Reduced Sarcoplasmic Reticulum Ca Research Articles

Reactive Oxygen Species–Activated Ca/Calmodulin Kinase IIδ Is Required for Late I Na Augmentation Leading to Cellular Na and Ca Overload

In heart failure Ca/calmodulin kinase (CaMK)II expression and reactive oxygen species (ROS) are increased. Both ROS and CaMKII can increase late I(Na) leading to intracellular Na accumulation and arrhythmias. It has been shown that ROS can activate CaMKII via oxidation. We tested whether CaMKIIδ is required for ROS-dependent late I(Na) regulation and whether ROS-induced Ca released from the sarcoplasmic reticulum (SR) is involved. 40 μmol/L H(2)O(2) significantly increased CaMKII oxidation and autophosphorylation in permeabilized rabbit cardiomyocytes. Without free [Ca](i) (5 mmol/L BAPTA/1 mmol/L Br(2)-BAPTA) or after SR depletion (caffeine 10 mmol/L, thapsigargin 5 μmol/L), the H(2)O(2)-dependent CaMKII oxidation and autophosphorylation was abolished. H(2)O(2) significantly increased SR Ca spark frequency (confocal microscopy) but reduced SR Ca load. In wild-type (WT) mouse myocytes, H(2)O(2) increased late I(Na) (whole cell patch-clamp). This increase was abolished in CaMKIIδ(-/-) myocytes. H(2)O(2)-induced [Na](i) and [Ca](i) accumulation (SBFI [sodium-binding benzofuran isophthalate] and Indo-1 epifluorescence) was significantly slowed in CaMKIIδ(-/-) myocytes (versus WT). CaMKIIδ(-/-) myocytes developed significantly less H(2)O(2)-induced arrhythmias and were more resistant to hypercontracture. Opposite results (increased late I(Na), [Na](i) and [Ca](i) accumulation) were obtained by overexpression of CaMKIIδ in rabbit myocytes (adenoviral gene transfer) reversible with CaMKII inhibition (10 μmol/L KN93 or 0.1 μmol/L AIP [autocamtide 2-related inhibitory peptide]). Free [Ca](i) and a functional SR are required for ROS activation of CaMKII. ROS-activated CaMKIIδ enhances late I(Na), which may lead to cellular Na and Ca overload. This may be of relevance in hear failure, where enhanced ROS production meets increased CaMKII expression.

A small leak may sink a great ship but what does it do to the heart?

A small leak may sink a great ship but what does it do to the heart?

Role of chronic ryanodine receptor phosphorylation in heart failure and β-adrenergic receptor blockade in mice

Increased sarcoplasmic reticulum (SR) Ca2+ leak via the cardiac ryanodine receptor/calcium release channel (RyR2) is thought to play a role in heart failure (HF) progression. Inhibition of this leak is an emerging therapeutic strategy. To explore the role of chronic PKA phosphorylation of RyR2 in HF pathogenesis and treatment, we generated a knockin mouse with aspartic acid replacing serine 2808 (mice are referred to herein as RyR2-S2808D+/+ mice). This mutation mimics constitutive PKA hyperphosphorylation of RyR2, which causes depletion of the stabilizing subunit FKBP12.6 (also known as calstabin2), resulting in leaky RyR2. RyR2-S2808D+/+ mice developed age-dependent cardiomyopathy, elevated RyR2 oxidation and nitrosylation, reduced SR Ca2+ store content, and increased diastolic SR Ca2+ leak. After myocardial infarction, RyR2-S2808D+/+ mice exhibited increased mortality compared with WT littermates. Treatment with S107, a 1,4-benzothiazepine derivative that stabilizes RyR2-calstabin2 interactions, inhibited the RyR2-mediated diastolic SR Ca2+ leak and reduced HF progression in WT and RyR2-S2808D+/+ mice. In contrast, β-adrenergic receptor blockers improved cardiac function in WT but not in RyR2-S2808D+/+ mice.Thus, chronic PKA hyperphosphorylation of RyR2 results in a diastolic leak that causes cardiac dysfunction. Reversing PKA hyperphosphorylation of RyR2 is an important mechanism underlying the therapeutic action of β-blocker therapy in HF.

