SR Ca Leak Research Articles

Dynamic calcium-calmodulin-dependent stabilization of leaky RyR2 in live cardiac myocytes.

Calmodulin (CaM) is a major regulator of cardiac ryanodine receptors (RyR2). CaM binding shifts RyR2 channel conformation to a state that suppresses diastolic SR Ca leak that can become pro arrhythmic as commonly observed in cardiac diseases such as heart failure (HF) or diabetic cardiomyopathy. In these diseases, CaM affinity for RyR2 is reduced, promoting a leaky pathological RyR2 structural state that is arrhythmogenic. Conversely, increased CaM binding can suppress RyR2-mediated Ca leak and promote a conformational shift to the stable physiological non-leaky state. Still, there are unknowns regarding the dynamic interplay between CaM-RyR2 during each heartbeat and in pathogenic RyR states in live cardiac myocytes. We hypothesize that high [Ca] elevations reached during Ca transients in a healthy beating myocyte would increase CaM-RyR binding throughout the cardiac cycle, promoting RyR stability. Further, the lower CaM-RyR2 affinity in disease, exacerbated by reduced Ca transient amplitude, resulting in lower CaM-RyR2 binding, destabilized RyR2s and enhance arrhythmogenic diastolic SR Ca leak. We used permeabilized cardiac myocytes from WT and HF mouse models as a controlled approach to measuring both the [Ca]i and [CaM] dependence of CaM-RyR2 binding via FRET between donor-labeled FKBP12.6 and acceptor-labeled CaM. Moreover, we simultaneously recorded [Ca]i transients within myocytes, enabling measurement of dynamic CaM-RyR2 saturation during periodic Ca waves that emulate paced Ca transients. We find that CaM-RyR2 FRET is reduced in the leaky pathological state and associated with alterations in Ca leak event frequencies. We infer that the healthy higher CaM-RyR2 affinity favors more complete saturation of RyR2 with CaM throughout the cardiac cycle, versus pathological states. These data suggest that a modest fraction of CaM-free RyR2s with pathological diastolic conformation might suffice to elicit proarrhythmic SR Ca leak in cardiac disease.

Oxidative activation of CaMKII mediates arrhythmias and contractile dysfunction in an obstructive sleep apnea mouse model

Abstract Background/Introduction Atrial arrhythmias frequently occur in patients with obstructive sleep apnea (OSA), but the underlying mechanisms remain insufficiently understood. We recently demonstrated that CaMKII-dependent pro-arrhythmic activity is increased in patients with OSA, but the mechanisms of CaMKII activation remain unknown. Interestingly, OSA can lead to increased reactive oxygen species production, which may facilitate CaMKII activation by oxidation at methionine residues 281/282. Purpose We tested if oxidation of CaMKII is involved in the development of arrhythmias and contractile dysfunction in vitro and in vivo in a mouse model of obstructive sleep apnea by tongue enlargement. Methods Experiments were performed using 41 wild-type (WT) and 25 genetically modified mice lacking oxidative CaMKII activation due to mutation of methionine 281/282 to valine (MMVV mice). Polytetrafluorethylene (PTFE, 100 μl) was injected into the tongue of 23 WT and 12 MMVV mice to induce OSA by sustained tongue enlargement as previously established. Whole body plethysmography was performed to confirm success of intervention. Echocardiography was performed at baseline and after 8 weeks to assess left-ventricular function. After 8 weeks, isolated atrial and ventricular cardiomyocytes were incubated with the Ca-sensitive dye FURA-2 AM (5 μM, 15 min) and analyzed with epifluorescence microscopy under regular electrical field stimulation (1 Hz). Results PTFE injection resulted in an increased frequency of inspiratory flow limitations (IFLs/h) from 27.4±5.95 to 59.6±6.22 in PTFE mice (p<0.001, fig. 1A). Interestingly, in WT PTFE mice, left ventricular ejection fraction (LVEF, in %) was reduced at 8 weeks post procedure from 57.0±1.36 to 51.5±1.85 (p=0.001, fig. 1B). Importantly, mice lacking oxidative CaMKII activation were protected from such decline in contractile function, and LVEF was higher at 8 weeks vs. WT PTFE (p<0.001, fig. 1B). Congruent with deterioration of contractility in vivo, the Ca transient amplitude after 30s pause (normalized to steady-state before pause) was decreased in ventricular cardiomyocytes of WT PTFE mice indicating increased SR Ca leak (p for interaction genotype x PTFE = 0.014, fig. 1C). In contrast to WT, the post-pause ratio was increased in MMVV PTFE mice (p=0.018, fig. 1C). In addition to contractile function, cellular arrhythmic events were analyzed as non-stimulated pro-arrhythmic events (NSEs) during steady-state electrical field stimulation at 1 Hz (fig. 1D, red arrows). NSE frequency was increased in atrial cardiomyocytes from WT PTFE mice with 0.050±0.005 compared to 0.018±0.003 s–1 in WT CTRL (p<0.001, fig. 1D). Intriguingly, MMVV mice were protected from NSEs after PTFE treatment (0.020±0.004, p<0.001 vs. WT PTFE, Fig. 1D). Conclusion In a mouse model of obstructive sleep apnea, contractile dysfunction and pro-arrhythmic activity are modulated by oxidative CaMKII activation, which may have therapeutic implications. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft; Medical Faculty at University of Regensburg

Enhanced Cardiac CaMKII Oxidation and CaMKII-Dependent SR Ca Leak in Patients with Sleep-Disordered Breathing.

