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

Abstract

Background: Doxorubicin-induced cardiomyopathy (DICM) is one of the complications that can limit treatment for a significant number of cancer patients. In animal models, the administration of statins can prevent the development of DICM. Therefore, the use of statins with anthracyclines potentially could enable cancer patients to complete their chemotherapy without added cardiotoxicity. The precise mechanism mediating the cardioprotection is not well understood. The purpose of this study is to determine the molecular mechanism by which rosuvastatin confers cardioprotection in a mouse model of DICM.

Methods: Rosuvastatin was intraperitoneally administered into adult male mice at 100 μg/kg daily for 7 days, followed by a single intraperitoneal doxorubicin injection at 10 mg/kg. Animals continued to receive rosuvastatin daily for an additional 14 days. Cardiac function was assessed by echocardiography. Optical calcium mapping was performed on retrograde Langendorff perfused isolated hearts. Ventricular tissue samples were analyzed by immunofluorescence microscopy, Western blotting, and quantitative polymerase chain reaction.

Results: Exposure to doxorubicin resulted in significantly reduced fractional shortening (27.4% ± 1.11% vs 40% ± 5.8% in controls; P < 0.001) and re-expression of the fetal gene program. However, we found no evidence of maladaptive cardiac hypertrophy or adverse ventricular remodeling in mice exposed to this dose of doxorubicin. In contrast, rosuvastatin-doxorubicin-treated mice maintained their cardiac function (39% ± 1.26%; P < 0.001). Mechanistically, the effect of rosuvastatin was associated with activation of Akt and phosphorylation of phospholamban with preserved sarcoplasmic/endoplasmic reticulum Ca²⁺ transporting 2 (SERCA2)-mediated Ca²⁺ reuptake. These effects occurred independently of perturbations in ryanodine receptor 2 function.

Conclusions: Rosuvastatin counteracts the cardiotoxic effects of doxorubicin by directly targeting sarcoplasmic calcium cycling.

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 analyzed by confocal immunofluorescence microscopy (I) at 14 d post-Dox. Green = wheat germ agglutinin for extracellular matrix (ECM); red = α-actinin; blue = 4′,6-diamidino-2-phenylindole for DNA. 100-130 cardiomyocytes quantified from 3 representative images. Data are mean ± SEM; n = 3 mice/group. (J) Quantification of mRNA levels from Dox-treated and rosuvastatin-Dox-treatedmice as analyzed by real-time reverse transcriptase quantitative polymerase chain reaction 14 d post-Dox treatment, normalized to β-actin. Data are mean ± SEM; n = 4. ∗P < 0.001 vs Con. (H, I) Representative immunoblot (K) and quantification (L) in LV protein extracts from con, Dox-treated, and statin-Dox-treated mice 14 d post-Dox, employing antibodies as indicated on the left (normalized to β-actin). Data are mean ± SEM; n = 3. #P < 0.005 Dox vs statin-Dox. ANP, atrial natriuretic peptide; Bad, Bcl-2 associated agonist of cell death; BNP, brain natriuretic peptide; Cl-Casp 3, cleaved caspase-3; CytoC, cytochrome C; Gata 4, GATA binding protein 4; Mef2, myocyte enhancer factor 2; Myh6, myosin heavy chain 6; Nkx2-5, NK2 Homeobox 5. See Supplemental Appendix S2 for original Western Blots.

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 analysis of variance performed for global comparison between the groups. For panels B-F, we used the following number of animals: Con group n = 5; Dox group n = 10; Statin-Dox group n = 10.

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.

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.