Induced Ca Release Research Articles

Ca2+-signalling pathways involved in P2X receptor-induced contractions of rat small pulmonary artery

P2X receptors are Ca2+-permeable, ligand-gated cation channels, which are activated by ATP. In arteries, including the rat small pulmonary artery (SPA), they elicit vasoconstriction (Chootip et al., 2002), which is dependent upon extracellular Ca2+. The aim of this study was to determine the relative contributions of Ca2+ influx via the P2X receptor pore and voltage-dependent CaV1.2 (L-type) Ca2+ channels, and the role of Ca2+-induced Ca2+ release (CICR) in P2X receptormediated contractions of rat SPA.

Ca2+ influx through L-type Ca2+ channels and Ca2+-induced Ca2+ release regulate cAMP accumulation and Epac1-dependent ERK 1/2 activation in INS-1 cells

We previously reported that INS-1 cells expressing the intracellular II-III loop of the L-type Ca2+ channel Cav1.2 (Cav1.2/II-III cells) are deficient in Ca2+-induced Ca2+ release (CICR). Here we show that glucose-stimulated ERK 1/2 phosphorylation (GSEP) is slowed and reduced in Cav1.2/II-III cells compared to INS-1 cells. This parallels a decrease in glucose-stimulated cAMP accumulation (GS-cAMP) in Cav1.2/II-III cells. Influx of Ca2+ via L-type Ca2+ channels and CICR play roles in both GSEP and GS-cAMP in INS-1 cells since both are inhibited by nicardipine or ryanodine. Further, the Epac1-selective inhibitor CE3F4 abolishes glucose-stimulated ERK activation in INS-1 cells, as measured using the FRET-based sensor EKAR. The non-selective Epac antagonist ESI-09 but not the Epac2-selective antagonist ESI-05 nor the PKA antagonist Rp-cAMPs inhibits GSEP in both INS-1 and Cav1.2/II-III cells. We conclude that L-type Ca2+ channel-dependent cAMP accumulation, that's amplified by CICR, activates Epac1 and drives GSEP in INS-1 cells.

Looking for answers to L-type calcium channels in the ageing brain (Commentary on Zanos etal.).

Looking for answers to L-type calcium channels in the ageing brain (Commentary on Zanos etal.).

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.

Cytosolic and nuclear calcium signaling in atrial myocytes: IP3-mediated calcium release and the role of mitochondria

In rabbit atrial myocytes Ca signaling has unique features due to the lack of transverse (t) tubules, the spatial arrangement of mitochondria and the contribution of inositol-1,4,5-trisphosphate (IP3) receptor-induced Ca release (IICR). During excitation-contraction coupling action potential-induced elevation of cytosolic [Ca] originates in the cell periphery from Ca released from the junctional sarcoplasmic reticulum (j-SR) and then propagates by Ca-induced Ca release from non-junctional (nj-) SR toward the cell center. The subsarcolemmal region between j-SR and the first array of nj-SR Ca release sites is devoid of mitochondria which results in a rapid propagation of activation through this domain, whereas the subsequent propagation through the nj-SR network occurs at a velocity typical for a propagating Ca wave. Inhibition of mitochondrial Ca uptake with the Ca uniporter blocker Ru360 accelerates propagation and increases the amplitude of Ca transients (CaTs) originating from nj-SR. Elevation of cytosolic IP3 levels by rapid photolysis of caged IP3 has profound effects on the magnitude of subcellular CaTs with increased Ca release from nj-SR and enhanced CaTs in the nuclear compartment. IP3 uncaging restricted to the nucleus elicites ‘mini’-Ca waves that remain confined to this compartment. Elementary IICR events (Ca puffs) preferentially originate in the nucleus in close physical association with membrane structures of the nuclear envelope and the nucleoplasmic reticulum. The data suggest that in atrial myocytes the nucleus is an autonomous Ca signaling domain where Ca dynamics are primarily governed by IICR.