Inhibition of Elevated Ca 2+ /Calmodulin-Dependent Protein Kinase II Improves Contractility in Human Failing Myocardium

Heart failure (HF) is known to be associated with increased Ca(2+)/calmodulin-dependent protein kinase (CaMK)II expression and activity. There is still controversial discussion about the functional role of CaMKII in HF. Moreover, CaMKII inhibition has never been investigated in human myocardium. We sought to investigate detailed CaMKIIδ expression in end-stage failing human hearts (dilated and ischemic cardiomyopathy) and the functional effects of CaMKII inhibition on contractility. Expression analysis revealed that CaMKIIδ, both cytosolic δ(C) and nuclear δ(B) splice variants, were significantly increased in both right and left ventricles from patients with dilated or ischemic cardiomyopathy versus nonfailing. Experiments with isometrically twitching trabeculae revealed significantly improved force frequency relationships in the presence of CaMKII inhibitors (KN-93 and AIP). Increased postrest twitches after CaMKII inhibition indicated an improved sarcoplasmic reticulum (SR) Ca(2+) loading. This was confirmed in isolated myocytes by a reduced SR Ca(2+) spark frequency and hence SR Ca(2+) leak, resulting in increased SR Ca(2+) load when inhibiting CaMKII. Ryanodine receptor type 2 phosphorylation at Ser2815, which is known to be phosphorylated by CaMKII thereby contributing to SR Ca(2+) leak, was found to be markedly reduced in KN-93-treated trabeculae. Interestingly, CaMKII inhibition did not influence contractility in nonfailing sheep trabeculae. The present study shows for the first time that CaMKII inhibition acutely improves contractility in human HF where CaMKIIδ expression is increased. The mechanism proposed consists of a reduced SR Ca(2+) leak and consequently increased SR Ca(2+) load. Thus, CaMKII inhibition appears to be a possible therapeutic option for patients with HF and merits further investigation.

Mitochondrial uncoupling downregulates calsequestrin expression and reduces SR Ca2+ stores in cardiomyocytes

Mitochondrial cardiomyopathy is associated with deleterious remodelling of cardiomyocyte Ca(2+) signalling that is partly due to the suppressed expression of the sarcoplasmic reticulum (SR) Ca(2+) buffer calsequestrin (CASQ2). This study was aimed at determining whether CASQ2 downregulation is directly caused by impaired mitochondrial function. Mitochondrial stress was induced in cultured neonatal rat cardiomyocytes by means of the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP). Ca(2+) transients and reactive oxygen species (ROS) were measured by confocal microscopy using the indicators fluo-4 and MitoSOX red, respectively. Mitochondrial stress led to concentration-dependent downregulation of calsequestrin (CASQ2) and changes in the Ca(2+) signals of the cardiomyocytes that were accompanied by reduction in SR Ca(2+) content and amplitude and duration of Ca(2+) sparks. Caspase 3, p38, and p53 inhibitors had no effect on FCCP-induced CASQ2 downregulation; however, it was attenuated by the ROS scavenger N-acetylcysteine (NAC). Importantly, NAC not only decreased FCCP-induced ROS production, but it also restored the Ca(2+) signals, SR Ca(2+) content, and Ca(2+) spark properties to control levels. Mitochondrial uncoupling results in fast transcriptional changes in CASQ2 expression that manifest as compromised Ca(2+) signalling, and these changes can be prevented by ROS scavengers. As impaired mitochondrial function has been implicated in several cardiac pathologies as well as in normal ageing, the mechanisms described here might be involved in a wide spectrum of cardiac conditions.

Inotropic Response of Cardiac Ventricular Myocytes to β-Adrenergic Stimulation With Isoproterenol Exhibits Diurnal Variation

