A Neonatal Mouse Model for Pressure Overload: Myocardial Response Corresponds to Severity

Figures

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  Figure 1. Effect of different degrees of pressure overload on the growth of neonatal mice. (A,B) Both 30-gauge (30G) and 32-gauge (32G) TAC affected the growth and development of mice; Representative images of 32G TAC mice (up), 30G TAC mice (middle) and sham mice (down); 7dp1d TAC: 7 days post 1 day TAC; 14dp1d TAC: 14 days post 1 day TAC; P1: postnatal day 1; P8: postnatal day 8; P15: postnatal day 15; n (number of mice) = 10, P1 sham; n = 10, P1 30G TAC; n = 10, P1 32G TAC; n = 15, P8 sham; n = 15, P8 30G TAC; n = 16, P8 32G TAC; n = 16, P15 sham; n = 16, P15 30G TAC; n = 15, P15 32G TAC. (C) Kaplan–Meier survival curve of sham, 30G TAC, 32G TAC, and 34-gauge (34G) TAC groups; n = 22, sham; n = 23, 30G TAC; n = 25, 32G TAC; n = 21, 34G TAC. The survival rate of the four groups was assessed by Kaplan–Meier analysis and a log-rank test. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Statistical significance was calculated using the unpaired two-tailed t-test.

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  Figure 2. Middle pressure overload significantly impaired heart function, while mild pressure overload did not. (A,B) Representative M-mode echocardiographic and aortic arch images of mice in the sham, 7dp1d TAC, and 14dp1d TAC groups with 30G and 32G constriction. (C) Representative images of M-mode echocardiograph and aortic arch in sham and 21 days post 1 day TAC (21dp1d TAC) groups with 30G constriction. (D) Color Doppler mode images of aortic arch blood flow in sham and TAC groups after 7 days of surgery. The arrow points to the turbulent blood flow area. (E–I) Ejection fraction (EF), fractional shortening (FS), left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), and corrected left ventricular mass (Corr.LV mass) were assessed by echocardiography; n = 7, 7dp1d sham; n = 11, 7dp1d 30G TAC; n = 10, 7dp1d 32G TAC; n = 7, 14dp1d sham; n = 8, 14dp1d 30G TAC; n = 8, 14dp1d 32G TAC; n = 7, 21dp1d sham; n = 7, 21dp1d 30G TAC. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Statistical significance was calculated using the unpaired two-tailed t-test.

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  Figure 3. Cardiomyocyte proliferation increased in the hearts of mice in the 30G TAC group but not in the 32G TAC group. (A) Timeline of 5-bromo-2-deoxyuridine (BrdU) injection and detection. 1dTAC: TAC performed on postnatal day 1; 3dp1d TAC: 3 days post 1 day TAC. (B) The nascent cardiomyocytes were identified by BrdU (red), 4′,6-diamidino-2-phenylindole (DAPI, blue) and α-actinin (green) co-localization in the hearts of mice in the sham, 30G TAC, and 32G TAC groups 3 days after TAC. Scale bar: 20 μm; n = 3 hearts per group. Each heart slide was randomly selected with five high-power fields for statistical analysis. (C,D) Immunostaining for phospho-histone H3 (pH3, green) and cardiac troponin I (cTNI, red) co-localization and Ki67 (green) and cTNI (red) co-localization to recognize cardiomyocyte proliferation in the heart sections of mice in the sham, 30G TAC, and 32G TAC groups 3 days after TAC. Scale bar: 100 μm; n = 3 hearts per group. Each heart slide was randomly selected with five to seven high-power fields for statistical analysis. (E,F) Cardiomyocyte proliferation was recognized by immunostaining for pH3, Ki67, and cTnI in the heart sections of mice in the sham, 30G TAC, and 32G…

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  Figure 4. The effect of different constrictions on myocardial hypertrophy and angiogenesis. (A) Immunostaining for CD31 (red), wheat germ agglutinin (WGA, green), and DAPI in heart sections. Scale bar: 20 μm. (B) Quantification of CD31+ capillaries/field; n = 3 hearts per group. Each heart slide was random selected with five high-power fields for statistical analysis. (C) Analysis of cardiomyocyte cross-sectional area in the three groups; n = 3 hearts per group. A total of 300 cardiomyocytes per group were analyzed. (D,E) Immunostaining of heart slides for CD31 and quantification of capillary density. Scale bar: 20 μm; n = 3 hearts per group. Each heart slide was random selected with five to eight high-power fields for statistical analysis. Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001, and ****P < 0.0001. Statistical significance was calculated using the unpaired two-tailed t-test.

