Aleksey V. Zima

Endothelin-1–Induced Arrhythmogenic Ca2+ Signaling Is Abolished in Atrial Myocytes of Inositol-1,4,5-Trisphosphate(IP3)–Receptor Type 2–Deficient Mice

Recent studies have suggested that inositol-1,4,5-trisphosphate-receptor (IP3R)–mediated Ca2+ release plays an important role in the modulation of excitation–contraction coupling (ECC) in atrial tissue and the generation of arrhythmias, specifically chronic atrial fibrillation (AF). IP3R type-2 (IP3R2) is the predominant IP3R isoform expressed in atrial myocytes. To determine the role of IP3R2 in atrial arrhythmogenesis and ECC, we generated IP3R2-deficient mice. Our results revealed that endothelin-1 (ET-1) stimulation of wild-type (WT) atrial myocytes caused an increase in basal [Ca2+]i, an enhancement of action potential (AP)-induced [Ca2+]i transients, an improvement of the efficacy of ECC (increased fractional SR Ca2+ release), and the occurrence of spontaneous arrhythmogenic Ca2+ release events as the result of activation of IP3R-dependent Ca2+ release. In contrast, ET-1 did not alter diastolic [Ca2+]i or cause spontaneous Ca2+ release events in IP3R2-deficient atrial myocytes. Under basal conditions the spatio-temporal properties (amplitude, rise-time, decay kinetics, and spatial spread) of [Ca2+]i transients and fractional SR Ca2+ release were not different in WT and IP3R2-deficient atrial myocytes. WT and IP3R2-deficient atrial myocytes also showed a significant and very similar increase in the amplitude of AP-dependent [Ca2+]i transients and Ca2+ spark frequency in response to isoproterenol stimulation, suggesting that both cell types maintained a strong inotropic reserve. No compensatory changes in Ca2+ regulatory protein expression (IP3R1, IP3R3, RyR2, NCX, SERCA2) or morphology of the atria could be detected between WT and IP3R2-deficient mice. These results show that lack of IP3R2 abolishes the positive inotropic effect of neurohumoral stimulation with ET-1 and protects from its arrhythmogenic effects.

Inositol‐1,4,5‐trisphosphate‐dependent Ca2+ signalling in cat atrial excitation–contraction coupling and arrhythmias

Inositol‐1,4,5‐trisphosphate (IP3)‐dependent Ca2+ release represents the major Ca2+ mobilizing pathway responsible for diverse functions in non‐excitable cells. In the heart, however, its role is largely unknown or controversial. In intact cat atrial myocytes, endothelin (ET‐1) increased basal [Ca2+]i levels, enhanced action potential‐evoked [Ca2+]i transients, caused [Ca2+]i transients with alternating amplitudes (Ca2+ alternans), and facilitated spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) in the form of Ca2+ sparks and arrhythmogenic Ca2+ waves. These effects were prevented by the IP3 receptor (IP3R) blocker aminoethoxydiphenyl borate (2‐APB), suggesting the involvement of IP3‐dependent SR Ca2+ release. In saponin‐permeabilized myocytes IP3 and the more potent IP3R agonist adenophostin increased basal [Ca2+]i and the frequency of spontaneous Ca2+ sparks. In the presence of tetracaine to eliminate Ca2+ release from ryanodine receptor (RyR) SR Ca2+ release channels, IP3 and adenophostin triggered unique elementary, non‐propagating IP3R‐dependent Ca2+ release events with amplitudes and kinetics that were distinctly different from classical RyR‐dependent Ca2+ sparks. The effects of IP3 and adenophostin were prevented by heparin and 2‐APB. The data suggest that IP3‐dependent Ca2+ release increases [Ca2+]i in the vicinity of RyRs and thus facilitates Ca2+‐induced Ca2+ release during excitation–contraction coupling. It is concluded that in the adult mammalian atrium IP3‐dependent Ca2+ release enhances atrial Ca2+ signalling and exerts a positive inotropic effect. In addition, by facilitating Ca2+ release, IP3 may also be an important component in the development of Ca2+‐mediated atrial arrhythmias.