Although >10% of cardiac gene expression displays diurnal variations, little is known of their impact on excitation-contraction coupling. To determine whether the time of day affects excitation-contraction coupling in rat ventricles. Left ventricular myocytes were isolated from rat hearts at 2 opposing time points, corresponding to the animals resting or active periods. Basal contraction and [Ca(2+)](i) was significantly greater in myocytes isolated during the resting versus active periods (cell shortening 12.4+/-0.3 versus 11.0+/-0.2%; P<0.05 and systolic [Ca(2+)](i) 422+/-12 versus 341+/-9 nmol/L; P<0.01. This corresponded to a greater sarcoplasmic reticulum (SR) Ca(2+) load (672+/-20 versus 551+/-13 nmol/L P<0.001). The increase in systolic [Ca(2+)](i) in response to isoproterenol (>3 nmol/L) was also significantly greater in resting versus active period myocytes, reflecting a greater SR Ca(2+) load at this time. This diurnal variation in response of Ca(2+)-homeostasis to isoproterenol translated to a greater incidence of arrhythmic activity in resting period myocytes. Inhibition of neuronal NO synthase during stimulation with isoproterenol, further increased systolic [Ca(2+)](i) and the percentage of arrhythmic myocytes, but this effect was significantly greater in active period versus resting period myocytes. Quantitative RT-PCR analysis revealed a 2.65-fold increase in neuronal NO synthase mRNA levels in active over resting period myocytes (P<0.05). The threshold for the development of arrhythmic activity in response to isoproterenol is higher during the active period of the rat. We suggest this reflects a reduction in SR Ca(2+) loading and a diurnal variation in neuronal NO synthase signaling.

A Leakage Leads to Failure

Cytosolic-free calcium (Ca2+) is a multifunctional intracellular messenger that regulates many different cellular processes in cardiac myocytes.1 A transient rise in intracellular free Ca2+ concentrations ([Ca2+]i) during excitation-contraction (E-C) coupling is required for initiating contraction of cardiac muscle. Membrane depolarization activates voltage-gated L-type Ca2+ channels within the transverse tubules (invaginations of the sarcolemma), and extracellular Ca2+ enters cardiac myocytes. The increased [Ca2+]i in the junctional space between transverse tubules and the sarcoplasmic reticulum (SR) triggers the Ca2+-induced Ca2+ release from the SR via ryanodine receptor (RyR2)/intracellular Ca2+ release channels. The resultant transient rise in global [Ca2+]i activates the myofilaments to produce cardiac contraction. Myocyte relaxation occurs when [Ca2+]i levels decline quickly through transport by the Ca2+ recycling proteins, such as SR Ca2+-ATPase (SERCA), which pumps Ca2+ back into SR, and the Na+/Ca2+ exchanger, which extrudes Ca2+ out of myocytes.1 In addition to its pivotal role in cardiac E-C coupling, [Ca2+]i is also a critical regulator of multiple signaling transduction pathways, including activation of protein kinases or protein phosphatases and modulation of gene transcription and expression (Figure).2 Figure. Remodeling of the Ca2+-dependent signalosome during cardiac hypertrophy and HF. Under conditions of persistent pathological stress on the heart, SR Ca2+ content is reduced and [Ca2+]i is increased. Both reduced Ca2+ pumping by SERCA and increased SR Ca2+ leak via RyR2 may be responsible for the reduced SR Ca2+ content and increased [Ca2+]i.3 Other possible sources of the hypertrophy-associated increase in [Ca2+]i may be from increases in influx of extracellular Ca …

Stress synchronizes calcium release and promotes SR calcium leak

Stress synchronizes calcium release and promotes SR calcium leak

Sodium Current-Induced Release of Calcium from the Sarcoplasmic Reticulum in Rabbit Ventricular Myocytes

The hypothesis that Na current (INa) can induce release of Ca from the sarcoplasmic reticulum (SR) by activating reverse Na-Ca exchange (NCX) has been debated since 1990. We tested this hypothesis with epi-fluorescence imaging of adult rabbit ventricular myocytes loaded with the Ca indicator fluo-4. Ca release was triggered with an action potential clamp with and without an initial voltage ramp from −80 to −40 mV, for a duration of 1.5s. We confirmed that this protocol selectively blocked INa without altering Ca influx through L-type Ca channels (LCCs) and SR Ca load. With 0 mM Na in the pipette (to reduce intracellular Na), inactivating INa reduced SR Ca release flux by 27%±4% (n=9). With 5 mM Na in the pipette, the Ca release upon inactivation of INa was reduced by 33%±5% (n=4). We suggest that increased activation of reverse NCX by increased intracellular Na concentration mainly produced by INa explains these findings. These conclusions are in agreement with studies on normal and NCX knockout mice, which show that INa affects SR Ca release only in normal, but not in NCX knockout mice. In similar experiments, we applied 100 nM TTX to selectively block brain isoforms of Na channels. In the presence of TTX, the SR Ca release flux was reduced by 35%±3% (n=6). This effect of INa on Ca release can be explained by early reverse NCX, activated by TTX sensitive INa, which could prime the dyadic cleft with Ca. Furthermore, the results can be explained if INa activation of NCX, and subsequent priming of the dyadic cleft with Ca, increases the coupling fidelity between LCCs and ryanodine receptors within a couplon. Thus the presence of INa increases the likelihood that couplons are activated.

