Ca Waves Research Articles

Abstract 17427: A Unique “Cellular Fibrillation” Status in Mouse Ventricular Myocytes

Different intracellular calcium (Ca i ) wave patterns have been observed in cardiac myocytes. However, their mechanisms and corresponding electrophysiological properties remain unclear. In the present study, we have generated various patterns of Ca i waves in isolated mouse ventricular myocytes under highly-activated Ca i cycling states, such as high external Ca concentration or in the present of -adrenergic stimulation (i.e. isoproterenol and Bay K8644). Our results revealed at least three distinct types of Ca wave dynamics: I) slow propagating planar waves; II) Repetitive single spiral waves; III) Multiple spiral/micro waves, in which average Ca i level remained high. All waves were completely blocked by tetracaine (1 mM), suggesting RyR leaking is the original source for the waves. Simultaneous recording of membrane potential under current-clamp mode revealed that these complex Ca i wave dynamics have several functional consequences. The cell exhibited individual or oscillatory EADs and/or DADs under the conditions with type I or type II Ca i waves. Interestingly, a unique “cellular fibrillation” status was associated with type III Ca i waves. Under this condition, the cell maintained a high Ca i level and a depolarized membrane potential (~ -20 mV) and thus lost its excitability and contractility. We also found that in the same cell, the Ca waves can be converted from one type to another by changing the Ca handling conditions. The multiple spiral/micro waves (type III) were transformed into slow-propagating, planar waves (type I) by a Ca/Na-free Tyrode's solution or by Gadolinium (Gd 3+ ), a nonselective TRPC channel blocker; but not by nifedipine (L-type Ca channel blocker), SEA0400 (Na-Ca exchange blocker) or TTX (Na channel blocker). Hyperpolarizing the cell membrane by external injection of an outward current also transformed type III to type I Ca waves, suggesting the facilitating role of membrane current in the generation of the cellular fibrillation status. Termination of the cellular fibrillation by blocking TRPC channel activity may provide new opportunities for the treatment of clinical ventricular fibrillation.

The role of cytoplasmic nanospaces in smooth muscle cell Ca2+ signalling

We address the importance of cytoplasmic nanospaces in Ca(2+) transport and signalling in smooth muscle cells and how quantitative modelling can shed significant light on the understanding of signalling mechanisms. Increasingly more convincing evidence supports the view that these nanospaces--nanometre-scale spaces between organellar membranes, hosting cell signalling machinery--are key to Ca(2+) signalling as much as Ca(2+) transporters and Ca(2+) storing organelles. Our research suggests that the origin of certain diseases is to be sought in the disruption of the proper functioning of cytoplasmic nanospaces. We begin with a historical perspective on the study of smooth muscle cell plasma membrane-sarcoplasmic reticulum nanospaces, including experimental evidence of their role in the generation of asynchronous Ca(2+) waves. We then summarize how stochastic modelling approaches have aided and guided our understanding of two basic functional steps leading to healthy smooth muscle cell contraction. We furthermore outline how more sophisticated and realistic quantitative stochastic modelling is now being employed not only to deepen our understanding but also to aid in the hypothesis generation for further experimental investigation.

Spontaneous Ca 2+ Signaling of Interstitial Cells in the Guinea Pig Prostate

We investigated whether prostate interstitial cells generate spontaneous Ca(2+) oscillation, a proposed mechanism underlying pacemaker potentials to drive spontaneous activity in stromal smooth muscle cells. Intracellular free Ca(2+) in portions of guinea pig prostate and freshly isolated, single prostate interstitial cells were visualized using fluo-4 Ca(2+) fluorescence. Spontaneous electrical activity was recorded in situ with intracellular microelectrodes. In whole tissue preparations spontaneous Ca(2+) flashes firing synchronously across all smooth muscle cells within the field of view resulted in muscle wall contractions. Nonpropagating Ca(2+) waves were also recorded in individual smooth muscle cells. Nifedipine (Sigma®) (1 μM) largely decreased or abolished these Ca(2+) flashes and suppressed slow wave discharge upon blockade of their superimposed action potentials. Isolated prostate interstitial cells were readily distinguished from smooth muscle cells by their spiky processes and lack of contraction during intracellular Ca(2+) increases. Prostate interstitial cells generated spontaneous Ca(2+) transients in the form of whole cell flashes, intracellular Ca(2+) waves or localized Ca(2+) sparks. All 3 Ca(2+) signals were abolished by nicardipine (1 μM), cyclopiazonic acid (10 μM), caffeine (Sigma) (10 mM) or extracellular Ca(2+) removal. Prostate interstitial cells generate spontaneous Ca(2+) transients that occur at a frequency comparable to Ca(2+) flashes in situ or slow waves relying on functional internal Ca(2+) stores. However, unlike other interstitial cells in the urinary tract, Ca(2+) influx through L-type Ca(2+) channels is fundamental to Ca(2+) transient firings in prostate interstitial cells. Thus, it is not possible to conclude that prostate interstitial cells are responsible for pacemaker potential generation.