Dual Role of Functionally Intact Dyadic Junctions in Cardiac Excitation-Contraction Coupling

Cardiac excitation-contraction (EC) coupling relies on Ca2+-induced Ca2+ release (CICR) enabled by an intimate relationship between L-type (CaV1.2) channels and ryanodine receptors (RyRs) at dyadic junctions. How CaV1.2 channels target in proximity to RyRs at dyads is unknown, but likely involves protein interactions mediated by one or more intracellular loops of CaV1.2 pore-forming α1C subunit. We hypothesized that over-expressing α1C intracellular loops that play a critical role in CaV1.2/RyR functional co-localization would disrupt CICR in cardiomyocytes. We over-expressed fluorescent-protein-tagged α1C intracellular loops and termini (NT, I-II, II-III, III-IV, CT) in adult rat cardiomyocytes and assessed their impact on field-stimulation-evoked rhod-2 reported Ca2+ transients and other determinants of CICR (ICaL, t-tubule structure, SR Ca2+ content, spontaneous Ca2+ sparks). Over-expressed I-II and CT uniquely disrupted EC coupling as characterized by two distinct signatures: a sharp augmentation in CICR failure, and an increased propensity for arrhythmic Ca2+ transients. Surprisingly, both I-II and CT paradoxically induced a substantial rise in frequency of spontaneous Ca2+ sparks. The effects of I-II on CICR and Ca2+ sparks were phenocopied by over-expressing the 18-residue AID peptide responsible for auxiliary CaVβ binding to the I-II loop in α1C. Over-expressing a mutated AID (YWI/AAA) that no longer binds CaVβ produced normal CICR and spontaneous Ca2+ sparks. The effects of CT on CICR and spontaneous Ca2+ sparks were phenocopied by the distal-CT that is unique to α1C, and contains binding sites for several signaling and scaffold proteins. Altogether, the results shed new light on molecular determinants important for CaV1.2 functional targeting in cardiomyocytes, and suggest a new unconventional role for functionally intact dyads– as being necessary to quell spontaneous openings of RyRs.

Effects of MH and CCD Mutations in the Central Region on RyR1 Channels

Type 1 ryanodine receptor (RyR1) is a Ca2+ release channel in the sarcoplasmic reticulum and the major target for muscle diseases, e.g., malignant hyperthermia (MH) and central core disease (CCD). It is widely believed that MH and CCD mutations cause hyperactivation of the Ca2+-induced Ca2+ release (CICR), resulting in abnormal Ca2+ homeostasis in skeletal muscle. However, it remains unclear how the disease-associated mutations affect CICR. We have recently characterized several disease-associated mutations in the amino-terminal region by live-cell Ca2+ imaging and [3H]ryanodine binding and found that these mutations divergently affect the gain (i.e., peak activity) and the sensitivity to activating Ca2+ of CICR. In this study, we extended this approach to 15 MH and MH/CCD mutations in the central region (1592-2508). The disease-associated mutations increased the gain and the sensitivity to activating Ca2+ in a site-dependent manner. The calculated CICR activity strongly correlated with the ER Ca2+ level, an index of Ca2+ leak. Importantly, the accelerated sensitivity to activating Ca2+ was linked to pathogenesis of CCD. Overall, the effects were similar to those of the amino-terminal mutations. The underlying molecular mechanism will be discussed.

The calcium stored in the sarcoplasmic reticulum acts as a safety mechanism in rainbow trout heart.

Cardiomyocyte contraction depends on rapid changes in intracellular Ca2+. In mammals, Ca2+ influx as L-type Ca2+ current (ICa) triggers the release of Ca2+ from sarcoplasmic reticulum (SR) and Ca2+-induced Ca2+ release (CICR) is critical for excitation-contraction coupling. In fish, the relative contribution of external and internal Ca2+ is unclear. Here, we characterized the role of ICa to trigger SR Ca2+ release in rainbow trout ventricular myocytes using ICa regulation by Ca2+ as an index of CICR. ICa was recorded with a slow (EGTA) or fast (BAPTA) Ca2+ chelator in control and isoproterenol conditions. In the absence of β-adrenergic stimulation, the rate of ICa inactivation was not significantly different in EGTA and BAPTA (27.1 ± 1.8 vs. 30.3 ± 2.4 ms), whereas with isoproterenol (1 μM), inactivation was significantly faster with EGTA (11.6 ± 1.7 vs. 27.3 ± 1.6 ms). When barium was the charge carrier, inactivation was significantly slower in both conditions (61.9 ± 6.1 vs. 68.0 ± 8.7 ms, control, isoproterenol). Quantification revealed that without isoproterenol, only 39% of ICa inactivation was due to Ca2+, while with isoproterenol, inactivation was Ca2+-dependent (∼65%) and highly reliant on SR Ca2+ (∼46%). Thus, SR Ca2+ is not released in basal conditions, and ICa is the main trigger of contraction, whereas during a stress response, SR Ca2+ is an important source of cytosolic Ca2+. This was not attributed to differences in SR Ca2+ load because caffeine-induced transients were not different in both conditions. Therefore, Ca2+ stored in SR of trout cardiomyocytes may act as a safety mechanism, allowing greater contraction when higher contractility is required, such as stress or exercise.

