Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Anesthetic preconditioning (APC), defined as brief exposure to inhalational anesthetics before cardiac ischemia-reperfusion (I/R), limits injury in both animal models and in humans. APC can result in the production of reactive oxygen species (ROS), and prior work has shown that APC can modify activation of NF-kappaB during I/R, with consequent reduction in the expression of inflammatory mediators. However, the role of NF-kappaB activation before I/R is unknown. Therefore, these experiments tested the hypothesis that APC-induced ROS results in activation of NF-kappaB before I/R, with consequent increased expression of antiapoptotic proteins such as Bcl-2 and decreased apoptosis. Experiments utilized an established perfused heart rat model of sevoflurane APC and I/R. The role of NF-kappaB was defined by a novel method of transient inhibition of the regulatory kinase IKK using the reversible inhibitor SC-514. In addition to functional measures of left ventricular developed and end-diastolic pressure, phosphorylation of IkappaBalpha and activation of NF-kappaB were measured along with cytosolic protein content of Bcl-2, release of cytochrome c, and degradation of caspase-3. APC resulted in ROS-dependent phosphorylation of IkappaBalpha and activation of NF-kappaB before I/R. APC also increased the expression of Bcl-2 before I/R. In addition to functional protection following I/R, APC resulted in lower release of cytochrome c and caspase-3 degradation. These protective effects of APC were abolished by transient inhibition of IkappaBalpha phosphorylation and NF-kappaB activation by SC-514 followed by washout. ROS-dependent activation of NF-kappaB by APC before I/R is a critical element in the protective effect of APC. APC reduces apoptosis and functional impairment by increasing Bcl-2 expression before I/R. Interventions that increase NF-kappaB activation before I/R should protect hearts from I/R injury.

Unlabelled

Sevoflurane preconditioning (SPC) in adult hearts reduces myocardial ischemia/reperfusion (I/R) injury, an effect that may be mediated by reductions in intracellular Ca(2+) ([Ca(2+)](i)) and/or mitochondrial Ca(2+) ([Ca(2+)](m)) accumulation during ischemia and reperfusion. Because the physiology, pharmacology, and metabolic responses of the newborn differ from adults, we tested the hypothesis that SPC protects newborn myocardium by limiting [Ca(2+)](i) and [Ca(2+)](m) by a K(ATP) channel-dependent mechanism. Fluorescence spectrofluorometry and nuclear magnetic resonance spectroscopy were used to measure [Ca(2+)](i), [Ca(2+)](m), and adenosine triphosphate (ATP) in 4- to 7-day-old Langendorff-perfused rabbit hearts. Three experimental groups were used to study the effect of SPC on [Ca(2+)](m)/[Ca(2+)](i), ATP, as well as hemodynamics and ischemic injury. The role of mitochondrial K(ATP) channels was assessed by exposing the SPC hearts to the mitochondrial K(ATP) channel blocker 5-hydroxydecanoic acid. Our results show that SPC significantly decreased [Ca(2+)](i) and [Ca(2+)](m) during I/R, as well as decreased creatine kinase release during reperfusion and resulted in higher ATP. 5-Hydroxydecanoic acid abolished the effect of SPC on [Ca(2+)], hemodynamics, ATP, and creatine kinase release. In conclusion, decreased [Ca(2+)](i) and [Ca(2+)](m) observed with SPC is associated with greater ATP recovery as well as diminished cell injury. Mitochondrial K(ATP) channel blockade attenuates the SPC effect during I/R, suggesting that these channels are involved in the protective effects of SPC in the newborn.

Implications

The results of this study support the hypothesis that sevoflurane preconditioning protects newborn hearts from calcium overload and ischemic injury via a mechanism dependent on mitochondrial KATP channels.

Key points

Heart 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.

Abstract

Heart 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.

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 (V_m) 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 V_m 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.

Sympathetic and parasympathetic control of the heart is a classic example of norepinephrine (NE) and acetylcholine (ACh) triggering opposing actions. Sympathetic NE increases heart rate and contractility through activation of β receptors, whereas parasympathetic ACh slows the heart through muscarinic receptors. Sympathetic neurons can undergo a developmental transition from production of NE to ACh and we provide evidence that mouse cardiac sympathetic nerves transiently produce ACh after myocardial infarction (MI). ACh levels increased in viable heart tissue 10-14 d after MI, returning to control levels at 21 d, whereas NE levels were stable. At the same time, the genes required for ACh synthesis increased in stellate ganglia, which contain most of the sympathetic neurons projecting to the heart. Immunohistochemistry 14 d after MI revealed choline acetyltransferase (ChAT) in stellate sympathetic neurons and vesicular ACh transporter immunoreactivity in tyrosine hydroxylase-positive cardiac sympathetic fibers. Finally, selective deletion of the ChAT gene from adult sympathetic neurons prevented the infarction-induced increase in cardiac ACh. Deletion of the gp130 cytokine receptor from sympathetic neurons prevented the induction of cholinergic genes after MI, suggesting that inflammatory cytokines induce the transient acquisition of a cholinergic phenotype in cardiac sympathetic neurons. Ex vivo experiments examining the effect of NE and ACh on rabbit cardiac action potential duration revealed that ACh blunted both the NE-stimulated decrease in cardiac action potential duration and increase in myocyte calcium transients. This raises the possibility that sympathetic co-release of ACh and NE may impair adaptation to high heart rates and increase arrhythmia susceptibility.

