant gain-of-function defect consistent with leaky Ca 2+ release channels and a significant rightward shift in the half-maximal inhibitory Mg 2+ concentration (IC 50 ). Treatment with the experimental drug JTV519 enhanced binding of calstabin2 to RyR2 and normalized channel function. Conclusions— Sympathetic activation during exercise induces ventricular arrhythmias above a threshold heart rate in RyR2 mutation carriers. Simulating the downstream effects of the sympathetic activation by PKA phosphorylation of RyR2 channels containing these FPVT missense mutations produced a consistent gain-of-function defect. RyR2 function and calstabin2 depletion were rescued by JTV519, suggesting stabilization of the RyR2 channel complex may represent a molecular target for the treatment and prevention of exercise-induced arrhythmias and sudden death in these patients. (Circulation. 2004;109:3208-3214.)

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is a relatively newly recognized disease. A clinical profile of patients with this condition was first published in 1982.1 In that report, it was observed that the majority of patients were male. Patients presented with ventricular tachycardia of left bundle-branch block morphology. An enlarged right ventricle due to fibrofatty infiltration of the right ventricular free wall and a familial association were noted. It was considered to be a rare disease of unknown cause. Since then, it has been diagnosed with increasing frequency and has been reported to account for 3% to 5% of unexplained sudden cardiac death under the age of 65 years.2,3 Evidence of the disease is found in 30% to 50% of family members who are studied by noninvasive tests, including ECG, echocardiography, and signal-averaged ECG.4,5 It has been suggested that MRI may also be used for diagnostic purposes, but this remains controversial.6,7 Six genetic loci have been reported to be associated with ARVD/C, and 3 genes have been identified: ryanodine receptor (RyR2),8 plakoglobin (JUP),9and desmoplakin (DSM).10 There is autosomal dominant transmission with all the genetic forms except Naxos disease8 and Carvahal syndrome,11 both of which have autosomal recessive transmission. These 2 autosomal recessive disorders are variants of ARVD/C that are associated with skin and hair abnormalities. The cardiac presentation in Carvahal syndrome is that of a left ventricular cardiomyopathy and arrhythmias. With the recognition that ARVD/C is frequently familial and can cause arrhythmic death, the clinical challenge is how to definitively identify individuals with ARVD/C who have mild or minimal structural abnormalities of the right ventricle. Definite identification of minimal structural abnormalities of the right ventricle can be difficult because of the irregular shape and asymmetrical contractile pattern …

Background— Chronic β-adrenergic receptor (β-AR) blockade improves cardiac contractility and prolongs survival in patients with heart failure; however, the mechanisms underlying these favorable responses are poorly understood. Stress-induced activation of the sympathetic nervous system results in protein kinase A (PKA)-mediated phosphorylation of the calcium (Ca 2+ ) release channel/cardiac ryanodine receptor (RyR2), required for cardiac excitation-contraction (EC) coupling, activating the RyR2 channel, and increasing cardiac contractility. The hyperadrenergic state of heart failure results in leaky RyR2 channels attributable to PKA hyperphosphorylation and depletion of the stabilizing FK506 binding protein, FKBP12.6. We tested the hypothesis that improved cardiac muscle function attributable to β-AR blockade is associated with restoration of normal RyR2 channel function in patients with heart failure. Methods and Results— We assessed the effects of β-AR blockade on left ventricular volume using isolated perfused hearts and β-agonist responsiveness using muscle strips from patients undergoing transplantation. Twenty-four human hearts were examined, 10 from patients with heart failure treated with β-AR blockers (carvedilol, metoprolol, or atenolol), 9 from patients with heart failure without β-AR blocker treatment, and 5 normal hearts. RyR2 PKA phosphorylation was determined by back-phosphorylation, FKBP12.6 in the RyR2 macromolecular complex was determined by coimmunoprecipitation, and channel function was assayed using planar lipid bilayers. β-AR blockers reduced left ventricular volume (reverse remodeling) and restored β-agonist response in cardiac muscle from patients with heart failure. Improved cardiac muscle function was associated with restoration of normal FKBP12.6 levels in the RyR2 macromolecular complex and RyR2 channel function. Conclusions— Improved cardiac muscle function during β-AR blockade is associated with improved cardiac Ca 2+ release channel function in patients with heart failure.