Synaptically activated Ca2+waves and NMDA spikes locally suppress voltage-dependent Ca2+signalling in rat pyramidal cell dendrites

Postsynaptic [Ca(2+)](i) changes contribute to several kinds of plasticity in pyramidal neurons. We examined the effects of synaptically activated Ca(2+) waves and NMDA spikes on subsequent Ca(2+) signalling in CA1 pyramidal cell dendrites in hippocampal slices. Tetanic synaptic stimulation evoked a localized Ca(2+) wave in the primary apical dendrites. The [Ca(2+)](i) increase from a backpropagating action potential (bAP) or subthreshold depolarization was reduced if it was generated immediately after the wave. The suppression had a recovery time of 30-60 s. The suppression only occurred where the wave was generated and was not due to a change in bAP amplitude or shape. The suppression also could be generated by Ca(2+) waves evoked by uncaging IP(3), showing that other signalling pathways activated by the synaptic tetanus were not required. The suppression was proportional to the amplitude of the [Ca(2+)](i) change of the Ca(2+) wave and was not blocked by a spectrum of kinase or phosphatase inhibitors, consistent with suppression due to Ca(2+)-dependent inactivation of Ca(2+) channels. The waves also reduced the frequency and amplitude of spontaneous, localized Ca(2+) release events in the dendrites by a different mechanism, probably by depleting the stores at the site of wave generation. The same synaptic tetanus often evoked NMDA spike-mediated [Ca(2+)](i) increases in the oblique dendrites where Ca(2+) waves do not propagate. These NMDA spikes suppressed the [Ca(2+)](i) increase caused by bAPs in those regions. [Ca(2+)](i) increases by Ca(2+) entry through voltage-gated Ca(2+) channels also suppressed the [Ca(2+)](i) increases from subsequent bAPs in regions where the voltage-gated [Ca(2+)](i) increases were largest, showing that all ways of raising [Ca(2+)](i) could cause suppression.

Subcellular Heterogeneity of Ryanodine Receptor Properties in Ventricular Myocytes with Low T-Tubule Density

RationaleIn ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI).ObjectiveTo characterize RyR functional properties in relation to TT proximity, at baseline and after MI.MethodsMyocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca2+ transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca2+ release, F>F50 within 20 ms) or their absence (delayed areas). Spontaneous Ca2+ release events during diastole, Ca2+ sparks, reflecting RyR activity and properties, were subsequently assigned to either category.ResultsIn CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na+/Ca2+ exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca2+ influx via Ca2+ channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca2+ content. Extremely long sparks (>47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca2+ removal by NCX at the membrane was significantly lower in MI.ConclusionTT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca2+ loss and raise SR Ca2+ content, but may promote Ca2+ waves.

Peroxisome Proliferator-Activated Receptor γ Agonists Accelerate Oligodendrocyte Maturation and Influence Mitochondrial Functions and Oscillatory Ca2+Waves

