α7 Nicotinic acetylcholine receptor mediates right ventricular fibrosis and diastolic dysfunction in pulmonary hypertension

Abstract

Right ventricular (RV) fibrosis is a key feature of maladaptive RV hypertrophy and dysfunction and is associated with poor outcomes in pulmonary hypertension (PH). However, mechanisms and therapeutic strategies to mitigate RV fibrosis remain unrealized. Previously, we identified that cardiac fibroblast α7 nicotinic acetylcholine receptor (α7 nAChR) drives smoking-induced RV fibrosis. Here, we sought to define the role of α7 nAChR in RV dysfunction and fibrosis in the settings of RV pressure overload as seen in PH. We show that RV tissue from PH patients has increased collagen content and ACh expression. Using an experimental rat model of PH, we demonstrate that RV fibrosis and dysfunction are associated with increases in ACh and α7 nAChR expression in the RV but not in the left ventricle (LV). In vitro studies show that α7 nAChR activation leads to an increase in adult ventricular fibroblast proliferation and collagen content mediated by a Ca2+/epidermal growth factor receptor (EGFR) signaling mechanism. Pharmacological antagonism of nAChR decreases RV collagen content and improves RV function in the PH model. Furthermore, mice lacking α7 nAChR exhibit improved RV diastolic function and have lower RV collagen content in response to persistently increased RV afterload, compared with WT controls. These finding indicate that enhanced α7 nAChR signaling is an important mechanism underlying RV fibrosis and dysfunction, and targeted inhibition of α7 nAChR is a potentially novel therapeutic strategy in the setting of increased RV afterload.

Keywords: Cardiology; Fibrosis; Heart failure; Ion channels; Pulmonology.

Figure 1. Characterization of pulmonary hypertension model.

(A) Schematic of the experimental design. (B and C) Invasively measured RVSP (B) and RVEDP (C) at 3 weeks (wks) (n = 5 control/3 PH) and 7 wks (n = 10/11). (D) RV weight normalized to the respective LV weight (n = 6/6). (E–G) Echocardiographic measurements at 3-wk (n = 9/11) and 7-wk (n = 6/5) time points of TAPSE (E) using M-Mode, RV free wall early diastolic velocity (e’) using tissue Doppler (F), and cardiac output (G). (H) Representative images of transverse heart sections from control and PH animals stained with Picrosirius red. Scale bar: 5 mm. (I) Collagen content measured using hydroxyproline assay from RV and LV homogenates at 3 wks (n = 6) and 7 wks (n = 6). Two-tailed t tests were performed between control and PH at 3wks and 7 wks; *P < 0.05. Con, control; PH, pulmonary hypertension; RV, right ventricle; RVEDP, right ventricular end diastolic pressure; RVSP, right ventricular systolic pressure; RV e’, right ventricle free wall early diastolic velocity (e’); TAPSE, tricuspid annular plane systolic excursion.

Figure 2. PH is associated with an increase in RV ACh/α7 nAChR.

(A) ELISA for ACh performed on homogenates from the RV at 3 wks (n = 5 Con/6 PH) and 7 wks (n = 12 Con/12 PH) and LV at 3 wks and 7 wks (n = 6 Con/6 PH). (B) AChE activity measured in homogenates from the RV and LV at both time points (n = 6 Con/6 PH). (C) Expression of α7 nAChR protein relative to Vinculin in RV at 3 wks (n = 6 Con/6 PH) and 7 wks (n = 13 Con/12 PH) normalized to controls. (D) Expression of α7 nAChR mRNA in freshly isolated RVCF and RV cardiomyocytes from control and 7 wk PH animals (n = 6 Con/6 PH). (E) Quantitated immunofluorescence intensity of α7 nAChR in nonpermeabilized isolated RVCF from 7-wk control and PH animals. (F) Summary schematic. Data are shown as mean ± SEM. Two-tailed t tests were performed between control and PH at 3 and 7 wks; Mann-Whitney U test for real-time PCR data. *P < 0.05. ACh, acetylcholine; AChE, acetylcholinesterase; CF, cardiac fibroblasts; CM, cardiomyocytes; Glyceraldehyde 3-phosphate dehydrogenase; IF, immunofluorescence; nAChR, nicotinic acetylcholine receptor; PH, pulmonary hypertension; RV, right ventricle.

Figure 3. PAH is associated with an increase in RV Collagen and ACh.

