Spontaneous Ca Waves Research Articles

Abstract Or126: Targeting ER stress can attenuate arrhythmia in catecholaminergic polymorphic ventricular tachycardia

Gain-of-function mutations of the sarcoplasmic reticulum (SR) Ca 2+ release channel, the ryanodine receptor (RyR2), are linked to the inherited arrhythmia syndrome catecholaminergic polymorphic ventricular tachycardia (CPVT). Increasing evidence suggests that RyR2 gain-of-function not only disturbs intracellular Ca 2+ homeostasis but drives remodeling of cell signaling and ultrastructure that markedly contributes to the arrhythmogenic phenotype. It is well established that disturbed SR Ca 2+ homeostasis can activate the endoplasmic reticulum (ER) stress response, yet whether RyR2 gain-of-function in CPVT drives ER stress is yet to be explored. The goal of our study was to determine the contribution of ER stress evoked by RyR2 gain-of-function to cardiac arrhythmogenesis. To test this, we created a new rat model of CPVT induced by RyR2-S2222L (+/-) mutation. Simultaneous whole cell patch clamp and Ca 2+ imaging demonstrated that under β-adrenergic stimulation, CPVT ventricular myocytes (VMs) exhibit a high propensity to spontaneous Ca 2+ waves (SCWs) and delayed afterdepolarizations. Importantly, CPVT VMs showed increased XBP1 splicing as a marker of ER stress, as well as increased intra-SR redox stress measured using biosensor ER_roGFPiE. Assessment of ER stress proteins that contribute to SR redox status revealed upregulation of H 2 0 2 -producer enzyme ERO1α, with no compensatory change in expression of H 2 O 2 -degrader Peroxiredoxin-4 (PRDX4). Adenoviral overexpression of PRDX4 in CPVT VMs not only normalized SR redox status but attenuated Ca 2+ mishandling, reducing RyR2 activity and the incidence of proarrhythmic spontaneous Ca 2+ waves. Of note, immunofluorescence and biochemical studies suggest a direct interaction between RyR2 and PRDX4. To test whether targeting ER stress was protective at the whole heart level, we delivered AAV9-αMHC-PRDX4 to CPVT rats and performed ex vivo optical mapping. While CPVT hearts exhibited triggered activity and increased incidence of VT, sustained VT was prevented in PRDX4-injected hearts. Collectively, these data strongly suggest that ER stress contributes to the arrhythmogenic phenotype of CPVT. Targeting ER stress protein PRDX4 has promising therapeutic potential to normalize SR homeostasis, stabilize RyR2 activity and attenuate Ca 2+ -dependent arrhythmogenesis.

Zbtb16 increases susceptibility of atrial fibrillation in type 2 diabetic mice via Txnip-Trx2 signaling

Atrial fibrillation (AF) is the most prevalent sustained cardiac arrhythmia, and recent epidemiological studies suggested type 2 diabetes mellitus (T2DM) is an independent risk factor for the development of AF. Zinc finger and BTB (broad-complex, tram-track and bric-a-brac) domain containing 16 (Zbtb16) serve as transcriptional factors to regulate many biological processes. However, the potential effects of Zbtb16 in AF under T2DM condition remain unclear. Here, we reported that db/db mice displayed higher AF vulnerability and Zbtb16 was identified as the most significantly enriched gene by RNA sequencing (RNA-seq) analysis in atrium. In addition, thioredoxin interacting protein (Txnip) was distinguished as the key downstream gene of Zbtb16 by Cleavage Under Targets and Tagmentation (CUT&Tag) assay. Mechanistically, increased Txnip combined with thioredoxin 2 (Trx2) in mitochondrion induced excess reactive oxygen species (ROS) release, calcium/calmodulin-dependent protein kinase II (CaMKII) overactivation, and spontaneous Ca2+ waves (SCWs) occurrence, which could be inhibited through atrial-specific knockdown (KD) of Zbtb16 or Txnip by adeno-associated virus 9 (AAV9) or Mito-TEMPO treatment. High glucose (HG)-treated HL-1 cells were used to mimic the setting of diabetic in vitro. Zbtb16-Txnip-Trx2 signaling-induced excess ROS release and CaMKII activation were also verified in HL-1 cells under HG condition. Furthermore, atrial-specific Zbtb16 or Txnip-KD reduced incidence and duration of AF in db/db mice. Altogether, we demonstrated that interrupting Zbtb16-Txnip-Trx2 signaling in atrium could decrease AF susceptibility via reducing ROS release and CaMKII activation in the setting of T2DM.Graphical

Alzheimer's disease-related presenilin 1 M146L mutation disrupts SERCA2a regulation and increases propensity of Ca waves in ventricular cardiomyocytes.

