SGLT2 promotes cardiac fibrosis following myocardial infarction and is regulated by miR-141

Abstract

Cardiac fibrosis is a primary event during myocardial infarction (MI) progression, which impairs cardiac function. The present study aimed to investigate the effect of SGLT2 on cardiac fibrosis following MI. To validate the role of SGLT2 in the regulation of cardiac fibrosis in vivo, an MI rat model was established. Echocardiography was performed to determine cardiac function at 4 weeks post-MI. MI model rats were transfected with short hairpin RNA (sh)-SGLT2 or sh-negative control lentiviruses to investigate the effect of SGLT2 on rat heart function post-MI. Subsequently, the effects of SGLT2 on the cardiac fibrosis of infarcted hearts were assessed by performing Masson's trichrome staining. To further clarify the effect of SGLT2 on cardiac fibroblast proliferation, TGFβ was used to stimulate primary cardiac fibroblasts in vitro. The results demonstrated that SGLT2 served a key role in cardiac fibrosis. SGLT2 expression levels in infarct tissues were significantly increased at week 1 post-MI compared with the sham group. Compared with the control group, SGLT2 knockdown attenuated cardiac fibrosis by inhibiting the expression of collagen I and collagen III in cardiac fibroblasts in vitro and in vivo. Furthermore, the results indicated that SGLT2 expression was modulated by miR-141 in cardiac fibroblasts. In summary, the present study indicated that upregulated SGLT2 expression in cardiac fibrosis following MI was regulated by miR-141 and SGLT2 that knockdown reduced cardiac fibrosis and improved cardiac function after MI.

Keywords: cardiac fibrosis; microRNA-141; myocardial infarction; sodium-glucose linked transporter 1.

Conflict of interest statement

The authors declare that they have no competing interests.

Copyright: © Li et al.

Figures

Figure 1

SGLT2 expression was analyzed in the infarcted myocardial tissues of MI model rats. SGLT2 (A) mRNA and (B) protein expression levels in infarcted areas of MI model rats at indicated times (n=5). SGLT2 mRNA expression levels in the (C) border and (D) far zones (n=5). **P<0.01, ***P<0.001 vs. sham. SGLT2, sodium-glucose linked transporter 2; MI, myocardial infarction; W, week; NS, not significant.

Figure 2

SGLT2 knockdown improves rat heart function following MI in vivo. SGLT2 (A) mRNA and (B) protein expression levels in infarcted areas treated with sh-SGLT2 or sh-NC (n=5). (C) Echocardiography results of rats in the different groups (n=5). At 4 weeks post-MI, ventricular parameters were measured and analyzed by echocardiography, including (D) LVEF, (E) LVEDV, (F) LVESV, (G) (-) LVdp/dtmax, (H) (+) LVdp/dtmax, (I) HR and (J) E/A ratio (n=5). *P<0.05, **P<0.01, ***P<0.001. SGLT2, sodium-glucose linked transporter 2; MI, myocardial infarction; sh, short hairpin RNA; NC, negative control; LVEF, left ventricular ejection fractions; LVEDV, left ventricular end diastolic volume; LVESV, left ventricular end systolic volume; (-)/(+) LVdp/dtmax, the maximum left ventricular change in pressure/time; HR, heart rate; E/A ratio, ratio of the peak early transmitral flow velocity to peak late transmitral flow velocity.

Figure 3

SGLT2 knockdown inhibits cardiac fibrosis following MI in vivo. (A) Representative photomicrographs of Masson trichrome staining of hearts of MI model rats treated with PBS, sh-NC or sh-SGLT2 (n=5, original magnification x12.5). (B) Fibrotic area was determined as the ratio of the average circumference of fibrotic scars (blue)/average inner circumference of the left ventricle (n=5). Collagen I and collagen III (C) mRNA and (D) protein expression levels in cardiac tissues (n=5). *P<0.05, **P<0.01, ***P<0.001. SGLT2, sodium-glucose linked transporter 2; MI, myocardial infarction; sh, short hairpin RNA; NC, negative control.

Figure 4

SGLT2 knockdown inhibits TGFβ-induced proliferation and collagen synthesis in cardiac fibroblasts. Primary cardiac fibroblasts were transfected with sh-NC or sh-SGLT2 and then treated with 20 ng/ml recombinant TGFβ for 72 h. (A) SGLT2 expression levels. (B) The MTT assay was performed to examine cell proliferation. (C) Protein and (D) mRNA expression levels and of collagen I and collagen III in primary cardiac fibroblasts. *P<0.05, **P<0.01, ***P<0.001. SGLT2, sodium-glucose linked transporter 2; TGFβ, transforming growth factor β; sh, short hairpin RNA; NC, negative control.

Figure 5

SGLT2 is targeted by miR-141. (A) The binding site between the 3'-UTR of SGLT2 mRNA and miR-141. SGLT2 3'-UTR-mutants were generated in which 6 complementary binding site nucleotides were altered. (B) miR-141 expression levels in the infarcted areas of MI model rats at indicated times. (C) Relative luciferase activities were determined by conducting a luciferase reporter assay. (D) Transfection efficiency of miR-141 mimics. SGLT2 (E) protein and (F) mRNA expression levels in primary cardiac fibroblasts transfected with miR-141 mimics. *P<0.05, **P<0.01, ***P<0.001. SGLT2, sodium-glucose linked transporter 2; miR, microRNA; UTR, untranslated region; miR, microRNA; W, week; WT, wild-type; MUT, mutant; NC, negative control.

Figure 6

TGFβ-induced proliferation and collagen synthesis in cardiac fibrosis is regulated by the miR-141/SGLT2 axis. Primary cardiac fibroblasts were transfected with miR-141 NC, miR-141 mimics or miR-141 mimics + pcDNA3.1-SGLT2 and treated with 20 ng/ml recombinant TGFβ for 72 h. (A) Transfection efficiency of pcDNA3.1-SGLT2. (B) The MTT assay was performed to assess cell proliferation. Collagen I and collagen III (C) mRNA and protein expression levels in primary cardiac fibroblasts. *P<0.05. TGFβ, transforming growth factor β; miR, microRNA; SGLT2, sodium-glucose linked transporter 2; NC, negative control; NS, not significant.