SR Ca Leak Research Articles

How Does Stochastic Ryanodine Receptor-Mediated Ca Leak Fail to Initiate a Ca Spark?

Spontaneous calcium (Ca) sparks are initiated by single ryanodine receptor (RyR) opening. Once one RyR channel opens, it elevates local [Ca] in the cleft space ([Ca]Cleft), which opens other RyR channels in the same Ca release unit (CaRU) via Ca-induced Ca-release. Experiments by Zima et al. (J. Physiol. 588:4743–4757, 2010) demonstrate that spontaneous Ca sparks occur only when intrasarcoplasmic-reticulum (SR) [Ca] ([Ca]SR) is above a threshold level, but that RyR-mediated SR Ca leak exists without Ca sparks well below this threshold [Ca]SR. We examine here how single RyR opening at lower [Ca]SR can fail to recruit Ca sparks at a CaRU, while still contributing to SR Ca leak. We assess this using a physiologically detailed mathematical model of junctional SR Ca release in which RyR gating is regulated by [Ca]SR and [Ca]Cleft. We find that several factors contribute to the failure of Ca sparks as [Ca]SR declines: 1), lower [Ca]SR reduces driving force and thus limits local [Ca]Cleft achieved and the rate of rise during RyR opening; 2), low [Ca]SR limits RyR open time (τO), which further reduces local [Ca]Cleft attained; 3), low τO and fast [Ca]Cleft dissipation after RyR closure shorten the opportunity for neighboring RyR activation; 4), at low [Ca]SR, the RyR exhibits reduced [Ca]Cleft sensitivity. We conclude that all of these factors conspire to reduce the probability of Ca sparks as [Ca]SR declines, despite continued RyR-mediated Ca leak. In addition, these same factors explain the much lower efficacy of L-type Ca channel opening to trigger local SR Ca release at low [Ca]SR during excitation-contraction coupling. Conversely, all of these factors are fundamentally important for increasing the propensity for pro-arrhythmic Ca sparks and waves in cardiac myocytes at high [Ca]SR.

Calcitonin gene‐related peptide restores disrupted excitation–contraction coupling in myotubes expressing central core disease mutations in RyR1

Central core disease (CCD) is a congenital human myopathy associated with mutations in the gene encoding the skeletal muscle ryanodine receptor (RyR1), resulting in skeletal muscle weakness and lower limb deformities. The muscle weakness can be at least partially explained by a reduced magnitude of voltage-gated Ca(2+) release (VGCR). To date, only a few studies have focused on identifying potential therapeutic agents for CCD. Therefore, in this work we investigated the potential use of the calcitonin gene related peptide (CGRP) to restore VGCR in myotubes expressing CCD RyR1 mutants. We also examined the influence of CCD mutants on Ca(2+)-dependent processes involved in myogenesis (myoblast fusion and sarcoendoplasmic reticulum Ca(2+)-ATPase isoform 2 (SERCA2) gene expression). C2C12 cells were transfected with cDNAs encoding either wild-type RyR1 or CCD mutants, and then exposed to CGRP (100 nm, 1-4 h). Expression of the I4897T mutant significantly inhibited SERCA2 gene expression and myoblast fusion, whereas the Y523S mutant exerted the opposite effect. Interestingly, both mutants clearly inhibited VGCR (50%), due to a reduction in SR Ca(2+) content. However, no major changes due to CGRP or CCD mutants were observed in I(CaL). Our data suggest that the Y523S mutant results in store depletion via decompensated SR Ca(2+) leak, while the I4897T mutant inhibits SERCA2 gene expression. Remarkably, in both cases CGRP restored VGCR, likely to have been by enhancing phospholamban (PLB) phosphorylation, SERCA activity and SR Ca(2+) content. Taken together, our data show that in the C2C12 model system, changes in excitation-contraction coupling induced by the expression of RyR1 channels bearing CCD mutations Y523S or I4897T can be reversed by CGRP.

