Dyadic Cleft Research Articles

Simulating cardiac Ca2+ release units: effects of RyR cluster size and Ca2+ buffers on diastolic Ca2+ leak.

Leak of Ca2+ out of the cardiac sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) during diastole is vital to regulate SR Ca2+ levels. This leak can become deleterious when large spontaneous RyR-mediated Ca2+ release events evoke proarrhythmic Ca2+ waves that can lead to delayed after-depolarizations. Here, we model diastolic SR Ca2+ leak at individual SR Ca2+ release sites using computer simulations of RyR arrays like those in the dyadic cleft. The results show that RyR arrays size has a significant effect on SR Ca2+ leak, with bigger arrays producing larger and more frequent Ca2+ release events. Moreover, big RyR arrays are more susceptible to small changes in the levels of dyadic Ca2+ buffers. Such changes in buffering shift Ca2+ leak from small Ca2+ release events (involving few open RyRs) to larger events (with many open RyRs). Moreover, by analyzing a large parameter space of possible buffering and SR Ca2+ loads, we find further evidence for the hypothesis that SR Ca2+ leak by RyR arrays can undergo a sudden phase transition.

Multiscale Modeling of Dyadic Structure-Function Relation in Ventricular Cardiac Myocytes

Cardiovascular disease is often related to defects of subcellular components in cardiac myocytes, specifically in the dyadic cleft, which include changes in cleft geometry and channel placement. Modeling of these pathological changes requires both spatially resolved cleft as well as whole cell level descriptions. We use a multiscale model to create dyadic structure-function relationships to explore the impact of molecular changes on whole cell electrophysiology and calcium cycling. This multiscale model incorporates stochastic simulation of individual L-type calcium channels and ryanodine receptor channels, spatially detailed concentration dynamics in dyadic clefts, rabbit membrane potential dynamics, and a system of partial differential equations for myoplasmic and lumenal free Ca2+ and Ca2+-binding molecules in the bulk of the cell.

Nano-scale morphology of cardiomyocyte t-tubule/sarcoplasmic reticulum junctions revealed by ultra-rapid high-pressure freezing and electron tomography

Detailed knowledge of the ultrastructure of intracellular compartments is a prerequisite for our understanding of how cells function. In cardiac muscle cells, close apposition of transverse (t)-tubule (TT) and sarcoplasmic reticulum (SR) membranes supports stable high-gain excitation-contraction coupling. Here, the fine structure of this key intracellular element is examined in rabbit and mouse ventricular cardiomyocytes, using ultra-rapid high-pressure freezing (HPF, omitting aldehyde fixation) and electron microscopy. 3D electron tomograms were used to quantify the dimensions of TT, terminal cisternae of the SR, and the space between SR and TT membranes (dyadic cleft). In comparison to conventional aldehyde-based chemical sample fixation, HPF-preserved samples of both species show considerably more voluminous SR terminal cisternae, both in absolute dimensions and in terms of junctional SR to TT volume ratio. In rabbit cardiomyocytes, the average dyadic cleft surface area of HPF and chemically fixed myocytes did not differ, but cleft volume was significantly smaller in HPF samples than in conventionally fixed tissue; in murine cardiomyocytes, the dyadic cleft surface area was higher in HPF samples with no difference in cleft volume. In both species, the apposition of the TT and SR membranes in the dyad was more likely to be closer than 10 nm in HPF samples compared to CFD, presumably resulting from avoidance of sample shrinkage associated with conventional fixation techniques. Overall, we provide a note of caution regarding quantitative interpretation of chemically-fixed ultrastructures, and offer novel insight into cardiac TT and SR ultrastructure with relevance for our understanding of cardiac physiology.

Activation of reverse Na+-Ca2+ exchanger by skeletal Na+ channel isoform increases excitation-contraction coupling efficiency in rabbit cardiomyocytes.

