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Sarcoplasmic reticulum (SR) Ca2+ cycling is tightly regulated by ryanodine receptor (RyR) Ca2+ release and sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) Ca2+ uptake during each excitation-contraction coupling cycle. We previously showed that RyR refractoriness plays a key role in the onset of SR Ca2+ alternans in the intact rabbit heart, which contributes to arrhythmogenic action potential duration (APD) alternans. Recent studies have also implicated impaired SERCA function, a key feature of heart failure, in cardiac alternans and arrhythmias. However, the relationship between reduced SERCA function and SR Ca2+ alternans is not well understood. Simultaneous optical mapping of transmembrane potential (Vm) and SR Ca2+ was performed in isolated rabbit hearts (n = 10) using the voltage-sensitive dye RH237 and the low-affinity Ca2+ indicator Fluo-5N-AM. Alternans was induced by rapid ventricular pacing. SERCA was inhibited with cyclopiazonic acid (CPA; 1-10 μM). SERCA inhibition (1, 5, and 10 μM of CPA) resulted in dose-dependent slowing of SR Ca2+ reuptake, with the time constant (tau) increasing from 70.8 ± 3.5 ms at baseline to 85.5 ± 6.6, 129.9 ± 20.7, and 271.3 ± 37.6 ms, respectively (p < 0.05 vs. baseline for all doses). At fast pacing frequencies, CPA significantly increased the magnitude of SR Ca2+ and APD alternans, most strongly at 10 μM (pacing cycle length = 220 ms: SR Ca2+ alternans magnitude: 57.1 ± 4.7 vs. 13.4 ± 8.9 AU; APD alternans magnitude 3.8 ± 1.9 vs. 0.2 ± 0.19 AU; p < 0.05 10 μM of CPA vs. baseline for both). SERCA inhibition also promoted the emergence of spatially discordant alternans. Notably, at all CPA doses, alternation of SR Ca2+ release occurred prior to alternation of diastolic SR Ca2+ load as pacing frequency increased. Simultaneous optical mapping of SR Ca2+ and Vm in the intact rabbit heart revealed that SERCA inhibition exacerbates pacing-induced SR Ca2+ and APD alternans magnitude, particularly at fast pacing frequencies. Importantly, SR Ca2+ release alternans always occurred before the onset of SR Ca2+ load alternans. These findings suggest that even in settings of diminished SERCA function, relative refractoriness of RyR Ca2+ release governs the onset of intracellular Ca2+ alternans.

Key pointsCardiac electrophysiology and Ca2+ handling change rapidly during the fight-or-flight response to meet physiological demands. Despite dramatic differences in cardiac electrophysiology, the cardiac fight-or-flight response is highly conserved across species. In this study, we performed physiological sympathetic nerve stimulation (SNS) while optically mapping cardiac action potentials and intracellular Ca2+ transients in innervated mouse and rabbit hearts. Despite similar heart rate and Ca2+ handling responses between mouse and rabbit hearts, we found notable species differences in spatio-temporal repolarization dynamics during SNS. Species-specific computational models revealed that these electrophysiological differences allowed for enhanced Ca2+ handling (i.e. enhanced inotropy) in each species, suggesting that electrophysiological responses are fine-tuned across species to produce optimal cardiac fight-or-flight responses.AbstractSympathetic activation of the heart results in positive chronotropy and inotropy, which together rapidly increase cardiac output. The precise mechanisms that produce the electrophysiological and Ca2+ handling changes underlying chronotropic and inotropic responses have been studied in detail in isolated cardiac myocytes. However, few studies have examined the dynamic effects of physiological sympathetic nerve activation on cardiac action potentials (APs) and intracellular Ca2+ transients (CaTs) in the intact heart. Here, we performed bilateral sympathetic nerve stimulation (SNS) in fully innervated, Langendorff-perfused rabbit and mouse hearts. Dual optical mapping with voltage- and Ca2+ -sensitive dyes allowed for analysis of spatio-temporal AP and CaT dynamics. The rabbit heart responded to SNS with a monotonic increase in heart rate (HR), monotonic decreases in AP and CaT duration (APD, CaTD), and a monotonic increase in CaT amplitude. The mouse heart had similar HR and CaT responses; however, a pronounced biphasic APD response occurred, with initial prolongation (50.9 ± 5.1 ms at t = 0 s vs. 60.6 ± 4.1 ms at t = 15 s, P

