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Cyclic adenosine 3',5'-monophosphate (cAMP) is a key second messenger in cardiomyocytes responsible for transducing autonomic signals into downstream electrophysiological responses. Previous studies have shown intracellular heterogeneity and compartmentalization of cAMP signaling. However, whether cAMP signaling occurs heterogeneously throughout the intact heart and how this drives sex-dependent functional responses are unknown. Here, we developed and validated a novel cardiac-specific fluorescence resonance energy transfer-based cAMP reporter mouse and a combined voltage-cAMP whole-heart imaging system. We showed that in male hearts, cAMP was uniformly activated in response to pharmacological β-adrenergic stimulation. In contrast, female hearts showed that cAMP levels decayed faster in apical versus basal regions, which was associated with nonuniform action potential changes and notable changes in the direction of repolarization. Apical phosphodiesterase (PDE) activity was higher in female versus male hearts, and PDE inhibition prevented repolarization changes in female hearts. Thus, our imaging approach revealed sex-dependent regional breakdown of cAMP and associated electrophysiological differences.

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.

BackgroundCardiac gene expression and arrhythmia occurrence have time-of-day variation; however, daily changes in cardiac electrophysiology, arrhythmia susceptibility, and Ca2+ handling have not been characterized. Furthermore, how these patterns change with age is unknown.MethodsHearts were isolated during the light (zeitgeber time [ZT] 4 and ZT9) and dark cycle (ZT14 and ZT21) from adult (12-18 weeks) male mice. Hearts from aged (18-20 months) male mice were isolated at ZT4 and ZT14. All hearts were Langendorff-perfused for optical mapping with voltage- and Ca2+-sensitive dyes (n=4-7/group). Cardiac gene and protein expression were assessed with real-time polymerase chain reaction (n=4-6/group) and Western blot (n=3-4/group).ResultsAdult hearts had the shortest action potential duration (APD) and Ca2+ transient duration (CaTD) at ZT14 (APD80: ZT4: 45.4±4.1 ms; ZT9: 45.1±8.6 ms; ZT14: 34.7±4.2 ms; ZT21: 49.2±7.6 ms, P

BackgroundEpidemiological 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.ObjectivesThe purpose of this study was to investigate the impact of an environmentally relevant concentration of SHS on cardiac electrophysiology and indicators of arrhythmia.MethodsMale 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.ResultsAt 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.ConclusionsIn 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.

BackgroundInterleukin 1β (IL-1β) is a key regulator of the inflammatory response after myocardial infarction (MI) by modulating immune cell recruitment, cytokine production, and extracellular matrix turnover. Elevated levels of IL-1β are associated with adverse remodeling, and inhibition of IL-1 signaling after MI results in improved contractile function.ObjectiveThe goal of this study was to determine whether IL-1 signaling also contributes to post-MI arrhythmogenesis.MethodsMI was created in 2 murine models of elevated inflammation: atherosclerotic on the Western diet or wild-type with a subseptic dose of lipopolysaccharide. The role of IL-1β was assessed with the IL-1 receptor antagonist anakinra (10 mg/(kg·d), starting 24 hours post-MI).ResultsIn vivo and ex vivo molecular imaging showed reduced myocardial inflammation after a 4-day course of anakinra treatment, despite no change in infarct size. At day 5 post-MI, high-speed optical mapping of transmembrane potential and intracellular Ca2+ in isolated hearts revealed that IL-1β inhibition improved conduction velocity, reduced action potential duration dispersion, improved intracellular Ca2+ handling, decreased transmembrane potential and Ca2+ alternans magnitude, and reduced spontaneous and inducible ventricular arrhythmias. These functional improvements were linked to increased expression of connexin 43 and sarcoplasmic reticulum Ca2+-ATPase.ConclusionThis study revealed a novel mechanism for IL-1β in contributing to defective excitation-contraction coupling and arrhythmogenesis in the post-MI heart. Our results suggest that inhibition of IL-1 signaling post-MI may represent a novel antiarrhythmic therapy.

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.

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.

BackgroundAtherosclerotic animal models show increased recruitment of inflammatory cells to the heart after myocardial infarction (MI), which impacts ventricular function and remodeling.ObjectiveThe purpose of this study was to determine whether increased myocardial inflammation after MI also contributes to arrhythmias.MethodsMI was created in 3 mouse models: (1) atherosclerotic (apolipoprotein E deficient [ApoE(-/-)] on atherogenic diet, n = 12); (2) acute inflammation (wild-type [WT] given daily lipopolysaccharide [LPS] 10 μg/day, n = 7); and (3) WT (n = 14). Sham-operated (n = 4) mice also were studied. Four days post-MI, an inflammatory protease-activatable fluorescent probe (Prosense680) was injected intravenously to quantify myocardial inflammation on day 5. Optical mapping with voltage-sensitive dye was performed on day 5 to assess electrophysiology and arrhythmia susceptibility.ResultsInflammatory activity (Prosense680 fluorescence) was increased approximately 2-fold in ApoE+MI and LPS+MI hearts vs WT+MI (P

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

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is an enzyme with important regulatory functions in the heart and brain, and its chronic activation can be pathological. CaMKII activation is seen in heart failure, and can directly induce pathological changes in ion channels, Ca(2+) handling and gene transcription. Here, in human, rat and mouse, we identify a novel mechanism linking CaMKII and hyperglycaemic signalling in diabetes mellitus, which is a key risk factor for heart and neurodegenerative diseases. Acute hyperglycaemia causes covalent modification of CaMKII by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAc modification of CaMKII at Ser 279 activates CaMKII autonomously, creating molecular memory even after Ca(2+) concentration declines. O-GlcNAc-modified CaMKII is increased in the heart and brain of diabetic humans and rats. In cardiomyocytes, increased glucose concentration significantly enhances CaMKII-dependent activation of spontaneous sarcoplasmic reticulum Ca(2+) release events that can contribute to cardiac mechanical dysfunction and arrhythmias. These effects were prevented by pharmacological inhibition of O-GlcNAc signalling or genetic ablation of CaMKIIδ. In intact perfused hearts, arrhythmias were aggravated by increased glucose concentration through O-GlcNAc- and CaMKII-dependent pathways. In diabetic animals, acute blockade of O-GlcNAc inhibited arrhythmogenesis. Thus, O-GlcNAc modification of CaMKII is a novel signalling event in pathways that may contribute critically to cardiac and neuronal pathophysiology in diabetes and other diseases.