Sarcoplasmic Reticulum Ca Leak Research Articles

On the “spark Frequency Vs. Leak Rate” Relationship in Ventricular Myocytes: A Study in the Rabbit

Recent studies have linked heart disease to mutated or hyperactive ryanodine receptors (RyRs) in ventricular myocytes, renewing the interest on how the sarcoplasmic reticulum (SR) Ca release process works during diastole. We addressed two questions of potential clinical relevance: (1) whether the SR Ca leak rate (Jleak) can be entirely explained by Ca sparks, and (2) whether the spark-dependent fraction of Jleak can be varied upon RyR phosphorylation. We used confocal microscopy to simultaneously measure Jleak and Ca sparks in Fluo 4-loaded rabbit ventricular myocytes. Control cells (C; n = 47) were paced at 0.5 Hz, while isoproterenol-treated cells (I; n = 14, [Isoproterenol] = 125 nM) were paced at 0.25 Hz to match the SR loads (C = 136.4 ± 5.6 μmoles/l cytosol; I = 126 ± 9.2 ; P = 0.47 in t-test with Welch correction). Jleak was quantified as in Shannon et al. (2002; Circ Res 91:594-600), but using a lower Km for the forward rate of uptake in the I group. Although Jleak did not significantly differ among the groups (C = 10.87 ± 0.93 μM/s; I = 13.12 ± 2.53 ; P = 0.42), the spark frequency was more than doubled in the isoproterenol-treated cells (C = 1.21 ± 0.15 sparks ∗ (100 μm)−1∗ s−1); I = 2.82 ± 0.51; P = 0.0082). These findings point to an increase in the spark-dependent fraction of Jleak upon RyR phosphorylation (for a given SR load), while suggesting an enhancement of Ca-induced RyR coupling relative to the influence of stabilizing RyR couplers.

Inhibition of RyR2-S2814 Phosphorylation Prevents Heart Failure by Reducing SR Ca Leak

Abnormal regulation of RyR2 by Ca2+/calmodulin-dependent protein kinase 2 (CaMKII) has been suggested as a cause of sarcoplasmic reticulum (SR) Ca2+ leakage and contractile dysfunction in heart failure. We hypothesized that CaMKII phosphorylation of RyR2 is crucial for heart failure development. We generated RyR2 knockin mice in which CaMKII phosphorylation site S2814 was mutated to alanine (S2814A) to prevent phosphorylation. Cardiac function and dimensions monitored by echocardiography were similar in S2814A and WT mice up to 12 months of age. WT (n=13) and S2814A mice (n=9) were subjected to transverse aortic constriction (TAC) to induce pressure overload. At 8 weeks after TAC, S2814A mice displayed a similar hypertrophic response and decrease in ejection fraction (EF) as WT mice. At 16 weeks after TAC, however, EF was significantly lower in WT (32.5 +/- 3.4%) compared to S2814A mice (43.0 +/- 2.9%) suggesting inhibition of heart failure development in the latter. This rescue effect was further verified by a lower lung-weight-to-tibia-length ratio and a decrease in expression levels of the cardiac stress genes ANF and BNP in S2814A mice compared to WT mice at 16 weeks after TAC. Ca2+ imaging in cardiomyocytes, isolated from S2814A and WT mice at 16 weeks after sham or TAC surgery, demonstrated an decreased incidence of spontaneous SR Ca2+ release (SCR) events in S2814A (29.7% of myocytes) compared to WT (61.3% of myocytes; P<0.01). Whereas CaMKII inhibitor KN93 reduced the incidence of SCR events in WT to 36.1% (P<0.01 vs. WT TAC), KN93 did not have an effect of SCR in S2814A myocytes (30.0%; P=NS vs. S2814A TAC). Together, our results demonstrate that blocking CaMKII phosphorylation of RyR2 prevents SR Ca2+ leak, and inhibits or delays the progression to congestive heart failure in S2814A mice.

