Induction of Wnt signaling antagonists and p21-activated kinase enhances cardiomyocyte proliferation during zebrafish heart regeneration

Abstract

Heart regeneration occurs by dedifferentiation and proliferation of pre-existing cardiomyocytes (CMs). However, the signaling mechanisms by which injury induces CM renewal remain incompletely understood. Here, we find that cardiac injury in zebrafish induces expression of the secreted Wnt inhibitors, including Dickkopf 1 (Dkk1), Dkk3, secreted Frizzled-related protein 1 (sFrp1), and sFrp2, in cardiac tissue adjacent to injury sites. Experimental blocking of Wnt activity via Dkk1 overexpression enhances CM proliferation and heart regeneration, whereas ectopic activation of Wnt8 signaling blunts injury-induced CM dedifferentiation and proliferation. Although Wnt signaling is dampened upon injury, the cytoplasmic β-catenin is unexpectedly increased at disarrayed CM sarcomeres in myocardial wound edges. Our analyses indicated that p21-activated kinase 2 (Pak2) is induced at regenerating CMs, where it phosphorylates cytoplasmic β-catenin at Ser 675 and increases its stability at disassembled sarcomeres. Myocardial-specific induction of the phospho-mimetic β-catenin (S675E) enhances CM dedifferentiation and sarcomere disassembly in response to injury. Conversely, inactivation of Pak2 kinase activity reduces the Ser 675-phosphorylated β-catenin (pS675-β-catenin) and attenuates CM sarcomere disorganization and dedifferentiation. Taken together, these findings demonstrate that coordination of Wnt signaling inhibition and Pak2/pS675-β-catenin signaling enhances zebrafish heart regeneration by supporting CM dedifferentiation and proliferation.

Keywords: PAK2 kinase; Wnt signaling; cardiomyocyte dedifferentiation; cardiomyocyte proliferation; heart regeneration; zebrafish.

Figure 1

Multiple secreted Wnt inhibitors sFrps and Dkks are induced following cardiac injury. (A) Expression levels of wnt inhibitors (dkk1b, dkk2, dkk3b, sfrp1a, sfrp1b, and sfrp2) and wnt ligands (wnt2ba, wnt4a, wnt6b, and wnt8a) during cardiac regeneration. raldh2 was used as a positive control (Kikuchi et al., 2011). Data are mean ± SEM from three biological replicates and three technical replicates. Student’s t-test, **P < 0.01. (B‒U) Confocal images displaying sFrp1, Dkk3, Dkk1b, and sFrp2 expression in uninjured and injured hearts at 3, 7, and 14 dpa. Dotted line indicates amputation line. Brackets indicate amputation plane. Scale bar, 100 µm. (B‒E) Small numbers of epicardial cells adjacent to the injury site induce sFrp1 expression (arrows) at 3 dpa, whereas sFrp1 expression is little detectable in uninjured ventricles. At 7 dpa, sFrp1 expression is expanded throughout the ventricular epicardium and detectable within the regenerate (arrows). By 14 dpa, sFrp1 expression is largely restricted to the epicardial sheet surrounding the regenerate (arrows). (F‒I) Dkk3 is little detectable in uninjured ventricles. At 3 dpa, Dkk3 is expressed in epicardial cells adjacent to the injury region. By 7 dpa, Dkk3 expression is enhanced and expanded in the epicardium enclosing the ventricle and the wound (arrows). Dkk3 expression remains in the epicardial sheet surrounding the regenerate (arrows) by 14 dpa. (J–Q) Whereas Dkk1b expression is not detectable in the uninjured ventricle in Tg(dkk1b:egfp) animals (J and N), some CMs express Dkk1b at the apical edge of the wound at 3 dpa (K and O). Enhanced Dkk1b expression is detectable in the apical edge cells of the regenerating myocardium at 7 dpa (L and P). Dkk1b expression remains in a limited number of CMs within the regenerate at 14 dpa (M and Q). (R‒U) sFrp2 expression is detectable in the wounded heart at 3 dpa, enhanced at the apical cell edge cells of the injured myocardium at 7 dpa, and gradually reduced by 14 dpa. Faint expression of sFrp2 expression is detected in uninjured hearts (R).

