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. Author manuscript; available in PMC: 2009 Oct 26.
Published in final edited form as: Circ Res. 2009 Apr 9;104(9):1076–1084. doi: 10.1161/CIRCRESAHA.109.196899

Genetic Fate Mapping Identifies Second Heart Field Progenitor Cells As a Source of Adipocytes in Arrhythmogenic Right Ventricular Cardiomyopathy

Raffaella Lombardi 1, Jinjiang Dong 1, Gabriela Rodriguez 1, Achim Bell 1, Tack Ki Leung 1, Robert J Schwartz 1, James T Willerson 1, Ramon Brugada 1, Ali J Marian 1
PMCID: PMC2767296  NIHMSID: NIHMS113913  PMID: 19359597

Abstract

The phenotypic hallmark of arrhythmogenic right ventricular cardiomyopathy, a genetic disease of desmosomal proteins, is fibroadipocytic replacement of the right ventricle. Cellular origin of excess adipocytes, the responsible mechanism(s) and the basis for predominant involvement of the right ventricle are unknown. We generated 3 sets of lineage tracer mice regulated by cardiac lineage promoters α-myosin heavy chain (αMyHC), Nkx2.5, or Mef2C. We conditionally expressed the reporter enhanced yellow fluorescent protein while concomitantly deleting the desmosomal protein desmoplakin in cardiac myocyte lineages using the Cre-LoxP technique. Lineage tracer mice showed excess fibroadiposis and increased numbers of adipocytes in the hearts. Few adipocytes in the hearts of αMyHC-regulated lineage tracer mice, but the majority of adipocytes in the hearts of Nkx2.5- and Mef2C-regulated lineage tracer mice, expressed enhanced yellow fluorescent protein. In addition, rare cells coexpressed adipogenic transcription factors and the second heart field markers Isl1 and Mef2C in the lineage tracer mouse hearts and in human myocardium from patients with arrhythmogenic right ventricular cardiomyopathy. To delineate the responsible mechanism, we generated transgenic mice expressing desmosomal protein plakoglobin in myocyte lineages. Transgene plakoglobin translocated to nucleus, detected by immunoblotting and immunofluorescence staining and coimmunoprecipitated with Tcf7l2, a canonical Wnt signaling transcription factor. Expression levels of canonical Wnt/Tcf7l2 targets bone morphogenetic protein 7 and Wnt5b, which promote adipogenesis, were increased and expression level of connective tissue growth factor, an inhibitor of adipogenesis, was decreased. We conclude adipocytes in arrhythmogenic right ventricular cardiomyopathy originate from the second heart field cardiac progenitors, which switch to an adipogenic fate because of suppressed canonical Wnt signaling by nuclear plakoglobin.

Keywords: adipocytes, progenitor cells, Wnt signaling, desmosomes, heart failure

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic disease characterized by the unique phenotype of fibroadipocytic replacement of cardiac myocytes, predominantly in the right ventricle.1–3 Clinical manifestations of ARVC include ventricular arrhythmias, typically originating from the right ventricle; sudden cardiac death, which often is its first manifestation; and right ventricular aneurysmal dilatation and failure.1,4 The left ventricle is also commonly involved in the advanced stages.1,5

ARVC is typically an autosomal dominant disease. Recessive forms in conjunction with palmoplantar keratoderma and woolly hair (Naxos disease) or predominant involvement of the left ventricle (Carvajal syndrome) are referred to as “cardiocutaneous syndromes.”6,7 Recently, mutations in 5 genes for ARVC, namely, DSP, JUP, PKP2, DSC2, and DSG2, encoding desmosomal proteins desmoplakin (Dsp), plakoglobin (PG), plakophilin 2, desmocollin 2, and desmoglein 2, respectively, have been identified.6,8–12 Hence, ARVC, at least in a subset, is a disease of desmosomes, intercellular junction structure responsible for cell-cell adhesion in epidermal cells and cardiac myocytes.

Pathogenesis of ARVC is not fully understood. Impaired myocyte to myocyte attachment because of defective desmosomes may explain cardiac dysfunction.13 It does not, however, explain the pathogenesis of the unique phenotype of fibro-fatty replacement of the myocardium and the cellular origin of excess adipocytes. Heart is a heterogeneous organ. It contains myocytes, fibroblasts, adipocytes, smooth muscle cells, endothelial cells, and pericytes, as well as circulating cells that implant in the myocardium. We posit the cell type that gives rise to adipocytes in ARVC must either express the mutant desmosomal protein or differentiate into adipocytes through a paracrine mechanism(s) emanating from cells expressing the mutant desmosomal protein. In the heart, the only cell type known to express desmosomal proteins is cardiac myocyte lineage. Adult cardiac myocytes are terminally differentiated and, hence, not plausible candidates to dedifferentiate to adipocytes.14 In contrast, cardiac progenitor cells expressing the desmosomal proteins might have the potential to differentiate to adipocytes. To test this hypothesis, we performed genetic fate-mapping experiments using the LoxP-Cre technology regulated by 3 cardiac lineage promoters. We extended the results of lineage tracing experiments to hearts of humans with autopsy-proven ARVC. We show adipocytes in the lineage tracer mice and in human hearts originate from the second heart field progenitor cells, which switch to an adipogenic fate because of suppressed canonical Wnt signaling by nuclear PG.