Presenilin 1 (PS1) is a transmembrane protein expressed in the endoplasmic reticulum (ER) of different cell types. It has been shown that Ca mishandling due to PS1 mutations contributes to the Alzheimer's disease (AD)-related neurodegeneration. PS1 is also expressed in the heart. Several clinical studies revealed that PS1 mutations are associated with cardiomyopathies. We previously showed that PS1 interacts with the cardiac ER Ca ATPase (SERCA2a) and regulates its function. Here, we studied the role of AD-related PS1M146L mutation in regulation of intracellular Ca homeostasis. Fluorescent resonance energy transfer (FRET) experiments in HEK293 cells transfected with fluorescently labeled SERCA2a and PS1M146L revealed that the mutation does not disrupt the interaction between these two proteins. Measurements of SERCA2a-mediated Ca transport showed that at low ER Ca loads ([Ca]ER≤0.15 mM), both PS1WT and PS1M146L enhance ER Ca uptake by a similar level (∼40%) compared to control (no PS1 expression). At high ER Ca loads ([Ca]ER≥0.35 mM), however, PS1WT reduced ER Ca uptake and ER Ca load, whereas PS1M146L was less effective in regulating SERCA2a function. We used in vivo gene delivery in mouse cardiac wall to study the effect of PS1WT and PS1M146L overexpression on Ca regulation in ventricular myocytes. We found that both PS1WT and PS1M146L localized predominantly at the sarcoplasmic reticulum. Analysis of cytosolic Ca dynamics revealed that PS1WT overexpression in cardiomyocytes increases diastolic Ca and decreases Ca transient amplitudes. In contrast, PS1M146L expression increased propensity of spontaneous Ca waves. These results illustrate that the lack of SERCA2a regulation by PS1M146L at high ER Ca loads can lead to ER Ca overload and Ca waves. These defects in Ca regulation might contribute to the onset of cardiomyopathies in patients with PS1 mutations.

Modulation of global Ca2+ handling by SR-Ca2+ release depending on IP3

In the heart muscle, Ca2+-induced Ca2+ release (CICR) is the main mechanism for Ca2+ increase required for the excitation-contraction coupling. However, a second mechanism for Ca2+ release from the sarcoplasmic reticulum induced by inositol 1,4,5-trisphosphate (IP3), called inositol IP3-induced intracellular Ca2+ release (IP3ICR), has been verified in cardiomyocytes. Occurrence of global Ca2+ transients as well as spontaneous Ca2+ waves (SCaW) are driven by CICR. Under pathophysiological remodeling conditions in which an up-regulation of IP3 receptors (IP3Rs) was observed, a modulatory role of IP3ICR on CICR seems plausible.

Mitochondrial calpain inhibition restores defective SR-mitochondrial crosstalk in CPVT rat myocytes

Cardiac RYR2-mediated sarcoplasmic Ca2+ (SR) release is essential for matching increased energy demand during fight-or-flight response with mitochondrial metabolic output by delivering Ca2+ into the mitochondrial matrix to activate Ca2+-dependent Krebs cycle dehydrogenases. RYR2 complex gain-of-function mutations associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) have been linked to mitochondrial structural damage and enhanced production of reactive oxygen species (ROS). Despite being critical for arrhythmogenesis in CPVT, the exact causes of these phenomena remain undetermined. Taking advantage of a new rat model of CPVT induced by heterozygous RYR2 gain-of-function mutation S2222L, we tested how RYR2 overactivity alters mitochondrial Ca2+ and ROS handling, and how these changes cause mitochondrial structural defects. Injection of epinephrine (1 mg/kg) and caffeine (120 mg/kg) induced bigamy and bidirectional VT in vivo in 100% of CPVT rats. Simultaneous whole-cell patch clamp and confocal Ca2+-imaging demonstrated that under β-adrenergic stimulation with isoproterenol (50 nM), CPVT ventricular myocytes (VMs) exhibited severe Ca2+ mishandling and high propensity for generation of spontaneous Ca2+ waves (SCWs) that cause arrhythmogenic afterdepolarizations. Diminished Ca2+ transient amplitude in CPVT VMs resulted in a significant reduction in mitochondrial matrix-[Ca2+], and thereby a mito-ROS surge, visualized using matrix-targeted biosensors mtRCaMP1h and MLS-HyPer, respectively. Importantly, using novel Ca2+-biosensors targeted to intermembrane space (IMS-GECO), we uncovered that [Ca2+] in this compartment reaches 1 µM, sufficient for activation of Ca2+-dependent protease μ-calpain. Adenoviral overexpression of IMS-targeted calpastatin, an endogenous calpain inhibitor, reduced mito-ROS, restored cytosolic Ca2+ transient amplitude and SR Ca2+ content, and reduced RYR2-mediated SCWs in CPVT VMs. These changes were paralleled by restored expression levels of OPA1, a mitochondrial structural protein responsible for tight cristae organization. Our data suggest that enhanced mito-ROS due to matrix-[Ca2+] reduction in CPVT VMs and unexpectedly high IMS-[Ca2+] promotes IMS-calpain-mediated degradation of OPA1, resulting in mitochondrial structural damage that contributes to proarrhythmic remodeling.

