Trigger Ca2 Research Articles

Inteplay of Trigger CA2+ Waves and CA2+ Transient Alternans in Atrial Myocytes

We previously documented triggered calcium (Ca) wave (TCW) development in atrial myocytes from large animals during rapid pacing. The goal of this study was to investigate the relationship between Ca transient (CaT) alternans and TCWs in atrial myocytes from normal and failing hearts. Ca cycling was measured via confocal microscopy in atrial myocytes isolated from normal and failing canine hearts then loaded with Ca-sensitive fluorescent dyes. Normal atrial myocytes (NAMs; n = 84) or heart failure atrial myocytes (HFAMs; n = 162) were stimulated at increasingly rapid rates leading to TCW development at rates ≥ 3Hz in ∼54% of NAMs, but often at lower rates and with greater incidence in ∼88% HFAMs. TCW development was contingent on retaining high SR load with diastolic Ca rising 10-20% above that at basal pacing (1Hz) and fractional Ca-release (FCR) being ≈ 0.5 early during rapid pacing epochs. TCWs failed to develop if such FCR was or became ≪ 0.5. However, if such FCR was or became ≫ 0.5, typically few if any TCWs developed or previously developed TCWs ceased, and either larger magnitude TCW-negative CaTs or CaT alternans developed. Overall, CaT alternans developed in ∼29% of NAMs and ∼24% of HFAMs. Additionally, large TCWs often exhibited a periodicity of about 1 per 4-5 pacing cycles, thus manifesting as large slow-rate CaTs superimposed on small rapid-rate CaTs. In summary, atrial myocytes are predisposed to TCW formation during rapid pacing. Significantly increasing FCR is effective in abrogating TCWs, but often leads to the development of CaT alternans in their stead. Our data suggest that TCWs could cause early afterdepolarizations and, along with Ca alternans, could promote dispersion of repolarization, thus promoting atrial arrhythmogenesis (such as atrial fibrillation) particularly in the setting of heart failure.

Signature and Pathophysiology of Non-canonical Pores in Voltage-Dependent Cation Channels.

Opening and closing of voltage-gated cation channels allows the regulated flow of cations such as Na(+), K(+), and Ca(2+) across cell membranes, which steers essential physiological processes including shaping of action potentials and triggering Ca(2+)-dependent processes. Classical textbooks describe the voltage-gated cation channels as membrane proteins with a single, central aqueous pore. In recent years, however, evidence has accumulated for the existence of additional ion permeation pathways in this group of cation channels, distinct from the central pore, which here we collectively name non-canonical pores. Whereas the first non-canonical pores were unveiled only after making specific point mutations in the voltage-sensor region of voltage-gated Na(+) and K(+) channels, recent evidence indicates that they may also be functional in non-mutated channels. Moreover, several channelopathies have been linked to mutations that cause the appearance of a non-canonical ion permeation pathway as a new pathological mechanism. This review provides an integrated overview of the biophysical properties of non-canonical pores described in voltage-dependent cation channels (KV, NaV, Cav, Hv1, and TRPM3) and of the (patho)physiological impact of opening of such pores.

Modeling calcium regulation of contraction, energetics, signaling, and transcription in the cardiac myocyte.

Calcium (Ca(2+)) plays many important regulatory roles in cardiac muscle cells. In the initial phase of the action potential, influx of Ca(2+) through sarcolemmal voltage-gated L-type Ca(2+) channels (LCCs) acts as a feed-forward signal that triggers a large release of Ca(2+) from the junctional sarcoplasmic reticulum (SR). This Ca(2+) drives heart muscle contraction and pumping of blood in a process known as excitation-contraction coupling (ECC). Triggered and released Ca(2+) also feed back to inactivate LCCs, attenuating the triggered Ca(2+) signal once release has been achieved. The process of ECC consumes large amounts of ATP. It is now clear that in a process known as excitation-energetics coupling, Ca(2+) signals exert beat-to-beat regulation of mitochondrial ATP production that closely couples energy production with demand. This occurs through transport of Ca(2+) into mitochondria, where it regulates enzymes of the tricarboxylic acid cycle. In excitation-signaling coupling, Ca(2+) activates a number of signaling pathways in a feed-forward manner. Through effects on their target proteins, these interconnected pathways regulate Ca(2+) signals in complex ways to control electrical excitability and contractility of heart muscle. In a process known as excitation-transcription coupling, Ca(2+) acting primarily through signal transduction pathways also regulates the process of gene transcription. Because of these diverse and complex roles, experimentally based mechanistic computational models are proving to be very useful for understanding Ca(2+) signaling in the cardiac myocyte.

