DOI: 10.5061/dryad.3n5tb2rqw
title: Hypertrophic cardiomyopathy-associated mutations drive stromal activation via EGFR-mediated paracrine signaling

description: Hypertrophic cardiomyopathy (HCM) is characterized by thickening of the
 left ventricular wall, diastolic dysfunction, and fibrosis, and is
 associated with mutations in genes encoding sarcomere proteins. While in
 vitro studies have used human induced pluripotent stem cell-derived
 cardiomyocytes (hiPSC-CMs) to study HCM, these models have not examined
 the multicellular interactions involved in fibrosis. Using engineered
 cardiac microtissues (CMTs) composed of HCM-causing MYH7-variant hiPSC-CMs
 and wild-type fibroblasts, we observed cell-cell cross-talk leading to
 increased collagen deposition, tissue stiffening, and decreased
 contractility dependent on fibroblast proliferation. hiPSC-CM conditioned
 media and single-nucleus RNA sequencing data suggested that fibroblast
 proliferation is mediated by paracrine signals from MYH7-variant
 cardiomyocytes. Furthermore, inhibiting epidermal growth factor receptor
 tyrosine kinase with erlotinib hydrochloride attenuated stromal
 activation. Last, HCM-causing MYBPC3-variant CMTs also demonstrated
 increased stromal activation and reduced contractility, but with distinct
 characteristics. Together, these findings establish a paracrine-mediated
 cross-talk potentially responsible for fibrotic changes observed in HCM.
description: <strong>snRNA-sequencing
 data</strong><strong> </strong>
 snRNA-seq dataset includes data on CMTs made with healthy wild
 type (WT) or hypertrophic cardiomyopathy (HCM)-causing (R403Q+/- mutation
 in myosin heavy chain) human induced pluripotent stem cell
 derived-cardiomyocytes (hiPSC-CMs) (derived from PGP1 hiPSC line) and
 ventricular cardiac fibroblasts (Lonza Cat. CC-2904) (1).  
 <em>Methods</em>: A total of 60,000 cells per tissue
 (ten tissues pooled per sample), consisting of 90% hiPSC-CMs and 10% vCFs,
 were mixed in an ECM solution consisting of 4 mg/ml of human fibrinogen
 (Sigma), 10% Matrigel (Corning), 0.4 unit of thrombin (Sigma) per mg of
 fibrinogen, 5 µM Y-27632 (Tocris), and 0.033 mg/mL aprotinin (Sigma). The
 cell-ECM mixture was pipetted into each tissue well, and after gel
 polymerization for ten minutes, tissue maintenance growth media containing
 high glucose DMEM (Fisher) supplemented with 10% FBS (Sigma), 1%
 penicillin-streptomycin (P/S) (Fisher), 1% Non-essential Amino Acids
 (Fisher), 1% Glutamax (Fisher), 5 µM Y-27632, 0.033 mg/mL aprotinin, and
 150 µg/mL L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate.
 Y-27632 was removed two days following seeding, and the growth media was
 replaced every other day.  CMTs were flash frozen in
 liquid nitrogen on day 7 (10 tissues pooled per sample) and stored in
 -80°C. Individual nuclei were isolated from frozen tissue samples.
 Briefly, nuclei were isolated (<em>2</em>) and RNA was
 reverse-transcribed and converted into cDNA libraries using a 10x Chromium
 Controller and Chromium Single Cell 3' v3.1 reagent kit (10x
 Genomics). Bar-coded libraries were pooled and sequenced (Illumina NovaSeq
 6000). Single nucleus RNAseq alignment and gene counts were performed
 using Cell Ranger 1.2 (10x Genomics) and Seurat (<em>3</em>)
 and R 4.1.0 and managed via RStudio. There were 15,129 total sequenced
 nuclei.  Principal component (PC) analysis was
 performed to determine the dimensionality of this dataset within Seurat
 and the number of PCs was selected using both permutation-based and
 heuristic methods. Cluster assignment was performed in an unsupervised
 manner using a shared nearest neighbor approach within Seurat. To validate
 the ideal number of PCs and clustering resolution in this dataset, repeat
 analysis was performed while varying both the dimensionality and
 resolution. Using the Clustree R package (<em>4</em>), the
 hierarchical evolution of cluster identity was determined. This analysis
 allows the user to investigate how clustering resolution impacts cell
 identity, in an effort to assign robust classification to cell clusters
 which remain consistent across resolution values. For each clustering
 resolution, marker genes for each cluster, <em>TTN</em> for
 cardiomyocytes and <em>FN1</em> for fibroblasts, were
 identified in order to assign relevant biological function to various
 subclusters. Dimensionality and resolution were adjusted until each
 identified cluster contained a non-zero amount of significantly expressed
 marker genes that were able to assign functional gene ontological terms;
 in this case, at cluster resolution 0.4, which was used for clustering
 analysis. At greater resolution values, cluster subsets returned either
 too few genes to assign functional classifications or non-coding genes of
 unknown function. Dotplot projections of single cells were generated with
 UMAP coordinates, using the number of dimensions determined from PCA as
 described above. For the purposes of this analysis, 30 dimensions were
 considered for both the initial PCA, and subsequent UMAP visualization. To
 assign cell clusters specific identities, canonical marker gene expression
 was analyzed to assign broad cell classes. To assign cell subclusters,
 such as CM1-CM6, unbiased genetic markers for each population were
 calculated using the Wilcoxon Rank Sum test within Seurat, at which point
 at most the top 100 genes were used as a Gene Ontology query to assign
 cellular functions to each cell subcluster. Genes that
 were upregulated R403Q+/- cardiomyocytes compared to WT cardiomyocytes
 with a p-value &lt; 0.01 and fold-change greater than 1.3 were input
 into Enrichr (5,6).  The top upregulated pathways associated with the top
 upregulated genes were obtained using WikiPathways (7).