Materials and Methods

An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.

Genetically Modified Mice

Dsp-floxed, Nkx2.5-Cre, Mef2C-Cre, and αMyHC-Cre mice have been published.13,15–17 R26-EYFP was a generous gift from Dr F. Costantini.18 We generated the PG transgenic mice by cloning a Flag-tagged full-length PG cDNA downstream to the conventional 5.5-kbp α-myosin heavy chain (αMyHC) promoter.

M-Mode, 2-Dimensional, and Doppler Echocardiography

We performed echocardiography in 7 to 12 mice per group under sodium pentobarbital-induced anesthesia.13,19

Gross and Histological Cardiac Phenotype

An investigator without knowledge of the genotypes analyzed morphological and histological phenotypes in 5 to 9 age-, sex-, and body weight–matched mice per group.13,19,20 We calculated myocyte cross-sectional area in ≈18 000 myocytes per mouse by digital morphometry of fresh frozen thin myocardial sections stained with Texas red– conjugated wheat germ agglutinin (Molecular Probes Inc, Eugene, Ore).19,20

Immunoblotting

We electrophoresed 30-μg aliquots of cardiac protein extracts on 12% SDS polyacrylamide gels, transferred the proteins to membranes and probed them with the specific antibodies. Following stripping, we reprobed the membranes with an anti–α-tubulin antibody.

Coimmunoprecipitation

We performed coimmunoprecipitation as published previously.19 In brief, we mixed 6-μg aliquots of anti-Tcf7l2 monoclonal antibody (Millipore–Upstate, Billerica, Mass) with 500-μg aliquots of total protein extracts. We precipitated the antibody–protein complex by adding 20-μL aliquots of Protein A/G PLUS-Agarose beads. The primary antibodies used in immunoblotting were monoclonal anti–β-catenin (Santa Cruz Biotechnology Inc, Santa Cruz, Calif), anti-FLAG (Sigma, St Louis, Mo), and anti–total (transgene+endogenous) PG (Invitrogen–Zymed, Carlsbad, Calif). The secondary antibody was donkey anti-mouse IgG horseradish peroxidase– conjugated (Santa Cruz Biotechnology Inc).

Separation of Cardiac Protein Subfractions

We extracted nuclear, cytosolic and membrane proteins using a commercial kit (Chemicon International, Danvers, Mass).21 In brief, we homogenized the hearts in 5 volumes of a cold buffer containing HEPES (pH 7.9), MgCl2, KCl, EDTA, sucrose, glycerol, sodium orthovanadate, and protease inhibitors. We collected the supernatant containing the cytosolic proteins after centrifugation at 18 000g. We resuspended the pellets in 100 μL of a cold buffer containing HEPES, MgCl2, NaCl, EDTA, glycerol, sodium orthovanadate, and proteinase inhibitors and centrifuged and collected the supernatant containing the nuclear proteins. We resuspended the residual pellets in 100 μL of a cold buffer containing HEPES, MgCl2, KCl, EDTA, sucrose, glycerol, sodium deoxycholate, NP-40, sodium orthovanadate, and protease inhibitors and centrifuged to collect the supernatant containing the membrane proteins.

To detect expression of Flag-tagged PG (transgene), we probed the membranes with a rabbit anti–DYKDDDDK-Tag antibody (Cell Signaling Technology Inc, Danvers, Mass). To detect the endogenous and the Flag-tagged PG simultaneously, we probed the membranes with a rabbit anti-PG antibody. The secondary antibody was donkey anti-rabbit IgG horseradish peroxidase– conjugated (Santa Cruz Biotechnology Inc).

Immunofluorescence

We embedded thin myocardial sections from mid-ventricle in optimal cutting temperature compound (Sakura Finetek Inc, Torrance, Calif) and flash-froze them in an isopentane-liquid nitrogen bath. We stained the sections with the primary antibodies in 5% donkey or goat normal serum (Santa Cruz Biotechnology Inc). The primary antibodies (Santa Cruz Biotechnology Inc, unless otherwise specified) were goat polyclonal anti-Islet1 (Isl1), rabbit polyclonal anti-Mef2C (Aviva System Biology LLC, San Diego, Calif), rabbit polyclonal anti– enhanced yellow fluorescent protein (EYFP), rabbit polyclonal anti–peroxisome proliferator-activated receptor (PPAR)-γ, goat polyclonal anti–C/EBP-α, rabbit polyclonal anti–C/EBP-α, rabbit polyclonal anti-PG, rabbit anti-Flag (Cell Signaling Technology Inc, Beverly, Mass), and rat monoclonal anti–F4/80 (Abcam, Cambridge, Mass), the latter as a macrophage-specific marker. The secondary antibodies were FITC-labeled donkey anti-rabbit IgG and anti-goat IgG, FITC-labeled goat anti-rabbit IgG, Texas red–labeled goat anti-rat IgG, and Texas red–labeled donkey anti-goat IgG. The sections were mounted in DAPI-containing Hard Set mounting medium (Vector Laboratories Inc, Burlingame, Calif) and examined under fluorescence microscopy.