A novel neuronal channelopathy could provide the trigger for Ca2+ dysregulation and sympathetic impairment seen in the pro-hypertensive rat

A novel neuronal channelopathy could provide the trigger for Ca2+ dysregulation and sympathetic impairment seen in the pro-hypertensive rat

Bombyx mori prothoracicostatic peptide receptor is allosterically activated via a Gα(i/o)-protein-biased signalling cascade by Drosophila sex peptide.

In insects, molting and metamorphosis are strictly regulated by ecdysteroids. Ecdysteroid synthesis is positively or negatively controlled by several neuropeptides. The prothoracicostatic peptide (PTSP) BmPTSP (Bombyx mori prothoracicostatic peptide), isolated from the larval brain of B. mori, has been demonstrated to inhibit ecdysteroid synthesis in the prothoracic glands (PGs) [Hua et al. (1999) J. Biol. Chem. 274, 31169-31173]. More recently, the newly recognized B. mori receptor for Drosophila melanogaster sex peptide (DmSP) has been identified as a receptor for BmPTSP. However, details on the signalling pathways and physiological functions of this receptor have remained elusive. In the present paper, we report the functional characterization of the BmPTSP receptor (BmPTSPR)/sex peptide (SP) receptor (SPR) using both mammalian and insect cells. Synthetic DmSP shows the potential to inhibit forskolin (FSK) or adipokinetic hormone (AKH)-induced cAMP-response element (CRE)-driven luciferase (Luc) activity in a manner comparable with synthetic BmPTSP1. However, DmSP displayed a much lower activity in triggering Ca²⁺ mobilization and internalization than did BmPTSP1. Additionally, 6-carboxy-fluorescein fluorophore (FAM)-labelled DmSP and BmPTSP3 were found to bind specifically to BmPTSPR/SPR. The binding of FAM-DmSP was displaced by unlabelled DmSP, but not by unlabelled BmPTSP1 and, vice versa, the binding of FAM-BmPTSP3 was blocked by unlabelled BmPTSP3, but not by unlabelled DmSP. Moreover, internalization assays demonstrated that BmPTSP1, but not DmSP, evoked recruitment of the Bombyx non-visual arrestin, Kurtz, to the activated BmPTSPR/SPR in the plasma membrane. This was followed by induction of internalization. This suggests that BmPTSP1 is probably an endogenous ligand specific for BmPTSPR/SPR. We therefore designate this receptor BmPTSPR. In contrast, DmSP is an allosteric agonist that is biased towards Gα(i/o)-dependent cAMP production and away from Ca²⁺ mobilization and arrestin recruitment.

Opposite Changes of Ca2+ Wave Threshold and Fractional SR Ca2+ Release during SERCA Stimulation in Cardiomyocytes

In cardiac muscle, PKA-dependent phosphorylation of of the RyRs is proposed to increase their Ca2+ sensitivity. This mechanism could be arrhythmogenic via facilitation of spontaneous Ca2+ waves. Surprisingly, the level of Ca2+ inside the SR ([Ca2+]SR) needed to initiate such waves has been reported to increase upon β-adrenergic stimulation, an observation which cannot be easily reconciled with elevated Ca2+ sensitivity of the RyRs. We tested the hypothesis that this change of Ca2+ wave threshold could occur indirectly, subsequent to SERCA disinhibition. Ca2+ currents and transients, or cytosolic and intra-SR Ca2+ waves were simultaneously recorded with confocal line-scans in intact and permeabilized mouse cardiomyocytes with rhod-2 and fluo-5-N, respectively. We analyzed changes of several Ca2+ signaling parameters during specific SERCA stimulation by ochratoxin A (OTA) and jasmone. SERCA stimulation resulted in a substantial increase of Ca2+ wave thresholds (30±5.1%) and reduced fractional Ca2+ release. Faster Ca2+ wave decay and SR refilling confirmed SERCA acceleration. In patch-clamped myocytes, a decrease of fractional Ca2+ release together with a slowing of Ca2+ current inactivation and reduced EC-coupling gain was observed. A faster Ca2+ transient decay corroborated the pharmacological SERCA stimulation. These results suggest that SERCA stimulation alone can elevate the intra-SR threshold for the generation of Ca2+ waves, independently of RyR phosphorylation. Unexpectedly, this occurs without noticeably increasing spontaneous and triggered fractional Ca2+ release. This phenomenon could result from an intra-SR mechanism limiting CICR. Supported by SNF.

