Abstract 14409: TRPA1 Channels Are a Source of Calcium-driven Cellular Mechano-arrhythmogenicity

Introduction: Pathologic changes in myocardial mechanics and hemodynamic load result in arrhythmias via mechanically-induced changes in electrophysiology or intracellular Ca 2+ (‘mechano-arrhythmogenicity’). While molecular mechanisms driving mechano-arrhythmogenicity are poorly defined, they are associated with disease-related alterations in voltage-Ca 2+ dynamics. Objective: Define mechanisms of mechano-arrhythmogenicity during alterations in voltage-Ca 2+ dynamics in rabbit ventricular myocytes. Methods: Rabbit (♀, NZW) LV myocytes were transiently stretched (8-16% change in sarcomere length, 100ms) during diastole or late repolarisation in control or during K ATP channel activation (pinacidil). Drugs were used to buffer Ca 2+ (BAPTA), stabilise RyR (dantrolene), non-selectively block stretch-activated channels (streptomycin), or specifically block (HC-030031) or activate (AITC) mechano-sensitive TRPA1 channels. Voltage-Ca 2+ dynamics were simultaneously monitored with fluorescent dyes (di-4-ANBDQPQ, Fluo-5F) and a single camera-optical splitter system and diastolic Ca 2+ was measured using Fura Red. Results: Pinacidil caused greater shortening of the AP than Ca 2+ transient (-144±17 vs -74±11ms; n =24 cells, N =7 rabbits; p <0.001) with no change in cell stiffness or contractility. Stretch during pinacidil application caused arrhythmias in both diastole and late repolarisation (8 and 10% of stretches; n =46, N =5), which voltage-Ca 2+ imaging revealed were Ca 2+ -driven. Arrhythmias were reduced with BAPTA (3 and 0% of stretches; p <0.05), streptomycin (4 and 2%; p <0.05), and HC-030031(2 and 1%; p <0.01), while dantrolene had no effect ( n =40, N =5 for each). Stretch in diastole during AITC application also caused arrhythmias (15%; p <0.001), which were blocked by HC-030031 (4%; p <0.001) or BAPTA (3%; p <0.001; n =40, N =5 each). Both AITC and pinacidil caused an increase in diastolic Ca 2+ (112±29 and 78±29% of control; p<0.05), which was reduced by HC-030031 with AITC (26±24%; p <0.05), but not with pinacidil ( n =25, N =5 each). Conclusions: TRPA1 activation increases mechano-arrhythmogenicity via a Ca 2+ -driven mechanism and may represent a novel anti-arrhythmic target in pathologies involving altered cardiac mechanics.

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Mechano-arrhythmogenicity is enhanced during late repolarisation in ischemia and driven by a TRPA1-, calcium-, and reactive oxygen species-dependent mechanism

10.1101/2021.08.20.457138 ◽

2021 ◽

Author(s):

Breanne Ashleigh Cameron ◽

T Alexander Quinn

Keyword(s):

Reactive Oxygen Species ◽

Ventricular Myocytes ◽

Reactive Oxygen ◽

Oxygen Species ◽

Cellular Mechanisms ◽

Sustained Activity ◽

Intracellular Ca ◽

Rabbit Ventricular Myocytes ◽

Induced Changes ◽

Dependent Mechanism

Background: Cardiac dyskinesis in regional ischemia results in arrhythmias through mechanically-induced changes in electrophysiology ('mechano-arrhythmogenicity') that involve ischemic alterations in voltage-calcium (Ca2+) dynamics, creating a vulnerable period (VP) in late repolarisation. Objective: To determine cellular mechanisms of mechano-arrhythmogenicity in ischemia and define the importance of the VP. Methods and Results: Voltage-Ca2+ dynamics were simultaneously monitored in rabbit ventricular myocytes by dual-fluorescence imaging to assess the VP in control and simulated ischemia (SI). The VP was longer in SI than in control (146±7 vs 54±8 ms; p<0.0001) and was reduced by blocking KATP channels with glibenclamide (109±6 ms; p<0.0001). Cells were rapidly stretched (10-18% increase in sarcomere length over 110-170 ms) with carbon fibres during diastole or the VP. Mechano-arrhythmogenicity, associated with stretch and release in the VP, was greater in SI than control (7 vs 1% of stretches induced arrhythmias; p<0.005) but was similar in diastole. Arrhythmias during the VP were more complex than in diastole (100 vs 69% had sustained activity; p<0.05). In the VP, incidence was reduced with glibenclamide (2%; p<0.05), by chelating intracellular Ca2+ (BAPTA; 2%; p<0.05), blocking mechano-sensitive TRPA1 (HC-030031; 1%; p<0.005), or by scavenging (NAC; 1%; p<0.005) or blocking reactive oxygen species (ROS) production (DPI; 2%; p<0.05). Ratiometric Ca2+ imaging revealed that SI increased diastolic Ca2+ (+9±1%, p<0.0001), which was not prevented by HC-030031 or NAC. Conclusion: In ischemia, mechano-arrhythmogenicity is enhanced specifically during the VP and is mediated by ROS, TRPA1, and Ca2+.