Additional control groups for immunofluorescence included myocardial sections stained with the corresponding IgG isotypes and secondary antibodies. Likewise, to further demonstrate the specificity of staining of cardiac adipocytes for expression of EYFP, we stained tissue sections from visceral and subcutaneous fats from the wild-type (WT) and lineage tracer mice using the same antibodies that were used for staining of cardiac sections.

To quantify the number of cardiac adipocytes expressing EYFP, we stained frozen thin myocardial sections from WT and lineage tracer mice for oil red O and the corresponding adjacent sections for expression EYFP and C/EBPα. We scored the number of cells that stained positive for oil red O and for coexpression of C/EBPα and EYFP in 14 to 44 thin sections per mouse and in 5 to 9 mice per group.

Human Myocardial Sections

The results of genetic fate-mapping experiments showing rare cardiac cells that coexpressed adipogenic and the second heart field markers and given that fibroadiposis in humans is an evolving and progressive phenotype, we postulated that human hearts with ARVC might contain rare cells in transition from a myogenic to an adipogenic fate. Therefore, we costained paraffin-embedded thin myocardial sections from 3 human patients with autopsy-proven ARVC who died of sudden cardiac death (ages 28 to 38 years) for coexpression of Isl1 or Mef2C with C/EBP-α or PPAR-γ. As a control, we stained thin sections of a normal heart for the above markers.

Statistical Analysis

Statistical calculations (STATA-Intercooled version 10.1, StataCorp LP, College Station, Tex) were as published.22 We compared the differences for normally distributed continuous variables among the 3 groups by ANOVA, followed by pairwise comparisons by Bonferroni method. We analyzed variables that violated the normality assumption and the nonparametric variables by Kruskal–Wallis test.

Results

Generation of Dsp-Deficient Lineage Tracer Mice

Using the LoxP-Cre technology, we generated 3 sets of lineage tracer mice regulated by cardiac lineage promoters Nkx2.5, an early and pan-specific cardiogenic marker; Mef2C, a second heart field-specific marker; and αMyHC, a relatively late marker of cardiogenesis (Figure 1). We screened the offspring for the presence of floxed alleles and the Cre recombinase by PCR (oligonucleotide primers are shown in Online Table I). Distribution of the genotypes of the offspring for Dsp-deficient mice deviated from the expected Mendelian inheritance, because no liveborn homozygous Dsp-deficient mouse was identified. The finding suggests embryonic lethality, as was reported previously in conditional deletion of Dsp in the heart and in the germ line.13,23 Therefore, the experiments were performed in heterozygous Dsp-deficient mice, which genetically represent human autosomal dominant ARVC.5,24,25 We confirmed expression of the reporter protein EYFP in the heart by immunoblotting using an anti-EYFP antibody (Figure 1).

Figure 1.

Figure 1

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Diagram illustrating generation of lineage tracer Dsp-deficient mice. Mice with floxed exon 2, the site of the initiation codon of Dsp (A), were crossed to EYFP mice (B), in which expression of EYFP is blocked by an upstream loxP-flanked STOP sequence. The offspring carrying both floxed transgenes (C) were crossed to Cre deleter mice (D), the latter being regulated by cardiac lineage promoters Nkx2.5, Mef2C, or αMyHC. E, Recombined Dsp and EYFP alleles. F, Expression of an ≈30-kDa band corresponding to the size of EYFP protein in Nkx-Cre:R26-EFYPF/F and Nkx2.5-Cre:DspW/F:R26-EYFPF/F mice.

Cardiac Phenotype in Control and Lineage Tracer Mice

We did not detect a discernible cardiac phenotype in mice expressing EYFP or Cre recombinase alone. Cardiac histology, analyzed by hematoxylin/eosin, Masson trichrome and oil red O staining of thin myocardial sections, was normal in the αMyHC-Cre, Nkx2.5-Cre, Mef2C-Cre, R26-EYFPF/F, and αMyHC- or Nkx2.5- or Mef2C-Cre:R26-EYFPF/F mice. Cardiac size and function, determined by M-mode, 2D, and Doppler echocardiography, were also normal in the above mice. Likewise, expression of EYFP in the background of Dsp deficiency did not influence cardiac phenotype because there were no significant differences in cardiac phenotype between Nkx2.5-Cre:DspW/F and Nkx2.5-Cre:DspW/F:R26-EYFPF/F or Mef2C-Cre:DspW/F and Mef2C-Cre:DspW/F:R26-EYFPF/F or αMyHC-Cre:DspW/F and αMyHC-Cre:DspW/F:R26-EYFPF/F mice. Therefore, for brevity, data on WT (control) and 3 sets of lineage tracer mice (αMyHC- or Nkx2.5- or Mef2C-Cre:DspW/F:R26-EYFPF/F) is presented along with relevant data on Nkx2.5-Cre:DspW/F and Mef2C-Cre:DspW/F, whenever appropriate.