Nicotine increases GABAergic input on rat dorsal raphe serotonergic neurons through alpha7 nicotinic acetylcholine receptor.

The dorsal raphe nucleus (DRN) contains large populations of serotonergic (5-HT) neurons. This nucleus receives GABAergic inhibitory afferents from many brain areas and from DRN interneurons. Both GABAergic and 5-HT DRN neurons express functional nicotinic acetylcholine receptors (nAChRs). Previous studies have demonstrated that nicotine increases 5-HT release and 5-HT DRN neuron discharge rate by stimulating postsynaptic nAChRs and by increasing glutamate and norepinephrine release inside DRN. However, the influence of nicotine on the GABAergic input to 5-HT DRN neurons was poorly investigated. Therefore, the aim of this work was to determine the effect of nicotine on GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) of 5-HT DRN neurons and the subtype of nAChR(s) involved in this response. Experiments were performed in coronal slices obtained from young Wistar rats. GABAergic sIPSCs were recorded from post hoc-identified 5-HT DRN neurons with the whole cell voltage patch-clamp technique. Administration of nicotine (1 μM) increased sIPSC frequency in 72% of identified 5-HT DRN neurons. This effect was not reproduced by the α4β2 nAChR agonist RJR-2403 and was not influenced by TTX (1 μM). It was mimicked by the selective agonist for α7 nAChR, PNU-282987, and exacerbated by the positive allosteric modulator of the same receptor, PNU-120596. The nicotine-induced increase in sIPSC frequency was independent on voltage-gated calcium channels and dependent on Ca(2+)-induced Ca(2+) release (CICR). These results demonstrate that nicotine increases the GABAergic input to most 5-HT DRN neurons, by activating α7 nAChRs and producing CICR in DRN GABAergic terminals.

Endogenous calcium buffering at photoreceptor synaptic terminals in salamander retina.

Calcium operates by several mechanisms to regulate glutamate release at rod and cone synaptic terminals. In addition to serving as the exocytotic trigger, Ca2+ accelerates replenishment of vesicles in cones and triggers Ca2+-induced Ca2+ release (CICR) in rods. Ca2+ thereby amplifies sustained exocytosis, enabling photoreceptor synapses to encode constant and changing light. A complete picture of the role of Ca2+ in regulating synaptic transmission requires an understanding of the endogenous Ca2+ handling mechanisms at the synapse. We therefore used the "added buffer" approach to measure the endogenous Ca2+ binding ratio (κendo ) and extrusion rate constant (γ) in synaptic terminals of photoreceptors in retinal slices from tiger salamander. We found that κendo was similar in both cell types-∼25 and 50 in rods and cones, respectively. Using measurements of the decay time constants of Ca2+ transients, we found that γ was also similar, with values of ∼100 s(-1) and 160 s(-1) in rods and cones, respectively. The measurements of κendo differ considerably from measurements in retinal bipolar cells, another ribbon-bearing class of retinal neurons, but are comparable to similar measurements at other conventional synapses. The values of γ are slower than at other synapses, suggesting that Ca2+ ions linger longer in photoreceptor terminals, supporting sustained exocytosis, CICR, and Ca2+ -dependent ribbon replenishment. The mechanisms of endogenous Ca2+ handling in photoreceptors are thus well-suited for supporting tonic neurotransmission. Similarities between rod and cone Ca2+ handling suggest that neither buffering nor extrusion underlie differences in synaptic transmission kinetics.

Cellular mechanism of premature ventricular contraction-induced cardiomyopathy.

