Ca Alternans Research Articles

Mitochondrial calcium uniporter complex activation protects against calcium alternans in atrial myocytes.

Cardiac alternans, defined as beat-to-beat alternations in action potential duration, cytosolic Ca transient (CaT) amplitude, and cardiac contraction is associated with atrial fibrillation (AF) and sudden cardiac death. At the cellular level, cardiac alternans is linked to abnormal intracellular calcium handling during excitation-contraction coupling. We investigated how pharmacological activation or inhibition of cytosolic Ca sequestration via mitochondrial Ca uptake and mitochondrial Ca retention affects the occurrence of pacing-induced CaT alternans in isolated rabbit atrial myocytes. Cytosolic CaTs were recorded using Fluo-4 fluorescence microscopy. Alternans was quantified as the alternans ratio (AR = 1 - CaTsmall/CaTlarge, where CaTsmall and CaTlarge are the amplitudes of the small and large CaTs of a pair of alternating CaTs). Inhibition of mitochondrial Ca sequestration via mitochondrial Ca uniporter complex (MCUC) with Ru360 enhanced the severity of CaT alternans (AR increase) and lowered the pacing frequency threshold for alternans. In contrast, stimulation of MCUC mediated mitochondrial Ca uptake with spermine-rescued alternans (AR decrease) and increased the alternans pacing threshold. Direct measurement of mitochondrial [Ca] in membrane permeabilized myocytes with Fluo-4 loaded mitochondria revealed that spermine enhanced and accelerated mitochondrial Ca uptake. Stimulation of mitochondrial Ca retention by preventing mitochondrial Ca efflux through the mitochondrial permeability transition pore with cyclosporin A also protected from alternans and increased the alternans pacing threshold. Pharmacological manipulation of MCUC activity did not affect sarcoplasmic reticulum Ca load. Our results suggest that activation of Ca sequestration by mitochondria protects from CaT alternans and could be a potential therapeutic target for cardiac alternans and AF prevention.NEW & NOTEWORTHY This study provides conclusive evidence that mitochondrial Ca uptake and retention protects from Ca alternans, whereas uptake inhibition enhances Ca alternans. The data suggest pharmacological mitochondrial Ca cycling modulation as a potential therapeutic strategy for alternans-related cardiac arrhythmia prevention.

Sympathetic Nervous Regulation of Calcium and Action Potential Alternans in the Intact Heart.

Rationale: Arrhythmogenic cardiac alternans are thought to be an important determinant for the initiation of ventricular fibrillation. There is limited information on the effects of sympathetic nerve stimulation (SNS) on alternans in the intact heart and the conclusions of existing studies, focused on investigating electrical alternans, are conflicted. Meanwhile, several lines of evidence implicate instabilities in Ca handling, not electrical restitution, as the primary mechanism underpinning alternans. Despite this, there have been no studies on Ca alternans and SNS in the intact heart. The present study sought to address this, by application of voltage and Ca optical mapping for the simultaneous study of APD and Ca alternans in the intact guinea pig heart during direct SNS.Objective: To determine the effects of SNS on APD and Ca alternans in the intact guinea pig heart and to examine the mechanism(s) by which the effects of SNS are mediated.Methods and Results: Studies utilized simultaneous voltage and Ca optical mapping in isolated guinea pig hearts with intact innervation. Alternans were induced using a rapid dynamic pacing protocol. SNS was associated with rate-independent shortening of action potential duration (APD) and the suppression of APD and Ca alternans, as indicated by a shift in the alternans threshold to faster pacing rates. Qualitatively similar results were observed with exogenous noradrenaline perfusion. In contrast with previous reports, both SNS and noradrenaline acted to flatten the slope of the electrical restitution curve. Pharmacological block of the slow delayed rectifying potassium current (IKs), sufficient to abolish IKs-mediated APD-adaptation, partially reversed the effects of SNS on pacing-induced alternans. Treatment with cyclopiazonic acid, an inhibitor of the sarco(endo)plasmic reticulum ATPase, had opposite effects to that of SNS, acting to increase susceptibility to alternans, and suggesting that accelerated Ca reuptake into the sarcoplasmic reticulum is a major mechanism by which SNS suppresses alternans in the guinea pig heart.Conclusions: SNS suppresses calcium and action potential alternans in the intact guinea pig heart by an action mediated through accelerated Ca handling and via increased IKs.

