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Participation of ventricular trabeculae in neonatal cardiac regeneration leads to ectopic recruitment of Purkinje-like cells

Nature Cardiovascular Research volume 3, pages 1140–1157 (2024)Cite this article

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A Publisher Correction to this article was published on 11 September 2024

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Abstract

Unlike adult mammals, newborn mice can regenerate a functional heart after myocardial infarction; however, the precise origin of the newly formed cardiomyocytes and whether the distal part of the conduction system (the Purkinje fiber (PF) network) is properly formed in regenerated hearts remains unclear. PFs, as well as subendocardial contractile cardiomyocytes, are derived from trabeculae, transient myocardial ridges on the inner ventricular surface. Here, using connexin 40-driven genetic tracing, we uncover a substantial participation of the trabecular lineage in myocardial regeneration through dedifferentiation and proliferation. Concomitantly, regeneration disrupted PF network maturation, resulting in permanent PF hyperplasia and impaired ventricular conduction. Proliferation assays, genetic impairment of PF recruitment, lineage tracing and clonal analysis revealed that PF network hyperplasia results from excessive recruitment of PFs due to increased trabecular fate plasticity. These data indicate that PF network hyperplasia is a consequence of trabeculae participation in myocardial regeneration.

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Fig. 1: Cardiomyocytes derived from embryonic trabeculae are overrepresented in regenerated regions after a neonatal MI.
Fig. 2: Cardiomyocytes derived from embryonic trabeculae enter cell cycle and express trabecular immature markers during the regeneration process.
Fig. 3: Persisting defects in the PF network of regenerated hearts.
Fig. 4: The hyperplastic PF network of regenerated hearts has an intermediate phenotype between contractile myocardium and healthy PF network.
Fig. 5: Perturbed electrical propagation in regenerated hearts.
Fig. 6: Hyperplasia of the PF network in response to MI depends on maximal level of Nkx2-5 following a recruitment mechanism.
Fig. 7: Excessive PFs leading to PF network hyperplasia following MI are recruited from perinatal trabeculae.
Fig. 8: Clonal analysis of Cx40-high expressing cells after cardiac regeneration.

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Data availability

All data supporting the findings of this study are found within the manuscript and its Supplementary Information and are available from the corresponding author on reasonable request. The smFISH data are available from Zenodo (https://doi.org/10.5281/zenodo.12773891) (ref. 74). Source data are provided with this paper.

Code availability

All codes used in this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank N. Lalevée (Aix-Marseille University, INSERM UMR 1263, C2VN) and B.J. Boukens (Department of Physiology, University Maastricht, Maastricht University Medical Center) for the help in setting up and analyzing the ECGs and the Turing Centre for Living Systems, for financing formations and providing a rich scientific environment. The France-BioImaging infrastructure is supported by the Agence Nationale de la Recherche (ANR) (ANR-10-INBS-04-01, ‘Investissements d’Avenir’). This work was supported by the Centre National de la Recherche Scientifique (L.M.) and Institut National de la Santé et de la Recherche Médicale INSERM (R.G.K.), by grants from the Association Française contre les Myopathies (No. 23711, L.M.) and from the ANR ‘PurkinjeNet’ (L.M.). L.B. is a recipient of a doctoral fellowship from the Ecole Normale Superieure and a doctoral fellowship extension from the Institut Marseille Maladies Rares. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Authors and Affiliations

  1. Aix-Marseille Université, CNRS UMR 7288, Developmental Biology Institute of Marseille, Marseille, France

    Lucie Boulgakoff, Rachel Sturny, Robert G. Kelly & Lucile Miquerol

  2. Charles University, First Faculty of Medicine, Institute of Anatomy, Prague, Czech Republic

    Veronika Olejnickova & David Sedmera

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  1. Lucie Boulgakoff
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Contributions

L.B. and L.M. conceived the project and designed the experiments; L.B. performed all experiments, image acquisition and data curation with the help of R.S. for surgical procedures; V.O. and D.S. performed optical mapping experiments; L.B. performed the statistical analysis. R.G.K. and L.M. provided funding; R.G.K. reviewed and edited the manuscript; and L.B. and L.M. wrote the manuscript.

