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

Figures

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  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.

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  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.

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  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.

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  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.

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  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…

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  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.

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  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…

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  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.