Single-Cell Sequencing of the Cardiovascular System: Challenges in Translation : Cardiology Discovery

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Single-Cell Sequencing of the Cardiovascular System: Challenges in Translation

Li, Zheng; Zhou, Bingying

Editor(s): Xu, Tianyu; Fu, Xiaoxia

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Cardiology Discovery 1(3):p 145-147, September 2021. | DOI: 10.1097/CD9.0000000000000027
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To date, single-cell sequencing technology has penetrated all the major areas of biomedical research, including the cardiovascular field. When combined with other single-cell techniques, one can easily study various combinations of the genomes, epigenomes, transcriptomes, and proteomes of single cells, which is known as the “single-cell omics” approach. The surge in cardiovascular single-cell studies in recent years has yielded paradigm-shifting observations, ranging from unraveling developmental cell fates to identifying novel therapeutic targets.

Single-cell profiling of the heart

The earliest studies employing single-cell RNA sequencing (scRNA-seq) were focused on the embryonic mouse heart. These studies proved immensely valuable in the elucidation of coordinated lineage segregation events and their functional commitments during heart development. Pioneering work by DeLaughter et al[1] and Li et al[2] demonstrated the power of scRNA-seq in delineating spatiotemporal gene expression dynamics and cellular heterogeneity in the developing mammalian heart. Subsequent studies pushed the boundaries of this technology even further by examining time points prior to the formation of the heart tube (ie, before embryonic day 8.0).[3–6] These studies identified critical roles of Mesp1 in cardiovascular lineage commitment, Isl1 and Nkx2-5 in cardiac progenitor cell fate transition and differentiation, and Hand2 in the specification of outflow tract cells. Despite the extensive research conducted on mouse cardiogenesis, most studies have exclusively focused on early heart development, during which the heart is known to undergo some of the most drastic changes. The alterations that take place in the later stages of heart development remain unclear. For example, how do cardiac cells continue to diversify or whether other molecular changes occur during various embryonic/fetal developmental stages. Targeted analyses of specific cell types later during heart development may enhance our understanding of the developing heart.[7,8]

Owing to limited access to clinical specimens and the inability to perform in vivo validation of heart function directly, human embryonic heart development is less understood. Nevertheless, Cui et al[9] performed scRNA-seq on embryonic/fetal hearts ranging from 5 to 25 weeks of gestation and showed stepwise involvement of and changes in different cardiac cell types during development. Through the combined use of scRNA-seq, spatial transcriptomics, and in situ sequencing, Asp et al[10] created the first spatiotemporal map of the human heart at 4.5 to 9 weeks of gestation. In addition to mapping the cell type distribution in the human embryonic heart, the authors also analyzed the roles of different cell types during embryonic development. In particular, a special subtype of cardiomyocytes that was highly enriched in Myoz2 expression was postulated to play an important role in cardiac function and pathology.

The large size of mature cardiomyocytes (averaging over 100 μm in length) has hampered the comprehensive characterization of postnatal development and of the adult heart. Nonetheless, alternatives, such as handpicking cardiomyocytes,[11,12] single-nucleus RNA sequencing (snRNA-seq),[13–18] or solely focusing on non-myocytes for analysis,[19–22] have produced exciting results. As the risk of developing cardiac diseases increases with age and because most cardiac disorders manifest as abnormalities or impairment of cardiomyocytes, the ability to sequence intact adult cardiomyocytes offers the unique advantage to study cellular and molecular dynamics during disease onset and progression at single-cell resolution, which can only be achieved, in higher-throughput, via the application of fluorescence-activated cell sorting[23,24] or other automated systems with large selection nozzles.[25–27] Another determining factor for the reliability of single cardiomyocyte analysis is the methodology used for cell isolation. Although Langendorff perfusion remains the gold standard for mammalian cardiomyocyte isolation, it is not readily applicable to human specimens.[28] Common chunk digestion methods fail to produce adult cardiomyocytes of sufficient quality and quantity. Recently developed methods based on myocardial slicing prior to isolation have produced satisfactory single-cell sequencing data.[25,29] Future efforts to further optimize the isolation procedure to obtain in vivo-like cardiomyocytes are warranted.

