Previous Cardiovascular Injury is a Prerequisite for Immune Checkpoint Inhibitor-Associated Lethal Myocarditis in Mice

Figures

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  Figure 1. (A) Schematic representation of the HTN + ICIs dual therapy mouse model. Mice were administered with L‐NAME for 5 weeks, represented by the black lines, and AngII 1 week after for 4 weeks, indicated by the purple lines. Blue circles represent the day of the weekly ICI dual treatment (anti‐CTL‐4 and anti‐PD‐1). Mice were analysed on days 7 and/or 21 of ICI treatment (black circles). (B) Comparative results from the CTRL group versus CTRL + ICIs group (day 7 and day 21), CTRL group versus HTN group (day 21) and HTN versus HTN + ICIs groups (day 7 and day 21). (1) In the absence of inflammatory stimuli, although some circulating cardiac autoantibodies (AAbs) might be present and increased by ICIs, cardiac tissue remains without signs of inflammation or damage. (2) Under sustained cardiac injury induced by L‐NAME and angiotensin II (ANG II), cardiac dysfunction and autoantigens might be released without evidence of myocarditis. (3) Increased mortality due to dual ICI therapy in the HTN group associated with evidence of myocarditis and increased IgG cardiac autoantigens. Created with BioRender.com.

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  Figure 2. ICIs increase mice mortality and cardiac remodelling from the first treatment dose. (A) Kaplan Meir curves of mice survival (n = 8–17). (B) Body weight gain (n = 6–9). (C) Heart weight/tibia length [HW/TL; (n = 6–13)]. (D) Myocyte area normalized to the control group (n = 4–9). The dotted line are guides to the eye for each experimental group. Data are presented as mean ± SEM. The log‐rank test was used for survival curves comparison and ANOVA test with post hoc Holm–Sidak multiple comparisons test. *P ≤ 0.05, *versus CTRL, #versus CTRL + ICIs and &versus HTN.

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  Figure 3. One dose of ICIs treatment triggers cardiac dysfunction associated with hypertrophy. (A) Representative images of PV‐Loop recordings with preload impediment by transiently occluding the inferior vena cava after 7 days of ICIs administration are shown. The first loop represents a steady‐state loop. The following hemodynamic parameters are displayed for each group: (B) stroke volume (μL), (C) cardiac output (mL/min), (D) ejection fraction (%), and (E) EDPVR slope. (F) BNP mRNA relative expression by qPCR from the cardiac apical section obtained from each group and normalized with GAPDH (n = 5–8). The dotted line are guides to the eye for each experimental group. Data are presented as mean ± SEM; ANOVA test with post hoc Holm–Sidak multiple comparisons test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

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  Figure 4. Inflammation is exacerbated from the first treatment dose with ICIs, triggering myocarditis with evidence of T‐cell infiltration and increased autoantibodies. mRNA relative expression by qPCR from the cardiac apical section obtained from each group and normalised with GAPDH gene expression (n = 5–8) were assessed for (A) TNF‐α and (B) IL‐6. (C) Representative images of histological H&E staining analysis of immune cell infiltration by myocarditis score determination, 20×. (D) Immunohistochemistry images of T cells (CD3 positive cells), 40×. (E) Autoantibodies from each group identified by indirect ELISA (1:80 dilution factor) are shown (n = 3–7). (F) Antibody titre on day 21 of different mice per group. The dotted line are guides to the eye for each experimental group. Data are presented as mean ± SEM; ANOVA test with Tukey's (gene expression) or post hoc Holm–Sidak (autoantibodies) multiple comparisons test. *P ≤ 0.05, versus CTRL, #versus CTRL + ICIs and &versus HTN.