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. Author manuscript; available in PMC: 2026 Jun 9.
Published in final edited form as: Circ Heart Fail. 2026 Jun 5;19(7):e013966. doi: 10.1161/CIRCHEARTFAILURE.125.013966

Epicardial contributions to fibro-inflammatory signaling in a Pkp2-deficient arrhythmogenic cardiomyopathy model

Daniel D Han 1,2, Alan C Brooks 1, Cameron D Baker 3, Ronald A Dirkx Jr 1, Deanne M Mickelsen 1, Benjamin Fisler 4, Kavya Phadke 5, John M Ashton 3, Mario Delmar 5, Eric M Small 1,2,6,7,8
PMCID: PMC13245365  NIHMSID: NIHMS2179330  PMID: 42246055

Abstract

Background:

Arrhythmogenic Cardiomyopathy (ACM) is an inherited disease that is characterized by lethal ventricular arrhythmias stemming from myocyte dysfunction. ACM is associated with considerable subepicardial fibrosis and inflammation with right ventricle predominance. Most cases of gene positive ACM are caused by a desmosome protein mutation, with plakophilin-2 (Pkp2) mutations being most common. We hypothesized Pkp2-deficiency in epicardium-derived cells (EPDCs) contributes to fibro-inflammatory signaling and ACM pathogenesis.

Methods:

We developed transgenic mice that lack Pkp2 in cardiomyocytes (Pkp2-cKO), in EPDC (Pkp2-eKO), or in both cardiomyocyte and EPDC (Pkp2-ceKO) via the tissue-specific expression of tamoxifen-inducible Cre recombinase. Non-myocyte populations were isolated 21 days post-tamoxifen injection for single cell RNA-sequencing (scRNA-seq). Immunohistochemistry, flow cytometry, qRT-PCR, and echocardiography were used to interrogate cardiac physiology and cellular composition.

Results:

We identified a population of epicardium-derived fibroblasts characterized by the expression of Ccl2, Ccl7, Thbs1, and Ptx3 that accumulated upon Pkp2 deletion in both cardiomyocytes and EPDC. Pkp2 deletion in cardiomyoctyes induced a modereate fibro-inflammatory EPDC phenotype, while deletion in EPDC did not elicit a pathological phenotype, suggesting cardiomyocyte involvement is necessary for ACM pathogenesis. Pro-inflammatory fibroblasts acquired the senescence-associated secretory phenotype (SASP), correlating with elevated Senescence Associated (SA)-βgal staining in the right ventricle. Gene expression, flow cytometry, and histological data also revealed an exaggerated inflammatory response in Pkp2-ceKO mice, that progresses from right to left ventricular predominance. Importantly, macrophages and B cells accumulate in both Pkp2-cKO and Pkp2-ceKO mice compared to controls. While B cell depletion delays the early inflammatory and fibrosis response, it did not alter end-stage cardiac physiology.

Conclusion:

Pkp2 deletion in EPDC facilitates the emergence of a fibro-inflammatory phenotype that may contribute to ACM pathogenesis.

Keywords: Pkp2, Arrhythmogenic Cardiomyopathy, Epicardium, Fibroblast, Senescence, Macrophage, B cell

Introduction

Arrhythmogenic Cardiomyopathy (ACM), also known as the Arrhythmogenic Right Ventricular Cardiomyopathy/Dysplasia (ARVC/D), is an inherited cardiac disorder that primarily affects young adults1. Most cases of gene positive ACM are caused by mutations in genes that encode desmosome proteins, structural components of the cardiomyocyte intercalated disk. Plakophilin-2 (Pkp2) mutations account for around 40% of the observed desmosome protein mutations in ACM patients. Mutations in other desmosome proteins including desmoglein-2 (Dsg2), desmocollin-2 (Dsc2), desmoplakin (Dsp), and plakoglobin (Jup) occur less frequently2. The primary clinical manifestation of ACM is ventricular arrhythmia and ultimately sudden cardiac death (SCD) if untreated. ACM is the most common cause of SCD after hypertrophic cardiomyopathy among patients who are <35 years old3. As many patients with ACM are asymptomatic, misdiagnosis of ACM is common and SCD often occurs before diagnosis4.

The most notable cardiac phenotype of ACM is the progressive buildup of fibrofatty deposits5. Fibrofatty deposits initially form in the free wall of the right ventricle and progress into the left ventricle to cause biventricular fibrosis5. Furthermore, clinical studies have revealed that fibrofatty deposits are initially observed in the sub-epicardium, expanding later into the myocardium towards the endocardium6,7. The epicardium is a source of progenitor cells that differentiate into various non-myocyte cardiac lineages during embryonic development, including cardiac fibroblasts, smooth muscle cells, and pericytes8. The epicardium also becomes re-activated after myocardial insult, and expresses pro-inflammatory paracrine signals, suggesting the epicardium and its descendants may play a role in cardiac inflammation and remodeling after injury9,10.