We have previously shown that natural (15-deoxy-Δ-prostaglandin J2) and synthetic (pioglitazone) agonists of peroxisome proliferator-activated receptor γ (PPAR-γ) strengthen the intrinsic cellular mechanisms protecting oligodendrocyte (OL) progenitors (OPs) from oxidative insults and promote their differentiation. Here, we demonstrate that repeated administrations of PPAR-γ agonists to OP cultures accelerate their differentiation to OLs, as indicated by increased numbers of O4- and O1-positive cells that show increased myelin basic protein expression, elaborated cholesterol-enrichedmembranes and have increased peroxisomes. Moreover, PPAR-γ agonist-treated OLs show increased activity of the mitochondrial respiratory chain Complex IV and an increased ability to respond to environmental signals, such as adenosine diphosphate (ADP), with oscillatory Ca waves; the latter closely correlated with the presence of mitochondria and were inhibited by the mitochondrial respiratory chain Complex I inhibitor rotenone. Because Ca oscillations and mitochondrial respiratory chain activity play crucial roles in OL differentiation, these findings suggest that PPAR-γ agonists could protect OLs and promote myelination through several mechanisms, including those involving mitochondrial functions. Our studies support the therapeutic potential of PPAR-γ agonists in brain diseases in which mitochondrial alteration, oxidative stress, and demyelination occur and point to the need for a better understanding of the role of PPAR-γ and its agonists in OL biology.

Arrhythmogenic adverse effects of cardiac glycosides are mediated by redox modification of ryanodine receptors

The therapeutic use of cardiac glycosides (CGs), agents commonly used in treating heart failure (HF), is limited by arrhythmic toxicity. The adverse effects of CGs have been attributed to excessive accumulation of intracellular Ca(2+) resulting from inhibition of Na(+)/K(+)-ATPase ion transport activity. However, CGs are also known to increase intracellular reactive oxygen species (ROS), which could contribute to arrhythmogenesis through redox modification of cardiac ryanodine receptors (RyR2s). Here we sought to determine whether modification of RyR2s by ROS contributes to CG-dependent arrhythmogenesis and examine the relevant sources of ROS. In isolated rat ventricular myocytes, the CG digitoxin (DGT) increased the incidence of arrhythmogenic spontaneous Ca(2+) waves, decreased the sarcoplasmic reticulum (SR) Ca(2+) load, and increased both ROS and RyR2 thiol oxidation. Additionally, pretreatment with DGT increased spark frequency in permeabilized myocytes. These effects on Ca(2+) waves and sparks were prevented by the antioxidant N-(2-mercaptopropionyl) glycine (MPG). The CG-dependent increases in ROS, RyR2 oxidation and arrhythmogenic propensity were reversed by inhibitors of NADPH oxidase, mitochondrial ATP-dependent K(+) channels (mito-K(ATP)) or permeability transition pore (PTP), but not by inhibition of xanthine oxidase. These results suggest that the arrhythmogenic adverse effects of CGs involve alterations in RyR2 function caused by oxidative changes in the channel structure by ROS. These CG-dependent effects probably involve release of ROS from mitochondria possibly mediated by NADPH oxidase.

Actin, more than just a housekeeping protein at the scene of fertilization

Since the first demonstration of sperm entry into the fertilized eggs of Mediterranean sea urchin Paracentrotus lividus by Hertwig (1876), enormous progress and insights have been made on this topic. However, the precise molecular mechanisms underlying fertilization are largely unknown. The two most dramatic changes taking place in the zygote immediately after fertilization are: (i) a sharp increase of intracellular Ca(2+) that initiates at the sperm interaction site and traverses the egg cytoplasm as a wave, and (ii) the concomitant dynamic rearrangement of the actin cytoskeleton. Traditionally, this has been studied most extensively in the sea urchin eggs, but another echinoderm, starfish, whose eggs are much bigger and transparent, has facilitated experimental approaches using microinjection and fluorescent imaging methodologies. Thus in starfish, it has been shown that the sperm-induced Ca(2+) increase in the fertilized egg can be recapitulated by several Ca(2+)-evoking second messengers, namely inositol 1,4,5-trisphosphate (InsP3), cyclic ADP-ribose (cADPr) and nicotinic acid adenine dinucleotide phosphate (NAADP), which may play distinct roles in the generation and propagation of the Ca(2+) waves. Interestingly, it has also been found that the dynamic rearrangement of the actin cytoskeleton in the fertilized eggs plays pivotal roles in guiding monospermic sperm entry and in the fine modulation of the intracellular Ca(2+) signaling. As it is well known that Ca(2+) regulates the structure of the actin cytoskeleton, our finding that Ca(2+) signaling can be reciprocally affected by the state of the actin cytoskeleton raises an intriguing possibility that actin and Ca(2+) signaling may form a 'positive feedback loop' that accelerates the downstream events of fertilization. Perturbation of the cortical actin networks also inhibits cortical granules exocytosis. Polymerizing actin bundles also compose the 'acrosome process,' a tubular structure protruding from the head of fertilizing sperm. Hence, actin, which is one of the most strictly conserved proteins in eukaryotes, modulates almost all major aspects of fertilization.

