microRNA-21 promotes cardiac fibrosis and development of heart failure with preserved left ventricular ejection fraction by up-regulating Bcl-2

Abstract

The morbidity and mortality of heart failure with preserved left ventricular ejection fraction (HFpEF) were similar to those of systolic heart failure, but the pathogenesis of HFpEF remains poorly understood. It was demonstrated that, in systolic heart failure, microRNA-21 (miR-21) could inhibit the apoptosis of cardiac fibroblasts, leading to cardiac hypertrophy and myocardial fibrosis, but the role of miR-21 in HFpEF remains unknown. By employing cell culture technique, rat myocardiocytes and cardiac fibroblasts were obtained. The expression of miR-21 in the two cell types under different conditions was compared and we found that the miR-21 expression was significantly higher in cardiac fibroblasts than in myocardiocytes. We established a rat HFpEF model and harvested the tissues of cardiac apex for pathological examination, Northern blotting and so forth. We found that miR-21 expression was significantly higher in model rats than in sham-operated rats, and the model rats developed the cardiac atrophy and cardiac fibrosis. After injection of miR-21 antagonist, the the cardiac atrophy and cardiac fibrosis were conspicuously ameliorated. Both in vivo and in vitro, inhibition of miR-21 expression resulted in reduced Bcl-2 expression while over-expression of miR-21 led to elevation of Bcl-2 expression. Our study suggested that miR-21 promoted the development of HFpEF by up-regulating the expression of anti-apoptotic gene Bcl-2 and thereby suppressing the apoptosis of cardiac fibrosis.

Keywords: Bcl-2; Cardiac fibrosis; HFpEF; miR-21.

Figure 1

Evaluation of HFpEF model. Eight weeks after TAC, assess the HFPEF model by echocardiography. The ultrasound examination measured diastolic function (LWEDD, VST, PWT, IVRT) and systolic function (EF), which reflected that diastolic functions of TAC group were impaired (A), as indicated by inverted E and A peaks and significant protracted IVRT (B). Meanwhile, the parameters of hemodynamic examination (LVEDV, ± LVdp/dtmax) were decreased absolutely in TAC group, which indicating the diastolic functions in TAC group were impaired (C).

Figure 2

High miR-21 expression in fibroblasts of HFpEF rats. In situ hybridization, miR-21 expression was higher in TAC group than in sham group, and it was more obvious in cardiac interstitial tissues (A). While, the positive area reflected the same finding as mentioned above (B) (*P<0.05). Northern-blot also exhibited that miR-21 was highly expressed in TAC rats (C, D). Detecting miR-21 by RT-PCR in cardiac fibroblasts, compared with sham group, miR-21 expression significantly higher in TAC group (E). In TAC group, the expression of miR-21 is more higher in cardiac fibroblasts than in myocardiocytes (F). miR-21 expression significantly higher in cardiac fibroblasts than in myocardiocytes.

Figure 3

The association between mir-21 expression and cardiac hypertrophy and fibrosis. The cardiomyocytes was irregularly arranged in TAC group (A) by HE staining. MUSSON staining showed that it had an obvious cardiac fibrosis in HFpEF rats (B). The cardiac index was increased in TAC group than in sham group (C), **P<0.01.

Figure 4

The association between mir-21 expression and cardiac hypertrophy and fibrosis. Three weeks after the injection of miR-21 antagonist via jugular vein, HE staining showed that the arrangement of cardiomyocytes of TAC group was improved (A). The fibrosis of TAC group was ameliorated (B). And, cardiac index of antagonist-treated rats was lower (C).

Figure 5

Regulation of Bcl-2 by miR-21. Cell fractions from rat hearts stained with antibodies against prolyl 4-hydroxylase (P4HB) and a2-actinin (ACTN2). The cardiac fibroblasts were full of the vision and the purity is up to 98% (A). After jugular injection of miR-21 antagonist into HFpEF rat model, western blotting revealed that Bcl-2 expression was lower at protein level (B), and RT-PCR showed that Bcl-2 expression in rat cardiac tissues was decreased (D). After induction of fibrosis by AngII, Bcl-2 expression in the fibroblasts transfected with anti-miR-21 was lowered at both gene (E) and protein level (C). RT-PCR showed that Bcl-2 expression was decreased 48 hours (F) and 72 hours (G) after transfection with pre-miR21, **P<0.01.