Ca Spark Frequency Research Articles

Ryanodine Receptor Sensitization Alters Local And Global Sarcoplasmic Reticulum Calcium Release Termination Threshold In Rabbit Ventricular Myocytes

Dynamic measurements of Ca within the sarcoplasmic reticulum ([Ca]SR) using low-affinity Ca indicators give critical insight into the role of [Ca]SR in Ca release termination. Here we used a low dose of caffeine to examine the effects of ryanodine receptor (RyR) sensitization on local and global SR Ca release in rabbit ventricular myocytes. In field stimulated myocytes (1 Hz), application of 250 μM caffeine caused an initial 44% increase in amplitude of action potential-induced [Ca]SR depletion. This resulted in unloading of the SR (27% decrease in steady state diastolic [Ca]SR) and a lowering of the termination level for global release (28% decrease in systolic [Ca]SR). A single stimulus protocol was used to examine the effects of caffeine on SR Ca release after varying [Ca]SR. At all [Ca]SR levels where release was observed, caffeine increased the [Ca]SR depletion amplitude by lowering the global termination level of release. We next studied the effects of caffeine on local SR Ca release events in permeabilized myocytes by simultaneously measuring cytosolic Ca sparks with associated local [Ca]SR depletions (Ca blinks). Under control conditions, Ca sparks terminated at a fixed [Ca]SR depletion threshold, irrespective of initial [Ca]SR. Application of 200 μM caffeine caused an immediate increase in Ca spark frequency (58%), amplitude (8%), duration (23%), and spatial width (13%), and decreased the Ca blink termination level below the control threshold level. Taken together, these data suggest that sensitization of the RyR produces an increase in SR Ca release by decreasing the [Ca]SR termination level for release at individual release junctions.

IP3 Receptor-mediated Ca Release Facilitates RyR-Ca Release To Cause Inotropy And Arrhythmogenicity In Mouse Ventricular Myocytes

Endothelin (ET-1)-induced IP3R-dependent Ca release in atrial myocytes is well established. ET-1-induced positive inotropy and arrhythmogenicity have also been shown in ventricular myocytes from rat and rabbit. Here we used transgenic mice overexpressing IP3R2 and IP3 sponge to study ET-1-induced inotropy and arrhythmogenicity in ventricular myocytes. 100 nM ET-1 (14 min) increased ventricular myocyte Ca transients (ΔCa) in IP3R2OX by 37% (0.5 Hz, fluo4 AM, n=11) vs WT and IP3 sponge ventricle (-6±10 %). ET-1 increased spontaneous calcium transient (SCT) frequency in IP3R2OX ventricles compared with WT and IP3 sponge (10±4 vs 1±1 SCT/min). Similar ΔCa results were obtained using the Ca indicator indo-1 (41±9 vs 19±12 % IP3R2OX vs WT, n=6). We found no differences between IP3R2OX (n=17) and WT (n=10) ventricular myocytes in control (0.5 Hz) ΔCa (350±70 vs 330±28 nM Cai) or SR Ca content as assessed with 10 mM caffeine (1368±273 vs 1385±78 nM Cai). Basal Ca spark frequency in saponin-permeabilized ventricular myocytes did not differ between IP3R2OX and WT (19±4 vs 16±4 sparks x s-1 x (100 μm)-1). However, direct application of 10 μM IP3 produced higher increase in Ca spark frequency on IP3R2OX (35 %, n=4) than WT (18 %). After 3 min of IP3 application SR Ca content was depleted to 80 % of control. These data suggest that IP3R-dependent inotropy is mediated via IP3R-dependent Ca release that facilitates Ca release through ryanodine receptors. This IP3-dependent inotropy is associated with spontaneous Ca release activity which may be arrhythmogenic (inducing action potentials). We conclude that IP3R expression and IP3-dependent Ca release play an important role in the generation of ventricular arrhythmias.

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.

