Investigation of the pathomechanism and potential therapeutic targets in radiation-induced heart disease in a rat model

Abstract

Radiation-induced heart disease (RIHD) is a potential late side-effect of thoracic radiotherapy resulting in left ventricular hypertrophy (LVH) and fibrosis due to a complex pathomechanism leading to heart failure (HF). MicroRNA-212 (miR-212) is a crucial regulator of pathologic LVH via forkhead box O3 (FOXO3)-mediated pathways in pressure-overload-induced HF. Angiotensin-II receptor blockers (ARBs), including losartan, are frequently used to control HF of various etiologies. Preclinical evidence is lacking on the anti-remodeling effects of ARBs in RIHD, while the results of clinical studies are controversial. We aimed to i) set up a rat model in the acute, early chronic, and late chronic phases of RIHD, ii) investigate whether miR-212 and iii) its selected target FOXO3 play a role in the development of RIHD, iv) and test the effects of losartan in our rat model of RIHD. In our studies, RIHD was induced by selective heart irradiation (50 Gy) in male Sprague-Dawley rats. In our model setup study, 1, 3, and 19 weeks after selective heart irradiation, transthoracic echocardiography was performed to monitor cardiac morphology and function. At the endpoints, qRT-PCR was performed to measure the gene expression changes of miR-212 and Foxo3 in all follow-up time points. Cardiac expression of FOXO3 and phospho-FOXO3 (pFOXO3) were investigated at the protein level by Western blot at week 19. In RIHD, diastolic dysfunction (DD) was present at every time point. Mild hypertrophy developed at week 3, and a marked LVH with interstitial fibrosis developed at week 19 in the irradiated hearts. In our RIHD model, cardiac miR-212 was overexpressed at weeks 3 and 19, and Foxo3 was repressed at the mRNA level only at week 19. In contrast, the total FOXO3 protein level failed to decrease in response to heart irradiation at week 19. In conclusion, miR-212 seems to play a role in the development of LVH via FOXO3-independent mechanisms in RIHD. In our treatment study, male Sprague-Dawley rats were studied in three groups: 1) control, 2) radiotherapy (RT) only, 3) RT treated with losartan (per os 10 mg/kg/day), and were followed for 1, 3, or 15 weeks. 15 weeks postirradiation, losartan alleviated the echocardiographic and histological signs of LVH and fibrosis and reduced the LV overexpression of chymase (Cma), connective tissue growth factor (Ctgf), and transforming growth factor-beta (Tgfb) measured by qRT-PCR; likewise, the level of the SMAD2/3 protein determined by Western blot decreased. In both RT groups, the pro- survival phospho-AKT/AKT and the phospho-ERK1,2/ERK1,2 ratios were increased at week 15. The antiremodeling effects of losartan seem to be associated with the repression of Cma and several elements of the TGF-β/SMAD signaling pathway in our RIHD model