Ventricular weight/body weight ratio was increased in the αMyHC-Cre:DspW/F:R26-EYFPF/F and Nkx2.5-Cre:DspW/F:R26-EYFPF/F mice but not in Mef2C-Cre:DspW/F:R26-EYFPF/F mice, as compared to WT mice (Figure I in the online data supplement). Likewise, ventricular weight/body weight ratio was also increased in the Nkx2.5-Cre:DspW/F (without EYFP) mice as was reported in the αMyHC-Cre:DspW/F mice.13 The αMyHC and Nkx2.5 regulated lineage tracer mice as well as the Nkx2.5-Cre:DspW/F mice exhibited left ventricular enlargement and dysfunction (Online Figure II and Online Table II). In contrast, left ventricular size and function were normal in Mef2C-Cre:DspW/F:R26-EYFPF/F (and Mef2C-Cre:DspW/F) mice (Online Figure II and Online Table II). The latter findings reflect specificity of the Mef2C-Cre in deleting Dsp in the second heart field, which gives rise to the right but not the left ventricle.

Myocardial histology in the lineage tracer mice was remarkable for excess fibroadiposis (Figure 2). Adipocytes, scored from oil red O–stained sections, comprised 0.16±0.02% of the total cells examined in the hearts of 3- to 6-month-old WT mice (N=5 mice, 10 800±1643 total cells per mouse). The corresponding percentages in the hearts of αMyHC-Cre–, Nkx2.5-Cre–, and Mef2C-Cre–regulated lineage tracer mice were 0.28±0.14% (N=7 mice, 10 000±4898 total cells per mouse), 0.44±0.26% (N=9 mice, 11 625±3739 total cells per mouse), and 0.63±0.32% (N=5 mice, 21 000±6363 total cells per mouse; all pair wise probability values versus WT mice were <0.05). As observed in human ARVC, fibroadiposis was more prominent at the epicardium in the lineage tracer mice (Figure 2). In accordance with the morphological data on cardiac size, cardiac myocyte cross-sectional area was also increased in the αMyHC-Cre:DspW/F:R26-EYFPF/F and Nkx2.5-Cre:DspW/F:R26-EYFPF/F but not in the Mef2C-Cre:DspW/F:R26-EYFPF/F mice, as compared with WT mice (Figure 2).

Figure 2.

Figure 2

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Fibroadiposis in the lineage tracer mice. Myocardial transverse sections stained for oil red O (ORO), hematoxylin/eosin (H&E), Masson trichrome (MT), and wheat germ agglutinin Texas red (WGA-TR) conjugate are shown from WT, αMyHC-Cre:DspW/F:R26-EYFPF/F, Nkx2.5-Cre:DspW/F:R26-EYFPF/F, and Mef2C-Cre:DspW/F:R26-EYFPF/F mice. Patchy areas of myocyte drop out and fibroadiposis were present in all 3 sets of lineage tracer mice. Fibroadiposis in the Mef2C-regulated lineage tracer mice was restricted to the right ventricle. Cardiac myocyte size was increased in αMyHC and Nkx2.5 but not in Mef2C-regulated lineage tracer mice (WT: 4458±182; αMyHC: 4830±151; Nkx2.5: 4891±96; Mef2C: 4659±42; N=3 to 6; pairwise probability values for αMyHC, Nkx2.5, and Mef2C tracer vs WT mice were 0.024, 0.009, and 0.458, respectively).

Lineage Tracing of Excess Adipocytes

To determine the origin of excess adipocytes in the heart of lineage tracer mice, we coimmunostained myocardial sections with antibodies against EYFP and C/EBP-α, an adipogenic transcription factor. EYFP and C/EBP-α were coexpressed in epicardial adipocytes in the hearts of Nkx-2.5– and Mef2C-regulated lineage tracer mice (Figure 3 and Online Figure III, A). The number of adipocytes, identified by C/EBPα stained cells, that stained positive for EYFP varied. On average, 78% of adipocytes in the hearts of Mef2C-Cre:DspW/F:EYFPF/F costained positive for EYFP (N=6 mice, 100±57/128±73 adipocytes per mouse). A similar percentage of adipocytes in the hearts of Nkx2.5-Cre:DspW/F:EYFPF/F mice also expressed EYFP (N=9, 40±32/51±33 adipocytes per mouse). In contrast, only few adipocytes (4/279, 1.4%) in the αMyHC-Cre:DspW/F:EYFPF/F lineage tracer mice hearts stained positive for EYFP expression.

Figure 3.

Figure 3

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Coexpression of EYFP and C/EBP-α in adipocytes in the hearts of lineage tracer mice. Oil red O and corresponding DAPI, C/EBP-α, and EYFP costained sections, along with merged images, are shown. C/EBP-α and EYFP are coexpressed in adipocytes in the hearts of Nkx2.5:DspW/F:R26-EYFPF/F and Mef2C-Cre:DspW/F:R26-EYFPF/F mice.

To establish the specificity of antibodies in detecting EYFP and C/EBPα, we stained myocardial sections with the IgG isotypes of the primary antibodies and the secondary antibodies. The isotypes and secondary antibodies did not react with EYFP and C/EBPα (Online Figure III, B). To further substantiate the specificity of the findings, we stained visceral and subcutaneous adipose tissues with the same antibodies against EYFP and C/EBPα. Adipocytes from visceral and subcutaneous adipose tissues did not express EYFP, further indicating cardiac specificity (Online Figure IV).

