Metabolic Syndrome Mediates ROS-miR-193b-NFYA-Dependent Downregulation of Soluble Guanylate Cyclase and Contributes to Exercise-Induced Pulmonary Hypertension in Heart Failure With Preserved Ejection Fraction

Abstract

Background: Many patients with heart failure with preserved ejection fraction have metabolic syndrome and develop exercise-induced pulmonary hypertension (EIPH). Increases in pulmonary vascular resistance in patients with heart failure with preserved ejection fraction portend a poor prognosis; this phenotype is referred to as combined precapillary and postcapillary pulmonary hypertension (CpcPH). Therapeutic trials for EIPH and CpcPH have been disappointing, suggesting the need for strategies that target upstream mechanisms of disease. This work reports novel rat EIPH models and mechanisms of pulmonary vascular dysfunction centered around the transcriptional repression of the soluble guanylate cyclase (sGC) enzyme in pulmonary artery (PA) smooth muscle cells.

Methods: We used obese ZSF-1 leptin-receptor knockout rats (heart failure with preserved ejection fraction model), obese ZSF-1 rats treated with SU5416 to stimulate resting pulmonary hypertension (obese+sugen, CpcPH model), and lean ZSF-1 rats (controls). Right and left ventricular hemodynamics were evaluated using implanted catheters during treadmill exercise. PA function was evaluated with magnetic resonance imaging and myography. Overexpression of nuclear factor Y α subunit (NFYA), a transcriptional enhancer of sGC β1 subunit (sGCβ1), was performed by PA delivery of adeno-associated virus 6. Treatment groups received the SGLT2 inhibitor empagliflozin in drinking water. PA smooth muscle cells from rats and humans were cultured with palmitic acid, glucose, and insulin to induce metabolic stress.

Results: Obese rats showed normal resting right ventricular systolic pressures, which significantly increased during exercise, modeling EIPH. Obese+sugen rats showed anatomic PA remodeling and developed elevated right ventricular systolic pressure at rest, which was exacerbated with exercise, modeling CpcPH. Myography and magnetic resonance imaging during dobutamine challenge revealed PA functional impairment of both obese groups. PAs of obese rats produced reactive oxygen species and decreased sGCβ1 expression. Mechanistically, cultured PA smooth muscle cells from obese rats and humans with diabetes or treated with palmitic acid, glucose, and insulin showed increased mitochondrial reactive oxygen species, which enhanced miR-193b-dependent RNA degradation of nuclear factor Y α subunit (NFYA), resulting in decreased sGCβ1-cGMP signaling. Forced NYFA expression by adeno-associated virus 6 delivery increased sGCβ1 levels and improved exercise pulmonary hypertension in obese+sugen rats. Treatment of obese+sugen rats with empagliflozin improved metabolic syndrome, reduced mitochondrial reactive oxygen species and miR-193b levels, restored NFYA/sGC activity, and prevented EIPH.

Conclusions: In heart failure with preserved ejection fraction and CpcPH models, metabolic syndrome contributes to pulmonary vascular dysfunction and EIPH through enhanced reactive oxygen species and miR-193b expression, which downregulates NFYA-dependent sGCβ1 expression. Adeno-associated virus-mediated NFYA overexpression and SGLT2 inhibition restore NFYA-sGCβ1-cGMP signaling and ameliorate EIPH.

Keywords: MIRN193 microRNA, human; nitric oxide; nuclear factor Y; pulmonary hypertension.

Figure 1.. Novel HFpEF rat and CpcPH model of Exercise Induced Pulmonary Hypertension

(A) Experimental timeline for lean, lean treated with sugen, obese (EIPH model), and obese treated with sugen rats (CpcPH model). (B) Representative image of implantation of polyethylene (PE) tube in right (RV) and left ventricle (LV). (C-D) At 22 weeks old of age, Body weight, HbA1c level, HOMA-iR and Tryiglyceride were measured (lean n=8, lean+sugen n=5, obese n=8, obese+sugen n=5). (E) Right ventricular systolic and end-diastolic pressure (RVSP and RVEDP), left ventricular systolic and end-diastolic pressure (LVSP and LVEDP) were measured at rest and during exercise (lean n=6–8, lean+sugen n=5, obese n=6–8, obese+sugen n=5). (F) Workload were calculated with the distance of test run and body weight (BW) (lean n=8, lean+sugen n=5, obese n=8, obese+sugen n=5). (G) Fulton index was measured as weight of RV/weight of LV + septum in lean (n=8), lean+sugen (n=5), obese (n=8), and obese+sugen rats (n=5). Results are expressed as mean±SEM. *P<0.05, **P<0.01, ***P<0.001. Comparisons of parameters were performed with 2-tailed Student’s t-test, Welch’s t-test, one-way ANOVA, repeated measure two-way ANOVA or mixed-effects model analysis followed by Tukey’s honestly significant difference (HSD) test for multiple comparisons.