Phospholamban regulates nuclear Ca2+ stores and inositol 1,4,5-trisphosphate mediated nuclear Ca2+ cycling in cardiomyocytes

AimsPhospholamban (PLB) is the key regulator of the cardiac Ca2+ pump (SERCA2a)-mediated sarcoplasmic reticulum Ca2+ stores. We recently reported that PLB is highly concentrated in the nuclear envelope (NE) from where it can modulate perinuclear Ca2+ handling of the cardiomyocytes (CMs). Since inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) mediates nuclear Ca2+ release, we examined whether the nuclear pool of PLB regulates IP3-induced nuclear Ca2+ handling. Methods and resultsFluo-4 based confocal Ca2+ imaging was performed to measure Ca2+ dynamics across both nucleus and cytosol in saponin-permeabilized CMs isolated from wild-type (WT) or PLB-knockout (PLB-KO) mice. At diastolic intracellular Ca2+ ([Ca2+]i = 100 nM), the Fab fragment of the monoclonal PLB antibody (anti-PLB Fab) facilitated the formation and increased the length of spontaneous Ca2+ waves (SCWs) originating from the nuclear region in CMs from WT but not from PLB-KO mice. We next examined nuclear Ca2+ activities at basal condition and after sequential addition of IP3, anti-PLB Fab, and the IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) at a series of [Ca2+]i. In WT mice, at 10 nM [Ca2+]i where ryanodine receptor (RyR2) based spontaneous Ca2+ sparks rarely occurred, IP3 increased fluorescence amplitude (F/F0) of overall nuclear region to 1.19 ± 0.02. Subsequent addition of anti-PLB Fab significantly decreased F/F0 to 1.09 ± 0.02. At 50 nM [Ca2+]i, anti-PLB Fab not only decreased the overall nuclear F/F0 previously elevated by IP3, but also increased the amplitude and duration of spark-like nuclear Ca2+ release events. These nuclear Ca2+ releases were blocked by 2-APB. At 100 nM [Ca2+]i, IP3 induced short SCWs originating from nucleus. Anti-PLB Fab transformed those short waves into long SCWs with propagation from the nucleus into the cytosol. In contrast, neither nuclear nor cytosolic Ca2+ dynamics was affected by anti-PLB Fab in CMs from PLB-KO mice in all these conditions. Furthermore, in WT CMs pretreated with RyR2 blocker tetracaine, IP3 and anti-PLB Fab still increased the magnitude of nuclear Ca2+ release but failed to regenerate SCWs. Finally, anti-PLB Fab increased low Ca2+ affinity mag-fluo 4 fluorescence intensity in the lumen of NE of nuclei isolated from WT but not in PLB-KO mice. ConclusionPLB regulates nuclear Ca2+ handling. By increasing Ca2+ uptake into lumen of the NE and perhaps other perinuclear membranes, the acute reversal of PLB inhibition decreases global Ca2+ concentration at rest in the nucleoplasm, and increases Ca2+ release into the nucleus, through mechanisms involving IP3R and RyR2 in the vicinity.

Calcium-Dependent Arrhythmogenic Foci Created by Weakly Coupled Myocytes in the Failing Heart.

Intercellular uncoupling and Ca2+ (Ca) mishandling can initiate triggered ventricular arrhythmias. Spontaneous Ca release activates inward current which depolarizes membrane potential (Vm) and can trigger action potentials in isolated myocytes. However, cell-cell coupling in intact hearts limits local depolarization and may protect hearts from this arrhythmogenic mechanism. Traditional optical mapping lacks the spatial resolution to assess coupling of individual myocytes. We investigate local intercellular coupling in Ca-induced depolarization in intact hearts, using confocal microscopy to measure local Vm and intracellular [Ca] simultaneously. We used isolated Langendorff-perfused hearts from control (CTL) and heart failure (HF) mice (HF induced by transaortic constriction). In CTL hearts, 1.4% of myocytes were poorly synchronized with neighboring cells and exhibited asynchronous (AS) Ca transients. These AS myocytes were much more frequent in HF (10.8% of myocytes, P<0.05 versus CTL). Local Ca waves depolarized Vm in HF but not CTL hearts, suggesting weaker gap junction coupling in HF-AS versus CTL-AS myocytes. Cell-cell coupling was assessed by calcein fluorescence recovery after photobleach during intracellular [Ca] recording. All regions in CTL hearts exhibited faster calcein diffusion than in HF, with HF-AS myocyte being slowest. In HF-AS, enhancing gap junction conductance (with rotigaptide) increased coupling and suppressed Vm depolarization during Ca waves. Conversely, in CTL hearts, gap junction inhibition (carbenoxolone) decreased coupling and allowed Ca wave-induced depolarizations. Synchronization of Ca wave initiation and triggered action potentials were observed in HF hearts and computational models. Well-coupled CTL myocytes are effectively voltage-clamped during Ca waves, protecting the heart from triggered arrhythmias. Spontaneous Ca waves are much more common in HF myocytes and these AS myocytes are also poorly coupled, enabling local Ca-induced inward current of sufficient source strength to overcome a weakened current sink to depolarize Vm and trigger action potentials.

Mechanism for Triggered Waves in Atrial Myocytes

Excitation-contraction coupling in atrial cells is mediated by calcium (Ca) signaling between L-type Ca channels and Ryanodine receptors that occurs mainly at the cell boundary. This unique architecture dictates essential aspects of Ca signaling under both normal and diseased conditions. In this study we apply laser scanning confocal microscopy, along with an experimentally based computational model, to understand the Ca cycling dynamics of an atrial cell subjected to rapid pacing. Our main finding is that when an atrial cell is paced under Ca overload conditions, Ca waves can then nucleate on the cell boundary and propagate to the cell interior. These propagating Ca waves are referred to as “triggered waves” because they are initiated by L-type Ca channel openings during the action potential. These excitations are distinct from spontaneous Ca waves originating from random fluctuations of Ryanodine receptor channels, and which occur after much longer waiting times. Furthermore, we argue that the onset of these triggered waves is a highly nonlinear function of the sarcoplasmic reticulum Ca load. This strong nonlinearity leads to aperiodic response of Ca at rapid pacing rates that is caused by the complex interplay between paced Ca release and triggered waves. We argue further that this feature of atrial cells leads to dynamic instabilities that may underlie atrial arrhythmias. These studies will serve as a starting point to explore the nonlinear dynamics of atrial cells and will yield insights into the trigger and maintenance of atrial fibrillation.