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Abstract 14416: Mechano-arrhythmogenicity is Enhanced in Late Repolarisation During Acute Ischemia by a Calcium- and TRPA1-dependent Mechanism

Circulation ◽

10.1161/circ.142.suppl_3.14416 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Breanne A Cameron ◽

T Alexander Quinn

Keyword(s):

Sarcomere Length ◽

Fluorescent Dyes ◽

Ventricular Myocytes ◽

Tissue Mechanics ◽

Acute Ischemia ◽

Cellular Mechanisms ◽

Sustained Activity ◽

Rabbit Ventricular Myocytes ◽

Acute Regional Ischemia ◽

Dependent Mechanism

Introduction: Altered tissue mechanics in acute regional ischemia contribute to arrhythmias by a mechano-sensitive, Ca 2+ -dependent mechanism. This is facilitated by uncoupling of voltage-Ca 2+ dynamics, creating a vulnerable period (VP) in late repolarisation for stretch-induced arrhythmias (‘mechano-arrhythmogenicity’). However, cellular mechanisms driving mechano-arrhythmogenicity in acute ischemia are unknown. Objective: Define cellular mechanisms of mechano-arrhythmogenicity in the VP during acute ischemia in rabbit ventricular myocytes. Methods: Rabbit (♀, NZW) LV myocytes were transiently stretched (8-16% change in sarcomere length, 100 ms) during diastole or the VP in normal Tyrode (NT) or simulated ischemia (SI) solution (hyperkalemia, acidosis, metabolic inhibition). Drugs were used to buffer Ca 2+ (BAPTA), stabilise RyR (dantrolene), block mechano-sensitive TRPA1 channels (HC-030031), or block (DPI) or increase (bi-product of di-4-ANBDQPQ excitation) ROS production. Voltage-Ca 2+ was simultaneously monitored with fluorescent dyes (di-4-ANBDQPQ, Fluo-5F) and a single camera-optical splitter system. Results: SI shortened AP duration (APD NT =384 vs APD SI =219ms; p <0.0001) more than Ca 2+ transient duration (CaTD NT =424 vs CaTD SI =357 ms; p <0.0001) and increased the length of the VP (=CaTD-APD; VP NT =54 vs VP SI =146ms; n =50 cells for N NT =6 and N SI =14 rabbits ; p <0.0001). Mechano-arrhythmogenicity (single ectopy and complex sustained activity) was increased in SI compared to NT, but only for stretch in the VP (7 vs 1% of stretches; n =50, N =6 each ; p <0.005), and arrhythmias in the VP were proportionally more complex than those that occurred with stretch in diastole (100 vs 69%; n =50, N =6; p <0.05). Arrhythmia incidence in the VP during SI was reduced by BAPTA (2% of stretches; p <0.05), HC-030031 (1%; p <0.005), and DPI (2%; p <0.05), while dantrolene had no effect ( n =50, N =6 each). Fluorescence imaging during SI further increased mechano-arrhythmogenicity in both the VP and diastole (29 and 14%; n =42, N =4; p <0.05). Conclusions: Acute ischemia enhances cellular mechano-arrhythmogenicity specifically in the VP through a mechanism involving Ca 2+ , ROS, and TRPA1, suggesting potential targets for anti-arrhythmic therapy.

