Overexpression of bone morphogenetic protein 10 in myocardium disrupts cardiac postnatal hypertrophic growth

Abstract

Postnatal cardiac hypertrophies have traditionally been classified into physiological or pathological hypertrophies. Both of them are induced by hemodynamic load. Cardiac postnatal hypertrophic growth is regarded as a part of the cardiac maturation process that is independent of the cardiac working load. However, the functional significance of this biological event has not been determined, mainly because of the difficulty in creating an experimental condition for testing the growth potential of functioning heart in the absence of hemodynamic load. Recently, we generated a novel transgenic mouse model (alphaMHC-BMP10) in which the cardiac-specific growth factor bone morphogenetic protein 10 (BMP10) is overexpressed in postnatal myocardium. These alphaMHC-BMP10 mice appear to have normal cardiogenesis throughout embryogenesis, but develop to smaller hearts within 6 weeks after birth. alphaMHC-BMP10 hearts are about half the normal size with 100% penetrance. Detailed morphometric analysis of cardiomyocytes clearly indicated that the compromised cardiac growth in alphaMHC-BMP10 mice was solely because of defect in cardiomyocyte postnatal hypertrophic growth. Physiological analysis further demonstrated that the responses of these hearts to both physiological (e.g. exercise-induced hypertrophy) and pathological hypertrophic stimuli remain normal. In addition, the alphaMHC-BMP10 mice develop subaortic narrowing and concentric myocardial thickening without obstruction by four weeks of age. Systematic analysis of potential intracellular pathways further suggested a novel genetic pathway regulating this previously undefined cardiac postnatal hypertrophic growth event. This is the first demonstration that cardiac postnatal hypertrophic growth can be specifically modified genetically and dissected out from physiological and pathological hypertrophies.

Fig. 1

Generation of αMHC-BMP10 transgenic mice. (A) Schematic diagram of construct. (B) Semi-quantitative RT-PCR comparison of the level of transgenic BMP10 and total BMP10 (transgenic and endogenous) transcripts in developing hearts and postnatal heart (a). PCR primers (p1-p4) are indicated in (A). (b) Using qRT-PCR to determine the total BMP10 expression level in the hearts further confirmed the transient expression of transgenic BMP10 at embryonic stage. The y axis indicates relative expression levels as normalized to Rpl7 transcripts. (C) In situ hybridization (C, a and C, b, blue staining indicates expression) and qRT-PCR (C, c) analyses to determine the spatial distribution of BMP10 expression in adult hearts. (D) αMHC-BMP10 transgenic mice have normal cardiac development. RA, right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle, NTG, nontransgenic control; TG, αMHC-BMP10 transgenic mice.

Fig. 2

Characterization of αMHC-BMP10 transgenic mice. (A) Comparison of the gross morphology of αMHC-BMP10 hearts and littermate controls at different ages. (B) Quantitative comparison of body weight (B, a), heart weight (B, b), and heart weight vs body weight ratio (B, c) between αMHC-BMP10 transgenic mice and sex matched littermate controls. (C) Analysis of the size of cardiomyocytes and the total number of cardiomyocytes in αMHC-BMP10 transgenic hearts (3 month old); C, a, cells were stained with Hoechst to visualize nuclei, and the cell image was photographed via phase microscopy and pseudocolored green to visualize the myocyte cytoplasm, and parameters for cell size were measured using ImagePro software; C, b, total cell count of cardiomyocytes in adult hearts (3 month old).

Fig. 2

Characterization of αMHC-BMP10 transgenic mice. (A) Comparison of the gross morphology of αMHC-BMP10 hearts and littermate controls at different ages. (B) Quantitative comparison of body weight (B, a), heart weight (B, b), and heart weight vs body weight ratio (B, c) between αMHC-BMP10 transgenic mice and sex matched littermate controls. (C) Analysis of the size of cardiomyocytes and the total number of cardiomyocytes in αMHC-BMP10 transgenic hearts (3 month old); C, a, cells were stained with Hoechst to visualize nuclei, and the cell image was photographed via phase microscopy and pseudocolored green to visualize the myocyte cytoplasm, and parameters for cell size were measured using ImagePro software; C, b, total cell count of cardiomyocytes in adult hearts (3 month old).

Fig. 3

Testing cardiac response of αMHC-BMP10 mice to hypertrophic stimuli. (A) Gross morphology (a) and quantitative comparison of heart/body weight ratio (b) of hearts from mice without (control) and with chronic swimming exercise (Sw). (B) Western blot analysis of activated Akt level in basal (non-exercised), exercised, and insulin-induced ventricular tissues of non-transgenic and αMHC-BMP10 mice. Akt activation is not altered in αMHC-BMP10 heart. (C) Gross morphology (a) and quantitative comparison of heart/body weight ratio (b) of hearts from mice without (control) and with isoproterenol treatment (Iso). (D) Comparison of the size of dispersed cardiomyocytes of non-treated control, swimming exercised, and isoproterenol treated mice. Similar degree of hypertrophic response was observed in nontransgenic and αMHC-BMP10 cardiomyocytes.

