Blockade of PAR-1 Signaling Attenuates Cardiac Hypertrophy and Fibrosis in Renin-Overexpressing Hypertensive Mice

Abstract

Background Although PAR-1 (protease-activated receptor-1) exerts important functions in the pathophysiology of the cardiovascular system, the role of PAR-1 signaling in heart failure development remains largely unknown. We tested the hypothesis that PAR-1 signaling inhibition has protective effects on the progression of cardiac remodeling induced by chronic renin-angiotensin system activation using renin-overexpressing hypertensive (Ren-Tg) mice. Methods and Results We treated 12- to 16-week-old male wild-type (WT) mice and Ren-Tg mice with continuous subcutaneous infusion of the PAR-1 antagonist SCH79797 or vehicle for 4 weeks. The thicknesses of interventricular septum and the left ventricular posterior wall were greater in Ren-Tg mice than in WT mice, and SCH79797 treatment significantly decreased these thicknesses in Ren-Tg mice. The cardiac fibrosis area and monocyte/macrophage deposition were greater in Ren-Tg mice than in WT mice, and both conditions were attenuated by SCH79797 treatment. Cardiac mRNA expression levels of PAR-1, TNF-α (tumor necrosis factor-α), TGF-β1 (transforming growth factor-β1), and COL3A1 (collagen type 3 α1 chain) and the ratio of β-myosin heavy chain (β-MHC) to α-MHC were all greater in Ren-Tg mice than in WT mice; SCH79797 treatment attenuated these increases in Ren-Tg mice. Prothrombin fragment 1+2 concentration and factor Xa in plasma were greater in Ren-Tg mice than in WT mice, and both conditions were unaffected by SCH79797 treatment. In isolated cardiac fibroblasts, both thrombin and factor Xa enhanced ERK1/2 (extracellular signal-regulated kinase 1/2) phosphorylation, and SCH79797 pretreatment abolished this enhancement. Furthermore, gene expression of PAR-1, TGF-β1, and COL3A1 were enhanced by factor Xa, and all were inhibited by SCH79797. Conclusions The results indicate that PAR-1 signaling is involved in cardiac remodeling induced by renin-angiotensin system activation, which may provide a novel therapeutic target for heart failure.

Keywords: cardiac fibrosis; cardiac hypertrophy; factor Xa; protease‐activated receptor; renin–angiotensin system.

Figure 1. Systolic blood pressure in renin‐overexpressing hypertensive (Ren‐Tg) mice and wild‐type (WT) mice at baseline and at 2 and 4 weeks after treatment with vehicle or PAR‐1 (protease‐activated receptor‐1) antagonist SCH79797 (25 μg/kg per day).

*P<0.01 vs WT mice treated with vehicle at the same time point. **P<0.05 vs Ren‐Tg mice treated with vehicle at the same time point.

Figure 2. PAR‐1 (protease‐activated receptor‐1) antagonist SCH79797 attenuates cardiac hypertrophy in renin‐overexpressing hypertensive (Ren‐Tg) mice.

A, Interventricular septum thickness and (B) left ventricular posterior wall thickness at baseline and at 2 and 4 weeks after treatment with vehicle or PAR‐1 antagonist SCH79797. C, Representative images by echocardiography at 4 weeks after treatment. D, The ratio of heart weight to body weight. *P<0.01 vs wild‐type (WT) mice treated with vehicle at the same time point. **P<0.05 vs Ren‐Tg mice treated with vehicle at the same time point. ***P<0.01 vs Ren‐Tg mice treated with vehicle at the same time point. ^† P<0.01 vs baseline in Ren‐Tg mice. ^†† P<0.05 vs baseline in Ren‐Tg mice.

Figure 3. PAR‐1 (protease‐activated receptor‐1) antagonist SCH79797 attenuates cardiac interstitial fibrosis.

A, Representative sections of left ventricle stained with Masson's trichrome in renin‐overexpressing hypertensive (Ren‐Tg) mice or wild‐type (WT) mice treated with SCH79797 or vehicle for 4 weeks. B, The area of cardiac interstitial fibrosis was evaluated.

Figure 4. PAR‐1 (protease‐activated receptor‐1) antagonist SCH79797 attenuates cardiac deposition of monocytes or macrophages stained with CD68.

A, Representative sections of left ventricle stained with CD68 in renin‐overexpressing hypertensive (Ren‐Tg) mice or wild‐type (WT) mice treated with SCH79797 or vehicle for 4 weeks. B, The CD68‐positive area was evaluated.

Figure 5. PAR‐1 (protease‐activated receptor‐1) antagonist SCH79797 attenuates the increase in cardiac mRNA expression levels related to proinflammatory and profibrotic processes.

Expressions of PAR‐1 (A), TNF‐α (tumor necrosis factor‐α) (B), and TGF‐β1 (transforming growth factor‐β1) (C). The ratio of β‐myosin heavy chain (β‐MHC) to α‐MHC (D). Expression of COL3A1 (collagen type 3 α1 chain) (E).

Figure 6. Concentrations of prothrombin fragment 1+2 (A) and factor Xa (B) in plasma. Ren‐Tg indicates renin‐overexpressing hypertensive; WT, wild‐type.

Figure 7. PAR‐1 (protease‐activated receptor‐1) antagonist SCH79797 (1 μmol/L) inhibits ERK1/2 (extracellular signal‐regulated kinase 1/2) phosphorylation in response to thrombin (1 U/mL) (A) or factor Xa (FXa; 20 nmol/L) (B) in isolated cardiac fibroblasts by western blot analysis. pERK indicates phosphorylated ERK1/2; tERK, total ERK1/2.

Figure 8. PAR‐1 (protease‐activated receptor‐1) antagonist SCH79797 (1 μmol/L) attenuates the increase in proinflammatory and fibrotic‐related gene expression in response to factor Xa (FXa; 20 nmol/L) in isolated cardiac fibroblasts.

Expressions of PAR‐1 (A), TNF‐α (tumor necrosis factor‐α) (B), TGF‐β1 (transforming growth factor‐β1) (C), and COL3A1 (collagen type 3 α1 chain) (D).

Figure 9. SCH79797 is not associated with either apoptosis or cell death.

A, The ratio of cleaved Casp3 (caspase‐3) to Casp3 was examined in either 0.3 or 1 μmol/L of SCH79797 or dimethyl sulfoxide (DMSO) as vehicle at 3, 6, and 24 hours in HEK293 cells. B and C, The survival rate of HEK293 cells incubated with 0.1, 0.3, or 1 μmol/L of SCH79797 for 3 or 24 hours assessed by trypan blue staining.