Extracellular ATP Triggers Action Potentials in Ventricular Cardiomyocytes

Myocardial ischemia causes a high frequency of ventricular arrhythmias. A long established metabolic response to hypoxia in the heart is the release of ATP. Here we tested the hypothesis that ATP released from cardiomyocytes during acute myocardial injury can contribute to ventricular ectopy by causing delayed afterdepolarizations and triggered action potentials. Isolated mouse ventricular cardiomyocytes were loaded with Fura2-AM to measure intracellular Ca. Spontaneous Ca waves (SCW) and triggered beats (TB) were quantified after a train of field-stimulated Ca transients (for 20s with 3 Hz) in presence of inotropic stimulation with isoproterenol (1 µM) and partial inhibition of the inward rectifier with Ba (5 µM) to facilitate TBs. Application of extracellular ATP (3-50 µM) significantly increased the incidence and frequency of TBs, whereas the rate of SCWs was not significantly changed. Notably, extracellular ATP application generated a different type of TBs that was not preceded by SCW, which were termed ATP-triggered beats (TBATP). Higher concentrations of ATP (> 50 µM) reduced the rates of TBATP. Simultaneous [Ca]i and membrane potential measurements using perforated patch method demonstrated that both regular TBs and TBATP were preceded by a delayed afterdepolarization (DAD) of similar shape. However, in presence of ATP, 34% of DAD that triggered beats occurred WITHOUT concomitant elevations of cytosolic [Ca]. In other words, general DADs and TBs were mediated by the classic Ca-dependent mechanism (i.e., Na-Ca exchange current in response to spontaneous SR Ca release), whereas ATP generated DADs occurred in the absence of the classic Ca-dependent pathway. In isolated Langendorff perfused mouse hearts, adding ATP (30 µM) to the perfusate triggered both atrial and ventricular premature beats. We conclude that low micromolar extracellular ATP activates an inward current that is sufficient to generate DADs and spontaneous action potentials in the absence of spontaneous SR Ca release. Although the molecular identity of ATP-activated inward current remains to be determined, the current study demonstrates that ATP release by acute ischemic stress could facilitate arrhythmia susceptibility via a Ca-independent mechanism.

Dyssynchronous calcium removal in heart failure-induced atrial remodeling.

We tested the hypothesis that in atrial myocytes from a rabbit left ventricular heart failure (HF) model, spatial inhomogeneity and temporal dyssynchrony of Ca removal during excitation-contraction coupling together with increased Na/Ca exchange (NCX) activity generate a substrate for proarrhythmic Ca release. Ca removal occurs via Ca reuptake into the sarcoplasmic reticulum and extrusion via NCX exclusively in the cell periphery since rabbit atrial myocytes lack transverse tubules. Ca removal kinetics were assessed by the time constant τ of decay of local peripheral subsarcolemmal (SS) and central (CT) action potential (AP)-induced Ca transients (CaTs) recorded in confocal line scan mode (using Fluo-4). Spatial and temporal dyssynchrony of Ca removal was quantified by CV TAU, defined as the standard deviation of local τ along the transverse cell axis divided by mean τ. In normal cells CT CaT decline was slower compared with the SS domain, while in HF cells decline was accelerated, became equal in SS and CT regions, and a significant increase of CV TAU indicated an increased Ca removal dyssynchrony. In HF atrial cells NCX upregulation was accompanied by an overall higher incidence of spontaneous Ca waves and a higher propensity of arrhythmogenic Ca waves, defined as waves that triggered APs due to NCX-mediated membrane depolarization. NCX inhibition normalized CV TAU in HF atrial cells and decreased the propensity of Ca waves. In summary, HF atrial myocytes show accelerated but dyssynchronous diastolic Ca removal and altered sarcoplasmic reticulum Ca-ATPase (SERCA) and NCX activity that result in increased susceptibility to arrhythmia.

Novel CPVT-Associated Calmodulin Mutation in CALM3 (CALM3-A103V) Activates Arrhythmogenic Ca Waves and Sparks.

Calmodulin (CaM) mutations are associated with severe forms of long QT syndrome and catecholaminergic polymorphic ventricular tachycardia (CPVT). CaM mutations are found in 13% of genotype-negative long QT syndrome patients, but the prevalence of CaM mutations in genotype-negative CPVT patients is unknown. Here, we identify and characterize CaM mutations in 12 patients with genotype-negative but clinically diagnosed CPVT. We performed mutational analysis of CALM1, CALM2, and CALM3 gene-coding regions, in vitro measurement of CaM-Ca(2+) (Ca)-binding affinity, ryanodine receptor 2-CaM binding, Ca handling, L-type Ca current, and action potential duration. We identified a novel CaM mutation-A103V-in CALM3 in 1 of 12 patients (8%), a female who experienced episodes of exertion-induced syncope since age 10, had normal QT interval, and displayed ventricular ectopy during stress testing consistent with CPVT. A103V modestly lowered CaM Ca-binding affinity (3-fold reduction versus WT-CaM), but did not alter CaM binding to ryanodine receptor 2. In permeabilized cardiomyocytes, A103V-CaM (100 nmol/L) promoted spontaneous Ca wave and spark activity, a cellular phenotype of ryanodine receptor 2 activation. Even a 1:3 mixture of A103V-CaM:WT-CaM activated Ca waves, demonstrating functional dominance. Compared with long QT syndrome D96V-CaM, A103V-CaM had significantly less effects on L-type Ca current inactivation, did not alter action potential duration, and caused delayed afterdepolarizations and triggered beats in intact cardiomyocytes. We discovered a novel CPVT mutation in the CALM3 gene that shares functional characteristics with established CPVT-associated mutations in CALM1. A small proportion of A103V-CaM is sufficient to evoke arrhythmogenic Ca disturbances via ryanodine receptor 2 dysregulation, which explains the autosomal dominant inheritance.