Frequent premature ventricular contractions (PVCs) are associated with increased risk of sudden cardiac death and can cause secondary cardiomyopathy. We sought to determine the mechanism(s) responsible for prolonged refractory period and left ventricular (LV) dysfunction demonstrated in our canine model of PVC-induced cardiomyopathy. Single myocytes were isolated from LV free wall of PVC and control canines and used for patch-clamp recording, intracellular Ca(2+) measurements, and immunocytochemistry/confocal microscopy. LV tissues adjacent to the area of myocyte isolation were used for the immunoblot quantification of protein expression. In the PVC group, LV ejection fraction decreased from 57.6% ± 1.5% to 30.4% ± 3.1% after ≥4 months of ventricular bigeminy. Compared to control myocytes, PVC myocytes had decreased densities of both outward (transient outward current [Ito] and inward rectifier current [IK1]) and inward (L-type Ca current [ICaL]) currents, but no consistent changes in rapid or slow delayed rectifier currents. The reduction in Ito, IK1, and ICaL was accompanied by decreased protein levels of their channel subunits. The extent of reduction in Ito, IK1, and ICaL varied among PVC myocytes, creating marked heterogeneity in action potential configurations and durations. PVC myocytes showed impaired Ca-induced Ca release from the sarcoplasmic reticulum (SR), without increase in SR Ca leak or decrease in SR Ca store. This was accompanied by a decrease in dyad scaffolding protein, junctophilin-2, and loss of Cav1.2 registry with Ca-releasing channels (ryanodine receptor 2). PVCs increase dispersion of action potential configuration/duration, a risk factor for sudden cardiac death, because of the heterogeneous reduction in Ito, IK1, and ICaL. The excitation-contraction coupling is impaired because of the decrease in ICaL and Cav1.2 misalignment with respect to ryanodine receptor 2.

Functional expression of adrenoreceptors in mesenchymal stromal cells derived from the human adipose tissue

Cultured mesenchymal stromal cells (MSCs) from different sources represent a heterogeneous population of proliferating non-differentiated cells that contains multipotent stem cells capable of originating a variety of mesenchymal cell lineages. Despite tremendous progress in MSC biology spurred by their therapeutic potential, current knowledge on receptor and signaling systems of MSCs is mediocre. Here we isolated MSCs from the human adipose tissue and assayed their responsivity to GPCR agonists with Ca2+ imaging. As a whole, a MSC population exhibited functional heterogeneity. Although a variety of first messengers was capable of stimulating Ca2+ signaling in MSCs, only a relatively small group of cells was specifically responsive to the particular GPCR agonist, including noradrenaline. RT-PCR and immunocytochemistry revealed expression of α1B-, α2A-, and β2-adrenoreceptors in MSCs. Their sensitivity to subtype-specific adrenergic agonists/antagonists and certain inhibitors of Ca2+ signaling indicated that largely the α2A-isoform coupled to PLC endowed MSCs with sensitivity to noradrenaline. The all-or-nothing dose-dependence was characteristic of responsivity of robust adrenergic MSCs. Noradrenaline never elicited small or intermediate responses but initiated large and quite similar Ca2+ transients at all concentrations above the threshold. The inhibitory analysis and Ca2+ uncaging implicated Ca2+-induced Ca2+ release (CICR) in shaping Ca2+ signals elicited by noradrenaline. Evidence favored IP3 receptors as predominantly responsible for CICR. Based on the overall findings, we inferred that adrenergic transduction in MSCs includes two fundamentally different stages: noradrenaline initially triggers a local and relatively small Ca2+ signal, which next stimulates CICR, thereby being converted into a global Ca2+ signal.

Cytoplasmic nanojunctions between lysosomes and sarcoplasmic reticulum are required for specific calcium signaling.

Herein we demonstrate how nanojunctions between lysosomes and sarcoplasmic reticulum (L-SR junctions) serve to couple lysosomal activation to regenerative, ryanodine receptor-mediated cellular Ca (2+) waves. In pulmonary artery smooth muscle cells (PASMCs) it has been proposed that nicotinic acid adenine dinucleotide phosphate (NAADP) triggers increases in cytoplasmic Ca (2+) via L-SR junctions, in a manner that requires initial Ca (2+) release from lysosomes and subsequent Ca (2+)-induced Ca (2+) release (CICR) via ryanodine receptor (RyR) subtype 3 on the SR membrane proximal to lysosomes. L-SR junction membrane separation has been estimated to be < 400 nm and thus beyond the resolution of light microscopy, which has restricted detailed investigations of the junctional coupling process. The present study utilizes standard and tomographic transmission electron microscopy to provide a thorough ultrastructural characterization of the L-SR junctions in PASMCs. We show that L-SR nanojunctions are prominent features within these cells and estimate that the junctional membrane separation and extension are about 15 nm and 300 nm, respectively. Furthermore, we develop a quantitative model of the L-SR junction using these measurements, prior kinetic and specific Ca (2+) signal information as input data. Simulations of NAADP-dependent junctional Ca (2+) transients demonstrate that the magnitude of these signals can breach the threshold for CICR via RyR3. By correlation analysis of live cell Ca (2+) signals and simulated Ca (2+) transients within L-SR junctions, we estimate that "trigger zones" comprising 60-100 junctions are required to confer a signal of similar magnitude. This is compatible with the 110 lysosomes/cell estimated from our ultrastructural observations. Most importantly, our model shows that increasing the L-SR junctional width above 50 nm lowers the magnitude of junctional [Ca (2+)] such that there is a failure to breach the threshold for CICR via RyR3. L-SR junctions are therefore a pre-requisite for efficient Ca (2+)signal coupling and may contribute to cellular function in health and disease.

