MicroRNA-208a increases myocardial fibrosis via endoglin in volume overloading heart

Abstract

MicroRNA-208a (mir-208a) is essential for cardiac hypertrophy and fibrosis. Endoglin, a co-receptor of transforming growth factor-β is also essential for cardiac fibrosis. Endoglin has been shown to be a target of mir-208a in the in vitro mechanical stress model. Volume overload can lead to heart failure and cardiac fibrosis. The role of mir-208a and endoglin in volume overload heart failure is well known. We sought to investigate the mechanism of regulation of mir-208a and endoglin in volume overload-induced heart failure. Aorta-caval (AV) shunt was performed in adult Sprague-Dawley rats to induce volume overload. Heart weight and heart weight/body weight ratio significantly increased in AV shunt animals. AV shunt significantly increased left ventricular end-diastolic dimension as compared to sham group. Mir-208a was significantly induced by AV shunt from 3 to 14 days. Endoglin, myosin heavy chain-β and brain natriuretic peptide were significantly induced by AV shunt from 3 to 14 days. Overexpression of mir-208a in the sham group without AV shunt significantly increased endoglin expression similar to the AV shunt group. Antagomir-208a attenuated the endoglin expression induced by AV shunt. Pretreatment with atorvastatin also attenuated the endoglin expression induced by AV shunt. AV shunt significantly increased myocardial fibrosis as compared to sham group. Overexpression of mir-208a in the sham group significantly increased myocardial fibrosis. Antagomir-208a and atorvastatin significantly attenuated the myocardial fibrosis induced by AV shunt. In conclusion, mir-208a increased endoglin expression to induce myocardial fibrosis in volume overloaded heart failure. Treatment with atorvastatin can attenuate the myocardial fibrosis induced by volume overload through inhibition of endoglin expression.

Figure 1. Aorta-caval shunt increases mir-208a expression in rat myocardium.

Pretreatment with atorvastatin significantly attenuated the increase of mir-208a expression induced by AV shunt. *P<0.001 vs. sham group (n = 5–6 per group). ^#P<0.01 vs. shunt 5D. ^‡P<0.001 vs. shunt 7D.

Figure 2. Aorta-caval shunt increases endoglin, β-myosin heavy chain (MHCβ) and brain natriuretic peptide (BNP) proteins expression in rat myocardium.

A. Representative western blot for endoglin, MHCβ, and BNP protein expression in rat myocardium after different days of shunting. B, Quantitative analysis of endoglin, MHCβ, and BNP protein levels. The values from myocardium after AV shunt have been normalized to matched α-tubulin measurement and then expressed as a ratio of normalized values to protein in sham group (n = 6 per group). *P<0.001 vs. sham.

Figure 3. Mir-208a mediates the myocardial endoglin expression in AV shunt rat.

A, Representative western blot for endoglin and MHCβ protein expression in the rat myocardium after 7 days of shunting. Mir-208 expression vector was transfected into left ventricular myocardium by low pressure-accelerated gene gun. Overexpression of mir-208a in the sham group significantly increased endoglin and MHCβ protein expression.*P<0.001 vs. sham. #P<0.001 vs. shunt 7D. (n = 6 per group). Pretreatment with atorvastatin significantly attenuated the increase of endoglin and MHCβ protein expression induced by AV shunt.

Figure 4. In situ hybridization assay detects the presence of mir-208a in the cardiac myocytes.

Representative microscopic images showing the presence of mir-208a (green color) in the cytoplasm of cardiac myocytes from left ventricular myocardium in AV shunt rats. The sham group or scrambled probe did not detect the presence of mir-208a.

Figure 5. Immunohistochemnical staining of left ventricular myocardium after induction of aorta-caval shunt with or without antagomir-208a treatment.

There are significantly increased immunoreactive signals for endoglin and MHCβ after overexpression of mir-208a and AV shunt for 7 days. Antagomir-208a significantly decreased the immunoreactive signal induced by AV shunt. Rare endoglin signals were seen in the sham group.

Figure 6. Antagomir-208a and atorvastatin decreases myocardial fibrosis induced by aorta-caval shunt.

A. Representative Masson's trichrome stain for cross-section of myocardium. Masson's trichrome staining was performed to delineate fibrosis tissue from viable myocardium. B. Quantitative analysis of fibrosis area. N = 6 per group. *P<0.001 vs. sham group. ^#P<0.001 vs. shunt 7D.