Similarities and Differences in Gating of the Two-Pore Channels TPC1 and TPC2

Endolysosomal Ca2+ homeostasis is implicated in several diseases and controls many endolysosomal functions. A key to understanding endolysosomal Ca2+ signaling to understand the function of the newly discovered endolysosomal PI(3,5)P2-regulated two-pore channels (TPCs) and their potential activation by NAADP. Our recent work concluded that the lysosomal TPC2 function as a NAADP and PI(3,5)P2-activated channel regulated by cytoplasmic Mg2+. The properties and gating of the mostly endosomal TPC1 are not known. Recording whole-organelle currents of enlarged endosomes expressing GFP-TPC1, we discovered that TPC1 is potently activated by PI(3,5)P2 and NAADP. Moreover, PI(3,5)P2 facilitates TPC1 activation by NAADP. Unlike, TPC2, the activity of TPC1 showed poor inhibition by Mg2+. Most notably, the concentration dependence of activation of TPC1 and TPC2 by NAADP are remarkably different. While activation of TPC2 by NAADP followed normal saturation dependence with no sign of inhibition by high NAADP concentration, activation of TPC1 by NAADP followed a bell shaped dependency, resembling the NAADP-mediated Ca2+ release in intact cells. These findings provide the first direct evidence for gating of TPC1 by NAADP and indicate that endosomal, rather than lysosomal, Ca2+ release is the key trigger Ca2+ pool that sensitizes the IP3-mediated Ca2+ release from the ER. The interaction between TPC1-expressing endosomes and the ER are being examined.

Abstract 18342: Inducible Cardiomyocyte-Specific Deletion of Stromal Interaction Molecules Results in Cardiomyopathy in Mice

Background: Stromal interaction molecules (STIM1 and STIM2) are sarco/endoplasmic calcium sensors ubiquitously expressed in mammalian cells. Upon Ca2+ store discharge STIM proteins oligomerize in proximity of the plasma membrane to activate the Ca2+ selective channels (Orai) and trigger Ca2+ entry. While this mechanism is widely accepted to be the main source of calcium in non-excitable cells, in cardiomyocytes the calcium-induced calcium-release is the major controller of myocardial function, and the presence of STIM in these cells appears somewhat puzzling. Here we investigated the role of STIM in cardiomyocytes and its role in regulating cardiac contractility and intracellular Ca2+ dynamics. Methods: We have generated an inducible cardiac-specific STIM1 knockout (STIM KO) mouse model to elucidate the role of STIM in the myocardium. Echocardiography was used to evaluate structure and function of the left ventricle. Heart rate was monitored continuously in conscious mice by telemetry. Cell dimensions, shortening, and relaxation were determined by videomicroscopy and calcium transients by fura 2. Results: Echocardiographic analyses revealed development of dilated cardiomyopathy with a significant reduction of left ventricular fractional shortening (39.91±4.05% vs. 22.41±7.92%, p<0.01). Histological and morphological analyses confirmed a dilated cardiomyopathy characterized by enlarged ventricular chambers. Electrocardiography showed higher heart rate in the STIM1 KO mice with no differences in P-wave duration and QRS interval while Q-T interval was reduced in the STIM1 KO mice as compared to the WT mice. Sarcomere length and cell shortening measurements in freshly isolated cardiomyocytes from STIM1 KO mice confirmed the reduced contractility associated with a reduction of time to 50% relaxation (283±13ms vs. 258±8ms, p<0.05) . Intracellular calcium transient analysis in STIM1 KO cardiomyocytes showed a higher peak amplitude and a time to 50% decay of calcium significantly accelerated. Conclusions: Our data demonstrate that STIM proteins play an important role in the maintaining normal cardiac function in the adult heart and reveal that STIM plays an important role during cardiac repolarization.