Altered intracellular Ca2+ regulation in chronic rat heart failure

intracellular Ca(2+) handling by the sarcoplasmic reticulum (SR) plays a crucial role in the pathogenesis of heart failure (HF). Despite extensive effort, the underlying causes of abnormal SR Ca(2+) handling in HF have not been clarified. To determine whether the diastolic SR Ca(2+) leak along with reduced Ca(2+) reuptake is required for decreased contractility, we investigated the cytosolic Ca(2+) transients and SR Ca(2+) content and assessed the expression of ryanodine receptor (RyR2), FK506 binding protein (FKBP12.6), SR-Ca(2+) ATPase (SERCA2a), and L-type Ca(2+) channel (LTCC) using an SD-rat model of chronic HF. We found that the cytosolic Ca(2+) transients were markedly reduced in amplitude in HF myocytes (DeltaF/F(0) = 12.3 +/- 0.8) compared with control myocytes (DeltaF/F(0) = 17.7 +/- 1.2, P < 0.01), changes paralleled by a significant reduction in the SR Ca(2+) content (HF: DeltaF/F(0) = 12.4 +/- 1.1, control: DeltaF/F(0) = 32.4 +/- 1.9, P < 0.01). Moreover, we demonstrated that the expression of FKBP12.6 associated with RyR2, SERCA2a, and LTCC was significantly reduced in rat HF. These results provide evidence for phosphorylation-induced detachment of FKBP12.6 from RyRs and down-regulation of SERCA2a and LTCC in HF. We conclude that diastolic SR Ca(2+) leak (due to dissociation of FKBP12.6 from RyR2) along with reduced SR Ca(2+) uptake (due to down-regulation of SERCA2a) and defective E-C coupling (due to down-regulation of LTCC) could contribute to HF.

Transient Receptor Potential Canonical Type 1 (TRPC1) Operates as a Sarcoplasmic Reticulum Calcium Leak Channel in Skeletal Muscle

Extensive studies performed in nonexcitable cells and expression systems have shown that type 1 transient receptor potential canonical (TRPC1) channels operate mainly in plasma membranes and open through phospholipase C-dependent processes, membrane stretch, or depletion of Ca(2+) stores. In skeletal muscle, it is proposed that TRPC1 channels are involved in plasmalemmal Ca(2+) influx and stimulated by store depletion or membrane stretch, but direct evidence for TRPC1 sarcolemmal channel activity is not available. We investigated here the functional role of TRPC1 using an overexpressing strategy in adult mouse muscle fibers. Immunostaining for endogenous TRPC1 revealed a striated expression pattern that matched sarcoplasmic reticulum (SR) Ca(2+) pump immunolabeling. In cells expressing TRPC1-yellow fluorescent protein (YFP), the same pattern of expression was observed, compatible with a longitudinal SR localization. Resting electric properties, action potentials, and resting divalent cation influx were not altered in TRPC1-YFP-positive cells. Poisoning with the SR Ca(2+) pump blocker cyclopiazonic acid elicited a contracture of the fiber at the level of the overexpression site in presence and absence of external Ca(2+) which was not observed in control cells. Ca(2+) measurements indicated that resting Ca(2+) and the rate of Ca(2+) increase induced by cyclopiazonic acid were higher in the TRPC1-YFP-positive zone than in the TRPC1-YFP-negative zone and control cells. Ca(2+) transients evoked by 200-ms voltage clamp pulses decayed slower in TRPC1-YFP-positive cells. In contrast to previous hypotheses, these data demonstrate that TRPC1 operates as a SR Ca(2+) leak channel in skeletal muscle.

Calcium/Calmodulin-Dependent Protein Kinase II Contributes to Cardiac Arrhythmogenesis in Heart Failure

Transgenic (TG) Ca/calmodulin-dependent protein kinase II (CaMKII)delta(C) mice have heart failure and isoproterenol (ISO)-inducible arrhythmias. We hypothesized that CaMKII contributes to arrhythmias and underlying cellular events and that inhibition of CaMKII reduces cardiac arrhythmogenesis in vitro and in vivo. Under baseline conditions, isolated cardiac myocytes from TG mice showed an increased incidence of early afterdepolarizations compared with wild-type myocytes (P<0.05). CaMKII inhibition (AIP) completely abolished these afterdepolarizations in TG cells (P<0.05). Increasing intracellular Ca stores using ISO (10(-8) M) induced a larger amount of delayed afterdepolarizations and spontaneous action potentials in TG compared with wild-type cells (P<0.05). This seems to be due to an increased sarcoplasmic reticulum (SR) Ca leak because diastolic [Ca](i) rose clearly on ISO in TG but not in wild-type cells (+20+/-5% versus +3+/-4% at 10(-6) M ISO, P<0.05). In parallel, SR Ca leak assessed by spontaneous SR Ca release events showed an increased Ca spark frequency (3.9+/-0.5 versus 2.0+/-0.4 sparks per 100 microm(-1).s(-1), P<0.05). However, CaMKII inhibition (either pharmacologically using KN-93 or genetically using an isoform-specific CaMKIIdelta-knockout mouse model) significantly reduced SR Ca spark frequency, although this rather increased SR Ca content. In parallel, ISO increased the incidence of early (54% versus 4%, P<0.05) and late (86% versus 43%, P<0.05) nonstimulated events in TG versus wild-type myocytes, but CaMKII inhibition (KN-93 and KO) reduced these proarrhythmogenic events (P<0.05). In addition, CaMKII inhibition in TG mice (KN-93) clearly reduced ISO-induced arrhythmias in vivo (P<0.05). We conclude that CaMKII contributes to cardiac arrhythmogenesis in TG CaMKIIdelta(C) mice having heart failure and suggest the increased SR Ca leak as an important mechanism. Moreover, CaMKII inhibition reduces cardiac arrhythmias in vitro and in vivo and may therefore indicate a potential role for future antiarrhythmic therapies warranting further studies.