Heart failure is a complex disorder involving maladaptive responses that result in defective regulation and function of multiple biological systems. Central to our understanding of heart failure and to the ability to design and test novel therapeutic approaches that will prolong survival and improve quality of life for the millions of individuals worldwide is the need to gain a better understanding of the molecular pathogenesis of the disorder. In the search for molecular physiological defects in failing hearts, it is logical to examine the mechanism of excitation-contraction (EC) coupling in which cardiomyocyte membrane depolarization, because of the cardiac action potential, is translated into mechanical contraction in the heart. This system requires the normal function of 3 key elements: (1) calcium (Ca2+) entry via the voltage-gated Ca2+ channel (VGCC) on the plasma membrane (transverse tubule); (2) Ca2+ release via the ryanodine receptor/Ca2+ release channel (RyR2); and (3) Ca2+ uptake via the Ca2+-ATPase on the sarcoplasmic reticulum (SR) (Figure 1). Figure 1. Regulation of key molecules in cardiac EC coupling by stress activated pathways. The normal fight or flight stress response activates 3 key molecules involved in cardiac EC coupling via PKA phosphorylation: (1) the trigger for cardiac EC coupling, the voltage-gated Ca2+ channel (VGCC); (2) the SR Ca2+ release channel RyR2; and (3) the Ca2+ uptake pathway (via PKA phosphorylation of phospholamban which reduces inhibition of the Ca2+-ATPase SERCA2a). These regulatory events conspire to increase systolic SR Ca2+ release and thereby increase contractility, providing increased cardiac output to meet metabolic demands of stressful conditions. In failing hearts, this system becomes defective because of the maladaptive chronic hyperadrenergic state of heart failure, resulting in PKA-hyperphosphorylated RyR2 channels that cause a diastolic SR Ca2+ leak1 that conspires with …

To the Editor: Catecholamine-induced polymorphic ventricular tachycardia (CPVT) is characterized by episodes of syncope, seizures, or sudden death in response to physical activity or emotional stress. The two modes of inheritance that have been described are autosomal dominant1 and autosomal recessive.2 Mutations in the ryanodine receptor 2 gene ( RYR2 ), which encodes a cardiac sarcoplasmic reticulum Ca2+ release channel, were shown to cause the autosomal dominant form of the disease.3 Recently, Priori et al …

Background — Mutations in the cardiac ryanodine receptor gene ( RyR2 ) underlie catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited arrhythmogenic disease occurring in the structurally intact heart. The proportion of patients with CPVT carrying RyR2 mutations is unknown, and the clinical features of RyR2 -CPVT as compared with nongenotyped CPVT are undefined. Methods and Results — Patients with documented polymorphic ventricular arrhythmias occurring during physical or emotional stress with a normal heart entered the study. The clinical phenotype of the 30 probands and of 118 family members was evaluated, and mutation screening on the RyR2 gene was performed. Arrhythmias documented in probands were: 14 of 30 bidirectional ventricular tachycardia, 12 of 30 polymorphic ventricular tachycardia, and 4 of 30 catecholaminergic idiopathic ventricular fibrillation; RyR2 mutations were identified in 14 of 30 probands (36% bidirectional ventricular tachycardia, 58% polymorphic ventricular tachycardia, 50% catecholaminergic idiopathic ventricular fibrillation) and in 9 family members (4 silent gene carriers). Genotype-phenotype analysis showed that patients with RyR2 CPVT have events at a younger age than do patients with nongenotyped CPVT and that male sex is a risk factor for syncope in RyR2 -CPVT (relative risk=4.2). Conclusions — CPVT is a clinically and genetically heterogeneous disease manifesting beyond pediatric age with a spectrum of polymorphic arrhythmias. β-Blockers reduce arrhythmias, but in 30% of patients an implantable defibrillator may be required. Genetic analysis identifies two groups of patients: Patients with nongenotyped CPVT are predominantly women and become symptomatic later in life; patients with RyR2 CPVT become symptomatic earlier, and men are at higher risk of cardiac events. These data provide a rationale for prompt evaluation and treatment of young men with RyR2 mutations.

