The role of intracellular Ca²⁺ (Ca₁²⁺) in triggering early afterdepolarizations (EADs), the origins of EADs and the mechanisms underlying Torsade de Pointes (TdP) were investigated in a model of long QT syndrome (Type 2). Perfused rabbit hearts were stained with RH327 and Rhod‐2/AM to simultaneously map membrane potential (V_m) and Ca₁²⁺ with two photodiode arrays. The I_Kr blocker E4031 (0.5 μM) together with 50% reduction of [K⁺]_o and [Mg²⁺]_o elicited long action potentials (APs), V_m oscillations on AP plateaux (EADs) then ventricular tachycardia (VT). Cryoablation of both ventricular chambers eliminated Purkinje fibres as sources of EADs. E4031 prolonged APs (0.28 to 2.3 s), reversed repolarization sequences (base→apex) and enhanced repolarization gradients (30 to 230 ms, n= 12) indicating a heterogeneous distribution of I_Kr. At low [K⁺]_o and [Mg²⁺]_o, E4031 elicited spontaneous Ca₁²⁺and V_m spikes or EADs (3.5 ± 1.9 Hz) during the AP plateau (n= 6). EADs fired ‘out‐of‐phase’ from several sites, propagated, collided then evolved to TdP. Phase maps (Ca₁²⁺vs. V_m) had counterclockwise trajectories shaped like a ‘boomerang’ during an AP and like ellipses during EADs, with V_m preceding Ca₁²⁺ by 9.2 ± 1.4 (n= 6) and 7.2 ± 0.6 ms (n= 5/6), respectively. After cryoablation, EADs from surviving epicardium (∼1 mm) fired at the same frequency (3.4 ± 0.35 Hz, n= 6) as controls. At the origins of EADs, Ca₁²⁺ preceded V_m and phase maps traced clockwise ellipses. Away from EAD origins, V_m coincided with or preceded Ca₁²⁺. In conclusion, overload elicits EADs originating from either ventricular or Purkinje fibres and ‘out‐of‐phase’ EAD activity from multiple sites generates TdP, evident in pseudo‐ECGs.

Heart rate is under the precise control of the autonomic nervous system. However, the wiring of peripheral neural circuits that regulate heart rate is poorly understood. Here, we develop a clearing-imaging-analysis pipeline to visualize innervation of intact hearts in 3D and employed a multi-technique approach to map parasympathetic and sympathetic neural circuits that control heart rate in mice. We identify cholinergic neurons and noradrenergic neurons in an intrinsic cardiac ganglion and the stellate ganglia, respectively, that project to the sinoatrial node. We also report that the heart rate response to optogenetic versus electrical stimulation of the vagus nerve displays different temporal characteristics and that vagal afferents enhance parasympathetic and reduce sympathetic tone to the heart via central mechanisms. Our findings provide new insights into neural regulation of heart rate, and our methodology to study cardiac circuits can be readily used to interrogate neural control of other visceral organs.

In congenital and acquired long QT type 2, women are more vulnerable than men to Torsade de Pointes. In prepubertal rabbits (and children), the arrhythmia phenotype is reversed; however, females still have longer action potential durations than males. Thus, sex differences in K⁺channels and action potential durations alone cannot account for sex-dependent arrhythmia phenotypes. The L-type calcium current (I_Ca,L) is another determinant of action potential duration, Ca²⁺overload, early afterdepolarizations (EADs), and Torsade de Pointes. Therefore, sex, age, and regional differences inI_Ca,Ldensity and in EAD susceptibility were analyzed in epicardial left ventricular myocytes isolated from the apex and base of prepubertal and adult rabbit hearts. In prepubertal rabbits, peakI_Ca,Lat the base was 22% higher in males than females (6.4±0.5 versus 5.0±0.2 pA/pF;P<0.03) and higher than at the apex (6.4±0.5 versus 5.0±0.3 pA/pF;P<0.02). Sex differences were reversed in adults:I_Ca,Lat the base was 32% higher in females than males (9.5±0.7 versus 6.4±0.6 pA/pF;P<0.002) and 28% higher than the apex (9.5±0.7 versus 6.9±0.5 pA/pF;P<0.01). Apex–base differences inI_Ca,Lwere not significant in adult male and prepubertal female hearts. Western blot analysis showed that Ca_v1.2α levels varied with sex, maturity, and apex–base, with differences similar to variations inI_Ca,L; optical mapping revealed that the earliest EADs fired at the base. Single myocyte experiments and Luo–Rudy simulations concur thatI_Ca,Lelevation promotes EADs and is an important determinant of long QT type 2 arrhythmia phenotype, most likely by reducing repolarization reserve and by enhancing Ca²⁺overload and the propensity forI_Ca,Lreactivation.

