Trigger Ca2 Research Articles

Ca2+ releasing process and nicotinic acid adenine dinucleotide phosphate

The paper is devoted to recognizing of nicotinic acid adenine dinucleotide phosphate (NAADP) as releaser of intracellular calcium from intracellular stores. It was revealed the current concepts of the mechanisms of NAADP synthesis inside the cell resuming involve enzyme CD38. The effect of NAADP was described in several cells and tissue preparation, as well as in sea urchin eggs. Briefly, it was characterized the acidic store of cells, that is sensitive to NAADP as it had been shown by different authors. Assumed mechanisms of Ca 2+ accumulating in acidic store predict involve proton gradient across membrane of acidic store. The channels structure of acidic store and endoplasmatic reticulum are considered as receptors to NAADP. Potential mechanisms for NAADP-induced calcium release was considered: direct model, trigger Ca 2+ -induced Ca 2+ -releasing model, promiscuous coupling model, conformational coupling model and also unifying hypothesis, that explains the differences between other mechanisms. Physiological processes in which NAADP is involved are described. Keywords : NAADP, acidic store, Ca 2+ , ryanodine receptor, two-pore channels.

PLCζ and the initiation of Ca2+ oscillations in fertilizing mammalian eggs

Mammalian eggs undergo a prolonged series of low frequency Ca2+ oscillations at fertilization. These Ca2+ oscillations are the immediate cause of egg activation. The Ca2+ oscillations in mouse eggs have been shown to be driven by increased InsP3 production. Substantial evidence now indicates that a sperm-derived phospholipase C-zeta (PLCζ) is the key molecule that causes these Ca2+ oscillations at fertilization. The fertilizing sperm is envisaged to introduce this essential molecule into the egg following gamete fusion. This review summarizes our current knowledge of how sperm PLCζ causes these oscillations and why it is so much more effective at triggering InsP3 production and Ca2+ oscillations in eggs, than other somatic isoforms of PLC. The molecular features of PLCζ and how they relate to the pattern of Ca2+ oscillations seen at fertilization are considered. We also discuss the evidence that PLCζ does not hydrolyze the conventional source of PI(4,5)P2 in the plasma membrane to make InsP3, but instead uses a distinct pool of PI(4,5)P2 present on intracellular vesicles. This leads us to suggest that sperm PLCζ may be targeted to these cytoplasmic vesicles by directly interacting with a specific but as yet unidentified egg PLCζ-binding protein.

The cardiac L-type calcium channel distal carboxy terminus autoinhibition is regulated by calcium

The L-type calcium channel (LTCC) provides trigger Ca(2+) for sarcoplasmic reticulum Ca-release, and LTCC function is influenced by interacting proteins including the LTCC distal COOH terminus (DCT) and calmodulin. DCT is proteolytically cleaved and reassociates with the LTCC complex to regulate calcium channel function. DCT reduces LTCC barium current (I(Ba,L)) in reconstituted channel complexes, yet the contribution of DCT to LTCC Ca(2+) current (I(Ca,L)) in cardiomyocyte systems is unexplored. This study tests the hypothesis that DCT attenuates cardiomyocyte I(Ca,L). We measured LTCC current and Ca(2+) transients with DCT coexpressed in murine cardiomyocytes. We also heterologously coexpressed DCT and Ca(V)1.2 constructs with truncations corresponding to the predicted proteolytic cleavage site, Ca(V)1.2Δ1801, and a shorter deletion corresponding to well-studied construct, Ca(V)1.2Δ1733. DCT inhibited I(Ba,L) in cardiomyocytes, and in human embryonic kidney (HEK) 293 cells expressing Ca(V)1.2Δ1801 and Ca(V)1.2Δ1733. Ca(2+)-CaM relieved DCT block in cardiomyocytes and HEK cells. The selective block of I(Ba,L) combined with Ca(2+)-CaM effects suggested that DCT-mediated blockade may be relieved under conditions of elevated Ca(2+). We therefore tested the hypothesis that DCT block is dynamic, increasing under relatively low Ca(2+), and show that DCT reduced diastolic Ca(2+) at low stimulation frequencies but spared high frequency Ca(2+) entry. DCT reduction of diastolic Ca(2+) and relief of block at high pacing frequencies and under conditions of supraphysiological bath Ca(2+) suggests that a physiological function of DCT is to increase the dynamic range of Ca(2+) transients in response to elevated pacing frequencies. Our data motivate the new hypothesis that DCT is a native reverse use-dependent inhibitor of LTCC current.