Divergent Effects of Disease-Associated Mutations on Type 2 Ryanodine Receptor Channel

Type 2 ryanodine receptor (RyR2) is a Ca2+ release channel in the sarcoplasmic reticulum and plays a pivotal role in excitation-contraction coupling in heart. RyR2 is the major target for arrhythmogenic diseases, e.g., catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular cardiomyopathy (ARVC). To date, over 150 mutations have been identified in the RyR2 gene of CPVT and ARVC patients. It is widely believed that CPVT and ARVC mutations cause hyperactivation of the Ca2+-induced Ca2+ release (CICR), resulting in abnormal Ca2+ homeostasis in cardiac muscle. CICR shows biphasic Ca2+ dependence against cytoplasmic Ca2+, thus the activity can be determined by three parameters: sensitivity to activating Ca2+, sensitivity to inactivating Ca2+, and the gain (i.e., peak activity). In addition, CICR is also regulated by luminal Ca2+; high luminal Ca2+ activates CICR, and vise versa. However, it remains unclear how the disease-associated mutations affect these parameters. In this study, we expressed RyR2 channels carrying several CPVT/ARVC mutations in HEK cells and tested their CICR by live-cell Ca2+ imaging and [3H]ryanodine binding. Our results suggest that the disease-associated mutations divergently affects the parameters of CICR depending on the sites for mutation. The underlying molecular mechanism will be discussed.

Dissecting Roles of Cav1.2 (α1C) Intracellular Loops in Cardiac Excitation-Contraction Coupling

Cardiac excitation-contraction (EC) coupling relies on Ca2+-induced Ca2+ release (CICR) enabled by an intimate relationship between L-type (CaV1.2) channels and ryanodine receptors (RyRs) at dyadic junctions. The determinants underlying CaV1.2/RyR functional proximity responsible for effective CICR are unknown, but likely entail protein interactions involving one or more intracellular loops of CaV1.2 pore-forming α1C subunit. We hypothesized that over-expressing α1C intracellular loops that play a critical role in CaV1.2/RyR communication would disrupt CICR in cardiomyocytes. We used adenoviruses to overexpress CFP-tagged α1C intracellular loops and termini (NT, I-II, II-III, III-IV, CT) in cardiomyocytes and determined their impact on field-stimulation-evoked rhod-2-reported Ca2+ transients. Over-expressed NT, II-III, and III-IV loops had minimal effect on CICR. By contrast, over-expressed I-II and CT produced dramatic disruption of EC coupling characterized by two distinct signatures: a sharp augmentation in CICR failure, and an increased propensity for arrhythmic Ca2+ transients. The defective EC coupling was not due to a gross misalignment of CaV1.2 and RyR as reported by co-immunolocalization assays. Surprisingly, overexpressed I-II had no impact on CaV1.2 current (ICa,L), whereas CT strongly suppressed ICa,L. The results identify a dominant role for I-II and CT in establishing CaV1.2/RyR functional interrelationship, and suggest a new approach to dissect molecular components and interactions critical for CICR in the heart.