Background: Sleep-disordered breathing (SDB) is associated with increased oxidant generation. Oxidized Ca/calmodulin kinase II (CaMKII) can contribute to atrial arrhythmias by the stimulation of sarcoplasmic reticulum Ca release events, i.e., Ca sparks. Methods: We prospectively enrolled 39 patients undergoing cardiac surgery to screen for SDB and collected right atrial appendage biopsies. Results: SDB was diagnosed in 14 patients (36%). SDB patients had significantly increased levels of oxidized and activated CaMKII (assessed by Western blotting/specific pulldown). Moreover, SDB patients showed a significant increase in Ca spark frequency (CaSpF measured by confocal microscopy) compared with control subjects. CaSpF was 3.58 ± 0.75 (SDB) vs. 2.49 ± 0.84 (no SDB) 1/100 µm−1s−1 (p < 0.05). In linear multivariable regression models, SDB severity was independently associated with increased CaSpF (B [95%CI]: 0.05 [0.03; 0.07], p < 0.001) after adjusting for important comorbidities. Interestingly, 30 min exposure to the CaMKII inhibitor autocamtide-2 related autoinhibitory peptide normalized the increased CaSpF and eliminated the association between SDB and CaSpF (B [95%CI]: 0.01 [−0.1; 0.03], p = 0.387). Conclusions: Patients with SDB have increased CaMKII oxidation/activation and increased CaMKII-dependent CaSpF in the atrial myocardium, independent of major clinical confounders, which may be a novel target for treatment of atrial arrhythmias in SDB.

P6283Inhibition of neuronal Na channel NaV1.8 reduces INaL and SR-Ca leak in isolated failing human and mouse ventricular cardiomyocytes

P6283Inhibition of neuronal Na channel NaV1.8 reduces INaL and SR-Ca leak in isolated failing human and mouse ventricular cardiomyocytes

CaMKII-dependent phosphorylation of RyR2 promotes targetable pathological RyR2 conformational shift

Diastolic calcium (Ca) leak via cardiac ryanodine receptors (RyR2) can cause arrhythmias and heart failure (HF). Ca/calmodulin (CaM)-dependent kinase II (CaMKII) is upregulated and more active in HF, promoting RyR2-mediated Ca leak by RyR2-Ser2814 phosphorylation. Here, we tested a mechanistic hypothesis that RyR2 phosphorylation by CaMKII increases Ca leak by promoting a pathological RyR2 conformation with reduced CaM affinity. Acute CaMKII activation in wild-type RyR2, and phosphomimetic RyR2-S2814D (vs. non-phosphorylatable RyR2-S2814A) knock-in mouse myocytes increased SR Ca leak, reduced CaM-RyR2 affinity, and caused a pathological shift in RyR2 conformation (detected via increased access of the RyR2 structural peptide DPc10). This same trio of effects was seen in myocytes from rabbits with pressure/volume-overload induced HF. Excess CaM quieted leak and restored control conformation, consistent with negative allosteric coupling between CaM affinity and DPc10 accessible conformation. Dantrolene (DAN) also restored CaM affinity, reduced DPc10 access, and suppressed RyR2-mediated Ca leak and ventricular tachycardia in RyR2-S2814D mice. We propose that a common pathological RyR2 conformational state (low CaM affinity, high DPc10 access, and elevated leak) may be caused by CaMKII-dependent phosphorylation, oxidation, and HF. Moreover, DAN (or excess CaM) can shift this pathological gating state back to the normal physiological conformation, a potentially important therapeutic approach.

CaMKII and Heart Failure Promote a Pathological Ryanodine Receptor Conformation that Reduces Calmodulin Binding and Enhances SR Ca2+ Leak