Ca2+ Cycling in Heart Cells from Ground Squirrels: Adaptive Strategies for Intracellular Ca2+ Homeostasis

Heart tissues from hibernating mammals, such as ground squirrels, are able to endure hypothermia, hypoxia and other extreme insulting factors that are fatal for human and nonhibernating mammals. This study was designed to understand adaptive mechanisms involved in intracellular Ca2+ homeostasis in cardiomyocytes from the mammalian hibernator, ground squirrel, compared to rat. Electrophysiological and confocal imaging experiments showed that the voltage-dependence of L-type Ca2+ current (I Ca) was shifted to higher potentials in ventricular myocytes from ground squirrels vs. rats. The elevated threshold of I Ca did not compromise the Ca2+-induced Ca2+ release, because a higher depolarization rate and a longer duration of action potential compensated the voltage shift of I Ca. Both the caffeine-sensitive and caffeine-resistant components of cytosolic Ca2+ removal were more rapid in ground squirrels. Ca2+ sparks in ground squirrels exhibited larger amplitude/size and much lower frequency than in rats. Due to the high I Ca threshold, low SR Ca2+ leak and rapid cytosolic Ca2+ clearance, heart cells from ground squirrels exhibited better capability in maintaining intracellular Ca2+ homeostasis than those from rats and other nonhibernating mammals. These findings not only reveal adaptive mechanisms of hibernation, but also provide novel strategies against Ca2+ overload-related heart diseases.

Tetracaine is a Potent Inhibitor of SR Ca Leak in Ventricular Cardiac Myocytes

Tetracaine is commonly used to inhibit RyR-dependent SR Ca leak, however ability to do so in intact cardiac myocytes has not been fully characterized. In single RyR channel bilayer a significant portion of leak would not be inhibited even by low millimolar concentrations of tetracaine. In this study we investigated the effectiveness of tetracaine on RYR-dependent SR Ca leak in ventricular myocytes. Experiments were performed in isolated rabbit myocytes loaded with fluo-4. Myocytes were perfused with 2mM Ca NT with 200µM caffeine in order to increase basal SR Ca leak in the presence of varying concentrations of tetracaine (1, 10, 30, 100µM). Myocytes were then rapidly perfused with 0 Na, 0 Ca NT with 1mM tetracaine following washout with same solution but with varying amounts of tetracaine. SR Ca leak was quantified as the shift of Ca from the cytosol to the SR upon blockage of the RyR with high tetracaine. The EC50 for tetracaine is significantly reduced in intact myocytes compared to single RyR channel. Among the potential explanations for this effect is the possibility that Ca flux regulation makes the RyR clusters in the SR junction more sensitive to blockage of release.

Suppression of Cardiac L-Type Ca Channel Activity Causes Cardiac Hypertrophy

Increased L type Ca channel (LTCC) activity was proposed to induce Ca dependent hypertrophy and heart failure by activating CaMK and calcineurin (CaN) signaling. Previous experiments showed that upregulation of LTCC activity induced profound cardiomyopathy. However, the enhanced Ca influx did not activate CaN or hypertrophy; but led to necrotic myocyte death. We hypothesized that reducing LTCC by genetic deletion of 50% of Cav1.2 would be cardioprotective like β-adrenergic receptor or LTCC blockers. Surprisingly, this deletion enhanced disease, CaN activation and hypertrophy. These myocytes with reduced Ca current exhibited significantly increased diastolic Ca. To further address these surprising findings we analyzed the Ca dynamics of mice with a standard null allele crossed with a cardiac specific flox allele (α1cf/-Cre), which led to an even greater deletion of the α1c protein in the heart. Here we measured Ca handling dynamics in α1cf/-Cre mice. Twitch amplitudes were significantly reduced (50%) and relaxation time was prolonged (16%). SR Ca load was reduced significantly (∼25%). These findings may be explained by increased diastolic SR Ca leak that we measured in myocytes from α1cf/-Cre mice. Thus, the deletion of the α1c protein in mouse cardiac myocytes leads to an increased basal [Ca]i by increasing Ca leak from the SR. Interestingly, the CaMKII inhibitor KN93 completely reversed the effects of the reduced LTCC: twitch amplitude and relaxation as well as SR Ca load were rescued. These results suggest that resting [Ca]i and the gain in ECC are increased to maintain contractility. This increase might drive hypertrophy by activating CaN and CaMKII.

