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. 2012;9(6):413-23.
doi: 10.7150/ijms.4514. Epub 2012 Jul 21.

MicroRNA 21 inhibits left ventricular remodeling in the early phase of rat model with ischemia-reperfusion injury by suppressing cell apoptosis

Yanjun Qin  1 , Yueqing Yu, Hua Dong, Xiaohua Bian, Xuan Guo, Shimin Dong
Affiliations

MicroRNA 21 inhibits left ventricular remodeling in the early phase of rat model with ischemia-reperfusion injury by suppressing cell apoptosis

Yanjun Qin et al. Int J Med Sci. 2012.

Abstract

Objective: To determine the role of microRNA 21(miR-21) on left ventricular remodeling of rat heart with ischemia-reperfusion (I/R) injury and to investigate the underlying mechanism of miR-21 mediated myocardium protection.

Methods: Rats were randomly divided into three groups: an I/R model group with Ad-GFP (Ad-GFP group), an I/R model group with Ad-miR-21 (Ad-miR-21 group) and a sham-surgery group. Changes in hemodynamic parameters were recorded at 1 week after I/R. Histological diagnosis was achieved by hematoxylin and eosin (H&E). Left ventricular (LV) dimensions, myocardial infarct size, LV/BW, collagen type Ⅰ, type Ⅲ and PCNA positive cells were measured. Primary cultures of neonatal rat cardiac ventricular myocytes were performed and cell ischemic injury was induced by hypoxia in a serum- and glucose-free medium, and reoxygenation (H/R). MiR-21 inhibitor and pre-miR-21 were respectively added to the culture medium for the miR-21 knockdown and for the miR-21 up-regulation. qRT-PCR was used to determine the miR-21 levels in cultured cells. Flow cytometry was performed to examine the cell apoptosis.

Results: In the Ad-miR-21 group, LV dimensions, myocardial infarct size, LV/BW, collagen type Ⅰ, type Ⅲ and PCNA positive cells all significantly decreased compared with the Ad-GFP group. At 1 week after I/R, the Ad-miR-21 significantly improved LVSP, LV +dp/dt(max), LV - dp/dt(min), and decreased heart rate (HR) and LVEDP compared with the Ad-GFP group. Compared with the Ad-GFP, the cell apoptotic rate significantly decreased in the Ad-miR-21 group. The miR-21 inhibitor exacerbated cardiac myocyte apoptosis and the pre-miR-21 decreased hypoxia/reoxygenation- induced cardiac myocyte apoptosis.

Conclusions: Ad-miR-21 improves LV remodeling and decreases the apoptosis of myocardial cells, suggesting the possible mechanism by which Ad-miR-21 functions in protecting against I/R injury.

Keywords: apoptosis; collagen; hemodynamic; ischemia-reperfusion; microRNA 21; rat.; ventricular remodeling.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

FIGURE 1
FIGURE 1
Cardiac hemodynamics measured at 1 week after I/R. HR is heart rate. BPM is beats per minute. LVSP is left ventricular systolic pressure. LV±dp/dt are the first derivatives (positive and negative) of LV pressure over time. LVEDP is left ventricular end-diastolic pressure. Results are expressed as mean ± SD, n=10. *P<0.05 compared with the Ad-GFP group. † P<0.05 compared with the sham group.
FIGURE 2
FIGURE 2
The ratio LV/BW measured 1 week after I/R. LV is left ventricular. BW is body weight. Results are expressed as the mean ± SD *P<0.05 compared with the Ad-GFP group. † P<0.05 compared with the sham group.
FIGURE 3
FIGURE 3
The effect of adenovirus-mediated miR-21 gene transfer on myocardial infarct size. A, Ad-miR-21 (8×108 pfu) reduced infarct size in rat hearts at 1 week after I/R. Note: Data presented as mean ± SD, n=10; *, p<0.05 compared with Ad-GFP control. B, representative TTC-stained heart slices from rats treated with control adenovirus, Ad-GFP, or Ad-miR-21. The infarcted area in the Ad-GFP-treated image was 48.5% and the infarcted area in the Ad-miR-21-treated image was 32.7%.
FIGURE 4
FIGURE 4
A representative collagen type Ⅰ, type Ⅲ and PCNA positive cells photomicrographs from the rat heart sections at 1 week after I/R (immunohistochemistry, original magnification ×400). The positive expression colored brown-yellow. B collagen content in noninfarcted area and infarcted area. Note: Data presented as mean ± SD, n = 6. C collagen type Ⅰ/ Ⅲ ratio in noninfarcted area and infarcted area. Note: Data presented as mean ± SD, n = 6. D the PCNA positive cells in the border area. * p<0.05 compared with the Ad-GFP control. † P<0.05 compared with the sham group.
FIGURE 5
FIGURE 5
The effect of miR-21 overexpression on cardiac cell apoptosis in the border area of the rat heart. A, representative apoptotic cells by flow cytometry 1 week after I/R. B, quantitative analysis of the apoptotic cells in heart sections. Note: Data presented as mean ± SD, n= 6, *, p<0.05 compared with the Ad-GFP group. † P<0.05 compared with the sham group.
FIGURE 6
FIGURE 6
The effect of miR-21 on ischemia-related cell injury in vitro. Cultured cardiac cell injury was induced by hypoxia for 4 h in a serum- and glucose-free medium followed by reoxygenation (H/R) for 3 h innormal culture medium. Cell apoptosis was determined by flow cytometry. A, miR-21 inhibitor, LNA-anti-miR-21 (30 nM) decreased, but pre-miR-21 (30 nM) increased miR-21 expression in cultured cardiac cells. n=5, *, p< 0.05 compared with the vehicle control. †, p< 0.05 compared with the LNA-anti-miR-21 group. B, hypoxia/reoxygenation resulted in an increase in apoptosis. Pre-miR-21 decreased the cardiac myocyte apoptosis. In contrast, cardiac myocyte apoptosis was exacerbated after treatment with LNA-anti-miR-21. n=5, *, p< 0.05 compared with the noninjured (without H/R) control. † , p< 0.05 compared with H/R treated with the vehicle control.
FIGURE 7
FIGURE 7
The effect of adenovirus-mediated miR-21 gene transfer on LV dimensions. A, representative heart sections from sham-opened, Ad-GFP, or Ad-miR-21-treated rats at 1 week after I/R. B, Ad-miR-21 (8×108 pfu) reduced internal diastolic diameter of left ventricles in rat hearts at 1 week after I/R. Note: Data presented as mean ± SD, n = 8; *, P< 0.05 compared with Ad-GFP control. † P<0.05 compared with the sham group.
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