Pkp2 is expressed in epicardial cells, and its deficiency alters the epicardial phenotype in vitro by reducing cell adhesions and stimulating epicardial to mesenchymal transition11,12. However, evidence that Pkp2-deficient epicardium-derived cells (EPDCs) contribute to ACM pathogenesis has not been demonstrated in vivo. Therefore, we generated mice lacking Pkp2 in cardiomyocytes (Pkp2-cKO) or in both cardiomyocytes and EPDCs (Pkp2-ceKO) to investigate potential contributions of Pkp2-deficient EPDCs in ACM pathogenesis. Using single-cell RNA sequencing (scRNA-seq) we defined the emergence of a pro-inflammatory fibroblast population, particularly in Pkp2-ceKO mice. The fibro-inflammatory program originating from EPDCs also induced cellular senescence and the induction of the senescence associated secretory phenotype (SASP). We further found Pkp2-ceKO mice exhibit an overabundance of macrophages, B cells, and T cells in the heart, progressing from RV to LV predominance. While ablating B cells in mice using a blocking antibody delayed the development of fibrosis, this approach did not alter end stage cardiac physiology. Taken together, our results establish the role of EPDCs in the emergence of a senescent and pro-inflammatory phenotype that may contribute to ACM disease pathogenesis.

Methods

Data availability.

Detailed methods can be found in Supplemental Methods. Primer sequences and antibody information are listed in Tables S1 and S2. scRNA-sequencing data has been deposited in the Gene Expression Omnibus (GEO) database under accession code GSE300141.

Animal Studies.

All experiments using animals were approved by the University Committee on Animal Resources at the University of Rochester.

Statistical analysis.

Replicates in this study were biological (not technical) unless specifically noted otherwise, and all data are reported as mean +/− SD. Statistical analyses were performed using Prism 9.1.1. (GraphPad). Normality was assessed by conducting D’Agostino& Pearson test, Anderson-Darling test, Shapiro-Wilk test, and Kolmogorov-Smirnov test for both Normality and Lognormality. If any of these tests failed, we have used non-parametric test. We have included all the data points and there was no exclusion of outliers. For comparison between two groups that were not normally distributed, non-parametric unpaired, two-tailed Mann Whiteny test was conducted. If normally distributed, the comparison was performed using unpaired, two-tailed t-tests with Welch’s correction for unequal standard deviations. To compare the differences among three or more groups of data that are not normally distributed, we used the Kruskal-Wallis test with Dunn’s post-hoc analysis. For groups of data that were normally distributed, one-way ANOVA was used with Holm- Šidák post-hoc analysis. Geisser-Greenhouse correction was used for repeated measures two-way ANOVA. Post-hoc comparisons were made using the Turkey test for two-way repeated measures ANOVA where three or more groups were compared. For non-continuous variables in scRNA-seq data analyses, Chi-square test was used for testing imbalance in proportionality across more than 2 categories. p < 0.05 was considered significant.

Results

Epicardium-endocardium gradient of fibro-inflammatory deposition following Pkp2 deletion

Using a previously developed mouse line that exhibits many of the clinical characteristics of ACM (arrhythmias, reduced LVEF, and sudden cardiac death) following cardiomyocyte (CM)-specific Pkp2 deletion (called Pkp2-cKO)13, we observed fibrotic foci originating at the epicardial surface (Figure S1A). To investigate the impact of Pkp2 deletion in EPDCs on fibrosis, we generated mouse lines harboring tamoxifen-inducible Cre recombinase under Myh6 responsive transgenes and constitutively active Cre recombinase under Wt1 responsive transgene (Wt1-CreBAC14 and Myh6-MerCreMer15), to cause recombination of floxed Pkp2 alleles in the epicardium and cardiomyocytes, respectively (Figure 1A, B). It is important to note that Wt1 CreBAC line allows Pkp2 to be knocked out in epicardial cells during development, when Wt1 is expressed9, 10, 14. These mice also harbor a RosamTmG allele to label the recombined cells with green fluorescence protein (GFP). Picro-sirius red stained heart sections showed comparable biventricular fibrosis in Pkp2-ceKO mice and Pkp2-cKO at 21 days post tamoxifen injection (dpi) (Figure 1C), which was significantly elevated compared to controls and Pkp2-eKO (epicardium deletion alone) mice (Figure 1C). Serial echocardiography also revealed comparable decline in cardiac physiology in Pkp2-ceKO and Pkp2-cKO, but not in Pkp2-eKO mice, which progresses from right to bi-ventricular predominance (Figure S1B). These findings reveal that both Pkp2-cKO and Pkp2-ceKO mice capture the fibrotic ACM pathophysiology.

Figure 1. Defining the single cell transcriptome of non-myocytes in a Pkp2 deficient mouse model of ACM.

Figure 1.

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A) Schematic describing Pkp2 gene deletion strategy in cardiomyocytes, epicardial cells, or both cell-types, compared to control. B) Schematic describing experimental timeline for tamoxifen injections, histology and scRNA-seq. C) Picrosirius Red stained heart sections (left) and quantification (right) in the left ventricle (LV) and right ventricle (RV) of various genotypes (Cre control n=6, Pkp2-eKO n=6, Pkp2-cKO n=5, Pkp2-ceKO n=6). Quantification of LV and RV fibrosis represented by mean +/− SD. Scale bar=1 mm. D) Hierarchical clustering based on the 10 most differentially expressed genes (left) and uniform manifold approximation and projection (UMAP, right top) defines non-myocyte identities from Cre Control, Pkp2-cKO and Pkp2-ceKO mice. Data is obtained from cells pooled from n=2 hearts per genotype. Epicardium-derived cells were identified as GFP expressing cells (right bottom).