Indole derivatives inhibited the formation of bacterial biofilm and modulated Ca2+ efflux in diatom

Marine biofouling is a serious environmental problem worldwide. As an effort to find environmental friendly antifoulants, indole derivatives were determined for their activities to inhibit the growth of bacteria and diatom. The minimum inhibitory concentrations (MICs) of indole derivatives against bacteria were very low, especially for 6-chloroindole. It was proved that 6-chloroindole obviously inhibited the growth of bacteria, interfered with the formation of bacterial biofilm, destroyed bacterial cell morphology and also inhibited the growth of diatom Cylindrotheca sp. as well. By using noninvasive micro-test technique (NMT), 6-chloroindole triggered algal cellular Ca2+ efflux. The highest value was 72.03pmolcm−2s−1, 10.6 times of the control group. The present studies indicated that indole derivatives might have the potential to be new antifouling agents because of their excellent antibacterial and anti-algal activities. At the same time, Ca2+ efflux might be one of the mechanisms that indole derivatives inhibited the growth of diatom.

Convergence of Ionotropic and Metabotropic Signal Pathways upon Activation of P2X Receptors in Vascular Smooth Muscle Cells

Ionotropic P2X receptors (P2XRs) are involved in sympathetic control of the vascular tone; they mediate entry of Ca2+ in smooth muscle cells (SMCs), which results in depolarization of the latter and activation of voltage-gated L-type calcium channels. In addition, Ca2+ ions, after their entry into the cell, trigger Ca2+ release from the sarcoplasmic reticulum (SR) of SMCs via ryanodine receptors (RyRs), and this amplifies calcium signals. We found earlier that Ca2+ release mediated by inositol triphosphate (IP3) receptors (IP3Rs) also provides a considerable contribution to P2XR-mediated calcium signaling. Thus, a metabotropic signal pathway is a component of the calcium signaling system triggered by ionotropic P2XRs. Using confocal detection of changes in the intracellular Ca2+ concentration ([Ca2+] i ) and applications of the inhibitors of calcium channels (nicardipine, 5 μM), sarco-endoplasmic Ca2+ ATPase SERCA (CPA, 10 μM), IP3Rs (2-APB, 30 μM), RyRs (tetracaine, 100 μM), and phosphalipase C (PLC; U-73122, 2.5 μM), we estimated relative contributions of the above-mentioned four components to increase in the [Ca2+] i induced by the action of an agonist of P2XRs, α,β-meATP. The contributions of transmembrane Ca2+ entry via channels of P2XRs and calcium channels were comparable (11.0 ± 1.4 %, n = 14 and 8.0 ± 1.4 %, n = 14, respectively). The contribution of Ca2+ release via IP3Rs was found to be three times greater than that via RyRs (41 ± 5 %, n = 26 and 14 ± 7 %, n = 16, respectively). Blocking of calcium channels resulted in a sevenfold decrease in the contribution of IP3R-mediated Ca2+ release (from 41.0 to 5.6%); in this case, the contribution of RyR-mediated Ca2+ release underwent no significant changes. This fact allows us to suppose that there is a functional relation between activation of calcium channels and functioning of a metabotropic PLC/IP3-mediated signal cascade. The efficiency of inhibition of α,β-meATP-induced calcium responses by the blocker of PLC, on the one hand, and by the IP3R blocker and nicardipine, on the other hand, is comparable, and this fact agrees with the above hypothesis. According to our data, P2XR activation-induced increase in [Ca2+] I results not only from P2XR-mediated Ca2+ entry that triggers Ca2+ release via RyRs but also from Ca2+ release via IP3Rs. The latter process is realized due to the functioning of the PLC-mediated pathway, is in close relation with activation of calcium channels, and provides a dominant contribution in Ca2+ release from the stores after activation of the above ionotropic receptors.

Sperm-specific post-acrosomal WW-domain binding protein (PAWP) does not cause Ca2+ release in mouse oocytes.