Detection of expression of EYFP in adipocytes from the Mef2C-regulated lineage tracer mice indicated an origin from the second heart field progenitors. We surmised that the hearts in the ARVC mice might contain cells in transition from a myogenic to an adipogenic fate. To test this hypothesis and further validate the finding in Mef2C-regulated lineage tracer mice, we costained myocardial sections with antibodies against Isl1 or Mef2C and C/EBP-α or PPAR-γ. We detected rare cells (0 to 6 cells per heart) in the hearts of Nkx2.5- and Mef2C-regulated lineage tracer mice that coexpressed Isl1 and PPAR-γ or Mef2C and C/EBP-α (Figure 4A and 4B). We did not detect expression of second heart field markers in the few EYFP-expressing adipocytes in the hearts of αMyHC-regulated lineage tracer mice.

Figure 4.

Figure 4

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Coexpression of second heart field and adipogenic transcription factors in the hearts of lineage tracer mice. A, Oil red O–stained and coimmunostained thin myocardial sections for DNA (DAPI), Islet-1 (Isl1), a second heart field marker; and PPAR-γ, an adipogenic transcription factor, are shown. Rare cells showing coexpression of Isl1 and PPAR-γ in the hearts of Nkx2.5:DspW/F:R26-EYFPF/F and Mef2C-Cre:DspW/F:R26-EYFPF/F lineage tracer mice were detected. B, Individual and merged images showing coexpression of second heart field marker Mef2C and adipogenic marker C/EBP-α are presented along with oil red O–stained image. As shown, Mef2C and C/EBP-α are coexpressed in a subset of adipocytes in the hearts of Nkx2.5:DspW/F:R26-EYFPF/F and Mef2C:DspW/F:R26-EYFPF/F lineage tracer mice.

Because Isl1 is not an exclusive cardiac lineage marker, to exclude the possibility that macrophages were the rare cells that expressed the second field markers, we costained thin myocardial sections with an antibody against F4/80, a macrophage-specific marker and EYFP. We included the corresponding oil red O–stained section and thin sections from spleen as controls. The results (Online Figure V) showed adipocytes in the heart of lineage tracer mice expressed EYFP but not F4/80 macrophage marker.

Detection of Expression of Isl1 and Mef2C in Adipocytes in Human Hearts With ARVC

To corroborate the results of lineage tracing studies in mice in human patients, we costained thin right ventricular sections from 3 human patients with autopsy-proven ARVC for cardiac progenitor markers and adipogenic transcription factors. In accordance with our findings in the genetic fate-mapping studies in mice, we detected rare cells (1 to 3 cells per heart) in the fibroadiposis area in the myocardium that coexpressed Isl1 or Mef2C and the adipogenic transcription factor C/EBP-α (Figure 5 and Online Figure VI).

Figure 5.

Figure 5

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Coexpression of second heart field and adipogenic markers in the hearts of human patients with autopsy-proven ARVC. Bright field and costained sections for Isl1, C/EBP-α, and DNA (A) or Mef2C, C/EBP-α, and DNA (B) in the heart of patients with ARVC who died suddenly. Rare cells coexpressing second heart field markers Isl1 or Mef2C and C/EBPα were detected in the human hearts with ARVC.

Nuclear PG Promotes Adipogenesis Through Suppression of the Canonical Wnt Signaling

We have implicated PG, an armadillo member of desmosomal protein with signaling function,26–28 in the pathogenesis of ARVC.13 Likewise, Wnt signaling is implicated in development of the second heart field (right ventricle), the predominant site of involvement in ARVC.29,30 To elucidate a mechanism for differentiation of second heart field progenitors to adipocytes in ARVC, we expressed Flag-tagged PG in cardiac myocytes through transgenesis under transcriptional regulation of the αMyHC promoter. Expression level of the Flag-tagged transgene PG comprised approximately 25% to 35% of the total PG levels in the heart (Figure 6). The PG transgenic mice showed excess adipocytes in the heart along with patchy areas of fibrosis, an intact desmosome structure and normal left ventricular function (Figure 6 and Online Figure VII).

Figure 6.

Figure 6

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Cardiac phenotype in PG transgenic mice. A, PG transgene construct showing 5-Flag–tagged full-length PG cDNA positioned downstream to a 5.5-kbp αMyHC promoter. B, Immunoblots showing expression of transgene and endogenous PG proteins detected using Flag-specific and pan-PG–specific antibodies in nontransgenic (NTG) and transgenic mice. C, Oil red O (ORO)-, hematoxylin/eosin (H&E)-, and Masson trichrome (MT)-stained thin myocardial sections show fibroadiposis in the heart of PG transgenic mice. Immunofluorescence staining (IF) shows localization of PG to desmosomes.