Single cell transcriptomics identifies pro-inflammatory fibroblasts in Pkp2-ceKO mice

Since EPDCs give rise to fibroblasts and Pkp2 is expressed in the epicardium11, we probed whether Pkp2 deletion in EPDCs alters their transcriptional phenotype. Hearts from mice of genotypes described above were digested via Langendorff Perfusion at 21 dpi and the non-myocyte fraction was subjected to single cell RNA sequencing (sc-RNAseq) using the 10X Genomics platform. We observed comparable RNA features, counts, and percent mitochondrial between the various genotypes (Figure S2A). Hierarchical clustering and uniform manifold approximation and projection (UMAP) captured the transcriptional signature of fibroblasts, endothelial cells, mural cells, and immune cells (Figure 1D and Table S3). Importantly, non-myocyte proportions defined here transcriptionally were nearly identical to previously reported non-myocyte proportions defined by flow cytometry16 (Figure S2B). EPDCs were identified based on expression of eGFP, revealing a majority of EPDCs are mesenchymal with minimal contributions to endothelial cell populations (Figure 1D, Figure S3A, B).

UMAP revealed the expression of Tcf21 and Pdgfrα across all cell populations to define epicardium-derived fibroblasts (Figure 2A). We defined 3 pro-fibrotic clusters (4, 5, and 6) based on high expression of Postn and various collagens and a reduction in Tcf21 expression (Figure 2A, B). Cluster 4 contains activated fibroblasts based on co-expression of Meox117 (Figure 2A), while cluster 6 is composed of activated pericytes based on the lack of Meox1 and the robust expression of Pdgfrb and Cspg418 (Figure 2B). Notably, cluster 5 exhibits a distinct pro-inflammatory phenotype based on the expression of pro-inflammatory cytokines including Ptx3, Ccl2, Ccl7, Thbs1, and Il6 (Figure 2B). When pseudobulk analysis of sc-RNAseq was performed across all the genotypes, we observed an inverse relationship between pro-inflammatory and fibrosis marker genes at 21dpi, with Pkp2-ceKO mice exhibiting higher levels of pro-inflammatory cytokines and lower levels of fibrosis markers, compared to Pkp2-cKO mice (Figure 2C, D). Indeed, Pathway Enrichment analysis revealed an over-representation of genes regulated by transcription factors related to immune pathways in Pkp2-ceKO such as Rela, Relb, Stat3, Brd4, Runx1, and Runx2 in Pkp2-ceKO non-myocytes (Figure S3C). Importantly, overlaying genotype onto UMAP space in conjunction with proportionality analyses correlated the emergence of a robust pro-inflammatory fibroblast phenotype in Pkp2-ceKO mice with a significant immune cell burden (Figure 2E and S4A). Indeed, raw counts reveal a near complete absence of pro-inflammatory fibroblasts in control mice, which expand to become a major component of the non-myocyte fraction in Pkp2-ceKO mice.

Figure 2. scRNA-seq reveals excessive fibro-inflammatory signaling in Pkp2-ceKO mice.

Figure 2.

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A) UMAP (top) and violin plots (bottom) characterize the expression of fibroblast markers (Tcf21, Postn, Meox1, Pdgfra) B) Violin plots reveal activated fibroblasts (Col1a1, Col3a1, Fap), pericytes (Pdgfrb, Cspg4) and pro-inflammatory fibroblasts (Ccl2, Ptx3, Thbs1, Ccl7, Il6). C, D) Pseudobulk analysis reveals the relative expression of various markers of fibrosis (C) and inflammation (D) in the various genotypes. E) UMAP (left) and proportionality histograms (right) reveal the distribution of cell types among different genotypes.

Sub-analysis of EPDCs revealed 9 populations with unique transcriptional signatures (Figure 3A–C and Table S4). Tcf21 and Pdgfrα defined all fibroblasts, and Postn, Meox1, Fap, and Acta2 defined activated fibroblasts (Fig. 3C, D). This analysis also better resolved the pro-inflammatory fibroblast population (Cluster 7), which is significantly enriched in Pkp2-ceKO mice, apparent in Pkp2-cKO, and nearly non-existent in controls (Figure 3B and S4B). Pro-inflammatory fibroblasts uniquely express Ccl2, Ptx3, Ccl7, Thbs1, and Il6 (Figure 3E). Pseudotime trajectory analysis revealed the most naïve (quiescent) fibroblast population as cluster 3, which expresses the most Tcf21. Lineage trajectory 2 terminates with cluster 7 pro-inflammatory fibroblasts, defined by gene ontology terms related to inflammatory signaling and apoptosis (Figure 3F). Additional pseudotime trajectories yielded quiescent fibroblasts (Cluster 0, Lineage 1), activated fibroblasts (Cluster 5, Lineage 3), and pericytes (Cluster 8, Lineage 4) (Figure S5 and S6). Consistent with the emergence of a pro-inflammatory fibroblast phenotype, cytokine array revealed an elevation in IFN-γ, Serpine1, Ptx3, M-CSF, and Postn in peripheral blood of Pkp2-cKO mice at 21 dpi compared to controls (Figure 3G and S7). Since most of the cytokine signals are likely to be locally produced in the damaged myocardium, our data suggests a systemic pro-inflammatory signal may originate from epicardium-derived fibroblasts.

Figure 3. Sub-analysis of epicardium-derived cells reveals emergence of a pro-inflammatory fibroblast upon Pkp2 deletion.

Figure 3.