Mature mammalian oocytes undergo a prolonged series of cytoplasmic calcium (Ca(2+)) oscillations at fertilization that are the cause of oocyte activation. The Ca(2+) oscillations in mammalian oocytes are driven via inositol 1,4,5-trisphosphate (IP3) generation. Microinjection of the sperm-derived phospholipase C-zeta (PLCζ), which generates IP3, causes the same pattern of Ca(2+) oscillations as observed at mammalian fertilization and it is thought to be the physiological agent that triggers oocyte activation. However, another sperm-specific protein, 'post-acrosomal WW-domain binding protein' (PAWP), has also been reported to elicit activation when injected into mammalian oocytes, and to produce a Ca(2+) increase in frog oocytes. Here we have investigated whether PAWP can induce fertilization-like Ca(2+) oscillations in mouse oocytes. Recombinant mouse PAWP protein was found to be unable to hydrolyse phosphatidylinositol 4,5-bisphosphate in vitro and did not cause any detectable Ca(2+) release when microinjected into mouse oocytes. Microinjection with cRNA encoding either the untagged PAWP, or yellow fluorescent protein (YFP)-PAWP, or luciferase-PAWP fusion proteins all failed to trigger Ca(2+) increases in mouse oocytes. The lack of response in mouse oocytes was despite PAWP being robustly expressed at similar or higher concentrations than PLCζ, which successfully initiated Ca(2+) oscillations in every parallel control experiment. These data suggest that sperm-derived PAWP is not involved in triggering Ca(2+) oscillations at fertilization in mammalian oocytes.

Cell death caused by quinazolinone HMJ-38 challenge in oral carcinoma CAL 27 cells: dissections of endoplasmic reticulum stress, mitochondrial dysfunction and tumor xenografts

BackgroundThis investigation clearly clarified the synthesized and antimitotic compound, 2-(3′-methoxyphenyl)-6-pyrrolidinyl-4-quinazolinone (HMJ-38), addressing its target and precise mechanism of action. We hypothesized that HMJ-38 might sensitize apoptotic death of human oral carcinoma CAL 27 cells in vitro and inhibit xenograft tumor growth in vivo. MethodsCell viability was assessed utilizing MTT assay. HMJ-38-treated cells represented DNA fragmentation using agarose gel electrophoresis as further evidenced using TUNEL staining. Flow cytometric analyses, immunoblotting and quantitative RT-PCR were applied for protein and gene expression. Antitumor xenograft study was employed. ResultsHMJ-38 concentration- and time-dependently reduced viability of CAL 27 cells. The effect of intrinsic molecules was signalized during HMJ-38 exposure with disruption of ΔΨm, MPT pore opening and the release of various events from mitochondria undergoing cell apoptosis. HMJ-38 also markedly facilitated G2/M phase arrest. HMJ-38 stimulated the activation of CDK1 activity that modulated phosphorylation on Ser70 of Bcl-2-mediated mitotic arrest and apoptosis. HMJ-38 triggered intracellular Ca2+ release and activated related pivotal hallmarks of ER stress. HMJ-38 in nude mice bearing CAL 27 tumor xenografts decreased tumor growth. Furthermore, HMJ-38 enhanced caspase-3 gene expression and protein level in xenotransplanted tumors. ConclusionsEarly roles of mitotic arrest, unfolded protein response and mitochondria-dependent signaling contributed to apoptotic CAL 27 cell demise induced by HMJ-38. In in vivo experiments, HMJ-38 also efficaciously suppressed tumor volume in a xenotransplantation model. General significanceThis finding might fully support a critical event for HMJ-38 via induction of apoptotic machinery and ER stress against human oral cancer cells.

Lipid Modulation of Calcium Flux through CaV2.3 Regulates Acrosome Exocytosis and Fertilization

Membrane lipid regulation of cell function is poorly understood. In early development, sterol efflux and the ganglioside GM1 regulate sperm acrosome exocytosis (AE) and fertilization competence through unknown mechanisms. Here, we show that sterol efflux and focal enrichment of GM1 trigger Ca(2+) influx necessary for AE through CaV2.3, whose activity has been highly controversial in sperm. Sperm lacking CaV2.3's pore-forming α1E subunit showed altered Ca(2+) responses, reduced AE, and a strong subfertility phenotype. Surprisingly, AE depended on spatiotemporal information encoded by flux through CaV2.3, not merely the presence/amplitude of Ca(2+) waves. Using studies in both sperm and voltage clamp of Xenopus oocytes, we define a molecular mechanism for GM1/CaV2.3 regulatory interaction, requiring GM1's lipid and sugar components and CaV2.3's α1E and α2δ subunits. Our results provide a mechanistic understanding of membrane lipid regulation of Ca(2+) flux and therefore Ca(2+)-dependent cellular and developmental processes such as exocytosis and fertilization.