Background β-Adrenergic receptor blockade is one of the most effective treatments for heart failure, a leading cause of mortality worldwide. The use of β-adrenergic receptor blockers in patients with heart failure is counterintuitive, however, because they are known to decrease contractility in normal hearts. The ryanodine receptor (RyR2) on cardiac sarcoplasmic reticulum is the key calcium release channel required for excitation-contraction coupling. In failing hearts, the stoichiometry and function of the RyR2 macromolecular complex is altered. Decreased levels of phosphatases (PP1 and PP2A) and hyperphosphorylation by protein kinase A result in dissociation of the regulatory protein FKBP12.6 and channels with increased open probability. Methods and Results Here, we show that systemic oral administration of a β-adrenergic receptor blocker reverses protein kinase A hyperphosphorylation of RyR2, restores the stoichiometry of the RyR2 macromolecular complex, and normalizes single-channel function in a canine model of heart failure. Conclusions These results may, in part, explain the improved cardiac function observed in heart failure patients treated with β-adrenergic receptor blockers.

Background —Catecholaminergic polymorphic ventricular tachycardia is a genetic arrhythmogenic disorder characterized by stress-induced, bidirectional ventricular tachycardia that may degenerate into cardiac arrest and cause sudden death. The electrocardiographic pattern of this ventricular tachycardia closely resembles the arrhythmias associated with calcium overload and the delayed afterdepolarizations observed during digitalis toxicity. We speculated that a genetically determined abnormality of intracellular calcium handling might be the substrate of the disease; therefore, we considered the human cardiac ryanodine receptor gene ( hRyR2 ) a likely candidate for this genetically transmitted arrhythmic disorder. Methods and Results —Twelve patients presenting with typical catecholaminergic polymorphic ventricular tachycardia in the absence of structural heart abnormalities were identified. DNA was extracted from peripheral blood lymphocytes, and single-strand conformation polymorphism analysis was performed on polymerase chain reaction–amplified exons of the hRyR2 gene. Four single nucleotide substitutions leading to missense mutations were identified in 4 probands affected by the disease. Genetic analysis of the asymptomatic parents revealed that 3 probands carried de novo mutations. In 1 case, the identical twin of the proband died suddenly after having suffered syncopal episodes. The fourth mutation was identified in the proband, in 4 clinically affected family members, and in none of 3 nonaffected family members in a kindred with 2 sudden deaths that occurred at 16 and 14 years, respectively, in the sisters of the proband. Conclusions —We demonstrated that, in agreement with our hypothesis, hRyR2 is a gene responsible for catecholaminergic polymorphic ventricular tachycardia.

Background —Fischer 344 (F344) rats are relatively resistant to hypoxia-induced right ventricular (RV) hypertrophy compared with the Wistar-Kyoto (WKY) strain. These 2 strains were used to examine the genetic basis for the differential response. Methods and Results —Male F 2 offspring from an F344×WKY intercross were exposed to hypoxia (10% O 2 ) for 3 weeks, and pulmonary artery pressure and cardiac chamber weights were measured. Genomic DNA was screened by use of polymorphic microsatellite markers across the whole genome (excluding the sex chromosomes). A quantitative trait locus (QTL) for RV weight was identified on rat chromosome 17 (lod score 6.5) that accounted for 22% of the total variance of RV weight in the F 2 population and was independent of pulmonary artery pressure. The peak was centered over marker D17Rat41, close to Chrm3 , with a 1-lod support interval of 5 cM. Comparison of homologous regions in mice and humans suggested that Ryr2, the cardiac isoform of the ryanodine receptor, colocalizes with our QTL. A panel of somatic cell hybrids and fluorescence in situ hybridization mapped Ryr2 close to the gene Chrm3 within our QTL. [ 3 H]Ryanodine binding to cardiac membranes from the parental strains showed a 21% reduction in B max in the WKY compared with the F344 strain, with no difference in K d . Conclusions —These data provide the first demonstration of a QTL linked to the RV response to hypoxia-induced pulmonary hypertension. The Ryr2 receptor gene lies within this QTL and merits further investigation as a candidate for this differential RV response.