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A) UMAP defines the identity of epicardium-derived non-myocyte lineages. B) UMAP (left) and proportionality histograms (right) define the relative distribution of cell phenotypes among different genotypes. Cluster 7 is significantly enriched in Pkp2-ceKO mice. C) Hierarchical clustering based on the 10 most differentially expressed genes defines nine epicardium-derived mesenchymal sub-types. D) UMAP defines the distribution of all fibroblasts (Pdgfra, Tcf21) and activated fibroblasts within cluster 4 (Meox1, Postn, Fap, Acta2). E) UMAP establishes localization of pro-inflammatory markers (Ccl2, Ptx3, Ccl7, Thbs1, Il6) in cluster 7, which is enriched in Pkp2-ceKO mice. F) Pseudotime trajectory (left) and Enrichr analysis (right) reveals a distinct lineage terminating in fibroblast cluster 7 that is characterized by gene ontology terms related to inflammation. G) Serum was obtained from control and Pkp2-cKO mice at 21 dpi. Cytokine Array reveals elevated levels of pro-inflammatory cytokines in Pkp2-cKO. Data represents serum pooled from n=3 (controls) and n=4 (Pkp2-cKO). IFN-gamma, Interferon Gamma; IGFBP-1, Insulin-like Growth Factor-binding-protein-1; M-CSF, Macrophage-Colony Stimulating Factor; TPO, thrombopoietin; PTX3, Pentraxin 3; POSTN, Periostin.

Pkp2 deletion induces senescence and the Senescence-Associated Secretory Phenotype (SASP) in non-myocytes

The senescence-associated secretory program (SASP) refers to secreted factors expressed by senescent cells that contribute to negative paracrine and systemic effects19. Senescence associated (SA)-βgal staining revealed an accumulation of senescent cells in the RV (but not LV) of Pkp2-cKO mice at 21 dpi; no positive stain was observed in controls (Figure 4A). Given Ccl2, Thbs1, Ptx3, Ccl7, Il6 are highly expressed in Pkp2-ce and -cKO mice, we utilized a published panel of SASP factors to identify whether the pro-inflammatory fibroblast population may express markers of cellular senescence19. The expression of Apoe and Tmem176b is detected in many non-myocytes, along with Cdkn1a, Mt1, and Mt2 (Figure 4B). Among EPDCs, the most robust SASP signature is in pro-inflammatory fibroblasts (Figure 4C and S8). Pseudobulk analysis of EPDCs from all genotypes revealed the enrichment of SASP and p53-dependent cellular senescence in Pkp2-ceKO mice (Figure 4D). It is important to note that Pkp2-ceKO mice exhibit higher expression of DNA damage markers (Atm, Atr, Hmga2), suggesting DNA damage may initiate p53-mediated senescence (Figure 4C).

Figure 4. Pro-inflammatory fibroblasts arising in Pkp2-cKO mice exhibit signs of cellular senescence.

Figure 4.

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A) Heart sections obtained from control and Pkp2-cKO hearts at 21 dpi were stained with SA -βgal as a marker of cellular senescence (n=3/genotype). Scale bar=100 μm. B, C) Violin plots (B) and UMAP (C) of non-myocyte scRNA-seq data reveal cellular distribution of senescence and Senescence-Associated Secretory Phenotype (SASP) markers. D) Pseudobulk analysis reveals relative expression of senescence and SASP markers between genotypes.

Increased immune cell burden in the Pkp2-ceKO heart

Induction of the pro-inflammatory SASP in Pkp2-ceKO mice correlates with increased immune cell burden (See Figure 2E). Here, we used Ptprc (Cd45) expression in Violin plots and UMAP to confirm the identity of all macrophages, B cells, and T cells (Figure 5A). High expression of Adgre1 (F4/80) and Cd68 define cluster 16, 17, and 18 as macrophages and monocytes (Figure 5B). Cluster 16, which is identified in equivalent numbers in Pkp2 mutant and control genotypes, contains resident macrophages based on high Cx3cr1 and low Ccr2 expression20,21 (Figure 5B). Additional resident macrophage markers, Mrc1, Cd163, Ccl24, and C1qA were also enriched in cluster 1622 (Figure 5C and S9A). In contrast, recruited macrophages that highly express Ccr2 (Cluster 17) are more abundant in Pkp2-ceKO hearts (Figure 5C and see Figure 2E). Recruited macrophages in cluster 17 also express Plac8, Ly6c2, Illb, (Figure 5C and S9A). The pseudobulk analysis of macrophage clusters between all genotypes confirmed macrophage burden in Pkp2-ceKO mice with higher gene expression of Cd68, Ly6c2, Ccr2, Spp1, Tgfb1, Il1b, Il6, Lgals3, Plac8 (Figure 5D). Finally, we identified the Pkp2-ceKO enriched cluster 19 as B cells based on expression of Cd19, Cd79a, Cd79b, and other B cell markers (Figure 5D, E and S9B); while Cluster 20 contains T cells based on high expression of Cd3e, Cd4, Cd8a, Ifng, Ccr7, and other T cell markers (Figure 5D, E and S9C).

Figure 5. Immune cell recruitment to the Pkp2 deficient heart.

Figure 5.