 <strong>Bulk RNA-sequencing data</strong>
 Bulk RNA-sequencing data include data on serum starved vCFs, vCFs
 treated with 100 ng/mL recombinant human epidermal growth factor (rhEGF),
 and conditioned media from hiPSC-CMs (1).
 <em>Methods</em>: vCFs were trypsinized, centrifuged,
 and flash-frozen after two days in serum-starvation (low glucose DMEM with
 0.1% fetal bovine serum) vs 100 ng/mL rhEGF or conditioned media from
 wild-type vs R403Q+/- hypertrophic cardiomyopathy hiPSC-CMs for three
 different batches. Cells were homogenized in Trizol Reagent (Life
 Technologies, Inc., Grand Island, NY) with TissueLyzer II (QIAGEN, Inc.,
 Valencia, CA) and RNA was isolated by conventional methods. RNA went
 through two rounds of mRNA purification (polyA-selection) using Dynabeads
 mRNA DIRECT Kit (Invitrogen, Carlsbad, CA). Double-stranded cDNA was
 generated using the Superscript III First-Strand Synthesis System
 (Invitrogen, Carlsbad, CA). The cDNA products were used to construct
 libraries with the Nextera XT DNA Sample Preparation Kit (Illumina, Inc.,
 San Diego, CA). Libraries were paired-end sequenced with a read length of
 75 base pairs (75PE) on the Illumina NextSeq 500. Reads were aligned to
 the hg38 Human Genome using Spliced Transcripts Alignment to a Reference
 (STAR) (<em>8</em>). Data were analyzed as previously
 described (<em>9</em>) and normalized to the total number of
 reads per kilobase of exon per million (RPKM).  Genes
 that were upregulated in rhEGF-treated vCFs compared to serum-starved vCFs
 with a p-value &lt; 0.01 and fold-change greater than 1.3 were input
 into Enrichr (5,6). For conditioned media experiments, gene expression
 values were averaged over three experimental repeats. Genes that had an
 average fold-change expression greater than 1.15 with a p-value &lt;
 0.01 for all three experiments were input into Enrichr. The top
 upregulated pathways and transcription factors associated with the top
 upregulated genes were obtained using WikiPathways (7) and ChEA (10)
 databases, respectively.   1. J.K.
 Ewoldt <em>et al. </em>Hypertrophic cardiomyopathy–associated
 mutations drive stromal activation via EGFR-mediated paracrine
 signaling.<em>Sci. Adv.</em><strong>10
 </strong>(2024). 2. E. R. Nadelmann, J. M.
 Gorham, D. Reichart, D. M. Delaughter, H. Wakimoto, E. L. Lindberg, M.
 Litviňukova, H. Maatz, J. J. Curran, D. Ischiu Gutierrez, N. Hübner, C. E.
 Seidman, J. G. Seidman, Isolation of Nuclei from Mammalian Cells and
 Tissues for Single-Nucleus Molecular Profiling. <em>Curr
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 Butler, M. J. Lee, A. J. Wilk, C. Darby, M. Zager, P. Hoffman, M.
