Rivaroxaban Suppresses the Progression of Ischemic Cardiomyopathy in a Murine Model of Diet-Induced Myocardial Infarction

Supplemental Fig. 1.. Evaluation of occluded coronary arteries Representative images of various extents of occluded coronary arteries (A, no plaque; B, < 50% occluded; C, > 50% occluded; D, 100% occluded). Bar = 50 µm.

Fig. 1.. Rivaroxaban treatment significantly improved the survival rate The survival rate of the rivaroxaban-treated group improved significantly compared with the non-treated ICM model (rivaroxaban group, n = 49; ICM model group, n = 126). **p < 0.01.

Fig. 2.. Rivaroxaban suppressed the progression of heart failure in ICM model mice (A) The heart weight/body weight ratio, heart weight/tibia length ratio, lung weight/body weight ratio, and lung weight/tibia length ratio were decreased significantly (n = 11–30 per group). (B) Echocardiography showed that fraction shortening in the rivaroxaban-treated group was prevented remarkably compared with the nontreated group (n = 10–11). (C) The mRNA expression of ANP and BNP was decreased markedly (n = 5–8). *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 3.. Effects of rivaroxaban on atherosclerosis in the aortic root and on occluded coronary arteries in ICM model mice Rivaroxaban alleviated atherosclerosis and occluded coronary arteries in ICM model mice. (A) Representative images of oil red O staining of atherosclerotic plaques in aortic roots. Rivaroxaban treatment alleviated lipid deposition as compared with the non-treated group (n = 5–7; bar = 500 µm). (B) HE staining indicated that occluded coronary arteries were decreased by rivaroxaban treatment compared with the non-treated group (n = 11–14; bar = 50 µm). (C) The mRNA expression of proinflammatory genes revealed that rivaroxaban decreased the inflammatory molecules in the heart (n = 8–9). *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 4.. Rivaroxaban treatment attenuated cardiac fibrosis in ICM model mice (A) Masson's staining showed that fibrotic areas were significantly alleviated by rivaroxaban treatment (n = 7–11; bar = 1 mm). (B) The mRNA expression of fibrosis-related genes was decreased by rivaroxaban treatment (n = 8). *p < 0.05, **p < 0.01

Fig. 5.. Cardiac PAR expression in non-MI mice, ICM mice, and rivaroxaban-treated ICM mice (A) The mRNA expression levels of PAR2 and PAR1 were significantly upregulated after a high-fat diet administration and downregulated markedly by rivaroxaban treatment (n = 5–8). (B) The protein level of PAR2 was reduced by rivaroxaban treatment consistently (n = 4). *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 6.. Effects of rivaroxaban in cardiac myocytes in vitro (A) Hypoxic stimulation induced a decrease in the cell viability of CMs. The cell viability of CMs under hypoxia was dose-dependently improved by the presence of rivaroxaban for 12 and 24 hours (n = 4–5 per group). (B) Hypoxic stimulation caused the apoptosis of CMs. The apoptotic CMs were reduced remarkably after rivaroxaban treatment for 24 hours. The ratio of TUNEL-positive cells/total cells (%) was calculated in 10 areas under a microscope and averaged. *p < 0.05, **p < 0.01, and ***p < 0.001. Riv: rivaroxaban.

Fig. 7.. Effects of rivaroxaban on PAR1, PAR2, and inflammation genes in cardiac myocytes (A) The mRNA expression levels of PAR1 and PAR2 in CMs were increased after stimulation under hypoxia for 24 hours. Rivaroxaban treatment reduced both PAR1 and PAR2, and PAR2 significantly (n = 4–6 per group). (B) Protein levels of PAR2 in CMs showed no significant changes (n = 3–4 per group). (C) The mRNA expression levels of pro-inflammatory genes in CMs were decreased significantly (n = 3–4 per group). *p < 0.05 and ***p < 0.001. Riv: rivaroxaban.

Fig. 8.. Rivaroxaban downregulated the expression of PAR2 in cardiac fibroblasts (A) The MTT assay demonstrated that hypoxic stimulation for 24 or 48 hours induced the reduction of proliferation of cardiac FBs. Rivaroxaban showed no effect on cell viability under hypoxic conditions for 24 or 48 hours (n = 5–6 per group). (B) The results of qPCR analysis demonstrated that hypoxic stimulation for 24 hours increased the mRNA expression of PAR1 and PAR2, which were decreased by the presence of rivaroxaban, significantly in PAR2 (n = 3). (C) The expression of the PAR2 protein level in cardiac FBs was increased by hypoxic stimulation for 24 hours, and was suppressed remarkably by the presence of rivaroxaban (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001. Riv: rivaroxaban.

Fig. 9.. Effects of rivaroxaban and a PAR2 antagonist on fibrosis and inflammation in cardiac fibroblasts Cardiac FBs exposed to hypoxia were treated with or without a PAR2 antagonist, FSLLRY (10 µM) for 24 hours or rivaroxaban (2 µg/ml) for 24 hours or FSLLRY (10 µM) for 24 hours, subsequently changed to rivaroxaban (2 µg/ml) for 24 hours. (A) The elevated expression level of PAR2 induced by hypoxic stimulation was downregulated by rivaroxaban or the PAR2 antagonist FSLLRY, or both. (B, C) The mRNA expressions of MMP9 and COL-1 in cardiac FBs were attenuated by the presence of rivaroxaban or FSLLRY or both. (D–F) The mRNA expressions of pro-inflammatory genes IL-6, IL-1β, and TNF-α were reduced by the presence of rivaroxaban or FSLLRY or both (n = 3–5 per group). *p < 0.05, **p < 0.01, and ***p < 0.001. Riv: rivaroxaban.