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Twitch-dependent SR Ca accumulation and release in rabbit ventricular myocytes

AJP Cell Physiology ◽

10.1152/ajpcell.1993.265.2.c533 ◽

1993 ◽

Vol 265 (2) ◽

pp. C533-C540 ◽

Cited By ~ 59

Author(s):

J. W. Bassani ◽

R. A. Bassani ◽

D. M. Bers

Keyword(s):

Steady State ◽

Ventricular Myocytes ◽

State Level ◽

Exposure Period ◽

Steady State Level ◽

Fluorescent Indicator ◽

Intracellular Ca ◽

Rabbit Ventricular Myocytes ◽

Time Required ◽

Ca Depletion

Using caffeine-induced contractures (Ccaf) and thapsigargin (TG), we estimated the fraction of sarcoplasmic reticulum (SR) Ca released at one twitch and also the number of twitches required to reload a Ca-depleted SR. Similar results were obtained for twitches or intracellular Ca (Cai) transient with the fluorescent indicator, indo 1. Sustained exposure to 10 mM caffeine completely depletes the SR of Ca in < 5 s (as assessed by a second Ccaf). After such Ca depletion, four to five twitches are necessary to reload the SR to the steady-state level (with a twitch constant, tau = 1.6 twitches). We also determined the time required for complete inhibition of the SR Ca-adenosinetriphosphatase (ATPase) by TG. After SR Ca depletion, 5 microM TG was applied for different periods of time before a train of "reloading" twitches. A TG exposure period of 90 s was sufficient to completely prevent Ccaf after these reloading twitches. When SR is Ca depleted, the twitch is larger in the presence of TG, indicating that the SR Ca-ATPase can limit the ability of Ca influx to activate contraction. To assess SR Ca released at one twitch in cells with normally Ca-loaded SR, 5 microM TG was applied for 90 s to prevent SR Ca reuptake. Then one or several twitches were activated (causing SR Ca release, but with reuptake completely blocked). After the twitch (or train), a Ccaf was used to assess remaining SR Ca.(ABSTRACT TRUNCATED AT 250 WORDS)

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CaM kinase augments cardiac L-type Ca2+ current: a cellular mechanism for long Q-T arrhythmias

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1999.276.6.h2168 ◽

1999 ◽

Vol 276 (6) ◽

pp. H2168-H2178 ◽

Cited By ~ 36

Author(s):

Yuejin Wu ◽

Leigh B. MacMillan ◽

R. Blair McNeill ◽

Roger J. Colbran ◽

Mark E. Anderson

Keyword(s):

Ventricular Myocytes ◽

Ryanodine Receptors ◽

Cam Kinase ◽

Early Afterdepolarizations ◽

Ultrastructural Studies ◽

Intracellular Ca ◽

Dependent Protein ◽

T Tubules ◽

Rabbit Ventricular Myocytes

Early afterdepolarizations (EAD) caused by L-type Ca2+ current ( I Ca,L) are thought to initiate long Q-T arrhythmias, but the role of intracellular Ca2+ in these arrhythmias is controversial. Rabbit ventricular myocytes were stimulated with a prolonged EAD-containing action potential-clamp waveform to investigate the role of Ca2+/calmodulin-dependent protein kinase II (CaM kinase) in I Ca,L during repolarization. I Ca,L was initially augmented, and augmentation was dependent on Ca2+ from the sarcoplasmic reticulum because the augmentation was prevented by ryanodine or thapsigargin. I Ca,Laugmentation was also dependent on CaM kinase, because it was prevented by dialysis with the inhibitor peptide AC3-I and reconstituted by exogenous constitutively active CaM kinase when Ba2+ was substituted for bath Ca2+. Ultrastructural studies confirmed that endogenous CaM kinase, L-type Ca2+ channels, and ryanodine receptors colocalized near T tubules. EAD induction was significantly reduced in current-clamped cells dialyzed with AC3-I (4/15) compared with cells dialyzed with an inactive control peptide (11/15, P = 0.013). These findings support the hypothesis that EADs are facilitated by CaM kinase.

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Sarcoplasmic reticulum and Na+/Ca2+exchanger function during early and late relaxation in ventricular myocytes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1997.273.6.h2765 ◽

1997 ◽

Vol 273 (6) ◽

pp. H2765-H2773 ◽

Cited By ~ 18

Author(s):

Atsushi Yao ◽

Hiroshi Matsui ◽

Kenneth W. Spitzer ◽

John H. B. Bridge ◽

William H. Barry

Keyword(s):