Carvedilol and its new analogs suppress arrhythmogenic store overload–induced Ca2+ release

Carvedilol is one of the most effective beta blockers for preventing ventricular tachyarrhythmias in heart failure, but the mechanisms underlying its favorable antiarrhythmic benefits remain unclear. Spontaneous Ca(2+) waves, also called store overload-induced Ca(2+) release (SOICR), evoke ventricular tachyarrhythmias in individuals with heart failure. Here we show that carvedilol is the only beta blocker tested that effectively suppresses SOICR by directly reducing the open duration of the cardiac ryanodine receptor (RyR2). This unique anti-SOICR activity of carvedilol, combined with its beta-blocking activity, probably contributes to its favorable antiarrhythmic effect. To enable optimal titration of carvedilol's actions as a beta blocker and as a suppressor of SOICR separately, we developed a new SOICR-inhibiting, minimally beta-blocking carvedilol analog, VK-II-86. VK-II-86 prevented stress-induced ventricular tachyarrhythmias in RyR2-mutant mice and did so more effectively when combined with either of the selective beta blockers metoprolol or bisoprolol. Combining SOICR inhibition with optimal beta blockade has the potential to provide antiarrhythmic therapy that can be tailored to individual patients.

Calcium Signaling in Smooth Muscle

Changes in intracellular Ca(2+) are central to the function of smooth muscle, which lines the walls of all hollow organs. These changes take a variety of forms, from sustained, cell-wide increases to temporally varying, localized changes. The nature of the Ca(2+) signal is a reflection of the source of Ca(2+) (extracellular or intracellular) and the molecular entity responsible for generating it. Depending on the specific channel involved and the detection technology employed, extracellular Ca(2+) entry may be detected optically as graded elevations in intracellular Ca(2+), junctional Ca(2+) transients, Ca(2+) flashes, or Ca(2+) sparklets, whereas release of Ca(2+) from intracellular stores may manifest as Ca(2+) sparks, Ca(2+) puffs, or Ca(2+) waves. These diverse Ca(2+) signals collectively regulate a variety of functions. Some functions, such as contractility, are unique to smooth muscle; others are common to other excitable cells (e.g., modulation of membrane potential) and nonexcitable cells (e.g., regulation of gene expression).

Novel Image Processing Methods for the Analysis of Calcium Dynamics in Glial Cells

Calcium (Ca(2+)) waves and Ca(2+) oscillations within cells initiate a wide range of physiological processes including control of cell signaling, gene expression, secretion, and cell migration. A thorough analysis of Ca(2+) waves in glial cells provides information not only about the subcellular location of signaling processing events but also about nonneuronal or intercellular signaling pathways, their timing, routes, spatial domains, and coordination. In this study, three novel image processing methods have been applied to the study of Ca(2+) dynamics in cells. These bring additional information to the methods already available in the literature, providing insight into the analysis of calcium dynamics in fluorescence recordings and defining bidimensional maps that give a complete and detailed description of calcium intracellular behavior. The application of these processing methods to glial cells highlighted the complex 2-D Ca(2+) dynamics phenomena, the location of calcium uptake and release microdomains on the endoplasmic reticulum, and the correlation between different calcium signals inside the cell. A perinuclear zone acting as a filter and regulator of intracellular calcium waves was detected: it acts as a controller of calcium fluxes between the cytoplasm and the nucleus.

Ca2+ signaling during maturation of cumulus–oocyte complex in mammals

Under the influence of gonadotropins or growth factors, a close cooperation develops between cumulus cells and the oocyte that is implicated in transmitting signals involved in maintaining or releasing the meiotic arrest in the oocyte. While cyclic adenosine 5'-monophosphate (cAMP) is a key molecule in maintaining the meiotic arrest, calcium (Ca(2+)) may play a role in controlling either spontaneous or gonadotropin-induced oocyte maturation, possibly by modulating intracytoplasmic cAMP concentrations via Ca(2+)-sensitive adenylate cyclases. This review focuses on the mechanisms related to the origin of the Ca(2+) wave that travels from the cumulus cells to the oocyte, and discusses the source of variations affecting the dynamics of this wave.

