Chronic nicotine exposure is associated with electrophysiological and sympathetic remodeling in the intact rabbit heart.

Nicotine is the primary addictive component of tobacco products. Through its actions on the heart and autonomic nervous system, nicotine exposure is associated with electrophysiological changes and increased arrhythmia susceptibility. To assess the underlying mechanisms, we treated rabbits with transdermal nicotine (NIC, 21 mg/day) or control (CT) patches for 28 days before performing dual optical mapping of transmembrane potential (RH237) and intracellular Ca2+ (Rhod-2 AM) in isolated hearts with intact sympathetic innervation. Sympathetic nerve stimulation (SNS) was performed at the first to third thoracic vertebrae, and ß-adrenergic responsiveness was additionally evaluated following norepinephrine (NE) perfusion. Baseline ex vivo heart rate (HR) and SNS stimulation threshold were higher in NIC versus CT (P = 0.004 and P = 0.003, respectively). Action potential duration alternans emerged at longer pacing cycle lengths (PCL) in NIC versus CT at baseline (P = 0.002) and during SNS (P = 0.0003), with similar results obtained for Ca2+ transient alternans. SNS shortened the PCL at which alternans emerged in CT but not in NIC hearts. NIC-exposed hearts tended to have slower and reduced HR responses to NE perfusion, but ventricular responses to NE were comparable between groups. Although fibrosis was unaltered, NIC hearts had lower sympathetic nerve density (P = 0.03) but no difference in NE content versus CT. These results suggest both sympathetic hypoinnervation of the myocardium and regional differences in ß-adrenergic responsiveness with NIC. This autonomic remodeling may contribute to the increased risk of arrhythmias associated with nicotine exposure, which may be further exacerbated with long-term use.NEW & NOTEWORTHY Here, we show that chronic nicotine exposure was associated with increased heart rate, increased susceptibility to alternans, and reduced sympathetic electrophysiological responses in the intact rabbit heart. We suggest that this was due to sympathetic hypoinnervation of the myocardium and diminished ß-adrenergic responsiveness of the sinoatrial node following nicotine treatment. Though these differences did not result in increased arrhythmia propensity in our study, we hypothesize that prolonged nicotine exposure may exacerbate this proarrhythmic remodeling.

Chronic nicotine exposure is associated with electrophysiological and sympathetic remodeling in the intact rabbit heart.

Nicotine is the primary addictive component in tobacco products. Through its actions on the heart and autonomic nervous system, nicotine exposure is associated with electrophysiological changes and increased arrhythmia susceptibility. However, the underlying mechanisms are unclear. To address this, we treated rabbits with transdermal nicotine (NIC, 21 mg/day) or control (CT) patches for 28 days prior to performing dual optical mapping of transmembrane potential (RH237) and intracellular Ca 2+ (Rhod-2 AM) in isolated hearts with intact sympathetic innervation. Sympathetic nerve stimulation (SNS) was performed at the 1 st - 3 rd thoracic vertebrae, and ß-adrenergic responsiveness was additionally evaluated as changes in heart rate (HR) following norepinephrine (NE) perfusion. Baseline ex vivo HR and SNS stimulation threshold were increased in NIC vs. CT ( P = 0.004 and P = 0.003 respectively). Action potential duration alternans emerged at longer pacing cycle lengths (PCL) in NIC vs. CT at baseline ( P = 0.002) and during SNS ( P = 0.0003), with similar results obtained for Ca 2+ transient alternans. SNS reduced the PCL at which alternans emerged in CT but not NIC hearts. NIC exposed hearts also tended to have slower and reduced HR responses to NE perfusion. While fibrosis was unaltered, NIC hearts had lower sympathetic nerve density ( P = 0.03) but no difference in NE content vs. CT. These results suggest both sympathetic hypo-innervation of the myocardium and diminished ß-adrenergic responsiveness with NIC. This autonomic remodeling may underlie the increased risk of arrhythmias associated with nicotine exposure, which may be further exacerbated with continued long-term usage. NEW & NOTEWORTHY: Here we show that chronic nicotine exposure was associated with increased heart rate, lower threshold for alternans and reduced sympathetic electrophysiological responses in the intact rabbit heart. We suggest that this was due to the sympathetic hypo-innervation of the myocardium and diminished ß- adrenergic responsiveness observed following nicotine treatment. Though these differences did not result in increased arrhythmia propensity in our study, we hypothesize that prolonged nicotine exposure may exacerbate this pro-arrhythmic remodeling.

