Cardiac stress, ventricle enlargement and increased cardiomyocyte number correlate with hypoxia in Endoglin-deficient fish. (A,B) RT-qPCR analysis of nppa and nppb (cardiac stress) (A), and egln3 and epoa (hypoxia) (B), in 5, 10, 15, 20, 25 and 30 dpf sibling and eng−/− fish (RT-PCR genotyping is presented below graphs). Target gene expression values were normalized to rpl13a. 5 dpf and 10 dpf, n=15; 15 dpf, n=8; 20 dpf, n=5; 25 dpf, n=4; 30 dpf, n=3. Data are mean±s.e.m. of technical replicates. nppa: 15 dpf, 20 dpf, 25 dpf and 30 dpf, *P=0.05. nppb: 15 dpf, *P=0.05; 20 dpf, *P=0.0383; 25 dpf and 30 dpf, *P=0.05. egln3: 15 dpf and 20 dpf, *P=0.05; 25 dpf, *P=0.03831; 30 dpf, *P=0.05. epoa: 15 dpf, 20 dpf, 25 dpf and 30 dpf, *P=0.05. ns, not significant (one-tailed Mann–Whitney test). dfp, days postfertilization. (C) Analysis of ventricle volume in 10, 12 and 15 dpf sibling and eng−/− fish in Tg(cmlc2:GFP) background (n=5). Ventricle volume is estimated by the simplified ellipsoid volume calculation formula V=0.523(width in mm)2(length in mm). *P=0.0159; ns, not significant (one-tailed Mann–Whitney test). (D) Flow cytometry analysis of cardiomyocyte (cmlc2:GFP+) fraction in whole organism at 5, 10, 15, 20 and 25 dpf. Cell suspensions at 5, 10, 15, 20 and 25 dpf were prepared from pools of 26, 18, nine, seven and seven eng−/− fish or siblings, respectively. Bottom graph represents fold change in cmlc2:GFP+ cells in eng−/− fish relative to siblings. Data are representative of three independent experiments.
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