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A) Localization of Ptprc in UMAP (top) and violin plots (bottom) from non-myocyte scRNA-seq reveals identity of hematopoietic cells B) Cell clusters defined by general macrophage/monocyte markers in UMAP (top) and violin plots (bottom). C) Cell clusters defined by activated macrophage markers in UMAP plots. D) Pseudobulk analysis of non-myocyte scRNA-seq data reveals relative expression of immune cell markers between all genotypes. E) Cell clusters defined by B cell (left) and T cell (right) markers in UMAP (top) and violin plots (bottom). F) CellChat defines intercellular communication from pro-inflammatory fibroblasts to immune cells (left) and from immune cells to pro-inflammatory fibroblasts (right).

To identify triggers of the pro-inflammatory phenotype, we performed CellChat between the pro-inflammatory fibroblasts and whole ventricle transcriptomes obtained via bulk RNA-seq comparing Pkp2-cKO hearts to control hearts13. However, this did not yield significant interactions other than collagen-related pathways (Figure S10). Therefore, fibroblast activation may not be driven by factors displaying differential expressions in cardiomyocytes at 21dpi, or may be driven by stress pathway that do not depend on alterations in gene expression such as damage associated molecular patterns (DAMPs).

Given the abundance of both pro-inflammatory cytokines and immune cells in Pkp2-ceKO heart, we again used CellChat to define interactions between all cell populations. This analysis identified immune cell recruitment/retention signals arising from pro-inflammatory EPDC cluster 11 (Figure 5F, S11 and S12). The key pathways include Thsb1-CD47, which is present in all immune cells, in addition to Ccl2-Ccr2 and Ccl7-Ccr2 signaling, which are present in the resident and recruited macrophage and T cells. An additional unique feature is the C3-Cr2 pathway, which is biased towards B cells and is often involved in the autoimmunity (Figure 5F, S11, and S12). Indeed, a prior report described features of autoimmunity and autoantibody formation in ACM.23

B cell depletion reduces early stage ventricular remodeling in Pkp2-ceKO mice

Based on flow cytometry, Pkp2-ceKO hearts displayed an increase in the CD45+ immune cell burden between 10 and 21 dpi (Figure 6A, B). Moreover, there was a trend towards increased B cell burden in Pkp2-ceKO mice at 21 dpi, although this did not reach statistical significance. (Figure 6B). The role of B cells in cardiac pathologies has gained considerable attention; however, conflicting reports suggest beneficial or detrimental effects of B cell in heart24,25. Therefore, we tested whether B cell depletion with anti-CD20 antibody infusion alters ACM pathogenesis in Pkp2-ceKO mice (Figure 6C). Flow cytometry confirmed B cell depletion following anti-CD20 infusion compared to IgG isotype control at 7 and 21 dpi (Figure 6D). Although the RV diameter trended smaller in the Pkp2-ceKO infused with anti-CD20, we did not observe a significant difference in any functional parameter based on echocardiography at 21 dpi (Figure 6E). In contrast, B-cell depletion elicited a significant decrease in the amount of LV fibrosis at 21 dpi (Figure 6F).

Figure 6. B cell depletion delays early inflammatory and fibrotic responses without improving end stage ACM pathophysiology.

Figure 6.

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A) Schematic describing experimental timeline of cardiac immune cell quantification. B) Heart cells obtained from Pkp2-ceKO hearts at 10 dpi and 21 dpi were subjected to flow cytometry (left) to define all hematopoietic cells (CD45+) and B cells (CD19+) (Pkp2-ceKO, 10dpi (n=5), 21 dpi (n=4)). Quantification of flow cytometry data represented by average +/− SD (right). Data analyzed by two-tailed Mann Whitney test. *P<0.05. C) Schematic describing experimental timeline for B Cell Depletion and analysis. D) Flow cytometry performed on peripheral blood (7 dpi) and cardiac non-myocytes (21 dpi) to define all hematopoietic cells (CD45+) and B cells (CD19+) (n=4 mice per group/timepoint). Quantification of flow cytometry data represented by average +/− SD (right). Data analyzed by two-tailed Mann Whitney test. *P<0.05. E) Cardiac physiology measurements obtained at 21dpi. Left ventricle ejection fraction (LVEF), Fractional Shortening (FS), LV Mass and RV diastolic diameter obtained by echocardiography (IgG, n=10; anti-CD20, n=11). Data represented by mean +/− SD (right). Data analyzed by two-tailed Mann Whitney test. NS, not significant. F) Heart sections obtained at 21dpi from Pkp2-ceKO mice infused with isotype IgG control or anti-CD20 antibodies were stained with Picrosirius red to visualize fibrosis (left) (IgG, n=3; anti-CD20, n=3). Quantification of LV and RV fibrosis represented by mean +/− SD (right). Data analyzed by two-tailed Mann Whitney test. *P<0.05. Scale bar = 1 mm.

We confirmed efficient Cre-dependent deletion of Pkp2 in both Pkp2-cKO and Pkp2-ceKO hearts by qRT-PCR (Figure 7A). The expression of the B cell marker Cd19 is significantly elevated in the LV of Pkp2-ceKO mice treated with IgG control, compared to Pkp2-cKO mice, while anti-CD20 treatment decreased Cd19 expression in the LV (Figure 7B). Fibrosis markers (Postn, Col1a1, Col1a2, and Col3a1) and pro-inflammatory markers (Ccl2, Thbs1, Ptx3, and Ccl7) are elevated in both the LV and RV of Pkp2-ceKO mice infused with IgG control; in contrast, Pkp2-cKO mice display an increase in these inflammatory and fibrosis markers primarily in the RV. B cell depletion led to a significant reduction in the expression of several fibrosis and inflammation markers in the LV of Pkp2-ceKO mice but did not reduce the fibrotic and inflammatory program in the RV (Figure 7C and 7D). Interestingly, senescence markers (Cdkn1a, Apoe, and Tmem176b) were also enriched in hearts obtained from Pkp2-cKO and Pkp2-ceKO mice infused with IgG control. Cdkn1a elevation in the LV was suppressed by anti-CD20 treatment, while Apoe and Tmem176b were only elevated in the RV and not impacted by B cell depletion (Figure 7E).