The relationship between arrhythmogenesis and impaired contractility in heart failure: role of altered ryanodine receptor function

In heart failure (HF), abnormal myocyte Ca(2+) handling has been implicated in cardiac arrhythmias and contractile dysfunction. In the present study, we investigated the relationships between Ca(2+) handling, reduced myocyte contractility, and enhanced arrhythmogenesis during HF progression in a canine model of non-ischaemic HF. Key Ca(2+) handling parameters were determined by measuring cytosolic and intra-sarcoplasmic reticulum (SR) [Ca(2+)] in isolated ventricular myocytes at different stages of HF. The progression of HF was associated with an early and continuous increase in ryanodine receptor (RyR2)-mediated SR Ca(2+) leak. The increase in RyR2 activity was paralleled by an increase in the frequency of diastolic spontaneous Ca(2+) waves (SCWs) in HF myocytes under conditions of β-adrenergic stimulation. In addition to causing arrhythmogenic-delayed afterdepolarizations, SCWs decreased the amplitude of subsequent electrically evoked Ca(2+) transients by depleting SR Ca(2+). At late stages of HF, Ca(2+) release oscillated essentially independent of electrical pacing. The increased propensity for the generation of SCWs in HF myocytes was attributable to reduced ability of the RyR2 channels to become refractory following Ca(2+) release. The progressive alterations in RyR2 function and Ca(2+) cycling in HF myocytes were associated with sequential modifications of RyR2 by CaMKII-dependent phosphorylation and thiol oxidation. These findings suggest that destabilized RyR2 activity due to excessive CaMKII phopshorylation and oxidation resulting in impaired post-release refractoriness is a common mechanism involved in arrhythmogenesis and contractile dysfunction in the failing heart.

Ca2+ wave probability is determined by the balance between SERCA2-dependent Ca2+ reuptake and threshold SR Ca2+ content

In this manuscript, we determined the roles of the sarcoendoplasmic reticulum Ca(2+) ATPase 2 (SERCA2) and the ryanodine receptor (RyR) in Ca(2+) wave development during β-adrenergic stimulation. SERCA2 knockout mice (KO) were used 6 days after cardio-specific gene deletion, with left ventricular SERCA2a abundance reduced by 54 ± 9% compared with SERCA2(flox/flox) controls (FF) (P < 0.05). Ca(2+) waves occurred in fewer KO than FF myocytes (40 vs. 68%, P < 0.05), whereas the addition of isoproterenol (ISO) induced waves in an equal percentage of myocytes (82 vs. 64%). SERCA2-dependent Ca(2+) reuptake was slower in KO (-ISO, KO vs. FF: 15.4 ± 1.2 vs. 21.1 ± 1.4 s(-1), P < 0.05), but equal during ISO (+ISO, KO vs. FF: 21.9 ± 3.3 vs. 27.7 ± 2.7 s(-1)). Threshold SR Ca(2+) content for wave development was lower in KO (-ISO, KO vs. FF: 126.6 ± 10.3 vs. 159.3 ± 7.1 µmol/L, P < 0.05) and was increased by ISO only in FF (+ISO, KO vs. FF: 131.7 ± 8.7 vs. 205.5 ± 20.4 µmol/L, P < 0.05). During ISO, Ca(2+)/calmodulin-dependent kinase II (CaMKII)-dependent phosphorylation of RyR in KO was 217 ± 21% of FF (P < 0.05), and SR Ca(2+) leak indicated higher RyR open probability in KO. CaMKII inhibition decreased Ca(2+) spark frequency in KO by 44% (P < 0.05) but not in FF. Mathematical modelling predicted that increased Ca(2+) sensitivity of RyR in KO could account for increased Ca(2+) wave probability during ISO. In ventricular cardiomyocytes with reduced SERCA2 abundance, Ca(2+) wave development following β-adrenergic stimulation is potentiated. We suggest that this is caused by a CaMKII-dependent shift in the balance between SERCA2-dependent Ca(2+) reuptake and threshold SR Ca(2+) content.