β-adrenergic receptor (β-AR) blockers may be administered during acute myocardial infarction (MI), as they reduce energy demand through negative chronotropic and inotropic effects and prevent ischemia-induced arrhythmogenesis. However, the direct effects of β-AR blockers on ventricular electrophysiology and intracellular Ca2+ handling during ischemia remain unknown. Using optical mapping of transmembrane potential (with RH237) and sarcoplasmic reticulum (SR) Ca2+ (with the low-affinity indicator Fluo-5N AM), the effects of 15 min of regional ischemia were assessed in isolated rabbit hearts (n = 19). The impact of β-AR inhibition on isolated hearts was assessed by pre-treatment with 100 nM propranolol (Prop) prior to ischemia (n = 7). To control for chronotropy and inotropy, hearts were continuously paced at 3.3 Hz and contraction was inhibited with 20 μM blebbistatin. Untreated ischemic hearts displayed prototypical shortening of action potential duration (APD80) in the ischemic zone (IZ) compared to the non-ischemic zone (NI) at 10 and 15 min ischemia, whereas APD shortening was prevented with Prop. Untreated ischemic hearts also displayed significant changes in SR Ca2+ handling in the IZ, including prolongation of SR Ca2+ reuptake and SR Ca2+ alternans, which were prevented with Prop pre-treatment. At 5 min ischemia, Prop pre-treated hearts also showed larger SR Ca2+ release amplitude in the IZ compared to untreated hearts. These results suggest that even when controlling for chronotropic and inotropic effects, β-AR inhibition has a favorable effect during acute regional ischemia via direct effects on APD and Ca2+ handling.

Key pointsHeart failure leads to dramatic electrophysiological remodelling as a result of numerous cellular and tissue-level changes. Important cellular changes include increased sensitivity of ryanodine receptors (RyRs) to Ca(2+) release and down-regulation of the inward rectifying K(+) current (IK1), both of which contribute to triggered action potentials in isolated cells. We studied the role of increased RyR sensitivity and decreased IK1 in contributing to focal arrhythmia in the intact non-failing rabbit heart using optical mapping and pharmacological manipulation of RyRs and IK1. Neither increased RyR sensitivity or decreased IK1 alone led to significant increases in arrhythmia following local sympathetic stimulation; however, in combination, these two factors led to a significant increase in premature ventricular complexes and focal ventricular tachycardia. These results suggest synergism between increased RyR sensitivity and decreased IK1 in contributing to focal arrhythmia in the intact heart and may provide important insights into novel anti-arrhythmic treatments in heart failure.AbstractHeart failure (HF) results in dramatic electrophysiological remodelling, including increased sensitivity of ryanodine receptors (RyRs) and decreased inward rectifying K(+) current (IK1), which predisposes HF myocytes to delayed afterdepolarizations and triggered activity. Therefore, we sought to determine the role of increased RyR sensitivity and decreased IK1 in contributing to focal arrhythmia in the intact non-failing heart. Optical mapping of transmembrane potential and intracellular Ca(2+) was performed in Langendorff-perfused rabbit hearts (n = 15). Local β-adrenergic receptor stimulation with noradrenaline (norepinephrine; NA, 50 μl, 250 μM) was applied to elicit focal activity (premature ventricular complexes (PVCs) or ventricular tachycardia (VT ≥ 3 beats)). NA was administered under control conditions (CTL) and following pretreatment with 50 μM BaCl2 to reduce IK1, or 200 μM caffeine (Caff) to sensitize RyRs, both alone and in combination. Local NA injection resulted in Ca(2+)-driven PVCs arising from the injection site in all hearts studied. No increase in NA-mediated PVCs was observed following pretreatment with either BaCl2 or Caff alone (CTL: 1.1 ± 0.7, BaCl2: 1.0 ± 0.7, Caff: 1.3 ± 0.8 PVCs/injection, P not significant). However, pretreatment with the combination of BaCl2 + Caff resulted in a significant increase in PVCs (2.3 ± 2.8 PVCs/injection, P < 0.05 vs. CTL, BaCl2, Caff). Additionally, pretreatment with BaCl2 + Caff led to sustained monomorphic VT arising from the NA application site in all hearts studied, which lasted up to 6 min following a single NA injection. VT was never observed under any other condition suggesting synergism between increased RyR sensitivity and decreased IK1 in contributing to focal activity. These findings may have important implications for the understanding and prevention of focal arrhythmia in HF.

