X-ray Radiotherapy Impacts Cardiac Dysfunction by Modulating the Sympathetic Nervous System and Calcium Transients

Abstract

Recent epidemiological studies have shown that patients with right-sided breast cancer (RBC) treated with X-ray irradiation (IR) are more susceptible to developing cardiovascular diseases, such as arrhythmias, atrial fibrillation, and conduction disturbances after radiotherapy (RT). Our aim was to investigate the mechanisms induced by low to moderate doses of IR and to evaluate changes in the cardiac sympathetic nervous system (CSNS), atrial remodeling, and calcium homeostasis involved in cardiac rhythm. To mimic the RT of the RBC, female C57Bl/6J mice were exposed to X-ray doses ranging from 0.25 to 2 Gy targeting 40% of the top of the heart. At 60 weeks after RI, Doppler ultrasound showed a significant reduction in myocardial strain, ejection fraction, and atrial function, with a significant accumulation of fibrosis in the epicardial layer and apoptosis at 0.5 mGy. Calcium transient protein expression levels, such as RYR2, NAK, Kir2.1, and SERCA2a, increased in the atrium only at 0.5 Gy and 2 Gy at 24 h, and persisted over time. Interestingly, 3D imaging of the cleaned hearts showed an early reduction of CSNS spines and dendrites in the ventricles and a late reorientation of nerve fibers, combined with a decrease in SEMA3a expression levels. Our results showed that local heart IR from 0.25 Gy induced late cardiac and atrial dysfunction and fibrosis development. After IR, ventricular CSNS and calcium transient protein expression levels were rearranged, which affected cardiac contractility. The results are very promising in terms of identifying pro-arrhythmic mechanisms and preventing arrhythmias during RT treatment in patients with RBC.

Keywords: breast cancer; cardiovascular disease; ionizing radiation; radiotherapy.

Figure 1

Impact of 30% top-heart X-ray irradiation on atrial and ventricular cardiac functions in mice 60 weeks after irradiation. (A) Ventricular cardiac functions were measured by echocardiography in M-mode: left ventricular ejection fraction (LVEF) (a) and diastole left ventricular wall depth (LW) (b). Data were presented as mean ± SEM (n = 5–6 per group). * p < 0.05, ** p < 0.01 vs. NIR mice by one-way ANOVA test followed by Dunnett’s multiple comparison post hoc test. (B) Schematic representation of specific myocardial regions and segments used for measurement of myocardial deformation by myocardial strain analysis (a). Measurement of left ventricular (b) dyssynchrony, (c–e) longitudinal deformation in specific myocardial regions, and (f) strain rate. Data were presented as mean ± SEM (n = 4–5 per group). * p < 0.05 and ** p < 0.01 vs. NIR mice by one-way ANOVA test followed by Dunnett’s multiple comparison post hoc test or Kruskal–Wallis test followed by Dunn’s multiple comparison post hoc test. (C) Electrocardiographic (ECG) analysis with summary of QRS interval (a), RR interval (b), heart rate (c), and P-wave duration (d). Data were presented as mean ± SEM (n = 3–5 per group). * p < 0.05, ** p < 0.01, and *** p < 0.005 vs. NIR mice by one-way ANOVA test followed by Dunnett’s multiple comparison post hoc test or Kruskal–Wallis test followed by Dunn’s multiple comparison post hoc test. (D) Post-pacing pause recorded following transesophageal atrial fibrillation. Left atrium diastole volume (mm³). Data were presented as mean ± SEM (n = 4–5 per group). * p < 0.05 vs. NIR mice by one-way ANOVA test followed by Dunnett’s multiple comparison post hoc test. (E) Micro-CT analysis of left atrium diastole volume 60 weeks post-IR. Data were presented as mean ± SEM (n = 4–6 per group). ** p < 0.01 vs. NIR mice by one-way ANOVA test followed by Dunnett’s multiple comparison post hoc test.

Figure 2

The 30% top-heart X-ray irradiation induced fibrosis and apoptosis mainly in atria. (A) Western blot analysis of the cleaved caspase-3 protein in atria and ventricles in heart of mice 24 h post-IR (a). GAPDH was used as an internal control. Data were shown as a percentage effect ± SEM compared to NIR mice (n = 4–6 per group from 2 independent experiments) (b). * p < 0.05 vs. NIR mice by one-way ANOVA test followed by Dunnett’s multiple comparison post hoc test. (B) Western blot analysis of the cleaved caspase-3 protein in atria and ventricles in heart of mice 60 weeks post-IR. GAPDH was used as an internal control (a). Data were shown as a percentage effect ± SEM compared to NIR mice (n = 3–5 per group from 2 independent experiments) (b). * p < 0.05 and *** p < 0.005 vs. NIR mice by one-way ANOVA test followed by Dunnett’s multiple comparison post hoc test. (C) Representative images demonstrating patterns of interstitial fibrosis by Picrosirius Red staining (collagen fibers stained red) on longitudinal cardiac tissue sections in the heart, atria, and ventricles from all experimental groups at 60 weeks post-IR (scale bar: 2.5 mm, 250 µM, and 100 µM, respectively) (a). Black squares indicate the location of the zoomed region of interest in the atria and ventricles. Arrows indicate interstitial fibrosis. Quantification of cardiac fibrosis is presented on the right. Data were presented as mean ± SEM (n = 3 per group) (b–d). * p < 0.05, ** p < 0.01 and *** p < 0.005 vs. NIR mice by one-way ANOVA test followed by Dunnett’s multiple comparison post hoc test.