Nonlinear onset of calcium wave propagation in cardiac cells.

Spontaneous calcium (Ca) waves in cardiac myocytes are known to underlie a wide range of cardiac arrhythmias. However, it is not understood which physiological parameters determine the onset of waves. In this study, we explore the relationship between Ca signaling between ion channels and the nucleation of Ca waves. In particular, we apply a master equation approach to analyze the stochastic interaction between neighboring clusters of ryanodine receptor (RyR) channels. Using this analysis, we show that signaling between clusters can be described as a barrier hopping process with exponential sensitivity to system parameters. A consequence of this feature is that the probability that Ca release at a cluster induces release at a neighboring cluster exhibits a sigmoid dependence on the Ca content in the cell. This nonlinearity originates from the regulation of RyR opening due to more than one Ca ion binding site, in conjunction with Ca mediated cooperativity between RyR channels in clusters. We apply a spatially distributed stochastic model of Ca cycling to analyze the physiological consequences of this nonlinearity, and show that it explains the sharp onset of Ca wave nucleation in cardiac cells. Furthermore, we show that this sharp onset can serve as a mechanism for Ca alternans under physiologically relevant conditions. Thus our findings identify the nonlinear features of Ca signaling which potentially underlie the onset of Ca waves and Ca alternans in cardiac cells.

Stretch-Activated Current Can Promote or Suppress Cardiac Alternans Depending on Voltage-Calcium Interaction

Cardiac alternans has been linked to the onset of ventricular fibrillation and ventricular tachycardia, leading to life-threatening arrhythmias. Here, we investigated the effects of stretch-activated currents (ISAC) on alternans using a physiologically detailed model of the ventricular myocyte. We found that increasing ISAC suppresses alternans if the voltage-Ca coupling is positive or the alternans is voltage driven. However, for electromechanically discordant alternans, which occurs when the alternans is Ca driven with negative voltage-Ca coupling, increasing ISAC promotes Ca alternans. In addition, if action potential duration-Ca transients show quasiperiodicity, we observe a biphasic effect of ISAC, i.e., suppressing quasiperiodic oscillation at small stretch but promoting electromechanically discordant alternans at larger stretch. Our results demonstrate how ISAC interacts with coupled voltage-Ca dynamical systems with respect to alternans.

Mitochondrial Permeability Transition Pore Opening Promotes Calcium Alternans and Waves in Ventricular Myocytes

The interactions between mitochondria and sarcoplasmic reticulum (SR) calcium (Ca) release form a positive feedback loop, which plays important roles in cardiac excitation-contraction coupling and arrhythmogenesis. We developed a spatially-distributed model for rabbit ventricular myocytes, which consists of a three-dimensional network of coupled mitochondria and Ca release units (CRUs). The L-type Ca channels, the ryanodine receptors, and the mitochondrial permeability transition pores (mPTPs) were simulated using random Markov transitions. We then carried out computer simulations using the detailed ventricular cell model to investigate the effects of mitochondrial depolarization on intracellular Ca cycling dynamics. We show that opening of the mPTPs that cause depolarization of a small portion of the mitochondria promotes Ca alternans and Ca waves due to Ca extrusion from mitochondria elevating SR Ca and enhancing CRU coupling. In experiments of isolated rabbit and mouse ventricular myocytes, we applied FCCP to cause mitochondrial depolarization (measured by TMRM) and mPTP opening (measured by calcein loss from the matrix). For rabbit myocytes at pacing cycle length (PCL) 400 ms, no Ca alternans was observed under control conditions, but Ca alternans appeared 2 minutes after FCCP treatment (50-100 nM) coincident with matrix calcein loss indicating mPTP opening. At the same PCL, spontaneous Ca release was observed 2 minutes after FCCP treatment. For mouse myocytes at PCL 200 ms, no Ca alternans was observed under control condition, but Ca alternans appeared 2 minutes after FCCP treatment. The FCCP-mediated Ca alternans was prevented by the mPTP inhibitor cyclosporine A (CsA). FCCP treatment also sped up Ca oscillations which slowed after CsA. These results show that mitochondrial depolarization and mPTP opening can result in Ca alternans and waves which are known to be arrhythmogenic, mechanistically linking metabolic stress to cardiac arrhythmogenesis.