Unveiling the intricacies of intracellular Ca2+ regulation in the heart.

Recent studies have provided valuable insight into the key mechanisms contributing to the spatiotemporal regulation of intracellular Ca2+ release and Ca2+ signaling in the heart. In this research highlight, we focus on the latest findings published in Biophysical Journal examining the structural organization of Ca2+ handling proteins and assessing the functional aspects of intracellular Ca2+ regulation in health and the detrimental consequences of Ca2+ dysregulation in disease. These important studies pave the way for future mechanistic investigations and multiscale understanding of Ca2+ signaling in the heart.

Mechanisms of spontaneous Ca2+ release-mediated arrhythmia in a novel 3D human atrial myocyte model: II. Ca2+ -handling protein variation.

Disruption of the transverse-axial tubule system (TATS) in diseases such as heart failure and atrial fibrillation occurs in combination with changes in the expression and distribution of key Ca2+ -handling proteins. Together this ultrastructural and ionic remodelling is associated with aberrant Ca2+ cycling and electrophysiological instabilities that underlie arrhythmic activity. However, due to the concurrent changes in TATs and Ca2+ -handling protein expression and localization that occur in disease it is difficult to distinguish their individual contributions to the arrhythmogenic state. To investigate this, we applied our novel 3D human atrial myocyte model with spatially detailed Ca2+ diffusion and TATS to investigate the isolated and interactive effects of changes in expression and localization of key Ca2+ -handling proteins and variable TATS density on Ca2+ -handling abnormality driven membrane instabilities. We show that modulating the expression and distribution of the sodium-calcium exchanger, ryanodine receptors and the sarcoplasmic reticulum (SR) Ca2+ buffer calsequestrin have varying pro- and anti-arrhythmic effects depending on the balance of opposing influences on SR Ca2+ leak-load and Ca2+ -voltage relationships. Interestingly, the impact of protein remodelling on Ca2+ -driven proarrhythmic behaviour varied dramatically depending on TATS density, with intermediately tubulated cells being more severely affected compared to detubulated and densely tubulated myocytes. This work provides novel mechanistic insight into the distinct and interactive consequences of TATS and Ca2+ -handling protein remodelling that underlies dysfunctional Ca2+ cycling and electrophysiological instability in disease. KEY POINTS: In our companion paper we developed a 3D human atrial myocyte model, coupling electrophysiology and Ca2+ handling with subcellular spatial details governed by the transverse-axial tubule system (TATS). Here we utilize this model to mechanistically examine the impact of TATS loss and changes in the expression and distribution of key Ca2+ -handling proteins known to be remodelled in disease on Ca2+ homeostasis and electrophysiological stability. We demonstrate that varying the expression and localization of these proteins has variable pro- and anti-arrhythmic effects with outcomes displaying dependence on TATS density. Whereas detubulated myocytes typically appear unaffected and densely tubulated cells seem protected, the arrhythmogenic effects of Ca2+ handling protein remodelling are profound in intermediately tubulated cells. Our work shows the interaction between TATS and Ca2+ -handling protein remodelling that underlies the Ca2+ -driven proarrhythmic behaviour observed in atrial fibrillation and may help to predict the effects of antiarrhythmic strategies at varying stages of ultrastructural remodelling.

Mechanisms of spontaneous Ca2+ release-mediated arrhythmia in a novel 3D human atrial myocyte model: I. Transverse-axial tubule variation.