Properties Of Sarcoplasmic Reticulum Ca Leak In Rabbit Ventricular And Atrial Myocytes

To investigate properties of sarcoplasmic reticulum (SR) Ca leak in ventricular and atrial myocytes, we simultaneously measured Ca sparks and intra-SR free Ca ([Ca]SR) after sarcolemma permeabilization. SR Ca leak (Δ[Ca]SR-total/s) was measured over a wide range of SR Ca loads after complete SERCA inhibition with thapsigargin. We found that in both tissues the ryanodine receptor (RyR) was the main contributor to SR Ca leak. RyR-mediated leak occurred in part as Ca sparks, but also as non-spark-mediated leak. Additionally, there was a component of SR Ca leak that was insensitive to RyR inhibitors. In contrast to ventricular cells, atrial SR had a slower total leak rate mainly due to a smaller contribution from RyR non-spark-mediated leak. As result of this, atrial myocytes had a higher SR Ca load under control conditions (1.4 mM) than ventricular (0.8 mM). RyR type-2 expression levels were similar in both types of cells suggesting that observed differences in SR leak are due to difference in RyR regulation. Activation of IP3 receptors (IP3R) increased total SR Ca leak rate more than 2-fold in atrial myocytes, but only slightly affected leak in ventricular myocytes. This finding agrees with higher (more than 3 times) IP3R type-2 and -3 expression levels in atrial than in ventricular myocytes. In conclusion, ventricular myocytes have a more “leaky” SR than atrial cells due to higher RyR activity at resting condition. However, SR Ca leak in atrial myocytes can be facilitated significantly during activation of IP3-dependent signaling pathways.

Does Deregulation Of Calcium Handling Precede Or Follow Alterations Of Cardiac Function During Progression To Heart Failure?

In heart failure (HF), abnormalities of in vivo cardiac function are closely correlated with reduced sarcoplasmic reticulum (SR) Ca release and myocyte contractility, supporting the concept that HF is a result of deranged Ca cycling. However, most studies addressing the role and mechanisms of altered Ca handling in HF have been performed at advanced stages of HF providing little information as to whether these deficiencies are causes or consequences of HF.In the present study we compared the time course of development of alterations in myocytes Ca handling, using a canine tachypacing model of chronic HF. In vivo function of left ventricle was assessed at several time points (1, 4, and >8 mo) with parallel studies in single ventricular myocytes of Ca handling utilizing Ca imaging in intact, permeabilized, and patch-clamped cells.LV fractional shortening progressively decreased by 50, 65, and 75 % of control values at 1, 4, and >8 month of tachypacing, respectively. The frequency of Ca sparks and the rate of SR Ca leak increased progressively with the duration of tachypacing, while diastolic [Ca]SR decreased with the duration of tachypacing. Na/Ca exchange activity was significantly augmented at 1 mo, and did not change thereafter. The SERCA-mediated Ca uptake and the density of peak Ca current were not changed up to >8 mo of tachypacing. Progressive decreases in the amplitude of depolarization-induced Ca transients and single-cell contractions were observed only starting at the 4th month of tachypacing.These results suggest that diminished SR Ca release follows rather than precedes deterioration of in vivo cardiac function, thus is not likely to be a cause of HF.

Phosphorylation of Ryanodine Receptor At Serine-2809 Modulates Sarcoplasmic Reticulum Ca Release in Rabbit Ventricular Myocytes