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.

In Silico Investigation into Cellular Mechanisms of Cardiac Alternans in Myocardial Ischemia.

Myocardial ischemia is associated with pathophysiological conditions such as hyperkalemia, acidosis, and hypoxia. These physiological disorders may lead to changes on the functions of ionic channels, which in turn form the basis for cardiac alternans. In this paper, we investigated the roles of hyperkalemia and calcium handling components played in the genesis of alternans in ischemia at the cellular level by using computational simulations. The results show that hyperkalemic reduced cell excitability and delayed recovery from inactivation of depolarization currents. The inactivation time constant τ f of L-type calcium current (I CaL) increased obviously in hyperkalemia. One cycle length was not enough for I CaL to recover completely. Alternans developed as a result of I CaL responding to stimulation every other beat. Sarcoplasmic reticulum calcium-ATPase (SERCA2a) function decreased in ischemia. This change resulted in intracellular Ca (Cai) alternans of small magnitude. A strong Na+-Ca2+ exchange current (I NCX) increased the magnitude of Cai alternans, leading to APD alternans through excitation-contraction coupling. Some alternated repolarization currents contributed to this repolarization alternans.

Abstract 19344: Involvement of Mitochondrial Permeability Transition Pore (mPTP) in Cardiac Arrhythmias: Evidence From Cyclophilin D Knockout Mice

Our previous studies have demonstrated that mitochondrial membrane potential ( ΔΨ m ) dissipation leads to mitochondrial Ca efflux via the mitochondrial permeability transition pore (mPTP), and consequently promotes spontaneous Ca waves (CaW) in isolated cardiomyocytes. In the present study, we have used a genetic mouse model which lacks cyclophilin D (CypD KO), a necessary regulator for mPTP opening, to assess the cardioprotective effect of mPTP inhibition on cellular CaWs, Ca alternans, and whole-heart inducible arrhythmias. Ventricular myocytes were isolated from wild type (WT) and CypD KO mice. Tetramethylrhodamine (TMRM) was used as an indicator of ΔΨ m. Mitochondrial calcein release was used as the index of mPTP opening. Cytosolic Ca was imaged using Fluo-4-AM. Spontaneous CaWs were induced in the presence of 4mM external Ca. The protonophore FCCP was used to depolarize Δ Ψ m . FCCP caused Δ Ψ m depolarization to the same extent in both WT and CypD KO myocytes, however, CypD KO cells exhibited significantly less mPTP opening than WT cells (p < 0.05). Consistent with these results, treatment with FCCP caused significant increases in WT CaW rate but no increase in CypD KO CaW rate. These results were further confirmed by treating WT cells with the mPTP blocker cyclosporin A (CsA). Furthermore, occurrence of Ca alternans after treatment with FCCP and programmed stimulation was observed in a significantly higher number of WT cells (11 of 13), than in WT cells treated with CsA (2 of 8; p < 0.05) or CypD KO cells (2 of 10; p < 0.01). At the whole-heart level, pseudo-Lead II ECGs were recorded from ex vivo, Langendorff-perfused WT and CypD KO hearts, and the incidences of T-wave alternans (a precursor of lethal arrhythimas) and arrhythmias induced by programmed S 1 -S 2 stimulation were compared. We observed T-wave alternans in 5 of 7 WT hearts, but none in 5 CypD KO hearts (p < 0.05) and in only 1 of 6 WT hearts treated with CsA (p <0.05). Consistent with these results, WT hearts exhibited a significantly higher average arrhythmia score than CypD KO (p <0.01) or WT hearts treated with CsA (p < 0.01). In conclusion, CypD deficiency-induced mPTP inhibition attenuates CaWs and Ca alternans during mitochondrial depolarization, and thereby protects against arrhythmogenesis in the heart.

