Interplay of cell–cell contacts and RhoA/MRTF‐A signaling regulates cardiomyocyte identity

Figures

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  Figure EV1. Expression of desmosomal, pro‐apoptotic, and adipocytic genes in PKP2mut CMs qRT–PCR analysis of PKP2 reveals similar expression levels in wt and PKP2mut CMs (n = 3).Left, Western blotting using an antibody directed against the N‐terminus of PKP2 demonstrates reduced expression of wild‐type PKP2 protein (PKP2 wt, 97 kDa) and absence of truncated A587fsX655‐PKP2 product (PKP2 mut, 72 kDa) in total lysates of PKP2mut CMs. Pan‐Cadherin is shown as a loading control. Right, densitometric readings for PKP2‐wt bands reveal an almost 50% reduction in PKP2mut myocytic lysates compared to wt ones. Values are expressed as the integrals (area × mean density) of each band normalized to Pan‐Cadherin and relative to wt; n = 3; *P < 0.01 vs. wt; t‐test.Immunofluorescence analysis indicates an interrupted desmoplakin expression (DSP, red) at the plasma membrane of PKP2mut CMs compared to wt cells. cTNT (green) marks cardiomyocytes. Nuclei are stained with Hoechst 33258 (blue). Scale bars, 12.5 μm.qRT–PCR analysis of pro‐apoptotic genes and white adipocytic markers shows similar expression levels in wt and PKP2mut CMs over time in culture in adipogenic medium. Brown/beige adipocytic markers were…

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  Figure 1. CMs with pathological PKP2 mutation convert into adipocytes in vitro AImmunostaining of cTNT (green) and PKP2 (red) in wt and PKP2mut CMs. Scale bars, 25 μm. Insets show a magnified image of PKP2 expression; scale bars, 10 μm.BImmunodetection of cTNT (blue) and lipid stain ORO (red) in wt and PKP2mut CMs over time in culture. Scale bars, 50 μm.C–EBar graphs show the percentage of cTNT+/ORO+ cells (C), cTNT+/ORO+ cells with organized (org), disorganized (disorg) and dissolving sarcomeres (loss) (D), and adipocyte‐like cells (E) in wt and PKP2mut CMs over time (n = 3; N = 45–300 cells per time point in each group; *P < 0.05, **P < 0.001 vs. wt at the same time point; t‐test).FqRT–PCR analysis of myocytic and adipocytic genes in wt and PKP2mut CMs over time in culture (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 vs. wt at the same time point, t‐test).GImmunodetection of cTNT (magenta) and lipid stain BODIPY (green) in wt and PKP2mut CMs after 8 weeks of culture. Scale bars, 50 μm. Lower panels show magnified images of areas indicated by the white box for BODIPY (green) and PPARγ (red). Scale bars, 25 μm.HGlycerol release of wt and PKP2mut CMs after 8 weeks in culture in the absence…

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  Figure 2. Cardiac muscle and fat derive from Isl1/Wt1‐expressing progenitors Immunofluorescence analysis of Isl1 derivatives in Isl1 Cre/+ ;R26 mTmG/+ mouse hearts at postnatal d28 (P28): Plin1 (magenta), membrane GFP (mG, green), and membrane Tomato (mT, red). The three panels on the right show a magnified view of the area indicated by the white box in the left panel. Arrows indicate Plin1+/mG+ adipocytes. la, left atrium; lv, left ventricle; ra, right atrium; rv, right ventricle. Scale bars, 500 μm (left panel), 25 μm (right panels); n = 3.Immunostaining of cTNT, Isl1 or Wt1 (all magenta), mG (green), and mT (red) in sagittal, sequential sections of Isl1 Cre/+ ;R26 mTmG/+ embryos at E9.5. The boxed region in panel a is shown at higher magnification in the consecutive section in bI. cI and cII are magnifications of the boxed area in bI (in consecutive sections). Isl1‐Cre‐mediated mG labeling was observed in CMs of the forming heart (a, box b), cardiac progenitors (bI, marked by the asterisk), and a proportion of Wt1+ proepicardial cells in the PEO (cI, indicated by arrows, and cII). Scale bars, 250 μm (a), 150 μm (bI), 25 μm (cI and cII); n = 3.Immunostaining for Isl1 and Wt1 in…