Ucp2 Modulates Cellular Excitation Contraction Coupling via Mitochondrial Calcium Uptake

Introduction: Mitochondrial Ca2+ handling modulates the spatial and temporal profile of intracellular Ca2+ signaling. It is mainly mediated by the mitochondrial calcium uniporter (MCU). Uncoupling protein-2 (UCP2) a mitochondrial membrane protein has been proposed to be fundamental for MCU activity. Whether cellular excitation contraction (EC) coupling is modulated by UCP2 remains to be determined. Methods: To investigate this scenario, we isolated intact cardiomyocytes from adult wild-type (WT) and B6.129S4-Ucp2tm1Lowl/J (UCP2-/-) mice to analyze dual Ca2+ transients from the mitochondrial ((Ca2+)m) and intracellular compartment ((Ca2+)c) in the whole cell configuration using Rhod2-AM and Fluo4 pentapotassium salt. We also tested whether the previously described inhibitory effect of ATP on mitochondrial Ca2+ uptake could be mediated by UCP2. To detect possible regulatory effects on sarcolemmal Ca2+ uptake in UCP2-/- mice, trigger Ca2+ influx was measured using Ryanodine. Results: In cardiomyocytes of WT mice (Ca2+)m was selectively decreased by Ruthenium360 (Ru360) while (Ca2+)c was upregulated. The (Ca2+)m was decreased in UCP2-/- myocytes compared to WT while (Ca2+)c was unchanged suggesting a compensational modulation of EC coupling. Trigger Ca2+ influx was reduced in UCP2-/- vs. WT myocytes, presumably to prevent cytosolic Ca2+ overload. Additionally, we observed an inhibitory effect of ATP on mitochondrial Ca2+ uptake in myocytes of WT mice while no effect was observed in UCP2-/- mice. Conclusion: Our experiments suggest a possible role of MCU and UCP2 interaction in controlling mitochondrial Ca2+ uptake as well as EC coupling.

Non-Linear Relationship Between T-Tubule Remodeling and Sr Calcium Release in Failing Rat Ventricle

T-tubule remodeling in heart failure (HF) may be responsible for abnormal intracellular Ca2+ cycling. The goal of this study was to investigate the relationship between T-tubule remodeling and Ca2+ release. Ca2+ transients and t-tubules were measured in individual myocytes in intact hearts from spontaneously hypertensive rats maintained on the stage of a confocal microscope. T-tubule organization was measured as the organizational index (OI) using a fast Fourier transform. T-tubule remodeling occurred in a progressive manner through stages ranging from a highly organized network to regional loss of t-tubules and development of longitudinal tubules, finally showing gross loss of tubules and occasional bright punctate staining. Ca2+ release reflected the loss of T-tubules as an increase in the standard deviation (Heterogeneity Index, HI) in the time to 50% of peak Ca2+ (TR50) along the myocyte, indicating that the regional loss of T-tubules is responsible for increased heterogeneity of Ca2+ release. Rapid pacing (BCL= 350msec) increased HI at low OI compared to slow basal pacing (700msec), indicating that Ca2+ release heterogeneities are exaggerated at higher heart rates. The relationship between OI and TD50 HI was non-linear; there was little effect on TR50 HI until OI fell from a normal above 0.75 to below 0.65, where TD50 HI increased sharply with OI. The relationship between OI and TR50 HI was well-described by an exponential function. Our data show that extensive T-tubule remodeling in HF is required before significant changes occur in Ca2+ release, suggesting inherent redundancy in insuring that Ca2+ cycling is maintained during early T-tubule loss. T-tubule remodeling causes a non-linear increase in the variability in Ca2+ release along the cell length as Ca2+-induced Ca2+ release efficiency decreases with distance from trigger Ca2+ provided by the closest L-type Ca2+ channels.

