IFN-γ-STAT1-ERK Pathway Mediates Protective Effects of Invariant Natural Killer T Cells Against Doxorubicin-Induced Cardiomyocyte Death

Figures

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  Figure 1. αGC Administration Activated iNKT Cells in DOX-Treated Hearts (A) Gene expression of Vα14Jα18 in the left ventricle (LV) of vehicle and α-galactosylceramide (αGC)-treated mice 7 days and 14 days after doxorubicin (DOX) injection (n = 10). They were normalized to Rps18 gene expression and expressed as ratio to control group. (B) Representative flow cytometric assessment of cardiac mononuclear cells obtained from the LV of vehicle and αGC-treated mice 14 days after DOX injection. Cardiac mononuclear cells from 8 different mice for each group were pooled and analyzed. αGC-loaded CD1d tetramer+ TCRβ+ cells were identified as invariant natural killer T (iNKT) cells. The inset numbers are a percentage of the gated region of the samples. Summary data for the number and proportion of iNKT cells. Data are expressed as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01: analysis of variance (ANOVA) with Tukey’s post hoc test or Kruskal-Wallis test.

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  Figure 2. αGC Administration Attenuated DOX-Induced Cardiac Dysfunction (A and B) Body weight (BW), heart rate (HR), and systolic blood pressure (sBP) in vehicle and αGC-treated mice 14 days after DOX injection (n = 8–12). (C) Representative echocardiographic images and left ventricular end-diastolic diameter (LVDd), left ventricular end-systolic diameter (LVDs), and left ventricular ejection fraction (LVEF) in the indicated groups (n = 8–11). (D) Whole heart weight to tibial length (TL) and LV weight to TL ratio in the indicated groups (n = 9). Data are expressed as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01: ANOVA with Tukey’s post hoc test or Kruskal-Wallis test. Abbreviations as in Figure 1.

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  Figure 3. αGC Administration Attenuated DOX-Induced Cardiomyocyte Injury and Apoptosis (A) Representative images of hematoxylin and eosin staining and quantification of the number of cytoplasmic vacuolization in the LV of vehicle and αGC-treated mice 14 days after DOX injection (n = 3). Black arrows indicate cytoplasmic vacuolization. (B) Representative images of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and quantification of the percentage of TUNEL-positive nuclei in the LV of indicated mice (n = 4–5). Red arrowheads indicate TUNEL-positive nuclei. (C) Representative immunoblots and quantitative analysis of Bcl-2-associated X protein (Bax) and B cell/CLL lymphoma 2 (Bcl-2) in the LV of indicated mice (n = 7). (D) Representative immunoblots and quantitative analysis of cleaved caspase 3 in the LV of indicated mice (n = 7). Data are expressed as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01: ANOVA with Tukey’s post hoc test. HPF = high-powered field; other abbreviations as in Figure 1.

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  Figure 4. αGC Administration Increased IFN-γ Production in DOX-treated Hearts Gene expression of Il1b(A), Il6(B), Tnfa(C), Il4(D), Il10(E), and Ifng(F) in the LV of vehicle and αGC-treated mice 7 and 14 days after DOX injection (n = 9–11). Data are expressed as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01: ANOVA with Tukey’s post hoc test or Kruskal-Wallis test. IFN = interferon; other abbreviations as in Figure 1.

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  Figure 5. αGC Administration Increased Phosphorylation of STAT1, ERK, and Akt in DOX-treated Hearts (A) Representative immunoblots of phosphorylation of signal transducers and activators of transcription 1 (STAT1), Akt, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 in the LV of vehicle and αGC-treated mice 7 days after DOX injection (n = 8–15). (B to F) Quantitative analysis of these signals in the LV of indicated mice. Data are expressed as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01: ANOVA with Tukey’s post hoc test or Kruskal-Wallis test. Abbreviations as in Figure 1.

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  Figure 6. Neutralization of IFN-γ Abolished Protective Effects of αGC on DOX-induced Cardiac Dysfunction and Apoptosis (A) Representative echocardiographic images and parameters such as LVDd, LVDs, and ejection fraction (EF) in DOX- and αGC + DOX–treated mice with or without anti-IFN-γ antibody (n = 5–9). (B) Representative images of TUNEL staining and quantification of the percentage of TUNEL-positive nuclei in the LV of indicated mice (n = 5). Red arrowheads indicate TUNEL-positive nuclei. (C) Representative immunoblots and quantitative analysis of Bax and Bcl-2 in the LV of indicated mice 8 days after DOX treatment (n = 5). (D) Representative immunoblots and quantitative analysis of cleaved caspase 3 in the LV of indicated mice 14 days after DOX treatment (n = 4–6). (E) Representative immunoblots and quantitative analysis of phosphorylation of STAT1, ERK, and Akt in the LV of indicated mice 8 days after DOX treatment (n = 6–8). Data are expressed as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01: ANOVA with Tukey’s post hoc test or Kruskal-Wallis test. Abbreviations as in Figures 1, 2, 3, 4, and 5.