Hemodynamic-mediated endocardial signaling controls in vivo myocardial reprogramming
- University of California, San Diego, United States
- Fudan University, China
- Massachusetts General Hospital and Harvard Medical School, United States
- Centre National de la Recherche Scientifique, UMR7104, INSERM U964, Université de Strasbourg, France
Figures
Figure 1 with 4 supplements
Endocardial Notch signaling controls myocardial reprogramming and cardiac regeneration.
(A–D, L–O) Confocal microscopy imaging of heat-shocked/HS (A, B, L, M) vmhc:mCherry-NTR, (C, D) vmhc:mCherry-NTR; hsp70l:dnM and (N, O) vmhc:mCherry-NTR; kdrl:Cre; hsp70l:RS-dnM hearts reveals that …
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Figure 1—source data 1
Source data for Figure 1 and Figure 1—figure supplements 1–3.
- https://doi.org/10.7554/eLife.44816.007
Figure 1—figure supplement 1
Metronidazole-ablated vmhc:mCherry-NTR ventricles display initial cardiomyocyte death, loss of ventricular tissue and impairment of contractile function but later exhibit cardiomyocyte proliferation and recovery of ventricular tissue and contractile function.
Following ventricular ablation, ventricular and atrial cardiomyocyte proliferation leads to cardiac recovery at 96 hpt. (A, B) Confocal maximum intensity projections of Tg(vmhc:mCherry-NTR) fish …
Figure 1—figure supplement 2
Reprogramming of atrial cardiomyocytes into ventricular cardiomyocytes during development.
(A) Schematic representation of the transgenic lines utilized in lineage tracing experiments. Treatment with 4-hydroxytamoxifen (4-OHT) activates the amhc:CreERT2 protein, which causes the …
Figure 1—figure supplement 3
Endocardial Notch signaling is transiently activated after myocardial injury.
Confocal microscopy imaging was performed on (A) Tp1:eGFP; vmhc:mCherry-NTR control or (B) ventricle-ablated hearts at 24 hr post-treatment/hpt (6 dpf). Green channel – (A’, B’) Tp1:eGFP. (C, D) …
Figure 1—figure supplement 4
Genetic inhibition of endocardial Notch signaling impairs myocardial reprogramming and regeneration.
(A–L) Whole-mount in situ hybridizations of heat-shocked (A, B, E, F, I, J) vmhc:mCherry-NTR or (C, D, G, H, K, L) vmhc:mCherry-NTR; hsp70l:dnM hearts show that dnMAML (dnM) Notch inhibition …
Figure 2 with 6 supplements
Ventricle-ablated hearts display altered oscillatory blood flow and Klf2a activation.
(A, B) High-speed confocal imaging was performed on (A) gata1:DsRed control hearts and (B) vmhc:mCherry-NTR; gata1:DsRed ventricle-ablated hearts at 24 hpt (six dpf). Arrows represent particle image …
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Figure 2—source data 1
Source data for Figure 2 and Figure 2—figure supplement 2.
- https://doi.org/10.7554/eLife.44816.011
Figure 2—figure supplement 1
Klf2a and Notch are activated in areas of the heart that exhibit increased oscillatory flow.
(A, B) Schematic spatial representation of the relative average fluorescence intensity of (A) Klf2a:H2B-GFP and (B) Tp1:d2GFP reveals that both signals are increased in the areas of the endocardium …
Figure 2—figure supplement 2
Post-injury Notch signaling is activated in Klf2a-positive endocardial cells.
(A, B) Confocal imaging performed on (A) control and (B) ventricle-ablated klf2a:H2B-GFP; vmhc:mCherry-NTR; Tp1:nls-mCherry hearts at 24 hpt (6 dpf) shows co-expression of Tp1:nls-mCherry (red) in kl…
Figure 2—video 1
Control hearts display anterograde intracardiac blood flow.
High-speed confocal imaging was performed on gata1:DsRed control hearts at 6 dpf. Video shows maximal projection reconstruction of 10 stacks at 24 fps (frames per second).
Figure 2—video 2
Ablated hearts exhibit retrograde intracardiac blood flow.
High-speed confocal imaging was performed on vmhc:mCherry-NTR; gata1:DsRed MTZ ventricle-ablated hearts at 24 hpt (6 dpf). Video shows maximal projection reconstruction of 10 stacks at 24 fps.
Figure 2—video 2
Particle image velocimetry (PIV) generated vectors confirm anterograde intracardiac blood flow in control hearts.
High-speed confocal imaging was performed on gata1:DsRed control hearts at 6 dpf. Video shows single planes at 24 fps. Vectors represent local estimations of flow velocity as measured by PIV.
Figure 2—video 4
Particle image velocimetry (PIV) generated vectors confirm retrograde intracardiac blood flow in ventricle-ablated hearts.
High-speed confocal imaging was performed on vmhc:mCherry-NTR; gata1:DsRed MTZ ventricle-ablated hearts at 24 hpt (6 dpf). Video shows single planes at 24 fps. Vectors represent local estimations of …
Figure 3 with 1 supplement
Reduced hemodynamic forces affect endocardial Notch and Klf2a post-injury activation.
Confocal imaging performed on (A–D) klf2a:H2B-GFP; vmhc:mCherry-NTR or (J–M) Tp1:d2GFP; vmhc:mCherry-NTR ventricle-ablated hearts at 24 hpt (6 dpf) reveals that (D, M) gata2a-/- mutant hearts as …
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Figure 3—source data 1
Source data for Figure 3.