Our prior work has shown that Na+ current (INa) affects sarcoplasmic reticular (SR) Ca2+ release by activating early reverse of the Na+-Ca2+ exchanger (NCX). The resulting Ca2+ entry primes the dyadic cleft, which appears to increase Ca2+ channel coupling fidelity. It has been shown that the skeletal isoform of the voltage-gated Na+ channel (Nav1.4) is the main tetrodotoxin (TTX)-sensitive Nav isoform expressed in adult rabbit ventricular cardiomyocytes. Here, I tested the hypothesis that it is also the principal isoform involved in the priming mechanism. Action potentials (APs) were evoked in isolated rabbit ventricular cells loaded with fluo-4, and simultaneously recorded Ca2+ transients before and after the application of either relatively low doses of TTX (100 nM), the specific Nav1.4 inhibitor μ-Conotoxin GIIIB or the specific Nav1.1 inhibitor ICA 121430. Although APs changes after the application of each drug reflected the relative abundance of each isoform, the effects of TTX and GIIIB on SR Ca2+ release (measured as the transient maximum upstroke velocity) were no different. Furthermore, this reduction in SR Ca2+ release was comparable with the value that we obtained previously when total INa was inactivated with a ramp applied under voltage clamp. Finally, SR Ca2+ release was unaltered by the same ramp in the presence of TTX or GIIB. In contrast, application of ICA had no effect of SR Ca2+ release. These results suggest that Nav1.4 is the main Nav isoform involved in regulating the efficiency of excitation-contraction coupling in rabbit cardiomyocytes by priming the junction via activation of reverse-mode NCX.NEW & NOTEWORTHY A number of studies suggest that the Na+-Ca2+ exchanger (NCX) activated by Na+ currents is involved in the process of excitation-contraction (EC) coupling in cardiac ventricular myocytes. Although insufficient to trigger sarcoplasmic Ca2+ release alone, the Ca2+ entering through reverse NCX during an action potential can prime the dyadic cleft and increase the Ca2+ current coupling fidelity. Using specific Na+ inhibitors in this study, we show that in rabbit ventricular cells the skeletal Na+ channel isoform (Nav1.4) is the main isoform responsible for this priming. Our study provides insights into a mechanism that may have an increased relevance where EC coupling is remodeled.

The architecture and function of cardiac dyads.

Cardiac excitation-contraction (EC) coupling, which links plasma membrane depolarization to activation of cardiomyocyte contraction, occurs at dyads, the nanoscopic microdomains formed by apposition of transverse (T)-tubules and junctional sarcoplasmic reticulum (jSR). In a dyadic junction, EC coupling occurs through Ca2+-induced Ca2+ release. Membrane depolarization opens voltage-gated L-type Ca2+ channels (LTCCs) in the T-tubule. The resulting influx of extracellular Ca2+ into the dyadic cleft opens Ca2+ release channels known as ryanodine receptors (RYRs) in the jSR, leading to the rapid increase in cytosolic Ca2+ that triggers sarcomere contraction. The efficacy of LTCC-RYR communication greatly affects a myriad of downstream intracellular signaling events, and it is controlled by many factors, including T-tubule and jSR structure, spatial distribution of ion channels, and regulatory proteins that closely regulate the activities of channels within dyads. Alterations in dyad architecture and/or channel activity are seen in many types of heart disease. This review will focus on the current knowledge regarding cardiac dyad structure and function, their alterations in heart failure, and new approaches to study the composition and function of dyads.

Phenytoin Reduces Activity of Cardiac Ryanodine Receptor 2; A Potential Mechanism for Its Cardioprotective Action.

Phenytoin is a hydantoin derivative that is used clinically for the treatment of epilepsy and has been reported to have antiarrhythmic actions on the heart. In a failing heart, the elevated diastolic Ca2+ leak from the sarcoplasmic reticulum can be normalized by the cardiac ryanodine receptor 2 (RyR2) inhibitor, dantrolene, without inhibiting Ca2+ release during systole or affecting Ca2+ release in normal healthy hearts. Unfortunately, dantrolene is hepatotoxic and unsuitable for chronic long-term administration. Because phenytoin and dantrolene belong to the hydantoin class of compounds, we test the hypothesis that dantrolene and phenytoin have similar inhibitory effects on RyR2 using a single-channel recording of RyR2 activity in artificial lipid bilayers. Phenytoin produced a reversible inhibition of RyR2 channels from sheep and human failing hearts. It followed a hyperbolic dose response with maximal inhibition of ∼50%, Hill coefficient ∼1, and IC50 ranging from 10 to 20 µM. It caused inhibition at diastolic cytoplasmic [Ca2+] but not at Ca2+ levels in the dyadic cleft during systole. Notably, phenytoin inhibits RyR2 from failing human heart but not from healthy heart, indicating that phenytoin may selectively target defective RyR2 channels in humans. We conclude that phenytoin could effectively inhibit RyR2-mediated release of Ca2+ in a manner paralleling that of dantrolene. Moreover, the IC50 of phenytoin in RyR2 is at least threefold lower than for other ion channels and clinically used serum levels, pointing to phenytoin as a more human-safe alternative to dantrolene for therapies against heart failure and cardiac arrythmias. SIGNIFICANCE STATEMENT: We show that phenytoin, a Na channel blocker used clinically for treatment of epilepsy, is a diastolic inhibitor of cardiac calcium release channels [cardiac ryanodine receptor 2 (RyR2)] at doses threefold lower than its current therapeutic levels. Phenytoin inhibits RyR2 from failing human heart and not from healthy heart, indicating that phenytoin may selectively target defective RyR2 channels in humans and pointing to phenytoin as a more human-safe alternative to dantrolene for therapies against heart failure and cardiac arrhythmias.

