Comparative Single-Nucleus Transcriptomic Analysis of the Intact Left Ventricle in Acomys cahirinus and Mus musculus

Figures

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  Figure 1. Overview of the experimental design. The scheme summarizes the post ischemic cardiac phenotypes that motivated the study, the sampling of intact left ventricles from adult A. cahirinus cahirinus and Mus musculus, nuclei isolation, single-nucleus RNA sequencing and subsequent analytical steps. These steps included quality control, data integration, cell type annotation, UMAP visualization, correlation analysis, assessment of interspecies separability using Augur, coexpression module detection and Gene Ontology enrichment analysis. RNA—Ribonucleic acid, UMAP—Uniform Manifold Approximation and Projection.

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  Figure 2. Interspecies integration of snRNA-seq data from the left ventricle of A. cahirinus and M. musculus, annotated with major cell populations. (A). UMAP projection of the integrated nuclear dataset annotated with the major myocardial cell populations. The identified populations included ventricular cardiomyocytes, fibroblasts, vascular, endocardial, and lymphatic endothelial cells, pericytes, smooth Muscle cells, epicardial cells, and immune populations, including macrophages, Lyve1+ macrophages, APC-like macrophages, neutrophils, T-lymphocytes, and B-lymphocytes. (B). Distribution of nuclei by species and individual sample on the shared UMAP projection. The upper panel shows the contribution of A. cahirinus and M. musculus, whereas the lower panel shows the distribution of individual biological replicates. (C). Proportions of annotated cell populations in each sample. Bars show the relative contribution of the major cell populations in M. musculus and A. cahirinus samples. (D). Expression of marker genes used to confirm cell-type annotations. Dot size represents the proportion of nuclei with detectable expression of the corresponding gene, and color intensity indicates the average…

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  Figure 3. Cell type-specific interspecies differences in transcriptomic profiles of the intact myocardium in A. cahirinus and M. musculus. (A). Interspecies distinctiveness of transcriptomic profiles in individual cell populations, as measured by Augur AUC values. (B). Number of differentially expressed genes between A. cahirinus and M. musculus in individual cell populations. Hatched segments indicate genes with higher expression in A. cahirinus, whereas gray segments indicate genes with higher expression in M. musculus. Values are not shown for neutrophils or smooth muscle cells due to insufficient numbers of nuclei for reliable pseudobulk statistical analysis. (C). Correlation matrices of transcriptomic profiles across individual samples for the complete nuclear dataset and individual cell populations. Color represents the correlation coefficient between samples. AK-1–AK-3—individual A. cahirinus samples, APC—antigen-presenting cell, AUC—area under the receiver operating characteristic curve, DE—differentially expressed, LEC—lymphatic endothelial cell, Mφ—macrophage, MK-1–MK-3—individual M. musculus samples, SMC—smooth muscle cell, VEC—vascular endothelial cell.

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  Figure 4. Coexpression modules associated with the major cell populations of the intact left ventricle in A. cahirinus and M. musculus. (A). Gene coexpression matrix with hierarchical clustering and identification of 11 coexpression modules. The key genes for each module are shown on the right. (B). UMAP projection of the integrated set of nuclei annotated with major myocardial cell populations. (C). Projection of coexpression module scores onto the UMAP projection of the integrated set of nuclei. The spatial distribution of the modules corresponds to their cellular localization. (D). Comparison of module scores across individual A. cahirinus and M. musculus samples. Dot size indicates the proportion of nuclei with a high score for the corresponding module, and color intensity indicates the mean module score in the group. (E). Distribution of selected module scores in the corresponding cell populations at the level of individual samples. Shown are the Fibroblast Matrix Synthesis module in fibroblasts, the Cardiomyocyte Excitation–Contraction module in ventricular cardiomyocytes, the EGFR-associated extracellular matrix (ECM) Remodeling module in fibroblasts, and the Neuro-Lymphatic…

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  Figure 5. Reintegration and comparative analysis of ventricular cardiomyocytes in the intact left ventricle of A. cahirinus and M. musculus. (A). UMAP projection of the ventricular cardiomyocyte population after focused reintegration, with cluster annotations. (B). UMAP projection of nuclei after the clusters were combined into three transcriptional states comprising contractile CM, matrix-associated CM, and alternative CM state. (C). Distribution of A. cahirinus and M. musculus nuclei in the UMAP embedding of the reintegrated ventricular cardiomyocyte subset. (D). Representation of selected markers in individual samples of A. cahirinus and M. musculus. Genes associated with matrix/subendocardial traits, the cardiomyocyte contractile program, the fibroblast/ECM component, Yap/Taz-Tead-related regulation, developmental traits, and the cardiac progenitor-associated program are shown. The size of the dot reflects the proportion of nuclei with detectable gene expression, and the intensity of the staining reflects the average expression in the group. (E). Distribution of the markers shown in Panel (D) across the three annotated states of ventricular cardiomyocytes, separately for A. cahirinus…

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  Figure 6. Summary of baseline interspecies differences in the intact left ventricle of A. cahirinus and M. musculus. (A). Global cross-species analysis showing comparable major myocardial cell populations and a distinct baseline transcriptional profile in A. cahirinus. (B). Three cardiomyocyte states identified by focused reintegration, including contractile CM, matrix-associated CM, and alternative CM state. (C). Summary of cardiomyocyte differences between species. Adult A. cahirinus retained a mature phenotype but differed from adult M. musculus in reference scores, functional specialization, and developmental, morphogenetic, and Hippo-associated features. (D). Relative positioning of adult A. cahirinus, adult M. musculus, and developing M. musculus according to Adult reference and Early-state reference scores. A. cahirinus differed from adult M. musculus without corresponding directly to embryonic or early postnatal mouse myocardium. CM—cardiomyocyte, ECM—extracellular matrix.