Intracellular calcium (Ca2+ ) cycling is tightly regulated in the healthy heart ensuring effective contraction. This is achieved by transverse (t)-tubule membrane invaginations that facilitate close coupling of key Ca2+ -handling proteins such as the L-type Ca2+ channel and Na+ -Ca2+ exchanger (NCX) on the cell surface with ryanodine receptors (RyRs) on the intracellular Ca2+ store. Although less abundant and regular than in the ventricle, t-tubules also exist in atrial myocytes as a network of transverse invaginations with axial extensions known as the transverse-axial tubule system (TATS). In heart failure and atrial fibrillation, there is TATS remodelling that is associated with aberrant Ca2+ -handling and Ca2+ -induced arrhythmic activity; however, the mechanism underlying this is not fully understood. To address this, we developed a novel 3D human atrial myocyte model that couples electrophysiology and Ca2+ -handling with variable TATS organization and density. We extensively parameterized and validated our model against experimental data to build a robust tool examining TATS regulation of subcellular Ca2+ release. We found that varying TATS density and thus the localization of key Ca2+ -handling proteins has profound effects on Ca2+ handling. Following TATS loss, there is reduced NCX that results in increased cleft Ca2+ concentration through decreased Ca2+ extrusion. This elevated Ca2+ increases RyR open probability causing spontaneous Ca2+ releases and the promotion of arrhythmogenic waves (especially in the cell interior) leading to voltage instabilities through delayed afterdepolarizations. In summary, the present study demonstrates a mechanistic link between TATS remodelling and Ca2+ -driven proarrhythmic behaviour that probably reflects the arrhythmogenic state observed in disease. KEY POINTS: Transverse-axial tubule systems (TATS) modulate Ca2+ handling and excitation-contraction coupling in atrial myocytes, with TATS remodelling in heart failure and atrial fibrillation being associated with altered Ca2+ cycling and subsequent arrhythmogenesis. To investigate the poorly understood mechanisms linking TATS variation and spontaneous Ca2+ release, we built, parameterized and validated a 3D human atrial myocyte model coupling electrophysiology and spatially-detailed subcellular Ca2+ handling governed by the TATS. Simulated TATS loss causes diastolic Ca2+ and voltage instabilities through reduced Na+ -Ca2+ exchanger-mediated Ca2+ removal, cleft Ca2+ accumulation and increased ryanodine receptor open probability, resulting in spontaneous Ca2+ release and promotion of arrhythmogenic waves and delayed afterdepolarizations. At fast electrical rates typical of atrial tachycardia/fibrillation, spontaneous Ca2+ releases are larger and more frequent in the cell interior than at the periphery. Our work provides mechanistic insight into how atrial TATS remodelling can lead to Ca2+ -driven instabilities that may ultimately contribute to the arrhythmogenic state in disease.

Enhanced calcium release at specialised surface sites compensates for reduced t-tubule density in neonatal sheep atrial myocytes.

Cardiac myocytes rely on transverse (t)-tubules to facilitate a rapid rise in calcium throughout the cell. However, despite their importance in triggering synchronous Ca2+ release, t-tubules are highly labile structures. They develop postnatally, increase in density during exercise training and are lost in diseases such as heart failure (HF). In the majority of settings, an absence of t-tubules decreases function. Here we show that despite reduced t-tubule density due to immature t-tubules, the newborn atrium is highly specialised to maintain Ca2+ release. To compensate for fewer t-tubules triggering a central rise in Ca2+, Ca2+ release at sites on the cell surface is enhanced in the newborn, exceeding that at all Ca2+ release sites in the adult. Using electron and super resolution microscopy to investigate myocyte ultrastructure, we found that newborn atrial cells had enlarged surface sarcoplasmic reticulum and larger, more closely spaced surface and central ryanodine receptor clusters. We suggest that these adaptations mediate enhanced Ca2+ release at the sarcolemma and aid propagation to compensate for reduced t-tubule density in the neonatal atrium.

Optimising Large Animal Models of Sustained Atrial Fibrillation: Relevance of the Critical Mass Hypothesis.

BACKGROUND: Large animal models play an important role in our understanding of the pathophysiology of atrial fibrillation (AF). Our aim was to determine whether prospectively collected baseline variables could predict the development of sustained AF in sheep, thereby reducing the number of animals required in future studies. Our hypothesis was that the relationship between atrial dimensions, refractory periods and conduction velocity (otherwise known as the critical mass hypothesis) could be used for the first time to predict the development of sustained AF. METHODS: Healthy adult Welsh mountain sheep underwent a baseline electrophysiology study followed by implantation of a neurostimulator connected via an endocardial pacing lead to the right atrial appendage. The device was programmed to deliver intermittent 50 Hz bursts of 30 s duration over an 8-week period whilst sheep were monitored for AF. RESULTS: Eighteen sheep completed the protocol, of which 28% developed sustained AF. Logistic regression analysis showed only fibrillation number (calculated using the critical mass hypothesis as the left atrial diameter divided by the product of atrial conduction velocity and effective refractory period) was associated with an increased likelihood of developing sustained AF (Ln Odds Ratio 26.1 [95% confidence intervals 0.2-52.0] p = 0.048). A receiver-operator characteristic curve showed this could be used to predict which sheep developed sustained AF (C-statistic 0.82 [95% confidence intervals 0.59-1.04] p = 0.04). CONCLUSION: The critical mass hypothesis can be used to predict sustained AF in a tachypaced ovine model. These findings can be used to optimise the design of future studies involving large animals.

Phosphodiesterase 5 inhibition improves contractile function and restores transverse tubule loss and catecholamine responsiveness in heart failure.