(A–C) Collagen (A), ACh (B), and α7 nAChR (C) protein expression assessed using Sircol assay, ELISA, and immunoblot, respectively, in explanted RV tissue from end-stage human PAH (n = 5) and controls (n = 4). (D) Immunofluorescent staining for α7 nAChR (green), vimentin (red), and nuclei (blue) in human RV tissue from control. Arrows demonstrate localization of α7 nAChR in CF (vimentin positive cells). Scale bar: 20 μm. Data are shown as mean ± SEM. Two-tailed t tests were performed between control and PAH. *P < 0.05. ACh, acetylcholine; nAChR, nicotinic acetylcholine receptor; PAH, pulmonary arterial hypertension; RV, right ventricle.

Figure 4. RV cardiomyocyte–derived ACh promotes cardiac fibroblast proliferation and collagen synthesis through α7 nAChR activation.

(A and B) RVCFs isolated from 7-wk control and PH rats treated with vehicle or 10 nM ACh for 24 hours and then assessed for cell proliferation by cell counts (A) and collagen content (B) with Sircol assay (n = 6). (C and D) Cell counts (n = 5) (C) and collagen content (n = 4) (D) of ARCF in response to 10 nM ACh with or without α7 nAChR antagonist α-BTX (100 nM) for 24 hours. (E and F) Cell counts (n = 5) (E) and collagen content (n = 4) (F) of ARCF in response to 10 nM ACh in the presence or absence of muscarinic receptor antagonist atropine (50 μM) for 24 hours. (G and H) Cell count (G) and collagen content (H) from ARCFs treated with conditioned media from isolated 7 wks Con/PH RV cardiomyocytes. Data are shown as mean ± SEM. ANOVA followed by Bonferroni comparison. *P < 0.05. ARCF, Adult rat cardiac fibroblasts; RVCF, right ventricular cardiac fibroblasts; ACh, acetylcholine; α-BTX, α-bungarotoxin; RVCM, right ventricular cardiac myocytes; CdM, conditioned media (from RV cardiomyocytes); CON, control; PH, pulmonary hypertension.

Figure 5. Cardiac fibroblast proliferation and collagen synthesis are mediated via α7 nAChR–EGFR transactivation.

(A) Representative fluorescence image of containing p-EGFR (Y¹⁰⁶⁸) (red) and nuclei (blue by Hoechst) in ARCF 30 minutes after vehicle, ACh (10 nM), or nicotine (600 nM) treatment in the presence or absence of Mec (20 μM). Scale bar: 20 μm. (B) Summary data from A (n = 9). (C) EGFR transactivation in ARCFs treated with vehicle, 600 nM nicotine, or 10 nM ACh for 30 minutes. EGFR activation was estimated by p-EGFR(Y¹⁰⁶⁸)/EGFR (n = 3). EGFR siRNA and scrambled siRNA-transfected cells were used to confirm the antibody specificities. (D) EGFR transactivation in HEK293T-overexpressing EGFR, α7 nAChR, and Myc-tagged NACHO, by nicotine (600 nM, 30 min) in the presence or absence of α-BTX. Cells stably overexpressing EGFP were used as a control. EGFR phosphorylation, overexpressed EGFR, α7 nAChR, and NACHO were detected by the antibodies against p-EGFR (Y¹⁰⁶⁸), GFP, α7 nAChR, and Myc, respectively. (E) Summary data from D (n = 6). p-EGFR/EGFR was normalized to that in cells without nicotine and α-BTX treatment. (F and G) Cell counts (n = 4) (F) and collagen content (n = 4) (G) of ARCF in response to ACh (10 nM) or nicotine (600 nM) in cells transfected with either EGFR specific or scrambled siRNA. (H and I) Cell counts (n = 5) (H) and collagen content (n = 5) (I) of ARCF in response to nicotine (600 nM) in presence or absence of EGFR inhibitor AG1478 (50 μM). Data are shown as mean ± SEM. ANOVA followed by Bonferroni comparison. *P < 0.05. ACh, acetylcholine; α-BTX, α-bungarotoxin; AG1478, EGFR inhibitor; Mec, mecamylamine; nAChR, nicotinic acetylcholine receptor.

Figure 6. Activation of α7 nAChR results in Ca²⁺-mediated EGFR transactivation.