Figure 2

Suppression of Wnt signaling enhances heart regeneration by increasing CM proliferation. (A) Schematic of heat shock experiments and Wnt agonist treatment for CM proliferation, qPCR, and AFOG analyses. Heat shock (HS): 37°C for 2 h for control and Tg(hsp70:dkk1b) animals at 4, 5, and 6 dpa for PCNA/Mef2C assay or over the time period from 4 to 21 dpa for AFOG analysis; 37°C for 4 h for control and Tg(hsp70:wnt8a) animals at 6 dpa for PCNA/Mef2C assay or over the time period from 6 to 30 dpa for AFOG analysis. EdU: injection at 5 and 6 dpa. CHIR99021: 20 µM (in DMSO) from 6 to 7 dpa. Control: 0.1% DMSO from 6 to 7 dpa. (B‒I) Confocal microscopy analyses for CM proliferation. Insets show high-magnification images in rectangles. Scale bar, 100 µm. Data are mean ± SEM from five hearts each. Student’s t-test, *P < 0.05, **P < 0.01. (B‒E and J) Confocal images of PCNA⁺Mef2C⁺ (B and C) and EdU⁺Mef2C⁺ (D and E) cells (arrowheads) in heat-shocked WT (Ctrl) and Tg(hsp70:dkk1b) hearts following resection and PCNA- or EdU-labeled CM proliferation index (J). (F‒I and K) Confocal images of PCNA⁺Mef2C⁺ cells (arrowheads) in heat-shocked Ctrl (F) and Tg(hsp70:wnt8a) (G) hearts after amputation or 0.1% DMSO-treated (H) and CHIR99021-treated (I) WT hearts following resection and PCNA-labeled CM proliferation index (K). (L and M) qPCR analysis for relative expression levels of axin2 and mycn in wounded ventricles of Ctrl and Tg(hsp70:dkk1b) (L) or Tg(hsp70:wnt8a) (M) animals. β-actin expression was used for normalization. Data are mean ± SEM from three biological replicates and three technical replicates. Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001. (N‒U) Representative images of AFOG-stained ventricle sections and regeneration scores. (N‒P and Q) Heat-shocked Ctrl and Tg(hsp70:dkk1b) animals at 21 dpa. (R‒T and U) Heat-shocked Ctrl and Tg(hsp70:wnt8a) animals at 30 dpa. Orange: muscle; blue: collagen; red: fibrin. Scale bar, 100 µm. Fisher’s exact test, *P < 0.05, **P < 0.01.

Figure 3

Induction of pS675-β-catenin at disassembled sarcomeres in the injured myocardium following cardiac damage. (A) In uninjured hearts, β-catenin is detectable throughout the myocardium stained with a sarcomeric Z-disk marker α-actinin. (B‒D) Following ventricular resection, β-catenin (B) and pS675-β-catenin (C) are induced at the apical cell edge of wounded myocardia. (D) Merged panel of B and C without α-actinin. Brackets, amputation area. (E) Bar chart depicting β-catenin and pS675-β-catenin levels following ventricular resection. Fluorescent intensities were measured at the injury border zone using Image J. β-catenin or pS675-β-catenin levels in control hearts are normalized as 1. Data are mean ± SEM from five hearts for each group. Student’s t-test, *P < 0.05. (F) High-magnification image of the dash-lined area in A displaying CMs in organized sarcomeric arrays in uninjured hearts. (G‒I) High-magnification images of the dash-lined window in C exhibiting the co-localization of pS675-β-catenin (H and I) with disassembled sarcomeres (G and H) in the injured myocardial cell edge. (I) Merged panel of G and H without α-actinin. 3D analyses display co-localizations of pS675-β-catenin (red) with dissociated sarcomere components (white) along the X-axis (H, x1‒x3) and the Y-axis (H, y1‒y3), as well as the cytoplasmic localization of pS675-β-catenin in Z sections (I, x1‒x3 and y1‒y3). Scale bar, 100 µm (A‒D) and 10 µm (F‒I).