Multiscale Modeling of Dyadic Structure-Function Relation in Ventricular Cardiac Myocytes

Cardiovascular disease is often related to defects of subcellular components in cardiac myocytes, specifically in the dyadic cleft, which include changes in cleft geometry and channel placement. Modeling of these pathological changes requires both spatially resolved cleft as well as whole cell level descriptions. We use a multiscale model to create dyadic structure-function relationships to explore the impact of molecular changes on whole cell electrophysiology and calcium cycling. This multiscale model incorporates stochastic simulation of individual L-type calcium channels and ryanodine receptor channels, spatially detailed concentration dynamics in dyadic clefts, rabbit membrane potential dynamics, and a system of partial differential equations for myoplasmic and lumenal free Ca2+ and Ca2+-binding molecules in the bulk of the cell. We found action potential duration, systolic, and diastolic [Ca2+] to respond most sensitively to changes in L-type calcium channel current. The ryanodine receptor channel cluster structure inside dyadic clefts was found to affect all biomarkers investigated. The shape of clusters observed in experiments by Jayasinghe et al. and channel density within the cluster (characterized by mean occupancy) showed the strongest correlation to the effects on biomarkers.

Cardiac CaMKII activation promotes rapid translocation to its extra-dyadic targets

Calcium-calmodulin dependent protein kinase IIδ (CaMKIIδ) is an important regulator of cardiac electrophysiology, calcium (Ca) balance, contraction, transcription, arrhythmias and progression to heart failure. CaMKII is readily activated at mouths of dyadic cleft Ca channels, but because of its low Ca-calmodulin affinity and presumed immobility it is less clear how CaMKII gets activated near other known, extra-dyad targets. CaMKII is typically considered to be anchored in cardiomyocytes, but while untested, mobility of active CaMKII could provide a mechanism for broader target phosphorylation in cardiomyocytes. We therefore tested CaMKII mobility and how this is affected by kinase activation in adult rabbit cardiomyocytes. We measured translocation of both endogenous and fluorescence-tagged CaMKII using immunocytochemistry, fluorescence recovery after photobleach (FRAP) and photoactivation of fluorescence. In contrast to the prevailing view that CaMKII is anchored near its myocyte targets, we found CaMKII to be highly mobile in resting myocytes, which was slowed by Ca chelation and accelerated by pacing. At low [Ca], CaMKII was concentrated at Z-lines near the dyad but spread throughout the sarcomere upon pacing. Nuclear exchange of CaMKII was also enhanced upon pacing- and heart failure-induced chronic activation. This mobilization of active CaMKII and its intrinsic memory may allow CaMKII to be activated in high [Ca] regions and then move towards more distant myocyte target sites.

Sarcoplasmic Reticulum Ca2+ Release Uses a Cascading Network of Intra-SR and Channel Countercurrents

In muscle, Ca2+ release from the sarcoplasmic reticulum (SR) into the cytosol is mediated through the ryanodine receptors (RyRs) and sustained by countercurrents that keep the SR membrane potential near 0 mV. Likewise, Ca2+ reuptake by the sarco/endoplasmic reticulum Ca2+ ATPase pump requires countercurrent. Although evidence has suggested that TRIC K+ channels and/or RyR K+ influx provide these countercurrents, the exact sources have not yet been determined. We used an equivalent circuit compartment model of a cardiac SR, the surrounding cytosol, and the dyadic cleft to probe the sources of countercurrent during a complete cardiac cycle. By removing and relocating TRIC K+ channels, as well as limiting when they are active, we explored the various possible sources of SR countercurrent under many conditions. Our simulations indicate that no single channel type is essential for countercurrent. Rather, a cascading network of countercurrents is present with anion fluxes within the SR redistributing charges throughout the full SR volume. This allows ion channels in the entire SR membrane, far from the Ca2+ fluxes through the RyRs in the junctional SR and sarco/endoplasmic reticulum Ca2+ ATPase pump in the nonjunctional SR, to mediate countercurrents that support Ca2+ release and reuptake. This multifactorial network of countercurrents allows Ca2+ release to be remarkably robust.