Subcellular Ca Channel Distribution and Ca Alternans in Atrial Myocytes

Unlike ventricular myocytes, atrial myocytes have a much less well-developed T-tubular network, especially in rodents. In this study, we carried out computer simulations to investigate the consequences of a T tubular network on the development of spatially discordant Ca alternans in atrial myocytes. Our study was motivated by experimental observations in isolated mouse atrial myocytes, which express both Cav1.2 and Cav1.3 channels. Knocking out Cav1.3, but not Cav1.2, promoted spatially discordant Ca alternans in which Ca amplitude alternates out-of-phase in different regions of the myocyte. Based on observations in sinoatrial myocytes that Cav1.2 channels are mainly distributed on the surface of the cell while Cav1.3 channels are more uniformly distributed throughout the whole cell, in our computer model, we coupled both types of channels to Ca release units on the surface of the cell, but only Cav1.3 to Ca release units in the interior of the cell. The cell model was paced with a clamped voltage waveform at a pacing cycle length of 300 msec. Under control, Ca transient was normal, and no alternans were observed. When Cav1.2 channels were removed, the Ca transient amplitude decreased, but alternans did not occur. When Cav1.3 channels were removed, however, Ca alternans occurred. The magnitude of the whole-cell Ca transient alternans varied over time, exhibiting a modulated pattern. The line scan showed spatially discordant alternans. These simulations show that the subcellular distribution of Ca channels in relation to Ca release units has important effects on the proclivity for spatially discordant Ca alternans. Specifically, a high fraction of orphaned RyR clusters in the center of the myocyte facilitates spatially discordant Ca alternans.

Urocortin 2 Regulates Sarcoplasmic Reticulum Calcium via Phosphorylation of Phospholamban and SERCA Activation and Protects against Pro-Arrhythmic Alternans in Cardiac Myocytes from Normal and Failing Hearts

We tested the effect of the cardioactive peptide Urocortin 2 (Ucn2; 100 nM) on action potential (AP)-induced Ca transients (CaT), sarcoplasmic reticulum (SR) Ca load and cardiac Ca alternans. Experiments were performed on single rabbit atrial and ventricular myocytes from normal and failing hearts. Chronic heart failure (HF) was induced by combined pressure- and volume-overload. Ca and mechanical alternans was induced by electrical pacing. Changes in cytosolic [Ca]i and [Ca]SR were monitored with fluorescent indicators, in conjunction with sarcomere length measurements. The average Ca alternans ratio (AR = 1-S/L; S: small amplitude CaT; L: large-amplitude CaT) was 0.51 in normal atrial myocytes, and 0.43 and 0.53 in normal and HF ventricular myocytes, respectively. Ucn2 increased SR Ca load in normal, and to a larger degree in HF ventricular cells, and enhanced AP-induced SR Ca depletion transients. Baseline phosphorylation levels of phospholamban (PLN) at Ser16 were increased in HF myocytes, and Ucn2 stimulation further augmented PLN phosphorylation in normal myocytes (∼10-fold) and to a lesser extent (∼5-fold) in HF ventricular myocytes. Ucn2 completely abolished Ca and mechanical alternans (within 2-3 min) in normal atrial as well as in normal and HF ventricular myocytes. In the presence of Astressin-2B (1 µM; inhibition of corticotropin-releasing factor receptor; CRFR2) and H89 (1 µM; protein kinase A inhibition) Ucn2 failed to rescue alternans. In conclusion, we propose that Ucn2 rescues cardiac alternans via CRFR2-mediated stimulation of PKA, phosphorylation of PLN and enhanced cytosolic Ca sequestration by SERCA.

Development of Ca Alternans in Atrial Myocytes is Modulated by Action Potential Morphology