Local calcium gradients during excitation–contraction coupling and alternans in atrial myocytes

Subcellular Ca2+ signalling during normal excitation‐contraction (E‐C) coupling and during Ca2+ alternans was studied in atrial myocytes using fast confocal microscopy and measurement of Ca2+ currents (ICa). Ca2+ alternans, a beat‐to‐beat alternation in the amplitude of the [Ca2+]i transient, causes electromechanical alternans, which has been implicated in the generation of cardiac fibrillation and sudden cardiac death. Cat atrial myocytes lack transverse tubules and contain sarcoplasmic reticulum (SR) of the junctional (j‐SR) and non‐junctional (nj‐SR) types, both of which have ryanodine‐receptor calcium release channels. During E‐C coupling, Ca2+ entering through voltage‐gated membrane Ca2+ channels (ICa) triggers Ca2+ release at discrete peripheral j‐SR release sites. The discrete Ca2+ spark‐like increases of [Ca2+]i then fuse into a peripheral ‘ring’ of elevated [Ca2+]i, followed by propagation (via calcium‐induced Ca2+ release, CICR) to the cell centre, resulting in contraction. Interrupting ICa instantaneously terminates j‐SR Ca2+ release, whereas nj‐SR Ca2+ release continues. Increasing the stimulation frequency or inhibition of glycolysis elicits Ca2+ alternans. The spatiotemporal [Ca2+]i pattern during alternans shows marked subcellular heterogeneities including longitudinal and transverse gradients of [Ca2+]i and neighbouring subcellular regions alternating out of phase. Moreover, focal inhibition of glycolysis causes spatially restricted Ca2+ alternans, further emphasising the local character of this phenomenon. When two adjacent regions within a myocyte alternate out of phase, delayed propagating Ca2+ waves develop at their border. In conclusion, the results demonstrate that (1) during normal E‐C coupling the atrial [Ca2+]i transient is the result of the spatiotemporal summation of Ca2+ release from individual release sites of the peripheral j‐SR and the central nj‐SR, activated in a centripetal fashion by CICR via ICa and Ca2+ release from j‐SR, respectively, (2) Ca2+ alternans is caused by subcellular alterations of SR Ca2+ release mediated, at least in part, by local inhibition of energy metabolism, and (3) the generation of arrhythmogenic Ca2+ waves resulting from heterogeneities in subcellular Ca2+ alternans may constitute a novel mechanism for the development of cardiac dysrhythmias.

Emerging roles of inositol 1,4,5-trisphosphate signaling in cardiac myocytes

Inositol 1,4,5-trisphosphate (IP(3)) is a ubiquitous intracellular messenger regulating diverse functions in almost all mammalian cell types. It is generated by membrane receptors that couple to phospholipase C (PLC), an enzyme which liberates IP(3) from phosphatidylinositol 4,5-bisphosphate (PIP(2)). The major action of IP(3), which is hydrophilic and thus translocates from the membrane into the cytoplasm, is to induce Ca(2+) release from endogenous stores through IP(3) receptors (IP(3)Rs). Cardiac excitation-contraction coupling relies largely on ryanodine receptor (RyR)-induced Ca(2+) release from the sarcoplasmic reticulum. Myocytes express a significantly larger number of RyRs compared to IP(3)Rs (~100:1), and furthermore they experience substantial fluxes of Ca(2+) with each heartbeat. Therefore, the role of IP(3) and IP(3)-mediated Ca(2+) signaling in cardiac myocytes has long been enigmatic. Recent evidence, however, indicates that despite their paucity cardiac IP(3)Rs may play crucial roles in regulating diverse cardiac functions. Strategic localization of IP(3)Rs in cytoplasmic compartments and the nucleus enables them to participate in subsarcolemmal, bulk cytoplasmic and nuclear Ca(2+) signaling in embryonic stem cell-derived and neonatal cardiomyocytes, and in adult cardiac myocytes from the atria and ventricles. Intriguingly, expression of both IP(3)Rs and membrane receptors that couple to PLC/IP(3) signaling is altered in cardiac disease such as atrial fibrillation or heart failure, suggesting the involvement of IP(3) signaling in the pathology of these diseases. Thus, IP(3) exerts important physiological and pathological functions in the heart, ranging from the regulation of pacemaking, excitation-contraction and excitation-transcription coupling to the initiation and/or progression of arrhythmias, hypertrophy and heart failure.