Reduced SERCA2 abundance decreases the propensity for Ca2+ wave development in ventricular myocytes

To describe the overall role of reduced sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) for Ca(2+) wave development. SERCA2 knockout [Serca2(flox/flox) Tg(alphaMHC-MerCreMer); KO] mice allowing inducible cardiomyocyte-specific disruption of the Serca2 gene in adult mice were compared with Serca(flox/flox) (FF) control mice. Six days after Serca2 gene disruption, SERCA2 protein abundance was reduced by 53% in KO compared with FF, whereas SERCA2 activity in field-stimulated, Fluo-5F AM-loaded cells was reduced by 42%. Baseline Ca(2+) content of the sarcoplasmic reticulum (SR) and Ca(2+) transient amplitude and rate constant of decay measured in whole-cell voltage-clamped cells were decreased in KO to 75, 81, and 69% of FF values. Ca(2+) waves developed in only 31% of KO cardiomyocytes compared with 57% of FF when external Ca(2+) was raised (10 mM), although SR Ca(2+) content needed for waves to develop was 79% of FF values. In addition, waves propagated at a 15% lower velocity in KO cells. Ventricular extrasystoles (VES) occurred with lower frequency in SERCA2 KO mice (KO: 3 +/- 1 VES/h vs. FF: 8 +/- 1 VES/h) (P < 0.05 for all results). Reduced SERCA2 abundance resulted in decreased amplitude and decay rate of Ca(2+) transients, reduced SR Ca(2+) content, and decreased propensity for Ca(2+) wave development.

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.

β-Adrenergic modulation of arrhythmogenesis and identification of targeted sites of antiarrhythmic therapy in Timothy (LQT8) syndrome: a theoretical study

Timothy syndrome (TS) is a malignant form of congenital long QT syndrome with a mode of arrhythmia onset often triggered by enhanced sympathetic tone. We sought to explore mechanisms by which beta-adrenergic stimulation (BAS) modulates arrhythmogenesis and to identify potential targeted sites of antiarrhythmic therapy in TS. Using a dynamic Luo-Rudy ventricular myocyte model incorporated with detailed intracellular Ca(2+) cycling, along with its one-dimensional multicellular strand, we simulated various clinical scenarios of TS, with stepwise increase in the percentage of G406R Ca(v)1.2 channels from 0 to 11.5 and 23%, and to 38.5 and 77%, respectively, for heterozygous and homozygous states of TS1 and TS2. Progressive prolongation of action potential duration (APD) and QT interval, accompanied by amplification of transmural dispersion of repolarization, steepening of APD restitution, induction of delayed afterdepolariztions (DADs), and both DAD and phase 3 early afterdepolariztion-mediated triggered activities, correlated well with the extent of G406R Ca(v)1.2 channel mutation. BAS amplified transmural dispersion of repolarization, steepened APD restitution, and facilitated inducibility of DAD-mediated triggered activity. Systematic analysis of intracellular Ca(2+) cycling revealed that sarcoplasmic reticulum Ca(2+) ATPase (uptake current) played an essential role in BAS-induced facilitation of DAD-mediated triggered activity and, in addition to L-type calcium current, it could be an effective site of antiarrhythmic therapy under the influence of BAS. Thus G406R Ca(v)1.2 channel mutation confers not only a trigger, but also a substrate for lethal ventricular arrhythmias, which can be exaggerated by BAS. It is suggested that, besides beta-adrenergic blockers and L-type calcium current channel blockers, an agent aimed at reduction of sarcoplasmic reticulum Ca(2+) ATPase uptake current may provide additional antiarrhythmic effect in patients with TS.

