MMI‐0100 Inhibits Cardiac Fibrosis in a Mouse Model Overexpressing Cardiac Myosin Binding Protein C

Figures

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  Figure 1. Inducible cardiomyocyte expression of cMyBP‐C40k results in cardiac fibrosis. A, Transgenic (tg) constructs used in this study. The “tet‐off” transgenic system was used to control cardiomyocyte‐specific 40‐kDa expression. B, Representative Masson's trichrome staining in 10‐week‐old mouse hearts (6 weeks after MyBP‐C40k induction). Transgene expression was silenced during embryonic development and until after weaning by doxycycline treatment of the mothers' food (4 weeks postbirth). Scale bar: 250 μm. C, Hydroxyproline levels were also determined to quantitate fibrosis at the apex. n=3, ***P<0.001. 40 kDa indicates the MyBP‐C40k cDNA sequences; Ntg, Dtg, nontransgenic, double transgenic, respectively; tetR/tetO, tetracycline repressor/operator sequences, respectively; tTA, tetracycline‐sensitive transactivator sequence; VP16, virus protein 16; αMHC, α myosin heavy chain promoter sequence.

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  Figure 2. A, Fibrosis progression after cMyBP‐C40k expression was examined by trichrome staining. Scale bar: 200 μm. B, Quantification of fibrosis area, n=4. C, Activation of fibrosis‐related signaling was detected using Western blot analyses of total cardiac protein. D, Quantitation of the Western blot, analyzed using comparisons between the Ntg and Dtg groups at each time point. *P<0.05, **P<0.01, ***P<0.001. Dtg indicates double‐transgenic; HNRNPA0, heterogeneous nuclear ribonucleoprotein A0; Ntg, nontransgenic.

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  Figure 3. Chronic MMI‐0100 treatment reduces cardiac fibrosis in cMyBP‐C40K hearts. A, Outline of the experimental design. Four weeks after transgene induction, control and cMyBP‐C40K mice were given phosphate buffered saline (PBS) or MMI‐0100 (50 μg/kg per day) intraperitoneally for up to 30 weeks. B, C, HNRNPA0 levels were quantitated. D, Masson's trichrome staining in 34 weeks (24‐week treatment) hearts. Scale bar: ×2, 2 mm; ×10, 500 μm. E, Hydroxyproline levels were determined to quantitate left ventricle fibrosis. n=3, *P<0.05. AP indicates apex; Dtg, double‐transgenic cMyBP‐C40K; GAPDH, glyceraldehyde phosphate dehydrogenase; HNRNPA0, heterogeneous nuclear ribonucleoprotein A0; LA, left atrium; LV, left ventricle; Ntg, nontransgenic; n.s., not significant; RV, right ventricle.

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  Figure 4. MMI‐0100 treatment reduces periostin and α‐smooth muscle actin (αSMA) expression in cMyBP‐C40k hearts. All mice received 24 weeks of MMI‐0100 treatment and were 34 weeks old when samples were obtained. A, αSMA and (B) Periostin mRNA expression levels were normalized to GAPDH and then to the phosphate buffered saline (PBS)–treated, nontransgenic (Ntg) group. C, Periostin and αSMA protein was also detected by using Western blots of cardiac protein. D and E, Quantitation of the Western analyses, *P<0.05, **P<0.01, ***P<0.001. F, αSMA positive cells were detected via immunofluorescence. Arrowhead: nonperivascular αSMA‐positive cells. G, Upregulation of periostin was detected via immunofluorescence. Scale bar: 250 μm. Double transgenic (Dtg), 4′,6‐diamidino‐2‐phenylindole (DAPI, nuclear staining; blue). αSMA indicates α‐smooth muscle actin; postn, periostin; TnI, troponin I.

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  Figure 5. MMI‐0100 treatment inhibits αSMA‐driven luciferase (αSMA‐luc) activity and transforming growth factor β1 (TGFβ) induced cardiac fibroblast migration in vitro. A, Neonatal rat cardiac fibroblasts were transfected with an αSMA promoter‐driven luciferase reporter (αSMA‐luc) construct and a CMV driven renilla luciferase reporter was co‐transfected as a control. Luciferase activity was measured 24 hours after TGFβ treatment with or without MMI‐0100 pre‐treatment. B, Stress fibers were visualized by F‐actin staining using phalloidin. White arrow: stress fiber positive cells. Scale bar: 100 μm. C, The percentage of cells containing stress fibers was calculated. More than 200 cells/treatment were examined for each treated culture. D, Representative image of in vitro cell migration assay. Neonatal rat cardiac fibroblasts were plated, grown to confluence and the plate subsequently scratched. Images were taken at 0 and 24 hours. E, Cell layer migration distance was measured. **P<0.01, ***P<0.001.

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  Figure 6. MMI‐0100 treatment alleviates cardiac hypertrophy. Mice were 34 weeks old when samples were obtained. A, Ratios of heart weight (HW) to body weight (BW) of 34‐week‐old nontransgenic (Ntg) and double transgenic (Dtg) mice treated with phosphate buffered saline (PBS) or MMI‐0100. *P<0.05, **P<0.01. B, Wheat germ agglutinin (green) staining was used to determine the cross‐sectional area (CSA) of the cardiomyocytes. Scale bar=50 μm. C, Cardiomyocyte CSA. Averaged data from 500 cells per heart, 3 hearts per group. D, Natriuretic peptides A and B (Nppa and Nppb, respectively) mRNA expression normalized to GAPDH and then to Ntg treated with PBS set arbitrarily to a value of 1. n=3, *P<0.05.

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  Figure 7. Thirty‐week MMI‐0100 treatment alleviates cardiac hypertrophy but has no effect on cardiac hemodynamics. A through F, Echocardiographic analysis of Ntg and Dtg cMyBP‐C40k‐expressing mice after long‐term PBS or MMI‐0100 treatment. n=7 to 11. Left ventricular mass (LV mass), body weight (BW), interventricular septum thickness end diastole (IVSd), left ventricular posterior wall thickness end diastole (LVPWd), left ventricular internal diameter end diastole (LVIDd), ejection fraction (EF), fractional shortening (FS). Groups at each time point were subjected to statistical analyses to determine if any differences were present. *P<0.05, **P<0.01, ***P<0.001. Dtg indicates double transgenic; Ntg, nontransgenic; PBS, phosphate buffered saline.

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  Figure 8. MMI‐0100 treatment prolongs survival of cMyBP‐C40k mice. Survival curve of nontransgenic (Ntg) and double transgenic (Dtg) mice treated with phosphate buffered saline (PBS) or MMI‐0100.