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

Abstract

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 β-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 β-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 β-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.

Keywords: action potential; arrhythmia; calcium; nicotine; sympathetic.

Figure 1.

Innervated rabbit heart preparation.

Figure 2.

Cotinine concentration, heart rate, and sympathetic nerve stimulation threshold in control and nicotine-exposed hearts. A: mean cotinine concentration at 0, 2, and 4 wk of nicotine exposure. n = 6–10/group. B: ex vivo baseline heart rate. Measurements taken before the addition of dyes and blebbistatin. C: stimulation frequency threshold for sympathetic nerve stimulation. Spinal cord was stimulated at T1–T2 with constant voltage and frequency was increased at 0.5-Hz increments until a >15% increase in heart rate was observed. n = 8–11/group. Data are represented as means ± SD. *P < 0.05 and **P < 0.01 by two-way ANOVA with mixed-effects analysis (A) or by Mann–Whitney test (B and C). X, males; O, females.

Figure 3.

Effect of sympathetic nerve stimulation (SNS) on control and nicotine-exposed hearts. Heart rate (A and B), action potential duration (APD₈₀, C and D), and calcium transient duration (CaTD₈₀, E and F) changes with SNS during sinus rhythm. Baseline data assessed just before SNS. n = 8–11/group. Data are represented as means ± SD. *P < 0.05 by two-way ANOVA with repeated measures (A, C, and E), Mann–Whitney test (B), or two-tailed, unpaired t test (D and F). X, males; O, females.

Figure 4.

Alternans threshold and magnitude in control and nicotine-exposed hearts. A: pacing cycle length (PCL) at which action potential duration (APD) alternans emerged at baseline and with sympathetic nerve stimulation (SNS). B: PCL at which Ca²⁺ alternans emerged. C and D: representative contour maps (i) and corresponding Ca²⁺ traces (ii) demonstrating Ca²⁺ alternans magnitude at baseline and with SNS at PCL = 200 ms (C: control; D: nicotine). Ca²⁺ trace averaged from entire field of view to demonstrate mean alternans magnitude. n = 8/group. Data are represented as means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-way ANOVA with repeated measures. X, males; O, females.

Figure 5.

Responses to norepinephrine (NE) in control and nicotine-exposed hearts. A and B: heart rate changes over time immediately before and after the addition of 500 nM NE to the perfusate. C: heart rate measurements after the addition of NE at 1 min (t1), 2 min (t2), and 3 min (t3). D: percent change from baseline to maximum heart rate with NE. E: maximum heart rate after NE added. F–I: action potential duration (APD; F and G) and calcium transient duration (CaTD; H and I) changes in response to NE in sinus rhythm. n = 7–11/group, except A–C where n = 4–11 depending on time point. Data are represented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by two-way ANOVA with mixed-effects analysis (A–C, F, and H) Mann–Whitney test (D), or t test (E, G, and I). X, males; O, females.

Figure 6.

Arrhythmia susceptibility in control and nicotine-exposed hearts. A: pseudo-ECG recording demonstrating a premature ventricular contraction (PVC) during sinus rhythm. Recordings were measured from the posterior thoracic cavity in the bath. B: average arrhythmia scores. n = 8–10/group. Data are represented as means ± SD. Significance tested by Mann–Whitney test. X, males; O, females.

Figure 7.

Fibrosis, sympathetic nerve density, and norepinephrine (NE) content in control and nicotine-exposed hearts. A: representative images of Masson’s trichrome staining; fibrosis indicated in blue and myocardial tissue in red. B: average fibrosis density in left ventricle. n = 5–7/group. C: representative immunofluorescence images of tyrosine hydroxylase (TH) labeling. D: sympathetic nerve fiber density in left ventricle. n = 6/group. E: NE content of left ventricle (LV) and right atria (RA). n = 7/group. Data are represented as means ± SD. *P < 0.05 and **P < 0.01 by Mann–Whitney test (B), two-tailed, unpaired t test (D), or two-way ANOVA with repeated measures (E). Scale bar = 100 µm. X, males; O, females.