Cardiac-Specific Overexpression of Phosphodiesterase 2 (PDE2) in Mouse is Cardioprotective

Phosphodiesterase 2 (PDE2) is a dual substrate enzyme, hydrolyzing both cAMP and cGMP. We showed that myocardial PDE2 is upregulated in human and experimental heart failure (HF) while PDE3 and PDE4 are reduced. To explore the pathophysiological consequences of enhanced PDE2 activity, transgenic mice with a heart-specific overexpression of the PDE2A3 isoform (PDE2-TG) were generated.PDE2 activity was measuredin heart extractsby radioenzymatic assay. Sarcomere shortening, Ca2+ transients and L-type Ca2+ current (ICa,L) were recorded in adult ventricular cardiomyocytes from wild-type (WT) and PDE2-TG mice. SR Ca2+ leak was estimated with tetracaine (RyR blocker) and spontaneous Ca2+ waves (SCW) were recorded during 30s pacing pause. Intracellular cAMP level was measured with FRET. Phosphorylation level of excitation-contraction coupling (ECC) proteins was determined by Western Blot. Heart function was investigated by echocardiography and ECG-telemetry. Isoprenaline (ISO) was used to compare β-adrenergic (β-AR) response of all parameters in WT and PDE2-TG mice.cAMP and cGMP-PDE2 activity was strongly increased in PDE2-TG as compared to WT mice. Resting and maximal heart rate was markedly reduced in PDE2-TG mice. The β-AR stimulation of cell contractility, Ca2+ transient, ICa,L and [cAMP]i was severely blunted. PDE2-TG mice showed reduced SR Ca2+ leak and SCW (both at the cellular and in vivo levels), without changes in Ca2+ load as compared to WT during β-AR activation. PDE2-TG mice showed a lower basal phosphorylation of ECC proteins and have a longer lifespan than their WT littermates. All effects of PDE2 overexpression were reversed by PDE2 inhibitor, Bay-607550.Our results demonstrate that PDE2 plays a critical role in the regulation of cardiac ECC. PDE2 overexpression appears to protect the cardiomyocytes by reducing Ca2+-leakage and arrhythmias during β-AR stimulation. PDE2 may represent a new therapeutic strategy in heart failure.

Inhibition of RYR2 Activity by Intracellular Flecainide Effectively Suppresses Arrhythmogenic Ca Waves in Intact Ventricular Myocytes from Casq2 -/- Mice

We reported previously that flecainide suppresses Ca waves in MEMBRANE-PERMEABILIZED ventricular myocytes from Casq2-/- mice and that flecainide is more potent (i.e., lower IC50) in Casq2-/-compared to WT myocytes. Other groups reported that in INTACT myocytes flecainide acts only by blocking Na+ channels and has no effect on RyR2. Here we test the effect of flecainide on INTACT ventricular myocytes from Casq2-/- and WT littermates using voltage-clamp. To eliminate any contribution of Na channels, all solutions contained 30 µM TTX. Complete block INa was confirmed for each cell tested. Spontaneous Ca waves were elicited by a pacing train followed by a holding potential of −70mV for 45 s. 6 µM flecainide or vehicle (H2O) was added to both external and pipette solutions. Under such conditions, flecainide had no significant effect on the average number of spontaneous Ca waves in WT myocytes (Vehicle: 3.1±0.6, n=24, Flecainide: 3.0±0.8, n=20, p=NS). In Casq2-/- myocytes, however, flecainide significantly reduced Ca waves (Vehicle: 6.6±1.2, n=23; Flecainide: 2.1±0.4, n=19, p 0.5 vs. Casq2-/- vehicle), suggesting that intracellular dialysis by the patch pipette removed flecainide from the cytosol. We conclude that intracellular concentrations of 6 µM flecainide effectively inhibit arrhythmogenic Ca waves in Casq2-/- but not in WT myocytes, indicating that flecainide action is dependent on the state of RyR2. Our results further demonstrate that flecainide suppression of Ca waves is independent from its Na channel block.