Ultrastructural and Functional Remodeling of the Coupling Between Ca 2+ Influx and Sarcoplasmic Reticulum Ca 2+ Release in Right Atrial Myocytes From Experimental Persistent Atrial Fibrillation

Persistent atrial fibrillation (AF) has been associated with structural and electric remodeling and reduced contractile function. To unravel mechanisms underlying reduced sarcoplasmic reticulum (SR) Ca(2+) release in persistent AF. We studied cell shortening, membrane currents, and [Ca(2+)](i) in right atrial myocytes isolated from sheep with persistent AF (duration 129+/-39 days, N=16), compared to matched control animals (N=21). T-tubule density, ryanodine receptor (RyR) distribution, and local [Ca(2+)](i) transients were examined in confocal imaging. Myocyte shortening and underlying [Ca(2+)](i) transients were profoundly reduced in AF (by 54.8% and 62%, P<0.01). This reduced cell shortening could be corrected by increasing [Ca(2+)](i). SR Ca(2+) content was not different. Calculated fractional SR Ca(2+) release was reduced in AF (by 20.6%, P<0.05). Peak Ca(2+) current density was modestly decreased (by 23.9%, P<0.01). T-tubules were present in the control atrial myocytes at low density and strongly reduced in AF (by 45%, P<0.01), whereas the regular distribution of RyR was unchanged. Synchrony of SR Ca(2+) release in AF was significantly reduced with increased areas of delayed Ca(2+) release. Propagation between RyR was unaffected but Ca(2+) release at subsarcolemmal sites was reduced. Rate of Ca(2+) extrusion by Na(+)/Ca(2+) exchanger was increased. In persistent AF, reduced SR Ca(2+) release despite preserved SR Ca(2+) content is a major factor in contractile dysfunction. Fewer Ca(2+) channel-RyR couplings and reduced efficiency of the coupling at subsarcolemmal sites, possibly related to increased Na(+)/Ca(2+) exchanger, underlie the reduction in Ca(2+) release.

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.

Silencing genes of sarcoplasmic reticulum proteins clarifies their roles in excitation–contraction coupling

Silencing genes of sarcoplasmic reticulum proteins clarifies their roles in excitation–contraction coupling

Loss of Intracellular and Intercellular Synchrony of Calcium Release in Systolic Heart Failure

Normal contraction of cardiac muscle requires a coordinated cellular mechanical response to the electric pacemaker signal that begins in the sinoatrial node and spreads through the conduction system and ultimately across the ventricular myocardium via gap junctions. In ventricular myocytes, depolarization by the action potential causes clusters of L-type calcium channels (LCCs) to open, allowing a small amount of Ca to enter the diadic cleft space between the sarcolemma and the sarcoplasmic reticulum (SR). When the Ca concentration in this space becomes sufficiently high to gate ryanodine receptors (RyRs), they open and release SR Ca into the cytoplasm, so that it can bind to and activate myofilaments, resulting in force development. Each cluster of LCCs on the sarcolemma, its opposing cluster of RyRs across the 10- to 12-nm diameter diadic cleft space, and associated SR regulatory proteins is a couplon.1 According to the local control theory of cardiac excitation-contraction (EC) coupling, couplons are activated independently. Independent activation of couplons depends on the extent of LCC activation, providing a mechanism for regulation of contractile force by stochastic recruitment of couplons.2 In systolic heart failure, numerous cellular defects in EC coupling and Ca regulation have been identified that contribute to contractile dysfunction (and ventricular arrhythmias), although there is a host of other major abnormalities leading to loss of force development, including fibrosis,3 defective energy metabolism,4 pH changes,5 heterogeneous conductance across connexin hemichannels,6 and defects in sarcomeric proteins.7 Thus, it is important to keep in mind that no single defect can explain the contractile dysfunction of systolic failure. Article see p 223 Recent studies of defective EC coupling in heart failure have addressed not only reductions in the extent and kinetics of Ca released in response to the action potential (ie, reduced recruitment of couplons and …