Heart failure (HF) is characterized by poor survival, a loss of catecholamine reserve and cellular structural remodeling in the form of disorganization and loss of the transverse tubule network. Indeed, survival rates for HF are worse than many common cancers and have not improved over time. Tadalafil is a clinically relevant drug that blocks phosphodiesterase 5 with high specificity and is used to treat erectile dysfunction. Using a sheep model of advanced HF, we show that tadalafil treatment improves contractile function, reverses transverse tubule loss, restores calcium transient amplitude and the heart's response to catecholamines. Accompanying these effects, tadalafil treatment normalized BNP mRNA and prevented development of subjective signs of HF. These effects were independent of changes in myocardial cGMP content and were associated with upregulation of both monomeric and dimerized forms of protein kinase G and of the cGMP hydrolyzing phosphodiesterases 2 and 3. We propose that the molecular switch for the loss of transverse tubules in HF and their restoration following tadalafil treatment involves the BAR domain protein Amphiphysin II (BIN1) and the restoration of catecholamine sensitivity is through reductions in G-protein receptor kinase 2, protein phosphatase 1 and protein phosphatase 2 A abundance following phosphodiesterase 5 inhibition.

Increased Vulnerability to Atrial Fibrillation Is Associated With Increased Susceptibility to Alternans in Old Sheep.

Background Atrial fibrillation ( AF ) is common in the elderly, but rare in the young; however, the changes that occur with age that promote AF are not fully understood. Action potential ( AP ) alternans may be involved in the initiation of AF . Using a translationally relevant model, we investigated whether age-associated atrial vulnerability to AF was associated with susceptibility to AP alternans. Methods and Results AF was induced in conscious young and old sheep using 50 Hz burst pacing. Old sheep were more vulnerable to AF . Monophasic and cellular AP s were recorded from the right atrium in vivo and from myocytes isolated from the left and right atrial appendages. AP alternans occurred at lower stimulation frequencies in old sheep than young in vivo (old, 3.0±0.1 Hz; young, 3.3±0.1 Hz; P<0.05) and in isolated myocytes (old, 1.6±0.1 Hz; young, 2.0±0.1 Hz; P<0.05). Simultaneous recordings of [Ca2+]i and membrane potential in myocytes showed that alternans of AP s and [Ca2+]i often occurred together. However, at low stimulation rates [Ca2+]i alternans could occur without AP alternans, whereas at high stimulation rates AP alternans could still be observed despite disabling Ca2+ cycling using thapsigargin. Conclusions We have shown, for the first time in a large mammalian model, that aging is associated with increased duration of AF and susceptibility to AP alternans. We suggest that instabilities in Ca2+ handling initiate alternans at low stimulation rates, but that AP restitution alone can sustain alternans at higher rates.

Dependence of cardiac transverse tubules on the BAR domain protein amphiphysin II (BIN-1).

RATIONALE: Transverse tubules (t-tubules) regulate cardiac excitation-contraction coupling and exhibit interchamber and interspecies differences in expression. In cardiac disease, t-tubule loss occurs and affects the systolic calcium transient. However, the mechanisms controlling t-tubule maintenance and whether these factors differ between species, cardiac chambers, and in a disease setting remain unclear. OBJECTIVE: To determine the role of the Bin/Amphiphysin/Rvs domain protein amphiphysin II (AmpII) in regulating t-tubule maintenance and the systolic calcium transient. METHODS AND RESULTS: T-tubule density was assessed by di-4-ANEPPS, FM4-64 or WGA staining using confocal microscopy. In rat, ferret, and sheep hearts t-tubule density and AmpII protein levels were lower in the atrium than in the ventricle. Heart failure (HF) was induced in sheep using right ventricular tachypacing and ferrets by ascending aortic coarctation. In both HF models, AmpII protein and t-tubule density were decreased in the ventricles. In the sheep, atrial t-tubules were also lost in HF and AmpII levels decreased. Conversely, junctophilin 2 levels did not show interchamber differences in the rat and ferret nor did they change in HF in the sheep or ferret. In addition, in rat atrial and sheep HF atrial cells where t-tubules were absent, junctophilin 2 had sarcomeric intracellular distribution. Small interfering RNA-induced knockdown of AmpII protein reduced t-tubule density, calcium transient amplitude, and the synchrony of the systolic calcium transient. CONCLUSIONS: AmpII is intricately involved in t-tubule maintenance. Reducing AmpII protein decreases t-tubule density, reduces the amplitude, and increases the heterogeneity of the systolic calcium transient.