Effects of Amino-Terminal Disease-Associated Mutations on the CICR Activity of RyR1 Channel

Type 1 ryanodine receptor (RyR1) is a Ca2+ release channel in the sarcoplasmic reticulum and plays a pivotal role in excitation-contraction coupling in skeletal muscle. RyR1 is the major target for muscle diseases, e.g., malignant hyperthermia (MH) and central core disease (CCD). To date, over 200 mutations have been identified in the RyR1 gene of MH and CCD patients. It is widely believed that MH and CCD mutations cause hyperactivation of the Ca2+-induced Ca2+ release (CICR), resulting in abnormal Ca2+ homeostasis in skeletal muscle. CICR shows biphasic Ca2+ dependence, thus the activity can be determined by three parameters: sensitivity to activating Ca2+, sensitivity to inactivating Ca2+, and the gain (i.e., peak activity). However, it remains unclear how the disease-associated mutations affects these parameters. In this study, we expressed several RyR1 channels carrying different MH/CCD mutations at amino-terminal region in HEK cells and tested their CICR by live-cell Ca2+ imaging and [3H]ryanodine binding. Our results suggest that the amino-terminal disease-associated mutations divergently affects the parameters of CICR depending on the sites for mutation. The underlying molecular mechanism will be discussed.

Calcium-induced calcium release mediates all-or-nothing responses of mesenchymal stromal cells to noradrenaline

By using Ca2+ imaging and Fluo-4 dye, we examined the capability of certain agonists of G-protein coupled receptors to stimulate Ca2+ signaling in cultured mesenchymal stromal cells (MSC) derived from the human adipose tissue. In particular, a small subpopulation (∼5%) MSC was found to respond to noradrenaline with Ca2+ transients. The all-or-nothing fashion was characteristic of adrenergic Ca2+ signaling in MSC, that is, while at low concentrations noradrenaline stimulated undetectable Ca2+ transients, virtually maximal responses were elicited by this agonist at any concentration above the threshold of 100–200 nM. In some experiments, MSC were loaded with the photosensitive Ca2+ chelator NP-EGTA to produce local or global jumps in cytosolic Ca2+ concentration by virtue of Ca2+ uncaging. Global uncaging eliciting a high enough Ca2+ jump triggered a Ca2+ transient in the MSC cytoplasm, which was similar to a noradrenaline response kinetically and by magnitude. When Ca2+ uncaging was produced locally, it initiated a Ca2+ signal that traveled along a cell with a speed that exceeded an expected one by two orders of magnitude, should Ca2+ signal transfer be mediated merely by passive Ca2+ diffusion in the presence of Ca2+ buffer. These findings implicated Ca2+-induced Ca2+ release (CICR) as a mechanism amplifying local Ca2+ signals in MSC. Of Ca2+ targets involved in CICR, the ryanodine receptor and IP3 receptor are only known. The inhibitory analysis revealed IP3 receptors to be principally responsible for CICR in MSC, whereas a contribution of ryanodine receptors was negligible. Altogether, our results suggest that an initial noradrenaline-dependent rise in cytosolic Ca2+ stimulates, should it reach the threshold level, IP3 receptors, thereby triggering an avalanche-like Ca2+ release from Ca2+ stores and underlying the all-or-nothing dependence of cellular responses on the agonist concentration.

Atrial Excitation-Contraction Coupling and Ca Wave Propagation in Normal and Failing Hearts

We compared excitation-contraction coupling and Ca wave propagation in normal and heart failure (HF) rabbit atrial myocytes. Cytosolic Ca transients (Fluo-4 or Rhod-2), sarcoplasmic reticulum (SR) [Ca] (Fluo-5N) and mitochondrial localization (Mitotracker Red) were recorded by confocal microscopy. SEA and Ru360 were used to inhibit sarcolemmal Na/Ca exchange (NCX) or mitochondrial Ca uptake, respectively. In normal and HF rabbit atrial myocytes transverse tubules are missing, and the peripheral junctional SR (j-SR) is separated from the central non-junctional SR (nj-SR) by a 1-2 µm wide gap that is largely devoid of mitochondria and SR structures. During action potential (AP) stimulation SR Ca release initiated from the j-SR and propagated in centripetal direction via Ca-induced Ca release (CICR) from nj-SR. The centripetal Ca propagation velocity was highest across the gap between j-SR and nj-SR, slowed during propagation across the nj-SR and increased after Ru360 application. In HF, the centripetal propagation velocity was higher and mitochondrial density was decreased. Spontaneous Ca waves propagated with a leading wave front located to the cell periphery, i.e. arising from j-SR Ca release. Nearly half of all Ca waves in normal cells, but <10% in normal cells pretreated with SEA and none in ventricular cells triggered an AP, followed by a whole-cell Ca transient (termed here 'arrhythmogenic Ca waves'). The incidence of spontaneous Ca waves, the fraction of arrhythmogenic Ca waves and NCX activity were significantly increased in HF. In summary, the lack of mitochondria in the gap between j-SR and nj-SR enables rapid propagation of CICR from the j-SR to the nj-SR. Decreased mitochondrial Ca uptake in HF contributes to an increased centripetal Ca propagation velocity. Atrial myocytes are prone to develop NCX-dependent arrhythmogenic Ca waves, which is further accentuated in HF.