The role of protein kinase A (PKA)-dependent phosphorylation of cardiac ryanodine receptor (RyR) is highly controversial. Here we studied a functional link between RyR phosphorylation at serine-2809 (PKA-specific site) and sarcoplasmic reticulum (SR) Ca release and leak in cardiomyocytes. We simultaneously measured intra-SR free Ca ([Ca]SR) with Fluo-5N and cytosolic Ca with Rhod-2 in permeabilized rabbit ventricular myocytes. RyR phosphorylation at site serine-2809 was measured with a phospho-specific antibody (Badrilla, UK). We found that cAMP (10 μM) increased Ca spark frequency by ∼2.6 times. This effect was associated with an increase in SR Ca load from 0.84 to 1.24 mM. PKA inhibitory peptide (10 μM) abolished cAMP-mediated increase of SR Ca load and spark frequency. When SERCA was completely blocked by thapsigargin, cAMP did not affect RyR-mediated Ca leak. The lack of cAMP effect on RyR function can be explained by almost maximal phosphorylation of RyR at serine-2809 after membrane permeabilization and also argues against the functional importance of another PKA-specific site (serine-2031) for SR Ca release. This high phosphorylation level of RyR could be due to a shift of the balance between protein kinase and phosphatase activity after permeabilization. Preventing this increase in phosphorylation with staurosporine (1 μM) decreased RyR-mediated SR Ca leak. Surprisingly, further dephosphorylation of RyR at serine-2809 with protein phosphatase 1 (PP1; 2 U/ml) markedly increased Ca leak. However, it is important to note that PP1 and staurosporine possibly affected other phosphorylation sites of RyR as well. In conclusion, our results provide direct evidence that RyR phosphorylation at serine-2809 modulates channel function and SR Ca release in rabbit ventricular myocytes.

Isoproterenol-Enhanced Diastolic Sarcoplasmic Reticulum Ca Leak in Ventricular Myocytes Requires Activation of Nitric Oxide Synthase

We have previously shown increased cardiac ryanodine receptor (RyR)-dependent diastolic SR Ca leak to be present in heart failure (HF) and in conditions when beta-adrenergic (β-AR) tone is high. This SR Ca leak could contribute to the cause of the observed decreased contractility in HF by limiting SR Ca load. Simultaneously, it could also lead to arrhythmogenic Ca-dependent inward depolarizing current commonly seen in failing hearts. We recently demonstrated that this leak increases in manner dependent on calcium-calmodulin-dependent protein kinase II (CaMKII) and completely independent of either protein kinase A (PKA) activation or an increase in bulk free Ca concentration ([Ca]i). Here we investigate this PKA- and [Ca]i-independent activation of CaMKII. We have found that while stimulating intact myocytes with the β-AR agonist isoproterenol (ISO, 250 nM) the CaMKII-dependent enhancement of SR Ca leak is abolished by treatment of the myocytes with nitric oxide synthase (NOS) inhibitor Nω-Nitro-L-arginine methyl ester (L-NAME, 100 μM). When SR Ca load was matched in each group (156 μM), myocytes treated with ISO alone had significantly higher leak (14.2 ± 2.0 μM) vs. those treated with ISO and L-NAME (3.8 ± 1.4 μM) or those left completely untreated (8.0 ± 0.91 μM). When SR Ca leak was matched (9.0 μM) we found the SR Ca load necessary to induce that leak was significantly lower in ISO-treated myocytes (91.6 ± 1.9 μM) vs. those treated with ISO and L-NAME (129.4 ± 16.3 μM) or those left untreated (127.4 ± 2.8 μM). This evidence indicates that NOS activation, and therefore generation of nitric oxide, is necessary for ISO-dependent activation of RyR by CaMKII.

Abstract 5326: S100A1 Prevents Arrhythmogenic Diastolic Sarcoplasmic Reticulum Calcium Leak In Ventricular Cardiomyocytes

Background: S100A1 is an inotropic calcium (Ca) sensor protein in cardiomyocytes interacting with the cardiac ryanodine receptor (RyR2). Previous studies of our group have shown that S100A1 protein can decrease Ca spark frequency in permeabilized ventricular cardiomyocytes (CMs). We therefore hypothesized that S100A1 might prevent arrhythmogenic sarcoplasmic reticulum (SR) Ca leak in CMs. Methods and Results: Ventricular rat cardiomyocytes were enzymatically isolated and transfected either with 10 MOI of a CMV-GFP control or CMV-S100A1/CMV-GFP adenovirus resulting in GFP fluorescence in &gt;99% of cultured GFP-CMs and S100A1-CMs as assessed by epifluorescent microscopy after 24-hours. Expression analysis revealed a 3-fold increase in S100A1 protein in S100A1-CM levels compared to GFP-CM. Confocal line-scan microscopy yielded a 50% reduction in Ca spark frequency in rhodamin2-AM loaded quiescent S100A1-CMs and epifluorescent imaging showed a 20% and 50% increase in the SR Ca content and systolic Ca transient amplitude, respectively, in FURA2-AM loaded 2Hz-stimulated S100A1-CM (37°C, 1.8 mM Ca) compared with GFP-CMs. Pathophysiological diastolic SR Ca leak was induced subjecting 2Hz-stimulated FURA2-AM loaded CMs to 0.5 mM caffeine and 10–7 M isoproterenol (caff/iso) as previously published by Isner and co-workers. As expected, this protocol resulted in permanent diastolic Ca waves in 100% (n=60/60) of 2Hz-stimulated iso/caff-treated GFP-CMs and was accompanied by significantly increased diastolic Ca levels and reduced systolic Ca transient amplitudes compared with iso-treated GFP-CMs. However, S100A1 efficiently protected more than 80% of iso/caff treated S100A1-CMs (n=49/60 cells, P&lt;0.05 vs. iso/caff GFP-CMs) from diastolic Ca waves and further alterations of CM Ca handling. Importantly, similar results were obtained in caff/iso-treated failing rat GFP- and S100A1-CMs (data not shown). Conclusions: Here we show for the first time that S100A1 can both diminish the physiological SR Ca leak and protect from arrhythmogenic Ca waves in CMs. Given its beneficial effects in the context of experimental HF animal models, S100A1 therapeutic inotropic actions might be complemented by an antiarrhythmic effect targeting dysfunctional RyR2.