Ca2+ spark-dependent and -independent sarcoplasmic reticulum Ca2+ leak in normal and failing rabbit ventricular myocytes

Sarcoplasmic reticulum (SR) Ca2+ leak is an important component of cardiac Ca2+ signalling. Together with the SR Ca2+‐ATPase (SERCA)‐mediated Ca2+ uptake, diastolic Ca2+ leak determines SR Ca2+ load and, therefore, the amplitude of Ca2+ transients that initiate contraction. Spontaneous Ca2+ sparks are thought to play a major role in SR Ca2+ leak. In this study, we determined the quantitative contribution of sparks to SR Ca2+ leak and tested the hypothesis that non‐spark mediated Ca2+ release also contributes to SR Ca2+ leak. We simultaneously measured spark properties and intra‐SR free Ca2+ ([Ca2+]SR) after complete inhibition of SERCA with thapsigargin in permeabilized rabbit ventricular myocytes. When [Ca2+]SR declined to 279 ± 10 μm, spark activity ceased completely; however SR Ca2+ leak continued, albeit at a slower rate. Analysis of sparks and [Ca2+]SR revealed, that SR Ca2+ leak increased as a function of [Ca2+]SR, with a particularly steep increase at higher [Ca2+]SR (>600 μm) where sparks become a major pathway of SR Ca2+ leak. At low [Ca2+]SR (<300 μm), however, Ca2+ leak occurred mostly as non‐spark‐mediated leak. Sensitization of ryanodine receptors (RyRs) with low doses of caffeine increased spark frequency and SR Ca2+ leak. Complete inhibition of RyR abolished sparks and significantly decreased SR Ca2+ leak, but did not prevent it entirely, suggesting the existence of RyR‐independent Ca2+ leak. Finally, we found that RyR‐mediated Ca2+ leak was enhanced in myocytes from failing rabbit hearts. These results show that RyRs are the main, but not sole contributor to SR Ca2+ leak. RyR‐mediated leak occurs in part as Ca2+ sparks, but there is clearly RyR‐mediated but Ca2+ sparks independent leak.

Termination of Cardiac Ca2+ Sparks: Role of Intra-SR [Ca2+], Release Flux, and Intra-SR Ca2+ Diffusion

Ca2+ release from cardiac sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) is regulated by dyadic cleft [Ca2+] and intra-SR free [Ca2+] ([Ca2+]SR). Robust SR Ca2+ release termination is important for stable excitation–contraction coupling, and partial [Ca2+]SR depletion may contribute to release termination. Here, we investigated the regulation of SR Ca2+ release termination of spontaneous local SR Ca2+ release events (Ca2+ sparks) by [Ca2+]SR, release flux, and intra-SR Ca2+ diffusion. We simultaneously measured Ca2+ sparks and Ca2+ blinks (localized elementary [Ca2+]SR depletions) in permeabilized ventricular cardiomyocytes over a wide range of SR Ca2+ loads and release fluxes. Sparks terminated via a [Ca2+]SR-dependent mechanism at a fixed [Ca2+]SR depletion threshold independent of the initial [Ca2+]SR and release flux. Ca2+ blink recovery depended mainly on intra-SR Ca2+ diffusion rather than SR Ca2+ uptake. Therefore, the large variation in Ca2+ blink recovery rates at different release sites occurred because of differences in the degree of release site interconnection within the SR network. When SR release flux was greatly reduced, long-lasting release events occurred from well-connected junctions. These junctions could sustain release because local SR Ca2+ release and [Ca2+]SR refilling reached a balance, preventing [Ca2+]SR from depleting to the termination threshold. Prolonged release events eventually terminated at a steady [Ca2+]SR, indicative of a slower, [Ca2+]SR-independent termination mechanism. These results demonstrate that there is high variability in local SR connectivity but that SR Ca2+ release terminates at a fixed [Ca2+]SR termination threshold. Thus, reliable SR Ca2+ release termination depends on tight RyR regulation by [Ca2+]SR.