Cardiac alternans is defined as cyclic, beat-to-beat variations in contraction force, action potential duration (APD) and cytosolic Ca transient (CaT) amplitude at constant stimulation rate.The bidirectional coupling of beat-to-beat regulation of cytosolic [Ca]i and membrane voltage (Vm) is a key causative factor for the development of alternans. The majority of previous studies have emphasized the role of [Ca]i→Vm coupling and disturbances in [Ca]i regulation as the primary cause of APD alternans. In this study we investigated how alternation in AP morphology feeds back on intracellular Ca handling (Vm→[Ca]i coupling) in single rabbit atrial myocytes. From AP recordings during pacing induced simultaneous CaT and APD alternans two AP waveforms were selected that represent a typical short and long AP of APD alternans. These AP waveforms were used as command voltages in subsequent voltage clamp experiments (AP voltage clamp). At pacing frequencies below alternans induction, using a series of short or long APs voltage clamp pulses, APD affected CaT amplitude where short APs caused slightly smaller amplitude CaTs. At higher frequencies CaT alternans could be induced with AP voltage clamp protocols in the absence of APD alternans, however the pacing frequency threshold where CaT alternans could be induced decreased with increasing APD, i.e. a long APD was capable of triggering CaT alternans at lower frequencies than a short AP. When cells were stimulated with an APD alternans protocol (APs of alternating duration as command voltage) the CaT alternans threshold was significantly lower. The data identify a contribution of Vm→[Ca]i coupling to the development and severity of CaT alternans and demonstrate that Vm→[Ca]i coupling affects Ca handling through changes in diastolic SR Ca load and voltage-dependent regulation of L-type Ca channels and Na/Ca exchange.

Two-dimensional imaging of fast intracellular Ca2+ release.

Asynchronous release of calcium (Ca(2+))-for example, the generation of Ca(2+) alternans in cardiac myocytes-is a phenomenon important in the development of cardiac arrhythmogenesis. The development of a failure to release Ca(2+) at individual release sites can be regarded as a major contributor to cardiac pathologies such as hypertrophy. Although confocal linescans provide sufficient temporal resolution to investigate the physiological and pathological cardiac excitation-contraction (EC) coupling, linescans can only image ∼1.5% of the cross section of myocytes, which raises doubts about how representative such recordings are, especially in light of nonhomogeneous uncoupling of Ca(2+) channels and ryanodine receptors. Nowadays, the speed of confocal microscopes has been greatly improved, enabling two-dimensional (2D) imaging at sufficient image rates (>100 frames/sec). To understand better the physiological and pathophysiological EC coupling of cardiomyocytes, we describe here a protocol to monitor fast intracellular Ca(2+) signals using fast 2D confocal scanning.

T-tubule disruption promotes calcium alternans in failing ventricular myocytes: Mechanistic insights from computational modeling

In heart failure (HF), T-tubule (TT) disruption contributes to dyssynchronous calcium (Ca) release and impaired contraction, but its role in arrhythmogenesis remains unclear. In this study, we investigate the effects of TT disruption and other HF remodeling factors on Ca alternans in ventricular myocytes using computer modeling. A ventricular myocyte model with detailed spatiotemporal Ca cycling modeled by a coupled Ca release unit (CRU) network was used, in which the L-type Ca channels and the ryanodine receptor (RyR) channels were simulated by random Markov transitions. TT disruption, which removes the L-type Ca channels from the associated CRUs, results in “orphaned” RyR clusters and thus provides increased opportunity for spark-induced Ca sparks to occur. This effect combined with other HF remodeling factors promoted alternans by two distinct mechanisms: 1) for normal sarco-endoplasmic reticulum Ca ATPase (SERCA) activity, alternans was caused by both CRU refractoriness and coupling. The increased opportunity for spark-induced sparks by TT disruption combined with the enhanced CRU coupling by Ca elevation in the presence or absence of increased RyR leakiness facilitated spark synchronization on alternate beats to promote Ca alternans; 2) for down-regulated SERCA, alternans was caused by the sarcoplasmic reticulum (SR) Ca load-dependent mechanism, independent of CRU refractoriness. TT disruption and increased RyR leakiness shifted and steepened the SR Ca release-load relationship, which combines with down-regulated SERCA to promote Ca alternans. In conclusion, the mechanisms of Ca alternans for normal and down-regulated SERCA are different, and TT disruption promotes Ca alternans by both mechanisms, which may contribute to alternans at different stages of HF.

Cardiac alternans and intracellular calcium cycling.