Novel Calmodulin Mutation (CALM3-A103V) Associated with CPVT Syndrome Activates Arrhythmogenic Ca Waves and Sparks

Introduction: Calmodulin (CaM) is an essential Ca2+ signaling protein in a wide range of biological processes. In the dyadic cleft CaM binds to RyR2, Cav1.2 and CaMKII. CaM mutations are associated with an autosomal dominant syndrome of sudden death that can present with clinical features of catecholaminergic polymorphic ventricular tachycardia (CPVT) or long QT syndrome (LQTS). CPVT-linked CaM mutations activate ryanodine receptor (RyR2) Ca release channels; whereas LQTS-CaMs have no effects on RyR2 channels but prolong the action potential by impairing L-type Ca current (LTCC) inactivation. Here we report a novel CaM mutation – A103V – in CALM3, in a 10 year old female with episodes of exertion-induced syncope and normal QT interval consistent with CPVT. Her mother is mutation positive and also had a positive exercise stress test.Methods: CaM-A103V Ca binding affinity was measured by spectrofluorometry. Ca handling and LTCC were tested in mouse ventricular myocytes dialyzed with WT or the mutant CaM and compared to a published CPVT CaM mutant (N54I).Results: A103V modestly lowered CaM Ca binding affinity (3-fold reduction vs WT-CaM). In permeabilized myocytes, A103V-CaMs at a physiological intracellular concentration (100nM) promoted significantly higher spontaneous Ca wave and spark activity, a typical cellular phenotype of CPVT. Even a 1:4 mixture of A103V-CaM:WT-CaM activated Ca waves, demonstrating functional dominance. In voltage-clamped cardiomyocytes dialyzed with WT or mutant CaM, CaM-A103V had significantly less effects on LTCC inactivation compared to published CaM mutants that cause LQTS.Conclusion: A103V CaM shares functional characteristics with the established CPVT-CaM N54I. A small proportion of A103V-CaM is sufficient to evoke arrhythmogenic Ca disturbances, which explains the autosomal dominant inheritance.

Abstract 16191: Phospholamban Regulates Perinuclear/nuclear Calcium Dynamics in Cardiomyocytes

Introduction: Phospholamban (PLB) regulates cardiac sarcoplasmic reticulum (SR) Ca 2+ -ATPase (SERCA2a), thus modulating SR Ca 2+ dynamics. Recent studies demonstrated that SERCA is involved in Ca 2+ uptake into the lumen of nuclear envelope (NE) of cardiomyocytes (CMs). However, the regulatory role of PLB on Ca 2+ uptake into NE remains unknown. Hypothesis: We hypothesize that PLB is also responsible for modulating nuclear Ca 2+ dynamics. Methods: Confocal immunofluorescence microscopy was used to determine subcellular expression of PLB. By using fluo-4 based confocal line-scan Ca 2+ imaging, we measured spontaneous Ca 2+ waves (SCWs) across both cytoplasmic and nuclear regions in isolated permeabilized mouse CMs. Results: Several anti-PLB antibodies strongly stained PLB at both SR and the perinuclear membranes in CMs. A PLB peptide (residues 1-31) eliminated all these anti-PLB antibody stains. To identify the functional role of PLB expressed in the perinuclear membranes, we took advantage of our recently established method that a Fab fragment of anti-PLB monoclonal antibody (Fab) reversed PLB inhibition specifically and increased SR Ca 2+ uptake and release. SCWs through the nuclear regions had typically relative low fluorescent amplitude (F/F 0 ) and slow decay time (t 1/2 ) compared to that in the cytoplasmic region. At the free intracellular Ca 2+ concentration ([Ca 2+ ] i ) of 400 nM, Fab (100 μg/mL) significantly enhanced F/F 0 and decreased t 1/2 of SCWs in both cytoplasmic and nuclear regions. After addition of Fab, F/F 0 of SCWs through the nuclear regions increased from 0.91±0.16 to 1.27±0.19 (n=9, p&lt;.05) while t 1/2 decreased from 137.6±18.5 ms to 105.0±11.3 ms, (p&lt;.05). Similar effects were also observed after phosphorylation of PLB by addition of 20 μM cAMP (F/F 0 =1.43±0.11 vs. 1.04±0.14 in control, p&lt;.05; t 1/2 =107.82±10.9 ms vs. 139.21±20.1 ms in control, n=6, p&lt;.05). At high [Ca 2+ ] i of 1000 nM where PLB does not inhibit SERCA2a, addition of cAMP or Fab had no significant effect on SCWs in both cytoplasmic and nuclear regions. Conclusions: We demonstrated that PLB is expressed in and around NE. Acute removal of PLB inhibition increased perinuclear/nuclear Ca 2+ uptake and release. PLB is critically involved in nuclear Ca 2+ signaling modulation.

Abstract 19344: Involvement of Mitochondrial Permeability Transition Pore (mPTP) in Cardiac Arrhythmias: Evidence From Cyclophilin D Knockout Mice