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  Figure EV2. Localization and quantification of mG‐labeled cells in Isl1 MerCreMer/+; R26 mTmG/+ and Wt1 Cre ERT 2/+;R26 mTmG/+ mouse embryos at E9.5 Temporal restriction of Cre‐mediated labeling of Isl1+ and Wt1+ progenitors and their derivatives using tamoxifen‐inducible Isl1 MerCreMer/+;R26 mTmG/+ and Wt1 CreERT2/+;R26 mTmG/+ mouse lines. Labeling was induced by tamoxifen treatment at E7.5, and embryos were analyzed at E9.5. Immunostaining of cTNT (red), Wt1 (magenta), Isl1 (magenta), and mG (green) in Isl1 MerCreMer/+;R26 mTmG/+ (upper panels) and Wt1 CreERT2/+;R26 mTmG/+ (lower panels). The boxed regions in the left panels are shown in higher magnification (in consecutive sections) in the four right panels a–d and a'–d' for Isl1 MerCreMer/+;R26 mTmG/+ and Wt1 CreERT2/+;R26 mTmG/+, respectively. Isl1 MerCreMer/+‐mediated mG labeling was absent in the PEO (a), but it was observed in epicardial cells (b), Isl1+ SHF progenitors (c), and CMs (d), as indicated by arrows. Wt1 CreERT2/+‐mediated mG labeling was detected in Wt1+ cells of the PEO (a') and in epicardial cells (b'), as indicated by arrows; mG expression was absent in Isl1+ SHF progenitors (c') and CMs (d'). Scale bars, 100 μm (left…

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  Figure EV3. Localization and quantification of mG‐labeled cells in Isl1 MerCreMer/+ ;R26 mTmG/+ and Wt1 Cre ERT 2/+ ;R26 mTmG/+ adult mouse hearts at P28 Temporal restriction of Cre‐mediated labeling of Isl1+ and Wt1+ progenitors and their derivatives using tamoxifen‐inducible Isl1 MerCreMer/+;R26 mTmG/+ and Wt1 CreERT2/+ ;R26 mTmG/+ mouse lines. Labeling was induced by tamoxifen treatment at E7.5, and adult mouse hearts were analyzed at postnatal day 28 (P28). Immunostaining of mG (green) and mT (red) of Isl1 MerCreMer/+ ;R26 mTmG/+ (left panel) and Wt1 CreERT2/+ ;R26 mTmG/+ (right panel). Nuclei are stained with Hoechst 33258. Scale bars, 100 μm. la, left atrium; lv, left ventricle; ra, right atrium; rv, right ventricle.Table depicting the regional distribution and the amount of the mG‐labeled CMs (as number of clusters and CM number/cluster) and adipocytes (as percentage of total AV groove adipocytes) in Isl1 MerCreMer/+ ;R26 mTmG/+ and Wt1 CreERT2/+ ;R26 mTmG/+ adult mouse hearts at P28. Shown are schematic representations of the distribution of mG‐expressing cells per each analyzed heart and as a summarized overview for each line. Size of CM clusters is illustrated. LA, left atrium;…

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  Figure 3. A single Isl1/Wt1‐expressing progenitor gives rise to both CMs and adipocyte lineages in vitro AScheme for isolation of Isl1+/YFP+ cardiac precursors from the blastocyst‐derived mouse Isl1 Cre/+ ;R26 YFP/+ ESCs and their clonal analysis.B, CAssessment of YFP expression by bright field/epifluorescence microscopy (B) and qRT–PCR analysis of cardiac progenitor markers (C) during EB differentiation of Isl1 Cre/+ ;R26 YFP/+ ESCs. Scale bars, 200 μm; n = 4; *P < 0.05, **P < 0.01 vs. d0; t‐test. Data are shown as means ± SEM.DFlow cytometry analysis of YFP expression and qRT–PCR analysis of cardiac progenitor markers in YFP+ and YFP− cells sorted from 4‐day‐old Isl1 Cre/+ ;R26 YFP/+ EBs. FITC, fluorescein isothiocyanate; PE, phycoerythrin. n = 3; *P < 0.05 vs. YFP−; t‐test. Data are shown as means ± SEM.EImmunostaining of cTNT (red) and PPARγ (red) in conjunction with lipid stain BODIPY (green) of sorted cells from 4‐day‐old Isl1 Cre/+ ;R26 YFP/+ EBs after differentiation in pro‐myogenic and pro‐adipogenic culture conditions. Scale bars, 50 μm. The insets show higher magnifications, scale bars, 25 μm.FImmunostaining of Isl1 (cyan), Wt1 (red), and YFP (green) in a representative clone…