Does Protein Kinase A–Mediated Phosphorylation of the Cardiac Ryanodine Receptor Play Any Role in Adrenergic Regulation of Calcium Handling in Health and Disease?

See related articles, pages 1743–1752 Cardiac myocyte ryanodine receptors (sarcoplasmic reticulum [SR] Ca release channel; cardiac ryanodine receptor [RyR2]) are localized in the junctional SR, in close proximity to L-type Ca channels (LTCCs) embedded in the membranes of the transverse (T)-tubules. This signaling microdomain has been termed the couplon,1 because it is here that excitation–contraction coupling takes place. As the heart fills with blood during diastole, RyR2 is stabilized in a closed state, allowing Ca uptake by the SR Ca ATPase to pump Ca from the cytoplasm into the SR, providing the primary source of Ca to activate the contractile apparatus during the next heart beat (systole). During systole, the cardiac action potential depolarizes the T-tubules and causes the opening of LTCCs. Ca influx through LTCCs elevates the [Ca] within the cytoplasmic space between the T-tubular and SR membrane, which promotes Ca binding to neighboring RyR2s, inducing some to open. Ca then moves out of the SR lumen into the subsarcolemmal, space and the additional elevation of [Ca] induces a regenerative opening of other RyRs in the couplon. The resulting localized Ca signal is called a Ca spark.2 The action potential synchronizes these local Ca release events throughout the cell (termed Ca-induced Ca release) to produce the global cardiac [Ca] transient.3 The stabilized closed state and Ca-dependent opening of RyR2 during diastole and systole are essential for normal cardiac diastolic and systolic function. Destabilizing the behavior of RyR2 would be expected to have a negative …

Spontaneous Ca waves in ventricular myocytes from failing hearts depend on Ca2+-calmodulin-dependent protein kinase II

Increased cardiac ryanodine receptor (RyR)-dependent diastolic SR Ca leak is present in heart failure and in conditions when adrenergic tone is high. Increasing Ca leak from the SR could result in spontaneous Ca wave (SCaW) formation. SCaWs activate the inward Na/Ca exchanger (NCX) current causing a delayed afterdepolarization (DAD), potentially leading to arrhythmia. Here we examine SCaWs in ventricular myocytes isolated from failing and healthy rabbit hearts. Myocytes from healthy hearts did not exhibit SCaWs under baseline conditions versus 43% of those exposed to isoproterenol (ISO). This ISO-induced increase in activity was reversed by inhibition of Ca-calmodulin-dependent protein kinase II (CaMKII) by KN93. Inhibition of cAMP-dependent protein kinase (PKA) by H89 had no observed effect. Of myocytes treated with forskolin 50% showed SCaW activity, attributable to a large increase in SR Ca load ([Ca]SRT) versus control. At similar [Ca]SRT (121μM) myocytes treated with ISO plus KN93 had significantly fewer SCaWs versus those treated with ISO or ISO plus H89 (0.2±0.28 vs. 1.1±0.28 and 1.29±0.39 SCaWs cell−1, respectively). In myocytes isolated from failing hearts ISO induced an increase in the percentage of cells generating SCaWs vs. baseline (74% vs. 11%) with no increase in [Ca]SRT. Inhibiting CaMKII reversed this effect (14%). At similar [Ca]SRT (71µM) myocytes treated with ISO or ISO plus H89 had significantly more SCaWs per cell vs. untreated (2.5±0.5; 1.6±0.7 vs. 0.36±0.3, respectively). Treatment with ISO plus KN93 completely abolished this effect. The evidence suggests the ISO-dependent increase in SCaW activity in both healthy and failing myocytes is CaMKII-dependent, implicating CaMKII in arrhythmogenesis.

