miR-451 Silencing Inhibited Doxorubicin Exposure-Induced Cardiotoxicity in Mice

Abstract

Oxidative stress and cardiomyocytes apoptosis were closely involved in the pathological process of doxorubicin- (Dox-) induced cardiac injury. MicroRNA-451 (miR-451) was mainly expressed in cardiomyocytes. However, the role of miR-451 in Dox-induced cardiac injury remained unclear. Our study aimed to investigate the effect of miR-451 on Dox-induced cardiotoxicity in mice. We established a Dox-induced cardiotoxicity model in the mice and manipulated miR-451 expression in the heart using a miR-451 inhibitor, which was injected every other day beginning at one day before Dox injection. Oxidative stress and apoptosis in the hearts were evaluated. miR-451 levels were significantly increased in Dox-treated mice or cardiomyocytes. miR-451 inhibition attenuated Dox-induced whole-body wasting and heart atrophy, reduced cardiac injury, restored cardiac function, and improved cardiomyocyte contractile function. Moreover, miR-451 inhibition reduced oxidative stress and cardiomyocytes apoptosis in vivo and in vitro. miR-451 inhibition increased the expression of calcium binding protein 39 (Cab39) and activated adenosine monophosphate activated protein kinase (AMPK) signaling pathway. A specific inhibitor of AMPK abolished the protection provided by miR-451 inhibition against cell injury in vitro. In conclusion, miR-451 inhibition protected against Dox-induced cardiotoxicity via activation of AMPK signaling pathway.

Figure 1

miR-451 inhibition prevented Dox-induced cardiac injury. (a) The level of miR-451 in the hearts (n=5). (b) The level of miR-451 in the H9c2 cells (n=5). (c) The level of miR-451 in the hearts (n=5). (d) Body weight in the Dox-treated mice (n=10). (e) The ratio of heart weight to tibial length in the indicated mice (n=10). (f) The level of NT-proBNP in the hearts (n=6). (g) The level of cardiac cTnI after Dox treatment (n=6). (h) The level of CK in the mice (n=6). ∗P<0.05 compared with the group with saline or PBS. ^#P<0.05 compared with mice after Dox injection.

Figure 2

miR-451 inhibition improved cardiac function in mice. (a-b) The alteration in +dP/dt and –dP/dt in mice (n=8). (c) Ejection fraction in the mice (n=8). (d-e) Cardiac output and stroke work in the mice (n=8). (f-g) Tau (Weiss and Glantz) in the mice (n=8). ∗P<0.05 compared with the group with saline. ^#P<0.05 compared with mice after Dox injection.

Figure 3

miR-451 inhibition improved cardiomyocyte contractile properties. (a) Resting cell length. (b) Peak shortening (normalized to cell length). (c-d) Maximal velocity of shortening (+dL/dt) and maximal velocity of relengthening (−dL/dt). ∗P<0.05 compared with the group with saline. ^#P<0.05 compared with mice after Dox injection. n=50 cells from 3 mice per group.

Figure 4

miR-451 inhibition attenuated oxidative stress and apoptosis in mice. (a) The level of 4-HNE in the hearts (n=6). (b) The level of GSH/GSSG in the hearts (n=6). (c) The level of cardiac MDA (n=6). (d) The activity of SOD in the hearts (n=6). (e) Cell apoptosis detected by TUNEL staining (n=5). (f) The activity of caspase3 in the hearts (n=6). ∗P<0.05 compared with the group with saline. ^#P<0.05 compared with mice after Dox injection.

Figure 5

miR-451 inhibition attenuated oxidative stress and apoptosis in H9c2 cells. (a) The level of miR-451 expression after a miR-451 inhibitor treatment (n=6). (b) The level of ROS in H9c2 cells (n=6). (c) The level of 4-HNE in H9c2 cells (n=6). (d) The MDA content in H9c2 cells (n=6). (e) Cell viability after Dox treatment (n=6). (f) The activity of caspase3 in H9c2 cells (n=6). ∗P<0.05 compared with the group with PBS. ^#P<0.05 compared with Dox-treated H9c2 cells.

Figure 6

miR-451 inhibition provided cardioprotection via activating AMPK signaling pathway. (a) The level of Cab39 in the H9c2 cells (n=6). (b) AMPK signaling pathway in the H9c2 cells (n=6). (c) The level of Cab39 in the hearts (n=6). (d) AMPK signaling pathway in the hearts (n=6). (e) Cell viability in H9c2 cells (n=6). ∗P<0.05 compared with the group with PBS or saline. ^#P<0.05 compared with Dox-treated H9c2 cells or hearts.

Figure 7

miR-451 inhibition cannot affect Dox-induced death of cancer cells. (a) The level of miR-451 expression after a miR-451 inhibitor treatment (n=6). (b) Cell viability (n=6). ∗P<0.05 compared with the group with NC inhibitor.