Recovery of left ventricular function following in vivo reexpression of cardiac myosin binding protein C

Figures

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  Figure 1.. Generation of Tet-Off inducible mice expressing WT cMyBP-C. (A) Mice, hemizygous for the tTA transgene (Sanbe et al., 2003) and homozygous for endogenous cMyBP-C (tTA+/0, cMyBP-C+/+), were backcrossed for five successive generations with cMyBP-C null (cMyBP-C−/−) mice to generate the tTA mouse line (tTA+/0, cMyBP-C−/−) used in this study. The same strategy was used to generate the tResp transgenic mouse line on the cMyBP-C null background (tResp+/0, cMyBP-C−/−). Neither the tTA mice nor the tResp mice expressed cMyBP-C (bottom). (B) The tTA and tResp lines were subsequently crossed to generate the double-transgenic mouse (FT-WT). Approximately 25% of the resulting progeny were hemizygous for both transgenes (tTA+/0, tResp+/0, cMyBP-C−/−) and expressed WT cMyBP-C (bottom). (C) PCR genotyping strategy used to generate FT-WT mice. Shown are PCR products amplified from genomic DNA isolated from WT (cMyBP-C+/+), cMyBP-C null (cMyBP-C−/−), tTA (tTA+/0, cMyBP-C−/−), tResp (tResp+/0, cMyBP-C−/−), and FT-WT (tTA+/0, tResp+/0, cMyBP-C−/−) mice.

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  Figure 2.. Selection of the inducible FT-WT mouse line. (A–D) All values represent means, and error bars represent ±SEM, with n = 3 animals per group. Echocardiographic analysis of LV EF (A), ET (B), IVRT (C), and relative expression of cMyBP-C (D) in WT, cMyBP-C null, and FT-WT (sublines 2252, 2254, and 2267) mice. *, P < 0.05 significant difference of FT-WT and cMyBP-C null mice versus WT mice.

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  Figure 3.. Gene expression in FT-WT hearts. (A–D) The relative expression of MYBPC3 (A), atrial natriuretic peptide (B), and brain natriuretic peptide mRNA (C) was normalized to β-actin (D) in FT-WT, WT and cMyBP-C null hearts. All values represent means, and error bars represent SEM, with values obtained in triplicate from three hearts per group. *, P < 0.05 significant difference of FT-WT and cMyBP-C null mice versus WT mice.

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  Figure 4.. FT-WT mice exhibit normal cardiac histology and correct sarcomeric incorporation of cMyBP-C. Representative high-magnification (×400) photomicrographs of hematoxylin and eosin (H&E)–stained sections (top row) and Masson’s trichrome (MTC)–stained sections (middle row), demonstrating the absence of overt fibrosis in the FT-WT myocardium. Representative confocal images (IF; bottom row) of both WT and FT-WT cardiac sections demonstrate the classic A-band doublet staining of cMyBP-C.

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  Figure 5.. Steady-state force development in FT-WT skinned myocardium. (A and B) Ca2+-activated force–pCa relationship (A) and Hill plot transformations of the force–pCa data (B) were measured in skinned trabeculae from WT (triangle), FT-WT (circle), and cMyBP-C null (square) mice. All values represent means, and error bars represent ±SEM, with summary data listed in Table 1.

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  Figure 6.. FT-WT skinned trabeculae exhibit normal cross-bridge cycling kinetics. (A and B) The Ca2+-dependence (A) and activation-dependence (B) of the rate of force redevelopment following rapid release and restretch were measured in skinned trabeculae from WT (triangle), FT-WT (circle), and cMyBP-C null (square) mice. All values represent mean, and error bars represent ±SEM.

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  Figure 7.. Reversible expression of cMyBP-C in FT-WT myocardium. (A) Representative Western blot of myofibrils isolated from WT, cMyBP-C null, and FT-WT myocardium: noninduced (FT-WT0), during 4 wk of dietary doxycycline supplementation (FT-WT4), and following withdrawal of doxycycline (FT-WT20). (B) Densitometric analysis of the expression of cMyBP-C relative to β-actin (intensity ratio) and normalized to WT values. All values represent mean, and error bars represent ±SEM, with n = 8 hearts per group. *, P < 0.05 significant difference compared with WT mice at baseline.

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  Figure 8.. Analysis of in vivo LV function of FT-WT mice during partial knockdown and subsequent reexpression of cMyBP-C. (A–D) Time-course echocardiographic analysis of EF (A), ET (B), IVRT (C), and LV internal dimension (D) during diastole in WT (triangle, n = 12), cMyBP-C null (square, n = 10), and FT-WT (closed circle, control diet; open circle, doxycycline [Dox] diet, n = 8) mice. All values represent means, and error bars represent ±SEM. *, P < 0.05 significant difference compared with noninduced FT-WT mice at baseline (t = 0), P < 0.05.