Cardiac alternans refers to a condition in which there is a periodic beat-to-beat oscillation in electrical activity and the strength of cardiac muscle contraction at a constant heart rate. Clinically, cardiac alternans occurs in settings that are typical for cardiac arrhythmias and has been causally linked to these conditions. At the cellular level, alternans is defined as beat-to-beat alternations in contraction amplitude (mechanical alternans), action potential duration (APD; electrical or APD alternans) and Ca(2+) transient amplitude (Ca(2+) alternans). The cause of alternans is multifactorial; however, alternans always originate from disturbances of the bidirectional coupling between membrane voltage (Vm ) and intracellular calcium ([Ca(2+) ]i ). Bidirectional coupling refers to the fact that, in cardiac cells, Vm depolarization and the generation of action potentials cause the elevation of [Ca(2+) ]i that is required for contraction (a process referred to as excitation-contraction coupling); conversely, changes of [Ca(2+) ]i control Vm because important membrane currents are Ca(2+) dependent. Evidence is mounting that alternans is ultimately caused by disturbances of cellular Ca(2+) signalling. Herein we review how two key factors of cardiac cellular Ca(2+) cycling, namely the release of Ca(2+) from internal stores and the capability of clearing the cytosol from Ca(2+) after each beat, determine the conditions under which alternans occurs. The contributions from key Ca(2+) -handling proteins (i.e. surface membrane channels, ion pumps and transporters and internal Ca(2+) release channels) are discussed.

Urocortin 2 Protects Against Pacing-Induced Alternans via Phosphorylation of Phospholamban in Cardiac Myocytes from Normal and Failing Hearts

Cardiac alternans is a high risk indicator for cardiac arrhythmias, stroke and sudden cardiac death. The cardioactive peptide Urocortin 2 (Ucn2) exhibits beneficial effects in normal and failing hearts, and elicits PKA-dependent positive inotropic and lusitropic effects in normal myocytes. Thus, we investigated if Ucn2 protects against pacing-induced alternans and elucidated the underlying mechanism.Experiments were performed on single rabbit atrial and ventricular myocytes from normal and failing hearts. Chronic heart failure was induced by combined pressure and volume overload. Ca alternans was induced by incrementally increasing the pacing frequency until stable Ca alternans occurred at room temperature. Global Ca transients were measured with the fluorescent Ca indicators Fluo-4 or Indo-1 and monitored simultaneously with mechanical alternans (sarcomere length). In some experiments, cytosolic Ca alternans and intra-SR Ca alternans were simultaneously recorded with the Ca indicators Rhod-2 and Fluo-5N, respectively.The average alternans ratio (AR = 1-(small-amplitude/large-amplitude)) in atrial myocytes was 0.79, and in normal and failing ventricular myocytes the ARs were 0.69 and 0.64, respectively. Ucn2 (100 nM) completely abolished Ca and mechanical alternans (within 2-3 min) in atrial and ventricular myocytes from normal and failing hearts. An increased sarcoplasmic reticulum (SR) Ca content, together with an enhanced SR Ca release flux, suggested that Ucn2 normalized alternans through effects on SR Ca ATPase (SERCA). Ucn2 increased significantly the level of cyclic adenosine monophosphate (cAMP) in normal cells (∼12-fold), and enhanced phosphorylation of phospholamban (PLB) at Ser16 in normal myocytes (∼10-fold) and to a lesser extent (∼5-fold) in failing myocytes. These data demonstrate that Ucn2 rescues alternans presumably via increased SERCA activity in atrial and ventricular myocytes and thus protects normal and failing hearts from proarrhythmic alternans.

Contribution of Ca-Regulated Ion Currents to the Action Potential Morphology During Cardiac Alternans

Cardiac alternans, described as periodic beat-to-beat alternations in contraction, action potential (AP) morphology or cytosolic Ca transient (CaT) amplitude, is recognized as high risk indicator for cardiac arrhythmias, stroke and sudden cardiac death.We investigated mechanisms of cardiac alternans in single rabbit atrial myocytes. CaTs were monitored simultaneously with membrane currents or APs recorded with the patch clamp technique, and revealed a strong correlation between CaT amplitude and AP duration (APD) during alternans. Investigation of [Ca]i-Vm interactions indicates that disturbances in Ca signaling are the primary events mediating activity of Ca-regulated ion channels and leading to the changes in AP morphology. Voltage-clamp in form of pre-recorded APs (AP clamp) was used to assess the contribution of Ca-regulated membrane currents by measuring the difference between currents recorded during large and small alternans CaTs. Ca-dependent currents exhibited a large outward component (2.9 ± 1.1 pA/pF in amplitude) during AP phases 1 and 2 that was followed by an inward current (0.5 ± 0.1 pA/pF in amplitude; n=7) during AP repolarization. We also investigated contributions of Na/Ca exchange (NCX), L-type Ca, Ca-activated Cl and small conductance Ca-activated K currents to the AP morphology during Ca alternans. ∼90 % of the initial outward current was blocked by substitution of Cl ions or application of Cl channel blocker (DIDS) indicating that this current is mainly carried by Ca-activated Cl channels. In summary, during Ca alternans the prominent prolongation of AP at APD10 to APD30 repolarization levels during the small CaT is due to reduced Ca-activated Cl current while the overall effect of other Ca-sensitive currents (NCX, L-type Ca, small conductance K) cause slight shortening of AP in the APD50 to APD90 range.