SubstanceP prevents 1-methyl-4-phenylpyridinium-induced cytotoxicity through inhibition of apoptosis via neurokinin-1 receptors in MES23.5 cells.

[Sar9, Met(O2)11] termed Substance P (SP), is an effective and selective agonist for the neurokinin‑1 (NK‑1) receptors, which are synthetic peptides, similar in structure to SP. SP is an important neurotransmitter or neuromodulator mediated by neurokinin receptors, namely the SP receptor in the central nervous system. The excitatory effects induced by SP may be selectively inhibited by a neurokinin‑1 receptor antagonist, such as SR140333B. It has been proposed that Parkinson's disease (PD) is primarily caused by the loss of trophic peptidergic neurotransmitter, possibly SP, which may lead to the degeneration of neurons. In previous studies, 1‑methyl‑4‑phenylpyridinium (MPP+) has been frequently utilized to establish animal or cell models of PD. In the present study, to further investigate the effects of SP in PD, MPP+ was employed to investigate the promising anti‑apoptotic effects of SP, and examine the underlying mechanisms of the pathology in the MES23.5 dopaminergic cell line. The results indicated that MPP+‑triggered apoptosis was prevented by treatment with SP. SP treatment also decreased the MPP+‑triggered Ca2+ influx, caspase‑3 re‑activity, reactive oxygen species production and mitochondrial membrane potential decrease. Treatment with MPP+ also induced phosphorylation of c‑Jun N‑terminal kinase and p38 mitogen‑activated protein kinase. In addition, treatment with SP inhibited the MPP+‑triggered neurotoxicity in MES23.5 cells. However, no changes were observed in SR140333B+SP+MPP+‑treated MES23.5 cell lines. In conclusion, SP could protect the cells from MPP+‑induced cytotoxicity by inhibiting the apoptosis via NK-1 receptors.

The rationale for the application of laserophoresis of biologically active compounds

Laserophoresis is a technique for the transcutaneous administration of biologically active compounds by means of low-intensity laser radiation (LFLR). It is currently regarded as a most promising method for the integrated application of a pharmaceutical substance and a physical factor. At present laserophoresis of various medicinal preparations is successfully used after preliminary experimental studies of their phoretic properties for the treatment of various inflammatory and dystrophic conditions as well as for the prevention of skin ageing. The most important route for the administration of the majority of drug preparations is through the shunts provided by perspiratory glands and hair follicles. Another essential factor determining the potential possibility of drug penetration through the skin is the characteristic of the substance chosen for the administration, such as its molecular weight, chemical structure, conformation, and hydrophilic properties. However, the most likely mechanism underlying the transport of the substance through the glandular cells of perspiratory glands and epithelial cells of hair follicles is pinocytosis, i.e. the process integrating exocytosis and endocytosis. To-day, the majority of the researchers lay emphasis on thermodynamic triggering of Ca2+-dependent processes as the primary mechanism behind the biological action of low-intensity laser radiation. Both exocytosis and endocytosis being the Ca2+-dependent processes, the liberation of Ca2+-ions under the influence of LFLR causes the activation of pinocytosis as a whole.