Immunostaining of thin myocardial sections from the Flag-tagged PG transgenic mice as well as immunoblotting of cell protein subfractions showed nuclear localization of the transgene PG in cardiac myocytes (Figure 7A and 7B). To determine whether nuclear PG (also known as γ-catenin) interacted with protein constituents of the canonical Wnt signaling, we analyzed binding of PG with Tcf7l2 transcription factor by coimmunoprecipitation. Immunoblotting of the immunoprecipitates using transgene-specific anti-Flag and pan-PG antibodies showed binding of the transgene PG to Tcf7l2 (Figure 7C). In contrast, binding of β-catenin to Tcf7l2 in the PG transgenic hearts was reduced, implying competitive interactions between PG and β-catenin for binding to Tcf7l2.27

Figure 7.

Figure 7

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Pathogenesis of ARVC in PG transgenic mice. A and B show nuclear localization of transgene PG in myocytes by immunofluorescence staining, detected using a Flag-specific antibody, and by immunoblotting of subcellular protein fractions, detected using Flag-specific and pan-PG–specific antibodies. C illustrates binding of transgene PG to Tcf7l2, as detected by coprecipitation with an anti-Tcf7l2 antibody, which was increased in the PG transgenic mice. The lower blot shows coprecipitation of β-catenin with an anti-Tcf7l2 antibody, which was reduced in the PG transgenic mice. D through G, Quantification of expression levels of selected canonical Wnt signaling by real-time PCR. Expression level of cMyc, a known target of the canonical Wnt signaling, was reduced by ≈50%. Relative expression levels of Wnt5b and BMP7, which promote adipogenesis and are normally inhibited by the canonical Wnt signaling, were increased 3- to 4-fold in the transgenic mice. In contrast, expression level of CTGF, a negative regulator of adipogenesis, was suppressed by ≈5-fold.

To determine the biological effects of nuclear localization and binding of PG to Tcf7l2 on canonical Wnt signaling, we determined expression levels of selected canonical Wnt/β-catenin signaling target genes by quantitative PCR (N=4 per group, probes and primers sequence are provided in Online Table I and Online Figure VIII). Concordant with reduced binding of β-catenin to Tcf7l2 in the PG transgenic mice, expression of c-Myc, a known target of activation of the canonical Wnt signaling, was suppressed (Figure 7D). In addition, expression levels of adipogenic factors Wnt5b and BMP7, which are normally inhibited by the canonical Wnt signaling, were increased by 3- to 4-fold (Figure 7E and 7F).31,32 Recent data implicate BMP7 as a major regulator of switch from myogenesis to adipogenesis.32,33 In contrast, expression level of connective tissue growth factor (CTGF), which is known to inhibit adipogenesis,34 was reduced by ≈5-fold (Figure 7G).

Discussion

Through a series of genetic fate-mapping experiments in mice, we show adipocytes in ARVC originate from the second heart field cardiac progenitor cells. We corroborate the findings in autopsy-proven human hearts with ARVC by showing coexpression of second heart field markers, along with adipogenic transcription factors in rare cells in fibroadipocytic areas in the heart. At a mechanistic level, we show nuclear localization of desmosomal protein PG is associated with suppression of the canonical Wnt/β-catenin signaling and a transcriptional switch to adipogenesis through activation of BMP7 and Wnt5b and suppression of CTGF.31–34 The paucity of EYFP-expressing adipocytes in the hearts of αMyHC-regulated lineage tracer mice and a significantly higher percentage of EYFP-expressing adipocytes in the hearts of Mef2C- or Nkx2.5-regulated lineage tracer mice indicate that the adipogenic switch occurs before commitment of the progenitors to a myocyte lineage. The results indicate that adipocytes in ARVC originate from the second heart field progenitor cells, which switch to adipogenesis because of suppressed canonical Wnt/β-catenin signaling imparted by the nuclear PG (Online Figure IX).

In human patients with ARVC, fibroadiposis predominantly involves the right ventricle, a feature that has also been an enigma.1 The right ventricle primarily originates from the second (anterior) heart field as opposed to the left ventricle, which originates from the primary heart field.35–37 The 2 heart fields are distinguished by expression of different sets of transcriptional factors and signaling molecules. Accordingly, Nkx2.5 is common to both heart fields; however, Tbx5 and Hand1 mark the primary heart field, whereas Isl1, Mef2C, and Hand2 characterize the second heart field.29,35–37 Identification of the second heart field progenitors as the cell source of adipocytes in ARVC offers a plausible explanation for the predominant involvement of the right ventricle in ARVC. Our findings are also in accordance with the recent data emphasizing the significance of the canonical Wnt signaling in the formation of the right ventricle from the second heart field.29,30,38 It is also noteworthy that the canonical Wnt signaling is considered a major regulator of a switch between adipogenesis and myogenesis.39 Furthermore, changes in expression levels of BMP7, Wnt5b, and CTGF, which are targets of the canonical Wnt signaling, favored adipogenesis.31–33 However, the characteristics of the subset of the second heart field progenitors and complete molecular mechanisms that govern their differentiation to an adipogenic fate in ARVC remain unknown.

