Sotatercept analog improves cardiopulmonary remodeling and pulmonary hypertension in experimental left heart failure

Abstract

Pulmonary hypertension due to left heart disease (PH-LHD) is the most frequent manifestation of PH but lacks any approved treatment. Activin receptor type IIA-Fc fusion protein (ActRIIA-Fc) was found previously to be efficacious in experimental and human pulmonary arterial hypertension (PAH). Here we tested the hypothesis that ActRIIA-Fc improves pulmonary vascular remodeling and alleviates PH in models of PH-LHD, specifically in subtypes of heart failure with reduced ejection fraction (PH-HFrEF) and preserved ejection fraction (PH-HFpEF). Treatment with murine ActRIIA-Fc reduced cardiac remodeling and improved cardiac function in two mouse models of left heart disease without PH, confirming that this inhibitor of activin-class ligand signaling can exert cardioprotective effects in heart failure. In a mouse model of PH-HFrEF with prolonged pressure overload caused by transverse aortic constriction, ActRIIA-Fc treatment significantly reduced pulmonary vascular remodeling, pulmonary fibrosis, and pulmonary hypertension while exerting beneficial structural, functional, and histological effects on both the left and right heart. Additionally, in an obese ZSF1-SU5416 rat model of PH-HFpEF with metabolic dysregulation, therapeutic treatment with ActRIIA-Fc normalized SMAD3 overactivation in pulmonary vascular and perivascular cells, reversed pathologic pulmonary vascular and cardiac remodeling, improved pulmonary and cardiac fibrosis, alleviated PH, and produced marked functional improvements in both cardiac ventricles. Studies in vitro revealed that treatment with ActRIIA-Fc prevents an abnormal, glucose-induced, activin-mediated, migratory phenotype in human pulmonary artery smooth muscle cells, providing a mechanism by which ActRIIA-Fc could exert therapeutic effects in experimental PH-HFpEF with metabolic dysregulation. Our results demonstrate that ActRIIA-Fc broadly corrects cardiopulmonary structure and function in experimental PH-LHD, including models of PH-HFrEF and PH-HFpEF, leading to alleviation of PH under diverse pathophysiological conditions. These findings highlight the important pathogenic contributions of activin-class ligands in multiple forms of experimental PH and support ongoing clinical evaluation of human ActRIIA-Fc (sotatercept) in patients with PH-HFpEF.

Keywords: ActRIIA-Fc; Group 2 pulmonary hypertension (PH); activin; heart failure with preserved ejection fraction; heart failure with reduced ejection fraction; pulmonary arterial hypertension; smooth muscle cell; vascular remodeling.

FIGURE 1

ActRIIA-Fc exerts structural, functional, and anti-fibrotic cardioprotective effects in a transverse aortic constriction (TAC) mouse model of left heart failure caused by sustained pressure overload. (A) Experimental approach used to assess effects of ActRIIA-Fc (RAP-011). Wild-type mice were subjected to TAC and treated twice weekly with RAP-011 (R011, 10 mg/kg, s.c.) or vehicle (veh, phosphate-buffered saline, PBS) for 3 weeks starting one day post-surgery. (B) Heart weight normalized to body weight (HW/BW), (C) fractional shortening, (D) ejection fraction, (E) myocardial performance index (MPI), (F) LV developed pressure (LVDP), and (G) peak rates of LV pressure rise (dP/dt_max) and decline (-dP/dt_min). Data are means ± SEM (n = 10–15 mice per group for day 21). (H) Representative images of LV sections stained with Masson’s trichrome blue to detect fibrosis (scale bar, 100 μm), and (I) quantification of percentage area occupied by fibrotic tissue. Data are means ± SEM (n = 10–15 mice per group). Analysis by one-way ANOVA and Dunnett’s post-hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

FIGURE 2

ActRIIA-Fc improves pulmonary vascular remodeling and reduces PH in a TAC-PH mouse model. (A) Experimental approach used to assess effects of ActRIIA-Fc (RAP-011). Wild-type mice were subjected to TAC and treated twice weekly with RAP-011 (R011, 10 mg/kg, s.c.) or vehicle (veh, PBS) for 4 weeks starting 2 weeks post-surgery to promote development of PH in the untreated state. (B) Left atrial pressure, (C) mean pulmonary arterial pressure (mPAP), and (D) RV systolic pressure (RVSP). (E) Images of representative lung sections stained with hematoxylin and eosin showing degree of vascular remodeling. Scale bar, 50 μm. (F) Quantification of vessel wall thickness as a percentage of vessel outer diameter. (G) Images of lung sections stained with Masson’s trichrome to detect fibrosis. Scale bar, 50 μm. (H) Quantification of fibrotic tissue area. Data are means ± SEM. Analysis by one-way ANOVA and Dunnett’s post-hoc test (**P < 0.01, ***P < 0.001, ****P < 0.0001).