Increased diastolic SR Ca2+ (Ca) leak via cardiac ryanodine receptors (RyR2) can cause cardiac arrhythmias and dysfunction in heart failure (HF). RyR2 phosphorylation (at Ser2814) by Ca/calmodulin-dependent protein kinase II (CaMKII) is thought to be a critical promoter of leaky RyR2 in HF and arrhythmias. A conformational change in RyR2 (“domain unzipping”) observed in HF can increase SR Ca leak. Calmodulin (CaM) quiets RyR2 gating, and CaM binding affinity for RyR2 is reduced in HF. Here, we tested the hypothesis that CaMKII-dependent phosphorylation of RyR2 increases Ca leak by favoring the same unzipped-conformation that exhibits reduced CaM-RyR2 binding affinity. Using non-phosphorylatable RyR2-S2814A and phosphomimetic RyR2-S2814D knock-in mice, fluorescence resonance energy transfer (FRET) was measured to directly detect binding of fluorescently labeled DPc10 (RyR2 unzipping peptide), FK506-binding protein12.6, and CaM to RyR2 in permeabilized cardiomyocytes. In S2814D vs. S2814A: (1) Ca sparks and waves were increased; (2) CaM-RyR2 binding affinity was reduced 3-fold, but FRETmax was unaltered; and (3) F-DPc10 accessibility was 2-fold faster, reflecting unzipped-conformation. Dantrolene (1 μM), which stabilizes the zipped conformation: (1) restored normal CaM-RyR2 binding affinity; (2) reduced DPc10 access (zipped-conformation); and (3) reduced frequency of Ca sparks and waves in S2814D. Furthermore, in rabbit non-ischemic HF myocytes, in which RyR2 is more highly phosphorylated by CaMKII, vs. age-matched control: (1) The CaM-RyR2 binding affinity was reduced by 2.5-fold; and (2) F-DPc10 accessibility was 1.5-fold faster. Dantrolene also restored CaM-RyR2 binding affinity and kept zipped conformation in rabbit HF. We conclude that CaMKII-dependent phosphorylation of RyR2 promotes a pathological RyR2 conformation with reduced CaM binding affinity and elevated Ca leakiness. Dantrolene reverses this conformational change and restores normal CaM binding affinity in S2814D mouse and rabbit HF.

Inhibition of CaMKII Attenuates Progressing Disruption of Ca(2+) Homeostasis Upon Left Ventricular Assist Device Implantation in Human Heart Failure.

In heart failure, left ventricular assist device (LVAD) implantation is performed to ensure sufficient cardiac output. Whereas some patients are subsequently weaned from LVAD support, other patients still need heart transplantation. To elucidate underlying mechanisms, we assessed the arrhythmogenic SR-Ca(2+) leak at the time of LVAD implantation (HF-Im) and heart transplantation (HF-Tx) and evaluated the effects of CaMKII-inhibition. Human left-ventricular cardiomyocytes were isolated, paced at 1 Hz for 10 beats to ensure SR-Ca(2+) loading and scanned for diastolic Ca(2+) sparks (confocal microscopy). In HF-Im, the high diastolic spark frequency (CaSpF) of 0.76 ± 0.12 × 100 μm(-1) × s(-1) could be reduced to 0.48 ± 0.10 × 100 μm(-1) × s(-1) by CaMKII inhibition (AIP, 1 μM). The amplitude of Ca(2+) sparks, width, and length was not significantly altered. In sum, CaMKII inhibition yielded a clear tendency toward a reduction of the SR-Ca(2+) leak (n cells/patients = 76/6 vs. 108/6, P = 0.08). In HF-Tx, we detected an even higher CaSpF of 1.00 ± 0.10 100 μm(-1) × s(-1) and a higher SR-Ca(2+) leak compared with HF-Im (increase by 81 ± 33%, n cells/patients = 156/7 vs. 130/7, P < 0.05), which fits to the further decreased LV function. Here, CaMKII inhibition likewise reduced CaSpF (0.35 ± 0.09 100 μm(-1) × s(-1,) P = 0.06) and significantly reduced spark duration (n sparks/patients = 58/3 vs. 159/3, P < 0.05). Conclusively, the SR-Ca(2+) leak was reduced by 69 ± 12% in HF-Tx upon CaMKII inhibition (n cells/patients = 53/3 vs. 91/3, P < 0.05). These data show that the SR-Ca(2+) leak correlates with the development of LV function after LVAD implantation and may represent an important pathomechanism. The fact that CaMKII inhibition reduces the SR-Ca(2+) leak in HF-Tx suggests that CaMKII inhibition may be a promising option to beneficially influence clinical course after LVAD implantation.

STIM1 enhances SR Ca2+ content through binding phospholamban in rat ventricular myocytes

In ventricular myocytes, the physiological function of stromal interaction molecule 1 (STIM1), an endo/sarcoplasmic reticulum (ER/SR) Ca(2+) sensor, is unclear with respect to its cellular localization, its Ca(2+)-dependent mobilization, and its action on Ca(2+) signaling. Confocal microscopy was used to measure Ca(2+) signaling and to track the cellular movement of STIM1 with mCherry and immunofluorescence in freshly isolated adult rat ventricular myocytes and those in short-term primary culture. We found that endogenous STIM1 was expressed at low but measureable levels along the Z-disk, in a pattern of puncta and linear segments consistent with the STIM1 localizing to the junctional SR (jSR). Depleting SR Ca(2+) using thapsigargin (2-10 µM) changed neither the STIM1 distribution pattern nor its mobilization rate, evaluated by diffusion coefficient measurements using fluorescence recovery after photobleaching. Two-dimensional blue native polyacrylamide gel electrophoresis and coimmunoprecipitation showed that STIM1 in the heart exists mainly as a large protein complex, possibly a multimer, which is not altered by SR Ca(2+) depletion. Additionally, we found no store-operated Ca(2+) entry in control or STIM1 overexpressing ventricular myocytes. Nevertheless, STIM1 overexpressing cells show increased SR Ca(2+) content and increased SR Ca(2+) leak. These changes in Ca(2+) signaling in the SR appear to be due to STIM1 binding to phospholamban and thereby indirectly activating SERCA2a (Sarco/endoplasmic reticulum Ca(2+) ATPase). We conclude that STIM1 binding to phospholamban contributes to the regulation of SERCA2a activity in the steady state and rate of SR Ca(2+) leak and that these actions are independent of store-operated Ca(2+) entry, a process that is absent in normal heart cells.