Ca SR Leak is Modulated by CaMKII Phosphorylation on RyR2-S2814

CaMKII has been shown to increase cardiac SR Ca leak through RyRs. RyR2-S2814 has been suggested as the phosphorylation site responsible for SR Ca leak triggering cardiac arrhythmias in heart failure. Here we test the requirement of S2814 for these effects, in knock-in mice expressing only RyR2-S2814A or -S2814D. Ca spark frequency (CaSpF) and SR load were studied in intact and permeabilized cardiomyocytes using confocal microscopy. At baseline CaSpF is higher in S2814D vs. WT (8.7±0.4 (n=9) vs 6.44±0.3, n=8, p<0.01) without altered SR Ca load. Activation of endogenous CaMKII (1.2 μM Ca-calmodulin) in WT increases CaSpF as described by Guo et al (Circ. Res, 2006), but only to the level seen in S2814D at baseline. Moreover, CaMKII did not further increase CaSpF in S2814D myocytes. In RyR2-S2814A CaMKII activation produces a very small CaSpF increase vs WT (19 vs 60%), and that response in S2814A is secondary to increased SR Ca load (by 15%, n=10, p<0.01). In intact myocytes (as above), basal CaSpF was highest in S2814D vs. WT and S2814A (which were similar). Baseline Ca transients were not different among the groups. The cAMP activated GTP exchange factor Epac may activate CaMKII to induce SR Ca leak (Pereira et al, J Physiol, 2007). In WT mice the Epac activator 8-CPT enhanced CaSpF and decreased both SR Ca load and Ca transient amplitude (consistent with a primary effect on SR Ca leak). However, 8-CPT had no effect on any of these parameters in either S2814A or S2814D myocytes. These data indicate that RyR2-S2814 is the critical RyR2 site responsible for CaMKII-dependent enhancement of SR Ca leak and potential arrhythmogenesis in heart failure, and confirm that Epac signaling can work through this same pathway.

Nitric Oxide Can Mediate Beta-Adrenergic- and CaMKII-Dependent Spontaneous Ca2+ Waves in Cardiac Myocytes, Independent of PKA Activation

Increased diastolic SR Ca leak can initiate spontaneous Ca waves (SCaWs). SCaWs activate inward Na/Ca exchanger current causing an arrhythmogenic delayed afterdepolarization. Here we examine SCaWs in ventricular myocytes isolated from rabbit hearts. Myocytes did not exhibit SCaWs at baseline conditions, but 43% did when exposed to isoproterenol (ISO). This ISO-induced increase in activity was reversed by inhibition of CaMKII by KN93, but not with PKA inhibition by H89. At similar [Ca]SRT (121 μM) myocytes treated with ISO plus KN93 had significantly fewer SCaWs versus those treated with ISO or ISO plus H89 (0.2±0.28 vs. 1.1±0.28 & 1.29±0.39 SCaWs cell−1, respectively). We attribute this increase in activity to the previously characterized CaMKII-dependent increase in RyR-dependent leak. We also find that SR Ca leak is increased by the nitric oxide (NO) donor, SNAP; and this NO-dependent effect is also completely reversed by KN-93. We also show the increase in leak to be dependent on nitric oxide synthase 1 (NOS1) activity. At comparable SR Ca load (132 μM) ISO treated myocytes have significantly higher leak vs. control (8.4 vs. 3.8 μM). The ISO-induced leak (at constant SR Ca load) was attenuated by the NOS1 inhibitor, SMLT, but not the NOS3 inhibitor, L-NIO (3.5 vs. 6.8 μM). Moreover, ISO causes an upward trend in myocyte [NO] (sensed by the NO-dependent dye, DAF-2 A), and the NOS inhibitor, L-NAME significantly attenuated the development of SCaWs. Together this data suggests a novel pathway in which β1-adrenergic receptor activation stimulates NO production via NOS1, which in turn activates CaMKII to increase RyR gating, SR Ca leak, SCaWs and delayed afterdepolarizations.