Role of PI3Kα and sarcolemmal ATP-sensitive potassium channels in epoxyeicosatrienoic acid mediated cardioprotection

Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid that have known cardioprotective properties. While the mechanism(s) remains unknown, evidence suggests that phosphoinositide 3-kinase (PI3K) and sarcolemmal ATP-sensitive potassium channels (pmK(ATP)) are important. However the role of specific PI3K isoforms and corresponding intracellular mechanisms remains unknown. To study this, mice hearts were perfused in Langendorff mode for 40 min of baseline and subjected to 20 or 30 min of global no-flow ischemia followed by 40 min of reperfusion. C57BL6 mice perfused with 11,12-EET (1 μM) had improved postischemic recovery, whereas co-perfusion with PI3Kα inhibitor, PI-103 (0.1 μM), abolished the EET-mediated effect. In contrast, blocking of PI3Kβ or PI3Kγ isoforms failed to inhibit EET-mediated cardioprotection. In addition to the improved post-ischemic recovery, increased levels of p-Akt, decreased calcineurin activity and decreased translocation of proapoptotic protein BAD to mitochondria were noted in EET-treated hearts. Perfusion of 11,12-EET to Kir6.2 deficient mice (pmK(ATP)) failed to improve postischemic recovery, decrease calcineurin activity and translocation of proapoptotic protein BAD, however increased levels of p-Akt were still observed. Patch-clamp experiments demonstrated that 11,12-EET could not activate pmK(ATP) currents in myocytes pre-treated with PI-103. Mechanistic studies in H9c2 cells demonstrate that 11,12-EET limits anoxia-reoxygenation triggered Ca(2+) accumulation and maintains mitochondrial ΔΨm compared to controls. Both PI-103 and glibenclamide (10 μM, pmK(ATP) inhibitor) abolished EET cytoprotection. Together our data suggest that EET-mediated cardioprotection involves activation of PI3Kα, upstream of pmK(ATP), which prevents Ca(2+) overload and maintains mitochondrial function.

Waveless mammalian muscle

Waveless mammalian muscle

Extracellular ATP triggers proteolysis and cytosolic Ca2+ rise in Plasmodium berghei and Plasmodium yoelii malaria parasites

BackgroundPlasmodium has a complex cell biology and it is essential to dissect the cell-signalling pathways underlying its survival within the host.MethodsUsing the fluorescence resonance energy transfer (FRET) peptide substrate Abz-AIKFFARQ-EDDnp and Fluo4/AM, the effects of extracellular ATP on triggering proteolysis and Ca2+ signalling in Plasmodium berghei and Plasmodium yoelii malaria parasites were investigated.ResultsThe protease activity was blocked in the presence of the purinergic receptor blockers suramin (50 μM) and PPADS (50 μM) or the extracellular and intracellular calcium chelators EGTA (5 mM) and BAPTA/AM (25, 100, 200 and 500 μM), respectively for P. yoelii and P. berghei. Addition of ATP (50, 70, 200 and 250 μM) to isolated parasites previously loaded with Fluo4/AM in a Ca2+-containing medium led to an increase in cytosolic calcium. This rise was blocked by pre-incubating the parasites with either purinergic antagonists PPADS (50 μM), TNP-ATP (50 μM) or the purinergic blockers KN-62 (10 μM) and Ip5I (10 μM). Incubating P. berghei infected cells with KN-62 (200 μM) resulted in a changed profile of merozoite surface protein 1 (MSP1) processing as revealed by western blot assays. Moreover incubating P. berghei for 17 h with KN-62 (10 μM) led to an increase in rings forms (82% ± 4, n = 11) and a decrease in trophozoite forms (18% ± 4, n = 11).ConclusionsThe data clearly show that purinergic signalling modulates P. berghei protease(s) activity and that MSP1 is one target in this pathway.

Triggering of Ca2+ signals by NAADP-gated two-pore channels: a role for membrane contact sites?

