BDNF-mediates Down-regulation of MicroRNA-195 Inhibits Ischemic Cardiac Apoptosis in Rats - PubMed (original) (raw)

BDNF-mediates Down-regulation of MicroRNA-195 Inhibits Ischemic Cardiac Apoptosis in Rats

Pengzhou Hang et al. Int J Biol Sci. 2016.

Abstract

Background: Our previous studies suggested that brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (TrkB) axis inhibited cardiomyocyte apoptosis in myocardial infarction (MI). However, the relationship between BDNF and microRNA (miRNA) in cardiomyocytes are unclear. The present study was performed to investigate the role of miR-195 and the interplay between BDNF and miR-195 in ischemic cardiomyocyte apoptosis.

Methods: Male Wistar rats were subjected to coronary artery ligation, and primary neonatal rat ventricular myocytes were treated with hypoxia or hydrogen peroxide (H2O2). BDNF level in rat ventricles was measured by enzyme linked immunosorbent assay (ELISA). miR-195 mimic, inhibitor or negative control was transfected into the cardiomyocytes. Cell viability and apoptosis were detected by MTT assay and TdT-mediated dUTP nick end labeling (TUNEL) staining, respectively. Cardiac function and apoptosis were detected in MI rats intravenously injected with antagomiR-195. Luciferase assay, Western blot and Real-time RT-PCR were employed to clarify the interplay between miR-195 and BDNF.

Results: miR-195 level was dynamically regulated in response to MI and significantly increased in ischemic regions 24 h post-MI as well as in hypoxic or H2O2-treated cardiomyocytes. Meanwhile, BDNF protein level was rapidly increased in MI rats and H2O2-treated cardiomyocytes. Apoptosis in both hypoxic and H2O2-treated cardiomyocytes were markedly reduced and cell viability was increased by miR-195 inhibitor. Moreover, inhibition of miR-195 significantly improved cardiac function of MI rats. Bcl-2 but not BDNF was validated as the direct target of miR-195. Furthermore, BDNF abolished the pro-apoptotic role of miR-195, which was reversed by its scavenger TrkB-Fc.

Conclusion: Up-regulation of miR-195 in ischemic cardiomyocytes promotes ischemic apoptosis by targeting Bcl-2. BDNF mitigated the pro-apoptotic effect of miR-195 in rat cardiomyocytes. These findings may provide better understanding of the pro-apoptotic role of miR-195 in MI and suggest that BDNF/miR-195/Bcl-2 axis may be beneficial for limiting myocardial ischemic injury.

Keywords: Apoptosis; Bcl-2.; Brain-derived neurotrophic factor; Myocardial ischemia; miR-195.

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Conflict of interest statement

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

Figures

Figure 1

Figure 1

miR-195 and BDNF levels were dynamically regulated in different regions of rat myocardium in response to ischemic injury. (A, B) Real-time PCR analysis indicates that miR-195 is increased in the infarcted and border zones 24 h after myocardial infarction (MI). (C) miR-195 is decreased in remote zone in rat myocardium 24 h after MI. (D) BDNF level in ischemic zone of rat ventricles. *p<0.05, **p<0.01 vs. sham, n = 3.

Figure 2

Figure 2

miR-195 mimic and inhibitor transfection validation. (A) Real-time PCR analysis indicated that miR-195 level was dramatically increased after transfecting with miR-195 mimic compared with control group. (B) miR-195 level was markedly decreased after transfecting with miR-195 inhibitor compared with control group. **p<0.01 vs. control, ***p<0.001 vs. control, n = 3.

Figure 3

Figure 3

miR-195 inhibitor attenuated hypoxia-induced cardiomyocyte apoptosis. (A) Real-time PCR analysis indicates that miR-195 is increased in hypoxia-treated cardiomyocytes. (B) Statistical results of TUNEL-positive cells per field. (C) Representative images of TUNEL staining of cardiomyocyte showing the apoptotic cells (apoptotic cells stained in green and nucleus stained in blue with DAPI). *p<0.05 vs. control, #p<0.05 vs. hypoxia, n = 5, scale bar: 100 μm.

Figure 4

Figure 4

miR-195 inhibitor protected against cardiomyocyte injury induced by hydrogen peroxide (H2O2). (A, B) Cultured neonatal rat cardiomyocytes were exposed to different concentration of of H2O2 (50, 100 μM) for 4 h, or different timepoints (4 h, 24 h) at 100 μM. (C) MTT assay suggested that miR-195 inhibitor restored cell viability after H2O2 treatment. *p<0.05, **p<0.01 vs Control, #p<0.05 vs H2O2, &p<0.05 vs miR-195 inhibitor, n = 5.