Unique properties of muscularis mucosae smooth muscle in guinea pig urinary bladder.

The muscularis mucosae, a type of smooth muscle located between the urothelium and the urinary bladder detrusor, has been described, although its properties and role in bladder function have not been characterized. Here, using mucosal tissue strips isolated from guinea pig urinary bladders, we identified spontaneous phasic contractions (SPCs) that appear to originate in the muscularis mucosae. This smooth muscle layer exhibited Ca(2+) waves and flashes, but localized Ca(2+) events (Ca(2+) sparks, purinergic receptor-mediated transients) were not detected. Ca(2+) flashes, often in bursts, occurred with a frequency (∼5.7/min) similar to that of SPCs (∼4/min), suggesting that SPCs are triggered by bursts of Ca(2+) flashes. The force generated by a single mucosal SPC represented the maximal force of the strip, whereas a single detrusor SPC was ∼3% of maximal force of the detrusor strip. Electrical field stimulation (0.5-50 Hz) evoked force transients in isolated detrusor and mucosal strips. Inhibition of cholinergic receptors significantly decreased force in detrusor and mucosal strips (at higher frequencies). Concurrent inhibition of purinergic and cholinergic receptors nearly abolished evoked responses in detrusor and mucosae. Mucosal SPCs were unaffected by blocking small-conductance Ca(2+)-activated K(+) (SK) channels with apamin and were unchanged by blocking large-conductance Ca(2+)-activated K(+) (BK) channels with iberiotoxin (IbTX), indicating that SK and BK channels play a much smaller role in regulating muscularis mucosae SPCs than they do in regulating detrusor SPCs. Consistent with this, BK channel current density in myocytes from muscularis mucosae was ∼20% of that in detrusor myocytes. These findings indicate that the muscularis mucosae in guinea pig represents a second smooth muscle compartment that is physiologically and pharmacologically distinct from the detrusor and may contribute to the overall contractile properties of the urinary bladder.

Multiscale Modeling of Calcium Cycling in Cardiac Ventricular Myocyte: Macroscopic Consequences of Microscopic Dyadic Function

In cardiac ventricular myocytes, calcium (Ca) release occurs at distinct structures (dyads) along t-tubules, where L-type Ca channels (LCCs) appose sarcoplasmic reticulum (SR) Ca release channels (RyR2s). We developed a model of the cardiac ventricular myocyte that simulates local stochastic Ca release processes. At the local Ca release level, the model reproduces Ca spark properties. At the whole-cell level, the model reproduces the action potential, Ca currents, and Ca transients. Changes in microscopic dyadic properties (e.g., during detubulation in heart failure) affect whole-cell behavior in complex ways, which we investigated by simulating changes in the dyadic volume and number of LCCs/RyR2s in the dyad, and effects of calsequestrin (CSQN) as a Ca buffer (CSQN buffer) or a luminal Ca sensor (CSQN regulator). We obtained the following results: 1), Increased dyadic volume and reduced LCCs/RyR2s decrease excitation-contraction coupling gain and cause asynchrony of SR Ca release, and interdyad coupling partially compensates for the reduced synchrony. 2), Impaired CSQN buffer depresses Ca transients without affecting the synchrony of SR Ca release. 3), When CSQN regulator function is impaired, interdyad coupling augments diastolic Ca release activity to form Ca waves and long-lasting Ca release events.

Bestrophin is important for the rhythmic but not the tonic contraction in rat mesenteric small arteries

