Identification of a hybrid myocardial zone in the mammalian heart after birth

Abstract

Noncompaction cardiomyopathy is characterized by the presence of extensive trabeculations, which could lead to heart failure and malignant arrhythmias. How trabeculations resolve to form compact myocardium is poorly understood. Elucidation of this process is critical to understanding the pathophysiology of noncompaction disease. Here we use genetic lineage tracing to mark the Nppa⁺ or Hey2⁺ cardiomyocytes as trabecular and compact components of the ventricular wall. We find that Nppa⁺ and Hey2⁺ cardiomyocytes, respectively, from the endocardial and epicardial zones of the ventricular wall postnatally. Interposed between these two postnatal layers is a hybrid zone, which is composed of cells derived from both the Nppa⁺ and Hey2⁺ populations. Inhibition of the fetal Hey2⁺ cell contribution to the hybrid zone results in persistence of excessive trabeculations in postnatal heart. Our findings indicate that the expansion of Hey2⁺ fetal compact component, and its contribution to the hybrid myocardial zone, are essential for normal formation of the ventricular walls.Fetal trabecular muscles in the heart undergo a poorly described morphogenetic process that results into a solidified compact myocardium after birth. Tian et al. show that cardiomyocytes in the fetal compact layer also contribute to this process, forming a hybrid myocardial zone that is composed of cells derived from both trabecular and compact layers.

Fig. 1

Nppa and Hey2 are expressed in trabecular and compact myocardium, respectively. a Whole-mount and sectional in situ hybridization of Nppa on E11.5–E13.5 hearts. b In situ hybridization of Hey2 on E11.5–E14.5 heart sections. c Whole-mount fluorescence view of E10.5–E12.5 Nppa-GFP embryos or hearts. Inserts are bright-field view of embryos or hearts. d Immunostaining for GFP as surrogate for Nppa and cardiomyocyte marker TNNI3 on Nppa-GFP heart sections showed GFP is restricted to trabecular myocardium. e Whole-mount ESR staining of E10.5–E12.5 Hey2-2A-CreER mice hearts. f Immunostaining for ESR (as surrogate for Hey2) and TNNI3 on Hey2-2A-CreER heart sections showed ESR expression is enriched in compact myocardium but not in atrial or trabecular myocardium. Scale bars, 200 µm. Magnified images are shown in Supplementary Fig. 3b. Each image is a representative of four individual samples

Fig. 2

Generation and characterization of Nppa-rtTA mouse line. a Schematic showing knock-in strategy of Nppa-rtTA mouse line by homologous recombination. b Schematic showing characterization of Nppa-rtTA by rtTA responding reporter mice TetO-LacZ or TetO-GFP. LacZ or GFP is expressed after doxycycline treatment (+Dox). c X-gal staining of E12.5 to E14.5 Nppa-rtTA;TetO-LacZ heart sections. d Whole-mount fluorescence view of Nppa-rtTA;TetO-GFP mouse hearts. Inserts are bright-field images of same hearts. Dotted lines indicate epicardium. e Immunostaining for GFP and TNNI3 on Nppa-rtTA;TetO-GFP heart sections showed GFP is highly enriched in trabecular myocardium (TM) but sparse in compact myocardium (CM). Each image is a representative of four individual samples. Scale bars, 500 µm (white or black); 100 µm (yellow)

Fig. 3

Trabecular myocardium develops into the inner myocardial wall. a Schematic diagram showing the genetic labeling strategy of Nppa⁺ cardiomyocytes by tet-on system. After doxycycline (Dox) treatment, rtTA binds to tetO promoter and drives Cre expression. Cre-loxp recombination leads to tdTomato expression. b Schematic diagram showing the time point for doxycycline induction and tissue analysis. c Immunostaining for tdTomato and TNNI3 on E15.5 heart sections. Arrowheads indicate a small number of Nppa⁺ cells (tdTomato⁺) in compact (Comp.) myocardium. Dotted line indicates border between trabecular (Trab.) and comp. myocardium. Quantification of the percentage of tdTomato⁺ cardiomyocytes (CM) in Trab. or Comp. layer is shown on the right panel. Data are mean ± s.e.m.; n = 4. d Whole-mount bright-field and fluorescence views of hearts collected from Nppa-rtTA;tetO-Cre;R26-tdTomato mice. Dotted lines indicate the outline of hearts. e Immunostaining for tdTomato and TNNI3 on heart sections. Dotted lines indicate epicardium. Nuclei were stained with DAPI. Each image is a representative of four individual samples. Scale bars, 100 µm in c; 500 µm in d, e