Our previous studies have demonstrated that mitochondrial membrane potential ( ΔΨ m ) dissipation leads to mitochondrial Ca efflux via the mitochondrial permeability transition pore (mPTP), and consequently promotes spontaneous Ca waves (CaW) in isolated cardiomyocytes. In the present study, we have used a genetic mouse model which lacks cyclophilin D (CypD KO), a necessary regulator for mPTP opening, to assess the cardioprotective effect of mPTP inhibition on cellular CaWs, Ca alternans, and whole-heart inducible arrhythmias. Ventricular myocytes were isolated from wild type (WT) and CypD KO mice. Tetramethylrhodamine (TMRM) was used as an indicator of ΔΨ m. Mitochondrial calcein release was used as the index of mPTP opening. Cytosolic Ca was imaged using Fluo-4-AM. Spontaneous CaWs were induced in the presence of 4mM external Ca. The protonophore FCCP was used to depolarize Δ Ψ m . FCCP caused Δ Ψ m depolarization to the same extent in both WT and CypD KO myocytes, however, CypD KO cells exhibited significantly less mPTP opening than WT cells (p &lt; 0.05). Consistent with these results, treatment with FCCP caused significant increases in WT CaW rate but no increase in CypD KO CaW rate. These results were further confirmed by treating WT cells with the mPTP blocker cyclosporin A (CsA). Furthermore, occurrence of Ca alternans after treatment with FCCP and programmed stimulation was observed in a significantly higher number of WT cells (11 of 13), than in WT cells treated with CsA (2 of 8; p &lt; 0.05) or CypD KO cells (2 of 10; p &lt; 0.01). At the whole-heart level, pseudo-Lead II ECGs were recorded from ex vivo, Langendorff-perfused WT and CypD KO hearts, and the incidences of T-wave alternans (a precursor of lethal arrhythimas) and arrhythmias induced by programmed S 1 -S 2 stimulation were compared. We observed T-wave alternans in 5 of 7 WT hearts, but none in 5 CypD KO hearts (p &lt; 0.05) and in only 1 of 6 WT hearts treated with CsA (p &lt;0.05). Consistent with these results, WT hearts exhibited a significantly higher average arrhythmia score than CypD KO (p &lt;0.01) or WT hearts treated with CsA (p &lt; 0.01). In conclusion, CypD deficiency-induced mPTP inhibition attenuates CaWs and Ca alternans during mitochondrial depolarization, and thereby protects against arrhythmogenesis in the heart.

Ablation of HRC alleviates cardiac arrhythmia and improves abnormal Ca handling in CASQ2 knockout mice prone to CPVT.

Cardiac calsequestrin (CASQ2) and histidine-rich Ca-binding protein (HRC) are sarcoplasmic reticulum (SR) Ca-binding proteins that regulate SR Ca release in mammalian heart. Deletion of either CASQ2 or HRC results in relatively mild phenotypes characterized by preserved cardiac structure and function, although CASQ2 knockout (KO), or Cnull, shows increased arrhythmia burden under conditions of catecholaminergic stress. We hypothesized that given the apparent overlap of functions of CASQ2 and HRC, simultaneous ablation of both would deteriorate the cardiac phenotype compared with the single knockouts. In contrast to this expectation, double knockout (DKO) mice lacking both CASQ2 and HRC exhibited normal cardiac ejection fraction and ultrastructure. Moreover, the predisposition to catecholamine-dependent arrhythmia that characterizes the Cnull phenotype was alleviated in the DKO mice. At the myocyte level, DKO mice displayed Ca transients of normal amplitude; additionally, the frequency of spontaneous Ca waves and sparks in the presence of isoproterenol were decreased markedly compared with Cnull. Furthermore, restitution of SR Ca release was slowed in DKO myocytes compared with Cnull cells. Our results suggest that rather than being functionally redundant, CASQ2 and HRC modulate cardiac ryanodine receptor-mediated (RyR2) Ca release in an opposing manner. In particular, while CASQ2 stabilizes RyR2 rendering it refractory in the diastolic phase, HRC enhances RyR2 activity facilitating RyR2 recovery from refractoriness.

Regulation of calcium clock-mediated pacemaking by inositol-1,4,5-trisphosphate receptors in mouse sinoatrial nodal cells.

Inositol-1,4,5-trisphosphate receptors (IP3 Rs) modulate pacemaking in embryonic heart, but their role in adult sinoatrial node (SAN) pacemaking is uncertain. We found that stimulation of IP3 Rs accelerates spontaneous pacing rate in isolated mouse SAN cells, whereas inhibition of IP3 Rs slows pacing. In atrial-specific sodium-calcium exchanger (NCX) knockout (KO) SAN cells, where the Ca(2+) clock is uncoupled from the membrane clock, IP3 R agonists and antagonists modulate the rate of spontaneous Ca(2+) waves, suggesting that IP3 R-mediated Ca(2+) release modulates the Ca(2+) clock. IP3 R modulation also regulates Ca(2+) spark parameters, a reflection of ryanodine receptor open probability, consistent with the effect of IP3 signalling on Ca(2+) clock frequency. Modulation of Ca(2+) clock frequency by IP3 signalling in NCX KO SAN cells demonstrates that the effect is independent of NCX. These findings support development of IP3 signalling modulators for regulation of heart rate, particularly in heart failure where IP3 Rs are upregulated. Cardiac pacemaking initiated by the sinus node is attributable to the interplay of several membrane currents. These include the depolarizing 'funny current' (If ) and the sodium-calcium exchanger current (INCX ). The latter is activated by ryanodine receptor (RyR)-mediated calcium (Ca(2+) ) release from the sarcoplasmic reticulum (SR). Another SR Ca(2+) release channel, the inositol-1,4,5-triphosphate receptor (IP3 R), has been implicated in the generation of spontaneous Ca(2+) release in atrial and ventricular cardiomyocytes. Whether IP3 R-mediated Ca(2+) release also influences SAN automaticity is controversial, in part due to the confounding influence of periodic Ca(2+) flux through the sarcolemma accompanying each beat. We took advantage of atrial-specific sodium-calcium exchanger (NCX) knockout (KO) SAN cells to study the influence of IP3 signalling on cardiac pacemaking in a system where periodic intracellular Ca(2+) cycling persists despite the absence of depolarization or Ca(2+) flux across the sarcolemma. We recorded confocal line scans of spontaneous Ca(2+) release in WT and NCX KO SAN cells in the presence or absence of an IP3 R blocker (2-aminoethoxydiphenyl borate, 2-APB), or during block of IP3 production by the phospholipase C inhibitor U73122. 2-APB and U73122 decreased the frequency of spontaneous Ca(2+) transients and waves in WT and NCX KO cells, respectively. Alternatively, increased IP3 production induced by phenylephrine increased Ca(2+) transient and wave frequency. We conclude that IP3 R-mediated SR Ca(2+) flux is crucial for initiating and modulating the RyR-mediated Ca(2+) cycling that regulates SAN pacemaking. Our results in NCX KO SAN cells also demonstrate that RyRs, but not NCX, are required for IP3 to modulate Ca(2+) clock frequency.