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Correspondence to Lucile Miquerol.

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Extended Data

Extended Data Fig. 1 Ventricular trabeculae during development and repartition of their derivatives in the mature heart.

(a) Longitudinal sections of hearts from Cx40-Cre-RFP mice. Cx40 is expressed in the trabeculae, as seen by RFP expression, thus, genetic labeling in Cx40-CreERT2 mice labels CM from the trabeculae. (b) Cartoons summarizing the geometry of the trabeculae and the repartition of their derivatives at fetal, perinatal and mature stages. At E14.5, the trabeculae (cyan) reach their higher density. Thereafter, they progressively coalesce and give rise to subendocardial CMs (light green). Around birth, the innermost part of the embryonic trabeculae is not yet compacted, we call this area ‘perinatal trabeculae’. The perinatal trabeculae give rise to the most subendocardial CMs (dark green) and to the PF network. The compact myocardium and its derivatives are shown in dark gray and gray, respectively. Concomitant with trabeculae compaction, bipotent progenitor from the trabeculae progressively segregate between conductive (purple) and contractile (green) fate. Final maturation extends until the third week of mice life.

Extended Data Fig. 2 Cardiomyocytes derived from perinatal trabeculae are overrepresented in regenerated regions following a neonatal MI.

(a) Experimental workflow. Specific expression of Cx40-Cre in perinatal trabeculae (blue) leads to a mosaic genetic labeling (green) following a E18.5 4’-Hydroxytamoxifen injection. Infarcted area (red) is progressively regenerated, and labeled CMs derived from perinatal trabeculae are overrepresented in regenerated regions (black line) in comparison to the control region (dotted line). (b) Transverse sections of Sham- and MI-operated hearts 21 days after injury. The region of interest (ROI) is drawn in color and the control region in dotted gray. (c) High magnification of the ROI after thresholding of the TdTomato signal. Myocardial wall is divided in 10 radial layers (thin lines and number) from the endocardium (inner) to outer regions. (d) Repartition of TdTomato positive CMs in the regenerated myocardium. The graph is expressed as a percentage of TdTomato area in each radial layer of the ROI, from subendocardial to outer regions, normalized by the total TdTomato area of control region, in MI (red curve) and sham (blue curve). Quantifications were conducted on serial sections bellow the ligation every 140 µm (around 20 sections per hearts). Mean values are displayed, error bar: standard error, Two-sided Wilcoxon’s test. MI N = 7; Sham N = 6. (e) Comparison of the lineage tracing of embryonic trabeculae (E14), and perinatal trabeculae (E18). Area of genetically labeled CMs in each radial layer, from the endocardium to outer regions after an injection of tamoxifen at E14 (light green) or 4’-Hydroxytamoxifen at E18 (dark green), in Sham (full line) or MI (dotted line). The graph is expressed as a percentage of labeling in each radial layer of the control region (e) or ROI (e’) and normalized by the total labeled area in control region. Mean values are displayed, error bar: standard error, Two-sided Wilcoxon’s test. For embryonic trabeculae: MI N = 15; Sham N = 11. For perinatal trabeculae: MI N = 7; Sham N = 6. (f) Cartoon summarizing the repartition on embryonic (light green) and perinatal (dark green) trabeculae in healthy and regenerated hearts.

Source data

Extended Data Fig. 3 Poor sarcomere assembly in the trabeculae-derived myocardium during regeneration.

Actinin immunofluorescence show low (empty arrowhead) to null (white arrowhead) sarcomere assembly in Cx40-GFP-positive CMs from the subendocardial BZ at 5dpi in MI compared to Sham- operated mice. Maximal intensity projection of 7.5µm-thick stacks.