Sarcoplasmic Reticulum ◽

Ventricular Myocytes ◽

Initial Rate ◽

Terminal Phase ◽

Adult Rabbit ◽

High K ◽

Intracellular Ca ◽

Rabbit Ventricular Myocytes ◽

The Difference ◽

Rate Of Decline

The relative importance of the Na+/Ca2+exchanger in the initial and terminal phases of relaxation and the decline in the [Ca2+]itransient was investigated in adult rabbit ventricular myocytes loaded with the Ca2+ indicator fluo 3. For electrically stimulated contractions, the peak intracellular Ca2+ concentration ([Ca2+]i) was 700 ± 87 nM and end-diastolic [Ca2+]iwas 239 ± 30 nM (0.25 Hz, 37°C, 1.08 mM extracellular Ca2+ concentration; n = 14). Abrupt inhibition of Na+/Ca2+exchange was produced by removal of extracellular Na+ (KCl substitution) and Ca2+ [2 mM Ca2+-free ethylene glycol-bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid] by means of a rapid switcher device (SW). Abrupt exposure to high K+ induced an action potential, although sufficient Ca2+ remained adjacent to the sarcolemma to induce a contraction (SW beat) and [Ca2+]itransient that were identical in amplitude to those induced by electrical stimulation (ES beat). The initial relaxation and decline in the [Ca2+]itransient was not significantly prolonged by abrupt elimination of the Na+/Ca2+exchanger, but the rate and extent of the terminal phase of the decline in the [Ca2+]itransient were significantly reduced. The first derivative of [Ca2+]iwith respect to time versus [Ca2+]iduring the decline of the [Ca2+]itransient attributable to sarcoplasmic reticulum (SR) function was estimated from the average SW transients, and that attributable to Na+/Ca2+exchange was estimated from the difference between SW and ES transients. By this analysis, the Na+/Ca2+exchanger produces 13% of the first half of the decline in [Ca2+]iand 45% of the second half of the decline. We conclude that abrupt inhibition of forward Na+/Ca2+exchange does not significantly affect the amplitude or the initial rate of decline of the [Ca2+]itransient and relaxation. However, its contribution to the reduction of [Ca2+]ibecomes apparent late during the [Ca2+]itransient, when cytosolic [Ca2+]ihas been reduced.

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A model of graded calcium release and L-type Ca2+ channel inactivation in cardiac muscle

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00168.2003 ◽

2004 ◽

Vol 286 (3) ◽

pp. H1154-H1169 ◽

Cited By ~ 21

Author(s):

Vladimir E. Bondarenko ◽

Glenna C. L. Bett ◽

Randall L. Rasmusson

Keyword(s):

Molecular Mechanisms ◽

Calcium Release ◽

Ventricular Myocytes ◽

Ryanodine Receptors ◽

Quantitative Description ◽

Channel Current ◽

Channel Inactivation ◽

Transmembrane Voltage ◽

Intracellular Ca ◽

Junctional Sarcoplasmic Reticulum

We have developed a model of Ca2+ handling in ferret ventricular myocytes. This model includes a novel L-type Ca2+ channel, detailed intracellular Ca2+ movements, and graded Ca2+-induced Ca2+ release (CICR). The model successfully reproduces data from voltage-clamp experiments, including voltage- and time-dependent changes in intracellular Ca2+ concentration ([Ca2+]i), L-type Ca2+ channel current ( ICaL) inactivation and recovery kinetics, and Ca2+ sparks. The development of graded CICR is critically dependent on spatial heterogeneity and the physical arrangement of calcium channels in opposition to ryanodine-sensitive release channels. The model contains spatially distinct subsystems representing the subsarcolemmal regions where the junctional sarcoplasmic reticulum (SR) abuts the T-tubular membrane and where the L-type Ca2+ channels and SR ryanodine receptors (RyRs) are localized. There are eight different types of subsystems in our model, with between one and eight L-type Ca2+ channels distributed binomially. This model exhibits graded CICR and provides a quantitative description of Ca2+ dynamics not requiring Monte-Carlo simulations. Activation of RyRs and release of Ca2+ from the SR depend critically on Ca2+ entry through L-type Ca2+ channels. In turn, Ca2+ channel inactivation is critically dependent on the release of stored intracellular Ca2+. Inactivation of ICaL depends on both transmembrane voltage and local [Ca2+]i near the channel, which results in distinctive inactivation properties. The molecular mechanisms underlying many ICaL gating properties are unclear, but [Ca2+]i dynamics clearly play a fundamental role.