Protein Phosphatase Inhibitor-1 Augments a Protein Kinase A-Dependent Increase in the Ca2+ Loading of the Sarcoplasmic Reticulum Without Changing Its Ca2+ Release

An increase in cytosolic protein phosphatases (PPs) de-phosphorylates phospholamban, decreasing the Ca(2+) uptake of the sarcoplasmic reticulum (SR). The effects of PP inhibitors on cellular Ca(2+) handling were investigated. Twitch Ca(2+) transients (CaTs) and cell shortening were measured in intact rat cardiac myocytes, and caffeine-induced Ca(2+) transients (CaffCaTs) and Ca(2+) sparks were studied in saponin-permeabilized cells. Calyculin A augmented isoproterenol-induced increases in CaTs and cell shortening without altering the diastolic [Ca(2+)](i) and twitch [Ca(2+)](i) decay. The protein kinase A catalytic subunit (PKA(cat)) increased the peak of CaffCaTs between 5 and 50 U/ml, and the addition of inhibitor-1 (I-1) augmented the increase. PKA(cat) increased Ca(2+) spark frequency and the addition of I-1 increased it further. PKA(cat) at 50 U/ml amplified the peak and prolonged the duration of Ca(2+) sparks, whereas the addition of I-1 did not alter them. An abrupt inhibition of SR Ca(2+) uptake following exposure to PKA(cat) caused a gradual decrease in Ca(2+) spark frequency, but the addition of I-1 did not accelerate the decline of Ca(2+) spark frequency or CaffCaTs. Inhibition of PPs augmented the inotropic effect of isoproterenol. Specific inhibition of PP1 could stimulate the Ca(2+) uptake of the SR with less significant effects on the Ca(2+) release.

Protein Kinase C-ε Regulates Local Calcium Signaling in Airway Smooth Muscle Cells

Protein kinase C (PKC) is known to regulate ryanodine receptor (RyR)-mediated local Ca(2+) signaling (Ca(2+) spark) in airway and vascular smooth muscle cells (SMCs), but its specific molecular mechanisms and functions still remain elusive. In this study, we reveal that, in airway SMCs, specific PKCepsilon peptide inhibitor and gene deletion significantly increased the frequency of Ca(2+) sparks, and decreased the amplitude of Ca(2+) sparks in the presence of xestospogin-C to eliminate functional inositol 1,4,5-triphosphate receptors. PKCepsilon activation with phorbol-12-myristate-13-acetate significantly decreased Ca(2+) spark frequency and increased Ca(2+) spark amplitude. The effect of PKCepsilon inhibition or activation on Ca(2+) sparks was completely lost in PKCepsilon(-/-) cells. PKCepsilon inhibition or PKCepsilon activation was unable to affect Ca(2+) sparks in RyR1(-/-) and RyR1(+/-) cells. Modification of RyR2 activity by FK506-binding protein 12.6 homozygous or RyR2 heterozygous gene deletion did not prevent the effect of PKCepsilon inhibition or activation. RyR3 homogenous gene deletion did not block the effect of PKCepsilon inhibition and activation, either. PKCepsilon inhibition promotes agonist-induced airway muscle contraction, whereas PKCepsilon activation produces an opposite effect. Taken together, these results indicate that PKCepsilon regulates Ca(2+) sparks by specifically interacting with RyR1, which plays an important role in the control of contractile responses in airway SMCs.

Defective domain-domain interactions within the ryanodine receptor as a critical cause of diastolic Ca2+ leak in failing hearts