Rationale:: Atrial fibrillation (AF) contributes significantly to morbidity and mortality in elderly and hypertensive patients and has been correlated to enhanced atrial fibrosis. Despite a lack of direct evidence that fibrosis causes AF, reversal of fibrosis is considered a plausible therapy. Objective:: To evaluate the efficacy of the antifibrotic hormone relaxin (RLX) in suppressing AF in spontaneously hypertensive rats (SHR). Methods and Results:: Normotensive Wistar-Kyoto (WKY) and SHR were treated for 2 weeks with vehicle (WKY+V and SHR+V) or RLX (0.4 mg/kg per day, SHR+RLX) using implantable mini-pumps. Hearts were perfused, mapped optically to analyze action potential durations, intracellular Ca ²⁺ transients, and restitution kinetics, and tested for AF vulnerability. SHR hearts had slower conduction velocity (CV; P <0.01 versus WKY), steeper CV restitution kinetics, greater collagen deposition, higher levels of transcripts for transforming growth factor-β, metalloproteinase-2, metalloproteinase-9, collagen I/III, and reduced connexin 43 phosphorylation ( P <0.05 versus WKY). Programmed stimulation triggered sustained AF in SHR (n=5/5) and SHR+V (n=4/4), but not in WKY (n=0/5) and SHR+RLX (n=1/8; P <0.01). RLX treatment reversed the transcripts for fibrosis, flattened CV restitution kinetics, reduced action potential duration at 90% recovery to baseline, increased CV ( P <0.01), and reversed atrial hypertrophy ( P <0.05). Independent of antifibrotic actions, RLX (0.1 µmol/L) increased Na ⁺ current density, I _Na (≈2-fold in 48 hours) in human cardiomyocytes derived from inducible pluripotent stem cells (n=18/18; P <0.01). Conclusions:: RLX treatment suppressed AF in SHR hearts by increasing CV from a combination of reversal of fibrosis and hypertrophy and by increasing I _Na . The study provides compelling evidence that RLX may provide a novel therapy to manage AF in humans by reversing fibrosis and hypertrophy and by modulating cardiac ionic currents.

Mice that overexpress the inflammatory cytokine tumor necrosis factor-α in the heart (TNF mice) develop heart failure characterized by atrial and ventricular dilatation, decreased ejection fraction, atrial and ventricular arrhythmias, and increased mortality (males > females). Abnormalities in Ca²⁺ handling, prolonged action potential duration (APD), calcium alternans, and reentrant atrial and ventricular arrhythmias were previously observed with the use of optical mapping of perfused hearts from TNF mice. We therefore tested whether altered voltage-gated outward K⁺ and/or inward Ca²⁺ currents contribute to the altered action potential characteristics and the increased vulnerability to arrhythmias. Whole cell voltage-clamp recordings of K⁺ currents from left ventricular myocytes of TNF mice revealed an ∼50% decrease in the rapidly activating, rapidly inactivating transient outward K⁺ current I_to and in the rapidly activating, slowly inactivating delayed rectifier current I_K,slow1, an ∼25% decrease in the rapidly activating, slowly inactivating delayed rectifier current I_K,slow2, and no significant change in the steady-state current I_ss compared with controls. Peak amplitudes and inactivation kinetics of the L-type Ca²⁺ current I_Ca,L were not altered. Western blot analyses revealed a reduction in the proteins underlying Kv4.2, Kv4.3, and Kv1.5. Thus decreased K⁺ channel expression is largely responsible for the prolonged APD in the TNF mice and may, along with abnormalities in Ca²⁺ handling, contribute to arrhythmias.