Conditional Up-Regulation of SERCA2a Exacerbates Ventricular and Atrial Arrhythmias in the Setting of Catecholaminergic Polymorphic Ventricular Tachycardia

SERCA2a gene transfer is an emerging therapy for treating contractile dysfunction in heart failure. While improving contractile performance, SERCA2a overexpression has been shown to exacerbate arrhythmias, although beneficial effects of SERCA2a up-regulation on cardiac rhythm have also been reported. To examine the role of SERCA2a and the consequences of its acute up-regulation in arrhythmogenesis, we conditionally overexpressed SERCA2a in a genetic mouse model featuring catecholaminergic polymorphic ventricular tachycardia (CPVT) due to loss of calsequestrin 2 (CASQ2). CASQ2 knock-out (KO) mice were crossbred with doxycycline (DOX)-inducible SERCA2a transgenic mice to generate KO-TG mice. In-vivo ECG studies showed that uninduced KO-TG (DOX-) mice developed both ventricular and atrial arrhythmias in response to catecholamine challenge (isoproterenol, ISO or a combination of ISO and caffeine). Induction of SERCA2a (DOX+) markedly exacerbated both ventricular and atrial arrhythmias in response to ISO. Besides ventricular bigeminy, KO-TG (DOX+) also displayed frequent bursts of sustained ventricular ectopic beats that were not present in KO-TG (DOX-). Moreover, episodes of atrial rhythm disturbances in KO-TG (DOX+) mice occurred even under baseline conditions (no ISO). ISO further promoted atrial tachy- and brady-arrhythmias in the KO-TG (DOX+) mice. Consistent with the in-vivo studies, confocal Ca imaging in both ventricular and atrial myocytes demonstrated that acute SERCA2a overexpression significantly increased the rate of occurrence of diastolic spontaneous and triggered Ca release events. Thus, our results suggest that acute overexpression of SERCA2a exacerbates both ventricular and atrial arrhythmias in settings of CPVT by further elevating diastolic Ca release.

Dynamics of Intrinsic Dendritic Calcium Signaling during Tonic Firing of Thalamic Reticular Neurons

The GABAergic neurons of the nucleus reticularis thalami that control the communication between thalamus and cortex are interconnected not only through axo-dendritic synapses but also through gap junctions and dendro-dendritic synapses. It is still unknown whether these dendritic communication processes may be triggered both by the tonic and the T-type Ca2+ channel-dependent high frequency burst firing of action potentials displayed by nucleus reticularis neurons during wakefulness and sleep, respectively. Indeed, while it is known that activation of T-type Ca2+ channels actively propagates throughout the dendritic tree, it is still unclear whether tonic action potential firing can also invade the dendritic arborization. Here, using two-photon microscopy, we demonstrated that dendritic Ca2+ responses following somatically evoked action potentials that mimic wake-related tonic firing are detected throughout the dendritic arborization. Calcium influx temporally summates to produce dendritic Ca2+ accumulations that are linearly related to the duration of the action potential trains. Increasing the firing frequency facilitates Ca2+ influx in the proximal but not in the distal dendritic compartments suggesting that the dendritic arborization acts as a low-pass filter in respect to the back-propagating action potentials. In the more distal compartment of the dendritic tree, T-type Ca2+ channels play a crucial role in the action potential triggered Ca2+ influx suggesting that this Ca2+ influx may be controlled by slight changes in the local dendritic membrane potential that determine the T-type channels’ availability. We conclude that by mediating Ca2+ dynamic in the whole dendritic arborization, both tonic and burst firing of the nucleus reticularis thalami neurons might control their dendro-dendritic and electrical communications.

The transverse-axial tubular system of cardiomyocytes.