Amitriptyline Activates Cardiac Ryanodine Channels and Causes Spontaneous Sarcoplasmic Reticulum Calcium Release

Patients taking amitriptyline (AMT) have an increased risk of sudden cardiac death, yet the mechanism for AMT's proarrhythmic effects remains incompletely understood. Here, we hypothesize that AMT activates cardiac ryanodine channels (RyR2), causing premature Ca(2+) release from the sarcoplasmic reticulum (SR), a mechanism identified by genetic studies as a cause of ventricular arrhythmias and sudden cardiac death. To test this hypothesis, we measured the effect of AMT on RyR2 channels from mice and sheep and on intact mouse cardiomyocytes loaded with the Ca(2+) fluorescent indicator Fura-2 acetoxymethyl ester. AMT induced trains of long channel openings (bursts) with 60 to 90% of normal conductance in RyR2 channels incorporated in lipid bilayers. The [AMT], voltage, and open probability (P(o)) dependencies of burst frequency and duration indicated that AMT binds primarily to open RyR2 channels. AMT also activated RyR2 channels isolated from transgenic mice lacking cardiac calsequestrin. Reducing RyR2 P(o) by increasing cytoplasmic [Mg(2+)] significantly inhibited the AMT effect on RyR2 channels. Consistent with the single RyR2 channel data, AMT increased the rate of spontaneous Ca(2+) releases and decreased the SR Ca(2+) content in intact cardiomyocytes. Intracellular [AMT] were approximately 5-fold higher than extracellular [AMT], explaining AMT's higher potency in cardiomyocytes at clinically relevant concentrations (0.5-3 muM) compared with its effect in lipid bilayers (5-10 muM). Increasing extracellular [Mg(2+)] attenuated the effect of AMT in intact myocytes. We conclude that the heretofore unrecognized activation of RyR2 channels and increased SR Ca(2+) leak may contribute to AMT's proarrhythmic and cardiotoxic effects, which may be counteracted by interventions that reduce RyR2 channel open probability.

Intracellular Ca Alternans: Coordinated Regulation by Sarcoplasmic Reticulum Release, Uptake, and Leak

Beat-to-beat alternation in the cardiac intracellular Ca (Cai) transient can drive action potential (AP) duration alternans, creating a highly arrhythmogenic substrate. Although a steep dependence of fractional sarcoplasmic reticulum (SR) Ca release on SR Ca load has been shown experimentally to promote Cai alternans, theoretical studies predict that other factors are also important. Here we present an iterated map analysis of the coordinated effects of SR Ca release, uptake, and leak on the onset of Cai alternans. Predictions were compared to numerical simulations using a physiologically realistic AP model as well as to AP clamp experiments in isolated patch-clamped rabbit ventricular myocytes exposed to 1), the Ca channel agonist BayK8644 (100 nM) to increase SR Ca load and release fraction, 2), overexpression of an adenoviral SERCA2a construct to increase SR Ca uptake, and 3), low-dose FK506 (20μM) or ryanodine (1μM) to increase SR Ca leak. Our findings show that SR Ca release, uptake, and leak all have independent direct effects that promote (release and leak) or suppress (uptake) Cai alternans. However, since each factor affects the other by altering SR Ca load, the net balance of their direct and indirect effects determines whether they promote or suppress alternans. Thus, BayK8644 promotes, whereas Ad-SERCA2a overexpression, ryanodine, and FK506 suppress, Cai alternans under AP clamp conditions.