A domain peptide (DP) matching the Gly(2460)-Pro(2495) region of the cardiac type-2 ryanodine receptor (RyR2), DPc10, is known to mimic channel dysfunction associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), owing to its interference in a normal interaction of the N-terminal (1-600) and central (2000-2500) domains (viz. domain unzipping). Using DPc10 and two other DPs harboring different mutation sites, we investigated the underlying mechanism of abnormal Ca(2+) cycling in failing hearts. Sarcoplasmic reticulum (SR) vesicles and cardiomyocytes were isolated from dog left ventricular muscles for Ca(2+) leak and spark assays. The RyR2 moiety of the SR was fluorescently labelled with methylcoumarin acetate (MCA) using DPs corresponding to the 163-195 and 4090-4123 regions of RyR2 (DP163-195 and DP4090-4123, respectively) as site-directed carriers. Both DPs mediated a specific MCA fluorescence labelling of RyR2. Addition of either DP to the MCA-labelled SR induced domain unzipping, as evidenced by an increased accessibility of the bound MCA to a large-size fluorescence quencher. Both SR Ca(2+) leak and Ca(2+) spark frequency (SpF) were markedly increased in failing cardiomyocytes. Upon introduction of DP163-195 or DP4090-4123 into normal SR or cardiomyocytes, both Ca(2+) leak and SpF increased to the levels comparable with those of failing myocytes. K201 (JTV519) suppressed all of the effects induced by DP163-195 (domain unzipping and increased Ca(2+) leak and SpF) or those in failing cardiomyocytes, but did not suppress the effects induced by DP4090-4123. Defective inter-domain interaction between N-terminal and central domains induces diastolic Ca(2+) leak, leading to heart failure and lethal arrhythmia. Mutation at the C-terminal region seen in CPVT does not seem to communicate with the aforementioned N-terminal and central inter-domain interaction, although spontaneous Ca(2+) leak is similarly induced.

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 >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<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.

Abstract 5302: Control of Reversible Phosphorylation of Ryanodine Receptor by MiR-1 : Mechanism of Cardiac Arrhythmia

MicroRNAs are small endogenous noncoding RNAs that regulate protein expression by hybridization to imprecise complementary sequences of target mRNAs. Changes in abundance of muscle-specific microRNA, miR-1 , have been implicated in cardiac disease, including arrhythmia and heart failure. However, the specific molecular targets and cellular mechanisms involved in the action of miR-1 in the heart are only beginning to emerge. In this study we investigated the effects of increased expression of miR-1 on excitation-contraction coupling and Ca cycling in adult rat ventricular myocytes by using methods of electrophysiology, confocal Ca imaging and quantitative immunoblotting. Adenoviral-mediated overexpressions of miR-1 in myocytes resulted in a marked increase in the amplitude of the inward Ca current and flatten cytosolic Ca transients voltage dependency. The frequency of spontaneous Ca sparks recorded in intact resting cells was enhanced, while the sarcoplasmic reticulum Ca content was reduced in miR-1 - overexpressing myocytes as compared with controls. In the presence of beta-adrenergic receptor agonist isoproterenol, rhythmically paced miR-1 -overexpressing myocytes exhibited spontaneous arrhythmogenic oscillations of intracellular Ca, events that occurred only rarely in control myocytes. The effects of miR-1 were completely reversed by the CaMKII inhibitor KN93. Immunological analysis with phospho-specific antibodies showed that while phosphorylation of phospholamban was not altered, miR-1 overexpression increased phosphorylation of the ryanodine receptor at Ser-2814 (CaMKII) but not at Ser-2808 (PKA). Overexpression of miR-1 was accompanied by a selective decrease in expression of the protein phosphatase PP2A regulatory subunit B56alpha involved in PP2A targeting to specialized subcellular domains. We conclude that miR-1 through translational inhibition of this mRNA target, causes CaMKII-dependent hyperphosphorylation of RyR2 via disrupting localization of PP2A activity to this channel, enhances RyR2 activity, and promotes arrhythmogenic SR Ca release. This mechanism could contribute to induction of arrhythmia in disease states accompanied by elevated miR-1 . This research has received full or partial funding support from the American Heart Association, AHA National Center.