Norepinephrine-Induced Adrenergic Activation Strikingly Increased the Atrial Fibrillation Duration through β1- and α1-Adrenergic Receptor-Mediated Signaling in Mice.

BackgroundAtrial fibrillation (AF) is the most common arrhythmias among old people. It causes serious long-term health problems affecting the quality of life. It has been suggested that the autonomic nervous system is involved in the onset and maintenance of AF in human. However, investigation of its pathogenesis and potential treatment has been hampered by the lack of suitable AF models in experimental animals.ObjectivesOur aim was to establish a long-lasting AF model in mice. We also investigated the role of adrenergic receptor (AR) subtypes, which may be involved in the onset and duration of AF.Methods and ResultsTrans-esophageal atrial burst pacing in mice could induce AF, as previously shown, but with only a short duration (29.0±8.1 sec). We found that adrenergic activation by intraperitoneal norepinephrine (NE) injection strikingly increased the AF duration. It increased the duration to more than 10 minutes, i.e., by more than 20-fold (656.2±104.8 sec; P<0.001). In this model, a prior injection of a specific β1-AR blocker metoprolol and an α1-AR blocker prazosin both significantly attenuated NE-induced elongation of AF. To further explore the mechanisms underlying these receptors’ effects on AF, we assessed the SR Ca2+ leak, a major trigger of AF, and consequent spontaneous SR Ca2+ release (SCR) in atrial myocytes. Consistent with the results of our in-vivo experiments, both metoprolol and prazosin significantly inhibited the NE-induced SR Ca2+ leak and SCR. These findings suggest that both β1-AR and α1-AR may play important roles in the development of AF.ConclusionsWe have established a long-lasting AF model in mice induced by adrenergic activation, which will be valuable in future AF study using experimental animals, such as transgenic mice. We also revealed the important role of β1- and α1-AR-mediated signaling in the development of AF through in-vivo and in-vitro experiments.

Oxidation of ryanodine receptor (RyR) and calmodulin enhance Ca release and pathologically alter, RyR structure and calmodulin affinity

Oxidative stress may contribute to cardiac ryanodine receptor (RyR2) dysfunction in heart failure (HF) and arrhythmias. Altered RyR2 domain–domain interaction (domain unzipping) and calmodulin (CaM) binding affinity are allosterically coupled indices of RyR2 conformation. In HF RyR2 exhibits reduced CaM binding, increased domain unzipping and greater SR Ca leak, and dantrolene can reverse these changes. However, effects of oxidative stress on RyR2 conformation and leak in myocytes are poorly understood. We used fluorescent CaM, FKBP12.6, and domain-peptide biosensor (F-DPc10) to measure, directly in cardiac myocytes, (1) RyR2 activation by hydrogen peroxide (H2O2)-induced oxidation, (2) RyR2 conformation change caused by oxidation, (3) CaM–RyR2 and FK506-binding protein (FKBP12.6)–RyR2 interaction upon oxidation, and (4) whether dantrolene affects 1–3. H2O2 was used to mimic oxidative stress. H2O2 significantly increased the frequency of Ca2+ sparks and spontaneous Ca2+ waves, and dantrolene almost completely blocked these effects. H2O2 pretreatment significantly reduced CaM–RyR2 binding, but had no effect on FKBP12.6–RyR2 binding. Dantrolene restored CaM–RyR2 binding but had no effect on intracellular and RyR2 oxidation levels. H2O2 also accelerated F-DPc10–RyR2 association while dantrolene slowed it. Thus, H2O2 causes conformational changes (sensed by CaM and DPc10 binding) associated with Ca leak, and dantrolene reverses these RyR2 effects. In conclusion, in cardiomyocytes, H2O2 treatment markedly reduces the CaM–RyR2 affinity, has no effect on FKBP12.6–RyR2 affinity, and causes domain unzipping. Dantrolene can correct domain unzipping, restore CaM–RyR2 affinity, and quiet pathological RyR2 channel gating. F-DPc10 and CaM are useful biosensors of a pathophysiological RyR2 state.