Total internal reflectance fluorescence imaging of genetically engineered ryanodine receptor-targeted Ca2+ probes in rat ventricular myocytes

The details of cardiac Ca2+ signaling within the dyadic junction remain unclear because of limitations in rapid spatial imaging techniques, and availability of Ca2+ probes localized to dyadic junctions. To critically monitor ryanodine receptors’ (RyR2) Ca2+ nano-domains, we combined the use of genetically engineered RyR2-targeted pericam probes, (FKBP-YCaMP, Kd=150nM, or FKBP-GCaMP6, Kd=240nM) with rapid total internal reflectance fluorescence (TIRF) microscopy (resolution, ∼80nm). The punctate z-line patterns of FKBP,22FK506 binding protein.-targeted probes overlapped those of RyR2 antibodies and sharply contrasted to the images of probes targeted to sarcoplasmic reticulum (SERCA2a/PLB), or cytosolic Fluo-4 images. FKBP-YCaMP signals were too small (∼20%) and too slow (2–3s) to detect Ca2+ sparks, but the probe was effective in marking where Fluo-4 Ca2+ sparks developed. FKBP-GCaMP6, on the other hand, produced rapidly decaying Ca2+ signals that: a) had faster kinetics and activated synchronous with ICa33ICa, L-type Ca2+ current. but were of variable size at different z-lines and b) were accompanied by spatially confined spontaneous Ca2+ sparks, originating from a subset of eager sites. The frequency of spontaneously occurring sparks was lower in FKBP-GCaMP6 infected myocytes as compared to Fluo-4 dialyzed myocytes, but isoproterenol enhanced their frequency more effectively than in Fluo-4 dialyzed cells. Nevertheless, isoproterenol failed to dissociate FKBP-GCaMP6 from the z-lines. The data suggests that FKBP-GCaMP6 binds predominantly to junctional RyR2s and has sufficient on-rate efficiency as to monitor the released Ca2+ in individual dyadic clefts, and supports the idea that β-adrenergic agonists may modulate the stabilizing effects of native FKBP on RyR2.

Calcium Activated Chloride Current in Mammalian Ventricular Myocytes

Background: Calcium activated Cl− current (ICl(Ca)) mediated by TMEM16A and/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profile of ICl(Ca) during an actual ventricular action potential (AP), however, is poorly understood. Our goal was to study the current profile under normal calcium cycling and AP voltage-clamp condition as well as in case of altered intracellular calcium concentration ([Ca2+]i). The expression of TMEM16A and/or Bestrophin-3 in canine and human left ventricular myocytes was examined. The possible spatial distribution of these proteins and their co-localization with Cav1.2 was also studied.Methods: Whole-cell configuration of the patch-clamp technique and action potential voltage-clamp were used to monitor ICl(Ca), detected as 9-anthracene carboxylic acid (9-AC)-sensitive current. FURA-2-AM dye was used to measure [Ca2+]i. Expression and cellular localization of Cav1.2, Bestrophin-3 and TMEM16A was analyzed with immunocytochemistry and confocal microscopy.Results: Under AP voltage-clamp conditions the profile of ICl(Ca) contained an early fast outward (1.62±0.06 A/F) and a late inward component (−0.16±0.02 A/F), overlapping early and terminal repolarizations, respectively. Both components were reduced by ryanodine (1.05±0.03 A/F; −0.07±0.03 A/F), while fully abolished by BAPTA (0.13±0.10 A/F; −0.08±0.02 A/F), but not EGTA (1.17±0.09 A/F; −0.13±0.02 A/F). Setting [Ca2+]i to the systolic level (1.1 µM) decreased ICl(Ca), while application of Bay K8644, isoproterenol, and faster stimulation rates increased the amplitude of ICl(Ca). Both L-type Ca2+ current and ICl(Ca) were eliminated by nisoldipine. TMEM16A and Bestrophin-3 showed strong co-localization with one another and also with Cav1.2 channels both canine myocytes and human ventricular myocardium.Conclusions: Activation of ICl(Ca) in canine ventricular cells requires calcium entry through neighboring L-type Ca2+ channels and is only augmented by SR Ca2+-release. Substantial activation of ICl(Ca) requires high Ca2+ in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA.