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  Figure 5. Apoptosis and hypertrophy increased in mice 7 days after the 30G and 32G TAC surgery. (A) The hearts of mice in the sham (left), 30G TAC (middle), and 32G TAC (right) groups. (B) The heart weight analysis in the three groups: n = 14, sham; n = 10, 30G TAC; n = 11, 32G TAC. (C) Representative heart sections stained with WGA (green) in the three groups. Scale bar: 20 μm. (D) Analysis of cardiomyocyte cross-sectional area in the three groups; n = 3 hearts per group. A total of 250-300 cardiomyocytes per group were analyzed. (E) Representative images of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL, green)-positive cells from the heart sections in the three groups. Scale bar: 100 μm. (F) Statistical chart of TUNEL-positive cells in the three groups; n = 3 hearts per group. Each heart slide was random selected with five high-power fields for statistical analysis. (G,H) Quantitative real-time polymerase chain reaction (qRT-PCR) for the expression of myosin heavy chain-β (MYH7) and atrial natriuretic peptide (ANP), respectively, in the ventricular tissue; n = 4, sham; n = 6, 30G TAC; n = 5, 32G TAC. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, and ****P…

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  Figure 6. Apoptosis and hypertrophy increased in mice 14 days after the 30G and 32G TAC. (A) Hearts of mice in the sham (left), 30G TAC (middle), and 32G TAC (right) groups. (B) The heart weight analysis in the three groups: n = 12, sham; n = 15, 30G TAC; n = 10, 32G TAC. (C) Representative heart sections stained with WGA (green) in the three groups. Scale bar: 20 μm. (D) Analysis of cardiomyocyte cross-sectional area in the three groups; n = 3 hearts per group. A total of 300 cardiomyocytes per group were analyzed. (E–H) Expression of ANP, brain natriuretic peptide (BNP), Acta1 and four and a half LIM domains 1 (Fhl1) in the ventricular tissue; n = 6, sham; n = 6, 30G TAC; n = 6, 32G TAC. (I) Representative images of TUNEL-positive cells from heart sections in the three groups. Scale bar: 50 μm. (J) Statistical chart of TUNEL-positive cells in the three groups; n = 3 hearts per group. Each heart slide was random selected with three high-power fields for statistical analysis. (K) Representative transmission electron microscopy (×5,000) images revealed normal sham heart (left), hypertrophic 30G TAC heart (middle), and 32G TAC heart (right) with hypotrophy and mitochondrial swelling. M,…

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  Figure 7. Significantly increased fibrosis in the hearts of mice in the 32G TAC group 7 days after the surgery. (A) Masson staining of sham, 30G TAC, and 32G TAC mouse hearts. Scale bar: 500 μm. (B–D) Representative Masson trichrome staining in the hearts of mice in the sham (B), 30G TAC (C), and 32G TAC (D) groups. The middle images are high-power fields of the corresponding left images (black square frame), showing interstitial fibrosis. The right images are high-power fields of the corresponding left images (green square frame) showing perivascular fibrosis. Scale bar: 500 μm (left images); scale bar: 50 μm (middle and right images). (E) Interstitial fibrosis in the three groups; n = 3 hearts per group. Each heart slide was random selected with five high-power fields for statistical analysis. (F–I) qRT-PCR for the expression of matrix metallopeptidase 9 (MMP9), tissue inhibitor of metalloproteinases 1(Timp1), fibronectin 1(Fn1), and collagen type I alpha 1 chain (COL1A1) in the ventricular tissue; n = 4, sham; n = 6, 30G TAC; n = 5, 32G TAC. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001. Statistical significance was calculated using unpaired two-tailed…

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  Figure 8. Hearts of mice in the 32G TAC group had more fibrosis markers compared with those in the sham and 30G TAC groups. (A–C) Representative Masson staining of hearts of mice in the sham (A), 30G TAC (B), and 32G TAC (C) groups. The middle images are high-power fields of the corresponding left images (black square frame), showing interstitial fibrosis. The right images are high-power fields of the corresponding left images, showing perivascular fibrosis (green square frame, up) and endomyocardial fibrosis (blue square frame, down). Scale bar: 100 μm (left images); scale bar: 50 μm (middle and right images). (D) Interstitial fibrosis in the three groups; n = 3 hearts per group. Each heart slide was random selected with five high-power fields for statistical analysis. (E,F) qRT-PCR for the expression of Fn1 and Timp1 in the ventricular tissue; n = 6, sham; n = 6, 30G TAC; n = 6, 32G TAC. (G) Western blot stripes from three independent hearts in the three groups are shown. (H–L) Quantification of protein expression of TGF-β/β-tubulin (H), p-Smad3/Smad3 (I), MMP-9/β-tubulin (J), COL1A1/β-tubulin (K), and COL3/β-tubulin (L) in the hearts of mice in the sham, 30G TAC, and 32G TAC groups 14…

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  Figure 9. Graphical diagram of the consequences of different pressure overloads in neonatal mice. Cardiomyocytes were effectively stimulated to proliferate under mild pressure overload with a reserved heart function. Moderate heart pressure overload caused cardiomyocyte hypertrophy and fibrosis, further influencing cardiomyocyte proliferation and heart function. The mice barely survived after severe pressure overload.