The circadian clock protects against ionizing radiation-induced cardiotoxicity

Abstract

Radiation therapy (RT) is commonly used to treat solid tumors of the breast, lung, and esophagus; however, the heart is an unintentional target of ionizing radiation (IR). IR exposure to the heart results in chronic toxicities including heart failure. We hypothesize that the circadian system plays regulatory roles in minimizing the IR-induced cardiotoxicity. We treated mice in control (Day Shift), environmentally disrupted (Rotating Shift), and genetically disrupted (Per 1/2 mutant) circadian conditions with 18 Gy of IR to the heart. Compared to control mice, circadian clock disruption significantly exacerbated post-IR systolic dysfunction (by ultrasound echocardiography) and increased fibrosis in mice. At the cellular level, Bmal1 protein bound to Atm, Brca1, and Brca2 promoter regions and its expression level was inversely correlated with the DNA damage levels based on the state of the clock. Further studies with circadian synchronized cardiomyocytes revealed that Bmal1 depletion increased the IR-induced DNA damage and apoptosis. Collectively, these findings suggest that the circadian clock protects from IR-induced toxicity and potentially impacts RT treatment outcome in cancer patients through IR-induced DNA damage responses.

Keywords: Bmal1; circadian clock; heart; radiation; toxicity.

Fig. 1.. The circadian clock preserves heart function from the chronic effects of IR treatment.

C57BL/6 female mice aged 8 to 12-weeks were used in all groups. Day Shift (D.S.) is the control group and represents the intact clock, while Rotating Shift (R.S.) and Per1/2^−/− groups are environmentally- and genetically-clock disrupted groups, respectively. (A) Rotating shift was initiated by subjecting wild-type mice to weekly alternating Light/Dark cycles beginning 15 days before IR treatment. These mice remained in this condition throughout the study. On Day 0, all groups received a single 18 Gy dose of IR, localized to target the heart region. (B) Heart function was measured by echocardiography under anesthesia pre- (Week −1) and post-IR treatment with representative images captured on short axis views. There were also measurements for untreated mice. The captured images were analyzed using the sizes of the interventricular septum (IVS, red arrow), left ventricle inner dimension (LVID, blue arrow), and left ventricular posterior wall (LVPW, yellow arrow) parameters during diastole and systole. Collectively, these parameters determine the (C) left ventricular ejection fraction (LVEF), the measure of heart function in pumping out blood, and (D) fractional shortening (FS), the degree of shortening of the left ventricular diameter between end-diastole and end-systole, as percentages. Dotted lines represent untreated while solid lines represent IR-treated mice. (E) Representative images of Masson’s trichrome stain to indicate fibrosis (yellow arrows showing blue stains) in heart tissue sections with quantification in (F). Statistical analysis was done using Repeated Measures Two-way ANOVA for heart function (n=6 per group) and student’s t test for fibrosis (n=3 per group). *=p<0.05, ***=p<0.001. Error bars = S.E.M.

Fig. 2.. The clock regulates IR-induced DNA damage and Bmal1 levels in heart tissues.

(A) Protein lysates from whole heart tissue samples collected from untreated and 6 weeks post-IR treated mice were probed by immunoblotting for Bmal1, pH2a.x, Histone 2A (H2a), and Rev-erbα expression. (B-E) Densitometry analysis, normalized to actin, of immunoblot images. D.S. means Day Shift, R.S. means Rotating Shift, and Per1/2^−/− means Per1/2 mutant mice. Statistical analysis was done using Two-way ANOVA. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. Error bars = S.E.M. (n=3 mice per group).

Fig. 3.. The core clock protein, Bmal1, transcriptionally binds to pro-survival genes of DNA repair in mouse heart.

(A) BMAL1 binds to the promoter region of its targeted genes from C57BL/6 mouse heart nuclear extracts in ChIP assay at −1500 bp (region 1) and −3000 bp (region 2) upstream of the transcription start site. (B) Quantification of ChIP experiment. (C) From ChIP-seq analysis in Table 1, the identification of canonical and noncanonical E-Boxes, the binding sites for Bmal1, found in the enriched promoter region sequences, along with their distances from transcriptional start sites. Statistical analysis was done using two-tailed student’s t test. *=p<0.05, **=p<0.01, ***=p<0.001. Error bars = S.E.M. (n=3 mice per group).

Fig. 4.. Schematic of the influence of the circadian clock on radiation-induced toxicity and treatment efficacy in cancers.

Circadian clock protects against IR-induced cardiotoxicity by regulating DDR signaling-mediated pro-survival mechanisms, but a disrupted clock by silencing or inhibiting Bmal1 via increased Rev-erbα activity leads to increased toxicities.