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Passive Ca buffering and SR Ca uptake in permeabilized rabbit ventricular myocytes

AJP Cell Physiology ◽

10.1152/ajpcell.1993.264.3.c677 ◽

1993 ◽

Vol 264 (3) ◽

pp. C677-C686 ◽

Cited By ~ 84

Author(s):

L. Hove-Madsen ◽

D. M. Bers

Keyword(s):

Ventricular Myocytes ◽

Ruthenium Red ◽

Cell Protein ◽

Uptake Curve ◽

Spatial Gradients ◽

Ca Uptake ◽

Intracellular Ca ◽

Rabbit Ventricular Myocytes ◽

The Difference ◽

Binding Curve

Passive Ca binding was measured with a Ca-selective minielectrode in suspensions of permeabilized rabbit ventricular myocytes equilibrated with 5 microM thapsigargin and 30 microM ruthenium red to prevent sarcoplasmic reticulum (SR) or mitochondrial Ca uptake. Passive Ca binding was obtained by titration of the myocytes with Ca and subtraction of Ca binding in a blank titration without myocytes. Passive Ca binding could be described by a Michaelis binding curve with two sites: K1 = 0.42 microM n1 = 1.27 nmol/mg cell protein and K2 = 79 microM, n2 = 4.13 nmol/mg cell protein. The passive Ca buffering over the physiological Ca concentration was approximately twice the value expected from the values compiled by Fabiato [A. Fabiato. Am. J. Physiol. 245 (Cell Physiol. 14): C1-C14, 1983]. The maximal SR Ca uptake in the presence of 30 microM ruthenium red was fit by an uptake curve with a maximal uptake of 5.16 nmol/mg cell protein and a K 1/2 of 1.0 microM. In the presence of 5 microM thapsigargin and no ruthenium red, a significant Ca uptake attributed to mitochondria was measured between 10 and 100 microM free Ca. Rapid changes in free Ca concentration ([Ca]) measured with a Ca electrode were slower than simultaneous measurements of free [Ca] with indo-1 in permeabilized myocytes. However, oxalate, which buffers Ca and maximizes SR Ca uptake, increased the uptake rate and eliminated the difference in free [Ca] measured with Ca electrode and indo-1. This suggests that spatial gradients of [Ca] exist in permeabilized myocytes without Ca buffering. The new estimates of the buffering of intracellular Ca in cardiac myocytes should be valuable in developing quantitative insights into cardiac Ca regulation.

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An ionic model of stretch-activated and stretch-modulated currents in rabbit ventricular myocytes

EP Europace ◽

10.1016/j.eupc.2005.03.019 ◽

2005 ◽

Vol 7 (s2) ◽

pp. S128-S134 ◽

Cited By ~ 36

Author(s):

Sarah N. Healy ◽

Andrew D. McCulloch

Keyword(s):

Action Potential ◽

Safety Factor ◽

Ventricular Myocytes ◽

Reversal Potential ◽

Ionic Model ◽

Rabbit Ventricular Myocytes ◽

Induced Changes ◽

Transmural Heterogeneity ◽

Action Potential Shortening

Abstract Aims To develop an ionic model of stretch-activated and stretch-modulated currents in rabbit ventricular myocytes consistent with experimental observations, that can be used to investigate the role of these currents in intact myocardium. Methods and results A non-specific cation-selective stretch-activated current Ins, was incorporated into the Puglisi–Bers ionic model of epicardial, endocardial and midmyocardial ventricular myocytes. Using the model, we predict a reduction in action potential duration at 20% repolarization (APD20) and action potential amplitude, an elevated resting transmembrane potential and either an increase or decrease in APD90, depending on the reversal potential of Ins. A stretch-induced decrease in IK1 (70%), plus a small Ins current (gns=10 pS), results in a reduction in APD20 and increase in APD90, and a reduced safety factor for conduction. Increasing IK1 (150%) plus a large Ins current (gns=40 pS), also leads to a reduction in APD20 and increase in APD90, but with a greater safety factor. Endocardial and midmyocardial cells appear to be the most sensitive to stretch-induced changes in action potential. The addition of the K+-specific stretch-activated current (SAC) IKo results in action potential shortening. Conclusion Transmural heterogeneity of IKo may reduce repolarization gradients in intact myocardium caused by intrinsic ion channel densities, nonuniform strains and electrotonic effects.

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Contribution of the late sodium current to intracellular sodium and calcium overload in rabbit ventricular myocytes treated by anemone toxin

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00520.2015 ◽

2016 ◽

Vol 310 (3) ◽

pp. H426-H435 ◽

Cited By ~ 13

Author(s):

Dmytro Kornyeyev ◽

Nesrine El-Bizri ◽

Ryoko Hirakawa ◽

Steven Nguyen ◽

Serge Viatchenko-Karpinski ◽

...