Ca2+‐calmodulin can activate and inactivate cardiac ryanodine receptors

Ca(2+)-calmodulin (Ca(2+)CaM) is widely accepted as an inhibitor of cardiac ryanodine receptors (RyR2); however, the effects of physiologically relevant CaM concentrations have not been fully investigated. We investigated the effects of low concentrations of Ca(2+)CaM (50-100 nmol.L(-1)) on the gating of native sheep RyR2, reconstituted into bilayers. Suramin displaces CaM from RyR2 and we have used a gel-shift assay to provide evidence of the mechanism underlying this effect. Finally, using suramin to displace endogenous CaM from RyR2 in permeabilized cardiac cells, we have investigated the effects of 50 nmol.L(-1) CaM on sarcoplasmic reticulum (SR) Ca(2+)-release. Ca(2+)CaM activated or inhibited single RyR2, but activation was much more likely at low (50-100 nmol.L(-1)) concentrations. Also, suramin displaced CaM from a peptide of the CaM binding domain of RyR2, indicating that, like the skeletal isoform (RyR1), suramin directly competes with CaM for its binding site on the channel. Pre-treatment of rat permeabilized ventricular myocytes with suramin to displace CaM, followed by addition of 50 nmol x L(-1) CaM to the mock cytoplasmic solution caused an increase in the frequency of spontaneous Ca(2+)-release events. Application of caffeine demonstrated that 50 nmol x L(-1) CaM reduced SR Ca(2+) content. We describe for the first time how Ca(2+)CaM is capable, not only of inactivating, but also of activating RyR2 channels in bilayers in a CaM kinase II-independent manner. Similarly, in cardiac cells, CaM stimulates SR Ca(2+)-release and the use of caffeine suggests that this is a RyR2-mediated effect.

Flecainide Inhibits Cardiac Ryanodine Channels And Spontaneous Sarcoplasmic Reticulum Calcium Release In Casq2 Null Myocytes

Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic syndrome due to cardiac Ca2+ release channel (RYR2) or cardiac calsequestrin (CASQ2) mutations. VT is caused by spontaneous Ca2+-release from the sarcoplasmic reticulum (SR) that generates after-depolarizations and triggered beats during catecholamine surge. We recently found that flecainide, a class 1c Na+ channel blocker, suppressed ventricular arrhythmia in Casq2 null (Casq−/−) mice, a model of CPVT. Here, we investigated the effect of flecainide on on cardiac Ca2+ handling in Casq2−/− myocytes loaded with Fura-2 AM, and on sheep RyR2 RyR2 channels reconstituted in lipid bilayers.Results: In isoproterenol-stimulated Casq2−/− myocytes, flecainide (6 μmol/l) reduced triggered beats by over 70% (p<0.001). Unexpected for a Na+ channel blocker, flecainide also reduced SR Ca2+ leak (Ca2+ fluorescence ratio: vehicle: 0.12±0.01 vs. flecainide: 0.08±0.01, n=54 per group, p=0.02) and suppressed the rate of spontaneous Ca2+ releases (SCRs) from the SR (SCRs/min: vehicle: 48±5 vs. flecainide: 29±5, n=45 per group, p=0.006), suggesting flecainide directly inhibits SR Ca2+ release. Lipid bilayer experiments confirmed a direct action of flecainide on RyR2 SR Ca2+ release channels: Flecainide induced brief partial closures of channels to a substate with a conductance equal to 20% of the fully open state. On average, flecainide as low as 5 μmol/L caused a 4-fold increase in the frequency of closed events and caused a significant reduction in the open probability from control levels. The effect of flecainide was concentration dependent (IC50 ∼ 50 μmol/l) and fully-reversible upon washout. Flecainide also inhibited RyR2 channels activated by high luminal Ca2+.Conclusion: We report a heretofore unrecognized inhibitory action of flecainide on RyR2 channels, which together with flecainide's inhibition of Na+ channels may explain flecainide's effectiveness in preventing CPVT.Supported by R01-HL88635.