Figure 4

Wnt signaling inhibition associates with CM dedifferentiation during heart regeneration. (A‒H) Immunofluorescence analyses showing increased emCMHC stained with N2.261 antibody and pS675-β-catenin at apical myocardial cells of the wound at 7 dpa (A‒D), which was reduced by wnt8a activation in injured Tg(hsp70:wnt8a) hearts (E‒H). N2.261 is not detectable in uninjured hearts (A and E) and overlaps mostly with induced pS675-β-catenin (D). (D and H) Merged panels of B and C and F and G, respectively. (I‒L) ISH analyses displaying gata4 expression at injured myocardial cell edges in heat-shocked Ctrl hearts (J) at 7 dpa, which was reduced in Tg(hsp:wnt8a) hearts (K) and increased in Tg(hsp:dkk1b) hearts (L) but hardly detectable in uninjured hearts (I). Brackets indicate amputation planes. Scale bar, 100 µm. (M and N) Bar charts depicting pS675-β-catenin (M) and emCMHC (N) levels in Ctrl (normalized as 1), Tg(hsp:wnt8a), and Tg(hsp:dkk1b) hearts. Fluorescent intensities were measured at the injury border zone using Image J. Data are mean ± SEM from five hearts for each group. Student’s t-test, *P < 0.05, **P < 0.01.

Figure 5

Inhibiting Pak2 activity using dominant-negative mutation or chemical inhibitor reduces pS675-β-catenin at wound edges in the regenerating heart. (A‒D) Immunostaining and ISH analyses displaying increased Pak2 and pak2a expression, respectively, at wounded myocardial cell edge in injured hearts at 3 dpa (B and D) compared to uninjured heart (A and C). Scale bar, 100 µm. (E) qPCR analysis for expression levels of pak1, pak2a, and pak2b in uninjured and resected WT hearts at 3 or 7 dpa. β-actin expression was used for normalization. Data are mean ± SEM from three biological replicates and three technical replicates. Student’s t-test, **P < 0.01. (F) Schematic showing heat shock experiment of Tg(hsp70:dnpak2a) and FRAX597 treatment for β-catenin and pS675-β-catenin assessment. Heat shock: 37°C for 2 h daily from 4 to 6 dpa. FRAX597: 1 µM (in DMSO) from 4 to 6 dpa. Control: 0.1% DMSO from 4 to 6 dpa. (G‒V) Confocal microscopy analyses for the levels of pS675-β-catenin and total β-catenin at wounded myocardial cell edges in hearts at 7 dpa. Scale bar, 100 µm. Data are mean ± SEM from five hearts for each group. Student’s t-test, *P < 0.05. (G‒O) pS675-β-catenin was diminished whereas total β-catenin was slight reduced in Tg(hsp:dnpak2a) hearts at 7 dpa. (P‒V) pS675-β-catenin was diminished and total β-catenin was reduced in the Pak2a inhibitor FRAX597-treated hearts at 7 dpa compared to 0.1% DMSO-treated control hearts. (T and U) Merged panels of P and R and Q and S, respectively. (W and X) ISH analysis showing the same β-catenin expression level in FRAX597-treated hearts (X) compared to DMSO-treated hearts (W). (Y and Z) Immunostaining analyses revealing a reduction of cytoplasmic pS675-β-catenin in FRAX597-treated hearts (Z) compared to DMSO-treated hearts (Y) at 7 dpa. Scale bar, 10 µm.