NAADP (nicotinic acid-adenine dinucleotide phosphate) is a potent Ca2+-mobilizing messenger implicated in many Ca2+-dependent cellular processes. It is highly unusual in that it appears to trigger Ca2+ release from acidic organelles such as lysosomes. These signals are often amplified by archetypal Ca2+ channels located in the endoplasmic reticulum. Recent studies have converged on the TPCs (two-pore channels) which localize to the endolysosomal system as the likely primary targets through which NAADP mediates its effects. 'Chatter' between TPCs and endoplasmic reticulum Ca2+ channels is disrupted when TPCs are directed away from the endolysosomal system. This suggests that intracellular Ca2+ release channels may be closely apposed, possibly at specific membrane contact sites between acidic organelles and the endoplasmic reticulum.

Depolarization-Induced Intracellular Ca2+ Release in Postganglionic Sympathetic Neurons from Adult Mice

Ca2+ influx through voltage-activated plasmalemmal Ca2+ channels provides a trigger for Ca2+ release from internal Ca2+ stores in mammalian postganglionic sympathetic neurons. Here we examined whether a Ca2+ influx-independent, depolarization-induced component contributes to the rise in intracellular Ca2+. Exposure of postganglionic sympathetic neurons isolated from adult mice to a high K+ (80 mM), normal Ca2+ (2 mM) solution for 30 s caused sustained membrane depolarizations from −58.9±3.4 mV to −12.8±0.8 mV (mean±SEM; 38 cells) and increases in fluo-4 ΔF/F0 (F indicates fluorescence intensity, and F0 indicates F at baseline), which rapidly resolved upon repolarization (left Figure; peak ΔF/F0 averaged 5.20±0.38). Superfusion with a high K+, Ca2+-free solution caused depolarizations of similar magnitude and small-amplitude increases in ΔF/F0 (mean peak ΔF/F0 = 0.41±0.03; 29 cells) with slow decay upon repolarization (right Figure). Thapsigargin (1 μM) or the IP3 receptor inhibitor 2-APB (20 μM), but not ryanodine (20 μM) or nifedipine (50 μM), abrogated Ca2+ rises evoked by high K+ in the absence of external Ca2+ without affecting the membrane voltage response. Thus, sympathetic neurons exhibit depolarization-induced Ca2+ release from IP3-sensitive stores, linking electrical activity and a rise in cytoplasmic Ca2+.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Disruption and stabilization of β-cell actin microfilaments differently influence insulin secretion triggered by intracellular Ca 2+ mobilization or store-operated Ca 2+ entry

Latrunculin depolymerizes and jasplakinolide polymerizes β-cell actin microfilaments. Both increase insulin secretion when Ca 2+ enters β-cells during depolarization by glucose, sulfonylureas or potassium. Mouse islets were held hyperpolarized with diazoxide, and stimulated with acetylcholine to test the role of microfilaments in insulin secretion triggered by intracellular Ca 2+ mobilization and store-operated Ca 2+ entry (SOCE). Jasplakinolide slightly attenuated Ca 2+ mobilization and did not affect SOCE, but consistently inhibited the attending insulin secretion. Latrunculin did not affect Ca 2+ changes induced by acetylcholine, but consistently increased insulin secretion, its effect being larger in response to Ca 2+ entry than to Ca 2+ mobilization. Microfilaments have thus a distinct impact on exocytosis of insulin granules depending on the source of triggering Ca 2+.

Visualization of flow-induced ATP release and triggering of Ca2+ waves at caveolae in vascular endothelial cells

Endothelial cells (ECs) release ATP in response to shear stress, a fluid mechanical force generated by flowing blood but, although its release has a crucial role in controlling a variety of vascular functions by activating purinergic receptors, the mechanism of ATP release has never been established. To analyze the dynamics of ATP release, we developed a novel chemiluminescence imaging method by using cell-surface-attached firefly luciferase and a CCD camera. Upon stimulation of shear stress, cultured human pulmonary artery ECs simultaneously released ATP in two different manners, a highly concentrated, localized manner and a less concentrated, diffuse manner. The localized ATP release occurred at caveolin-1-rich regions of the cell membrane, and was blocked by caveolin-1 knockdown with siRNA and the depletion of plasma membrane cholesterol with methyl-β-cyclodexrin, indicating involvement of caveolae in localized ATP release. Ca(2+) imaging with Fluo-4 combined with ATP imaging revealed that shear stress evoked an increase in intracellular Ca(2+) concentration and the subsequent Ca(2+) wave that originated from the same sites as the localized ATP release. These findings suggest that localized ATP release at caveolae triggers shear-stress-dependent Ca(2+) signaling in ECs.

