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. 2023 Oct 27;148(23):1870–1886. doi: 10.1161/CIRCULATIONAHA.123.064332

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© 2023 The Authors.

Circulation is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDerivs License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made.

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Figure 6. — MDM2 regulates capillary formation in Myh6^R404Q/WT mice before the development of ventricular hypertrophy. A, Representative hematoxylin-eosin–stained heart cross-sections of postnatal days 25 (P25) and 60 (P60) WT and Myh6^R404Q/WT mice. Scale bars=1 mm. Echocardiography assessment of (B) interventricular septal thickness at end diastole (IVSd) and (C) left ventricular posterior wall thickness at end diastole (LVPWd) in WT (n=10–11) and Myh6^R404Q/WT (n=19–24) mice at P25, P60, or postnatal day 180 (P180). D, Representative images of WGA (red)–stained left ventricular tissue from WT and Myh6^R404Q/WT mice at P25 or P60. Scale bar=75 μm. E, Cardiomyocyte cross-sectional areas from WGA-stained left ventricular tissue from WT (n=5–6) and Myh6^R404Q/WT (n=5–6) mice at postnatal day 7 (P7), P25, or P60. Minimum 50 cardiomyocytes per sample. F, Representative immunohistochemistry images for CD31 (green) costained with WGA (red) in left ventricular tissue from P7 WT and Myh6^R404Q/WT mice. Nuclei are blue (DAPI). Scale bars=50 μm. G, Capillary-to-cardiomyocyte ratios in left ventricular tissue from WT (n=5–6) and Myh6^R404Q/WT (n=5–6) mice at postnatal day 2, P7, or P25. Minimum 200 cardiomyocytes per sample. H, Representative fluorescence images for the intravascularly injected endothelial cell stain T-lectin (green) in left ventricular tissue from P7 WT and Myh6^R404Q/WT mice. Scale bars=80 μm. I, Capillaries per mm² in left ventricular tissue from P7 WT (n=6) and Myh6^R404Q/WT (n=6) mice; 3 cross-sectional images per sample were analyzed. J through M, Immunoblots and quantification for MDM2, HIF1α, and HIF2α in left ventricular tissue lysates from P7 WT (n=6–9) and Myh6^R404Q/WT (n=6–9) mice normalized to β-actin and relative to WT. N, Capillary-to-cardiomyocyte ratios were calculated from left ventricular tissue in P7 WT (n=6), Mdm2^fl/+/Myh6:Cre (n=6), Myh6^R404Q/WT (n=6), and Myh6^R404Q/WTMdm2^fl/+/Myh6:Cre (n=6) mice. Minimum 200 cardiomyocytes per sample. O, Coronary flow reserve in P25 WT (n=5), Mdm2^fl/+/Myh6:Cre (n=5), Myh6^R404Q/WT (n=6), and Myh6^R404Q/WTMdm2^fl/+/Myh6:Cre (n=5) mice. All results are shown as mean±SEM. Student or Welch t test used for I, K, L, and M; 2-way ANOVA with Tukey multiple comparison test used for B, C, E, G, N, and O. CD31 indicates cluster of differentiation 31; DAPI, 4′,6-diamidino-2-phenylindole; HIF1α, hypoxia-inducible factor 1 alpha; HIF2α, hypoxia-inducible factor 2 alpha; MDM2, murine double minute 2; Mdm2^fl/+Cre, Mdm2 heterozygous floxed and Myh6:Cre; Mybpc3^-/-_, cardiac myosin binding protein 3 homozygous deletion; Myh6:Cre, myosin heavy chain 6:Cre recombinase; Myh6^R404Q/WT, myosin heavy chain 6 arginine to glutamine substitution at amino acid 404 heterozygous; T-lectin, tomato lectin; WGA, wheat germ agglutinin; and WT, wild-type.