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Individual adult ventricular cardiomyocytes are either mono- or multi-nucleated and undergo morphological changes during cardiac hypertrophy. However, corresponding transcriptional signatures, reflecting potentially different functions or the ability for cell-cycle entry, are not known. The aim of this study was to determine the transcriptional profile of mono- and multi-nucleated adult cardiomyocytes by single-cell RNA-sequencing (scRNA-seq) and to investigate heterogeneity among cardiomyocytes under baseline conditions and in pressure-induced cardiac hypertrophy. We developed an array-based approach for scRNA-seq of rod-shaped multi-nucleated cardiomyocytes from both healthy and hypertrophic hearts. Single-cell transcriptomes of mono- or multi-nucleated cardiomyocytes were highly similar, although a certain degree of variation was noted across both populations. Non-image-based quality control allowing inclusion of damaged cardiomyocytes generated artificial cell clusters demonstrating the need for strict exclusion criteria. In contrast, cardiomyocytes isolated from hypertrophic heart after transverse aortic constriction showed heterogeneous transcriptional signatures, characteristic for hypoxia-induced responses. Immunofluorescence analysis revealed an inverse correlation between HIF1α+ cells and CD31-stained vessels, suggesting that imbalanced vascular growth in the hypertrophied heart induces cellular heterogeneity. Our study demonstrates that individual mono- and multi-nucleated cardiomyocytes express nearly identical sets of genes. Homogeneity among cardiomyocytes was lost after induction of hypertrophy due to differential HIF1α-dependent responses most likely caused by none-homogenous vessel growth.
Formation and segregation of cell lineages forming the heart have been studied extensively but the underlying gene regulatory networks and epigenetic changes driving cell fate transitions during early cardiogenesis are still only partially understood. Here, we comprehensively characterize mouse cardiac progenitor cells (CPCs) marked by Nkx2-5 and Isl1 expression from E7.5 to E9.5 using single-cell RNA sequencing and transposase-accessible chromatin profiling (ATAC-seq). By leveraging on cell-to-cell transcriptome and chromatin accessibility heterogeneity, we identify different previously unknown cardiac subpopulations. Reconstruction of developmental trajectories reveal that multipotent Isl1+ CPC pass through an attractor state before separating into different developmental branches, whereas extended expression of Nkx2-5 commits CPC to an unidirectional cardiomyocyte fate. Furthermore, we show that CPC fate transitions are associated with distinct open chromatin states critically depending on Isl1 and Nkx2-5. Our data provide a model of transcriptional and epigenetic regulations during cardiac progenitor cell fate decisions at single-cell resolution.