The dual control of insulin secretion by glucose involves triggering and amplifying pathways in β-cells

This review outlines the two pathways that interact in β-cells to ensure temporal and amplitude control of insulin secretion by glucose. The most well known triggering pathway involves the following steps: acceleration of glucose metabolism, closure of ATP-sensitive potassium channels, depolarization, influx of Ca 2+ through voltage-gated calcium channels, and a rise in the concentration of cytosolic ionized Ca 2+ that triggers exocytosis of insulin-containing granules. This classic sequence is, however, incomplete. Additional mechanisms, involving other channels, are necessarily implicated in the production of the triggering Ca 2+ signal. It is also clear that the effect of glucose on insulin secretion would be poor if Ca 2+-induced exocytosis was not markedly augmented (approximately doubled) through a metabolic amplifying pathway, mechanistically distinct from neurohormonal amplifying pathways. This metabolic amplifying pathway is physiologically relevant for both phases of glucose-induced insulin secretion and for the potentiation, by glucose, of insulin secretion triggered by non-metabolized secretagogues (e.g. arginine). Three important challenges for future studies will be to identify the additional targets mediating control of the triggering Ca 2+ signal by glucose, to elucidate the cellular mechanisms of the metabolic amplifying pathway and to determine the contribution of each pathway in the alterations of insulin secretion in type 2 diabetic patients.

Selective Activation of the Transcription Factor NFAT1 by Calcium Microdomains near Ca2+ Release-activated Ca2+ (CRAC) Channels

NFATs are a family of Ca(2+)-dependent transcription factors that play a central role in the morphogenesis, development, and physiological activities of numerous distinct cell types and organ systems. Here, we visualize NFAT1 movement in and out of the nucleus in response to transient activation of store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels in nonexcitable cells. We show that NFAT migration is exquisitely sensitive to Ca(2+) microdomains near open CRAC channels. Another Ca(2+)-permeable ion channel (TRPC3) was ineffective in driving NFAT1 to the nucleus. NFAT1 movement is temporally dissociated from the time course of the Ca(2+) signal and remains within the nucleus for 10 times longer than the duration of the trigger Ca(2+) signal. Kinetic analyses of each step linking CRAC channel activation to NFAT1 nuclear residency reveals that the rate-limiting step is transcription factor exit from the nucleus. The slow deactivation of NFAT provides a mechanism whereby Ca(2+)-dependent responses can be sustained despite the termination of the initial Ca(2+) signal and helps explain how gene expression in nonexcitable cells can continue after the primary stimulus has been removed.

Sinoatrial Nodal (SAN) Cells from Center or Peripheral SAN Area are not Functionally Different