Figure 6

Induction of phospho-mimetic β-catenin (S675E) in adult CMs enhances sarcomere disassembly and CM dedifferentiation. (A) Schematic diagram of double transgenes of TRE3G:ctnnb2(S675E)^CMi. DOX treatment allows binding of TetON-3G to the TRE3G promoter, enabling the tissue-specific transcription of ctnnb2(S675E) in CMs. (B) Experimental strategy to induce myocardial ctnnb2(S675E) overexpression during the course of heart regeneration. Ctnnb2(S675E) induction (mScarlet-I/cTnT), sarcomere disassembly (α-actinin/mScarlet-1), CM dedifferentiation (emCMHC), and proliferation (PCNA/Mef2C) were examined at 7 dpa in vehicle-treated and TOX-treated TRE3G:ctnnb2(S675E)^CMi animals. (C and D) Brightfield (C) or red fluorescence (D) images of whole-mount hearts. mScarlet-I signal was only detected in myocardium of DOX-treated hearts. Scale bar, 500 µm. (E‒H) Representative confocal fluorescence images of cardiac sections immunostained for cTnT (green) and mScarlet-I (red). Scale bar, 100 µm. (I, J, and O) Myocardial wound edge regions in heart sections were co-stained for α-actinin (green) and mScarlet-I (red). Scale bar, 50 µm. (I1, I2, J1, and J2) Magnified panels show high-magnification images of the boxed regions in I and J. (O) Quantification of organized sarcomere Z-disks in α-actinin-marked CMs (100 × 100 pixels). (K‒N, P, and Q) Confocal image analyses of emCMHC expression (K and L) and PCNA⁺Mef2C⁺ cells (arrowheads, M and N). Brackets indicate amputation planes. Insets show high-magnification images of PCNA⁺Mef2C⁺ cells in boxed regions. (P) Fluorescent intensities depicting emCMHC levels were measured at the injury border zone using Image J. emCMHC levels in vehicle-treated hearts are normalized as 1. (Q) PCNA-labeled CM proliferation indices. Scale bar, 100 µm. For bar chart analyses in O‒Q, data are mean ± SEM from five hearts for each group. Student’s t-test, **P < 0.01.

Figure 7

Inhibition of Pak2 activity impairs CM dedifferentiation and proliferation. (A) Schematic of heat shock experiments and FRAX597 treatment for PCNA/Mef2C analyses. Heat shock: 37°C for 2 h daily from 4 to 6 dpa for PCNA/Mef2C assay or over the time period from 4 to 30 dpa for AFOG analysis; treatment: 1 µM FRAX597 or 0.1% DMSO from 4 to 6 dpa. (B‒D) Confocal image analyses displaying PCNA⁺Mef2C⁺ cells (arrowheads, B and C) and PCNA-labeled CM proliferation index (D) in Ctrl and Tg(hsp:dnpak2a) hearts at 7 dpa. Insets indicate high-magnification images in dash-lined areas. Data are mean ± SEM from five hearts for each group. Student’s t-test, **P < 0.01. (E‒G) Representative images (E and F) and quantification of regeneration scores (G) of ventricle sections from heat-shocked Ctrl and Tg(hsp70:dnpak2a) animals at 30 dpa and stained with AFOG. Orange: muscle; blue: collagen; red: fibrin. Fisher’s exact test. **P < 0.01. (H‒K) Confocal image analyses displaying PCNA⁺Mef2C⁺ cells (arrowheads) and quantification of CM proliferation indices (K) in DMSO-treated (H, Ctrl), Pak2 inhibitor (FRAX597)-treated (I), and FRAX597-treated Tg(hsp:dkk1b) (J) hearts at 7 dpa. Insets indicate high-magnification images of dash-lined area. Data are represented as mean ± SEM from five hearts for each group. Statistical significance was calculated using one-way ANOVA followed by Tukey’s test. n.s., none significance; *P < 0.05, ***P < 0.001. (L‒O) Immunostaining analyses displaying reduction of pS675-β-catenin (L and M) and emCMHC (N and O) at apical myocardial cells of the wounded edge in FRAX597-treated hearts compared to DMSO-treated hearts at 7 dpa. (L′ and M′) High-magnification images of dash-lined windows in L and M, respectively, exhibiting disassembled sarcomeres in DMSO-treated hearts (L′) and relatively normal striated sarcomeres in FRAX597-treated hearts (M′). (P and Q) ISH analysis displaying a reduction of gata4 at the wounded myocardial cell edge in FRAX597-treated hearts compared to DMSO-treated hearts at 7 dpa. Brackets indicate amputation planes. Scale bar, 100 µm (except 10 µm in L′ and M′). (R) A working model for Wnt/Pak2/pS675-β-catenin signaling events regulating heart regeneration. Cardiac injury induces multiple Wnt antagonists that restrain Wnt signaling throughout the wounded heart, enabling CM dedifferentiation and proliferation. In the context of Wnt signaling inhibition, Pak2 is upregulated in cardiac wounds, where it phosphorylates cytoplasmic β-catenin at the Ser 675 residue and increases its stability in disassembled sarcomeres, enhancing CM renewal and heart regeneration.