Significance statement

Sympathetic neurons normally make norepinephrine (NE), which increases heart rate and the contractility of cardiac myocytes. We found that, after myocardial infarction, the sympathetic neurons innervating the heart begin to make acetylcholine (ACh), which slows heart rate and decreases contractility. Several lines of evidence confirmed that the source of ACh was sympathetic nerves rather than parasympathetic nerves that are the normal source of ACh in the heart. Global application of NE with or without ACh to ex vivo hearts showed that ACh partially reversed the NE-stimulated decrease in cardiac action potential duration and increase in myocyte calcium transients. That suggests that sympathetic co-release of ACh and NE may impair adaptation to high heart rates and increase arrhythmia susceptibility.

Background

Epidemiological evidence suggests that a majority of deaths attributed to secondhand smoke (SHS) exposure are cardiovascular related. However, to our knowledge, the impact of SHS on cardiac electrophysiology, [Formula: see text] handling, and arrhythmia risk has not been studied.

Objectives

The purpose of this study was to investigate the impact of an environmentally relevant concentration of SHS on cardiac electrophysiology and indicators of arrhythmia.

Methods

Male C57BL/6 mice were exposed to SHS [total suspended particles (THS): [Formula: see text], nicotine: [Formula: see text], carbon monoxide: [Formula: see text], or filtered air (FA) for 4, 8, or 12 wk ([Formula: see text]]. Hearts were excised and Langendorff perfused for dual optical mapping with voltage- and [Formula: see text]-sensitive dyes.

Results

At slow pacing rates, SHS exposure did not alter baseline electrophysiological parameters. With increasing pacing frequency, action potential duration (APD), and intracellular [Formula: see text] alternans magnitude progressively increased in all groups. At 4 and 8 wk, there were no statistical differences in APD or [Formula: see text] alternans magnitude between SHS and FA groups. At 12 wk, both APD and [Formula: see text] alternans magnitude were significantly increased in the SHS compared to FA group ([Formula: see text]). SHS exposure did not impact the time constant of [Formula: see text] transient decay ([Formula: see text]) at any exposure time point. At 12 wk exposure, the recovery of [Formula: see text] transient amplitude with premature stimuli was slightly (but nonsignificantly) delayed in SHS compared to FA hearts, suggesting that [Formula: see text] release via ryanodine receptors may be impaired.

Conclusions

In male mice, chronic exposure to SHS at levels relevant to social situations in humans increased their susceptibility to cardiac alternans, a known precursor to ventricular arrhythmia. https://doi.org/10.1289/EHP3664.

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.

Myocardial infarction (MI) can result in sympathetic nerve loss in the infarct region. However, the contribution of hypo-innervation to electrophysiological remodeling, independent from MI-induced ischemia and fibrosis, has not been comprehensively investigated. We present a novel mouse model of regional cardiac sympathetic hypo-innervation utilizing a targeted-toxin (dopamine beta-hydroxylase antibody conjugated to saporin, DBH-Sap), and measure resulting electrophysiological and Ca²⁺ handling dynamics. Five days post-surgery, sympathetic nerve density was reduced in the anterior left ventricular epicardium of DBH-Sap hearts compared to control. In Langendorff-perfused hearts, there were no differences in mean action potential duration (APD₈₀) between groups; however, isoproterenol (ISO) significantly shortened APD₈₀ in DBH-Sap but not control hearts, resulting in a significant increase in APD₈₀ dispersion in the DBH-Sap group. ISO also produced spontaneous diastolic Ca²⁺ elevation in DBH-Sap but not control hearts. In innervated hearts, sympathetic nerve stimulation (SNS) increased heart rate to a lesser degree in DBH-Sap hearts compared to control. Additionally, SNS produced APD₈₀ prolongation in the apex of control but not DBH-Sap hearts. These results suggest that hypo-innervated hearts have regional super-sensitivity to circulating adrenergic stimulation (ISO), while having blunted responses to SNS, providing important insight into the mechanisms of arrhythmogenesis following sympathetic nerve loss.

Rationale

Sarcoplasmic reticulum (SR) Ca(2+) cycling is key to normal excitation-contraction coupling but may also contribute to pathological cardiac alternans and arrhythmia.

Objective

To measure intra-SR free [Ca(2+)] ([Ca(2+)]SR) changes in intact hearts during alternans and ventricular fibrillation (VF).

Methods and results

Simultaneous 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<0.05). Ryanodine receptor (RyR) refractoriness played a key role in the onset of SR Ca(2+) alternans, with SR Ca(2+) release alternans routinely occurring without changes in diastolic [Ca(2+)]SR. Sensitizing RyR with caffeine (200 μmol/L) significantly reduced the pacing threshold for both SR Ca(2+) and APD alternans (188±15 and 173±12 ms; P<0.05 versus baseline). Caffeine also reduced the magnitude of spatially discordant SR Ca(2+) alternans, but not APD alternans, the pacing threshold for discordance, or threshold for VF. During VF, [Ca(2+)]SR was high, but RyR remained nearly continuously refractory, resulting in minimal SR Ca(2+) release throughout VF.

Conclusions

In intact hearts, RyR refractoriness initiates SR Ca(2+) release alternans that can be amplified by diastolic [Ca(2+)]SR alternans and lead to APD alternans. Sensitizing RyR suppresses spatially concordant but not discordant SR Ca(2+) and APD alternans. Despite increased [Ca(2+)]SR during VF, SR Ca(2+) release was nearly continuously refractory. This novel method provides insight into SR Ca(2+) handling during cardiac alternans and arrhythmia.