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

Abstract

The loss of cardiac myosin binding protein C (cMyBP-C) results in left ventricular dilation, cardiac hypertrophy, and impaired ventricular function in both constitutive and conditional cMyBP-C knockout (MYBPC3 null) mice. It remains unclear whether the structural and functional phenotypes expressed in the MYBPC3 null mouse are reversible, which is an important question, since reduced expression of cMyBP-C is an important cause of hypertrophic cardiomyopathy in humans. To investigate this question, we generated a cardiac-specific transgenic mouse model using a Tet-Off inducible system to permit the controlled expression of WT cMyBP-C on the MYBPC3 null background. Functional Tet-Off mice expressing WT cMyBP-C (FT-WT) were generated by crossing tetracycline transactivator mice with responder mice carrying the WT cMyBP-C transgene. Prior to dietary doxycycline administration, cMyBP-C was expressed at normal levels in FT-WT myocardium, which exhibited similar levels of steady-state force and in vivo left ventricular function as WT mice. Introduction of dietary doxycycline for four weeks resulted in a partial knockdown of cMyBP-C expression and commensurate impairment of systolic and diastolic function to levels approaching those observed in MYBPC 3 null mice. Subsequent withdrawal of doxycycline from the diet resulted in the reexpression of cMyBP-C to levels comparable to those observed in WT mice, along with near-complete recovery of in vivo ventricular function. These results show that the cardiac phenotypes associated with MYBPC3 null mice are reversible. Our work also validates the use of the Tet-Off inducible system as a means to study the mechanisms underlying hypertrophic cardiomyopathy.

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.

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.

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.

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.

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.

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.

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.

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.