InsP3R Activation Facilitates Ca2+ Wave Propagation in Ventricular Myocytes

The contribution and significance of sarcoplasmic reticulum (SR) Ca2+ release driven by InsP3-R activation (IP3ICR) in cardiac excitation-contraction coupling (ECC) is still a matter of debate. In particular it's role in the Ca2+-induced Ca2+ release (CICR) mechanism in non-hypertrophic ventricular myocytes seems to be ambiguous. Here we characterize the contribution of IP3ICR in Ca2+ wave propagation driven by CICR in ventricular myocytes. We hypothesized a functional crosstalk and/or cooperativity between both SR-Ca2+ release channels, e.g. IP3ICR may trigger or facilitate CICR via RyR activation and/or RyR sensitization. IP3ICR was activated in a highly targeted way by UV-flash photolysis of a membrane-permeant caged InsP3 while simultaneously imaging Ca2+ with laser-scanning confocal microscopy in ventricular myocytes acutely isolated from mouse hearts. Recently, it has been shown that cellular ECC remodeling under pathophysiological stress leads to increased expression of InsP3-Rs and IP3ICR. To boost this situation to the extreme and in order to unmask a potential role of IP3ICR in ECC we used an InsP3-R overexpressing mouse model. Photolytic InsP3 release resulted in an immediate increase in Ca2+ wave propagation (from 63±7.3 μm/s in control to 79±3.7 μm/s after flash, n=10). This response was sensitive to the InsP3-R blocker Xestospongin C and was not seen in WT mice. After repetitive photolytic InsP3 release in a time window of >5 min, Ca2+ wave propagation reverted back to control. In addition, spontaneous Ca2+ wave appearance decreased or dissapeared. This suggests that Ca2+ wave propagation under conditions of high functional InsP3-R expression are accelerated by regional RyR sensitization mediated by IP3ICR. However, over long term RyR Ca2+ desensitisation and SR-Ca2+ store depletion by InsP3-Rs openings may lead to Ca2+ wave termination. Supported by SNF and Novartis Res. Foundation.

Structural and Functional Arrangements of Atrial Myocytes that Facilitate Excitation-Contraction Coupling

Rabbit atrial myocytes lack transverse tubules and only the peripheral sarcoplasmic reticulum (SR) is in contact with the surface membrane (junctional SR; j-SR) and exposed to Ca influx through voltage-gated sarcolemmal Ca channels that initiates Ca release which propagates centripetally by Ca-induced Ca release from non-junctional SR (nj-SR). In this study we examined the structural and functional characteristics of excitation-contraction coupling (ECC) unique to atrial myocytes. Subsarcolemmal (SS) Ca release from j-SR preceded release at central (CT) nj-SR regions, and SS release was of higher amplitude compared to CT release. Using simultaneous measurements of cytosolic ([Ca]i) and intra-SR ([Ca]SR) Ca, we show that depletions of SS [Ca]SR were smaller compared to CT [Ca]SR depletions during electrical stimulation. Atrial myocytes revealed an approximately 1-2 µm wide gap devoid of mitochondria and SR structures between the j-SR and the nj-SR that the Ca signal must traverse to activate release from nj-SR. The spread of Ca from the j-SR to the nj-SR occurred very rapidly and was aided by the absence of mitochondria which might buffer the diffusion of Ca. In contrast to SS release, the continuation of Ca release through the nj-SR to the cell center occurred much slower in a Ca wave-like fashion, and the rise of [Ca]i detected at individual release sites of the nj-SR preceded the depletion of [Ca]SR. During this latency period a transient elevation of [Ca]SR was observed that acted as an intra-SR Ca sensitization wave that may facilitate the spread of excitation through the nj-SR via the luminal Ca-sensitivity of the ryanodine receptor Ca release channel. In summary, we have identified key structural and functional characteristics of atrial myocytes that allow for efficient ECC in the intact heart.