RationaleSarcoplasmic reticulum (SR) Ca(2+) cycling is key to normal excitation-contraction coupling but may also contribute to pathological cardiac alternans and arrhythmia.ObjectiveTo measure intra-SR free [Ca(2+)] ([Ca(2+)]SR) changes in intact hearts during alternans and ventricular fibrillation (VF).Methods and resultsSimultaneous optical mapping of Vm (with RH237) and [Ca(2+)]SR (with Fluo-5N AM) was performed in Langendorff-perfused rabbit hearts. Alternans and VF were induced by rapid pacing. SR Ca(2+) and action potential duration (APD) alternans occurred in-phase, but SR Ca(2+) alternans emerged first as cycle length was progressively reduced (217±10 versus 190±13 ms; P

Aims Long QT syndrome (LQTS) carries a risk of life-threatening polymorphic ventricular tachycardia (Torsades de Pointes, TdP) and is a major cause of premature sudden cardiac death. TdP is induced by R-on-T premature ventricular complexes (PVCs), thought to be generated by cellular early-afterdepolarisations (EADs). However, EADs in tissue require cellular synchronisation, and their role in TdP induction remains unclear. We aimed to determine the mechanism of TdP induction in rabbit hearts with acquired LQTS (aLQTS). Methods and results Optical mapping of action potentials (APs) and intracellular Ca2+ was performed in Langendorff-perfused rabbit hearts (n = 17). TdP induced by R-on-T PVCs was observed during aLQTS (50% K+/Mg++ & E4031) conditions in all hearts (P < 0.0001 vs. control). Islands of AP prolongation bounded by steep voltage gradients (VGs) were consistently observed before arrhythmia and peak VGs were more closely related to the PVC upstroke than EADs, both temporally (7 ± 5 ms vs. 44 ± 27 ms, P < 0.0001) and spatially (1.0 ± 0.7 vs. 3.6 ± 0.9 mm, P < 0.0001). PVCs were initiated at estimated voltages of ∼ −40 mV and had upstroke dF/dtmax and Vm-Ca2+ dynamics compatible with I CaL activation. Computational simulations demonstrated that PVCs could arise directly from VGs, through electrotonic triggering of I CaL. In experiments and the model, sub-maximal L-type Ca2+ channel (LTCC) block (200 nM nifedipine and 90% gCaL, respectively) abolished both PVCs and TdP in the continued presence of aLQTS. Conclusion These data demonstrate that I CaL activation at sites displaying steep VGs generates the PVCs which induce TdP, providing a mechanism and rationale for LTCC blockers as a novel therapeutic approach in LQTS. [ABSTRACT FROM AUTHOR]

Atrial fibrillation (AF) from elevated adrenergic activity may involve increased atrial L-type Ca2+ current (ICaL) by noradrenaline (NA). However, the contribution of the adrenoceptor (AR) sub-types to such ICaL-increase is poorly understood, particularly in human. We therefore investigated effects of various broad-action and sub-type-specific α- and β-AR antagonists on NA-stimulated atrial ICaL. ICaL was recorded by whole-cell-patch clamp at 37 °C in myocytes isolated enzymatically from atrial tissues from consenting patients undergoing elective cardiac surgery and from rabbits. NA markedly increased human atrial ICaL, maximally by ~ 