Background —The response of ventricular myocytes to pressure overload is heterogeneous and not spatially coordinated. We investigated whether or not the alterations in SERCA and RyR gene expression are homogeneous within the myocardium. Methods and Results —The cellular distribution of mRNAs and proteins encoding the 2 sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA) isoforms (SERCA 2a and 2b) and 2 Ca 2+ release channels (the ryanodine receptor, RyR, and the IP 3 receptor, IP 3 R) were analyzed by in situ hybridization and immunofluorescence, respectively. Analyses were performed during early (1 and 5 days) and late (1 month) stages of cardiac hypertrophy induced in rat by thoracic aortic stenosis (AS). The results indicated that 1 and 5 days after AS, the cellular distribution of SERCA 2a and RyR2 mRNAs in right ventricle and atrium was similar to controls but the mRNA levels appeared to decrease in some areas of the left ventricle (LV). One month after AS, the distribution of SERCA 2a mRNA and protein became heterogeneous throughout the LV, whereas RyR2 mRNA and protein levels were decreased in a homogeneous manner. SERCA 2b, poorly expressed in both cardiomyocytes and vessels of controls, was increased 4-fold 1 month after AS in coronary arteries only. In both sham (Sh) and AS, SERCA 3 and IP 3 R mRNAs were mainly found in the vessels. Conclusions —In severe hypertrophy, decreased accumulation of SERCA 2a was heterogeneous and not compensated by an induction of SERCA 2b in the cardiomyocytes. Decrease in RyR2 expression was more homogeneous and not compensated by an increased IP 3 R expression.

Background The purpose of this study was to determine whether abnormal Ca 2+ release through ryanodine-sensitive Ca 2+ channels in the sarcoplasmic reticulum might contribute to the abnormal [Ca 2+ ] i homeostasis that has been described in failing human myocardium. Methods and Results Occupancy of low-affinity ryanodine binding sites on ryanodine-sensitive Ca 2+ channels stimulates oxalate-supported, ATP-dependent Ca 2+ accumulation in sarcoplasmic reticulum–derived microsomes by inhibiting concurrent Ca 2+ efflux through these channels. We examined the effects of 0.5 mmol/L ryanodine on 45 Ca 2+ accumulation in microsomes prepared from nonfailing (n=8) and failing (n=10) human left ventricular myocardium. In the absence of ryanodine, 45 Ca 2+ accumulation reached similar levels in microsomes from nonfailing and failing hearts. Incubation with 0.5 mmol/L ryanodine caused a 52.2±6.5% increase in peak 45 Ca 2+ accumulation in microsomes from nonfailing hearts and a 24.3±4.1% increase in microsomes from failing hearts. The density of high-affinity ryanodine binding sites and the inhibition of [ 3 H]ryanodine dissociation from these sites by 0.1 mmol/L ryanodine were similar in microsomes from nonfailing and failing hearts. Conclusions These results, which demonstrate a diminished stimulation of Ca 2+ accumulation by ryanodine in sarcoplasmic reticulum–derived microsomes from failing human myocardium that could be explained by an uncoupling of the occupancy of low-affinity ryanodine binding sites from the reduction in the open probability of these channels or by concurrent Ca 2+ efflux through a ryanodine-insensitive mechanism, are evidence that increased efflux of Ca 2+ from the sarcoplasmic reticulum may contribute to the abnormal [Ca 2+ ] i homeostasis described in failing human myocardium.