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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 3. — The noncanonical degradation of HIF1α in the Mybpc3^-/- myocardium is regulated by cardiomyocyte MDM2. A, Hif1α gene expression in left ventricular tissue RNA from postnatal day 7 (P7) WT (n=6) and Mybpc3^−/− (n=6) mice. Hif1α gene expression was normalized to Rpl32 and fold change relative to WT. B and C, Immunoblots and protein quantification for HIF1α in left ventricular tissue lysates from P7 WT (n=6), Mybpc3^−/− (n=6), and BTZ-injected Mybpc3^−/− (n=6) mice normalized to β-actin and relative to WT. D and E, Representative in situ proximity ligation assay images and quantification for Ub-modified HIF1α in left ventricular tissue from BTZ injected P7 WT (n=4) and Mybpc3^−/− (n=4) mice. HIF1α–Ub complexes are red and nuclei are blue (DAPI). Three nonoverlapping left ventricular images per sample; scale bars=25 μm. F and G, Immunoblot and quantification for VHL in left ventricular tissue lysate from P7 WT (n=7) and Mybpc3^−/− (n=7) mice normalized to beta-actin and relative to WT. H and I, Representative in situ proximity ligation assay images and quantification for HIF1α and VHL protein complexes in left ventricular tissue from P7 WT (n=4) and Mybpc3^−/− (n=4) mice. HIF1α–VHL complexes are shown in red and nuclei are blue (DAPI); 3 nonoverlapping left ventricular images per sample; scale bars=25 μm. J and K, Immunoblot and quantification for MDM2 in left ventricular tissue from P7 WT (n=9) and Mybpc3^−/− (n=8) mice normalized to β-actin and relative to WT. L, Schematic of cardiomyocyte selective reduction of MDM2 in Mybpc3^-/- mice generated by crossing Mdm2^fl/fl, Mybpc3^-/-, and Myh6:Cre mouse lines to create Mybpc3^−/−Mdm2^fl/+/Myh6:Cre. M, Immunoblot for MDM2, HIF1α, HIF2α, MYPBC3, and β-actin in left ventricular tissue from P7 WT, Mybpc3^−/−, Mybpc3^-/-/Myh6:Cre, and Mybpc3^−/−Mdm2^fl/+/Myh6:Cre mice. N and O, Representative in situ proximity ligation assay images and quantification for MDM2 and HIF1α protein complexes in left ventricular tissue from P7 WT (n=4), Mybpc3^−/− (n=4), and Mybpc3^−/−Mdm2^fl/+/Myh6:Cre (n=4) mice. MDM2–HIF1α complexes are shown in red and nuclei are blue (DAPI). Three nonoverlapping left ventricular images per sample; scale bars=25 μm. P and Q, Representative in situ proximity ligation assay images and quantification for Ub-modified HIF1α in left ventricular tissue from BTZ-injected P7 WT (n=4), Mybpc3^−/− (n=4), and Mybpc3^−/−Mdm2^fl/+/Myh6:Cre (n=4) mice. HIF1α–Ub complexes are red and nuclei are blue (DAPI). Three nonoverlapping left ventricular images per sample; scale bars=25 μm. R, Immunoprecipitation (IP) for HIF1α was performed on left ventricular tissue lysates from BTZ-injected P7 WT, Mybpc3^−/− and Mybpc3^−/−Mdm2^fl/+/Myh6:Cre mice and then immunoblots were performed for K48-linked Ub and HIF1α. The input left ventricular tissue lysates also underwent immunoblotting for HIF1α and β-actin. All results are shown as mean±SEM. Student t test was used for A, E, G, I, and K; 1-way ANOVA with Tukey or Dunnett T3 multiple comparison test were used for C, O, and Q. BTZ indicates bortezomib; DAPI, 4′,6-diamidino-2-phenylindole; HC, heavy chain; HIF1α, hypoxia-inducible factor 1 alpha; HIF2α, hypoxia-inducible factor 2 alpha; IB, immunoblot; LC, light chain; MDM2, murine double minute 2; Mdm2^fl/+, Mdm2 heterozygous floxed; Mybpc3^-/-_, cardiac myosin binding protein 3 homozygous deletion; Myh6:Cre, myosin heavy chain 6:Cre recombinase; Poly-Ub, K48-linked ubiquitin; Rpl32; ribosomal protein L32; Ub, Ubiquitin; VHL, Von Hippel-Lindau; and WT, wild-type.