Keyword(s):

Ventricular Myocytes ◽

Sodium Current ◽

Ion Homeostasis ◽

Strong Positive Correlation ◽

Patch Clamp Technique ◽

Late Sodium Current ◽

Whole Cell Patch Clamp ◽

Anemonia Sulcata ◽

Rabbit Ventricular Myocytes ◽

Induced Changes

Pathological enhancement of late Na+ current ( INa) can potentially modify intracellular ion homeostasis and contribute to cardiac dysfunction. We tested the hypothesis that modulation of late INa can be a source of intracellular Na+ ([Na+]i) overload. Late INa was enhanced by exposing rabbit ventricular myocytes to Anemonia sulcata toxin II (ATX-II) and measured using whole cell patch-clamp technique. [Na+]i was determined with fluorescent dye Asante NaTRIUM Green-2 AM. Pacing-induced changes in the dye fluorescence measured at 37°C were more pronounced in ATX-II-treated cells than in control (dye washout prevented calibration). At 22–24°C, resting [Na+]i was 6.6 ± 0.8 mM. Treatment with 5 nM ATX-II increased late INa 8.7-fold. [Na+]i measured after 2 min of electrical stimulation (1 Hz) was 10.8 ± 1.5 mM and 22.1 ± 1.6 mM ( P < 0.001) in the absence and presence of 5 nM ATX-II, respectively. Inhibition of late INa with GS-967 (1 μM) prevented Na+i accumulation. A strong positive correlation was observed between the late INa and the pacing-induced increase of [Na+]i ( R2 = 0.88) and between the rise in [Na+]i and the increases in cytosolic Ca2+ ( R2 = 0.96). ATX-II, tetrodotoxin, or GS-967 did not affect [Na+]i in quiescent myocytes suggesting that late INa was solely responsible for triggering the ATX-II effect on [Na+]i. Experiments with pinacidil and E4031 indicate that prolongation of the action potential contributes to as much as 50% of the [Na+]i overload associated with the increase in late INa caused by ATX-II. Enhancement of late INa can cause intracellular Na+ overload in ventricular myocytes.

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Revisiting the ionic mechanisms of early afterdepolarizations in cardiomyocytes: predominant by Ca waves or Ca currents?

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00742.2011 ◽

2012 ◽

Vol 302 (8) ◽

pp. H1636-H1644 ◽

Cited By ~ 69

Author(s):

Zhenghang Zhao ◽

Hairuo Wen ◽

Nadezhda Fefelova ◽

Charelle Allen ◽

Akemichi Baba ◽

...

Keyword(s):

Ventricular Myocytes ◽

Ryanodine Receptors ◽

Convincing Evidence ◽

Rate Dependency ◽

Early Afterdepolarizations ◽

Relative Contribution ◽

Intracellular Ca ◽

Ionic Mechanisms ◽

Rabbit Ventricular Myocytes ◽

Bayk 8644

Early afterdepolarizations (EADs) have been implicated in severe cardiac arrhythmias and sudden cardiac deaths. However, the mechanism(s) for EAD genesis, especially regarding the relative contribution of Ca2+ wave (CaW) vs. L-type Ca current ( ICa,L), still remains controversial. In the present study, we simultaneously recorded action potentials (APs) and intracellular Ca2+ images in isolated rabbit ventricular myocytes and systematically compared the properties of EADs in the following two pharmacological models: 1) hydrogen peroxide (H2O2; 200 μM); and 2) isoproterenol (100 nM) and BayK 8644 (50 nM) (Iso + BayK). We assessed the rate dependency of EADs, the temporal relationship between EADs and corresponding CaWs, the distribution of EADs over voltage, and the effects of blockers of ICa,L, Na/Ca exchangers, and ryanodine receptors. The most convincing evidence came from the AP-clamp experiment, in which the cell membrane clamp was switched from current clamp to voltage clamp using a normal AP waveform without EAD; CaWs disappeared in the H2O2 model, but persisted in the Iso + BayK model. We postulate that, although CaWs and reactivation of ICa,L may act synergistically in either case, reactivation of ICa,L plays a predominant role in EAD genesis under oxidative stress (H2O2 model), while spontaneous CaWs are a predominant cause for EADs under Ca2+ overload condition (Iso + BayK model).

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