Fig. 4

Compact myocardium contributes to the middle and outer myocardial wall. a Immunostaining for EdU and TNNI3 on E15.5 heart sections. Dotted line in magnified image denotes border between trabecular myocardium (Trab. M) and compact myocardium (Comp. M). b Quantification of the percentage of EdU⁺ cardiomyocytes in Trab. M or Comp. M. *P < 0.05; Data are mean ± s.e.m.; n = 4. c Schematic diagram showing the time point for tamoxifen (Tam) induction and tissue analysis. d Immunostaining for tdTomato, GFP, and TNNI3 on Hey2-2A-CreER;R26-tdTomato;Nppa-GFP heart section. Dotted line indicate border between Trab. and Comp. myocardium. Arrowheads indicate tdTomato+ cardiomyocytes (CM) in trab. layer. Quantification of the percentage of labeled CM in Trab. or Comp. is shown on the right panel. Data are mean ± s.e.m.; n = 4. e Whole-mount bright-field and fluorescence views of hearts collected from Hey2-2A-CreER;R26-tdTomato mice. f Immunostaining for tdTomato and TNNI3 on heart sections. Each image is a representative of four individual samples. Scale bars, 500 µm

Fig. 5

Fetal compact myocardium expands into pre-existing trabecular layer to form hybrid myocardial zone. a Immunostaining for tdTomato and TNNI3 on P7 Hey2-2A-CreER;R26-tdTomato heart sections. The boxed regions in the inner, middle, and outer myocardial wall are magnified. Tamoxifen was induced at E12.5. b Transverse sections of P7 heart from base to apex. c Quantification of the percentage of tdTomato⁺ cardiomyocytes (CM) in the inner, middle and outer myocardial wall. n = 4. d Immunostaining for tdTomato and TNNI3 on transverse sections shows different contribution of tdTomato⁺ CMs in the inner i, middle m, and outer o myocardial wall. e Cartoon figure showing the fate mapping of embryonic trabecular and compact myocardium into the postnatal stage. Yellow lines encircles hybrid myocardium. LV left ventricle, Pa papillary muscle. f, g Immunostaining for tdTomato, TNNI3, and Ki67 (f) or pHH3 (g) on P7 Hey2-2A-Cre;R26-tdTomato heart sections. Quantification of the percentage of proliferating cardiomyocytes (CM) in tdTomato⁺ or tdTomato⁻ populations. n.s. non-significant; n = 4. Scale bars, 500 µm (white) in a, d; 100 µm (yellow) in a, d; 100 µm in f, g

Fig. 6

Fate mapping of fetal compact myocardium. a Schematic diagram showing strategy for generation of Hey2-CreER knock-in allele. b Schematic diagram showing the time point for tamoxifen induction (Tam) and tissue analysis. c Whole-mount bright-field and fluorescence views of hearts collected from Hey2-CreER;R26-tdTomato mice. d Immunostaining for tdTomato and TNNI3 on heart sections. Nuclei were stained with DAPI. e Magnified images showing inner myocardial wall (IMW), middle myocardial wall (MMW) and outer myocardial wall (OMW). Each image is a representative of four individual samples. Scale bars, 500 µm (white); 100 µm (yellow)

Fig. 7

Sema3a⁺ cells contribute to the innermost layer of ventricle wall in postnatal heart. a Schematic showing strategy for generation of Sema3a-CreER knock-in allele by homologous recombination. b Experimental strategy for tamoxifen administration and tissue analysis. c Immunostaining for ESR and TNNI3 on E12.5 and E13.5 Sema3a-CreER hearts. Most Sema3a⁺ (ESR⁺) cells were detected in inner trabecular layer (yellow arrowheads) but not in outer trabecular layer (white arrowheads). d, e Immunostaining for tdTomato and TNNI3 on heart section from P0, P7, and P8w (8 weeks) Sema3a-CreER;R26-tdTomato hearts. Sema3a-derived cells are restricted in the innermost layer of ventricle wall (IMW) in postnatal heart. f Cartoon image showing cell fate of Sema3a⁺ cells in postnatal heart. Scale bars, 100 µm in c; 500 µm in d, e; Each image is representative of four individual samples