Extended Data Fig. 4 No damage PF network observed in Sham-operated mice.

a) Workflow: Cx40-GFP mice are used to study the damages caused by Sham surgery 1 to 3 days, or 21 days after surgery. (b) Open ventricles of Sham-operated mice 2 and 21 days after surgery. The PF network is visualized thanks to Cx40-GFP. (c) No damage of the PF network was observed at the luminal length at any level (successive transverse sections) in Sham-operated mice. (d) Transverse sections at 2 and 21 days after surgery. The PF network is shown in green with Cx40-GFP at 2dpi and Cntn2 at 21 dpi. Although some infiltrated immune cells (CD45) are seen in the myocardium at 2dpi, no accumulation can be observed, and no loss of Cx40-GFP expression in subendocardial myocardium can be seen neither – contrary to MI-operated mice. At 21 post Sham surgery, WGA staining can be observed in-between CMs, although no WGA rich area can be seen, contrary to MI-operated mice.

Extended Data Fig. 5 Permanent hyperplasia of the PF network and functional defects.

(a) Cntn2-positive area is measured on successive transverse sections spaced 140 µm apart. The ventricular lumen is delimitated automatically using the fluorescent background of the tissue at 21 dpi. Cntn2 staining is thresholded after a gaussian blur of 2 µm. The normalized Cntn2 area per section is expressed as the ratio of Cntn2-positive area divided by the luminal perimeter. (b) Plot showing the normalized Cntn2 area along the apico-basal axis (a-j), in Sham (Bleu) and MI (Red) as measured in (a). MI N = 19; Sh N = 17 Error bars: standard deviation. (c) Whole-mount open left ventricles six months after Sham or MI surgery in Cx40-GFP mice. Hyperplasia of the PF network is visible thanks to Cx40-GFP in all 3 MI. (d) The intermediate phenotype in regenerated heats is permanent. Transverse sections of regenerated hearts 6 months post-injury show heterogenous expression of conductive adhesion molecules (Cntn2) and fast-conducting gap junction (Cx40) in the hyperplastic PF network. White arrowheads: PFs, empty arrow heads: intermediate cells. (e) Longitudinal study of cardiac conduction system function thanks to electrocardiogram recordings in leads II at 3dpi, 9dpi, 21 dpi and 6 months post-injury. Note the recovery of the QRS amplitude following myocardium regeneration, however, ventricular conduction velocity remains slow as evidenced by prolonged QRS duration in MI compared to Sham at all stages. MI N = 5; Sh N = 5.

Source data

Extended Data Fig. 6 Downregulation of most PF-enriched genes in the hyperplastic PF network of regenerated hearts.

a) Transverse section of Sham and MI heart at 21 dpi, processed with 100-plex smFISH. The PF network, ROI shown in purple, was delimited manually, including cells with Cntn2 transcripts (magenta dots). In the MI, only the PF network from the BZ of the infarct was analyzed. The contractile myocardium, ROI shown in gray, includes all the imaged tissue, except the PF region. b) The density of RNA of each PF-enriched gene (transcripts per mm² of tissue), detected by smFISH, was quantified in the contractile myocardium and PF network of Sham and MI at 21 dpi. c) Density of transcripts from CM genes (transcripts per mm²), detected by smFISH, was quantified in the contractile myocardium and PF network (Cntn2+) of Sham and MI at 21 dpi. N: Sham=3, MI = 3. Box plots show the median, the 25th and 75th percentile, and the whiskers denote the minimum and maximum values, respectively. Normality was tested by Shapiro–Wilk test and rejected if p value < 0.01. Homoscedasticity was tested by F- test and rejected if p value < 0.01. Two-sided Student-t-test was used when normality and homoscedasticity were validated. Else, in case of heteroscedasticity the Welch Two samples T-Test was used, and, in case of non-normality, the Two-sided Wilcoxon Rank Test was used. LV: Left ventricle, PM: papillary muscles, ROI, region of interest.

Source data

Extended Data Fig. 7 Dual contribution of trabeculae during neonatal cardiac regeneration.

Trabeculae participate in the regeneration of the contractile myocardium, and are thus found in increased proportions in regenerated myocardium. However, this contribution to the myocardium involves a perdurance of an immature phenotype in the trabeculae during regeneration which prolongs the plasticity between the contractile and conductive fates and results in ectopic conductive commitment. Excessive production of conductive cells is accompanied by incomplete conductive maturation producing a hyperplastic and immature PF network and resulting in altered ventricular conduction.

