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. Author manuscript; available in PMC: 2021 May 19.
Published in final edited form as: Sci Transl Med. 2019 Oct 9;11(513):eaaw6419. doi: 10.1126/scitranslmed.aaw6419

Figure 2. Ect2 levels regulate cardiomyocyte cytokinesis and Ect2 gene inactivation lowers endowment and is lethal in mice.

Figure 2.

(A) Live cell imaging of neonatal rat cardiomyocytes (NRVM, P2-P3, 52 cardiomyocytes) shows that cleavage furrow regression precedes formation of binucleated cardiomyocytes, corresponding to Video S1. Cleavage furrow ingression is between 300–335 min, regression at 355 min, and formation of a binucleated cardiomyocyte at 510 min. (B) Transcriptional profiling of single cycling (+) and not cycling (−) cardiomyocytes at embryonic day 14.5 (E14.5) and 5 days after birth (P5) reveals that of 61 Dbl-homology family RhoGEF, Ect2 is significantly repressed in cycling P5 cardiomyocytes (P <0.05). (C) Binucleating cardiomyocytes exhibit lower RhoA activity (RhoA-GTP) at the cleavage furrow (E15.4: 59 midbodies; P2: n = 56 midbodies). (D-G) NRVM were transduced with Adv-CMV-GFP-Ect2. Live cell imaging shows appropriate and dynamic localization of GFP-ECT2 in cycling NRVM (D, corresponding to Video S4), reduced cytokinesis failure and reduced generation of binucleated cardiomyocytes. (F, G) Overexpression of GFP-Ect2 does not alter cardiomyocyte S- (F, GFP: n = 407; GFP-Ect2: n = 285) or M-phase (G, GFP: n = 432; GFP-Ect2: n = 443). (H-P) Ect2 gene inactivation in the αMHC-Cre; Ect2F/F mice at P1 (H-O) showed increased binucleated cardiomyocytes (H, Ect2F/wt n = 6, Ect2F/F n = 6 hearts) without change of DNA content per nucleus (I, Ect2F/wt n = 642 cardiomyocytes, Ect2F/F n = 647 cardiomyocytes), and a 50% lower cardiomyocyte endowment (J, Ect2F/wt n = 12, Ect2F/F n = 5 hearts). The reduced endowment triggers compensatory cardiomyocyte hypertrophy (K, Ect2F/wt n = 1,138 cardiomyocytes from 6 hearts, Ect2F/F n = 1,015 cardiomyocytes from 6 hearts), without change of heart weight (L, Ect2F/wt n = 14, Ect2F/F n = 6 hearts). (M, N) The lower cardiomyocyte endowment leads to myocardial dysfunction at P0 (M, left ventricular endocardium outlined in yellow, Ect2wt/wt n = 4, Ect2F/F n = 3 mice) and lethality before P2 (N, Video S5), but does not alter the M-phase (O, Ect2F/wt n = 6, Ect2F/F n = 6 hearts) and cell cycle entry (P, BrdU uptake, Ect2flox gene inactivation with αMHC-MerCreMer, tamoxifen DOL 0, 1, 2, followed by 3 days culture). Statistical significance was tested with Student’s t- test if not specified, and Fisher’s exact test (N). Scale bars 30 μm (E, P), 50 μm (F, G), 100 μm (K).