Fig. 3

Testing cardiac response of αMHC-BMP10 mice to hypertrophic stimuli. (A) Gross morphology (a) and quantitative comparison of heart/body weight ratio (b) of hearts from mice without (control) and with chronic swimming exercise (Sw). (B) Western blot analysis of activated Akt level in basal (non-exercised), exercised, and insulin-induced ventricular tissues of non-transgenic and αMHC-BMP10 mice. Akt activation is not altered in αMHC-BMP10 heart. (C) Gross morphology (a) and quantitative comparison of heart/body weight ratio (b) of hearts from mice without (control) and with isoproterenol treatment (Iso). (D) Comparison of the size of dispersed cardiomyocytes of non-treated control, swimming exercised, and isoproterenol treated mice. Similar degree of hypertrophic response was observed in nontransgenic and αMHC-BMP10 cardiomyocytes.

Fig. 4

Pathological and functional analyses of αMHC-BMP10 mice. (A) H&E (a and b) for regular cardiac histology and Masson’s trichrome staining for potential cardiac fibrosis (c) of histological sections of αMHC-BMP10 and nontransgenic hearts at different ages. Significant reduction in ventricular chamber, thickening of ventricular wall, and narrowing in sub-aortic region (black arrows) is found in adult αMHC-BMP10 heart (b and c). However, there is no extensive fibrosis found in αMHC-BMP10 hearts at all ages examined (c). (B) Echocardiograph analysis of non-transgenic and αMHC-BMP10 mice (3 month old male). Representative 2-dimensional and M-mode images are in panel-a and panel-b. Measurement of various parameters and statistic analysis are summarized in panel-c. (C) qRT-PCR analysis of representative cardiac markers on non-transgenic and αMHC-BMP10 hearts (4 week old). The y-axis indicates relative expression levels as normalized to Rpl7 transcripts

Fig. 4

Pathological and functional analyses of αMHC-BMP10 mice. (A) H&E (a and b) for regular cardiac histology and Masson’s trichrome staining for potential cardiac fibrosis (c) of histological sections of αMHC-BMP10 and nontransgenic hearts at different ages. Significant reduction in ventricular chamber, thickening of ventricular wall, and narrowing in sub-aortic region (black arrows) is found in adult αMHC-BMP10 heart (b and c). However, there is no extensive fibrosis found in αMHC-BMP10 hearts at all ages examined (c). (B) Echocardiograph analysis of non-transgenic and αMHC-BMP10 mice (3 month old male). Representative 2-dimensional and M-mode images are in panel-a and panel-b. Measurement of various parameters and statistic analysis are summarized in panel-c. (C) qRT-PCR analysis of representative cardiac markers on non-transgenic and αMHC-BMP10 hearts (4 week old). The y-axis indicates relative expression levels as normalized to Rpl7 transcripts

Fig. 5

qRT-PCR, immunohistochemical and Western blot analyses of potential signaling pathways involved in BMP10 signaling. (A) A representative PCR amplification/cycle graph for SYBR green fluorescence signals (a) and comparison of Alk3 and Alk6 in normal postnatal heart (2 week old) (b). Alk 3 mRNA level is over 1,000 fold higher than Alk 6 in the heart. (B) Immunohistochemical staining analysis of αMHC-BMP10 hearts (6 week old) using anti-pSmad1 antibody (note, this antibody also cross-reacts with pSmad5 and pSmad8) and anti-pSmad2 antibody (note this antibody also cross-reacts with pSmad3). Arrows point to positive staining signals. pSmad1/5/8 is dramatically activated in αMHC-BMP10 hearts. (C and D) Western blot analysis of Smads and TAK1-MAPK activation in αMHC-BMP10 hearts (1 month old). BMP10 specifically activates Smad1/5/8. Each lane represents different cardiac sample. Genotypes of these cardiac samples are as indicated in the figures.

Fig. 5

qRT-PCR, immunohistochemical and Western blot analyses of potential signaling pathways involved in BMP10 signaling. (A) A representative PCR amplification/cycle graph for SYBR green fluorescence signals (a) and comparison of Alk3 and Alk6 in normal postnatal heart (2 week old) (b). Alk 3 mRNA level is over 1,000 fold higher than Alk 6 in the heart. (B) Immunohistochemical staining analysis of αMHC-BMP10 hearts (6 week old) using anti-pSmad1 antibody (note, this antibody also cross-reacts with pSmad5 and pSmad8) and anti-pSmad2 antibody (note this antibody also cross-reacts with pSmad3). Arrows point to positive staining signals. pSmad1/5/8 is dramatically activated in αMHC-BMP10 hearts. (C and D) Western blot analysis of Smads and TAK1-MAPK activation in αMHC-BMP10 hearts (1 month old). BMP10 specifically activates Smad1/5/8. Each lane represents different cardiac sample. Genotypes of these cardiac samples are as indicated in the figures.