Altered Ca Homeostasis and Ca Alternans in Left Atrial Myocytes of Spontaneously Hypertensive Rats

Chronic hypertension can cause atrial fibrillation. The cellular mechanisms responsible for hypertension-induced atrial tachyarrhythmias, however, are unknown. Here we tested the hypothesis that, in spontaneously hypertensive rats (SHR), altered atrial Ca homeostasis may induce atrial tachyarrhythmias.At 6-8 months of age, SHR had significantly increased systolic blood pressure (185±6 mmHg, N=18; vs WKY: 129±4 mmHg, N=16, P<0.01), accompanied by compensatory left ventricular hypertrophy, but left atrial (LA) hypotrophy. At the cellular level, the area of the ventricular myocytes was increased in SHR, while LA myocytes were not different in size. Basal Ca transients (CaTs) of LA myocytes (1 Hz stimulation) showed no major differences between WKY and SHR. Further evaluation, however, revealed increased SR Ca load in SHR (amplitude of caffeine-induced CaT, Fura-2 ratio: 1.07±0.13, n=10; vs WKY: 0.72±0.04, n=18, P<0.01), reduced fractional SR Ca release (SHR: 0.20±0.03, n=10; vs WKY: 0.29±0.03, n=18, P<0.05) and reduced L-type Ca current (SHR: −4.3±0.2 pA/pF, n=12; vs WKY: −5.2±0.1 pA/pF, n=10, P<0.05) accompanied by reduced expression (−33%; P<0.01) of the alpha 1C subunit of the L-type calcium channel. Experimental simulation of tachycardia by increasing stimulation frequency (1-2-4 Hz) induced significantly more arrhythmogenic Ca alternans in SHR (2 Hz: 41%, 4 Hz: 72% of cells) than in WKY (2 Hz: 7%, 4 Hz 37% of cells; both P<0.05 vs SHR) LA myocytes.These data indicate that, at 6-8 months of age, SHR hearts display morphological changes at the ventricular but not at the atrial level. Subcellular changes in Ca homeostasis occur in LA myocytes at the sarcolemma and the SR, which are associated with a much higher incidence of arrhythmogenic Ca alternans. This may explain the increased propensity of SHR to develop atrial tachyarrhythmias.

Formation of Spatially Discordant Alternans Due to Fluctuations and Diffusion of Calcium

Spatially discordant alternans (SDA) of action potential duration (APD) is a phenomenon where different regions of cardiac tissue exhibit an alternating sequence of APD that are out-of-phase. SDA is arrhythmogenic since it can induce spatial heterogeneity of refractoriness, which can cause wavebreak and reentry. However, the underlying mechanisms for the formation of SDA are not completely understood. In this paper, we present a novel mechanism for the formation of SDA in the case where the cellular instability leading to alternans is caused by intracellular calcium (Ca) cycling, and where Ca transient and APD alternans are electromechanically concordant. In particular, we show that SDA is formed when rapidly paced cardiac tissue develops alternans over many beats due to Ca accumulation in the sarcoplasmic reticulum (SR). The mechanism presented here relies on the observation that Ca cycling fluctuations dictate Ca alternans phase since the amplitude of Ca alternans is small during the early stages of pacing. Thus, different regions of a cardiac myocyte will typically develop Ca alternans which are opposite in phase at the early stages of pacing. These subcellular patterns then gradually coarsen due to interactions with membrane voltage to form steady state SDA of voltage and Ca on the tissue scale. This mechanism for SDA is distinct from well-known mechanisms that rely on conduction velocity restitution, and a Turing-like mechanism known to apply only in the case where APD and Ca alternans are electromechanically discordant. Furthermore, we argue that this mechanism is robust, and is likely to underlie a wide range of experimentally observed patterns of SDA.