Biosensors to Measure Inositol 1,4,5-Trisphosphate Concentration in Living Cells with Spatiotemporal Resolution

Phosphoinositides participate in many signaling cascades via phospholipase C stimulation, which hydrolyzes phosphatidylinositol 4,5-bisphosphate, producing second messengers diacylglycerol and inositol 1,4,5-trisphosphate (InsP3). Destructive chemical approaches required to measure [InsP3] limit spatiotemporal understanding of subcellular InsP3 signaling. We constructed novel fluorescence resonance energy transfer-based InsP3 biosensors called FIRE (fluorescent InsP3-responsive element) by fusing plasmids encoding the InsP3-binding domain of InsP3 receptors (types 1–3) between cyan fluorescent protein and yellow fluorescent protein sequences. FIRE was expressed and characterized in COS-1 cells, cultured neonatal cardiac myocytes, and incorporated into an adenoviral vector for expression in adult cardiac ventricular myocytes. FIRE-1 exhibits an ∼11% increase in the fluorescence ratio (F530/F480) at saturating [InsP3] (apparent Kd = 31.3 ± 6.7 nm InsP3). In COS-1 cells, neonatal rat cardiac myocytes and adult cat ventricular myocytes FIRE-1 exhibited comparable dynamic range and a 10% increase in donor (cyan fluorescent protein) fluorescence upon bleach of yellow fluorescent protein, indicative of fluorescence resonance energy transfer. In FIRE-1 expressing ventricular myocytes endothelin-1, phenylephrine, and angiotensin II all produced rapid and spatially resolved increases in [InsP3] using confocal microscopy (with free [InsP3] rising to ∼30 nm). Local entry of intracellular InsP3 via membrane rupture by a patch pipette (containing InsP3)in myocytes expressing FIRE-1 allowed detailed spatiotemporal monitoring of intracellular InsP3 diffusion. Both endothelin-1-induced and direct InsP3 application (via pipette rupture) revealed that InsP3 diffusion into the nucleus occurs with a delay and blunted rise of [InsP3] versus cytosolic [InsP3]. These new biosensors allow studying InsP3 dynamics at high temporal and spatial resolution that will be powerful in under-standing InsP3 signaling in intact cells.

Effects of cytosolic NADH/NAD+ levels on sarcoplasmic reticulum Ca2+ release in permeabilized rat ventricular myocytes

In the heart ischaemic conditions induce metabolic changes known to have profound effects on Ca2+ signalling during excitation–contraction coupling. Ischaemia also affects the redox state of the cell. However, the role of cytosolic redox couples, such as the NADH/NAD+ redox system, for the regulation of Ca2+ homeostasis has remained elusive. We studied the effects of NADH and NAD+ on sarcoplasmic reticulum (SR) Ca2+ release in permeabilized rat ventricular myocytes as well as on Ca2+ uptake by SR microsomes and ryanodine receptor (RyR) single channel activity. Exposure of permeabilized myocytes to NADH (2 mm; [Ca2+]cyt= 100nm) decreased the frequency and the amplitude of spontaneous Ca2+ sparks by 62% and 24%, respectively. This inhibitory effect was reversed by NAD+ (2 mm) and did not depend on mitochondrial function. The inhibition of Ca2+ sparks by NADH was associated with a 52% decrease in SR Ca2+ load. Some of the effects observed with NADH may involve the generation of superoxide anion (O2−·) as they were attenuated to just a transient decrease of Ca2+ spark frequency by superoxide dismutase (SOD). O2−· generated in situ from the xanthine/xanthine oxidase reaction caused a slowly developing decrease of Ca2+ spark frequency and SR Ca2+ load by 44% and 32%, respectively. Furthermore, in studies with cardiac SR microsomes NADH slowed the rate of ATP‐dependent Ca2+ uptake by 39%. This effect also appeared to depend on O2−· formation. Single channel recordings from RyRs incorporated into lipid bilayers revealed that NADH (2 mm) inhibited the activity of RyR channels by 84%. However, NADH inhibition of RyR activity was O2−·‐independent. In summary, an increase of the cytoplasmic NADH/NAD+ ratio depresses SR Ca2+ release in ventricular cardiomyocytes. The effect appears to be mediated by direct NADH inhibition of RyR channel activity and by indirect NADH inhibition (O2−· mediated) of SR Ca2+‐ATPase activity with a subsequent decrease in SR Ca2+ content.