Tricyclic antidepressant amitriptyline alters sarcoplasmic reticulum calcium handling in ventricular myocytes

Tricyclic antidepressants such as amitriptyline (AMT) have been reported to have adverse side effects on cardiac performance. AMT effects on Ca handling in ventricular myocytes, however, are not well understood. Therefore, we investigated AMT action on sarcoplasmic reticulum (SR) Ca release in ventricular myocytes, ryanodine receptor (RyR) activity, and Ca uptake by SR microsomes. In permeabilized myocytes, AMT transiently increased free luminal Ca concentration ([Ca]) followed by marked depletion. AMT (10 microM) caused a rapid and a transient increase of Ca spark frequency, followed by a significant suppression of spark activity. The latter was associated with a decrease of Ca spark amplitude and SR Ca load to 87 and 60%, respectively. AMT (10 microM) completely abolished propagation of spontaneous Ca waves. Higher concentrations of AMT (0.1-1 mM) evoked SR Ca release reminiscent of the effect of caffeine (20 mM) and caused almost complete depletion of SR Ca content. Studies on single calsequestrin-free RyR channels revealed that AMT increased the mean open time and open probability (Po) in a dose-dependent fashion (dissociation constant = 4.2 microM). High concentrations of AMT (> 25 microM) evoked frequent long openings with Po reaching very high levels (> 0.70). In studies with cardiac SR microsomes, AMT slowed the rate of ATP-dependent Ca uptake. We conclude that AMT affects SR Ca handling in ventricular myocytes by multiple mechanisms, including direct stimulation of RyRs and inhibition of SR Ca uptake. These effects could contribute to AMT cardiotoxicity.

Nitric Oxide Mediates Stretch-Induced Ca2+ Release via Activation of Phosphatidylinositol 3-Kinase-Akt Pathway in Smooth Muscle

BackgroundHollow smooth muscle organs such as the bladder undergo significant changes in wall tension associated with filling and distension, with attendant changes in muscle tone. Our previous study indicated that stretch induces Ca2+ release occurs in the form of Ca2+ sparks and Ca2+ waves in urinary bladder myocytes. While, the mechanism underlying stretch-induced Ca2+ release in smooth muscle is unknown.Methodology/Principal FindingsWe examined the transduction mechanism linking cell stretch to Ca2+ release. The probability and frequency of Ca2+ sparks induced by stretch were closely related to the extent of cell extension and the time that the stretch was maintained. Experiments in tissues and single myocytes indicated that mechanical stretch significantly increases the production of nitric oxide (NO) and the amplitude and duration of muscle contraction. Stretch- induced Ca2+ sparks and contractility increases were abrogated by the NO inhibitor L-NAME and were also absent in eNOS knockout mice. Furthermore, exposure of eNOS null mice to exogenously generated NO induced Ca2+ sparks. The soluble guanylyl cyclase inhibitor ODQ did not inhibit SICR, but this process was effectively blocked by the PI3 kinase inhibitors LY494002 and wortmannin; the phosphorylation of Akt and eNOS were up-regulated by 204±28.6% and 258±36.8% by stretch, respectively. Moreover, stretch significantly increased the eNOS protein expression level.Conclusions/SignificanceTaking together, these results suggest that stretch-induced Ca2+ release is NO dependent, resulting from the activation of PI3K/Akt pathway in smooth muscle.

Stimulation of P2 purinergic receptors increases calcium (Ca) spark frequency in ventricular cardiac myocytes (CMs) from transgenic (TG) mice with cardiac specific overexpression of the human P2X4 receptor

Small local increases in cytosolic Ca occur in resting CMs. These are referred to as Ca sparks, and have been determined to be unitary sarcoplasmic reticulum (SR) Ca release events. Studies of CMs from TG mice have shown that SR Ca load, Ca transient amplitude and contractility are increased by purinergic agonists. Our objective was to determine if the elevation in SR Ca load in TG CMs was related to alterations in Ca spark events recorded from TG CMs. CMs acutely isolated from the hearts of TG or wild type (WT) mice were loaded with Fluo‐4 AM. CMs were superfused with a modified Tyrode's solution (22°C), in the presence or absence of the P2 receptor agonist 2MeS‐ATP (3 μM). Ca sparks were recorded with a Zeiss LSM 510. Under basal conditions, Ca spark frequency in TG CMs did not differ from WT (1.49 ± 0.32 vs 0.83 ± 0.31 sparks/100μm/s ± SEM, 2‐way ANOVA, SNK posttest, p = 0.14, n = 24 & 25). The frequency of sparks in the presence of 2MeS‐ATP was significantly greater in TG than in WT CMs (3.10 ± 0.32 vs 1.10 ± 0.31, p < 0.01, n = 24 & 25). In 7 of 25 WT CMs P2 stimulation significantly increased Ca spark frequency, from 1.00 ± 0.28 to 2.81 ± 0.40 (p < 0.01) sparks/100μm/s. Ca spark properties such as amplitude, width, duration and time to peak remained unchanged in all conditions. Thus, the P2 agonist‐induced increase in SR Ca load in TG CMs is related to an increase in Ca spark frequency, but not to altered Ca spark properties. Research support from NIH‐NHLBI.