SAN cells (SANC) determine the rate and rhythmicity of action potentials (AP) that emanate from the SAN to drive the heartbeat. Perspectives gleaned from SAN tissue have been interpreted to indicate that cells from the central SAN, i.e. smaller cells, control the SAN AP firing rate, while data from both SAN and isolated SANC argue that calcium cycling protein density is independent of SAN area and SANC size, and that spontaneous AP cycle length is independent of cell size. Since it is well documented that the gap junction protein, Connexin 43 (Cx43), is largely expressed in the peripheral vs. central SAN areas, we measured the properties of single SANC, employing Cx43 immunolabeling to distinguish cells isolated from the central (Cx43-negative) or peripheral (Cx43-positive) SAN areas.Freshly isolated adult rabbit SANC from the central area (Cx43-negative) are, on average, smaller (612.9±2.5μm2, n=340) than peripheral SANC (818.6±23.7μm2, n=188, p<0.001), but there is no difference in the spontaneous AP firing rate (3.07±0.13Hz, n=13, for Cx43-negative and 3.28±0.12Hz, n=23, for Cx43-positive). The AP amplitude and Maximum Diastolic Depolarization also did not differ, but compared to Cx43-positive SANC, the AP of Cx43-negative SANC has a slower AP upstroke (dV/dtmax (V/S): 8.12±1.31 vs. 13.4±0.62, p<0.001) and a longer repolarization time (APD75 (ms): 120.7±4.9 vs. 102.4±2.9, p<0.01). Linear regression analyses failed to detect any significant correlations between any AP parameter and the cell size for both. Preliminary data does not show significant differences between Cx43-positive or negative SANC in the AP triggered global Ca2+-transient or spontaneous diastolic Local Ca2+ Releases. Our results indicate that although different in size, single isolated central and peripheral SANC, in the basal state, are not functionally different from each other in respect to average spontaneous AP cycle length.

T-Tubule Remodeling Causes Ca 2+ Cycling Defects during the Progression to Heart Failure in the Intact Spontaneously Hypertensive Rat Heart

Severe disruption of the t-tubule network in cardiac myocytes has been found in nearly all animal and human models of heart failure. T-tubule disorganization would be expected to cause alterations in the EC coupling because of a separation of junctional SR from trigger Ca entry and the increase in orphaned ryanodine receptors. However, little is currently known about changes in t-tubules during the early stages of disease development, before heart failure (HF) develops. The goal of this study was to measure the relationship between cardiac function, t-tubule organization and Ca2+ cycling defects in whole hearts during the progression to HF.

Blink and You'll See It

See related article, pages 210–218 Nearly 2 decades ago, local Ca2+ release events during diastole were first observed and named Ca2+ sparks.1 These were originally attributed to large releases from single ryanodine receptor (RyR) channels.1 Subsequently, Lipp and Niggli proposed that Ca2+ sparks could be the summation of smaller releases recruited from within the same cluster and coined the term calcium quarks.2 Arduous experiments from this group and others have indeed recorded very small release events proposed to be from smaller numbers of RyRs; however, such reports have remained limited.3,4 In this issue of Circulation Research , evidence is presented to show how small release events from within a RyR cluster are a key component of sarcoplasmic reticulum (SR) Ca2+ release.5 Physiologically, during depolarization, Ca2+ sparks are triggered throughout the cells by a small influx of Ca2+ from the sarcolemmal Ca2+ channels. The resultant synchronous rise in Ca2+ is observed as the systolic Ca2+ transient. This global Ca2+ release has been used to derive a measure of RyR function termed the “gain” of coupling between trigger Ca2+ influx and Ca2+ release. This yields a whole-cell measure of RyR function; changes in this gain infer changes in the RyR behavior, which may be attributable to several factors, including the SR Ca2+ load, or the sensitivity of the channels themselves (see Heinzel et al6 for review). As a more direct measure for RyR behavior, diastolic Ca2+ sparks have been characterized, in particular in studies of pathological conditions. Measurements of diastolic RyR behavior have been used to calculate loss of Ca2+ from the SR and how this would affect the SR Ca2+ load (eg, in Neef et al7 …