Subcellular Mechanisms of Early Impaired Calcium Homeostasis with Chronic Beta1-Adrenergic Stimulation in Mice

Chronic beta-adrenergic stimulation leads to heart failure (HF). In mice overexpressing beta1-adrenoceptors (TG), increased diastolic Ca load in cardiomyocytes at early age is pivotal for the development of HF. The mechanisms underlying intracellular Ca dysregulation are unclear. We examined cytosolic Ca transients (Fluo4-AM, field stimulation), Na-Ca-exchanger (NCX) function and protein expression, cytosolic Na (SBFI) and T-tubular structures (Di8-ANEPPS) in cardiomyocytes from young (8-16 wks) TG mice and wildtype (WT) littermates.Results: Systolic [Ca] amplitude was unchanged, time to peak [Ca] (140±5 vs. 127±3 ms) and [Ca] decay (time constant, tau, 223±16 vs. 182±9 ms) were significantly prolonged in TG vs. WT. Diastolic Ca leak from the SR (quantified as tetracaine-sensitive change in diastolic [Ca] or diastolic Ca spark frequency) was unchanged. However, cytosolic Ca removal by NCX during coffein application was significantly slower (tau, 3683±337 in TG vs. 2304±272 ms in WT), indicating reduced forward mode NCX activity. NCX protein expression was unchanged. Preliminary results indicate increased cytosolic [Na] in young TG. Furthermore, confocal line scans revealed delayed (> 15 ms until half-maximal) systolic Ca release in 24.7±2.6 (TG) vs. 4.6±1.4% (WT) of the intracellular regions (n=32 and 31 cells, resp., p<0.01). The extent of dyssynchronous Ca release correlated with time to peak systolic [Ca] (R=0.51, P<0.001) and was associated with a lower density and increased irregularity of T-tubules in TG (22.8±1.6% of cell volume in TG vs. 26.1±2.5% in WT). In summary, in early HF remodeling with chronic beta1-adrenergic stimulation, slowed cytosolic Ca clearance isnot related to increased diastolic SR Ca leak but associated with decreased NCX forward mode activity, which may be related to increased cytosolic [Na]. Reduced T-tubule density with dyssynchronous, slowed systolic Ca release additionally contribute to increased cytosolic Ca load.

Redox modification of ryanodine receptors underlies calcium alternans in a canine model of sudden cardiac death

Although cardiac alternans is a known predictor of lethal arrhythmias, its underlying causes remain largely undefined in disease settings. The potential role of, and mechanisms responsible for, beat-to-beat alternations in the amplitude of systolic Ca(2+) transients (Ca(2+) alternans) was investigated in a canine post-myocardial infarction (MI) model of sudden cardiac death (SCD). Post-MI dogs had preserved left ventricular (LV) function and susceptibility to ventricular fibrillation (VF) during exercise. LV wedge preparations from VF dogs were more susceptible to action potential (AP) alternans and the frequency-dependence of Ca(2+) alternans was shifted towards slower rates in myocytes isolated from VF dogs relative to controls. In both groups of cells, cytosolic Ca(2+) transients ([Ca(2+)](c)) alternated in phase with changes in diastolic Ca(2+) in sarcoplasmic reticulum ([Ca(2+)](SR)), but the dependence of [Ca(2+)](c) amplitude on [Ca(2+)](SR) was steeper in VF cells. Abnormal ryanodine receptor (RyR) function in VF cells was indicated by increased fractional Ca(2+) release for a given amplitude of Ca(2+) current and elevated diastolic RyR-mediated SR Ca(2+) leak. SR Ca(2+) uptake activity did not differ between VF and control cells. VF myocytes had an increased rate of reactive oxygen species production and increased RyR oxidation. Treatment of VF myocytes with reducing agents normalized parameters of Ca(2+) handling and shifted the threshold of Ca(2+) alternans to higher frequencies. Redox modulation of RyRs promotes generation of Ca(2+) alternans by enhancing the steepness of the Ca(2+) release-load relationship and thereby providing a substrate for post-MI arrhythmias.

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.