CaMKIIδ Isoforms Differentially Affect Calcium Handling but Similarly Regulate HDAC/MEF2 Transcriptional Responses

The delta(B) and delta(C) splice variants of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), which differ by the presence of a nuclear localization sequence, are both expressed in cardiomyocytes. We used transgenic (TG) mice and CaMKII expression in cardiomyocytes to test the hypothesis that the CaMKIIdelta(C) isoform regulates cytosolic Ca(2+) handling and the delta(B) isoform, which localizes to the nucleus, regulates gene transcription. Phosphorylation of CaMKII sites on the ryanodine receptor (RyR) and on phospholamban (PLB) were increased in CaMKIIdelta(C) TG. This was associated with markedly enhanced sarcoplasmic reticulum (SR) Ca(2+) spark frequency and decreased SR Ca(2+) content in cardiomyocytes. None of these parameters were altered in TG mice expressing the nuclear-targeted CaMKIIdelta(B). In contrast, cardiac expression of either CaMKIIdelta(B) or delta(C) induced transactivation of myocyte enhancer factor 2 (MEF2) gene expression and up-regulated hypertrophic marker genes. Studies using rat ventricular cardiomyocytes confirmed that CaMKIIdelta(B) and delta(C) both regulate MEF2-luciferase gene expression, increase histone deacetylase 4 (HDAC4) association with 14-3-3, and induce HDAC4 translocation from nucleus to cytoplasm, indicating that either isoform can stimulate HDAC4 phosphorylation. Finally, HDAC4 kinase activity was shown to be increased in cardiac homogenates from either CaMKIIdelta(B) or delta(C) TG mice. Thus CaMKIIdelta isoforms have similar effects on hypertrophic gene expression but disparate effects on Ca(2+) handling, suggesting distinct roles for CaMKIIdelta isoform activation in the pathogenesis of cardiac hypertrophy versus heart failure.

Abstract 1082: Cytoskeletal Protein 4.1R Affects Repolarisation And Regulates Calcium Handling In The Heart