(A) Averaged time course of the changes in [Ca²⁺]_c in ARCF in response to 600 nM nicotine treatment (added at 30 seconds) in the presence (red squares, n = 63) or absence of 100 nM α-BTX (blue circles, n = 34). Data normalized to before nicotine stimulation. (B) EGFR transactivation in HEK293T overexpressing EGFR, α7 nAChR, and NACHO in presence of a cell-permeable Ca²⁺ chelator BAPTA-AM. (C) Summary data from B (n = 8). (D) Proposed mechanism of α7 nAChR–activated proliferation and enhanced collagen content in CF. Data are shown as mean ± SEM. ANOVA followed by Bonferroni comparison. *P < 0.05. ACh, acetylcholine; α-BTX, α-bungarotoxin; nAChR, nicotinic acetylcholine receptor; BAPTA, 1;2-bis(o-aminophenoxy)ethane-N;N;N′;N′-tetraacetic acid.

Figure 7. Treatment with nAChR blocker mecamylamine improves RV function in experimental pulmonary hypertension.

(A) Study design for mecamylamine (Mec) treatment. PH rats were randomized into vehicle (PH-Veh) or Mec (20 mg/kg, i.p., PH-Mec) treatment daily for 3 wks (n = 9 PH-Veh/11 PH-Mec). (B) RV weight normalized to the respective LV weight (n = 9 PH-Veh/11 PH-Mec). (C) Representative images of the RV from vehicle- and Mec-treated animal stained with Picrosirius red. Scale bar: 20 μm. (D) Collagen content in RV homogenates from vehicle- and Mec-treated PH rats (n = 7 PH-Veh/9 PH-Mec). (E) Representative immunofluorescence staining for pEGFR (Y¹⁰⁶⁸) in the RV from vehicle- and Mec-treated rats. Wheat germ agglutinin to outline the cardiomyocytes in white (Alexa 647), vimentin for cardiac fibroblasts in red (Alexa 594), pEGFR in green (Alexa488), and Hoechst for nuclei in blue. Red asterisks show colocalization of pEGFR with fibroblasts. Scale bar: 20 μm. (F) Quantification of area of pEGFR immunofluorescence in the RV (n = 7 PH-Veh/9 PH-Mec). (G and H) Invasively measured RV systolic pressures (n = 9 PH-Veh/11 PH-Mec) (G) and RV end diastolic pressures (n = 9 PH-Veh/10 PH-Mec) (H). (I and J) Representative examples of pressure-volume relationship for vehicle- (I) and Mec-treated (J) PH rats. (K–M) Arterial Elastance (Ea) as a measure of RV afterload (n = 6 PH-Veh/8 PH-Mec) (K), end-diastolic pressure-volume relationship (Eed) as an indicator of RV stiffness (n = 6 PH-Veh/8 PH-Mec) (L), and end-systolic pressure-volume relationship (Ees) as a measure of RV contractility (n = 6 PH-Veh/8 PH-Mec) (M) derived from the PV loop analyses. Data are shown as mean ± SEM. Two-tailed unpaired t tests or Mann-Whitney U test between vehicle and Mec treatment. *P < 0.05. Ea, arterial elastance; Eed, end-diastolic pressure-volume relationship; Ees, end-systolic pressure-volume relationship; LV, left ventricle; Mec, mecamylamine; PH, pulmonary hypertension; RV, right ventricle; RVSP, RV systolic pressure; RVEDP, RV end diastolic pressure; or RV contractility; WGA, wheat germ agglutinin.

Figure 8. RV collagen content is dependent on α7 nAChR in RV pressure overload.

(A) Study design of PAB model with fixed RV afterload; WT and α7 nAChR–KO mice were randomized to PAB or sham surgery and followed for 2 wks. (B and C) Gradient across the banding site (n = 14–18) assessed by echocardiography (B) and RV systolic pressure assessed by invasive hemodynamics (n = 13–18) (C). (D) RV weight normalized to respective LV weight (n = 10–18). (E) ACh levels in RV homogenates (n = 6). (F) RV end diastolic pressure. (G) Representative images of RV from WT and KO mice after sham or PA banding surgery stained with Picrosirius red. Scale bar: 20 μm. (H) RV collagen content by Sircol assay (n = 7–11). Data are shown as mean ± SEM. Two-way ANOVA followed by Bonferroni comparison test. *P < 0.05. PAB, pulmonary artery banding; RVSP, RV systolic pressure; RVEDP, RV end diastolic pressure.