FIGURE 3

ActRIIA-Fc reduces left ventricle (LV) remodeling and improves left heart function in the TAC-PH mouse model. (A) Heart weight normalized to body weight (HW/BW), (B) interventricular septum thickness in diastole (IVSd), (C) LV mass (LVM), (D) fractional shortening, (E) LV ejection fraction, (F) mitral inflow velocity (E) and mitral annular velocity (E’) ratio (E/E’), and (G) isovolumic relaxation time (IVRT). Data are means ± SEM (n = 10–15 mice per group for day 42). (H) Representative images of LV sections stained with Masson’s trichrome blue to detect fibrosis (scale bar, 50 μm) and (I) quantification of percentage area occupied by fibrotic tissue. Data are means ± SEM (n = 10–15 mice per group). Analysis by one-way ANOVA and Dunnett’s post-hoc test (*P < 0.05, ***P < 0.001, ****P < 0.0001). RAP-011 (R011), vehicle (veh, PBS).

FIGURE 4

Therapeutic treatment with ActRIIA-Fc improves pulmonary remodeling and cardiopulmonary function in the obese ZSF1-Su rat model of PH-HFpEF. (A) Experimental approach used to evaluate therapeutic effects of ActRIIA-Fc (RAP-011, R011) in an obese ZSF1-Su rat model of PH-HFpEF. Rats were treated at 8 weeks of age with a single dose of SU5416 (100 mg/kg, s.c). After allowing 6 weeks for development of PH, rats were treated with RAP-011 (10 mg/kg, s.c., twice weekly), sildenafil (30 mg/kg, p.o., twice daily), or vehicle (Veh, PBS) for 8 weeks. (B) Effects of RAP-011 (R011) or sildenafil (Sild) on RV systolic pressure (RVSP) and Fulton index [RV/(LV + S)], a measure of RV hypertrophy. (C) Images of representative lung sections stained with hematoxylin and eosin, with insets showing degree of vascular remodeling. Scale bar, 200 μm. (D) Vessel wall thickness as a percentage of vessel outer diameter. (E) Images of lung sections stained with Masson’s trichrome to detect fibrosis. Scale bar, 50 μm. (F) Quantification of fibrotic tissue area. Data are means ± SEM. Analysis by one-way ANOVA and Tukey’s (B) or Dunnett’s (D, F) post-hoc test. *P < 0.05; ***P < 0.001; ****P < 0.0001. Lean, lean control rats.

FIGURE 5

ActRIIA-Fc improves remodeling and function of both left and right heart in obese ZSF1-Su rats. Effects of ActRIIA-Fc (RAP-011, R011) on (A) left heart parameters LV mass, mitral inflow velocity (E) and mitral annular velocity (E’) ratio (E/E’), and isovolumic relaxation time (IVRT); (B) right heart parameters RV free-wall thickness (RVFWT), tricuspid annular plane systolic excursion (TAPSE), and RV fractional area change (RVFAC); (C) myocardial performance index (MPI); and (D) quantification of fibrosis in LV and RV. Data are means ± SEM (n = 5–13 rats per group). Analysis by one-way ANOVA and Dunnett’s post-hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

FIGURE 6

ActRIIA-Fc inhibits canonical and non-canonical signaling associated with the TGF-β superfamily pathway in the lung of obese ZSF1-Su rats. (A) Representative images of lung sections immunostained for phospho-Smad3 (pSmad3) in obese ZSF1-Su rats and lean controls after treatment with ActRIIA-Fc (RAP-011, R011) or vehicle (veh, PBS) as in Figure 1A. Scale bar, 50 μm. (B) Percentage of pSmad3-positive cells in lungs based on assessment of 30 high-magnification fields per rat. (C) Lung homogenates immunoblotted for phospho–extracellular-signal-regulated kinase (pERK), total ERK, phospho-c-Jun N-terminal kinase (pJNK), total JNK, and β-actin. Data are means ± SEM. Analysis by one-way ANOVA and Dunnett’s post-hoc test. *P < 0.05.

FIGURE 7

ActRIIA-Fc inhibits glucose-induced release of activin A and migration by human pulmonary artery smooth muscle cells (PASMCs) mediated through the SGLT pathway. (A) Glucose-induced activin A release by hPASMCs. (B) Effect of SGLT inhibitor (SGLTi) sotagliflozin on glucose-induced release of activin A by hPASMCs. (C) Effect of human ActRIIA-Fc (ACE-011) on glucose-induced release of activin A by hPASMCs. (D) ACE-011 inhibits activin A-induced migration of hPASMCs. (E) Effects of SGLTi or ACE-011 on glucose-induced migration of hPASMCs. N or NG, normal glucose concentration (5 mM); H or HG, high glucose concentration (25 mM). Data are means ± SEM. Analysis by one-way ANOVA and Dunnett’s post-hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (F) Effect of RAP-011 (R011) on dysregulated expression of glucose transporters in lung of obese ZSF1-Su rats. Lean, lean control rats; veh, vehicle (PBS). (G) Schematic overview of proposed mechanisms by which ActRIIA-Fc normalizes aberrant PASMC activity induced by high glucose levels in the obese ZSF1-Su rat. For clarity, activin A and its receptors are depicted as monomeric, and the non-covalent complex between mature activin A and its prodomain is not shown. Yellow circles identify phosphorylated proteins and dashed lines indicate mechanisms not yet delineated. I, activin receptor-like kinase 4 or 7; II, ActRIIA or ActRIIB; SGLT, sodium-glucose cotransporter; SGLTi, SGLT inhibitor.