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Quality control in scRNA-Seq can discriminate pacemaker cells - the mtDNA bias

ABSTRACT: Single-cell RNA-Sequencing (scRNA-Seq) provides high-resolution insights into complex tissues. Cardiac tissue, however, poses a major challenge due to the delicate isolation process and the large size of mature cardiomyocytes. Regardless of the experimental technique, captured cells are often impaired and some capture sites may contain multiple or no cells at all. All this refers to “low quality” potentially leading to data misinterpretation. Common standard quality control parameters involve the number of detected genes and transcripts per cell and the fraction of transcripts from mitochondrial genes (%mtDNA). While cutoffs for transcripts and genes per cell are usually user-defined for each experiment or individually calculated, a fixed threshold of 5% mtDNA is standard and often set as default in scRNA-Seq software. However, this parameter is highly dependent on the tissue type. In the heart, mitochondrial transcripts comprise almost 30% of total mRNA due to high energy demands. Here, we demonstrate that a 5%-threshold not only causes an unacceptable exclusion of cardiomyocytes but also introduces a bias that particularly discriminates pacemaker cells. This effect is apparent for our in vitro generated induced-sinoatrial-bodies (iSABs; highly enriched physiologically functional pacemaker cells), and also evident in a public dataset of cells isolated from embryonal murine sinoatrial node tis-sue [2]. Taken together, we recommend omitting this filtering parameter for scRNA-Seq in cardiovascular applications whenever possible.

ORGANISM(S): Mus musculus

PROVIDER: GSE174233 | GEO | 2021/05/28

REPOSITORIES: GEO

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Project description:to study the effect of induced expression of TBX3 within the atrium of the heart Background: Treatment of congenital or acquired disorders of the sinus node or atrioventricular node requires insight into the molecular mechanisms for the development and homeostasis of these pacemaker tissues. In the developing heart, transcription factor TBX3 is required for pacemaker and conduction system development. Here, we explore the role of TBX3 in the adult heart and investigate whether TBX3 is able to reprogram terminally differentiated working cardiomyocytes into pacemaker cells. This would be an attractive approach in biological pacemaker formation. Methods and results: TBX3 expression was ectopically induced in cardiomyocytes of adult transgenic mice. Expression analysis revealed an efficient switch from the working myocardial expression profile to that of the pacemaker myocardium. This included suppression of genes encoding gap junction subunits (Cx40, Cx43), the cardiac Na+ channel (NaV1.5; INa) and inwardly rectifying K+ ion channels (Kir-genes; IK1). Concordantly, we observed conduction slowing in these hearts, and reductions in INa and IK1 in cardiomyocytes isolated from these hearts. The reduction in IK1 resulted in a more depolarized maximum diastolic potential, thus enabling spontaneous diastolic depolarization. Neither ectopic pacemaker activity nor pacemaker current, If, were observed. Lentiviral expression of TBX3 in ventricular cardiomyocytes resulted in conduction slowing and development of heterogeneous phenotypes, including depolarized and spontaneously active cardiomyocytes. Conclusions: TBX3 partially reprograms terminally differentiated working cardiomyocytes into pacemaker-like cells and induces important pacemaker properties. The ability of TBX3 to reduce intercellular coupling to overcome current-to-load mismatch and the ability to reduce IK1 density to enable diastolic depolarization, are very promising TBX3 characteristics for biological pacemaker formation strategies. 5 TBX3 expressing left atrial appendage samples (Tamoxifen-treated Myh6MCM;CTBX3 adult male mice) and 6 controls ((Tamoxifen-treated Myh6MCM adult male mice)

Project description:BACKGROUND: Single-cell RNA sequencing (scRNA-seq) is widely used in cancer research and organ development since its powerful ability to analyze cellular heterogeneity. However, its application in cardiomyocytes is rare mainly because the cardiomyocytes are too large and fragile to withstand traditional single-cell approaches. METHODS: Through designing the isolation procedure of neonatal mouse cardiac cells, we perform scRNA-seq with samples from 27 neonatal mice. Individual cell transcriptomes were subsequently clustered based on transcriptional profiles of highly variable genes. Then these clusters were used as the basis for between-cluster differential gene expression analyses and between-cluster interaction analyses. At last, we analyzed the intersection of these clusters with genetic and pharmacologic data. RESULTS: We provide detailed cellular atlases of the heart at single-cell resolution across four different stages after birth. We have luckily obtained tens of thousands of cardiomyocytes, to our knowledge, the most extensive reference framework so far. Moreover, we have discovered unexpected erythrocytes-like cardiomyocyte-terminal cardiomyocytes, comprising more than a third of all the cardiomyocytes. Only a few genes are highly expressed in these cardiomyocytes. They are highly differentiated cardiomyocytes that function as contraction pump. In addition, we have identified two cardiomyocyte-like conducting cells, lending support to the theory that the sinoatrial node pacemaker cells are specialized cardiomyocytes. Furthermore, we provide a substantial blueprint for comprehensive interactions between cardiomyocytes and other cardiac cells. We linked specific cell types to adverse drug reactions and congenital heart diseases and analyzed the roles of cell subtypes in adverse drug reactions and cardiac diseases. CONCLUSIONS: This mouse cardiac cell atlas improves our understanding of the heart and provides a valuable reference in response to varying physiological conditions and diseases.

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Quality control in scRNA-Seq can discriminate pacemaker cells - the mtDNA bias

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