Sex Differences Of RyR2 Expression And Phosphorylation In Heart Failure

Sex differences in the expression and phosphorylation of Ca handling proteins could underlie sex differences in contractile function and ventricular arrhythmias in heart failure (HF). We previously showed down-regulation of cardiac RyR (RyR2) and increased CaMKII- & PKA -dependent phosphorylation of RyR2 associated with increased CaMKII-dependent SR Ca leak in our arrhythmogenic rabbit model of HF. Here, we assessed sex differences in RyR2 expression and phosphorylation in HF. We found total RyR2 protein expression in control F was 27% lower (vs control M, n=3,3, p<0.05); but with HF, both M & F exhibited a down-regulation of RyR2 vs control M (41% and 49%, respectively, n=4,4, p<0.05). Phosphorylation of RyR2 at the serine-2809 (PKA- & CaMKII-) and serine-2815 (CaMKII) sites was increased in both HF-M & HF-F (73% & 43%, and 67% & 135%, respectively, vs control M, n=4,4,4,4, p<0.05). Moreover, CaMKII and PKA backphosphorylation (-P) of RyR2 were assessed in immunoprecipitated RyR2 proteins with specific RyR2 antibody. CaMKII-P of RyR2 was significantly increased with HF in both sexes vs control M (71% and 87%, respectively, n=5,5,5,5, p<0.05), while PKA-P of RyR2 was increased (by 228%) only in HF-F (p < 0.001). HF-M (but not HF-F) exhibited increased global expression of CaMKIIδ (cardiac isoform) by 141% (vs control M, n=4,4,4,4, p<0.05), but co-immunoprecipitated CaMKIIδ with RyR2 antibody was increased in both HF-M & HF-F (vs control M, 38% and 43%, respectively, n=3,3,3,3, p<0.05). In contrast, co-immunoprecipitated PKA with RyR2 was increased in only HF-F (26%, p< 0.001), while global expression of PKA was unchanged in both sexes. Thus, there are sex differences in RyR2 hyperphosphorylation with HF. HF-F have increased co-localized CaMKIIδ and PKA with RyR2, while HF-M have increased global CaMKIIδ and increased co-localized CaMKIIδ (but not PKA) with RyR2.

Differential Regulation Of The L-type Ca Channels And SERCA Pump By Type 3 Phosphodiesterase

Inhibitors of phosphodiesterase type 3 (PDE3) are positive inotropic agents that are used for short-term management of acutely decompensated heart failure. However, the molecular mechanisms mediating the cardiac effects of PDE3 inhibitors are not clearly defined. The aim of the present study was to investigate the signaling pathways involved in the effects of PDE3 inhibition on cardiac excitation-contraction coupling in a large animal model. Confocal microscopy and patch-clamp techniques were used to monitor intracellular Ca cycling in isolated canine ventricular myocytes. PKA type 2 activity in live cells was recorded using FRET-based genetically encoded fluorescent probe. Application of cilostamide, a specific PDE3 inhibitor, resulted in a significant increase in the amplitude of depolarization-induced Ca transient. This effect had two phases: transient, developed within 10 min, and sustained, characterized by a smaller increase in the Ca transient persisting up to 3 hours. The same time-course was observed for cilostamide-mediated increase in Ca current. Cilostamide produced only a transient increase in SERCA and type 2 PKA activities. The rate of SR Ca leak measured in the presence of SERCA inhibitor, thapsigargin, was not altered by cilostamide, suggesting that ryanodine receptor function was not modified by PDE3 inhibition. Although inhibition of type 4 phosphodiesterase (PDE4) alone did not affect any of the recorded functional readouts, PDE4 inhibitors potentiated the effects of cilostamide and converted the cilostamide-mediated transient effects into sustained responses. These results suggest that PDE3 inhibition can produce the long-lasting effect on cardiac cytosolic Ca transients. This effect is predominantly mediated by an increase in the Ca current amplitude that occurs via type 1 PKA-dependent phosphorylation. The data also suggest that concomitant inhibition of PDE3 and PDE4 results in persistent stimulation of SERCA and type 2 PKA activities.

