Fig 2: Circulating Hpx levels in two patient cohorts.(A and B) Change in plasma Hpx as measured between 6 weeks and 3 months following initiation of anthracyclines was associated with change in GLS (absolute value) at 3 months in patients with breast cancer enrolled in the discovery and validation cohorts, respectively. (C) Absolute value of plasma Hpx at baseline (before anthracyclines) and at 6 weeks following the initiation of anthracyclines in the validation cohort, as measured by ELISA. (D) Baseline plasma Hpx concentration was associated with change in GLS at 3 months. All patients were treated with dose-dense doxorubicin and cyclophosphamide (ddAC). Patients denoted in red developed symptoms of heart failure. Pearson’s correlation coefficient was used to compare percent change in ∆Hpx or baseline Hpx to percent change in ∆GLS. ns indicates that there was no significant difference between the groups.

Fig 3: Circulating Hpx is elevated in mice treated with Dox.(A) Dox-induced chronic cardiomyopathy model in mice. i.p., intraperitoneally. (B) Heart weight (HW)/tibia length (TL) ratio in saline-treated (control) and Dox-treated (Dox) groups. (C) Representative images of control and Dox mice obtained during conscious echocardiography. (D) Cardiac function at 5 to 8 weeks after Dox treatment: FS, LVIDd, and LVIDs in control and Dox groups. FS, fractional shortening. (E) Absolute levels of mouse plasma Hpx following treatment in control and Dox groups. (F to H) Absolute levels of mouse plasma Hpx after treatment were associated with change in % FS, LVIDd, and LVIDs. Data were expressed as means ± SEM. Welch’s t test was used to compare the difference between control (n = 14) and Dox-treated (n = 13) groups. Pearson’s correlation coefficient was used in (F) to (H).

Fig 4: Hpx deficiency exacerbates anthracycline cardiac toxicity.(A) Survival curves of WT and Hpx−/− mice treated with saline or Dox. (B) Representative images of WGA staining in the heart. Scale bars, 100 μm. (C) Cardiomyocyte cross-sectional area in WT mice or Hpx−/− mice treated with saline or Dox, respectively. n = 4 per group. (D and E) Cardiac Nppb (n = 9, 7, 8, and 6, respectively) and Bax/Bcl2 ratio (n = 8, 9, 9, and 9, respectively) measured by RT-qPCR within 24 hours after completion of the 2-week Dox regimen. Data were expressed as means ± SEM. Ordinary one-way ANOVA followed by the Tukey-Kramer test was used to compare the means of experimental groups.

Fig 5: Hpx may be transported to the heart by circulating macrophages.(A) Hpx mRNA levels in the liver and heart in mice treated with saline (control; n = 8) and Dox (n = 8) mice within 24 hours after completion of the 2-week regimen. (B) Cardiac Hpx protein level within 24 hours after completion of the 2-week regimen (n = 8). (C) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Hpx, and LRP-1 detected by Western blot in adult MHECs treated with saline (control) or Dox at a concentration of 200, 400, and 800 ng/ml for 48 hours. The left-most lane is a liver sample that was used as a positive control. DAPI, 4',6-diamidino-2-phenylindole. (D) Circulating Hpx-expressing cells were CD45+ immune cells, as determined by fluorescence-activated cell sorting (FACS). FSC-A, forward scatter area. (E) Hpx-expressing cells were CD11b+CD64+ macrophages in the heart and circulation as determined by FACS. Data were expressed as means ± SEM. Welch’s t test was used to compare the difference between control and Dox-treated groups.