We have previously characterized a cGMP-dependent Ca(2+)-activated Cl(-) current in vascular smooth muscle cells (SMCs) and have shown its dependence on bestrophin-3 expression. We hypothesize that this current is important for synchronization of SMCs in the vascular wall. In the present study, we aimed to test this hypothesis by transfecting rat mesenteric small arteries in vivo with siRNA specifically targeting bestrophin-3. The arteries were tested 3 days after transfection in vitro for isometric force development and for intracellular Ca(2+) in SMCs. Bestrophin-3 expression was significantly reduced compared with arteries transfected with mutated siRNA. mRNA levels for bestrophin-1 and -2 were also significantly reduced by bestrophin-3 down-regulation. This is suggested to be secondary to specific bestrophin-3 down-regulation since siRNAs targeting different exons of the bestrophin-3 gene had identical effects on bestrophin-1 and -2 expression. The transfection affected neither the maximal contractile response nor the sensitivity to norepinephrine and arginine-vasopressin. The amplitude of agonist-induced vasomotion was significantly reduced in arteries down-regulated for bestrophins compared with controls, and asynchronous Ca(2+) waves appeared in the SMCs. The average frequency of vasomotion was not different. 8Br-cGMP restored vasomotion in arteries where the endothelium was removed, but oscillation amplitude was still significantly less in bestrophin-down-regulated arteries. Thus, vasomotion properties were consistent with those previously characterized for rat mesenteric small arteries. Data from our mathematical model are consistent with the experimental results. This study demonstrates the importance of bestrophins for synchronization of SMCs and strongly supports our hypothesis for generation of vasomotion.

Cellular Mechanism Underlying Burn Serum-Generated Bidirectional Regulation of Excitation-Contraction Coupling in Isolated Rat Cardiomyocytes

Myocardial depressant factors have long been recognized to be present in burn serum (BS) and contribute to burn-generated cardiac contractile dysfunction. However, much of the cellular and molecular mechanism for its role in the development of the cardiac deficiency remains unknown. In this study, we investigated the effect of BS on myocardial contractility and Ca handling in single rat cardiomyocytes. The results revealed that BS (5% by volume) bidirectionally regulated cardiac excitation-contraction (EC) coupling. The action potential-elicited Ca transient and cell shortening were increased by 28.0% ± 9.7% and 34.7% ± 12.5% within 20 min after BS stimulation (the upregulation phase), but decreased by 20.5% ± 6.8% and 32.3% ± 5.1% at 60 min after BS stimulation (the downregulation phase). There was a 32.0% ± 5.8% reduction in sarcoplasmic reticulum (SR) Ca content at the downregulation phase, whereas no alteration was detected at the upregulation phase. The incidences of spontaneous Ca sparks and Ca waves were significantly increased after BS stimulation, no matter at the upregulation or downregulation phase. The hyperactive Ca sparks and Ca waves could be completely abolished by antioxidative treatment (vitamin A, 0.2 mM; and vitamin E, 1 mM) and partially reversed by NOS inhibitor L-NAME (100 μM), but not by blocking Ca influx with nifedipine (1 μM). With the normalization of Ca sparks, BS-induced alterations of action potential-elicited Ca transient and contractility were prevented by antioxidative therapy. Taken together, we propose that BS-associated bidirectional regulation of EC coupling is attributed largely to oxidative stress-induced hyperactivity of ryanodine receptors, increasing EC coupling through enhancing intracellular Ca release initially, but subsequently decreasing EC coupling by partially depleting SR Ca content through enhancement of Ca spark-mediated SR leak.

Ethanol Enhances Carbachol-induced Protease Activation and Accelerates Ca2+ Waves in Isolated Rat Pancreatic Acini

Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute cases and 70-90% of chronic cases, yet the mechanisms leading to alcohol-associated pancreatic injury are unclear. An early and critical feature of pancreatitis is the aberrant signaling of Ca(2+) within the pancreatic acinar cell. An important conductor of this Ca(2+) is the basolaterally localized, intracellular Ca(2+) channel ryanodine receptor (RYR). In this study, we examined the effect of ethanol on mediating both pathologic intra-acinar protease activation, a precursor to pancreatitis, as well as RYR Ca(2+) signals. We hypothesized that ethanol sensitizes the acinar cell to protease activation by modulating RYR Ca(2+). Acinar cells were freshly isolated from rat, pretreated with ethanol, and stimulated with the muscarinic agonist carbachol (1 μM). Ethanol caused a doubling in the carbachol-induced activation of the proteases trypsin and chymotrypsin (p < 0.02). The RYR inhibitor dantrolene abrogated the enhancement of trypsin and chymotrypsin activity by ethanol (p < 0.005 for both proteases). Further, ethanol accelerated the speed of the apical to basolateral Ca(2+) wave from 9 to 18 μm/s (p < 0.0005; n = 18-22 cells/group); an increase in Ca(2+) wave speed was also observed with a change from physiologic concentrations of carbachol (1 μM) to a supraphysiologic concentration (1 mM) that leads to protease activation. Dantrolene abrogated the ethanol-induced acceleration of wave speed (p < 0.05; n = 10-16 cells/group). Our results suggest that the enhancement of pathologic protease activation by ethanol is dependent on the RYR and that a novel mechanism for this enhancement may involve RYR-mediated acceleration of Ca(2+) waves.