Analysis of Calcium Alternans in a Cardiac Myocyte Model that Uses Moment Equations to Represent Heterogenous Junctional SR Calcium

The recently introduced “probability density approach” to modeling local control of CICR in cardiac myocytes [Williams et al. Biophys. J. 92(7):2311-28, 2007] and associated moment closure technique [95(4):1689-703, 2008] can reproduce whole cell voltage-clamp protocols high-gain Ca release that is graded with changes in membrane potential. This modeling formalism represents heterogeneous local Ca signals in a population of diadic subspaces and junctional sarcoplasmic reticulum (jSR) depletion domains using a system of differential-algebraic equations for the time-evolution of the zeroth, first, and second moments of probability density functions for jSR [Ca] jointly distributed with calcium release unit (CaRU) state. Here we show that a cardiac myocyte model that uses moment equations to represent heterogenous jSR Ca can exhibit Ca alternans when periodically stimulated by depolarizing voltage pulses, and makes predictions regarding the distribution of jSR [Ca] across a large population CaRUs as a function of stimulation frequency and cellular parameters such as the rate of diffusive transfer between network and junctional SR. Factors promoting alternating Ca responses in the moment closure model are analyzed and compared to analogous mechanism in a minimal “common pool” model with comparatively simple SR and PM fluxes. We derive load-release and release-reuptake curves for both models, and investigate how model parameters influence these relations and the existence and stability of steady-state periodic Ca responses during repetitive depolarizing voltage pulses. Specifically, we find that increasing SR Ca leak, RyR sensitivity, and maximum release flux decreases the steepness of load-release curves, shifts load-release curves to smaller SR loads, and increases the critical simulation frequency resulting in Ca alternans.

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 1078: β-Adrenergic Stimulation of Ca/Calmodulin-Dependent Protein Kinase II (CaMKII) Overexpressing Myocytes Increases Sarcoplasmic Reticulum (SR) Calcium Leak Causing KN-93 Sensitive Arrhythmias

CaMKII is associated with hypertrophy, heart failure and alters intracellular Ca homeostasis. An increased SR Ca leak due to phosphorylation of SR Ca release channels by CaMKII leads to decreased SR Ca content and impaired contractility. This loss of Ca from the SR may also contribute to arrhythmias. We investigated whether β-adrenergic stimulation with isoproterenol (ISO) normalizes SR Ca content and whether inhibiting CaMKII reduces arrhythmias. CaMKII-overexpressing rabbit and mouse myocytes were investigated. Cell shortening, Ca fluorescence (fluo-3) and the incidence of arrhythmias were assessed. An arrhythmia-score differentiated between: early-spike-arrhythmias (ESA), late-spike-arrhythmias (LSA) and permanent arrhythmias (PA). ISO (37°C) had significantly different effects on myocytes with acute (24 h, rabbit, n=34) or chronic (22 w, mouse, n=34) CaMKII overexpression vs corresponding control myocytes (LacZ, n=21 or WT n=34). CaMKII overexpression lead to an ISO concentration-dependent (10 −10 -10 −5 mol/L) inotropic but compared to WT (or LacZ, respectively) impaired shortening and Ca transients (two-way ANOVA, P &lt;0.05). A similar difference between CaMKII-overexpressing (n=17) and WT (n=19) myocytes was also seen during a shortening-frequency protocol (stepwise increase from 0.1– 4 Hz, two-way ANOVA, P &lt;0.05). Arrhythmias spontaneously occurred in CaMKII-overexpressing mouse myocytes. With β-inotropic stimulation (10 −6 mol/L ISO) arrhythmias were increased 6.4-fold. Appearance of ESA and PA could be significantly reduced by KN-93 (1 μmol/L). At a basal stimulation rate of 1 Hz and 10 −7 mol/L ISO, PA could be dramatically reduced by half from control-level 21.43% (KN-92, inactive derivative, n=42) down to 10.87% (KN-93, n=46) arrhythmic events. ESA could be reduced almost 4-fold from 16.67% (KN-92) to 4.35% in the presence of KN-93. We conclude from these data that increasing ISO concentrations exerts positive inotropic effects but cannot normalize altered Ca handling in CaMKII-overexpressing myocytes. This may be due to an increased SR Ca leak under these conditions thus contributing to the arrhythmias observed. CaMKII inhibition clearly can reduce arrhythmias in the presence of β-adrenergic stimulation with ISO.