The 4.1 proteins are a family of multifunctional adaptor proteins that organise signalling/transport/cell adhesion molecules. They are capable of interaction with the spectrin-actin network thereby conferring mechanical stability to the cell membrane, with several ion transporters associated to this macromolecular complex. Protein 4.1R is expressed in the heart and upregulated in deteriorating human heart failure. However, no data exists on the roles of protein 4.1R in myocardial regulation and function. In particular, it is unknown whether this protein can influence cardiac contractility and/or electrophysiology. 4.1R-deficient mice (KO) were studied using echocardiography and ECG monitoring with radiotelemetry. Left ventricular dimensions were increased in KO mice (LV diameter - Dia (cm): WT = 0.43 ± 0.01 [6] (mean ± SEM [n]); KO = 0.49 ± 0.01 [6]; p < 0.01) - Sys cm): WT = 0.29 ± 0.01 [6]; KO = 0.34 ± 0.02 [6]; p < 0.05) with no changes in ejection fraction and fractional shortening. ECG analysis revealed reduced heart rate (RR interval (ms): WT = 113±5 [6]; KO = 139 ± 601 [6]; p < 0.01) accompanied by prolonged QT interval (corrected (ms): WT = 46 ± 2 [6]; KO = 52 ± 1 [6]; p < 0.05). The action potential duration (APD) measured in isolated ventricular myocytes was prolonged in KO (APD 90% at 1Hz (ms) WT = 146 ± 20 [21]; KO = 231 ± 29 [28]; p < 0.05). Ca transients, elicited by 1Hz field-stimulation and measured using the fluorescent indicator indo-1, were larger (amplitude (ratio units r.u.): WT = 0.07 ± 0.006 [22]; KO = 0.1 ± 0.006 [33]; p<0.05) and slower to decay in the KO group (time to 50% decline (ms): WT = 78 ± 3 [22]; KO = 91 ± 3 [33]; p < 0.05). This was associated with increased SR Ca content, (20 mM caffeine-induced indo-1 transient amplitude (r.u.): WT = 0.09 ± 0.01 [7]; KO = 0.13 ± 0.01 [16]; p < 0.05) and increased frequency of Ca sparks, measured by confocal microscopy using Fluo-4 (sparks/100μm/s. WT = 0.52 ± 0.08 [111]; KO = 1.21 ± 0.14 [124]; p < 0.001). We conclude that protein 4.1 R affects repolarisation of cardiac myocytes. This may have a role in bringing about QT prolongation in KO mice. The prolonged APD, together with effects on Ca handling proteins, may alter Ca regulation and cell contractility. The specific mechanisms controlling these effects are under investigation.

Indirect coupling between Cav1.2 channels and ryanodine receptors to generate Ca2+ sparks in murine arterial smooth muscle cells

In arterial vascular smooth muscle cells (VSMCs), Ca(2+) sparks stimulate nearby Ca(2+)-activated K(+) (BK) channels that hyperpolarize the membrane and close L-type Ca(2+) channels. We tested the contribution of L-type Ca(v)1.2 channels to Ca(2+) spark regulation in tibial and cerebral artery VSMCs using VSMC-specific Ca(v)1.2 channel gene disruption in (SMAKO) mice and an approach based on Poisson statistical analysis of activation frequency and first latency of elementary events. Ca(v)1.2 channel gene inactivation reduced Ca(2+) spark frequency and amplitude by approximately 50% and approximately 80%, respectively. These effects were associated with lower global cytosolic Ca(2+) levels and reduced sarcoplasmic reticulum (SR) Ca(2+) load. Elevating cytosolic Ca(2+) levels reversed the effects completely. The activation frequency and first latency of elementary events in both wild-type and SMAKO VSMCs weakly reflected the voltage dependency of L-type channels. This study provides evidence that local and tight coupling between the Ca(v)1.2 channels and ryanodine receptors (RyRs) is not required to initiate Ca(2+) sparks. Instead, Ca(v)1.2 channels contribute to global cytosolic [Ca(2+)], which in turn influences luminal SR calcium and thus Ca(2+) sparks.

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.

Mechanisms of impaired calcium handling underlying subclinical diastolic dysfunction in diabetes.

Isolated diastolic dysfunction is found in almost half of asymptomatic patients with well-controlled diabetes and may precede diastolic heart failure. However, mechanisms that underlie diastolic dysfunction during diabetes are not well understood. We tested the hypothesis that isolated diastolic dysfunction is associated with impaired myocardial Ca(2+) handling during type 1 diabetes. Streptozotocin-induced diabetic rats were compared with age-matched placebo-treated rats. Global left ventricular myocardial performance and systolic function were preserved in diabetic animals. Diabetes-induced diastolic dysfunction was evident on Doppler flow imaging, based on the altered patterns of mitral inflow and pulmonary venous flows. In isolated ventricular myocytes, diabetes resulted in significant prolongation of action potential duration compared with controls, with afterdepolarizations occurring in diabetic myocytes (P < 0.05). Sustained outward K(+) current and peak outward component of the inward rectifier were reduced in diabetic myocytes, while transient outward current was increased. There was no significant change in L-type Ca(2+) current; however, Ca(2+) transient amplitude was reduced and transient decay was prolonged by 38% in diabetic compared with control myocytes (P < 0.05). Sarcoplasmic reticulum Ca(2+) load (estimated by measuring the integral of caffeine-evoked Na(+)-Ca(2+) exchanger current and Ca(2+) transient amplitudes) was reduced by approximately 50% in diabetic myocytes (P < 0.05). In permeabilized myocytes, Ca(2+) spark amplitude and frequency were reduced by 34 and 20%, respectively, in diabetic compared with control myocytes (P < 0.05). Sarco(endo)plasmic reticulum Ca(2+)-ATPase-2a protein levels were decreased during diabetes. These data suggest that in vitro impairment of Ca(2+) reuptake during myocyte relaxation contributes to in vivo diastolic dysfunction, with preserved global systolic function, during diabetes.