Figure 7. Cardiac chamber specific changes in fibro-inflammatory program upon Pkp2 deletion and B-cell depletion.

Figure 7.

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The entire LV and RV freewall was obtained from various treatment groups at 21dpi for qRT-PCR to define gene expression changes. A) Pkp2 expression confirms tamoxifen mediated deletion. B) Cd19 expression represents B cell abundance. C) Expression of activated fibroblast markers and various collagens to define fibrosis phenotype. D) Expression of pro-inflammatory genes representing cluster 11 fibroblasts. E) Expression of markers of cellular senescence. Data represented by average +/− SD. Data analyzed by Two-way ANOVA with Tukey post-hoc test. *P<0.05 **P<0.01, ***P<0.001, ****P<0.0001.

At 21 dpi in our Pkp2 deletion model, the disease has progressed to the point of significant RV deterioration, while the LV exhibits early signs of cardiac pathology. qRT-PCR highlights the abundant expression of recruited immune cell markers such as Adgre1, Ccr2, Cx3cr1, Spp1, and Trem2 in the RV compared to LV (Figure 8A, B and see Figure 7D). In contrast, genes encoding secreted pro-inflammatory factors such as Ccl2, Ccl7, Ptx3, and Thbs1 are enriched in the LV compared to the RV (Figure 8B and see Fig. 7D). This data reinforces the concept that the pro-inflammatory phenotype is a transient and early response that precedes the recruitment of inflammatory cells, demonstrated here as a wave from the RV to the LV. The prevention of LV fibrosis and inflammation with anti-CD20 infusion suggests B cells may regulate early inflammatory and fibrotic responses captured by LV dysfunction at 21 dpi; however, its effects may eventually be masked by macrophage and T cell burden during the later stages of ACM pathogenesis, which is captured here by RV remodeling26.

Figure 8. Temporal progression of inflammatory response from RV to LV revealed by qRT-PCR.

Figure 8.

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The entire LV and RV freewall was obtained from various treatment groups at 21dpi for qRT-PCR to define gene expression changes. A) Macrophage marker gene expression. B) Macrophage and pro-inflammatory cytokine genes exhibit inverse expression in LV compared to RV. Data represented by average+/− SD. Data analyzed by Two-way ANOVA with Tukey post hoc test. *P<0.05 **P<0.01, ***P<0.001, ****P<0.0001.

Discussion

In this study, we identified a population of senescent and pro-inflammatory fibroblasts that emerge following deletion of Pkp2 in cardiomyocytes and epicardial cells. In addition, we observed an accumulation of macrophages, T cells, and B Cells in Pkp2-ceKO mice. While B cell depletion did not improve end stage cardiac physiology or RV fibrosis following Pkp2 deletion, it did ameliorate LV fibrosis, suggesting a potential early therapeutic window to rescue LV function in ACM. Taken together, this study defines a novel role for non-myocytes in amplifying inflammatory and senescent signaling and immune cell recruitment in Pkp2-deficient ACM, which regulates fibrotic burden.

While ACM disease pathology initiates in the sub-epicardium of the RV5–7, the mechanistic role for EPDCs in ACM has been understudied11. A recent report generated Wt1-specific haploinsufficient DSP knockout mice and lineage traced EPDCs with Cre-inducible eGFP. scRNA-seq revealed dysregulation of epithelial-mesenchymal transition (EMT) and increased TGFβ1 signaling27. Another report revealed the activation of TFAP2-dependent epithelial-to-mesenchymal transition upon Pkp2 deletion in human-induced pluripotent stem cell (hiPSCs)-derived epicardial cells, which stimulated the expression of ECM and adipogenic genes in vitro12. Here, we utilized a novel compound transgenic mouse line to delete Pkp2 in both the epicardium and cardiomyocytes, providing the first in vivo assessment of both epicardial and cardiomyocyte involvement in Pkp2-deficient ACM pathogenesis. EMT markers are significantly enriched in epicardium-derived fibroblasts and pericytes, supporting a role for TFAP2A in control of fibrosis in ACM. Furthermore, Pkp2 knockdown in the human iPSC-derived epicardial cells also increased EMT and enhanced pro-inflammatory interferon and fibrotic signaling, further suggesting the epicardial involvement in ACM pathogenesis28. Interestingly, although mesenchymal markers like Vim, Tagln, Fn1, and Mmps are highly elevated in Pkp2-ceKO EPDCs, Pkp2-cKO displayed more robust expression of transcriptional activators of the EMT program like Snai1, Snai2, and Twist1, suggesting that Pkp2-ceKO mice may exhibit more advanced disease presentation.