2.5-fold, with EC75 310 nM. Propranolol (β1 + β2-AR antagonist, 0.2 microM) substantially decreased NA (310 nM)-stimulated ICaL, in human and rabbit. Phentolamine (α1 + α2-AR antagonist, 1 microM) also decreased NA-stimulated ICaL. CGP20712A (β1-AR antagonist, 0.3 microM) and prazosin (α1-AR antagonist, 0.5 microM) each decreased NA-stimulated ICaL in both species. ICI118551 (β2-AR antagonist, 0.1 microM), in the presence of NA + CGP20712A, had no significant effect on ICaL in human atrial myocytes, but increased it in rabbit. Yohimbine (α2-AR antagonist, 10 microM), with NA + prazosin, had no significant effect on human or rabbit ICaL. Stimulation of atrial ICaL by NA is mediated, based on AR sub-type antagonist responses, mainly by activating β1- and α1-ARs in both human and rabbit, with a β2-inhibitory contribution evident in rabbit, and negligible α2 involvement in either species. This improved understanding of AR sub-type contributions to noradrenergic activation of atrial ICaL could help inform future potential optimisation of pharmacological AR-antagonism strategies for inhibiting adrenergic AF. [ABSTRACT FROM AUTHOR]

Although transmural heterogeneity of action potential duration (APD) is established in single cells isolated from different tissue layers, the extent to which it produces transmural gradients of repolarization in electrotonically coupled ventricular myocardium remains controversial. The purpose of this study was to examine the relative contribution of intrinsic cellular gradients of APD and electrotonic influences to transmural repolarization in rabbit ventricular myocardium. Transmural optical mapping was performed in left ventricular wedge preparations from eight rabbits. Transmural patterns of activation, repolarization, and APD were recorded during endocardial and epicardial stimulation. Experimental results were compared with modeled data during variations in electrotonic coupling. A transmural gradient of APD was evident during endocardial stimulation, which reflected differences previously seen in isolated cells, with the longest APD at the endocardium and the shortest at the epicardium (endo: 165 [+ or -] 5 vs. epi: 147 [+ or -] 4 ms; P < 0.05). During epicardial stimulation, this gradient reversed (epi: 162 [+ or -] 4 vs. endo: 148 [+ or -] 6 ms; P < 0.05). In both activation sequences, transmural repolarization followed activation and APD shortened along the activation path such that significant transmural gradients of repolarization did not occur. This correlation between transmural activation time and APD was recapitulated in simulations and varied with changes in intercellular coupling, confirming that it is mediated by electrotonic current flow between cells. These data suggest that electrotonic influences are important in determining the transmural repolarization sequence in rabbit ventricular myocardium and that they are sufficient to overcome intrinsic differences in the electrophysiological properties of the cells across the ventricular wall. action potential remodeling; electrophysiology doi: 10.1152/ajpheart.00518.2010.