K201 modulates excitation–contraction coupling and spontaneous Ca2+ release in normal adult rabbit ventricular cardiomyocytes

The drug K201 (JTV-519) increases inotropy and suppresses arrhythmias in failing hearts, but the effects of K201 on normal hearts is unknown. The effect of K201 on excitation-contraction (E-C) coupling in normal myocardium was studied by using voltage-clamp and intracellular Ca(2+) measurements in intact cells. Sarcoplasmic reticulum (SR) function was assessed using permeabilised cardiomyocytes. Acute application of <1 micromol/L K201 had no significant effect on E-C coupling. K201 at 1 micromol/L decreased Ca(2+) transient amplitude (to 83+/-7%) without affecting I(Ca,L) or the SR Ca(2+) content. At 3 micromol/L K201 caused a larger reduction of Ca(2+) transient amplitude (to 60+/-7%) with accompanying reductions in I(Ca,L) amplitude (to 66+/-8%) and SR Ca(2+) content (74+/-9%). Spontaneous SR Ca(2+) release during diastole was induced by increasing intracellular [Ca(2+)]. At 1 micromol/L K201 reduced the frequency of spontaneous Ca(2+) release. The effect of K201 on SR-mediated Ca(2+) waves and Ca(2+) sparks was examined in beta-escin-permeabilised cardiomyocytes by confocal microscopy. K201 (1 micromol/L) reduced the frequency and velocity of SR Ca(2+) waves despite no change in SR Ca(2+) content. At 3 micromol/L K201 completely abolished Ca(2+) waves and reduced the SR Ca(2+) content (to approximately 73%). K201 at 1 micromol/L reduced Ca(2+) spark amplitude and frequency. Assays specific to SR Ca(2+)-ATPase and RyR2 activity indicated that K201 inhibited both SR Ca(2+) uptake and release. K201 modifies E-C coupling in normal cardiomyocytes. A dual inhibitory action on SERCA and RyR2 explains the ability of K201 to suppress spontaneous diastolic Ca(2+) release during Ca(2+) overload without significantly affecting Ca(2+) transient amplitude.

Does the adenosine A2A receptor stimulate the ryanodine receptor?

A recent paper in Cardiovascular Research from Hove-Madsen and colleagues [1] has investigated the role of the adenosine A2A receptor in human atrial myocytes. Elegant confocal images show that this receptor is localized at the level of the Z line in the myocyte. Their paper then proceeds to address an important physiological question; the role of this receptor. No effect was found on the amplitude or voltage-dependence of the L-type Ca current. However agonists of the adenosine A2A receptor increased the frequency of both Ca sparks and Ca waves, phenomena that result from Ca induced Ca release (CICR) from the sarcoplasmic reticulum (SR). There was no change in either the amount of Ca extruded from the cell during each Ca wave or the SR Ca content (as assessed by the integral of the caffeine evoked Na–Ca exchange, NCX, current). Based on these …

Two distinct signaling pathways for regulation of spontaneous local Ca2+ release by phospholipase C in airway smooth muscle cells