It is important to note that Pkp2-deletion in EPDC alone did not lead to transcriptional, histological or functional changes, indicating cardiomyocyte damage and dysfunction is necessary for the emergence of a fibro-inflammatory response in ACM5,14. The primary initiation of ACM pathogenesis is likely due to the remodeling of the cardiomyocyte intercalated disc. Myocyte detachment eventually contributes to programmed cell death and progressive fibro-fatty replacement. TGF-β1 originating from PKP2 deficient cardiomyocytes may stimulate fibroblast activation in ACM29. Recent report showed that NF-kB signaling is activated in cardiac myocytes in Dsg2 mutant form of ACM and the small molecule inhibition of NF-kB by preserves cardiomyocyte integrity and contractile function30. Cardiomyocyte damage in ACM is known to increase myocardial and serum inflammatory mediators31, which suggest both regional and global inflammatory stress. Cytokine stimulation is known to increase senescence and production of SASP, but also DNA damage and oxidative stress induce senescence32. Given that in our model of Pkp2-cKO mice showed increased DNA damage and oxidative stress, it further emphasizes the role of cardiomyocyte damage in influencing the production of senescent and pro-inflammatory fibroblasts.

We detect high levels of pro-inflammatory cytokines such as Ccl2, Ccl5, Ptx3, and Thbs1 in Pkp2-ceKO mice. Likewise, Dsg2 mutant mice are reported to exhibit increased Ccl2/Ccr2, Ccl3/Ccr5, and Cxcl5/Cxcr2 mRNA expression and that there is an elevation of macrophages and T cells in the fibrotic scars33. These data support the recent concept that ACM pathogenesis may resemble aspects of myocarditis, which are both amplified by NF-kB signaling26,34,35. Indeed, a recent report identified Ptx3 as a biomarker of myocarditis and inflammatory cardiomyopathy36. It is important to note the presence of Ccl2+ and Thbs1+ fibroblasts are also found in other forms of heart disease including both ischemic and non-ischemic cardiomyopathy37. The presence of Ccl2+, Thbs1+ cells has been noted in a left ventricle pressure-overload, and deleting the NF-kB activator IKKbeta in these pro-inflammatory fibroblast population attenuated Ly6Chi monocyte recruitment and reduced cardiac dysfunction38. Moreover, the normal human heart contains Ptx3+, Osmr+, and Il6st+ fibroblasts that also express Ccl2, Ccl5, Thbs1, Thbs2, Mt1 and Mt2, which may recruit c1qa+ activated macrophages39. Given NF-kB is important in ACM pathogenesis, minimizing the amplification of inflammatory drive by fibroblasts may ameliorate ACM progression.

We found that, at 21 dpi, the Pkp2-deficient RV has higher recruited macrophage burden than the LV but expresses lower levels of inflammatory cytokines. This suggests a fulminant fibro-inflammatory response has developed in the RV, but is just being initiated in the LV. Ccr2+ macrophages are known to amplify pro-inflammatory macrophage recruitment and increase cardiac damage40. Moreover, Ccr2+, Spp1+ and Trem2+ macrophages induce atrial and ventricular arrhythmias in mice41. In contrast, CCR2- macrophages reduce myocardial remodeling, demonstrating the opposing role of resident and recruited macrophages in myocardial stress and damage22,42. This temporal delay in inflammatory processes in RV vs LV introduces an interesting aspect of ACM pathogenesis that may shed light on potential therapeutic strategies to minimize the biventricular damage.

In our study, we identified increased expression of SASP factors such as Mt1, Mt2, Tmem176b, and Apoe, and various pro-inflammatory cytokines in EPDCs. SASP induces senescence in neighboring cells via paracrine signaling43,44, and pro-inflammatory fibroblasts can induce senescence in other cells of the heart45. Here, we find SASP is induced in various non-myocytes, which may explain the widespread SA-βgal stain in the RV of Pkp2-cKO. In addition to SASP and SA-βgal, various criteria define cellular senescence, including elevated expression of cell cycle inhibitors like Cdkn1a and Cdkn2a, nuclear lamina damage, DNA damage, chromatin abnormalities, ribosomal stress, oxidative damage, and lysosomal alterations46. Pkp2-deficient cardiomyocytes exhibit significant nuclear deformation and DNA damage along with oxidative damage47. Previous reports highlighted activation of the p53 pathway and increased DNA damage in ACM48. In our study, we confirm the increased expression of Cdkn1a in both Pkp2-ceKO and Pkp2-cKO hearts, further supporting the accumulation of senescence in ACM. It is important to note that senescent populations in the heart contribute to multiple pathologies45. The long-term effects of fibroblast senescence may be detrimental resulting in pathological fibrosis from fibroblast senescence49,50. In addition, senescent cardiomyocytes contribute to pathological remodeling and dysfunction after MI, and Cdkn2a knockdown in myocytes prevents SASP and cardiac dysfunction51. Interestingly, senescent myofibroblasts are reported to couple with cardiomyocytes via Cx43 to prolong the action potential duration and cause conduction block, which increases risk of conduction failure and arrhythmogenicity in old mice with MI52. These results emphasize the various roles cellular senescence may play in cardiac pathophysiology, potentially including arrhythmogenesis.