CaMKII-Dependent Phosphorylation of RyR2 Causes Domain Unzipping and Reduced Calmodulin Binding, But Dantrolene Reverses These Effects

Increased diastolic SR Ca2+ (Ca) leak via cardiac ryanodine receptors (RyR2) can cause cardiac arrhythmias and dysfunction in heart failure (HF). CaMKII-dependent RyR2 phosphorylation (at S2814) is thought to be a critical promoter of leaky RyR2 in HF. A conformational change in RyR2 (“domain unzipping”) observed in HF can also increase SR Ca leak. Calmodulin (CaM) quiets RyR2 gating, and the CaM affinity for RyR2 is reduced in HF. Further, we have shown reciprocal negative coupling between CaM binding to RyR2 and accessibility of the unzipping RyR peptide DPc10 (Oda et al. 2013). That is, unzipping with DPc10 reduced CaM binding and increased Ca leak, whereas CaM quieted leak and inhibited DPc10 access. Here, we tested the hypothesis that RyR2 phosphorylation by CaMKII increases Ca leak by favoring the same unzipped-conformation that exhibits reduced CaM affinity. We either activated endogenous myocyte CaMKII or used knock-in mice expressing non-phosphorylatable RyR2-S2814A or phosphomimetic RyR2-S2814D. We used FRET to directly detect binding of fluorescently labeled DPc10, FKBP12.6, and CaM to RyR2 in permeabilized cardiomyocytes. In phosphomimetic S2814D vs. S2814A myocytes, CaM-RyR2 affinity was reduced 3-fold (Kd= 72 ± 9 nM vs. 20 ± 2 nM), but FRETmax was unaltered. Access of DPc10 was also 2-fold faster in S2814D versus S2814A myocytes. Dantrolene (1 μM), which stabilizes the zipped conformation, restored CaM-RyR2 binding affinity in S2814D, slowed DPc10 wash-in, and lowered Bmax. Forced RyR2-S2814D saturation with 500 nM CaM also slowed DPc10 wash-in and reduced Bmax. We conclude that RyR2 phosphorylation by CaMKII causes pathological conformation changes (unzipping) that reduce RyR2-CaM affinity and ability to quiet SR Ca leak. Dantrolene effectively reverses these effects.

Nitric Oxide-Dependent Activation of CaMKII Increases Diastolic Sarcoplasmic Reticulum Calcium Release in Cardiac Myocytes in Response to Adrenergic Stimulation

Spontaneous calcium waves in cardiac myocytes are caused by diastolic sarcoplasmic reticulum release (SR Ca2+ leak) through ryanodine receptors. Beta-adrenergic (β-AR) tone is known to increase this leak through the activation of Ca-calmodulin-dependent protein kinase (CaMKII) and the subsequent phosphorylation of the ryanodine receptor. When β-AR drive is chronic, as observed in heart failure, this CaMKII-dependent effect is exaggerated and becomes potentially arrhythmogenic. Recent evidence has indicated that CaMKII activation can be regulated by cellular oxidizing agents, such as reactive oxygen species. Here, we investigate how the cellular second messenger, nitric oxide, mediates CaMKII activity downstream of the adrenergic signaling cascade and promotes the generation of arrhythmogenic spontaneous Ca2+ waves in intact cardiomyocytes. Both SCaWs and SR Ca2+ leak were measured in intact rabbit and mouse ventricular myocytes loaded with the Ca-dependent fluorescent dye, fluo-4. CaMKII activity in vitro and immunoblotting for phosphorylated residues on CaMKII, nitric oxide synthase, and Akt were measured to confirm activity of these enzymes as part of the adrenergic cascade. We demonstrate that stimulation of the β-AR pathway by isoproterenol increased the CaMKII-dependent SR Ca2+ leak. This increased leak was prevented by inhibition of nitric oxide synthase 1 but not nitric oxide synthase 3. In ventricular myocytes isolated from wild-type mice, isoproterenol stimulation also increased the CaMKII-dependent leak. Critically, in myocytes isolated from nitric oxide synthase 1 knock-out mice this effect is ablated. We show that isoproterenol stimulation leads to an increase in nitric oxide production, and nitric oxide alone is sufficient to activate CaMKII and increase SR Ca2+ leak. Mechanistically, our data links Akt to nitric oxide synthase 1 activation downstream of β-AR stimulation. Collectively, this evidence supports the hypothesis that CaMKII is regulated by nitric oxide as part of the adrenergic cascade leading to arrhythmogenesis.

