Figure 2. Cardiomyocyte-specific knockout of RBPJ increases myocardial angiogenesis.

(a–c) Microvessel density quantification (a) on histological sections (b,c). Experimental genotypes are cKO (Myl2^Cre/+, Rbpj^flox/flox) and WT (Myl2^+/+, Rbpj^flox/flox) sham (−) or TAC (T), and iNICD (Myh6^rtTA, tetO-NICD) with (+) or without (−) doxycycline (DOX). n=4, mice for all conditions except WT/+TAC were n=3. Scale bar, 20 μm. (d,e) Electron microscopy of WT (d) and cKO (e) capillaries at 48 months. Scale bar, 2 μm (f,g) Perfusion analysis by ILB4 and CD31 co-localization after FITC-ILB4 injection. n=3 WT, and 5 cKO mice. Scale bar, 50 μm. (h–k) Co-localization between fibrinogen (green) and CD31 (red) in WT (h), cKO (i) and control WT-infarcted heart (MI) reflecting capillary integrity. (k) n=3. Scale bar, 20 μm. (l) Gene expression analysis of 29 secreted angiogenic factors and 3 Notch target genes in ventricles from cKO, WT and doxycycline-induced iNICD mice by qRT-PCR normalized to ActB. Volcano plots portray the cKO to WT ratio or iNICD to WT ratio relative to P-value. Red and green points indicate statistically significant (P≤0.05, n=4) induction or repression of gene expression, respectively; blue points indicate no statistically significant difference. (m,n) VEGFA₁₆₅ protein quantification by ELISA in WT, cKO and iNICD heart tissue (m) and conditioned media from WT and cKO-cultured ACM n=4 (n). (o) Conditioned media from WT and cKO ACM were applied to HMEC1 cells and network formation quantified incubation in the absence or presence of the VEGF receptor inhibitor carbozantinib (2 μM). n=3. (p) Histone marks in the endogenous Vegfa locus. ChIP with control IgG (white bars) and anti-H3K9/18Ac or anti-H3K4me³ (black bars) from isolated ACM was amplified using primers spanning RBPJ and HIF consensus binding motifs (schematic) and results normalized to input DNA. n=3. (q) Repression of Vegfa promoter-luciferase (50 ng) activity by RBPJ (1 ng) and reversion by co-transfection with escalating doses (0.5, 1 and 10 ng) of the SHARP RBPJ-binding domain (2002-3664) (ref. ¹⁸). n=3. (r) Loss of RBPJ leads to acquisition of active chromatin modifications and induction of Vegfa transcription. Data are mean±SEM *P<0.05; ns, not significant; n, biological replicates.