A characteristic histological feature of striated muscle cells is the presence of deep invaginations of the plasma membrane (sarcolemma), most commonly referred to as T-tubules or the transverse-axial tubular system (TATS). TATS mediates the rapid spread of the electrical signal (action potential) to the cell core triggering Ca(2+) release from the sarcoplasmic reticulum, ultimately inducing myofilament contraction (excitation-contraction coupling). T-tubules, first described in vertebrate skeletal muscle cells, have also been recognized for a long time in mammalian cardiac ventricular myocytes, with a structure and a function that in recent years have been shown to be far more complex and pivotal for cardiac function than initially thought. Renewed interest in T-tubule function stems from the loss and disorganization of T-tubules found in a number of pathological conditions including human heart failure (HF) and dilated and hypertrophic cardiomyopathies, as well as in animal models of HF, chronic ischemia and atrial fibrillation. Disease-related remodeling of the TATS leads to asynchronous and inhomogeneous Ca(2+)-release, due to the presence of orphan ryanodine receptors that have lost their coupling with the dihydropyridine receptors and are either not activated or activated with a delay. Here, we review the physiology of the TATS, focusing first on the relationship between function and structure, and then describing T-tubular remodeling and its reversal in disease settings and following effective therapeutic approaches.

264 CHARACTERISATION OF CARDIOMYOCYTES DERIVED FROM HUMAN INDUCED PLURIPOTENT STEM (IPS) CELLS

<h3>Objective</h3> The ability to derive cardiomyocytes from human induced pluripotent stem (iPS) cells provides a novel approach to study inherited cardiac channelopathies. Fibroblasts from patients can be reprogrammed to iPS cells and differentiated into cardiomyocytes that carry the patient9s exact genetic background. Although this technique has enormous potential for modeling inherited channelopathies the derived cardiomyocytes have not been fully characterised and compared to adult and foetal cells. Knowledge about their maturity will be critical if assessments of cellular function are to provide insight into disease processes that manifest in the adult. <h3>Methods</h3> Healthy control human iPS cells were differentiated into cardiomyocytes using an unguided differentiation protocol. Embryoid bodies from undifferentiated iPS cells were cultured in suspension for 4 days before being plated onto 0.1% gelatin. Spontaneously contracting areas were disaggregated and single cardiomyocytes were characterised using immunocytochemistry, TEM, electrophysiology and Ca²⁺ imaging. Cells were stained for Troponin T, Troponin I and α-actinin. Ca²⁺ release into the cytoplasm was detected by fluo-4 and fluorescence intensity was traced before and after Tetrodotoxin (TTX) treatment. Sodium channel activity was measured in a low sodium buffer using perforated patch-clamping. Ultrastructure of cells was analysed with transmission electron microscopy while the presence of t-tubules was investigated using the fluorescent dye Di-8-ANEPPS. Cardiac cells derived from iPS cells were then compared to primary cardiomyocytes isolated from human foetal and adult cardiac tissue. <h3>Results</h3> iPS derived cardiomyocytes express a wide range of cardiac markers as well as Tetrodotoxin resistant voltage activated sodium channels that inactivate and recover from inactivation and exhibit action potential triggered Ca²⁺-induced-Ca²⁺-release. Spontaneous contraction and lack of t-tubules were observed in derived cardiomyocytes and immature human foetal cardiomyocytes. <h3>Conclusion</h3> iPS cells give rise to mostly immature cardiomyocytes, that express typical cardiac proteins and have functional cardiac sodium channels. This differentiation system may be utilised to investigate patients with and without known genetic mutations to provide a better understanding of the pathophysiology of inherited cardiac channelopathies.

Bidirectional Ca2+ signaling occurs between the endoplasmic reticulum and acidic organelles

The endoplasmic reticulum (ER) and acidic organelles (endo-lysosomes) act as separate Ca(2+) stores that release Ca(2+) in response to the second messengers IP3 and cADPR (ER) or NAADP (acidic organelles). Typically, trigger Ca(2+) released from acidic organelles by NAADP subsequently recruits IP3 or ryanodine receptors on the ER, an anterograde signal important for amplification and Ca(2+) oscillations/waves. We therefore investigated whether the ER can signal back to acidic organelles, using organelle pH as a reporter of NAADP action. We show that Ca(2+) released from the ER can activate the NAADP pathway in two ways: first, by stimulating Ca(2+)-dependent NAADP synthesis; second, by activating NAADP-regulated channels. Moreover, the differential effects of EGTA and BAPTA (slow and fast Ca(2+) chelators, respectively) suggest that the acidic organelles are preferentially activated by local microdomains of high Ca(2+) at junctions between the ER and acidic organelles. Bidirectional organelle communication may have wider implications for endo-lysosomal function as well as the generation of Ca(2+) oscillations and waves.