Hyperphosphorylation of RyRs Underlies Triggered Activity in Transgenic Rabbit Model of LQT2 Syndrome

Loss-of function mutations in HERG potassium channels are associated with ventricular tachycardia and sudden cardiac death caused by stress. We aimed to investigate changes in Ca homeostasis in cardiomyocytes derived from LQT2 hearts and to determine whether these changes contribute to arrhythmogenic early afterdepolarizations (EADs) characteristic of LQT2 myocytes under β-adrenergic stimulation. Parameters of Ca handling were measured in ventricular myocytes isolated from normal and LQT2 hearts using confocal Ca imaging and whole-cell patch clamp. Ca imaging revealed no LQT2-mediated changes in amplitude of Ca transients and SR Ca content under basal conditions. Experiments in saponin-permeabilized cells using SR-entrapped low-affinity Ca indicator revealed enhanced RyR-mediated SR Ca leak and SERCA-mediated Ca uptake in LQT2 myocytes. Correspondingly, western blot analyses using phospho-specific antibodies showed that phosphorylation of both RyR and phospholamban is significantly higher in LQT2 CMs vs. controls at both PKA and CaMKII sites. In the presence of isoproterenol (50 nM) LQT2 CMs exhibited diminished Ca transient amplitudes and SR Ca content compared to controls. Stimulation of LQT2 CMs with isoproterenol resulted in prolongation of plateau of action potentials accompanied by aberrant Ca releases and phase 3 EADs, in contrast to controls. Importantly, preincubation of LQT2 CMs with CaMKII inhibitor KN93 (10 min, 500 nM) prevented ISO-mediated changes in AP duration, disturbances in Ca handling and EADs. Analysis of CaMKII activity revealed no differences between LQT2 and control heart tissues, while Western-blot analysis demonstrated ∼30% decrease in the abundance of catalytic subunit of protein phosphatase type 1 (PP1). These data suggest that hyperactive RyRs due to adaptive reduction in PP1 activity and thereby RyR hyperphosphorylation is a key contributor to enhanced triggered activity in hereditary LQT2 syndrome.

Cardiac Sarcoplasmic Reticulum Calcium Leak: Basis and Roles in Cardiac Dysfunction

Synchronized SR calcium (Ca) release is critical to normal cardiac myocyte excitation-contraction coupling, and ideally this release shuts off completely between heartbeats. However, other SR Ca release events are referred to collectively as SR Ca leak (which includes Ca sparks and waves as well as smaller events not detectable as Ca sparks). Much, but not all, of the SR Ca leak occurs via ryanodine receptors and can be exacerbated in pathological states such as heart failure. The extent of SR Ca leak is important because it can (a) reduce SR Ca available for release, causing systolic dysfunction; (b) elevate diastolic [Ca]i, contributing to diastolic dysfunction; (c) cause triggered arrhythmias; and (d) be energetically costly because of extra ATP used to repump Ca. This review addresses quantitative aspects and manifestations of SR Ca leak and its measurement, and how leak is modulated by Ca, associated proteins, and posttranslational modifications in health and disease.

Redox modification of ryanodine receptors by mitochondria‐derived reactive oxygen species contributes to aberrant Ca2+ handling in ageing rabbit hearts

Ageing is associated with a blunted response to sympathetic stimulation and an increased risk of arrhythmia and sudden cardiac death. Aberrant calcium (Ca(2+)) handling is an important contributor to the electrical and contractile dysfunction associated with ageing. Yet, the specific molecular mechanisms underlying abnormal Ca(2+) handling in ageing heart remain poorly understood. In this study, we used ventricular myocytes isolated from young (5-9 months) and old (4-6 years) rabbit hearts to test the hypothesis that changes in Ca(2+) homeostasis are caused by post-translational modification of ryanodine receptors (RyRs) by mitochondria-derived reactive oxygen species (ROS) generated in the ageing heart. Changes in parameters of Ca(2+) handling were determined by measuring cytosolic and intra-sarcoplasmic reticulum (SR) Ca(2+) dynamics in intact and permeabilized ventricular myocytes using confocal microscopy. We also measured age-related changes in ROS production and mitochondria membrane potential using a ROS-sensitive dye and a mitochondrial voltage-sensitive fluorescent indicator, respectively. In permeablized myocytes, ageing did not change SERCA activity and spark frequency but decreased spark amplitude and SR Ca(2+) load suggesting increased RyR activity. Treatment with the antioxidant dithiothreitol reduced RyR-mediated SR Ca(2+) leak in permeabilized myocytes from old rabbit hearts to the level comparable to young. Moreover, myocytes from old rabbits had more depolarized mitochondria membrane potential and increased rate of ROS production. Under β-adrenergic stimulation, Ca(2+) transient amplitude, SR Ca(2+) load, and latency of pro-arrhythmic spontaneous Ca(2+) waves (SCWs) were decreased while RyR-mediated SR Ca(2+) leak was increased in cardiomyocytes from old rabbits. Additionally, with β-adrenergic stimulation, scavenging of mitochondrial ROS in myocytes from old rabbit hearts restored redox status of RyRs, which reduced SR Ca(2+) leak, ablated most SCWs, and increased latency to levels comparable to young. These data indicate that an age-associated increase of ROS production by mitochondria leads to the thiol-oxidation of RyRs, which underlies the hyperactivity of RyRs and thereby shortened refractoriness of Ca(2+) release in cardiomyocytes from the ageing heart. This mechanism probably plays an important role in the increased incidence of arrhythmia and sudden death in the ageing population.

