Alpha7 nicotinic acetylcholine receptor mediates chronic nicotine inhalation-induced cardiopulmonary dysfunction

Abstract

Cigarette smoking remains the leading modifiable risk factor for cardiopulmonary diseases; however, the effects of nicotine alone on cardiopulmonary function remain largely unknown. Previously, we have shown that chronic nicotine vapor inhalation in mice leads to the development of pulmonary hypertension (PH) with right ventricular (RV) remodeling. The present study aims to further examine the cardiopulmonary effects of nicotine and the role of the α7 nicotinic acetylcholine receptor (α7-nAChR), which is widely expressed in the cardiovascular system. Wild-type (WT) and α7-nAChR knockout (α7-nAChR-/-) mice were exposed to room air (control) or nicotine vapor daily for 12 weeks. Consistent with our previous study, echocardiography and RV catheterization reveal that male WT mice developed increased RV systolic pressure with RV hypertrophy and dilatation following 12-week nicotine vapor exposure; in contrast, these changes were not observed in male α7-nAChR-/- mice. In addition, chronic nicotine inhalation failed to induce PH and RV remodeling in female mice regardless of genotype. The effects of nicotine on the vasculature were further examined in male mice. Our results show that chronic nicotine inhalation led to impaired acetylcholine-mediated vasodilatory response in both thoracic aortas and pulmonary arteries, and these effects were accompanied by altered endothelial nitric oxide synthase phosphorylation (enhanced inhibitory phosphorylation at threonine 495) and reduced plasma nitrite levels in WT but not α7-nAChR-/- mice. Finally, RNA sequencing revealed up-regulation of multiple inflammatory pathways in thoracic aortas from WT but not α7-nAChR-/- mice. We conclude that the α7-nAChR mediates chronic nicotine inhalation-induced PH, RV remodeling and vascular dysfunction.

Keywords: alpha7-nAChR; eNOS/NO signaling; endothelial dysfunction; nicotine; pulmonary hypertension; right ventricular remodeling.

Figure 1.. Chronic nicotine inhalation-induced pulmonary hypertension is mediated by α7-nAChR in male mice

(A) Chronic nicotine inhalation leads to increased right ventricular systolic pressure (RVSP) in wild-type (n=10 air; n=10 nicotine) but not α7-nAChR^−/− (n=7 air; n=10 nicotine) male mice. *P<0.05, **P<0.01. (B) Chronic nicotine inhalation results in a trend of increase in Fulton index (P=0.077) in wild-type (n=10 air; n=10 nicotine) but not α7-nAChR^−/− (n=8 air; n=10 nicotine) male mice; LV, left ventricle; RV, right ventricle; S, interventricular septum. (C) Chronic nicotine inhalation does not alter RVSP in wild-type (n=5 air; n=5 nicotine) or α7-nAChR^−/− (n=5 air; n=7 nicotine) female mice. (D) Chronic nicotine inhalation does not alter Fulton index in wild-type (n=5 air; n=8 nicotine) or α7-nAChR^−/− (n=5 air; n=8 nicotine) female mice. The above data were analyzed by two-way ANOVA followed by Tukey–Kramer post hoc test.

Figure 2.. Chronic nicotine inhalation-induced RV remodeling is mediated by α7-nAChR in male mice

(A) Chronic nicotine inhalation leads to increased RV free wall thickness (FWT) at diastole (d) in wild-type (n=15 air; n=19 nicotine) but not α7-nAChR^−/− (n=7 air; n=11 nicotine) male mice; ****P<0.0001. (B) Chronic nicotine inhalation leads to increased RV internal diameter (RVID) at diastole (d) in wild-type (n=15 air; n=19 nicotine) but not α7-nAChR^−/− (n=7 air; n=11 nicotine) male mice; **P<0.01, ****P<0.0001. (C) Chronic nicotine inhalation does not alter RV free wall thickness (FWT) at diastole (d) in wild-type (n=8 air; n=10 nicotine) or α7-nAChR^−/− (n=8 air; n=11 nicotine) female mice. (D) Chronic nicotine inhalation does not alter RV internal diameter (RVID) at diastole (d) in wild-type (n=8 air; n=10 nicotine) or α7-nAChR^−/− (n=8 air; n=11 nicotine) female mice. The above data were analyzed by two-way ANOVA followed by Tukey–Kramer post hoc test.