Spontaneous local Ca(2+) release events have been observed in airway smooth muscle cells (SMCs), but the underlying mechanisms are largely unknown. Considering that each type of SMCs may use its own mechanisms to regulate local Ca(2+) release events, we sought to investigate the signaling pathway for spontaneous local Ca(2+) release events in freshly isolated mouse airway SMCs using a laser scanning confocal microscope. Application of ryanodine to block ryanodine receptors (RyRs) abolished spontaneous local Ca(2+) release events, indicating that these events are RyR-mediated Ca(2+) sparks. Inhibition of inositol 1,4,5-triphosphate receptors (IP(3)Rs) by 2-aminoethoxydiphenyl-borate (2-APB) or xestospongin-C significantly blocked the activity of Ca(2+) sparks. Under patch clamp conditions, dialysis of IP(3) to activate IP(3)Rs increased the activity of local Ca(2+) events in control cells but had no effect in ryanodine-pretreated cells. The RyR agonist caffeine augmented the frequency of Ca(2+) sparks in cells pretreated with and without 2-APB or xestospongin-C. The specific phospholipase C (PLC) blocker U73122 decreased the activity of Ca(2+) sparks and prevented xestospongin-C from producing the inhibitory effect. The protein kinase C (PKC) activator 1-oleoyl-2-acetyl-glycerol or phorbol-12-myristate-13-acetate inhibited Ca(2+) sparks, whereas the PKC inhibitor chelerythrine, PKCvarepsilon inhibitory peptide, or PKCvarepsilon gene knockout produced an opposite effect. Collectively, our data suggest that the basal activation of PLC regulates the activity of RyR-mediated, spontaneous Ca(2+) sparks in airway SMCs through two distinct signaling pathways: a positive IP(3)-IP(3)R pathway and a negative diacylglycerol-PKCvarepsilon pathway.

Cytosolic energy reserves determine the effect of glycolytic sugar phosphates on sarcoplasmic reticulum Ca2+ release in cat ventricular myocytes

Localization of glycolytic enzymes in close proximity to Ca(2+) transport systems of the sarcoplasmic reticulum (SR) in cardiac cells suggests an important functional role for glycolysis in intracellular [Ca(2+)] regulation and, consequently, excitation-contraction coupling. Here, we investigated the mechanisms of regulation of SR Ca(2+) release by glycolytic sugar phosphate intermediates in cat ventricular myocytes. Experiments with permeabilized myocytes revealed that with normal cytosolic energy reserves (mm: ATP 5, ADP 0.01, phosphocreatine (CrP) 10) fructose-1,6-bisphosphate (FBP; 1 mm) and fructose-6-phosphate (F6P; 1 mm) caused a transient increase of Ca(2+) spark frequency by 62 and 42%, respectively. This effect of sugar phosphates was associated with a 13% decrease in SR Ca(2+) load. Pretreatment of the cells with an inhibitor of glycolysis, iodoacetate (IAA; 0.5 mm), did not prevent the effects of FBP and F6P on Ca(2+) sparks. Recording of single ryanodine receptor (RyR) channel activity indicated that FBP and F6P significantly increased RyR open probability. Reduction of cytosolic energy reserves decreased Ca(2+) spark activity. Increasing [ADP] to 0.4 mm or removal of CrP ([ATP] was kept constant) caused a slowly developing decrease of Ca(2+) spark frequency by 29 and 42%, respectively. Changing [ADP] and [CrP] simultaneously decreased Ca(2+) spark frequency by 66%. This inhibition of Ca(2+) sparks was associated with a 40% decrease in SR Ca(2+) load. The subsequent addition of FBP (1 mm) partially restored Ca(2+) spark frequency and SR Ca(2+) load. This recovery of Ca(2+) sparks was blocked completely by IAA. These data suggest that at physiological ATP, ADP and CrP levels accumulation of sugar phosphates from glycolysis can stimulate SR Ca(2+) release. This effect does not require the activity of downstream glycolytic enzymes, but rather is the result of direct activation of RyRs. However, under conditions associated with depletion of cellular energy reserves (e.g. myocardial ischaemia), ATP generated from glycolysis may play an important role in maintaining myocardial Ca(2+) homeostasis by improving SR Ca(2+) uptake.