Oxidation of Ryanodine Receptor following Ischemia/Reperfusion Increases the Propensity of Ca Waves during Beta-Adrenergic Receptor Stimulation

Beta-Adrenergic receptor (beta-AR) stimulation generates the main positive inotropic response in the heart. However, after ischemia/reperfusion (I/R), beta-AR stimulation can generate arrhythmias. I/R also increased intracellular reactive oxygen species (ROS) production. We investigated whether ryanodine receptor (RyR) oxidation by ROS contributes to the transition from positive inotropic to arrythmogenic effect in ventricular myocytes after I/R. Measurements of contractile and electrical activity from ex vivo rabbit hearts revealed that global I/R produces severe tachi-arrhythmias. Ventricular myocytes isolated from ischemic hearts were characterized by increased both SR Ca leak rates and fractional SR Ca release compared to cells from non-ischemic hearts. Furthermore, myocytes from ischemic hearts showed increased ROS production, decreased level of free thiols in RyRs (RyR oxidation), and increased level of oxidized glutathione (GSSG). Pretreatment of myocytes from ischemic hearts with the reducing agent mercaptopropionylglycine attenuated the oxidization of free thiols in RyRs and normalized systolic SR Ca release and diastolic Ca leak. In myocytes from ischemic hearts, isoproterenol (ISO; 10 nM) led to the occurrence of spontaneous Ca waves, whereas in cells from non-ischemic hearts the same dose of ISO only caused a positive inotropic effect. Treatment of myocytes from non-ischemic hearts with H2O2 (0.1 mM) increased SR Ca leak and fractional SR Ca release to similar levels observed in myocytes from ischemic hearts. Moreover, application of ISO (10 nM) to myocytes from non-ischemic hearts pretreated with H2O2 increased the propensity of Ca waves. These results indicate that augmentation of ROS production following I/R causes RyR oxidation. This post-translational modification of the RyR plays a critical role in the transition from positive inotropic to arrhythmogenic effect during beta-AR stimulation.

Redox Modification of Ryanodine Receptors underlies Abnormal Calcium Handling in Aging Rabbit Hearts

Aging alters the electrical activity of the heart and provides a pro-arrhythmic substrate that increases the risk of arrhythmia and sudden cardiac death (Cooper et al. 2012). Although it is known that aberrant calcium dynamics may be a source of electrical abnormalities, the underlying mechanisms that affect age-associated alterations in triggered activity remain unclear. In the present study, we used ventricular myocytes isolated from young (5-9 months) and old (4-5 years) rabbit hearts to test the hypothesis that changes in Ca2+ homeostasis are caused by post-translational modification of ryanodine receptors (RyRs) caused by increased production of reactive oxygen species (ROS) in the aging heart. Changes in parameters of Ca2+ handling were determined by measuring cytosolic and intra-sarcoplasmic reticulum (SR) Ca2+ dynamics in intact and permeabilized ventricular myocytes using confocal microscopy. Aging did not change SERCA activity and spark frequency but decreased spark amplitude and SR Ca2+ content in permeabilized cells, suggesting an increased activity of RyRs. Under stimulation withβ-adrenergic agonist isoproterenol, Ca2+ transient amplitude, SR Ca2+ load, and latency of pro-arrhythmic spontaneous Ca2+ waves (SCWs) were decreased in cardiomyocytes derived from old rabbits; whereas, the rate of ROS production was increased. Treatment with the antioxidant DTT attenuated RyR-mediated SR Ca leak in aged permeabilized myocytes to levels comparable to young. Moreover, pretreatment with the mitochondria-specific ROS scavenger mito-TEMPO ablated SCWs in isoproterenol-treated old cardiomyocytes to levels comparable to young. This data suggests that increased rate of ROS production by mitochondria lead to the thiol-oxidation of RyRs, which underlies the hyperactivity of RyRs and thereby shortened refractoriness of Ca2+ release in cardiomyocytes from the aging heart. This mechanism likely plays an important role in the increased risk of arrhythmia and sudden cardiac death in the aging population.