Sarcoplasmic reticulum (SR) Ca2+ cycling is tightly regulated by ryanodine receptor (RyR) Ca2+ release and sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) Ca2+ uptake during each excitation–contraction coupling cycle. We previously showed that RyR refractoriness plays a key role in the onset of SR Ca2+ alternans in the intact rabbit heart, which contributes to arrhythmogenic action potential duration (APD) alternans. Recent studies have also implicated impaired SERCA function, a key feature of heart failure, in cardiac alternans and arrhythmias. However, the relationship between reduced SERCA function and SR Ca2+ alternans is not well understood. Simultaneous optical mapping of transmembrane potential (Vm) and SR Ca2+ was performed in isolated rabbit hearts (n = 10) using the voltage-sensitive dye RH237 and the low-affinity Ca2+ indicator Fluo-5N-AM. Alternans was induced by rapid ventricular pacing. SERCA was inhibited with cyclopiazonic acid (CPA; 1–10 μM). SERCA inhibition (1, 5, and 10 μM of CPA) resulted in dose-dependent slowing of SR Ca2+ reuptake, with the time constant (tau) increasing from 70.8 ± 3.5 ms at baseline to 85.5 ± 6.6, 129.9 ± 20.7, and 271.3 ± 37.6 ms, respectively (p < 0.05 vs. baseline for all doses). At fast pacing frequencies, CPA significantly increased the magnitude of SR Ca2+ and APD alternans, most strongly at 10 μM (pacing cycle length = 220 ms: SR Ca2+ alternans magnitude: 57.1 ± 4.7 vs. 13.4 ± 8.9 AU; APD alternans magnitude 3.8 ± 1.9 vs. 0.2 ± 0.19 AU; p < 0.05 10 μM of CPA vs. baseline for both). SERCA inhibition also promoted the emergence of spatially discordant alternans. Notably, at all CPA doses, alternation of SR Ca2+ release occurred prior to alternation of diastolic SR Ca2+ load as pacing frequency increased. Simultaneous optical mapping of SR Ca2+ and Vm in the intact rabbit heart revealed that SERCA inhibition exacerbates pacing-induced SR Ca2+ and APD alternans magnitude, particularly at fast pacing frequencies. Importantly, SR Ca2+ release alternans always occurred before the onset of SR Ca2+ load alternans. These findings suggest that even in settings of diminished SERCA function, relative refractoriness of RyR Ca2+ release governs the onset of intracellular Ca2+ alternans. [ABSTRACT FROM AUTHOR]

Key points: Early‐afterdepolarizations (EADs) are abnormal action potential oscillations and a known cause of cardiac arrhythmias. Ventricular EADs involve reactivation of a Ca2+ current (ICaL) in its 'window region' voltage range. However, electrical mechanisms of atrial EADs, a potential cause of atrial fibrillation, are poorly understood.Atrial cells were obtained from consenting patients undergoing heart surgery, as well as from rabbits. ICaL was blocked with nifedipine and then a hybrid patch clamp/mathematical‐modelling technique, 'dynamic clamping', was used to record action potentials at the same time as injecting an artificial, modifiable, ICaL (ICaL,D‐C).Progressively widening the ICaL,D‐C window region produced EADs of various types, dependent on window width. EAD production was strongest upon moving the activation (vs. inactivation) side of the window.EADs were then induced by a different method: increasing ICaL,D‐C amplitude and/or K+ channel‐blockade (4‐aminopyridine). Narrowing of the ICaL,D‐C window by ∼10 mV abolished these EADs.Atrial ICaL window narrowing is worthy of further testing as a potential anti‐atrial fibrillation drug mechanism. Atrial early‐afterdepolarizations (EADs) may contribute to atrial fibrillation (AF), perhaps involving reactivation of L‐type Ca2+ current (ICaL) in its window region voltage range. The present study aimed (i) to validate the dynamic clamp technique for modifying the ICaL contribution to atrial action potential (AP) waveform; (ii) to investigate the effects of widening the window ICaL on EAD‐propensity; and (iii) to test whether EADs from increased ICaL and AP duration are supressed by narrowing the window ICaL. ICaL and APs were recorded from rabbit and human atrial myocytes by whole‐cell‐patch clamp. During AP recording, ICaL was inhibited (3 µm nifedipine) and replaced by a dynamic clamp model current, ICaL,D‐C (tuned to native