MicroRNA-532-3p regulates mitochondrial fission through targeting apoptosis repressor with caspase recruitment domain in doxorubicin cardiotoxicity

Abstract

Doxorubicin (DOX) is a wide-spectrum antitumor drug, but its clinical application is limited by its cardiotoxicity. However, the mechanisms underlying DOX-induced cardiomyopathy remain mostly unclear. Here we observed that apoptosis repressor with caspase recruitment domain (ARC) was downregulated in mouse heart and cardiomyocytes upon DOX treatment. Furthermore, enforced expression of ARC attenuated DOX-induced cardiomyocyte mitochondrial fission and apoptosis. ARC transgenic mice demonstrated reduced cardiotoxicity upon DOX administration. DOX-induced mitochondrial fission required the activity of dynamin-related protein 1 (Drp1). In elucidating the molecular mechanism by which ARC was downregulated upon DOX treatment, miR-532-3p was found to directly target ARC and participated in DOX-induced mitochondrial fission and apoptosis. MiR-532-3p was not involved in DOX-induced apoptosis in cancer cells. Taken together, these findings provide novel evidence that miR-532-3p and ARC constitute an antiapoptotic pathway that regulates DOX cardiotoxicity. Therefore, the development of new therapeutic strategies based on ARC and miR-532-3p is promising for overcoming the cardiotoxicity of chemotherapy for cancer therapy.

Figure 1

ARC is involved in DOX-induced mitochondrial fission and apoptosis in cardiomyocytes. (a and b) ARC mRNA levels (a) and protein levels (b) in neonatal rat cardiomyocytes treated with 2 μM DOX at the indicated time. *P<0.05 versus control (untreated). (c–f) Enfored expression of ARC by infecting adenoviral ARC (c) attenuated DOX (2 μM)-induced mitochondrial fission (d), cell death (e) and caspase-3 activation (f). Representative photos showed mitochondrial fission (d, left). Blue, DAPI (4,6-diamidino-2-phenylindole)-stained nuclei; red, MitoTracker Red CMXRos-stained mitochondria. Scale bar, 10 μm. The percentage of cells undergoing mitochondrial fission were counted (d, right). (g–j) Mitochondrial fission (h), cell death (i) and caspase-3 activation (j) were increased in response to low-dose DOX (0.2 μM) upon knockdown of endogenous ARC using its small interfering RNA (siRNA) (g). Data are expressed as the mean±S.D., n=3. *P<0.05

Figure 2

ARC regulates mitochondrial fission and apoptosis in vivo. (a and b) ARC mRNA levels (a) and protein levels (b) in ARC transgenic mice (Tg) and wild-type mice (WT) administered with DOX. The quantitative analysis of protein level was shown (b, up). n=3 mice per group. (c and d) Mitochondrial fission was analyzed in heart tissues of ARC Tg and WT mice administered with DOX. Scale bar, 2 μm; arrows indicate fission mitochondria (c). Percentage of mitochondrial fission (d). n=5 mice per group. (e) TUNEL (terminal deoxinucleotidyl transferase-mediated dUTP-fluorescein nick-end labeling) assay was used to detect apoptotic cells in heart tissues. Green, TUNEL-positive nuclei; bule, DAPI (4,6-diamidino-2-phenylindole)-stained nuclei; red, cardiomyocytes labeled with antibody to α-actinin, scale bar, 20 μm. n=5 mice per group. Data are presented as mean±S.D., *P<0.05

Figure 3

ARC attenuates DOX cardiotoxicity in mice. (a–d) ARC Tg mice are resistant to LV remodeling after DOX treatment. (a) Cross-sectional areas were analyzed by TRITC-conjugated wheat agglutinin staining. (b) The myocardial fibrosis was determined by Masson trichrome staining. Scale bar, 20 μM. (c and d) The expression levels of ANP and β-myosin heavy chain were detected by qRT-PCR. (e–g) ARC Tg or WT mice exposed to DOX or saline as described in panel (a), and echocardiography was used to test heart function. LVIDs, systolic left ventricular internal diameters; LVIDd, diastolic left ventricular internal diameters; FS, fractional shortening of left ventricular diameter. Data are presented as mean±S.D., n=8 mice per group. *P<0.05