- https://doi.org/10.7554/eLife.44816.018
Figure 3—figure supplement 1
Inhibiting hemodynamic flow leads to reduced cardiac Klf2a and Notch signaling.
(A–D) Confocal imaging performed on klf2a:H2B-GFP; vmhc:mCherry-NTR uninjured hearts at 24 hpt (6 dpf) reveals decreased klf2a:H2B-GFP expression in (B) blebbistatin (Blebb) and (C) tricaine (Tric) …
Figure 4 with 1 supplement
Hemodynamic forces control regeneration and myocardial reprogramming.
(A–D) Confocal microscopy imaging performed on vmhc:mCherry-NTR ventricle-ablated hearts reveals that (B) blebbistatin or (C) tricaine treatment as well as (D) the gata2a-/- mutant allele inhibit CM …
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Figure 4—source data 1
Source data for Figure 4.
- https://doi.org/10.7554/eLife.44816.021
Figure 4—figure supplement 1
Inhibiting hemodynamic flow perturbs post-injury re-activation of cardiac factors.
Whole-mount in situ hybridizations show increased (A) gata4 (n = 9/11), (E) hand2 (n = 10/10) and (I) nkx2.5 (n = 7/8) expression in vmhc:mCherry-NTR ventricle-ablated (ethanol/EtOH-treated) control …
Figure 5 with 4 supplements
The mechanosensitive channel Trpv4 regulates endocardial Notch activation and myocardial regeneration through Klf2a.
(A–D) Confocal imaging of vmhc:mCherry-NTR; klf2a:H2B-GFP hearts shows that klf2a:H2B-GFP expression is activated in (B) wild-type (wt) ventricle-ablated hearts at 24 hpt (6 dpf) compared to (A) …
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Figure 5—source data 1
Source data for Figure 5.
- https://doi.org/10.7554/eLife.44816.025
Figure 5—figure supplement 1
klf2a and trpv4 mutants display reduced endocardial Notch signaling.
(A–C) Confocal microscopy imaging performed on vmhc:mCherry-NTR, Tp1:d2GFP control hearts reveals reduced Tp1:d2GFP activation in (B) klf2a-/- and (C) trpv4-/- hearts at 24 hpt (6 dpf) when compared …
Figure 5—figure supplement 2
klf2a and trpv4 mutants show impaired post-injury re-activation of cardiogenesis transcription factors.
(A–R) Whole-mount in situ hybridizations show increased (B) gata4 (n = 21/24), (H) hand2 (n = 13/15) and (N) nkx2.5 (n = 12/13) expression in vmhc:mCherry-NTR ventricle-ablated wild-type (wt) hearts …
Figure 5—video 1
High-speed bright-field imaging performed on wild-type control heart at 2 dpf shows normal blood flow.
https://doi.org/10.7554/eLife.44816.026
Figure 5—video 2
High speed bright-field imaging performed on trpv4 -/- mutant heart at 2 dpf shows normal blood flow.
https://doi.org/10.7554/eLife.44816.027
Figure 6 with 1 supplement
Erbb2 and BMP signaling regulate cardiac regeneration and atrial-to-ventricular trans-differentiation.
(A–D) Confocal microscopy performed on vmhc:mCherry-NTR ventricle-ablated hearts reveals that (B) dorsomorphin (DM) and (C) AG1478-treated as well as (D) erbb2 loss-of-function mutation (erbb2-/-) …
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Figure 6—source data 1
Source data for Figure 6.
- https://doi.org/10.7554/eLife.44816.030
Figure 6—figure supplement 1
Inhibiting BMP or Erbb2 signaling impairs reactivation of early cardiogenesis transcription factors.
Whole-mount in situ hybridizations show increased (B) gata4 (n = 19/25), (J) hand2 (n = 10/12) and (R) nkx2.5 (n = 12/13) expression in vmhc:mCherry-NTR ventricle-ablated hearts at 48 hpt (7 dpf) …
Figure 7 with 2 supplements
Blood flow regulates myocardial Erbb2 and BMP signaling through endocardial Notch.
(A–F) Confocal imaging of BRE:d2GFP; vmhc:mCherry-NTR hearts at 48 hpt (7 dpf) shows that (B) BRE:d2GFP is activated after ventricular ablation (n = 21/21) when compared to (A) uninjured control …
Figure 7—figure supplement 1
Myocardial specific activation of BMP signaling.
(A–F) Confocal single-stacks corresponding to the projections shown in Figure 7A–F show (B–B’’’) BRE:d2GFP is activated in the myocardium of ablated hearts at 48 hpt (seven dpf) (n = 21/21), in …
Figure 7—figure supplement 2
Endocardial specific inhibition of Notch signaling blocks post-injury activation of bmp10 and nrg1.
Whole-mount in situ hybridizations of heat-shocked (HS) (A, B, E, F) vmhc:mCherry-NTR; hsp70l:RS-dnM or (C, D, G, H) vmhc:mCherry-NTR; kdrl:Cre; hsp70l:RS-dnM hearts show that dnMAML endocardial …
Author response image 1
Wild-type control and trpv4 -/- mutant hearts display similar blood flow patterns.
High speed imaging was performed on wild-type control and trpv4 -/- mutant hearts at 2-3 dpf. Arrows represent particle image velocimetry (PIV) generated vectors from blood flow.
Videos
Author response video 1
trpv4 mutant video.
https://doi.org/10.7554/eLife.44816.037
Author response video 2
Wild type video.
https://doi.org/10.7554/eLife.44816.038Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.44816.034