ICaL characteristics), computed in real‐time (every 50 µs) based on myocyte membrane potential. ICaL,D‐C‐injection restored the nifedipine‐suppressed AP plateau. Widening the window ICaL,D‐C, symmetrically by stepwise simultaneous equal shifts of half‐voltages (V0.5) of ICaL,D‐C activation (negatively) and inactivation (positively), generated EADs (single, multiple or preceding repolarization failure) in a window width‐dependent manner, as well as AP alternans. A stronger EAD‐generating effect resulted from independently shifting activation V0.5 (asymmetrical widening) than inactivation V0.5; for example, a 15 mV activation shift produced EADs in nine of 17 (53%) human atrial myocytes vs. 0 of 18 from inactivation shift (P < 0.05). In 11 rabbit atrial myocytes in which EADs were generated either by increasing the conductance of normal window width ICaL,D‐C or subsequent 4‐aminopyridine (2 mm), window ICaL,D‐C narrowing (10 mV) abolished EADs of all types (P < 0.05). The present study validated the dynamic clamp for ICaL, which is novel in atrial cardiomyocytes, and showed that EADs of various types are generated by widening (particularly asymmetrically) the window ICaL, as well as abolished by narrowing it. Window ICaL narrowing is a potential therapeutic mechanism worth pursuing in the search for improved anti‐AF drugs. Key points: Early‐afterdepolarizations (EADs) are abnormal action potential oscillations and a known cause of cardiac arrhythmias. Ventricular EADs involve reactivation of a Ca2+ current (ICaL) in its 'window region' voltage range. However, electrical mechanisms of atrial EADs, a potential cause of atrial fibrillation, are poorly understood.Atrial cells were obtained from consenting patients undergoing heart surgery, as well as from rabbits. ICaL was blocked with nifedipine and then a hybrid patch clamp/mathematical‐modelling technique, 'dynamic clamping', was used to record action potentials at the same time as injecting an artificial, modifiable, ICaL (ICaL,D‐C).Progressively widening the ICaL,D‐C window region produced EADs of various types, dependent on window width. EAD production was strongest upon moving the activation (vs. inactivation) side of the window.EADs were then induced by a different method: increasing ICaL,D‐C amplitude and/or K+ channel‐blockade (4‐aminopyridine). Narrowing of the ICaL,D‐C window by ∼10 mV abolished these EADs.Atrial ICaL window narrowing is worthy of further testing as a potential anti‐atrial fibrillation drug mechanism. [ABSTRACT FROM AUTHOR]

Volume overload leads to development of eccentric cardiac hypertrophy and heart failure. In our previous report, we have shown myocyte hypertrophy with no fibrosis and decrease in gap junctional coupling via connexin43 in a rat model of aorto-caval fistula at 21 weeks. Here we set to analyze the electrophysiological and protein expression changes in the left ventricle and correlate them with phenotypic severity based upon ventricles to body weight ratio. ECG analysis showed increased amplitude and duration of the P wave, prolongation of PR and QRS interval, ST segment elevation and decreased T wave amplitude in the fistula group. Optical mapping showed a prolongation of action potential duration in the hypertrophied hearts. Minimal conduction velocity (CV) showed a bell-shaped curve, with a significant increase in the mild cases and there was a negative correlation of both minimal and maximal CV with heart to body weight ratio. Since the CV is influenced by gap junctional coupling as well as the autonomic nervous system, we measured the amounts of tyrosine hydroxylase (TH) and choline acetyl transferase (ChAT) as a proxy for sympathetic and parasympathetic innervation, respectively. At the protein level, we confirmed a significant decrease in total and phosphorylated connexin43 that was proportional to the level of hypertrophy, and similarly decreased levels of TH and ChAT. Even at a single time-point, severity of morphological phenotype correlates with progression of molecular and electrophysiological changes, with the most hypertrophied hearts showing the most severe changes that might be related to arrhythmogenesis. [ABSTRACT FROM AUTHOR]