Sarcoplasmic reticulum Ca2+, Mg2+, K+, and Cl− concentrations adjust quickly as heart rate changes

During systole, Ca2+ is released from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) while, simultaneously, other ions (specifically K+, Mg2+, and Cl−) provide counter-ion flux. These ions move back into the SR during diastole through the SERCA pump and SR K+ and Cl− channels. In homeostasis, all ion concentrations in different cellular regions (e.g., junctional and non-junctional SR, dyadic cleft, and cytosol) are the same at the beginning and end of the cardiac cycle. Here, we used an equivalent circuit compartment model of the SR and the surrounding cytoplasm to understand the heart rate dependence of SR ion homeostasis. We found that the Ca2+, Mg2+, K+, and Cl− concentrations in the SR and the cytoplasm self-adjust within just a few heartbeats with only very small changes in Mg2+, K+, and Cl− concentrations and membrane voltages (just a few percent). However, those small changes were enough to compensate for the large heart-rate-dependent changes in SR and cytoplasmic Ca2+ concentrations in the new steady state. The modeling suggests that ion adaptation to increases in heart rate is inherent to the system and that physiological changes that increase contractility and cardiac output are accommodated by the same self-adjusting mechanism of producing small changes in ion driving forces. Our findings also support the long-held hypothesis that SR membrane potentials are small (~1–2mV).

Calcium/calmodulin-dependent kinase II and nitric oxide synthase 1-dependent modulation of ryanodine receptors during β-adrenergic stimulation is restricted to the dyadic cleft.

Key points The dyadic cleft, where coupled ryanodine receptors (RyRs) reside, is thought to serve as a microdomain for local signalling, as supported by distinct modulation of coupled RyRs dependent on Ca2+/calmodulin‐dependent kinase II (CaMKII) activation during high‐frequency stimulation.Sympathetic stimulation through β‐adrenergic receptors activates an integrated signalling cascade, enhancing Ca2+ cycling and is at least partially mediated through CaMKII.Here we report that CaMKII activation during β‐adrenergic signalling is restricted to the dyadic cleft, where it enhances activity of coupled RyRs thereby contributing to the increase in diastolic events. Nitric oxide synthase 1 equally participates in the local modulation of coupled RyRs.In contrast, the increase in the Ca2+ content of the sarcoplasmic reticulum and related increase in the amplitude of the Ca2+ transient are primarily protein kinase A‐dependent.The present data extend the concept of microdomain signalling in the dyadic cleft and give perspectives for selective modulation of RyR subpopulations and diastolic events. In cardiac myocytes, β‐adrenergic stimulation enhances Ca2+ cycling through an integrated signalling cascade modulating L‐type Ca2+ channels (LTCCs), phospholamban and ryanodine receptors (RyRs). Ca2+/calmodulin‐dependent kinase II (CaMKII) and nitric oxide synthase 1 (NOS1) are proposed as prime mediators for increasing RyR open probability. We investigate whether this pathway is confined to the high Ca2+ microdomain of the dyadic cleft and thus to coupled RyRs. Pig ventricular myocytes are studied under whole‐cell voltage‐clamp and confocal line‐scan imaging with Fluo‐4 as a [Ca2+]i indicator. Following conditioning depolarizing pulses, spontaneous RyR activity is recorded as Ca2+ sparks, which are assigned to coupled and non‐coupled RyR clusters. Isoproterenol (ISO) (10 nm) increases Ca2+ spark frequency in both populations of RyRs. However, CaMKII inhibition reduces spark frequency in coupled RyRs only; NOS1 inhibition mimics the effect of CaMKII inhibition. Moreover, ISO induces the repetitive activation of coupled RyR clusters through CaMKII activation. Immunostaining shows high levels of CaMKII phosphorylation at the dyadic cleft. CaMKII inhibition reduces I CaL and local Ca2+ transients during depolarizing steps but has only modest effects on amplitude or relaxation of the global Ca2+ transient. In contrast, protein kinase A (PKA) inhibition reduces spark frequency in all RyRs concurrently with a reduction of sarcoplasmic reticulum Ca2+ content, Ca2+ transient amplitude and relaxation. In conclusion, CaMKII activation during β‐adrenergic stimulation is restricted to the dyadic cleft microdomain, enhancing LTCC‐triggered local Ca2+ release as well as spontaneous diastolic Ca2+ release whilst PKA is the major pathway increasing global Ca2+ cycling. Selective CaMKII inhibition may reduce potentially arrhythmogenic release without negative inotropy.