Figure 2.. Right ventricular dysfunction after exercise in ZSF-1 obese and obese+sugen models.

(A) Representative images from M mode and pulse-wave (PW) doppler mode ultrasonography. Yellow scale bar, 100 msec. Blue scale bar, 2 mm (M mode), or 100 cm/s (PW mode). (B-C) Left ventricular Ejection fraction (LVEF), Fraction shortening (LVFS), Internal diastolic or systolic diameter (LVIDd or LVIDs), end-diastolic posterior wall (LVPWd), end-diastolic interventricular septal wall thickness (IVSd), E wave/A wave ratio (E/A), cardiac output (CO), and cardiac index (CI) were measured at rest and during exercise. (D) Representative images of M mode and PW doppler mode ultrasonography used to calculate right ventricular end-diastolic diameter (RVDd), pulmonary artery acceleration time (PAAT) per ejection time (ET), and tricuspid annular plane systolic excursion (TAPSE). Yellow scale bar, 100 msec. Blue scale bar, 3 mm (RVDd or TAPSE), or 100 cm/s (PAAT/ET). (E-F) RVDd, PAAT/ET, TAPSE, and TPRi were measured at rest and after exercise. Rats per group; lean n=8, lean+sugen n=5, obese n=8, obese+sugen n=5. Results are expressed as mean±SEM. *P<0.05, **P<0.01, ***P<0.001. Statistical analyses were performed as described in Figure 1 legend.

Figure 3.. Pulmonary artery dysfunction contributes to the pathophysiology in EIPH and in CpcPH during exercise

(A) Representative immunostaining for α-smooth muscle actin (αSMA) of the pulmonary arteries in lean, obese, obese+sugen rats. Scale bars, 100 μm. Medial index (%) of distal pulmonary arteries with a diameter of 20–100 μm was calculated as described in Methods in lean (n=8), lean+sugen (n=5), obese (n=8), and obese+sugen rats (n=5). (B) Representative images of MRI of left pulmonary artery and heart at rest and during dobutamine (5μg/kg/min) challenge in lean, lean+sugen, obese, and obese+sugen rats. White bars indicate 1 mm. Quantification of area of left pulmonary artery at second branch and area of right ventricular in lean (n=6–7), lean+sugen (n=5), obese (n=6), and obese+sugen rats (n=5). (C) Hemodynamics (RVSP, RVEDP, LVSP, and LVEDP) and cardiac index (CI) during dobutamine challenge in lean (n=6–7), lean+sugen (n=5), obese (n=6), and obese+sugen rats (n=5). (D-E) Schematic representation of the mechanisms underlying pulmonary artery and right ventricular dysfunction in EIPH in obese and obese+sugen rats. Results are expressed as mean±SEM. *P<0.05, **P<0.01, ***P<0.001. Statistical analyses were performed as described in Figure 1 legend.

Figure 4.. Increased mitochondrial derived ROS and decreased sGC enzyme expression and activity in PAVSMCs

(A) Myographic studies on isolated pulmonary arteries of lean and obese rats with acetylcholine (Ach) or sodium nitroprusside (SNP) (each n=6). (B) Representative immunofluorescence images and quantification of 4HNE of distal pulmonary arteries. lean n=8, obese n=8, and obese+sugen (n=5) at rest or after treadmill exercise. (C) Quantification of intra-cellular ROS (CellROX fluorescence) in cultured PAVSMCs from lean and obese rats, in which Rotenone (10μM) and MitoTEMPO (25 or 50μM) were added 10 minutes before analysis (each n=6). (D) Quantification of mitochondrial ROS (MitoSOX fluorescence) or membrane potential (TMRM fluorescence) in cultured PAVSMCs from lean and obese rats (each n=6). (E) Quantification of cGMP of plasma level in lean (n=7), obese (n=6), and obese+sugen rats (n=5). (F) Representative Western blot and quantification of sGCβ1, sGCα1, phosphorylation ser1177 eNOS, total eNOS and GAPDH in isolated pulmonary arteries of lean (diameter of PAs: 500–700μm) (n=8), obese (n=8), and obese+sugen rats (n=5). (G) Representative immunofluorescence image and quantification of sGCβ1 of distal pulmonary arteries. lean n=8, obese n=8, and obese+sugen (n=5) at rest or after treadmill exercise. (H) cGMP level in PAVSMCs of lean, obese, obese+sugen rats treated with NO donor DETA NONOate (10μM, 24 hours) (each n=6). (I-J) Lean and obese rats were treated with Nitrites via drinking water (100mg/L) or vehicle for 1-week. Quantification of cGMP of plasma level (each n=6). Right ventricular systolic pressure (RVSP) was measured at rest and during exercise (each n=6). (K) Quantification of relative mRNA expression of sGCβ1, sGCα1, NFYA, NFYB, NFYC, FoxO1, 3 and 4, and HDAC3 in cultured PAVSMCs from obese rats compared to lean rats (n=3, each) (*P<0.05, **P<0.01, ***P<0.001 vs Ln). (L) Representative Western blot and quantification of sGCβ1, NFYA, and GAPDH in cytoplasm in lean, obese and obese+sugen PAVSMCs (n=6, each). (M) Representative Western blot and quantification of sGCβ1, NFYA, and GAPDH in rat PAVSMCs treated with hydrogen peroxidase (H₂O₂, 25μM, 24 hours) (n=3, each). Results are expressed as mean±SEM. *P<0.05, **P<0.01, ***P<0.001. †† P<0.01, ††† P<0.001 vs same group at condition. Statistical analyses were performed as described in Figure 1 legend.