Ca 2+ Signaling Domains Responsible For Cardiac Hypertrophy and Arrhythmias

See related articles, pages 514–521 and 522–530 Ca2+ activates and regulates multiple processes in every cell type. In the mammalian heart, cyclic fluctuations in cytosolic [Ca2+] induce and regulate the strength of cardiac contraction (termed “contractile” [Ca2+]). In addition, changes in Ca2+ appear to be centrally involved in normal and pathological signaling (termed “signaling” [Ca2+]) that regulates myocyte growth, hypertrophy, apoptosis, and necrosis.1 Whether or not contractile and signaling [Ca2+] are derived from common or distinct sources and are constrained to unique cellular microdomains is not established.2 What is clear is that cardiovascular diseases including hypertension and myocardial infarction are associated with alterations in contractile and possibly signaling [Ca2+] that are centrally involved in pathological cardiac hypertrophy, heart failure progression,1 and lethal cardiac arrhythmias.3 Defining the sources of signaling Ca2+ involved in the induction of pathological hypertrophy and the bases of dysregulated contractile [Ca2+] in cardiovascular disease should identify novel ways to treat heart disease. In this issue of Circulation Research , 2 independent reports address fundamental aspects of alterations in signaling and contractile [Ca2+]. Chiang et al4 have studied the idea that Ca2+ influx through voltage operated α1H (CaV3.2) T-type Ca2+ channels (TTCCs) is the source of the signaling [Ca2+] that activates the calcineurin (Cn)-NFAT (nuclear factor of activated T cells) signaling cascade and induces pathological cardiac hypertrophy in pressure overload. In a separate report, Terentyev et al5 explore the idea that microRNA (miR)-1, a muscle-specific microRNA that increases in abundance in cardiac disease,6 causes dysregulated contractile [Ca2+] and induces single cell arrhythmias. These 2 reports are provocative and, if independently confirmed, will have identified novel …

Abstract 1534: Structural and Functional Remodeling of the Coupling between Sarcolemmal Ca 2+ Channels and the Ryanodine Receptor in Atrial Fibrillation

Background: Permanent atrial fibrillation (AF) in humans has been associated with structural and electrical remodelling. Data on contractile remodelling and control of sarcoplasmic reticulum (SR) Ca 2+ release via the ryanodine receptor (RyR) in permanent AF are however limited. Methods : Ewes were atrially paced at 600 bmp for a median of 23 weeks resulting in permanent AF (mean of 89.6±6 days, N=13) and compared to matched, non-instrumented control animals (CTRL, N=17). Atrial myocytes were isolated and cell shortening (field stimulation), membrane currents (whole cell voltage clamp) and [Ca 2+ ] i (Fluo-3) were measured. Protein expression was analyzed by immunoblotting. T-tubule density was quantified from confocal Z-stack images of cells stained with di-8-ANEPPS. Data are shown as mean±SEM of at least 17 cells and 5 animals. Results: Myocyte shortening and underlying [Ca 2+ ] i transients were profoundly reduced in AF (by 54.8% and 62%, p&lt;0.01). This reduced cell shortening could be corrected by increasing [Ca 2+ ] i during caffeine-induced Ca 2+ release from the SR (L/L 0 9.7±0.5% in AF vs. 10.8±0.5% in CTRL). SR Ca 2+ content (integrated Na + /Ca 2+ exchange current during caffeine application) was not different (CTRL 1.9±0.3 pC/pF vs. AF 2±0.4 pC/pF), but calculated fractional SR Ca 2+ release during a depolarizing step to +10 mV was reduced in AF (by 20.6%, p&lt;0.05). Peak Ca 2+ current density was modestly decreased (at &lt;10 mV by 23.9%, p&lt;0.01). T-tubules were present in the CTRL atrial myocytes though their density was much lower than in ventricular cells (11.4% vs. 20.6%, p&lt;0.05). T-tubule density was robustly reduced in AF vs. CTRL (by 45%, p&lt;0.01) with a reduction of myocyte surface:volume ratio (by 26%, p&lt;0.01). The organization of RyR was apparently unchanged but protein expression was reduced (by 18.6%, p&lt;0.05). Conclusion: In permanent AF, reduced SR Ca 2+ release is a major factor in the reduced cell contraction. Loss of T-tubules contributes to uncoupling of sarcolemmal Ca 2+ channels to RyR and reduced fractional SR Ca 2+ release despite preserved SR Ca 2+ content.