Expression of Normally Repressed Myosin Heavy Chain 7b in the Mammalian Heart Induces Dilated Cardiomyopathy

Figures

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  Figure 1. There is no baseline cardiac phenotype in young global‐MYH7b null mice. A, Left ventricular weights normalized to tibia length (LV/TL mg/mm) show no gross morphometric differences between WT and global‐MYH7b null (MYH7b null) in either male or female mice at 1.5 months of age. Animal numbers: male WT n=8, male MYH7b null n=20, female WT n=16, female MYH7b null n=12. B, Cardiac contractility as measured by percentage of fractional shortening (%FS) shows cardiac contractility is unaffected in MYH7b null males and slightly enhanced in MYH7b null females. Animal numbers are the same as reported in (A). C, Gene expression profiling by reverse transcriptase quantitative polymerase chain reaction (RT‐qPCR) shows no increases in markers of cardiac pathology in MYH7b null left ventricular tissue. %FS indicates percentage of fractional shortening; LV indicates left ventricle; NTG, non‐transgenic; TL, tibial length; WT, wild type. *P value of ≤0.05 by Student t‐test. Animal numbers: male WT, n=8; male MYH7b null, n=10; female WT, n=10; female MYH7b null, n=9.

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  Figure 2. MYH7b is expressed in mouse hearts and is incorporated into sarcomeres. A, Myosin separating gel showing transgenic protein expression from 2 separate female mice (2 months of age) for both NTG and α‐MYH7b transgenic mice. Transgene expression was ≈30% of the total myosin in the α‐MYH7b LV compared with NTG controls. B, Western blot analysis of myofibril isolation preps from both male and female 1.5‐month‐old mice showing exogenously expressed MYH7b (Myc‐tag, upper blot) is incorporated into intact myofibrils to the same extent as endogenously expressed myosin (Anti‐Sarcomeric MyHC, lower blot). C and D, Quantitation of both exogenous and endogenous myosin incorporation into intact myofibrils. Results are shown as proportion of each myosin relative to the total myosin in each fraction. E, (a) Immunofluorescence image of a transverse cross section of the LV and right ventricle (RV) showing abundant and widespread distribution of the MYH7b protein; scale bar=1 mm. (b) Higher magnification figure showing incorporation of MYH7b into sarcomeres as evidenced by the striated pattern. Inset box shows higher magnification of the sarcomere; scale bar=20 μm. Samples were stained using an…

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  Figure 3. MYH7b expression results in morphometric changes in 1.5‐month‐old mice. A, Morphological measurements comparing LV, RV, and atrial weights normalized to tibial length in male and female NTG and α‐MHY7b mice at 1.5 months of age. B, Gross morphological images of ventricular cross sections of male and female NTG and α‐MYH7b transgenic mice confirm the presence of cardiac dilation in male α‐MHY7b mice. C and D, Echocardiographic profiles of young 1.5‐month‐old male and female mice show no significant functional changes (%FS) but early cardiac dilation (LV Vol(d) μL) in male α‐MYH7b mice. E, Gene expression analysis showing increased expression of fetal genes in both male and female α‐MYH7b transgenic mice at 1.5 months. %FS indicates percentage of fractional shortening; LV indicates left ventricle; NTG, non‐transgenic; TL, tibial length. *P≤0.05; **P≤0.01, ***P≤0.001, ****P≤0.0001. Animal numbers: male NTG, n=13; male α‐MYH7b, n=17; female NTG, n=7; female α‐MYH7b, n=21.

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  Figure 4. MYH7b expression results in progressive cardiac dilation and LV dysfunction. A, Morphological measurements comparing LV, RV, and atrial weights normalized to tibial length (TL) in male and female NTG and α‐MHY7b mice. B, Gross morphological images of ventricular cross sections of male and female NTG and α‐MYH7b mice, confirming presence of cardiac dilation in both sexes by 18 months of age. Echocardiographic profiles of 18‐month‐old male and female mice showing (C) significant functional changes (%FS) and (D) cardiac dilation (diastolic left ventricular volume [LV Vol(d)] μL) in both male and female α‐MYH7b mice. %FS indicates percentage of fractional shortening; LV indicates left ventricle; NTG, non‐transgenic; RV, right ventricle; TL, tibial length. *P≤0.05; **P≤0.01, ***P≤0.001, ****P=0.0001. Animal numbers: male NTG n=12, male α‐MYH7b n=6, female NTG n=10, female α‐MYH7b n=17.

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  Figure 5. Contractile function in isolated myocytes from 1.5‐month‐old mice. A, Percentage of sarcomere shortening at frequencies ranging from 0.2 to 4 Hz in isolated myocytes from both male and female NTG and α‐MYH7b mice. B, Time from stimulus to peak contraction and (C) time from peak to 90% relaxation at frequencies ranging from 0.2 to 4 Hz in isolated myocytes from both male and female NTG and α‐MYH7b transgenic mice. D, Sarcomere length (in μm) at frequencies ranging from 0.2 to 4 Hz in isolated myocytes from both male and female NTG and α‐MYH7b mice. All measurements were performed at steady‐state. Data reported as mean±SD from 16 cardiomyocytes from 4 NTG male mice (blue curves), 21 cardiomyocytes from 5 α‐MYH7b male mice (light blue curves), 28 cardiomyocytes from 5 NTG female mice (red curves), and 19 cardiomyocytes from 5 α‐MYH7b female mice (pink curves). Data were analyzed using 2‐way ANOVA and differences between genotypes are reported in the figure (***P≤0.001, ****P≤0.0001). NTG indicates non‐transgenic.