Figure 3

Early impact of 30% top-heart X-ray irradiation on cardiac SNS morphology. (A) Images of iDISCO+ cleared hearts of mice 24 h post-IR acquired by light-sheet fluorescence microscopy (a). Quantification of TH-positive fibers in the whole heart using automated filament tracer in IMARIS software in mice 24 h after irradiation: total number of filaments (b), filament volume (c), filament length (d), number of dendritic segments on filaments (e), spine density on a dendritic segment (f), and branch level (g). Data were presented as mean ± SEM (n = 3–4 per group). Scale bar: 1000 µm. * p < 0.05, ** p < 0.01, **** p < 0.001 vs. NIR mice by one-way ANOVA test followed by Dunnett’s multiple comparison post hoc test. (B) Cardiac catecholamines levels in mice 24 h post-IR. Catecholamines were measured by ELISA in heart samples. Data were shown as a percentage effect ± SEM compared to NIR mice (n = 4–5 per group). ** p < 0.01 vs. NIR mice by one-way ANOVA test followed by Dunnett’s multiple comparison post hoc test. (C) Cardiac calcium concentration in mice 24 h post-IR. Calcium concentrations were measured by a colorimetric assay in heart samples. Data were shown as a percentage effect ± SEM compared to NIR mice (n = 3–5 per group). * p < 0.05 vs. NIR mice by Kruskal–Wallis test followed by Dunn’s multiple comparison post hoc test.

Figure 4

General IMARIS analysis of the cardiac sympathetic nervous system 60 weeks post-irradiation. Images of iDISCO+ cleared hearts of mice 60 weeks post-IR acquired by light-sheet fluorescent microscopy (a). Quantification of TH-positive fibers in the whole heart using the automated filament tracer in IMARIS software in mice 24 h after irradiation: total number of filaments (b), filament volume (c), filament length (d), number of dendritic segments on filaments (e), spine density on a dendritic segment (f), and branch level (g). Data were presented as mean ± SEM (n = 2–3 per group). Scale bar: 1000 µm.

Figure 5

The 30% top-heart X-ray irradiation induced a delayed reorganization of the cardiac SNS. (A) IMARIS analysis images (a) and quantification of TH-positive fibers inside the heart (b) using an automated filament tracer in IMARIS software in mice 60 weeks post-IR. Data were presented as mean ± SEM (n = 2–3 per group). * p < 0.05 vs. NIR mice by Kruskal–Wallis test followed by Dunn’s multiple comparison post hoc test. (B) Western blot analysis of the Sema3a in hearts of mice 60 weeks post-IR (a). GAPDH was used as an internal control. Data were shown as a percentage effect ± SEM compared to NIR mice (n = 3–6 per group from 2 independent experiments) (b). * p < 0.05 and ** p < 0.01 vs. NIR mice by one-way ANOVA test followed by Dunnett’s multiple comparison post hoc test. (C) Western blot analysis of ADRB1 protein in hearts of mice 60 weeks post-IR (a). GAPDH was used as an internal control. Data were shown as a percentage effect ± SEM compared to NIR mice (n = 3–7 per group from 2 independent experiments) (b). * p < 0.05, ** p < 0.01 and *** p < 0.005 vs. NIR mice by one-way ANOVA test followed by Dunnett’s multiple comparison post hoc test. (D) Cardiac catecholamine levels in mice 60 weeks post-IR. Catecholamines were measured by ELISA in heart samples and data were shown as a percentage effect ± SEM compared to NIR mice (n = 3–4 per group).

Figure 6

Effect of 30% top-heart X-ray irradiation on calcium homeostasis. (A) Western blot analysis of the calcium proteins in atria and ventricles of mice 24 h post-IR. RYR2 (a), SERCA2a (b), PLNp/PLNm ratio (c), NCX (d), NKA (e) and Kir2.1 (f). GAPDH was used as an internal control. Data were shown as a percentage effect ± SEM compared to NIR mice (n = 5–8 per group from 2 independent experiments). * p < 0.05 and ** p < 0.01, **** p < 0.001 vs. NIR mice by one-way ANOVA test followed by Dunnett’s multiple comparison post hoc test. (B) Western blot analysis of the calcium proteins in atria and ventricles of mice 60 weeks after irradiation. RYR2 (a), SERCA2a (b), PLNp/PLNm ratio (c), NCX (d), NKA (e) and Kir2.1 (f). GAPDH was used as an internal control. Data were shown as a percentage effect ± SEM compared to NIR mice (n = 3–6 per group from 2 independent experiments). * p < 0.05 and ** p < 0.01 vs. NIR mice by one-way ANOVA test followed by Dunnett’s multiple comparison post hoc test.

Figure 7

Schematic summary of the impact of ionizing radiation on the top of the heart.

Figure 8

Study design and localized irradiation procedure. The study design was divided into three major time points, namely, localized irradiation of the hear with SARRP, the early time point 24 h after irradiation, and the late time point 60 weeks after irradiation. Female C57BL/6J mice were divided into 4 experimental groups: non-irradiated control (NIR) mice, and 0.25, 0.5, and 2 Gy irradiated mice. At each time point, hearts were collected. Different experiments were then carried out, such as cardiac function, cardiac morphology, CSNS morphology, and cardiac molecular analysis.