MiRNA-Mediated Fibrosis in the Out-of-Target Heart following Partial-Body Irradiation

Abstract

Recent reports have shown a link between radiation exposure and non-cancer diseases such as radiation-induced heart disease (RIHD). Radiation exposures are often inhomogeneous, and out-of-target effects have been studied in terms of cancer risk, but very few studies have been carried out for non-cancer diseases. Here, the role of miRNAs in the pathogenesis of RIHD was investigated. C57Bl/6J female mice were whole- (WBI) or partial-body-irradiated (PBI) with 2 Gy of X-rays or sham-irradiated (SI). In PBI exposure, the lower third of the mouse body was irradiated, while the upper two-thirds were shielded. From all groups, hearts were collected 15 days or 6 months post-irradiation. The MiRNome analysis at 15 days post-irradiation showed that miRNAs, belonging to the myomiR family, were highly differentially expressed in WBI and PBI mouse hearts compared with SI hearts. Raman spectral data collected 15 days and 6 months post-irradiation showed biochemical differences among SI, WBI and PBI mouse hearts. Fibrosis in WBI and PBI mouse hearts, indicated by the increased deposition of collagen and the overexpression of genes involved in myofibroblast activation, was found 6 months post-irradiation. Using an in vitro co-culture system, involving directly irradiated skeletal muscle and unirradiated ventricular cardiac human cells, we propose the role of miR-1/133a as mediators of the abscopal response, suggesting that miRNA-based strategies could be relevant for limiting tissue-dependent reactions in non-directly irradiated tissues.

Keywords: Raman spectroscopy; abscopal effect; cardiac fibrosis; miR-1; miRNome; mir-133a.

Figure 1

(a) Lead parallelepiped to shield the anterior two-thirds of the mouse body. (b) Position of the NE 2571 ionization chamber into the cap to estimate the scatter dose to the shielded heart. (c) Workflow of experimental endpoints after mice irradiation.

Figure 2

Histogram showing the most significant REACTOME pathways associated with the statistically significant miRNAs (after genes enrichment) altered in WBI vs. SI mouse hearts (listed in Table S2). The single line represents specific gene ontology (GO) terms obtained from the ClueGo analysis. The colors indicates specific clusters of the differentially expressed genes between PBI and WBI samples. Red asterisks refer to significance in the percentage of pathway deregulation (* p < 0.05, ** p < 0.01).

Figure 3

Venn diagram showing overlap between the statistically significant miRNAs perturbed in in PBI (left side) and WBI mouse hearts (right side). All miRNAs in common were down-regulated.

Figure 4

Histogram showing the most significant REACTOME pathways associated with the statistically significant miRNAs in common between PBI and WBI mouse hearts (listed in Table 2). The single line represents specific gene ontology (GO) terms obtained from the ClueGo analysis. The colors indicate specific clusters of the differentially expressed genes between PBI and WBI samples. Red asterisks refer to significance in the percentage of pathway deregulation (* p < 0.05, ** p < 0.01).

Figure 5

(a) PCA scatterplot of Raman spectral data from SI (green), PBI (blue) and WBI (red) mice 15 days post-irradiation. (b) PC loading from PCA of Raman spectral data from SI, PBI and WBI mice 15 days post-irradiation showing spectral features responsible for the separation between the groups (red arrows).

Figure 6

(a) PCA scatterplot of Raman spectral data from SI (green), PBI (magenta) and WBI (black) mice 6 months post-irradiation. (b) PC loading from PCA of Raman spectral data from SI, PBI and WBI mice 6 months post-irradiation showing the spectral features responsible for the separation among the groups (red arrows). (c) Relative weightings of actin, DNA and TGF-β from least squares fitting of Raman spectra from SI, PBI and WBI groups 6 months post-irradiation. Error bars represent the standard error. *** p ≤ 0.001.

Figure 7

(a–c) Representative images of Masson’s trichrome staining of cardiac sections obtained from SI (a), WBI (b) and PBI (c) mice 6 months after exposure, in which collagen is stained blue. (d) Collagen-positive area quantified using HistoQuest 2.0.2.0249 software. Evaluation of vimentin (e), fibronectin 1 (f), α-SMA (g) and collagen 3A1 (h) expression levels, determined with qPCR analysis, 6 months after exposure. Each dataset represents the mean ± SEM of three independent biological replicates. * p < 0.05; ** p < 0.01 (Student’s t-test). Bars = 10 μm.

Figure 8

Intracellular levels of miR-1 (a) and miR-133a (b) in SkMCs after irradiation with 2 and 6 Gy, at different time points post-irradiation. (c) Schematic representation of the co-culture system. Intracellular levels of miR-1 (d) and miR-133a (e) in bystander and irradiated NHCF-Vs. Evaluation of fibronectin 1 (FN1 (f)), collagen 1A (COL1A1 (g)), collagen 3A1 (COL3A1 (h)) and connective tissue growth factor (CTGF (i)) expression levels in bystander and irradiated NHCF-Vs. Each dataset represents the mean ± SEM of three independent biological replicates. Values obtained with sham-irradiated cells (0 Gy) were taken as 1. * p < 0.05; ** p < 0.01; *** p < 0.001; (Student’s t-test). RX = irradiated cells. ByS = bystander cells.