 Stoeckius, E. Papalexi, E. P. Mimitou, J. Jain, A. Srivastava, T. Stuart,
 L. M. Fleming, B. Yeung, A. J. Rogers, J. M. McElrath, C. A. Blish, R.
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description: # **Hypertrophic cardiomyopathy-associated mutations drive stromal
 activation via EGFR-mediated paracrine signaling**
 [https://doi.org/10.5061/dryad.3n5tb2rqw](https://doi.org/10.5061/dryad.3n5tb2rqw) ## **Summary** This dataset includes single nucleus RNA-sequencing (snRNA-seq) of engineered cardiac microtissues (CMTs) made with wild-type (WT) healthy or hypertrophic cardiomyopathy (HCM)-causing (R403Q+/- mutation in myosin heavy chain) human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CMs) and bulk RNA sequencing data of ventricular cardiac fibroblasts (vCFs) treated with conditioned media from WT and R403Q+/- mutant hiPSC-CMs or recombinant human epidermal growth factor (rhEGF). We found that the top pathways associated with genes up-regulated in both cardiomyocyte and fibroblast cell populations within R403Q+/− tissues included pathways related to hypertrophy, senescence, and inflammatory cytokine signaling. vCFs treated with R403Q+/− hiPSC-CM conditioned media revealed an increase in gene expression in pathways relating to the release of extracellular vesicles (EVs) from cardiomyocytes, alterations in the yes-associated protein 1 (YAP1)/ECM signaling axis, and type I collagen synthesis. Transcription factors associated with genes more highly expressed in vCFs treated with R403Q+/− hiPSC-CM conditioned media include ones known to regulate cell proliferation, including nuclear receptor subfamily 3 group C member 1 (*NR3C1*) and T-box transcription factor (*TBX3*), and inflammation, including *NR3C1*, peroxisome proliferator activated receptor delta (*PPARD*), CCAAT enhancer binding protein delta (*CEBPD*), and nuclear factor-erythroid 2 related factor 2 (*NFE2L2*). In addition, when we administered rhEGF to serum-starved vCFs, gene expression pathways related to DNA replication, cell cycle progression, and inflammation were up-regulated.  ## **Description of the data and file structure** ### **snRNA-sequencing data** **allcluster_markers0.4.xlsx** This includes data from all nuclei from both WT and R403Q+/- CMTs. *p_val*: p-value that is not adjusted for multiple test correction *avg_logFC*: average log2(fold-change). A positive value means gene is more highly expressed in that cluster. *pct.1*: The percentage of cells in which the gene is detected in the cluster. *pct.2*: The percentage of cells in which the gene is detected on average in other clusters. *p_val_adj*: Adjusted p-value based on Bonferroni correction cluster: There were 9 defined cell clusters (0-8): cardiomyocyte (cm) 1-6 and fibroblast (cf) 1-3, respectively. *genes*: Ensembl gene ID *n/a* cells indicate places where there were no detectable reads for the gene, and therefore the fold-change could not be calculated. ** ** **cm1_wtvs403.xlsx, cm2_wtvs403.xlsx, cm3_wtvs403.xlsx, cm4_wtvs403.xlsx, cm5_wtvs403.xlsx, cm6_wtvs403.xlsx, cf1_wtvs403.xlsx, cf2_wtvs403.xlsx, cf3_wtvs403.xlsx** Each file is based on a different cell cluster identified in allcluster_markers0.4.xlsx. cm1 = cluster 0, cm2 = cluster 1, cm3 = cluster 2, cm4 = cluster 3, cm5 = cluster 4, cm6 = cluster 5, cf1 = cluster 6, cf2 = cluster 7, and cf3 = cluster 8. *p_val*: p-value that is not adjusted for multiple test correction *avg_logFC*: average log2(fold-change of WT over R403Q+/- sample). A positive value means gene is more highly expressed in the WT sample.   *pct.1*: The percentage of cells in which the gene is detected in the cluster. *pct.2*: The percentage of cells in which the gene is detected on average in other clusters. *p_val_adj*: Adjusted p-value based on Bonferroni correction *genes*: Ensembl gene ID   **cm_all_wtvs403.xlsx, cf_all_wtvs403.xlsx** These datasets include all identified cardiomyocytes (cm) or vCFs with removal of titin (*TTN*) positive fibroblasts (cf). *p_val*: p-value that is not adjusted for multiple test correction *avg_logFC*: average log2(fold-change of WT over R403Q+/- sample). A positive value means gene is more highly expressed in the WT sample.   *pct.1*: The percentage of cells in which the gene is detected in the cluster. *pct.2*: The percentage of cells in which the gene is detected on average in other clusters. *p_val_adj*: Adjusted p-value based on Bonferroni correction *genes*: Ensembl gene ID   ### **Bulk RNA-sequencing data** ###### **vCF\_Bulk\_RNA-sequencing.xlsx** *Gene*: Ensembl gene ID *Chr*: Chromosome *Start*: Genome start coordinate *End*: genome end coordinate *Description*: Description of gene *norm*: read counts normalized to 1 × 106 total sample reads for the indicated sample *reads*: gene read counts for the indicated sample *fold( *: fold-change of the normalized gene expression level of the samples listed *CF_egf*: vCFs treated with 100 ng/mL rhEGF *CF_starve*: vCFs treated with serum starvation *1-19_WT*: Batch 1 of conditioned media from WT hiPSC-CMs *1-19_403*: Batch 1 of conditioned media from R403Q+/- hiPSC-CMs *1-23_WT*: Batch 2 of conditioned media from WT hiPSC-CMs *1-23_403*: Batch 2 of conditioned media from R403Q+/- hiPSC-CMs *3-19_WT*: Batch 3 of conditioned media from WT hiPSC-CMs *3-19_403*: Batch 3 of conditioned media from R403Q+/- hiPSC-CMs