Coronary microcirculation damage in anthracycline cardiotoxicity

Figures

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  Figure 1. Study design and animal model. Large-white castrated male pigs weighing 30–35 kg were used in this study. Animals were divided into six groups. A Control group of untreated animals (n=10), ExPr.1 group: pigs received 1 IC doxorubicin sacrificed 48 h later (n=5). ExPr.2 group: pigs received 1 IC doxorubicin sacrificed 2 weeks later (n=5), ExPr.3 group: pigs received three IC doxorubicin doses followed by 2 dose-free weeks before sacrifice (n=10). ExPr.4 group: pigs received 3 bi-weekly IC doxorubicin doses followed by 12 dose-free weeks (n=5). ExPr.5 group: animals received five bi-weekly IC doxorubicin doses followed by eight dose-free weeks (n=5). CMR scans and coronary physiology assessment were performed before doxorubicin infusion. Infused and remote myocardial areas were collected at sacrifice for further analysis. Animals in the Expr.3 (only 5), 4, and 5 are the same that were included in a previous publication, where LVEF was already reported.11

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  Figure 2. Global and regional cardiac function assessment during anthracycline-induced cardiotoxicity development. (A) CMR-LVEF in early and long-term stages for all ExPr groups. (B) CMR-wall thickening analysis for the infused and remote myocardial areas in early and long-term stages for all ExPr groups. Sample size for panels (A) and (B): baseline=30, 48 h=10, 2w=25, 4w=20, 6w=20, 16w=5 each. (C) Circumferential myocardial strain by echocardiography in ExPr. 3 in infused and remote areas during early stages. (D) CMR-diastolic function evaluation by left atrium reservoir analysis in ExPr. 3 during early stages. Sample size for panels (C) and (D)=5. Asterisks indicate statistically significant differences compared with Baseline: *P<0.05, **P<0.01, ***P<0.001. For panels (A) and (D): one-way ANOVA was performed to detect differences between Baselines and the rest of the time points (paired, corrected by Bonferroni). For panels (B) and (C): two-way ANOVA was performed to detect differences between time points and infused-remote areas within the same time point (both paired, corrected by Bonferroni). Boxplots represent median and interquartile range, while whiskers show maximum and minimum…

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  Figure 3. Cardiac microvascular function assessment during anthracycline-induced cardiotoxicity development. (A) CMR-quantitative perfusion for the infused and remote myocardial areas in early and long-term stages for all ExPr groups (sample size: baseline=14, 48 h=5, 2w=10, 4w=9, 6w=9, 16w=5 each). (B) CFR for the infused and remote myocardial areas in early and long-term stages for all ExPr groups (sample size for infused area: baseline=30, 10m=5, 48 h=5, 2w=25, 4w=19, 6w=15, 16w=5 each. Sample size for remote area: baseline=20, 10m=0, 48h=0, 2w=20, 4w=14, 6w=10, 16w=2 only for 5 doses). (C) Representative CMR-based cardiac perfusion maps for baseline, 3 doses at 6 and 16 weeks and 5 doses at 16 weeks groups. Asterisks indicate statistically significant differences compared with baseline: *P<0.05, **P<0.01, ***P<0.001. Hashes indicate statistically significant differences compared with respective remote: #P<0.05, ##P<0.01, ###P<0.001. Two-way ANOVA was performed to detect differences between time points and infused-remote areas within the same time point (both paired, corrected by Bonferroni). Boxplots represent median and interquartile range, while whiskers show maximum and minimum…

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  Figure 4. Pathology evaluation of microvasculature during anthracycline-induced cardiotoxicity development. (A) Stacked barplots indicating artery’s percentage evaluated with each damage score for each group and myocardial area (n=5 animals, ∼11 arteries scored per individual). (B) Representative arteries from infused and remote areas stained by Masson’s trichrome for baseline, 3 doses at 6 and 16 weeks and 5 doses at 16 weeks groups. Asterisks indicate statistically significant differences compared with baseline: *P<0.05, **P<0.01, ***P<0.001. Hashes indicate statistically significant differences compared with respective remote: #P <0.05, ##P<0.01, ###P<0.001. Chi-squared test were performed between to compare proportions with baseline and their respective remote area.

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  Figure 5. Capillary niche evaluation during anthracycline-induced cardiotoxicity development. (A) Capillary density for each group and myocardial area (n=5). (B) Asterisks indicate statistically significant differences compared with baseline: *P<0.05, **P<0.01, ***P<0.001. Hashes indicate statistically significant differences compared with respective remote: #P<0.05, ##P<0.01, ###P<0.001. Two-way ANOVA was performed to detect differences between baseline and the rest of the groups (unpaired); and between infused-remote areas within the same group (paired, corrected by Bonferroni). Boxplots represent median and interquartile range, while whiskers show maximum and minimum values. Circles represent individual data.

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  Figure 6. Ex vivo coronary artery vascular reactivity evaluation on myography during early subclinical anthracycline-induced cardiotoxicity. Absolute vascular response (in mN) after increasing doses of (A) papaverine, (B) bradykinin, and (C) acetylcholine. Boxplots and individual dots for (D) KCl contraction (mN), (E) ACh relaxation (%), (F) stiffness (1/s), and (G) diameter. N=5 animals per group. Asterisks indicate statistically significant differences between both groups: *P<0.05, **P<0.01, ***P<0.001. Differences between control and doxorubicin animal arteries subjected to several drugs [panels (A)–(C)] were detected with ANOVA analysis comparing the two multinomial logistic models. Unpaired Students’ t-test was performed to detect differences between both groups in panels (D)–(G).