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  Figure 4. Cooperative role of WT1 and PPARγ in cardiomyocyte‐to‐adipocyte conversion AImmunostaining of endogenous WT1 (magenta), F‐actin (Phalloidin, green), and PPARγ (red) in wt and PKP2mut CMs at 0 and 28 days in culture. Arrows indicate PKP2mut cells with nuclear translocation of WT1, sarcomere disarray, and PPARγ activation. Scale bars, 25 μm.B, CImmunocytochemistry of wt CMs after 28‐day infection with lentiviral constructs encoding the nuclear‐tagged form of WT1 (NLS‐WT1) plus GFP or GFP alone showing GFP (green), cTNT (red) and PPARγ (magenta) (B), and GFP (green, left) and cTNT (blue) in conjunction with lipid stain ORO (red, right) (C). Scale bars, 25 μm (B) and 50 μm (C).D–FImmunocytochemistry of wt CMs after lentiviral overexpression of PPARγ alone (PPARγOE, D), PPARγ and NLS‐WT1 (NLS‐WT1/PPARγOE, E) or PPARγ, and shRNA targeting WT1 (shWT1/PPARγOE, F) at 14 (top) and 21 (bottom) days after infection. All constructs co‐express GFP. Representative images show GFP (green, left) and cTNT (blue) in conjunction with ORO stain (red, right) in the same cells. Scale bars, 50 μm.G, HqRT–PCR analysis of myocytic and adipocytic genes in PPARγOE (G), NLS‐WT1/PPARγOE (H), and shWT1/PPARγOE…

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  Figure EV4. Upregulated expression and aberrant subcellular localization of WT1 in iPSC‐derived PKP2mut CMs and native cardiac muscle cells from ARVC patient heart samples AqRT–PCR analysis of WT1 reveals elevated expression levels in iPSC‐derived PKP2mut compared to wt CMs; n = 3; *P < 0.05 vs. wt; t‐test. Data are shown as means ± SEM.BImmunostaining of cTNT (green) and WT1 (magenta) in wt and PKP2mut iPSC‐derived CMs. Note the filamentous‐like pattern of WT1 cytosolic expression. Nuclei are stained with Hoechst 33258 (blue). Scale bars, 25 μm.C, DAnalysis of adult human myocardium from patients affected and non‐affected by ARVC after immunostaining for cTNT (green) and WT1 (magenta). Nuclei are stained with Hoechst 33258 (blue). Low magnification of phase‐contrast images merged with immunoflourescence signals are shown in (C). Note intramyocardial fat infiltrations in ARVC conditions. Scale bars, 50 μm. High magnification images demonstrate nuclear localization of WT1 in CMs of ARVC patients (arrows), whereas in non‐ARVC individuals only cytosolic WT1 could be detected (D). Scale bars, 25 μm. Source data are available online for this figure.

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  Figure EV5. Forced expression of PPARγ and WT1 drives adipocytic conversion of mouse adult CMs in vivo AScheme of experimental setup for injection of adeno‐associated virus serotype 9 (AAV9) encoding NLS‐WT1 (AAV9‐NLS‐WT1) and PPARγ (AAV9‐NLS‐ PPARγ) in Myh6 Cre/+ ;R26 mTmG/+ mice and their analysis after 1 or 5 weeks. 2.5 × 1012 virus particles were injected intravenously via tail vein. Representative immunostainings of heart sections after 1‐week injection of each virus alone or in combination show CMs expressing the lineage marker mG (green) and the Wt1 (cyan) and PPARγ (red) transgenes. Nuclei are stained with Hoechst 33258. Arrowheads and arrows indicate mG+ CMs infected with one or both viruses, respectively. Scale bars, 50 μm. Quantification of transgene expressing mG+ CMs reveals high rate of co‐transduction. Three random heart slices per mouse and three mice per virus condition were analyzed.B, CAnalysis of heart sections 5 weeks after infection with AAV9‐NLS‐WT1 and AAV9‐PPARγ. (B) shows a representative phase‐contrast (PH) image merged with BODIPY fluorescence signal (green) visualizing a lipid‐filled cell within the myocardium of a mouse that received both viruses. Higher…