Intracellular calcium leak due to FKBP12.6 deficiency in mice facilitates the inducibility of atrial fibrillation

Although defective Ca(2+) homeostasis may contribute to arrhythmogenesis in atrial fibrillation (AF), the underlying molecular mechanisms remain poorly understood. Studies in patients with AF revealed that impaired diastolic closure of sarcoplasmic reticulum (SR) Ca(2+)-release channels (ryanodine receptors, RyR2) is associated with reduced levels of the RyR2-inhibitory subunit FKBP12.6. The objective of the present study was to test the hypothesis that Ca(2+) leak from the SR through RyR2 increases the propensity for AF in FKBP12.6-deficient (-/-) mice. Surface electrocardiogram and intracardiac electrograms were recorded simultaneously in FKBP12.6-/- mice and wild-type (WT) littermates. Right atrial programmed stimulation was performed before and after injection of RyR2 antagonist tetracaine (0.5 mg/kg). Intracellular Ca(2+) transients were recorded in atrial myocytes from FKBP12.6-/- and WT mice. FKBP12.6-/- mice had structurally normal atria and unaltered expression of key Ca(2+)-handling proteins. AF episodes were inducible in 81% of FKBP12.6-/-, but in only 7% of WT mice (P <.05), and were prevented by tetracaine in all FKBP12.6-/- mice. SR Ca(2+) leak in FKBP12.6-/- myocytes was 53% larger than in WT myocytes, and FKBP12.6-/- myocytes showed increased incidence of spontaneous SR Ca(2+) release events, which could be blocked by tetracaine. The increased vulnerability to AF in FKBP12.6-/- mice substantiates the notion that defective SR Ca(2+) release caused by abnormal RyR2 and FKBP12.6 interactions may contribute to the initiation or maintenance of atrial fibrillation.

Sarcoplasmic reticulum Ca2+ leak in heart failure: mere observation or functional relevance?

Heart failure (HF) is a chronic multi-factorial disease characterized by sarcoplasmic reticulum (SR) dysfunction that manifests as severely reduced contractility and increased risk of arrhythmia. Several lines of evidence have revealed the existence of defective ryanodine receptor (RyR2)-mediated Ca(2+) leak in HF, although its relevance as a causative factor rather than a phenotypic consequence of the disease is questioned. This review will consider the relative contribution of RyR2-mediated Ca(2+) leak to the profound cellular, transcriptional and electrical remodelling associated with HF. In particular, it will focus on our current understanding of the role of defective phosphorylation of RyR2 as a both a chronic mediator of excitation-contraction coupling (ECC) dysfunction and as a potent catalyst of RyR2-dependent arrhythmogenesis. A hypothetical concept that SR Ca(2+) leak fundamentally underlies the increased arrhythmogenic susceptibility in HF, but that it may not directly contribute to contractile dysfunction, which may involve maladaptive perturbations in metabolism and energy utilization, is also discussed.

The cAMP binding protein Epac modulates Ca2+ sparks by a Ca2+/calmodulin kinase signalling pathway in rat cardiac myocytes

cAMP is a powerful second messenger whose known general effector is protein kinase A (PKA). The identification of a cAMP binding protein, Epac, raises the question of its role in Ca(2+) signalling in cardiac myocytes. In this study, we analysed the effects of Epac activation on Ca(2+) handling by using confocal microscopy in isolated adult rat cardiomyocytes. [Ca(2+)](i) transients were evoked by electrical stimulation and Ca(2+) sparks were measured in quiescent myocytes. Epac was selectively activated by the cAMP analogue 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-CPT). Patch-clamp was used to record the L-type calcium current (I(Ca)), and Western blot to evaluate phosphorylated ryanodine receptor (RyR). [Ca(2+)](i) transients were slightly reduced by 10 microm 8-CPT (F/F(0): decreased from 4.7 +/- 0.5 to 3.8 +/- 0.4, P < 0.05), an effect that was boosted when cells were previously infected with an adenovirus encoding human Epac. I(Ca) was unaltered by Epac activation, so this cannot explain the decreased [Ca(2+)](i) transients. Instead, a decrease in the sarcoplasmic reticulum (SR) Ca(2+) load underlies the decrease in the [Ca(2+)](i) transients. This decrease in the SR Ca(2+) load was provoked by the increase in the SR Ca(2+) leak induced by Epac activation. 8-CPT significantly increased Ca(2+) spark frequency (Ca(2+) sparks s(-1) (100 microm)(-1): from 2.4 +/- 0.6 to 6.9 +/- 1.5, P < 0.01) while reducing their amplitude (F/F(0): 1.8 +/- 0.02 versus 1.6 +/- 0.01, P < 0.001) in a Ca(2+)/calmodulin kinase II (CaMKII)-dependent and PKA-independent manner. Accordingly, we found that Epac increased RyR phosphorylation at the CaMKII site. Altogether, our data reveal a new signalling pathway by which cAMP governs Ca(2+) release and signalling in cardiac myocytes.