Dietary Omega-3 Fatty Acids Promote Arrhythmogenic Remodeling of Cellular Ca2+ Handling in a Postinfarction Model of Sudden Cardiac Death

It has been proposed that dietary omega-3 polyunsaturated fatty acids (n-3 PUFAs) can reduce the risk of ventricular arrhythmias in post-MI patients. Abnormal Ca2+ handling has been implicated in the genesis of post-MI ventricular arrhythmias. Therefore, we tested the hypothesis that dietary n-3 PUFAs alter the vulnerability of ventricular myocytes to cellular arrhythmia by stabilizing intracellular Ca2+ cycling. To test this hypothesis, we used a canine model of post-MI ventricular fibrillation (VF) and assigned the animals to either placebo (1 g/day corn oil) or n-3 PUFAs (1-4 g/day) groups. Using Ca2+ imaging techniques, we examined the intracellular Ca2+ handling in myocytes isolated from post-MI hearts resistant (VF-) and susceptible (VF+) to VF. Frequency of occurrence of diastolic Ca2+ waves (DCWs) in VF+ myocytes from placebo group was significantly higher than in placebo-treated VF- myocytes. n-3 PUFA treatment did not decrease frequency of DCWs in VF+ myocytes. In contrast, VF- myocytes from the n-3 PUFA group had a significantly higher frequency of DCWs than myocytes from the placebo group. In addition, n-3 PUFA treatment increased beat-to-beat alterations in the amplitude of Ca2+ transients (Ca2+ alternans) in VF- myocytes. These n-3 PUFAs effects in VF- myocytes were associated with an increased Ca2+ spark frequency and reduced sarcoplasmic reticulum Ca2+ content, indicative of increased activity of ryanodine receptors. Thus, dietary n-3 PUFAs do not alleviate intracellular Ca2+ cycling remodeling in myocytes isolated from post-MI VF+ hearts. Furthermore, dietary n-3 PUFAs increase vulnerability of ventricular myocytes to cellular arrhythmia in post-MI VF- hearts by destabilizing intracellular Ca2+ handling.

Effects of Redox Environment on Calcium Alternans in Isolated Rabbit Cardiomyocytes

Cardiac alternans is a multifactorial phenomenon linked to cardiac arrythmias. At the cellular level cardiac alternans is defined by beat-to-beat alternations in contraction amplitude (mechanical alternans), action potential duration (electrical or action potential duration alternans) and Ca transient amplitude (Ca alternans) at constant stimulation frequency. The aim of this project was to characterize the effect of changes in the cellular redox environment on Ca alternans in cardiac myocytes. Single myocytes (from New Zealand White rabbits) were isolated enzymatically by retrograde Langendorff perfusion. Ca alternans were induced by incrementally increasing the pacing frequency (electrical field stimulation) until stable Ca alternans occurred. The frequency at which stable Ca alternans were observed varied from cell to cell and ranged from >1 to 2.5 Hz at room temperature. Global cytosolic Ca transients were measured with Indo-1. In some experiments, cytosolic Ca alternans and intra-SR Ca alternans were simultaneously measured with the fluorescent Ca indicators Rhod-2 and Fluo-5N, respectively. Confocal microscopy was used to measure Ca sparks with Fluo-4.Reducing agents dithiothreitol and reduced glutathione partially abolished Ca and mechanical alternans by restoring diastolic Ca and Ca transient amplitudes. A decreased sarcoplasmic reticulum (SR) Ca release flux but not Ca content, together with a decreased Ca spark frequency, suggest that reducing agents normalized alternans through effects on the SR Ca release channel (ryanodine receptor type-2). Addition of a membrane permeant superoxide dismutase mimetic, Tempol, had little effect on Ca alternans, suggesting the possible role of dithiotreitol directly acting on the ryanodine receptor. These data highlight that the redox state of the cell may be important in the generation of Ca and mechanical alternans during oxidative stress.