Fig. 8

Clonal analysis of single Hey2⁺ cardiomyocyte from fetal to postnatal stage. a Schematic figure showing four alternative recombination results (nGFP, YFP, RFP, and mCFP) by crossing Hey2-CreER with R26-Confetti line. b Schematic figure showing experimental strategy. c, f Whole-mount fluorescence view of E15.5 and P7 Hey2-CreER;R26-Confetti hearts. d, g Sectional view of clones in E14.5/E15.5 and P7 Hey2-CreER;R26-Confetti heart sections. Arrowheads indicate single color colonies. Dotted lines in g indicate epicardium. e, h Quantification of the percentage of clone numbers in the inner, middle, and outer layer in each heart. n = 18 for E14.5/E15.5; n = 51 for P7. i Cartoon image showing sparse labeling of Hey2⁺ cells at embryonic stage and their expansion and contribution to the middle and outer myocardium layer of postnatal heart. Scale bars, 1 mm in c, f; 100 µm in d, g

Fig. 9

Inhibition of fetal compact myocardial expansion results in prominent trabeculae and thin compacted layer of postnatal heart. a Schematic figure showing generation of Yap1 gene deletion in Hey2⁺ cells by tamoxifen injection at embryonic stage. b Immunostaining for YAP1 and TNNI3 on postnatal day 7 (P7) control and mutant heart sections. Although YAP1 is detected in the inner myocardial wall (IMW) of both control and mutant hearts, YAP1 is reduced in the outer myocardial wall (OMW) of mutant heart compared with that in the control heart. c Hematoxylin and eosin (H&E) staining of heart sections from P7 control (left) and mutant (right) mice. Inserts indicate whole-mount images of hearts. d Immunostaining for TNNI3 and pHH3 or EdU on control and mutant heart sections. Yellow arrowheads indicate proliferating cardiomyocytes in the magnified inserts. e Quantification on the percentage of pHH3⁺ or EdU⁺ cardiomyocytes is shown on the right panel. f Echocardiographic analysis of heart function showed a significant reduction of ejection fraction and fractional shortening in the mutant, compared with the control. *P < 0.05; n = 4 for mutant and n = 5 for control. g Cartoon image showing hybrid myocardial zone in the postnatal heart derived from trabecular layer and compact myocardium. The hybrid zone (yellow) is interposed between the inner myocardial wall (blue) produced mainly by trabecular coalescence and the outer myocardial wall (red) produced by compact myocardial expansion. RV right ventricle, LV left ventricle, VS ventricular septum; Pa. papillary muscle. Scale bars, 1 mm (black or yellow); 100 µm (white)

Fig. 10

Deletion of Yap1 in fetal trabecular myocardium does not result in thin compact myocardium. a Schematic figure showing generation of Nppa-CreER knock-in allele. b Whole-mount fluorescence view of E15.5 Nppa-CreER;R26-tdTomato heart. Tamoxifen was injected at E12.5. c Immunostaining for tdTomato and TNNI3 on heart section showed Nppa-CreER mainly targets trabecular myocardium. d Schematic figure showing strategy for Yap1 knockout and experimental design. e Immunostaining for YAP1 and TNNI3 on control or mutant heart sections shows a significant reduction of YAP1⁺ cardiomyocytes (arrowheads) in the mutant compared with the control. f Immunostaining for pHH3, TNNI3 and PECAM on the E15.5 mutant or control heart sections. g Quantification of the percentage of pHH3⁺ cardiomyocytes in trabecular myocardium of the control and mutant heart samples. *P < 0.05; n = 4. h Hematoxylin and eosin (H&E) staining of heart sections from P7 control and mutant hearts. Inserts indicate whole-mount images of hearts. Scale bars, 1 mm in h; 200 µm in b; 100 µm in c, e, f