Our focus on cardiac progenitor cells as the cell source of adipocytes is based on the premise that in ARVC caused by desmosomal mutations the stimulus for adipocytic differentiation has to originate from cells that express the desmosomal proteins. There are other potential sources that could be categorized into 2 sets. First, cells other than the myogenic lineage in the heart may express the mutant desmosomal proteins and, hence, could directly differentiate into adipocytes. This seems unlikely, because the only cell type in the heart known to express desmosomal proteins is the myocyte lineage. Second, cells that do not express the desmosomal proteins could differentiate into adipocytes through paracrine mechanisms emanating from desmosome-defective myocytes. In preliminary studies, we have cocultured myocytes isolated from the hearts of Dsp-deficient mice with cardiac fibroblasts and have not detected enhanced differentiation of the fibroblasts to adipocytes. Nevertheless, cellularly, the heart is a heterogeneous organ, and, hence, the possible differentiation of cells such as pericytes, fibrocytes, or circulating cells that seed at the myocardium and differentiate to adipocytes cannot be completely dismissed.

In conclusions, we have traced the origin of excess adipocytes in desmosomal ARVC to second heart field progenitor cells, a finding that was also corroborated in human hearts with ARVC. We implicate suppression of the canonical Wnt signaling by nuclear PG as a mechanism for switching differentiation of cardiac progenitor cells to adipocytes. The findings also explain the predominant involvement of the right ventricle in ARVC.

Supplementary Material

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Online Figure I. Box plots showing median, 25%, 75%, outliers (•) and adjacent values of ventricular weight/body weight (VW/BW) ratio. VW/BW ratio was increased significantly in the αMyHC- (N=12) and Nkx2.5-regulated (N=24) but not in Mef-2C-regulated (N=12) Dsp-deficient (with and without EYFP), as compared to wild type (N=26) mice. The pair wise p values are corrected for multiple comparisons by Bonferroni’ method.

Online FigureII. Echocardiographic phenotype. Representative M-mode echocardiograms of the left ventricle from wild type, αMyHC-Cre:DspW/F:R26-EYFPF/F , Nkx2.5-Cre:DspW/F:R26- EYFPF/F , and Mef2C-Cre:DspW/F:R26-EYFPF/F mice are shown. Compared with non-transgenic mice (Panel A), Left ventricle diastolic and systolic diameters were increased and systolic function was reduced in αMyHC--Cre:DspW/F:R26-EYFPF/F and Nkx2.5-Cre:DspW/F:R26-EYFPF/F mice (Panels B and C). In contrast cardiac dimensions and function was preserved in Mef2C-Cre:DspW/F:R26-EYFPF/F mice (Panel D).

Online FigureIII. Panel A. Oil Red O, DAPI, C/EBP-α and EYFP co-stained sections in wild type (non-transgenic) mice are shown as controls. As shown, immunostaining did not show expression of C/EBP-α and EYFP in the heart of wild type mice.

Panel B. Additional controls including stained sections with IgG isotypes from the corresponding host animals for the primary antibodies are shown to further substantiate the specificity of the findings.

Online FigureIV. Oil Red O, DAPI, C/EBP-α and EYFP co-stained sections in visceral fat tissues from Nkx-Cre:DspW/F:R26-EYFPF/F and Mef2C-Cre:DspW/F:R26-EYFPF/F lineage tracer mice are shown as controls. As shown, EYFP is not expressed in visceral fat tissues from the lineage tracer mice.

Online FigureV. Exclusion of macrophages as a cell source of excess adipocytes in ARVC. A. Absence of expression of F4/80, a specific marker of macrophage in adipocytes in Nkx2.5-Cre:DspW/F:R26- EYFPF/F mice hearts. The adipocytes show expression of EYFP. B. Sections of spleen from Nkx2.5-Cre:DspW/F:R26-EYFPF/F mice showing expression of F4/80, as a positive control for antibody. C. Myocardial sections stained for Oil Red O and specific IgG isotypes, as controls for the specificity of the antibodies tested.

Online FigureVI. Bright field and co-stained sections for C/EBP-α and DNA, Mef2C and DNA or Isl1 and DNA in a normal heart, as controls.

Online Figure VII. M-Mode echocardiograms from wild type and plakoglobin (PG) transgenic mice. The time scale represent 200 msec intervals. As shown cardiac size and function were normal in the PG transgenic mice and similar to that in the wild type (non-transgenic) mice.

Online FigureVIII. Quantitative PCR amplification plots showing expression levels of GAPDH as control and four targets of the canonical Wnt signaling in non-transgenic and wild type PG transgenic mice. Amplification plots for GAPDH were practically superimposed in the control and transgenic mice. In contrast, expression levels of Wnt5b and BMP7, both known to be inhibited by the canonical Wnt signaling and both involved in adipogenesis, were increased by 3 to 4 folds. In accord with a shift to adipogenesis, relative expression level of CTGF, an inhibitor of adipogenesis was decreased by 5-fold. Similarly, relative expression level of c-Myc, activated by the canonical Wnt signaling, was down regulated by approximately 2-fold.

