Deletion of Nkx2-5 in trabecular myocardium reveals the developmental origins of pathological heterogeneity associated with ventricular non-compaction cardiomyopathy
Fig 2
Morphology of adult hypertrabeculation induced by conditional deletion of Nkx2-5 in cardiomyocytes during trabecular development.
(A) Short-axis cine images recorded by CMR at end-diastole show anatomical structures of LV of 3 months-old control (CTL), Nkx2-5ΔTrbE10 (ΔTrbE10) and Nkx2-5ΔTrbE14 (ΔTrbE14) mutant mice. Stars indicate the papillary muscles, and arrowheads indicate excessive trabeculations. RV: right ventricle; IVS: interventricular septum; LV: left ventricle. Scale bar = 1mm. (B) Eosin staining on transversal sections of the mid-ventricle from control, Nkx2-5ΔTrbE10 and Nkx2-5ΔTrbE14 adult hearts. The dotted lines delimit the boundary between trabecular and compact zones in LV. (C) Immunofluorescence with endoglin, SMA and VEGFR2 antibodies to delineate endocardium (Enghigh/VEGFR2-) and vascular endothelial cells (Englow/VEGFR2+) on transversal sections of the mid-ventricle from control, Nkx2-5ΔTrbE10, and Nkx2-5ΔTrbE14 adult hearts. Scale bar = 500μm. On the right panel, high magnifications of the left ventricular lumen show the numerous endocardial islets detected in Nkx2-5ΔTrbE10 and Nkx2-5ΔTrbE14 mutants (arrows). Scale bar = 100μm. (D) Perfusion of WGA-Cy3 lectin in the ventricle of control and Nkx2-5ΔTrbE10 hearts. (E) Immunofluorescence with endoglin, VEGFR2 and perfused WGA to detect the endocardium. Scale bar = 1mm. Below, high magnification of Eng+/VEGFR2- endocardial islets stained with perfused WGA-Cy3 (arrows). Scale bar = 100μm. (F-G) Quantification of the degree of excessive trabeculations by measuring the total length of the endocardium layer. These measures were performed for 3 sections at the level of the apex and at mid-ventricle from control (N = 3), Nkx2-5ΔTrbE10 (** p < 0,005, N = 3) and Nkx2-5ΔTrbE14 (*p < 0,05, N = 3) adult hearts (F), and counting the number of endocardial islets per section (G) (**p < 0,002, N = 3 for Nkx2-5ΔTrbE10 vs control and $ p < 0,02, N = 3 vs Nkx2-5ΔTrbE14).