An Adult Mouse Model of Dilated Cardiomyopathy Caused by Inducible Cardiac-Specific Bis Deletion

Figures

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  Figure 1. Cardiac specific depletion of BCL-2 interacting cell death suppressor (BIS) by α-MHC-MerCreMer strategy. (A) Scheme of tamoxifen (TAM) injection and echocardiography (Echo). (B) Western blot analysis of BIS expression in several tissues from double transgenic mice (Bisf/f: α-MHC-MerCreMer-positive) before and 28 days after tamoxifen injection (upper) and in the hearts and skeletal muscles from control (Bisf/f, F/F) and tamoxifen-inducible cardiomyocyte-specific BIS-KO (Bis-iCKO) (iCKO) mice 28 days after the first tamoxifen injection (lower). (C) Survival curves for F/F (red) and iCKO (blue) mice. Survival was analyzed every week after tamoxifen injection (n = 12 for both groups 6 males and 6 females). (D) Morphology of whole hearts from F/F and iCKO mice on day 28; scale bar, 2 mm. (E) Heart weight to body weight ratio for F/F and iCKO mice was determined 28 days after the first tamoxifen injection (n = 7 for both groups, 4 males and 3 females). Data are presented as the mean ± standard deviation (SD). ** p < 0.01. (F) Hematoxylin and eosin staining of serial sections of the hearts from F/F and iCKO mice 28 days after tamoxifen injections; scale bar, 1 mm.

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  Figure 2. Tamoxifen-inducible depletion of BIS in the cardiomyocytes resulted in the development of dilated cardiomyopathy (DCM) in adult mice. (A) Echocardiographic tracing of F/F and iCKO mice before (D0) and after tamoxifen injection (D7, D14, D21 and D28). (B,C) Values of left ventricular ejection fraction (LVEF) and fractional shortening (FS) were obtained using echocardiography (left panels; n = 5 for both groups, 3 males and 2 females). LVEF delta and FS delta changes on day 28 are shown in the right panels. (D,E) Left ventricular internal diastolic dimension (LVIDd) and left ventricular internal systolic dimension (LVISd) were progressively increased in iCKO mice. Data are presented as the mean ± standard deviation (SD). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the values from F/F mice on corresponding days. (F) Time-dependent BIS expression levels in iCKO mice after tamoxifen injection were determined using Western blotting assay (left) and qRT–PCR (right). ** p < 0.01 compared with the values on D0.

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  Figure 3. Ultrastructure analysis and expression profiles of cardiac mRNA in Bis-iCKO mice. (A) Representative transmission electron microscopy images of the myocardium of F/F and iCKO mice 28 days after tamoxifen injection. Scale bar, 1.0 μm (left) and 0.5 μm (right). (B) Quantitative analysis of the mRNA levels of cardiac failure markers and profibrotic genes in F/F and iCKO mice 28 days after tamoxifen injection (n = 5 for both groups). Data are presented as the mean ± standard deviation (SD). * p < 0.05, ** p < 0.01, and *** p < 0.001.

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  Figure 4. Decreased expression of HSPB8 and subsequent accumulation of insoluble DESMIN in the cardiomyocytes in Bis-iCKO mice. Immunoblots for HSPB8, HSP70, and DESMIN in total fractions from the hearts of F/F and iCKO mice (left). Increased levels of DESMIN in the insoluble fraction in the hearts of iCKO mice were demonstrated using Western blotting (right).