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  Figure 5. Transcriptome and histone mark analyses reveal aberrant regulation of adipogenesis and impaired RhoA‐ and MRTF/SRF‐dependent gene programs in PKP2mut CMs AFunctional annotation of genome‐wide differentially expressed genes in PKP2mut CMs with gene set enrichment analysis (GSEA).BGSEA of gene sets for adipogenesis (left). For each GSEA, the normalized enrichment score (NES) and P‐value are specified. Relative expression of the “leading edge” genes of the three adipogenic GSEAs is shown in heatmaps on the right. Expression level is represented as a gradient from low (blue) to high (red).CGenome browser representation for H3K4me3, H3K27me3, and H3K27ac tracks at the PPARγ locus in wt CMs identified by ChIP‐Seq (top). qPCR analyses of H3K4me3‐, H3K27me3‐, and H3K27ac‐ChIP on p1 and p2 peaks (shaded in gray) at the PPARγ locus in wt and PKP2mut CMs and human adipocytes (bottom); n = 2; *P < 0.05 vs. wt; t‐test.D, EPlots of the BERENJENO_TRANSFORMED_BY_RHOA_FOREVER_UP (D) and SRF_C and SRF_01 (E) GSEAs. NES and P‐value are shown. Relative expression of the “leading edge” genes of the GSEA is shown in the concomitant heatmaps. Direct MRTF‐SRF downstream target genes are highlighted in…

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  Figure 6. Decreased RhoA signaling downstream of defective cell–cell contacts leads to reduced nuclear MRTF‐A localization and derepression of PPARγ AImmunostaining of cTNT (cyan), PKP2 (red), and RhoA (green) in wt and PKP2mut CMs. Scale bars, 12.5 μm.BRhoA activity in wt and PKP2mut CMs at 0 and 1 day in culture; n = 3; *P < 0.05, **P < 0.001 vs. wt at the same time point; t‐test.CImmunostaining of F‐actin (Phalloidin, green) and actin (red) in wt and PKP2mut CMs at 0 and 7 days in culture. Scale bars, 12.5 μm. Bar graph shows quantification of G‐actin/F‐actin ratio in wt and PKP2mut CMs; N = 10 (wt) and N = 9 (PKP2mut) cells; *P < 0.01 vs. wt at the same time point; Mann–Whitney U‐test.D, ELive imaging of MRTF‐A subcellular localization in wt and PKP2mut CMs by transient expression of doxycycline‐inducible MRTF‐A‐GFP. Images in (D) show representative CMs at d0 in 20% serum (left) and after subsequent treatment with adipogenic medium for 1 h (middle). Images in (E) show representative CMs in adipogenic medium at 7 days, before (left) and after treatment with 20 nM LMB for 1 h (middle). Small panels on the right show magnified views of the boxed areas. Arrows indicate dynamic changes of…

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  Figure 7. Pharmacological and genetic perturbation of RhoA‐mediated cytoskeleton remodeling induces myocyte–adipocyte conversion A, BImmunostaining of F‐actin (Phalloidin, green) and PKP2 (red) in wt CMs treated and non‐treated with 30 μM ROCK inhibitor Y‐27632 for 14 days (A). Immunofluorescence analysis of WT1 (magenta), F‐actin (Phalloidin, green), and PPARγ (red) in wt CMs treated and non‐treated with 30 μM Y‐27632 for 28 days. Arrow indicates a cell with severe remodeling of the actin cytoskeleton, nuclear shuttling of WT1, and PPARγ expression (B). Scale bars, 25 μm.CImmunodetection of cTNT (blue) and lipid stain ORO (red) in wt CMs untreated and treated with 30 μM Y‐27632 for 28 days. Scale bars, 50 μm. Bar graph shows the percentage of adipocyte‐like cells in treated and untreated conditions; n = 3; N = 600 (untreated) and N = 592 (Y‐27632) cells; *P < 0.05 vs. untreated cells; t‐test.D, EImmunostaining of cTNT (green) and NMIIB (red) (D) or cTNT (cyan), PKP2 (red), and RhoA (green) (E) in wt and MYH10mut CMs. Scale bars, 12.5 μm.FImmunostaining of F‐actin (Phalloidin, green) and actin (red) and quantification of G‐actin/F‐actin ratio in wt and MYH10mut CMs. Scale bars, 12.5 μm. N…