Extended Data Table 1 Repartition of genetically labeled CMs in regenerated myocardium
Full size table
Extended Data Table 2 Clonal analysis of Cx40 expressing cells at P1 after cardiac regeneration
Full size table

Supplementary information

Reporting Summary

Supplementary Table

Resolve genes list.

Supplementary Video 1

Epicardial activation movie control.

Supplementary Video 2

Epicardial activation movie MI.

Supplementary Video 3

Endocardial activation movie control.

Supplementary Video 4

Endocardial activation movie MI.

Source data

Source Data Fig. 1

Statistical source data. Figure 1c: repartition of YFP cardiomyocytes, derived from embryonic trabeculae, in ROI.

Source Data Fig. 2

Statistical source data. Figure 2c: EdU incorporation in the trabecular lineage.

Source Data Fig. 3

Statistical source data. Tab 1 (associated with Fig. 3e): damaged PF network at T0 (1–3 dpi) and 21 dpi. Tab 2 (associated with Fig. 3h): volume of PF network (Cntn2+) at 21 dpi. Tab 3 (associated with Fig. 3i): number of Cntn2+ cells at 21 dpi.

Source Data Fig. 4

Statistical source data. Tab 1 (associated with Fig. 4c): transcripts per mm2 of PF-enriched genes in the PF network (Cntn2+) and contractile myocardium of sham and MI at 21 dpi. Tab 2 (associated with Fig. 4i): PF shape.

Source Data Fig. 5

Statistical source data. Tab 1 (associated with Fig. 5d): ECG recording at 21 dpi (lead II). Tab 2 (associated with Fig. 5e): QRS duration at 21 dpi measured on lead II. Tab 3 (associated with Fig. 5f): angle (deg) and amplitude (mV) of the main activation wave at 21 dpi.

Source Data Fig. 6

Statistical source data. Tab 1 (associated with Fig. 6c): PF proliferation measured as the percentage of EdU-positive nuclei among all PF nuclei. Tab 2 (associated with Fig. 6f): QRS duration at 21 dpi measured on lead II. Tab 3 (associated with Fig. 6g): angle (deg) and amplitude (mV) of the main activation wave at 21 dpi.

Source Data Fig. 7

Statistical source data. (associated with Fig. 7c): percentage of YFP labeling among PFs at 21 dpi.

Source Data Fig. 8

Statistical source data. Tab 1: records of all quantified clones. Tab 2 (associated with Fig. 8c): proportion of clones of each category. Tab 3 (associated with Fig. 8d): proportion of clones within the mixed category. Tab 4 (associated with Fig. 8e): clone size in each clone category. Tab 5 (associated with Fig. 8f): number of daughter cells from each cell type per 100 mother cells.

Source Data Extended Data Fig. 2

Tab 1 (associated with Extended Data Fig. 2d): repartition of tdTomato CMs, derived from perinatal trabeculae, in ROI. Tab 2 (associated with Extended Data Fig. 2e): repartition of labeled cardiomyocytes, derived from embryonic (Tam E14) or perinatal trabeculae (4-OHT E18), in control region and ROI.

Source Data Extended Data Fig. 5

Tab 1 (associated with Extended Data Fig. 5b): normalized PF network area (Cntn2+) from Base to Apex at 21 dpi. Tab 2 (associated with Extended Data Fig. 5e): longitudinal following by ECG recording in lead II.

Source Data Extended Data Fig. 6

Tab 1 (associated with Extended Data Fig. 6b): transcripts per mm2 of PF-enriched genes in the PF network (Cntn2+) and contractile myocardium of sham and MI at 21 dpi. Tab 2 (associated with Extended Data Fig. 6c): transcripts per mm2 of CMs genes in the PF network (Cntn2+) and contractile myocardium of sham and MI at 21 dpi.

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Boulgakoff, L., Sturny, R., Olejnickova, V. et al. Participation of ventricular trabeculae in neonatal cardiac regeneration leads to ectopic recruitment of Purkinje-like cells. Nat Cardiovasc Res 3, 1140–1157 (2024). https://doi.org/10.1038/s44161-024-00530-z

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