Ryanodine Receptor S2808 Phosphorylation in Heart Failure

In this issue of Circulation Research , Zhang et al1 provide direct and clear evidence that phosphorylation of S2808 in the cardiac ryanodine receptors (RyR2) has no impact on either normal cardiac function, sympathetic stimulation of the heart, or progression of heart failure (HF) after myocardial infarction (MI). They used mice in which the S2808 site was rendered nonphosphorylatable in S2808A knock-in mice, and the results directly contradict results published by the group of A.R. Marks.2–4 This has been a highly controversial area. This new study compels me to assess this pathway in a balanced way, but recognizing that my opinions are influenced by work that my group has done in this area. My goal here is not to microdissect each controversial piece of data in dozens of relevant papers (see recent reviews and tables5,6), but rather to provide my informed opinion about key aspects of this issue in a balanced manner. Let us first consider the model that importantly raised the issue of SR Ca leak as a potential pathophysiological cardiac effect in HF. Article, see p 831 In 2000 the Marks group published an exciting and seminal paper that has fueled intensive research over the past 11 years.7 They proposed that in HF, PKA hyperphosphorylates RyR2-S2808, causing FKBP12.6 dissociation from RyR2, which directly enhances SR Ca leak/depletion and systolic dysfunction in HF (Figure). Tenets later added to this core model were a down-regulation of phosphatases associated with the RyR2 in HF (which would facilitate the PKA-dependent RyR2 hyperphosphorylation) and that this diastolic SR Ca leak could also contribute to triggered arrhythmias. An extensive series of papers by the same group since then have provided highly consistent and compelling support for this working hypothesis, extending the impact of this intriguing PKA/RyR-S2808/FKBP12.6/RyR …

Ankyrin-B reduction enhances Ca spark-mediated SR Ca release promoting cardiac myocyte arrhythmic activity

Ankyrin-B (AnkB) loss-of-function may cause ventricular arrhythmias and sudden cardiac death in humans. Cardiac myocytes from AnkB heterozygous mice (AnkB(+/-)) show reduced expression and altered localization of Na/Ca exchanger (NCX) and Na/K-ATPase (NKA), key players in regulating [Na](i) and [Ca](i). Here we investigate how AnkB reduction affects cardiac [Na](i), [Ca](i) and SR Ca release. We found reduced NCX and NKA transport function but unaltered [Na](i) and diastolic [Ca](i) in myocytes from AnkB(+/-) vs. wild-type (WT) mice. Ca transients, SR Ca content and fractional SR Ca release were larger in AnkB(+/-) myocytes. The frequency of spontaneous, diastolic Ca sparks (CaSpF) was significantly higher in intact myocytes from AnkB(+/-) vs. WT myocytes (with and without isoproterenol), even when normalized for SR Ca load. However, total ryanodine receptor (RyR)-mediated SR Ca leak (tetracaine-sensitive) was not different between groups. Thus, in AnkB(+/-) mice SR Ca leak is biased towards more Ca sparks (vs. smaller release events), suggesting more coordinated openings of RyRs in a cluster. This is due to local cytosolic RyR regulation, rather than intrinsic RyR differences, since CaSpF was similar in saponin-permeabilized myocytes from WT and AnkB(+/-) mice. The more coordinated RyRs openings resulted in an increased propensity of pro-arrhythmic Ca waves in AnkB(+/-) myocytes. In conclusion, AnkB reduction alters cardiac Na and Ca transport and enhances the coupled RyR openings, resulting in more frequent Ca sparks and waves although the total SR Ca leak is unaffected. This could enhance the propensity for triggered arrhythmias in AnkB(+/-) mice.

Dual role of junctin in the regulation of ryanodine receptors and calcium release in cardiac ventricular myocytes

Junctin, a 26 kDa intra-sarcoplasmic reticulum (SR) protein, forms a quaternary complex with triadin, calsequestrin and the ryanodine receptor (RyR) at the junctional SR membrane. The physiological role for junctin in the luminal regulation of RyR Ca(2+) release remains unresolved, but it appears to be essential for proper cardiac function since ablation of junctin results in increased ventricular automaticity. Given that the junctin levels are severely reduced in human failing hearts, we performed an in-depth study of the mechanisms affecting intracellular Ca(2+) homeostasis in junctin-deficient cardiomyocytes. In concurrence with sparks, JCN-KO cardiomyocytes display increased Ca(2+) transient amplitude, resulting from increased SR [Ca(2+)] ([Ca(2+)](SR)). Junctin ablation appears to affect how RyRs 'sense' SR Ca(2+) load, resulting in decreased diastolic SR Ca(2+) leak despite an elevated [Ca(2+)](SR). Surprisingly, the β-adrenergic enhancement of [Ca(2+)](SR) reverses the decrease in RyR activity and leads to spontaneous Ca(2+) release, evidenced by the development of spontaneous aftercontractions. Single channel recordings of RyRs from WT and JCN-KO cardiac SR indicate that the absence of junctin produces a dual effect on the normally linear response of RyRs to luminal [Ca(2+)]: at low luminal [Ca(2+)] (<1 mmol l(-1)), junctin-devoid RyR channels are less responsive to luminal [Ca(2+)]; conversely, high luminal [Ca(2+)] turns them hypersensitive to this form of channel modulation. Thus, junctin produces complex effects on Ca(2+) sparks, transients, and leak, but the luminal [Ca(2+)]-dependent dual response of junctin-devoid RyRs demonstrates that junctin normally acts as an activator of RyR channels at low luminal [Ca(2+)], and as an inhibitor at high luminal [Ca(2+)]. Because the crossover occurs at a [Ca(2+)](SR) that is close to that present in resting cells, it is possible that the activator-inhibitor role of junctin may be exerted under periods of prevalent parasympathetic and sympathetic activity, respectively.