Essential Amino Acids-Rich Diet Increases Cardiomyocytes Protection in Doxorubicin-Treated Mice

Figure 1. (A–C) Representative Sirius-red staining for collagen before and after Doxo administration according to diets under normal (left) and polarized light (right with black background). (A,B) Hearts in StD-Doxo-treated mice increase the degree of fibrosis. Differently, the hearts of animals fed only EAAs show finer collagen fibers (C) than those observed in StD-fed animals. Doxo treatment did not increase the fibrosis (D). Scale bar 20 µm.

Figure 2. Representative TEM pictures of cardiomyocytes according to diet and Doxo treatment. Nucleus (A) and cytoplasm (B) of cardiomyocytes in mice fed with StD alone. After Doxo treatment we frequently observed irregular nuclei with bits-like signs. Sometimes these nuclei appeared very fragmented, with thicker condensed chromatin near the inner surface of the nuclear envelope (C). Furthermore, we observed mitochondria with irregular shapes and sizes and impaired distribution (D). Mice fed EAAs alone show a regular shape of the nucleus (E), and inside the cytoplasm, mitochondria are regularly oriented and distributed (F). After Doxo administration, the nuclear morphology appears to be like the control group, without bits-like signs and/or fragmentation, but with mild evidence of condensed chromatin (G). The mitochondria morphology and distribution (H) improves almost as much as with the EAAs diet. N = nucleus. Scale bar 2 μm (A,C,E,G) or 1 μm (B,D,F,H).

Figure 3. Klotho immunostaining intensity is expressed as optical density (A). In (A), the upper right image represents tissue incubated without primary antibody (blank control). Basal levels of klotho were faint in StD-fed mice (B), but increased strongly in EAA-fed mice (D). After Doxo administration, in both diets, the levels were comparable with that basal (C,E). ANOVA F = 22.09 p < 0.000. Scale bar 50 μm. p < 0.05 vs. * StD, ^ StD + Doxo.

Figure 4. RIP1 immunostaining intensity is expressed as optical density (A). (B,C) RIP1 immunostaining before (B) and after (C) Doxo treatment in StD-fed mice. Doxo increases strongly the RIP1 staining. (D,E) Immunostaining staining before (D) and after (E) Doxo treatment in EAA-fed mice. The staining intensity increases in a non-homogeneous manner after Doxo. Indeed, many cells show faint staining. So, the staining intensity before and after treatment was non-significant. ANOVA F = 35.40 p < 0.000. Scale bar 50 μm. p < 0.05 vs. * StD, ^ StD + Doxo.

Figure 5. RIP3 immunostaining intensity is expressed as optical density (A). (A) The upper right image represents tissue incubated without primary antibodies (blank control). (B,C) RIP3 immunostaining before (B) and after (C) Doxo treatment in StD-fed mice shows a strong increase after chemotherapy (C). In the hearts of EAA-fed mice, the levels of RIP3 did not change before (D) or after chemotherapy (E). ANOVA F = 6.28 p < 0.002. Scale bar 50 μm. p < 0.05 vs. * StD, ^ StD + Doxo.

Figure 6. NFkB immunostaining intensity is expressed as a number of stained nuclei/100 μ2 (A). Stained nuclei increase strongly in StD-fed animals after Doxo administration. The EAA diet favors the increase of nuclear NFkB which does not change after chemotherapy. (B–E) Representative pictures of stained nuclei (arrows) according to diets and chemotherapy. ANOVA F = 9.71 p < 0.000. Scale bar 10 μm. p < 0.05 vs. * StD.

Figure 7. TNFα immunostaining intensity is expressed as optical density (A). In StD-fed animals, the immunoreaction increases strongly after chemotherapy (B,C). On the opposite side, the levels of TNFα do change before and after treatment in EAA-fed mice (D,E). ANOVA F = 14.84 p < 0.000. Scale bar 50 μm. p < 0.05 vs. * StD, ^ StD + Doxo.

Figure 8. IL1 immunostaining intensity is expressed as optical density (A). In StD-fed animals, IL1 staining increases strongly after chemotherapy (B,C). On the opposite side, its level does change in EAA-fed mice (D,E). ANOVA F = 20.05 p < 0.000. Scale bar 50 μm. p < 0.05 vs. * StD, ^ StD + Doxo.

Figure 9. SOD1 immunostaining intensity is expressed as optical density (A). In StD-fed animals, SOD1 staining increases after chemotherapy (B,C). The EAA diet favors the early increase in SOD (D), which remains constant even after treatment with chemotherapy (E). ANOVA F = 44.19 p < 0.000. Scale bar 50 μm. p < 0.05 vs. * StD, ^ StD + Doxo.

Figure 10. Glutathione peroxidase (Glu-px) immunostaining intensity is expressed as optical density (A). In StD-fed animals, Glu-px staining increases after chemotherapy (B,C). The EAA diet favors the early moderate increase in Glu-px (D), which increases much more after treatment with Doxo (E). ANOVA F = 41.85 p < 0.000. Scale bar 50 μm. p < 0.05 vs. * StD, ^ StD + Doxo.

Figure 11. Citrate synthase (Cit-syn) immunostaining is expressed as number of stained dots/100 μm2 of cytoplasm (A). In StD-fed animals, Cit-syn staining decreases strongly after chemotherapy (B,C). The EAA diet favors the maintenance of Cit-syn level even after Doxo treatment (D,E). ANOVA F = 20.15 p < 0.000. Scale bar 15 μm. p < 0.05 vs. * StD, ° EAA, ^ StD + Doxo.

Figure 12. Schematic representation of the effects of StD and a special diet containing a mixture of all EAAs in the stoichiometric ratio on cardiac damages induced by Doxo. EAAs play a fundamental role in favoring the expression of Klotho and limiting the oxidative and inflammatory stress, and death of cardiomyocytes after treatment with chemotherapy. Therefore, an excess of EAAs in the diet has a protective action against the heart damages induced by Doxo.