β-Adrenergic Enhancement of Sarcoplasmic Reticulum Calcium Leak in Cardiac Myocytes Is Mediated by Calcium/Calmodulin-Dependent Protein Kinase

Enhanced cardiac diastolic Ca leak from the sarcoplasmic reticulum (SR) ryanodine receptor may reduce SR Ca content and contribute to arrhythmogenesis. We tested whether beta-adrenergic receptor (beta-AR) agonists increased SR Ca leak in intact rabbit ventricular myocytes and whether this depends on protein kinase A or Ca/calmodulin-dependent protein kinase II (CaMKII) activity. SR Ca leak was assessed by acute block of the ryanodine receptor by tetracaine and assessment of the consequent shift of Ca from cytosol to SR (measured at various SR Ca loads induced by varying frequency). Cytosolic [Ca] ([Ca](i)) and SR Ca load ([Ca](SRT)) were assessed using fluo-4. beta-AR activation by isoproterenol dramatically increased SR Ca leak. However, this effect was not inhibited by blocking protein kinase A by H-89, despite the expected reversal of the isoproterenol-induced enhancement of Ca transient amplitude and [Ca](i) decline rate. In contrast, inhibitors of CaMKII, KN-93, or autocamtide-2-related inhibitory peptide II or beta-AR blockade reversed the isoproterenol-induced enhancement of SR Ca leak, and CaMKII inhibition could even reduce leak below control levels. Forskolin, which bypasses the beta-AR in activating adenylate cyclase and protein kinase A, did not increase SR Ca leak, despite robust enhancement of Ca transient amplitude and [Ca](i) decline rate. The results suggest that beta-AR stimulation enhances diastolic SR Ca leak in a manner that is (1) CaMKII dependent, (2) not protein kinase A dependent, and 3) not dependent on bulk [Ca](i).

Abstract 575: Both Phosphorylation and Dephosphorylation of Ryanodine Receptor Enhance Sarcoplasmic Reticulum Calcium Leak in Canine Ventricular Myocytes

In recent years, there has been an increase of emphasis on the possibility that alterations in phosphorylation of the cardiac ryanodine receptor (RyR2) may contribute to the defective intracellular Ca release in heart failure (HF). However, the specific mechanisms proposed by different laboratories remain controversial. We studied the effects of exogenous and endogenous kinases and phosphatases on sarcoplasmic reticulum (SR) Ca release by measuring [Ca] changes in the cytosolic and SR luminal ([Ca]SR) compartments, using the Ca indicators rhod-2 and fluo-5N, respectively, in permeabilized canine ventricular myocytes. Application of PP1 (10 u/ml) led to an acute increase in the frequency of spontaneous Ca release events (Ca sparks) followed by a decline in intra-SR [Ca] consistent with the possibility that activation of RyR2s contributes to unloading of the SR by releasing Ca. Indeed, PP1 caused a marked acceleration of the RyR2-mediated SR Ca leak directly measured as time-dependent decline of [Ca]SR in the presence of the inhibitor of the SR CaATPase (SERCA2), thapsigargin. Application of cAMP (20 uM) and PKA (10 u/ml) resulted in a significant increase in Ca spark frequency that was paralleled by an increase in [Ca]SR. Direct measurements of the RyR2-mediated leak in the presence of thapsigargin revealed a small but significant acceleration in the leak following cAMP or PKA application, implying that the potentiation of sparks in the absence of thapsigargin may involve direct effects upon RyR2s in addition to effects mediated by SERCA2-dependent increase in [Ca]SR. As determined by a phosphospecific antibody, RyR2 phosphorylation on Ser-2809 was increased by cAMP and reduced by PP1. Collectively, these results suggest that under baseline phosphorylation conditions, both phosphorylation and dephosphorylation stimulate RyR2 activity resulting in an increased SR Ca leak. Considering the multimeric nature of the RyR2 and the presence of multiple sets of phophorylation sites, these results can be rationalized by suggesting that the effects of kinases and phosphotases on RyR2 function are mediated by different degrees of RyR2 phosphorylation at a certain set of sites (e.g. Ser-2809) or by phosphorylation/dephosphorylation of different sets of sites.