In our scRNA-seq, we observe an over-abundance of B cells in Pkp2-ceKO mice. Although B cell depletion did not yield a difference in cardiac physiology, it did ameliorate LV fibrosis at 21 dpi. This result suggests B cell depletion may modulate inflammatory and fibrotic cascades in the early damage response. B cells are known to be mostly of peripheral origin, but some studies report subsets of B lymphocytes that reside within the heart53,54. It has also been noted that B cells may play a protective role in reducing inflammation and fibrosis from acute cardiac insult54. However, a previous study analyzed B cell interactions with other immune cells, and found only minor dysregulation of inflammatory pathways in myocardial B cells during ACM55. Taken together, these data may explain why B cell depletion does not show a more robust impact in later stage ACM when the disease has progressed beyond the RV.

Study Limitations

There are number of limitations of the study that should be addressed. First, the Pkp2 KO mouse model does not capture all aspects of the ACM pathology. For example, instead of the fibrofatty deposits characteristic of human ACM, Pkp2-cKO and Pkp2-ceKO mice exhibit fibrosis without fat accumulation. Other models of ACM, such as Dsp KO mice capture the fibrofatty deposition in the myocardium, suggesting potential differences in the molecular pathways elicited by various ACM mutations. Another possibility is that rapid disease progression in the Pkp2-cKO mouse may obscure more subtle aspects of the disease phenotype. Second, scRNA-seq data was obtained from only male mice given the male predominance of disease and constraints on resources. However, validation studies were performed in female mice suggesting our observations are generalizable between sexes. Third, we did not perform scRNA-seq on cardiomyocytes, which prevented a direct comparison of communication between cardiomyocytes and non-myocytes by CellChat. However, integrating our previous bulk RNA-seq data from whole ventricle tissue to our current scRNA-seq suggests myocyte contributions either occur before 21 dpi or are via a mechanism that is independent of gene dysregulation; potentially via DAMP production.

Conclusions

Our study reveals a role for epicardium-derived fibroblasts in Pkp2-deficient ACM. Pkp2 deletion leads to an accumulation of senescent and pro-inflammatory fibroblasts, which correlates with the recruitment and activation of immune cells in the heart. This study also sheds light on the epicardium as an initial driver of inflammation in ACM and suggests a temporal window when anti-inflammatory interventions may ameliorate LV remodeling.

Supplementary Material

Supplemental_Publication_Material

Supplemental Methods

Table S1–4

Figures S1–12

Clinical Perspective:

What is New?

  • Pkp2-deficient arrhythmogenic cardiomyopathy (ACM) involves the accumulation of sub-epicardial fibrosis, suggesting epicardial cells (EPDCs) may contribute to tissue remodeling.

  • Using single cell transcriptomics, we defined how non-myocyte phenotypes changed in mice lacking Pkp2 in EPDCs and cardiomyocytes.

  • Pkp2-deficient epicardium-derived fibroblasts become senescent and acquire the pro-inflammatory senescence-associated secretory phenotype (SASP).

  • The pro-inflammatory phenotype shifted from right to left ventricle predominance as disease progresses, correlating with excessive burden of CCR2+ macrophages, B-cells, and T-cells.

  • Although B-cell depletion limited the initial left ventricular fibrotic response, it did not prevent end-stage heart failure.

What are the clinical implications?

  • We identified a novel mechanism in ACM pathogenesis where Pkp2-deficient EPDCs produce cytokines that may increase immune cell burden.

  • Through better understanding the timing and immunological basis of ACM pathogenesis, in conjunction with the importance of epicardial contributions to inflammation, this study may help to accelerate ACM treatment strategies that target the inflammatory response.

Acknowledgement:

We thank the University of Rochester Medical Center Flow Cytometry Core for assistance with cardiac immune cell quantification, and the Genomics Research Center for their assistance with scRNA-seq.

Sources of Funding:

This study was supported by funding from the National Institutes of Health to EMS (R01HL169961, R01HL160758, and CTSA UL1 TR002001), to EMS and MD (R01HL136179), and to MD (R35HL160840); and from the American Heart Association to EMS (23EIA1029488). Daniel D. Han is a trainee in the Medical Scientist Training Program funded by NIH T32 GM152318 and GM007356. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Science or NIH.

Nonstandard Abbreviations and Acronyms:

ACM

Arrhythmogenic Cardiomyopathy

CM

Cardiomyocyte

CVD

Cardiovascular Disease

Dpi

Days post-tamoxifen injection

EPDCs

Epicardial Progenitor Derived Cells

DAMP

Damage-associated molecular pattern

DSC2

Desmocollin-2

DSG2

Desmoglein-2

DSP

Desmoplakin

GFP

Green Fluorescent Protein

JUP

Plakoglobin

LV

Left Ventricle

Pkp2

Plakophilin-2

Pkp2-cKO

Cardiomyocyte-specific knockdown of Plakophilin-2

Pkp2-ceKO

Cardiomyocyte and Epicardium-specific knockdown of Plakophilin-2

Pkp2-eKO

Epicardium-specific knockdown of Plakophilin-2

RV

Right Ventricle

SASP

Senescence-Associated Secretory Phenotype

SCD

Sudden Cardiac Death

scRNA-seq

Single-Cell RNA Sequencing

Footnotes

Disclosures:

The authors declare that there are no known disclosures.

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

Supplemental_Publication_Material
NIHMS2179330-supplement-Supplemental_Publication_Material.pdf (12.6MB, pdf)

Data Availability Statement

Detailed methods can be found in Supplemental Methods. Primer sequences and antibody information are listed in Tables S1 and S2. scRNA-sequencing data has been deposited in the Gene Expression Omnibus (GEO) database under accession code GSE300141.

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