Electromechanical coupling in the cardiac myocyte; stretch-arrhythmia feedback

The macroscopic hallmarks of the normal heartbeat are rapid onset of contraction and rapid relaxation and an inotropic response to both increased end diastolic volume and increased heart rate. At the microscopic level, the calcium ion (Ca(2+)) plays a crucial role in normal cardiac contraction. This paper reviews the cycle of Ca(2+) fluxes during the normal heartbeat, which underlie the coupling between excitation and contraction (ECC) and permit a highly synchronized action of cardiac sarcomeres. Length dependence of the response of the regulatory sarcomeric proteins mediates the Frank-Starling Law of the heart. However, Ca(2+) transport may go astray in heart disease and both jeopardize the exquisite mechanism of systole and diastole and triggering arrhythmias. The interplay between weakened and strong segments in nonuniform cardiac muscle may further lead to mechanoelectric feedback-or reverse excitation contraction coupling (RECC) mediating an early diastolic Ca(2+) transient caused by the rapid force decrease during the relaxation phase. These rapid force changes in nonuniform muscle may cause arrhythmogenic Ca(2+) waves to propagate by activation of neighbouring SR by diffusing Ca(2+) ions.

A mathematical model of spontaneous calcium release in cardiac myocytes

In cardiac myocytes, calcium (Ca) can be released from the sarcoplasmic reticulum independently of Ca influx from voltage-dependent membrane channels. This efflux of Ca, referred to as spontaneous Ca release (SCR), is due to Ryanodine receptor fluctuations, which can induce spontaneous Ca sparks, which propagate to form Ca waves. This release of Ca can then induce delayed after-depolarizations (DADs), which can lead to arrhythmogenic-triggered activity in the heart. However, despite its importance, to date there is no mathematical model of SCR that accounts for experimentally observed features of subcellular Ca. In this article, we present an experimentally based model of SCR that reproduces the timing distribution of spontaneous Ca sparks and key features of the propagation of Ca waves emanating from these spontaneous sparks. We have coupled this model to an ionic model for the rabbit ventricular action potential to simulate SCR within several thousand cells in cardiac tissue. We implement this model to study the formation of an ectopic beat on a cable of cells that exhibit SCR-induced DADs.

Self-Organization of Pacemaking Sites for Calcium Waves and Oscillations in Cardiac Myocytes

Calcium (Ca) sparks are elementary events of intracellular Ca signaling, which tend to occur randomly. Ca waves and whole-cell Ca oscillations occur under Ca overload and disease conditions. How Ca waves emerge from Ca sparks is not completely understood. We developed a three-dimensional model for Ca cycling which contains 100x20x10=20,000 identical Ca release units (CRUs), simulating the CRU network corresponding to a complete cardiac myocyte with dimensions of 100x20x10 micrometers. Using this model, we can generate the well known Ca signaling hierarchy: Ca quarks, Ca sparks, macro-sparks, abortive waves, and full Ca waves. We can also induce spiral waves within the cell, a wave phenomenon widely observed in myocyte experiments. Besides the well known experimental observation that increasing Ca loading promotes these wave dynamics, we also make the following observations: 1) The diffusion rate of Ca is a key parameter. Spontaneous Ca waves occur only when the diffusion rate is above a critical value. 2) When the model is homogeneous, Ca waves originate from different locations via a self-organizing process. This self-organizing process is influenced by, but does not require, heterogeneity. 3) When the model contains heterogeneities, such as heterogeneous Ca release channel distribution, Ca waves can originate from different locations or occur repeatedly from the same location. In real cardiac rabbit ventricular myocytes loaded with Fluo-4 AM to image intracellular Ca, Ca waves typically originate from different locations after successive rapid pacing episodes. In conclusion, our results indicate that Ca waves in cardiac myocytes originate predominantly as a result of self-organizing processes rather than pre-existing heterogeneities.