Sarcolemmal Ca2+-entry through L-type Ca2+ channels controls the profile of Ca2+-activated Cl− current in canine ventricular myocytes

Ca2+-activated Cl− current (ICl(Ca)) mediated by TMEM16A and/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profile of ICl(Ca) during an actual ventricular action potential (AP), however, is poorly understood.We aimed to study the profile of ICl(Ca) systematically under physiological conditions (normal Ca2+ cycling and AP voltage-clamp) as well as in conditions designed to change [Ca2+]i. The expression of TMEM16A and/or Bestrophin-3 in canine and human left ventricular myocytes was examined. The possible spatial distribution of these proteins and their co-localization with Cav1.2 was also studied.The profile of ICl(Ca), identified as a 9-anthracene carboxylic acid-sensitive current under AP voltage-clamp conditions, contained an early fast outward and a late inward component, overlapping early and terminal repolarizations, respectively. Both components were moderately reduced by ryanodine, while fully abolished by BAPTA, but not EGTA. [Ca2+]i was monitored using Fura-2-AM. Setting [Ca2+]i to the systolic level measured in the bulk cytoplasm (1.1μM) decreased ICl(Ca), while application of Bay K8644, isoproterenol, and faster stimulation rates increased the amplitude of ICl(Ca). Ca2+-entry through L-type Ca2+ channels was essential for activation of ICl(Ca). TMEM16A and Bestrophin-3 showed strong co-localization with one another and also with Cav1.2 channels, when assessed using immunolabeling and confocal microscopy in both canine myocytes and human ventricular myocardium.Activation of ICl(Ca) in canine ventricular cells requires Ca2+-entry through neighboring L-type Ca2+ channels and is only augmented by SR Ca2+-release. Substantial activation of ICl(Ca) requires high Ca2+ concentration in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA.

Activation of Reverse Na/Ca Exchanger by Skeletal Na Channel Isoform Increases Excitation-Contraction Coupling Efficiency in Rabbit Cardiomyocytes

Our prior work has shown that TTX-sensitive sodium current (INa) affects sarcoplasmic reticular (SR) Ca release. This current activates early reverse NCX. The resulting Ca entry primes the dyadic cleft, which appears to increase Ca channel coupling fidelity (we refer to this as the priming mechanism). The skeletal isoform Nav1.4 is the main TTX-sensitive Na channel expressed in adult rabbit ventricular cardiomyocytes. Here we tested the hypothesis that this is the principal isoform involved in the priming mechanism. We evoked action potentials (AP) in isolated rabbit ventricular cells loaded with fluo-4, and simultaneously recorded Ca transients before and after the application of either relatively low doses of TTX (100 nM) or the specific Nav1.4 inhibitor μ-Conotoxin GIIIB (1 μM). 100 nM of TTX will effectively block all skeletal and the neuronal Navs (Nav1.1 is also present with a low level of expression). However this concentration will have a negligible (5%) effect on the cardiac isoform. There was a more prominent AP upstroke velocity reduction in TTX (73±1%, n=4) than in GIIB (81±1%, n=6, P<0.05). This can be expected given the relative contribution of each Nav isoform to the phase 0 of the AP. However, the effects of either drug on SR Ca release (measured as the transient maximum upstroke velocity) were identical (69±0.03% in TTX (n=5) vs. 69±3% in GIIIB (n=6), P<0.05). Furthermore, this reduction in SR Ca release is comparable to the value that we obtained previously when total INa was inactivated with a ramp. This result suggests that the Nav skeletal isoform is the main Nav isoform involved in regulating the efficiency of excitation-contraction coupling in rabbit cardiomyocytes.