Figure 5

Figure 5

miR-195 inhibitor attenuated H2O2-induced cardiomyocyte apoptosis. (A) Real-time PCR analysis indicates that miR-195 is increased in H2O2-treated cardiomyocytes. (B) Western blot bands of BDNF and TrkB in control and H2O2-treated cardiomyocytes. (C, D) Statistical results of protein level of BDNF and TrkB, *p<0.05, n = 5. (E) Statistical results of TUNEL-positive cells per field. (F) Representative images of TUNEL staining of cardiomyocyte showing the apoptotic cells (apoptotic cells stained in green and nucleus stained in blue with DAPI). *p<0.05 vs. control, #p<0.05 vs. H2O2, &p<0.05 vs. +miR-195 mimic, n = 5, scale bar: 100 μM.

Figure 6

Figure 6

Improvement of cardiac function by antagomiR-195 in MI rats. (A) Ejection fractions. (B) Fractional shortening. (C) Left ventricular systolic diameter (LVDs). (D) Left ventricular diastolic diameter (LVDd). *p<0.05, **p<0.01 vs. sham, #p<0.05 vs. MI+NC, n = 5.

Figure 7

Figure 7

AntagomiR-195 inhibited cardiac injury and apoptosis by upregulating Bcl-2 in MI rats. (A) Representative HE staining pictures, scale bar: 50 μM. (B) Representative images of TUNEL staining in rat myocardium (apoptotic cells stained in brown), scale bar: 50 μM. (C) Statistical results of TUNEL-positive cells per field. (D) Western blot bands of Bcl-2 in sham, MI+NC and MI+antagomiR-195 rat hearts. (E) Statistical results of protein level of Bcl-2. *p<0.05 vs. sham, #p<0.05 vs. MI+NC, n = 5.

Figure 8

Figure 8

Target validation of miR-195. (A) Sequence alignment show between miR-195 and the binding sites in the 3'UTR of the Bdnf gene. (B) Representative western blot bands of BDNF. (C) Statistical results of protein level of BDNF in miR-195 mimic and NC group, n = 3. (D) The interaction between miR-195 and its binding sites in the 3'UTR of Bdnf was examined by luciferase assay in HEK293 cells, n = 3. (E) Representative western blot bands of Bcl-2. (F) Statistical results of protein level of Bcl-2 in miR-195 mimic and NC group, *p<0.05, vs. control, n = 3.

Figure 9

Figure 9

BDNF inhibited miR-195 expression and protected cardiomyocytes against H2O2-induced apoptosis. (A) Real-time PCR analysis indicates that miR-195 level is reduced by BDNF and restored by TrkB-Fc, *p<0.05, vs. control, #p<0.05 vs H2O2, &p<0.05 vs +BDNF, n = 5. (B) MTT assay showed that BDNF improved cell viability in H2O2-treated cardiomyocytes and was reversed by TrkB-Fc, *p<0.05, vs. control, #p<0.05 vs H2O2, &p<0.05 vs +BDNF, n = 5. (C) The quantitative presentation of apoptotic cells by Annexin V-FITC/propidium iodide (PI) staining, *p<0.05, vs. control, #p<0.05 vs H2O2, &p<0.05 vs +BDNF, n = 3. (D) Representative Annexin V-FITC/PI staining pictures.

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References

    1. Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, Cushman M. et al. Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association. Circulation. 2016;133:e38–e360. - PubMed
    1. Saraste A, Pulkki K, Kallajoki M, Henriksen K, Parvinen M, Voipio-Pulkki LM. Apoptosis in human acute myocardial infarction. Circulation. 1997;95:320–323. - PubMed
    1. Olivetti G, Quaini F, Sala R, Lagrasta C, Corradi D, Bonacina E, Gambert SR, Cigola E, Anversa P. Acute myocardial infarction in humans is associated with activation of programmed myocyte cell death in the surviving portion of the heart. J Mol Cell Cardiol. 1996;28:2005–2016. - PubMed
    1. Piro FR, di Gioia CR, Gallo P, Giordano C, d'Amati G. Is apoptosis a diagnostic marker of acute myocardial infarction? Arch Pathol Lab Med. 2000;124:827–831. - PubMed
    1. Palojoki E, Saraste A, Eriksson A, Pulkki K, Kallajoki M, Voipio-Pulkki LM, Tikkanen I. Cardiomyocyte apoptosis and ventricular remodeling after myocardial infarction in rats. Am J Physiol Heart Circ Physiol. 2001;280:H2726–H2731. - PubMed

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