Statins Protect Against Early Stages of Doxorubicin-induced Cardiotoxicity Through the Regulation of Akt Signaling and SERCA2

Figures

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  Figure 1. The development of the doxorubicin (Dox)-induced cardiomyopathy model. (A, B) Quantification of fractional shortening (A) by echocardiography (B) in mice treated with Dox or pretreated with rosuvastatin at 10 μg/kg/d (10R) or 100 μg/kg/d (100R) at 14 days post Dox treatment. For the remainder of the study, statin mice were pretreated with 100 μg/kg/d rosuvastatin. n = 6. ∗P < 0.01 vs control (Con). (C, D) Echocardiographic assessment of cardiac function left ventricular end-diastolic dimension (LVEDD) and left ventricular end-systolic dimension (LVESD), 14 days post-Dox from Control (Con), Dox, and rosuvastatin-treated mice. Data are mean ± standard error of the mean (SEM); n = 6. (E) Heart weight corrected for body weight. Data are mean ± SEM. n = 6. (F) Heart weight corrected for tibia length. Data are mean ± SEM; n = 6. (G) Representative micrographs of Masson trichrome-stained cardiac sections in long-axis view. The white-yellow dotted line represents the border between the left ventricle (LV) and the interventricular septum (IVS), and the border between the right ventricle (RV) and the IVS. (H, I) Quantification (H) of cardiomyocyte (CM) cross-sectional area within the LV as…

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  Figure 2. Calcium dysregulation in doxorubicin (Dox)-induced cardiomyopathy that is corrected by rosuvastatin (Statin). (A) Representative recordings of ventricular epicardial calcium signals recorded from Langendorff-perfusion hearts at 13 Hz, in Control, Dox, and Statin-Dox groups showing calcium amplitude alternans. (B) Calcium amplitude alternans ratio of ventricular epicardial calcium signals at various pacing frequencies. ∗P = 0.017 comparing the Con vs Dox groups. (C) The spontaneous calcium elevations (SCaEs) in Con, Dox, and Statin-Dox animals at various pacing frequencies. ∗P = 0.007, comparing the Con vs Dox groups, and ± P < 0.0001, comparing the Con vs Statin-Dox groups. (D, E) Repolarization Ca2+ transient duration to reach 50% (D: calcium transient duration [CaTD]50) or 80% (E: CaTD80) at various pacing frequencies. ∗P = 0.004 (CaTD50), comparing the Con vs Dox groups, and †P = 0.001 (CaTD50) and †P = 0.002 (CaTD80), comparing the Statin-Dox vs Dox groups. (F) Representative tracing of superimposed single beat from 3 groups to show the CaTD. All panels were done at various pacing rates and values are mean ± standard error of the mean. P values were derived from 2-way…

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  Figure 3. Rosuvastatin (Statin) prevents the inactivation of Akt/ phospholamban (PLN) signaling post-doxorubicin (Dox) treatment. (A, B) Immunoblot (top) and quantification (bottom) of protein expression of intracellular signaling effectors in control (Con), Dox, and Statin-Dox mice at 14 days post-Dox treatment using antibodies as indicated on the left. Western blots were repeated once with similar results. Data are means ± standard error of the mean. n = 3. ∗P < 0.01 vs Con #P < 0.01 vs Dox. σP < 0.01 vs Con. (C, D) Immunoblot (C) and quantification (D-F) of protein expression of acute changes in intracellular signaling mice after an insulin challenge. Western blots were repeated once done with similar results. Data are means ± standard error of the mean. n = 3. ∗#P < 0.01 vs 0 min. cam KIId, calcium-calmodulin-dependent protein kinases; mTOR, mammalian target of rapamycin; NCX1, sodium-calcium exchanger 1; p-mTOR, phosphorylated mTOR; PKA, pPLN, phospholamban; RyR2, ryanodine receptor 2; Serca2, sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2; S473, serine 473. Supplemental Appendix S2 for original Western Blots.

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  Figure 4. Schematic diagram of mechanisms mediating the protective effect of statins in doxorubicin-induced cardiomyopathy. cam KIId, calcium-calmodulin-dependent protein kinases; DDR, DNA damage response; PLN, phospholamban; PP1, protein phosphatase I; ROS, reactive oxygen species; RyR2, ryanodine receptor 2; Serca2, sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2.