Novel Calmodulin Mutation (CALM3-A103V) Associated with CPVT Syndrome Activates Arrhythmogenic Ca Waves and Sparks

Introduction: Calmodulin (CaM) is an essential Ca2+ signaling protein in a wide range of biological processes. In the dyadic cleft CaM binds to RyR2, Cav1.2 and CaMKII. CaM mutations are associated with an autosomal dominant syndrome of sudden death that can present with clinical features of catecholaminergic polymorphic ventricular tachycardia (CPVT) or long QT syndrome (LQTS). CPVT-linked CaM mutations activate ryanodine receptor (RyR2) Ca release channels; whereas LQTS-CaMs have no effects on RyR2 channels but prolong the action potential by impairing L-type Ca current (LTCC) inactivation. Here we report a novel CaM mutation – A103V – in CALM3, in a 10 year old female with episodes of exertion-induced syncope and normal QT interval consistent with CPVT. Her mother is mutation positive and also had a positive exercise stress test.Methods: CaM-A103V Ca binding affinity was measured by spectrofluorometry. Ca handling and LTCC were tested in mouse ventricular myocytes dialyzed with WT or the mutant CaM and compared to a published CPVT CaM mutant (N54I).Results: A103V modestly lowered CaM Ca binding affinity (3-fold reduction vs WT-CaM). In permeabilized myocytes, A103V-CaMs at a physiological intracellular concentration (100nM) promoted significantly higher spontaneous Ca wave and spark activity, a typical cellular phenotype of CPVT. Even a 1:4 mixture of A103V-CaM:WT-CaM activated Ca waves, demonstrating functional dominance. In voltage-clamped cardiomyocytes dialyzed with WT or mutant CaM, CaM-A103V had significantly less effects on LTCC inactivation compared to published CaM mutants that cause LQTS.Conclusion: A103V CaM shares functional characteristics with the established CPVT-CaM N54I. A small proportion of A103V-CaM is sufficient to evoke arrhythmogenic Ca disturbances, which explains the autosomal dominant inheritance.

A multiscale computational model of spatially resolved calcium cycling in cardiac myocytes: from detailed cleft dynamics to the whole cell concentration profiles.

Mathematical modeling of excitation-contraction coupling (ECC) in ventricular cardiac myocytes is a multiscale problem, and it is therefore difficult to develop spatially detailed simulation tools. ECC involves gradients on the length scale of 100 nm in dyadic spaces and concentration profiles along the 100 μm of the whole cell, as well as the sub-millisecond time scale of local concentration changes and the change of lumenal Ca2+ content within tens of seconds. Our concept for a multiscale mathematical model of Ca2+ -induced Ca2+ release (CICR) and whole cardiomyocyte electrophysiology incorporates stochastic simulation of individual LC- and RyR-channels, spatially detailed concentration dynamics in dyadic clefts, rabbit membrane potential dynamics, and a system of partial differential equations for myoplasmic and lumenal free Ca2+ and Ca2+-binding molecules in the bulk of the cell. We developed a novel computational approach to resolve the concentration gradients from dyadic space to cell level by using a quasistatic approximation within the dyad and finite element methods for integrating the partial differential equations. We show whole cell Ca2+-concentration profiles using three previously published RyR-channel Markov schemes.

Cytosolic Ca2+ buffering determines the intra-SR Ca2+ concentration at which cardiac Ca2+ sparks terminate

Single ryanodine receptor (RyR) Ca2+ flux amplitude (iCa-RyR) decreases as intra-sarcoplasmic reticulum (SR) Ca2+ levels fall during a cardiac Ca2+ spark. Since iCa-RyR drives the inter-RyR Ca2+-induced Ca2+ release (CICR) that underlies the spark, decreasing iCa-RyR may contribute to spark termination because RyRs that spontaneously close may stay closed. To test this possibility, we simultaneously measured local cytosolic and intra-SR ([Ca2+]cyto and [Ca2+]SR) during Ca2+ sparks in permeabilized rabbit ventricular myocytes. Local cytosolic or intra-SR Ca2+ dynamics were manipulated using Ca2+ buffers. Buffer manipulations applied in cells had no effect on individual RyR channels reconstituted in planar lipid bilayers. Presence of a fast cytosolic Ca2+ buffer (BAPTA) significantly suppressed Ca2+ spark activity and sparks terminated earlier at a higher than usual [Ca2+]SR level (∼80% vs. ∼62%). When cytosolic Ca2+ buffer power was reduced (i.e. cytosolic EGTA level decreased), sparks terminated later and at a lower than usual [Ca2+]SR level (∼45% vs. ∼62%). When intra-SR Ca2+ buffer power was increased, sparks also terminated later and at a lower than usual [Ca2+]SR (∼48% vs. ∼62%). These results suggest that cytosolic local control of inter-RyR CICR by iCa-RyR plays a substantial role during the spark termination process. Thus, alterations in local cytosolic Ca2+ handling dynamics in the dyadic cleft (Ca2+ buffering, extrusion, etc.) likely influence Ca2+ spark termination.