Figure 4

MiR-532-3p participates in the regulation of ARC expression. (a) Analysis of ARC 3′UTR potential binding site for miR-532-3p by RNAhybrid. Potential complementary residues are shown in red. (b) The miR-532-3p levels in neonatal rat cardiomyocytes treated with 2 μM DOX at the indicated time. *P<0.05 versus control (untreated). (c) MiR-532-3p levels in mice administered with DOX or saline as described in Materials and Methods. n=5 mice per group. (d) ARC mRNA and protein levels in neonatal rat cardiomyocytes overexpressing miR-532-3p by transfecting with miR-532-3p mimics. (e) Kncokdown of endogenous miR-532-3p by transfecting its antagomirs attenuated decrease of ARC mRNA and protein levels upon DOX (2 μM) for 12 h in cardiomyocytes. (f) Luciferase activity detected in HEK-293 transfected with synthesized miR-532-3p mimic or mimic control, along with luciferase reporter constructs as indicated. (g) Luciferase activity of luciferase construct ARC 3′UTR-Wt is decreased upon DOX treatment in cardiomyocytes. Data are expressed as the mean±S.D., n=3 except in panel (c). *P<0.05

Figure 5

MiR-532-3p can regulate mitochondrial fission and apoptosis in cardiomyocytes treated by DOX. (a and b) Mitochondrial fission (a) and cell death (b) were not changed in neonatal rat cardiomyocytes overexpressing miR-532-3p for the indicated times. (c and d) Enfored expression of miR-532-3p enhanced mitochondrial fission (c) and cell death (d) in cardiomyocytes upon DOX (0.2 μM) treatment. Representative photos showed mitochondrial fission (c, left). Blue, DAPI (4,6-diamidino-2-phenylindole)-stained nuclei; red, MitoTracker Red CMXRos-stained mitochondria. Scale bar, 10μM. The percentage of cells undergoing mitochondrial fission were counted (c, right). (e and f) Inhibition of endogenous miR-532-3p using miR-532-3p antagomir prevented mitochondrial fission (e) and cell death (f) in cardiomyocytes treated with DOX (2 μM). Data are expressed as the mean±S.D., n=3. *P<0.05

Figure 6

MiR-532-3p regulates mitochondrial fission and apoptosis by targeting ARC. (a and b) MiR-532-3p-increased sensitivity to DOX treatment was abolished by ARC. Mitochondrial fission (a) and cell death (b) were measured in neonatal rat cardiomyocytes upon 0.2 μM DOX treatment. (c and d) MiR-532-3p antagomir-attenuated DOX treatment was abolished by endogenous ARC knockdown. Mitochondrial fission (c) and cell death (d) were measured in cardiomyocytes upon 2 μM DOX treatment. (e) Enforced expression of miR-532-3p did not affect cell death in neonatal mouse cardiomyocytes isolated from ARC Tg mice upon DOX (0.2 μM) treatment. Data are expressed as the mean±S.D., n=3. *P<0.05

Figure 7

MiR-532-3p is not involved in DOX-induced apoptosis in cancer cells. (a) The miR-532-3p levels in cardiomyocytes, Hela, SGC-7901, SW-480 and HEPG-2 cells. *P<0.05 versus cardiomyocytes. (b and c) The miR-532-3p levels in Hela (b) and SGC-7901 (c) cells treated with 2 μM DOX for the indicated times. (d and e) Cell death in Hela (d) and SGC-7901 (e) cells transfected with miR-532-3p antagomir and treated with 2 μM DOX. Data are expressed as the mean±S.D., n=3