0270 : CaMKII inhibition prevents cardiac arrhythmias elicited by phosphodiesterases 3 and 4 inhibitors

β-adrenoceptors (β-AR) stimulation increases cardiac function by rising cAMP that activates protein kinase A (PKA) and enhances Ca2+-induced Ca2+- release by phosphorylating proteins such as ryanodine receptors and phospholamban, which are also targets of the Ca2+/Calmodulin Kinase II (CaMKII). Any dysregulation of the β-AR pathway promotes arrhythmias. Local cAMP concentration in heart is mainly regulated by phosphodiesterases (PDE) type 3 and 4. Here, we investigated the proarrhythmic effects of PDE3 and PDE4 inhibition and evaluated the relative contribution of PKA and CaMKII to these mechanisms. An IonOptix system was used to record intracellular Ca2+ transients in isolated adult rat and pig ventricular myocytes (ARVMs and APVMs) loaded with 1μM Fura-2AM and paced at 1Hz. In ARVMs, PDE4 inhibition with Ro20-1724 (Ro, 10μM) potentiated the inotropic effect of the β-AR agonist isoproterenol (Iso, 1nM) but induced spontaneous Ca2+ waves (SCWs) recorded during 10s pacing pauses (2.3±0.2 SCWs/10s, n=15; p<0.001). Sarcoplasmic reticulum (SR) Ca2+ load increased by 30±5% (n=13; p<0.001) in Iso, but doubled (p<0.001) in Iso+Ro leading to SR Ca2+ leak (measured in a 0Na+,0Ca2+ solution±1mM tetracaine, n=7; p<0.001). PKA inhibition by H89 (10μM) suppressed SCWs, SR Ca2+ leak and the inotropic effect of Iso±Ro. The CaMKII inhibitor KN93 (10μM), unlike its inactive analogue KN92, reduced SR Ca2+ leak by 85% (n=16; p<0.001) and SCWs incidence by 72% (n=8; p<0.001) without affecting the inotropic effect of Iso+Ro. KN93 blunted the SR Ca2+ leak induced by PDE3 inhibition with cilostamide (Cil, 1μM, n=10; p<0.001) by 81% but preserved Ca2+ transients amplitude. Similarly, CaMKII inhibition prevented SCWs evoked by Cil and Ro in APVMs. These results show that PDE inhibitors exert inotropic effects via PKA but lead to SCWs via both PKA and CaMKII activation. They also suggest the potential use of CaMKII inhibitors as adjuncts to PDE inhibitors to limit their proarrhythmic effects.

Calcium-Voltage Coupling in the Genesis of Early and Delayed Afterdepolarizations in Cardiac Myocytes

Early afterdepolarizations (EADs) and delayed afterdepolarizations (DADs) are voltage oscillations known to cause cardiac arrhythmias. EADs are mainly driven by voltage oscillations in the repolarizing phase of the action potential (AP), while DADs are driven by spontaneous calcium (Ca) release during diastole. Because voltage and Ca are bidirectionally coupled, they modulate each other’s behaviors, and new AP and Ca cycling dynamics can emerge from this coupling. In this study, we performed computer simulations using an AP model with detailed spatiotemporal Ca cycling incorporating stochastic openings of Ca channels and ryanodine receptors to investigate the effects of Ca-voltage coupling on EAD and DAD dynamics. Simulations were complemented by experiments in mouse ventricular myocytes. We show that: 1) alteration of the Ca transient due to increased ryanodine receptor leakiness and/or sarco/endoplasmic reticulum Ca ATPase activity can either promote or suppress EADs due to the complex effects of Ca on ionic current properties; 2) spontaneous Ca waves also exhibit complex effects on EADs, but cannot induce EADs of significant amplitude without the participation of ICa,L; 3) lengthening AP duration and the occurrence of EADs promote DADs by increasing intracellular Ca loading, and two mechanisms of DADs are identified, i.e., Ca-wave-dependent and Ca-wave-independent; and 4) Ca-voltage coupling promotes complex EAD patterns such as EAD alternans that are not observed for solely voltage-driven EADs. In conclusion, Ca-voltage coupling combined with the nonlinear dynamical behaviors of voltage and Ca cycling play a key role in generating complex EAD and DAD dynamics observed experimentally in cardiac myocytes, whose mechanisms are complex but analyzable.