Reduced junctional Na+/Ca2+-exchanger activity contributes to sarcoplasmic reticulum Ca2+ leak in junctophilin-2-deficient mice.

Expression silencing of junctophilin-2 (JPH2) in mouse heart leads to ryanodine receptor type 2 (RyR2)-mediated sarcoplasmic reticulum (SR) Ca(2+) leak and rapid development of heart failure. The mechanism and physiological significance of JPH2 in regulating RyR2-mediated SR Ca(2+) leak remains elusive. We sought to elucidate the role of JPH2 in regulating RyR2-mediated SR Ca(2+) release in the setting of cardiac failure. Cardiac myocytes isolated from tamoxifen-inducible conditional knockdown mice of JPH2 (MCM-shJPH2) were subjected to confocal Ca(2+) imaging. MCM-shJPH2 cardiomyocytes exhibited an increased spark frequency width with altered spark morphology, which caused increased SR Ca(2+) leakage. Single channel studies identified an increased RyR2 open probability in MCM-shJPH2 mice. The increase in spark frequency and width was observed only in MCM-shJPH2 and not found in mice with increased RyR2 open probability with native JPH2 expression. Na(+)/Ca(2+)-exchanger (NCX) activity was reduced by 50% in MCM-shJPH2 with no detectable change in NCX expression. Additionally, 50% inhibition of NCX through Cd(2+) administration alone was sufficient to increase spark width in myocytes obtained from wild-type mice. Additionally, superresolution analysis of RyR2 and NCX colocalization showed a reduced overlap between RyR2 and NCX in MCM-shJPH2 mice. In conclusion, decreased JPH2 expression causes increased SR Ca(2+) leakage by directly increasing open probability of RyR2 and by indirectly reducing junctional NCX activity through increased dyadic cleft Ca(2+). This demonstrates two novel and independent cellular mechanisms by which JPH2 regulates RyR2-mediated SR Ca(2+) leak and heart failure development.

The Role of Dyadic Organization in Regulation of Sarcoplasmic Reticulum Ca2+ Handling during Rest in Rabbit Ventricular Myocytes

The dyadic organization of ventricular myocytes ensures synchronized activation of sarcoplasmic reticulum (SR) Ca2+ release during systole. However, it remains obscure how the dyadic organization affects SR Ca2+ handling during diastole. By measuring intraluminal SR Ca2+ ([Ca2+]SR) decline during rest in rabbit ventricular myocytes, we found that ∼76% of leaked SR Ca2+ is extruded from the cytosol and only ∼24% is pumped back into the SR. Thus, the majority of Ca2+ that leaks from the SR is removed from the cytosol before it can be sequestered back into the SR by the SR Ca2+-ATPase (SERCA). Detubulation decreased [Ca2+]SR decline during rest, thus making the leaked SR Ca2+ more accessible for SERCA. These results suggest that Ca2+ extrusion systems are localized in T-tubules. Inhibition of Na+-Ca2+ exchanger (NCX) slowed [Ca2+]SR decline during rest by threefold, however did not prevent it. Depolarization of mitochondrial membrane potential during NCX inhibition completely prevented the rest-dependent [Ca2+]SR decline. Despite a significant SR Ca2+ leak, Ca2+ sparks were very rare events in control conditions. NCX inhibition or detubulation increased Ca2+ spark activity independent of SR Ca2+ load. Overall, these results indicate that during rest NCX effectively competes with SERCA for cytosolic Ca2+ that leaks from the SR. This can be explained if the majority of SR Ca2+ leak occurs through ryanodine receptors in the junctional SR that are located closely to NCX in the dyadic cleft. Such control of the dyadic [Ca2+] by NCX play a critical role in suppressing Ca2+ sparks during rest.