Autophagy inhibitor 3-methyladenine alleviates overload-exercise-induced cardiac injury in rats

Abstract

Overload-exercise (OE) causes myocardial injury through inducing autophagy and apoptosis. In this study we examined whether an autophagy inhibitor 3-methyladenine (3-MA) could alleviate OE-induced cardiac injury. Rats were injected with 3-MA (15 mg/kg, iv) or saline before subjected to various intensities of OE, including no swim (control), 2 h swim (mild-intensity exercise, MIE), 2 h swim with 2.5% body weight overload (moderate OE; MOE), 5% overload (intensive OE; IOE) or 2.5% overload until exhausted (exhaustive OE; EOE). After OE, the hearts were harvested for morphological and biochemiacal analysis. The cardiac morphology, autophagosomes and apoptosis were examined with H&E staining, transmission electron microscopy and TUNEL analysis, respectively. Autophagy-related proteins to (LC3-II/I and Beclin-1) and apoptosis-related proteins (Bcl-2/Bax) were assessed using Western blotting. Our results showed that compared with the control, MIE did not change the morphological structures of the heart tissues that exhibited intact myocardial fibers and neatly arranged cardiomyocytes. However, IOE resulted in irregular arrangement of cardiomyocytes and significantly increased width of cardiomyocytes, whereas EOE caused more swollen and even disrupted cardiomyocytes. In parallel with the increased OE intensity (MOE, IOE, EOE), cardiomyocyte autophagy and apoptosis became more and more prominent, evidenced by the increasing number of autophagosomes and expression levels of LC3-II/I and Beclin-1 as well as the increasing apoptotic cells and decreasing Bcl-2/Bax ratio. 3-MA administration significantly attenuated OE-induced morphological changes of cardiomyocytes as well as all the autophagy- and apoptosis-related abnormalities in MOE, IOE and EOE rats. Thus, the autophagy inhibitor 3-MA could alleviate OE-induced heart injury in rats.

Figure 1

3-MA reduced OE-induced heart injury in rats. (A) Representative histologic images showing the morphology of cardiomyocytes in the left ventricle of the heart in various OE groups treated with/without 3-MA. (B) Quantitative data of the cardiomyocyte width. ^*P<0.05 vs saline+Con, saline+MIE or saline+MOE. ^#P<0.05 vs 3-MA+Con or 3-MA+MIE. ^$P<0.05 vs saline+IOE or saline+EOE. The data are expressed as the mean±SD. n=3/group. Magnification: ×400, Scale bar: 40 μm. 3-MA: 3-methyladenine; MIE: mild-intensity exercise; MOE: moderate-overload exercise; IOE: intensive-overload exercise; EOE: exhausting-overload exercise.

Figure 2

3-MA reduced OE-induced autophagosome formation in the left ventricle. (A) Representative TEM plots showing the formation of autophagosomes (white arrow) in the left ventricle of the heart in various OE groups treated with/without 3-MA. a1 is the enlarged view of box a; b1 is the enlarged view of box b. Scale bar: 2 μm. (B) Quantification of autophagosomes. ^*P<0.05 vs saline+Con, saline+MIE or saline+MOE. ^#P<0.05 vs 3-MA+Con, 3-MA+MIE or 3-MA+MOE. ^$P<0.05 vs saline+IOE or saline+EOE. The data are expressed as the mean±SD. n=3/group. N: nucleus. 3-MA: 3-methyladenine; MIE: mild-intensity exercise; MOE: moderate-overload exercise; IOE: intensive-overload exercise; EOE: exhausting-overload exercise.

Figure 3

3-MA prevented OE-induced elevated ratio of LC-3 II/I and increased expression of beclin-1 in the left ventricle. (A) Representative Western blot bands and summarized data of LC3-I and LC3-II. (B) Representative bands and summarized data of beclin-1. ^*P<0.05 vs saline+Con. ^#P<0.05 vs 3-MA+Con. ^$P<0.05, vs saline+MIE or 3-MA+MIE. ^&P<0.05 vs saline+MOE, saline+IOE or saline+EOE. The data are expressed as the mean±SD. n=3/group. 3-methyladenine; MIE: mild-intensity exercise; MOE: moderate-overload exercise; IOE: intensive-overload exercise; EOE: exhausting-overload exercise.

Figure 4

3-MA decreased OE-induced cell apoptosis in rats. (A) Representative images showing the TUNEL positive cells which were labeled in brown (arrows). Scale bar: 100 μm. (B) Summarized data. ^*P<0.05 vs saline+Con or saline+MIE; ^#P<0.05 vs 3-MA+Con or 3-MA+MIE. ^$P<0.05 vs saline+MOE or 3-MA+MOE. ^&P<0.05 vs saline+MOE, saline+IOE or saline+EOE. The data are expressed as the mean±SD. n=3/group. 3-MA: 3-methyladenine; MIE: mild-intensity exercise; MOE: moderate-overload exercise; IOE: intensive-overload exercise; EOE: exhausting-overload exercise.

Figure 5

3-MA prevented OE-induced reduction of Bcl-2/Bax in the left ventricle. Top panels: Representative Western blot bands. The histogram shows the summarized data of Bcl-2/Bax. ^*P<0.05 vs saline+Con or saline+MIE. ^#P<0.05 vs 3-MA+Con or 3-MA+MIE. ^$P<0.05 vs 3-MA+MOE. ^&P<0.05 vs saline+MOE, saline+IOE or saline+EOE. The data are expressed as the mean±SD. n=3/group. 3-MA: 3-methyladenine; MIE: mild-intensity exercise; MOE: moderate-overload exercise; IOE: intensive-overload exercise; EOE: exhausting-overload exercise.