Figure 5.. Transcription factor NFYA controls sGC expression in PAVSMCs and is down regulated by metabolic stress and ROS

(A) Representative Western blot and quantification of sGCβ1, NFYA, NFYB, NFYC, and GAPDH in PAVSMCs treated with Si-Control or Si-NYFA (each n=3). Each plot represents the PAVSMCs sample cultured from one individual rat. (B-C) Quantification of intra-cellular ROS (CellROX fluorescence), mitochondrial ROS (MitoSOX fluorescence), and membrane potential (TMRM fluorescence) in rat PAVSMCs treated with Palmitate acid (P, 0.2 mM) and Glucose (G, 25 mM), Insulin (I, 120 nM) stimulation for 24 hours, in which Rotenone (10μM) and MitoTEMPO (50μM) were added 10 minutes before analysis (each n=6). (D) Representative Western blot and quantification of sGCβ1, NFYA and GAPDH in lean rat PAVSMCs treated with superoxide dismutase (SOD, 400 U/ml), Catalase (1 kU/ml) or PGI for 24 hours (n=6, each). (E-F) Transcription binding sites for promoter region of sGCβ1 (CCAAT sequence). Chromatin immunoprecipitation (ChIP) showing enrichment of NYFA at sGCβ1 promoter in lean and obese PAVSMCs treated with PGI, SOD or Catalase (each n=3). (G) RNA degradation of NFYA for 12 or 24 hours in lean and obese rat PAVSMCs treated with PGI, hydrogen peroxidase (H₂O₂, 25 μM), superoxide dismutase (SOD, 400 U/ml), or catalase (1 kU/ml) (n=3). Results are expressed as mean±SEM. *P<0.05, **P<0.01, ***P<0.001. † P<0.05, †† P<0.01 vs same group at condition. Statistical analyses were performed as described in Figure 1 legend.

Figure 6:. miR-193b promotes ROS-dependent degradation of NFYA, reducing sGC transcription

(A) Quantification of miR-193b expression in pulmonary arteries (diameter of PAs: 500–700μm) from lean (n=8), obese (n=8), and obese+sugen rats (n=5). (B) miR-193b expression in lean and obese rat PAVSMCs treated with hydrogen peroxidase (H₂O₂, 25 μM), superoxide dismutase (SOD, 400 U/ml), catalase (1 kU/ml) or mitoTEMPO (50μM) (n=6). (C) mRNA degradation rate of NFYA for 24 hours in lean and obese rat PAVSMCs treated with PGI or Antagomir-193b (n=6). (D-E) Expression of sGCβ1 mRNA and protein level in lean and obese rat PAVSMCs treated with PGI or AntagomiR-193b (n=6). (F) Representative Western blot and quantification of H3K9ac and Histone 3 in lean PAVSMCs treated with PGI or mitoTEMPO or obese and obese+sugen PAVSMCs treated with mitoTEMPO (n=3–4). (G-H) Transcription binding sites for promoter region of miR-193b. Chromatin immunoprecipitation (ChIP) showing enrichment of H3K9ac at miR-193b promoter in lean and obese PAVSMCs treated with PGI, SOD, Catalase, or mitoTEMPO (each n=3). (I) Schematic representation of the molecular mechanisms underlying reactive oxygen species (ROS) and sGCβ1/NFYA/miR-193 expression, resulting in pulmonary arterial relaxation dysfunction. Results are expressed as mean±SEM. *P<0.05, **P<0.01, ***P<0.001. † P<0.05, †† P<0.01, ††† P<0.001 vs Lean PAVSMCs at same condition. Statistical analyses were performed as described in Figure 1 legend.