Figure 3.. Chronic nicotine inhalation impairs endothelium-dependent vasodilation of thoracic aorta in wild-type but not α7-nAChR^−/− male mice

(A) Thoracic aortas isolated from wildtype male mice exposed to nicotine (n=12) exhibit reduced vasodilatory response to acetylcholine (ACh) compared with those from air-exposed males (n=11); *P<0.05, **P<0.01. The EC₅₀ of ACh was not impacted by nicotine and is represented for each response curve as a dashed vertical line. (B and C) Chronic nicotine inhalation does not alter vasodilatory response to sodium nitroprusside (SNP) or vasoconstrictive response to phenylephrine (Phe) in thoracic aorta. Air, n=9–11; nicotine, n=10–12. (D–F) Thoracic aortas isolated from α7-nAChR^−/− male mice exposed to nicotine (n=3–7) and air (n=3–6) exhibit similar vasoreactivity to ACh, SNP, and Phe. Response curves were analyzed by two-way ANOVA followed by Bonferroni’s multiple comparisons test and EC₅₀ was analyzed by two-tailed Student’s t-test.

Figure 4.. Chronic nicotine inhalation impairs vasoreactivity of pulmonary artery in wild-type but not α7-nAChR^−/− male mice

(A) Pulmonary arteries isolated from wild-type male mice exposed to nicotine (n=10) exhibit reduced vasodilatory response to acetylcholine (ACh) compared with those exposed to air (n=10); **P<0.01. The EC₅₀ of ACh was significantly increased by nicotine and is represented for each response curve as a dashed vertical line; †P<0.05. (B) Pulmonary arteries isolated from wild-type male mice exposed to nicotine (n=6) exhibit similar vasodilatory response to sodium nitroprusside (SNP) compared with those exposed to air (n=6). (C) Pulmonary arteries isolated from wild-type male mice exposed to nicotine (n=10) exhibit enhanced vasoconstrictive response to phenylephrine (Phe) compared with those exposed to air (n=10), and pretreatment with L-NAME (10 μM) abolishes the heightened response (right, n=9/group); *P<0.05. (D–F) Pulmonary arteries isolated from α7-nAChR^−/− male mice exposed to nicotine (n=7–8) and air (n=5–6) exhibit similar vasoreactivity to ACh, SNP, and Phe. Response curves were analyzed by two-way ANOVA followed by Bonferroni’s multiple comparisons test and EC₅₀ was analyzed by two-tailed Student’s t-test.

Figure 5.. Chronic nicotine inhalation leads to altered eNOS phosphorylation and reduced plasma nitrite levels in wild-type but not α7-nAChR^−/− male mice

(A) Chronic nicotine inhalation leads to increased inhibitory phosphorylation of eNOS at Thr⁴⁹⁵ without significant alteration of the stimulatory phosphorylation at Ser¹¹⁷⁷ in wildtype male mice; **P<0.01. (B) Chronic nicotine inhalation does not alter the levels of total or phosphorylated eNOS in α7-nAChR^−/− male mice. (C and D) Chronic nicotine inhalation leads reduced plasma nitrite levels in wild-type (n=14 air; n=15 nicotine) but not α7-nAChR^−/− (n=8 air; n=17 nicotine) male mice; *P<0.05. Data were analyzed by two-tailed Student’s t-test.

Figure 6.. Chronic nicotine inhalation alters aortic gene expression in male mice

(A) RNA sequencing (n=3/group) reveals that chronic nicotine inhalation in wild-type male mice results in 179 differentially regulated genes (149 up-regulated and 30 down-regulated), while only 7 non-overlapping genes are found to be differentially regulated by nicotine in α7-nAChR^−/− male mice. (B) Top-10 signaling pathways differentially regulated by nicotine in wild-type male mice. (C) Heatmap of genes identified in the pathways differentially regulated by nicotine in wild-type male mice.