Modeling CICR in rat ventricular myocytes: voltage clamp studies

BackgroundThe past thirty-five years have seen an intense search for the molecular mechanisms underlying calcium-induced calcium-release (CICR) in cardiac myocytes, with voltage clamp (VC) studies being the leading tool employed. Several VC protocols including lowering of extracellular calcium to affect Ca2+ loading of the sarcoplasmic reticulum (SR), and administration of blockers caffeine and thapsigargin have been utilized to probe the phenomena surrounding SR Ca2+ release. Here, we develop a deterministic mathematical model of a rat ventricular myocyte under VC conditions, to better understand mechanisms underlying the response of an isolated cell to calcium perturbation. Motivation for the study was to pinpoint key control variables influencing CICR and examine the role of CICR in the context of a physiological control system regulating cytosolic Ca2+ concentration ([Ca2+]myo).MethodsThe cell model consists of an electrical-equivalent model for the cell membrane and a fluid-compartment model describing the flux of ionic species between the extracellular and several intracellular compartments (cell cytosol, SR and the dyadic coupling unit (DCU), in which resides the mechanistic basis of CICR). The DCU is described as a controller-actuator mechanism, internally stabilized by negative feedback control of the unit's two diametrically-opposed Ca2+ channels (trigger-channel and release-channel). It releases Ca2+ flux into the cyto-plasm and is in turn enclosed within a negative feedback loop involving the SERCA pump, regulating[Ca2+]myo.ResultsOur model reproduces measured VC data published by several laboratories, and generates graded Ca2+ release at high Ca2+ gain in a homeostatically-controlled environment where [Ca2+]myo is precisely regulated. We elucidate the importance of the DCU elements in this process, particularly the role of the ryanodine receptor in controlling SR Ca2+ release, its activation by trigger Ca2+, and its refractory characteristics mediated by the luminal SR Ca2+ sensor. Proper functioning of the DCU, sodium-calcium exchangers and SERCA pump are important in achieving negative feedback control and hence Ca2+ homeostasis.ConclusionsWe examine the role of the above Ca2+ regulating mechanisms in handling various types of induced disturbances in Ca2+ levels by quantifying cellular Ca2+ balance. Our model provides biophysically-based explanations of phenomena associated with CICR generating useful and testable hypotheses.

A21 Altered calcium kinetics in skeletal muscle fibres of the R6/2 mouse model of HD

Background and aimsSome of the most obvious peripheral tissue changes in Huntington's disease (HD) are found in skeletal muscle. Alterations in morphology, gene expression pattern, energy metabolism and differentiation have...

Activation of reverse Na+-Ca2+exchange by the Na+current augments the cardiac Ca2+transient: evidence from NCX knockout mice

The hypothesis that Na(+) influx during the action potential (AP) activates reverse Na(+)-Ca(2+) exchange (NCX) and subsequent entry of trigger Ca(2+) is controversial. We tested this hypothesis by monitoring intracellular Ca(2+) before and after selective inactivation of I(Na) prior to a simulated action potential in patch-clamped ventricular myocytes isolated from adult wild-type (WT) and NCX knockout (KO) mice. First, we inactivated I(Na) using a ramp prepulse to 45 mV. In WT cells, inactivation of I(Na) decreased the Ca(2+) transient amplitude by 51.1 +/- 4.6% (P < 0.001, n = 14) and reduced its maximum release flux by 53.0 +/- 4.6% (P < 0.001, n = 14). There was no effect on diastolic Ca(2+). In striking contrast, Ca(2+) transients in NCX KO cardiomyocytes were unaffected by the presence or absence of I(Na) (n = 8). We obtained similar results when measuring trigger Ca(2+) influx in myocytes with depleted sarcoplasmic reticulum. In WT cells, inactivation of I(Na) decreased trigger Ca(2+) influx by 37.8 +/- 6% and maximum rate of flux by 30.6 +/- 7.7% at 2.5 mm external Ca(2+) (P < 0.001 and P < 0.05, n = 9). This effect was again absent in the KO cells (n = 8). Second, exposure to 10 mum tetrodotoxin to block I(Na) also reduced the Ca(2+) transients in WT myocytes but not in NCX KO myocytes. We conclude that I(Na) and reverse NCX modulate Ca(2+) release in murine WT cardiomyocytes by augmenting the pool of Ca(2+) that triggers ryanodine receptors. This is an important mechanism for regulation of Ca(2+) release and contractility in murine heart.