Online Figure IX. Pathogenesis of excess adipocytes in ARVC. Mutations in desmosomal proteins by disrupting proper desmosome assembly free plakoglobin (PG) to translocate from the desmosome to the nucleus. In the nucleus PG, also known as γ-catenin because of structural and functional similarityto it, suppresses the canonical Wnt signaling through Lef/Tcf transcription factors. The net effect is removal of the inhibitory effects of the canonical Wnt signaling on expression of BMP7 and Wnt5b, known promoters of adipogenesis and suppression of expression of CTGF, known inhibitor of adipogenesis. Together they promote differentiation of cardiac progenitor cells to adipocytes.

Acknowledgments

The αMyHC-Cre, Mef2C-Cre, and R26-EYFP mice were kind gifts from Drs Michael D. Schneider, Brian Black, and F. Costantini, respectively.

Sources of Funding Supported by National Heart, Lung, and Blood Institute grants R01-HL68884 and R01–088498 and a Burroughs Wellcome Award in Translational Research (1005907).

Footnotes

Disclosures None.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01

Online Figure I. Box plots showing median, 25%, 75%, outliers (•) and adjacent values of ventricular weight/body weight (VW/BW) ratio. VW/BW ratio was increased significantly in the αMyHC- (N=12) and Nkx2.5-regulated (N=24) but not in Mef-2C-regulated (N=12) Dsp-deficient (with and without EYFP), as compared to wild type (N=26) mice. The pair wise p values are corrected for multiple comparisons by Bonferroni’ method.

Online FigureII. Echocardiographic phenotype. Representative M-mode echocardiograms of the left ventricle from wild type, αMyHC-Cre:DspW/F:R26-EYFPF/F , Nkx2.5-Cre:DspW/F:R26- EYFPF/F , and Mef2C-Cre:DspW/F:R26-EYFPF/F mice are shown. Compared with non-transgenic mice (Panel A), Left ventricle diastolic and systolic diameters were increased and systolic function was reduced in αMyHC--Cre:DspW/F:R26-EYFPF/F and Nkx2.5-Cre:DspW/F:R26-EYFPF/F mice (Panels B and C). In contrast cardiac dimensions and function was preserved in Mef2C-Cre:DspW/F:R26-EYFPF/F mice (Panel D).

Online FigureIII. Panel A. Oil Red O, DAPI, C/EBP-α and EYFP co-stained sections in wild type (non-transgenic) mice are shown as controls. As shown, immunostaining did not show expression of C/EBP-α and EYFP in the heart of wild type mice.

Panel B. Additional controls including stained sections with IgG isotypes from the corresponding host animals for the primary antibodies are shown to further substantiate the specificity of the findings.

Online FigureIV. Oil Red O, DAPI, C/EBP-α and EYFP co-stained sections in visceral fat tissues from Nkx-Cre:DspW/F:R26-EYFPF/F and Mef2C-Cre:DspW/F:R26-EYFPF/F lineage tracer mice are shown as controls. As shown, EYFP is not expressed in visceral fat tissues from the lineage tracer mice.

Online FigureV. Exclusion of macrophages as a cell source of excess adipocytes in ARVC. A. Absence of expression of F4/80, a specific marker of macrophage in adipocytes in Nkx2.5-Cre:DspW/F:R26- EYFPF/F mice hearts. The adipocytes show expression of EYFP. B. Sections of spleen from Nkx2.5-Cre:DspW/F:R26-EYFPF/F mice showing expression of F4/80, as a positive control for antibody. C. Myocardial sections stained for Oil Red O and specific IgG isotypes, as controls for the specificity of the antibodies tested.

Online FigureVI. Bright field and co-stained sections for C/EBP-α and DNA, Mef2C and DNA or Isl1 and DNA in a normal heart, as controls.

Online Figure VII. M-Mode echocardiograms from wild type and plakoglobin (PG) transgenic mice. The time scale represent 200 msec intervals. As shown cardiac size and function were normal in the PG transgenic mice and similar to that in the wild type (non-transgenic) mice.

Online FigureVIII. Quantitative PCR amplification plots showing expression levels of GAPDH as control and four targets of the canonical Wnt signaling in non-transgenic and wild type PG transgenic mice. Amplification plots for GAPDH were practically superimposed in the control and transgenic mice. In contrast, expression levels of Wnt5b and BMP7, both known to be inhibited by the canonical Wnt signaling and both involved in adipogenesis, were increased by 3 to 4 folds. In accord with a shift to adipogenesis, relative expression level of CTGF, an inhibitor of adipogenesis was decreased by 5-fold. Similarly, relative expression level of c-Myc, activated by the canonical Wnt signaling, was down regulated by approximately 2-fold.

Online Figure IX. Pathogenesis of excess adipocytes in ARVC. Mutations in desmosomal proteins by disrupting proper desmosome assembly free plakoglobin (PG) to translocate from the desmosome to the nucleus. In the nucleus PG, also known as γ-catenin because of structural and functional similarityto it, suppresses the canonical Wnt signaling through Lef/Tcf transcription factors. The net effect is removal of the inhibitory effects of the canonical Wnt signaling on expression of BMP7 and Wnt5b, known promoters of adipogenesis and suppression of expression of CTGF, known inhibitor of adipogenesis. Together they promote differentiation of cardiac progenitor cells to adipocytes.

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