NADH Oxidase Activity of Rat Cardiac Sarcoplasmic Reticulum Regulates Calcium-Induced Calcium Release

Abstract— NADH and Ca2+ have important regulatory functions in cardiomyocytes related to excitation-contraction coupling and ATP production. To elucidate elements of these functions, we examined the effect of NADH on sarcoplasmic reticulum (SR) Ca2+ release and the mechanisms of this regulation. Physiological concentrations of cytosolic NADH inhibited ryanodine receptor type 2 (RyR2)–mediated Ca2+-induced Ca2+ release (CICR) from SR membranes (IC50=120 &mgr;mol/L) and significantly lowered single channel open probability. In permeabilized single ventricular cardiomyocytes, NADH significantly inhibited the amplitude and frequency of spontaneous Ca2+ release. Blockers of electron transport prevented the inhibitory effect of NADH on CICR in isolated membranes and permeabilized cells, as well as on the activity of RyR2 channels reconstituted in lipid bilayer. An endogenous NADH oxidase activity from rat heart copurified with SR enriched with RyR2. A significant contribution by mitochondria was excluded as NADH oxidation by SR exhibited >9-fold higher catalytic activity (8.8 &mgr;mol/mg protein per minute) in the absence of exogenous mitochondrial complex I (ubiquinone) or complex III (cytochrome c) electron acceptors, but was inhibited by rotenone and pyridaben (IC50=2 to 3 nmol/L), antimycin A (IC50=13 nmol/L), and diphenyleneiodonium (IC50=28 &mgr;mol/L). Cardiac junctional SR treated with [3H](trifluoromethyl)diazirinyl-pyridaben specifically labeled a single 23-kDa PSST-like protein. These data indicate that NADH oxidation is tightly linked to, and essential for, negative regulation of the RyR2 complex and is a likely component of an important physiological negative-feedback mechanism coupling SR Ca2+ fluxes and mitochondrial energy production.

IP3‐dependent nuclear Ca2+ signalling in the mammalian heart

In cardiac myocytes the type‐2 inositol 1,4,5‐trisphosphate receptor (IP3R2) is the predominant isoform expressed. The IP3R2 channel is localized to the SR and to the nuclear envelope. We studied IP3‐dependent nuclear Ca2+ signals ([Ca2+]Nuc) in permeabilized atrial myocytes and in isolated cardiac nuclei. In permeabilized myocytes IP3 (20 μm) and the more potent IP3R agonist adenophostin (5 μm) caused an elevation of [Ca2+]Nuc. An IP3‐dependent increase of [Ca2+]Nuc was still observed after pretreatment with tetracaine to block Ca2+ release from ryanodine receptors (RyRs), and the effect of IP3 was partially reversed or prevented by the IP3R blockers heparin and 2‐APB. Isolated nuclei were superfused with an internal solution containing the Ca2+ indicator fluo‐4 dextran. Exposure to IP3 (10 μm) and adenophostin (0.5 μm) increased [Ca2+]Nuc by 25 and 27%, respectively. [Ca2+]Nuc increased to higher levels than [Ca2+]Cyt immediately adjacent to the outer membrane of the nuclear envelope, suggesting that a significant portion of nuclear IP3 receptors are facing the nucleoplasm. When nuclei were pretreated with heparin or 2‐APB, IP3 failed to increase [Ca2+]Nuc. Isolated nuclei were also loaded with the membrane‐permeant low‐affinity Ca2+ probe fluo‐5N AM which compartmentalized into the nuclear envelope. Exposure to IP3 and adenophostin resulted in a decrease of the fluo‐5N signal that could be prevented by heparin. Stimulation of IP3R caused depletion of the nuclear Ca2+ stores by approximately 60% relative to the maximum depletion produced by the ionophores ionomycin and A23187. The fluo‐5N fluorescence decrease was particularly pronounced in the nuclear periphery, suggesting that the nuclear envelope may represent the predominant nuclear Ca2+ store. The data indicate that IP3 can elicit Ca2+ release from cardiac nuclei resulting in localized nuclear Ca2+ signals.