Figure 7.. Forced sGC activity and NFYA expression rescue exercise induced pulmonary hypertension in CpcPH rats

(A) Representative Western blot and quantification of sGCβ1, total NFYA (endogenous 44kDa and DDK-fusion 50kDa), DDK, and GAPDH in PAVSMCs infected with rAAV6-GFP or rAAV6-NFYA-DDK (each n=3). Each plot represents the PAVSMCs sample cultured from one individual rat. (B) cGMP level in PAVSMCs of obese treated with rAAV6-NFYA-DDK or rAAV6-GFP and NO donor DETA NONOate (10μM, 24 hours) (each n=6). (C) Administration of rAAV6-GFP or rAAV6-NFYA-DDK to obese+sugen rats. Representative immunofluorescence images and quantification of NFYA, CD31, αSMA, and DAPI of pulmonary arteries (each n=4). (D) Flow cytometry showing the percent of DDK in CD31 or αSMA positive cells (each n=4). (E) Representative Western blot and quantification of sGCβ1, total NFYA (endogenous 44kDa and DDK-fusion 50kDa), phosphorylation ser 177 eNOS, total eNOS and GAPDH in isolated pulmonary arteries of rats (diameter of PAs: 500–700μm) (each n=4). (F) Quantification of plasma levels of cGMP of the rats (each n=6). (G-H) Right ventricular systolic and end-diastolic pressure (RVSP and RVEDP, each n=6), left ventricular systolic and end-diastolic blood pressure (LVSP and LVEDP, each n=4), and Workload (each n=6) were measured at rest and during exercise (obese+sugen rats infected with rAAV6-GFP or rAAV6-NFYA-DDK). (I-K) Left ventricular Ejection fraction (LVEF), Fraction shortening (LVFS), E wave/A wave ratio (E/A), cardiac index (CI), RVDd, PAAT/ET, TAPSE, and TPRi were measured at rest and during exercise. Rats per group; obese+sugen rats infected with rAAV6-GFP or rAAV6-NFYA-DDK (each n=6). Results are expressed as mean±SEM. *P<0.05, **P<0.01, ***P<0.001. Statistical analyses were performed as described in Figure 1 legend.

Figure 8.. Upstream treatment of metabolic syndrome with SGLT2 inhibition rescues mitochondrial ROS in PAVSMCs and restores the NFYA-sGC-cGMP signaling to improve CpcPH

Treatment with SGLT2 inhibitor (Empagliflozin 10 mg/kg/day, via drinking water) in obese rats treated with sugen (Obese+sugen). (A-B) Body weight and HbA1c level were measured from 8 to 22 weeks old. At 22 weeks old, triglyceride plasma level was measured (lean n=5, obese+sugen n=8, obese+sugen+SGLT2 inhibitor n=10). (C) Representative immunofluorescence images and quantification of 4HNE (4-hydroxynonenal), αSMA and DAPI of pulmonary arteries in lean (n=5), obese+sugen (n=8), and obese+sugen treated with SGLT2 inhibitor (n=10). (D) Quantification of miR-193b expression in pulmonary arteries from lean (n=5), obese+sugen (n=8), and obese+sugen treated with SGLT2 inhibitor (n=10). (E-F) Representative Western blot and quantification of sGCβ1, NFYA, GAPDH, H3K9 ac, and Histone 3 in isolated pulmonary arteries (diameter of PAs: 500–700μm) of lean (n=5), obese+sugen (n=8), and obese+sugen treated with SGLT2 inhibitor (n=10). (G) Quantification of plasma levels of cGMP in lean (n=5), obese+sugen (n=8), and obese+sugen treated with SGLT2 inhibitor (n=10). (H) Right ventricular systolic and end-diastolic pressure (RVSP and RVEDP), left ventricular systolic and end-diastolic blood pressure (LVSP and LVEDP) were measured at rest and during exercise (lean n=5, obese+sugen n=8, obese+sugen+SGLT2 inhibitor n=10). (I) Workload were calculated with the distance of test run (lean n=5, obese+sugen n=8, obese+sugen+SGLT2 inhibitor n=10). (J) Representative images of MRI of left pulmonary artery and heart at rest and during dobutamine (5μg/kg/min) challenge in lean, obese+sugen, and obese+sugen treated with SGLT2 inhibitor. White bars indicate 1 mm. Quantification of area of left pulmonary artery at second branch and area of RV in lean (n=5), obese+sugen (n=8), and obese+sugen treated with SGLT2 inhibitor (n=8). Results are expressed as mean±SEM. *P<0.05, **P<0.01, ***P<0.001. Statistical analyses were performed as described in Figure 1 legend.