Regulation of Ca2+ and Na+ in Normal and Failing Cardiac Myocytes

Ca(2+) in cardiac myocytes regulates contractility and relaxation, and Ca(2+) and Na (+)regulation are linked via Na(+)/Ca(2+) exchange (NCX). Heart failure (HF) is accompanied by contractile dysfunction and arrhythmias, both of which may be due to altered cellular Ca(2+) handling. Smaller Ca(2+) transient and sarcoplasmic reticulum (SR) Ca(2+) content cause systolic dysfunction in HF. The reduced SR Ca(2+) content is due to: (a) reduced SR Ca(2+)-ATPase function (which also contributes to diastolic dysfunction), (b) increased expression and function of NCX (which competes with SR Ca(2+)-ATPase during relaxation, but preserves diastolic function), and (c) enhanced diastolic SR Ca(2+) leak. Relative contributions of these may vary with HF etiology and stage. Triggered arrhythmias (e.g., delayed afterdepolarizations [DADs]) are prominent in HF. DADs are due to spontaneous SR Ca(2+) release and consequent activation of transient inward NCX current, which in HF allows DADs to more readily trigger arrhythmogenic action potentials. Thus NCX and Na(+) are critical in systolic and diastolic function and arrhythmias. [Na(+)](i) is elevated in HF, which may limit SR unloading and provide some Ca(2+) influx during the HF action potential, thus limiting the depression of systolic function. High [Na(+)](i) in HF is due to enhanced Na(+) influx. Cellular Na(+)/K(+)-ATPase (NKA) function appears unaltered, despite reduced NKA expression. This dichotomy led us to test NKA regulation by phospholemman (PLM). We find that PLM regulates NKA in a manner analogous to phospholamban regulation of SR Ca(2+)-ATPase (i.e., inhibition that is relieved by PLM phosphorylation). We measured intermolecular FRET between PLM and NKA, which is reduced upon PLM phosphorylation. The lower expression level of more phosphorylated PLM in HF may explain the above dichotomy. Thus, altered Ca(2+) and Na(+) handling contributes to altered contractile function and arrhythmogenesis in HF.

Ca 2+ /Calmodulin–Dependent Protein Kinase Modulates Cardiac Ryanodine Receptor Phosphorylation and Sarcoplasmic Reticulum Ca 2+ Leak in Heart Failure

Abnormal release of Ca from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction and arrhythmogenesis in heart failure (HF). We previously demonstrated decreased Ca transient amplitude and SR Ca load associated with increased Na/Ca exchanger expression and enhanced diastolic SR Ca leak in an arrhythmogenic rabbit model of nonischemic HF. Here we assessed expression and phosphorylation status of key Ca handling proteins and measured SR Ca leak in control and HF rabbit myocytes. With HF, expression of RyR2 and FK-506 binding protein 12.6 (FKBP12.6) were reduced, whereas inositol trisphosphate receptor (type 2) and Ca/calmodulin-dependent protein kinase II (CaMKII) expression were increased 50% to 100%. The RyR2 complex included more CaMKII (which was more activated) but less calmodulin, FKBP12.6, and phosphatases 1 and 2A. The RyR2 was more highly phosphorylated by both protein kinase A (PKA) and CaMKII. Total phospholamban phosphorylation was unaltered, although it was reduced at the PKA site and increased at the CaMKII site. SR Ca leak in intact HF myocytes (which is higher than in control) was reduced by inhibition of CaMKII but was unaltered by PKA inhibition. CaMKII inhibition also increased SR Ca content in HF myocytes. Our results suggest that CaMKII-dependent phosphorylation of RyR2 is involved in enhanced SR diastolic Ca leak and reduced SR Ca load in HF, and may thus contribute to arrhythmias and contractile dysfunction in HF.

Abnormal intrastore calcium signaling in chronic heart failure.

Diminished Ca release from the sarcoplasmic reticulum (SR) is an important contributor to the impaired contractility of the failing heart. Despite extensive effort, the underlying causes of abnormal SR Ca release in heart failure (HF) remain unknown. We used a combination of simultaneous imaging of cytosolic and SR intraluminal [Ca] in isolated cardiomyocytes and recordings from single-ryanodine receptor (RyR) channels reconstituted into lipid bilayers to investigate alterations in intracellular Ca handling in an experimental model of chronic HF. We found that diastolic free [Ca] inside the SR was dramatically reduced because of a Ca leak across the SR membrane, mediated by spontaneous local release events (Ca sparks), in HF myocytes. Additionally, the magnitudes of intrastore Ca depletion signals during global and focal Ca release events were blunted, and [Ca]SR recovery was slowed after global but not focal Ca release in HF myocytes. At the single-RyR level, the sensitivity of RyRs to activation by luminal Ca was greatly enhanced, providing a molecular mechanism for the maintained potentiation of Ca sparks (and increased Ca leak) at reduced intra-SR [Ca] in HF. This work shows that the diminished SR Ca release characteristic of failing myocardium could be explained by increased sensitivity of RyRs to luminal Ca, leading to enhanced spark-mediated SR Ca leak and reduced intra-SR [Ca].