These technological advances have propelled the emergence of 3 major comprehensive studies of the adult human heart. The first full characterization of the human heart was accomplished in the laboratories of Wang et al.[25] This study comprehensively characterized 12 healthy hearts, 6 failed hearts, and 2 hearts that had undergone left ventricular assist device therapy. Interestingly, the authors discovered that non-myocytes, rather than cardiomyocytes, were responsible for changes in cardiac function. It is worth mentioning that this study is currently the only large-scale study using intact adult human cardiomyocytes for single-cell analysis. Another study led by global investigators also depicted the cellular landscape of the adult human heart by combining snRNA-seq of cardiomyocytes and scRNA-seq of endothelial and immune cell populations.[18] Due to the massive number of cells sequenced, the researchers were able to capture less abundant cardiac cell types, including neuronal cells and adipocytes. Likewise, snRNA-seq of all cardiac cell types was performed to assess the cellular and transcriptional diversity of the nonfailing human heart.[17] Integration of snRNA-seq data with results from genome-wide association studies revealed the most relevant cell types in cardiac diseases. For example, genes implicated in cardiomyopathies and arrhythmia syndromes were enriched in cardiomyocytes, whereas druggable genes were most commonly found in adipocytes.

Translational single-cell cardiac research

From a clinical standpoint, we would expect scRNA-seq to advance our understanding of the underlying mechanisms of cardiovascular pathophysiology to pave way for the development of novel therapeutic strategies. The immune system has received increased attention from researchers in the field because of its widespread involvement in cardiovascular disorders. scRNA-seq of aortic immune cells in atherosclerotic mouse models facilitates the characterization of immune cell repertoire and classification of aortic macrophage heterogeneity.[30,31] By integrating single-cell proteomic and transcriptomic approaches, Fernandez et al[32] uncovered distinct features of T cells and macrophages in human atherosclerotic plaques, enabling the design of individualized therapeutics. By profiling CD45+ cells from a pressure overload-induced heart failure model, Martini et al[22] uncovered extensive immune activation across various cell types, thereby offering possible explanations for clinical observations and hinting at diagnostic as well as therapeutic applications. Similarly, scRNA-seq of CD45+ cells in a mouse model of autoimmune myocarditis revealed the contribution of Hif1a to the inflammatory response, indicating its potential as a therapeutic target.[33] scRNA-seq analysis of leucocytes in a mouse model of myocardial infarction (MI) revealed interferon regulatory factor 3 and type I interferon as potential therapeutic targets for post-MI cardioprotection.[20]

A major strength of single-cell technology lies in its ability to dissect global intercellular communication during disease. Ren et al[26] illustrated cellular alterations at multiple stages of pressure overload-induced pathological cardiac hypertrophy and proposed cell type- and stage-specific intervention strategies that have been experimentally validated. In a study by Wang et al,[25] the authors profiled normal hearts and identified critical cell interaction networks regulating cardiac function. Atypical chemokine receptor 1 (ACKR1)+ endothelial cells were found to maintain heart homeostasis, and their injection into infarcted mouse hearts markedly attenuated deterioration of cardiac function, suggesting translational opportunities.

Exploiting experimental findings for clinical benefits remains a challenge in the field of clinical translation. Despite assuming impeccable data quality from all studies, only a limited fraction carries diagnostic or therapeutic implications because many of them are, by nature, not translational. For instance, studying heart regeneration in zebrafish, although informative, may not always reflect processes that occur during mammalian heart regeneration. Nor do investigate pre-adult mouse models help accurately predict responses that occur during the very limited post-injury regeneration observed in the heart of an elderly individual. Currently, many purely mechanistic and characterization studies are performed in these models. For example, ErbB2 signaling was shown to be essential for mammalian cardiomyocyte regeneration based on single-cell analysis.[34] Nevertheless, whether it can drive cardiomyocyte cell cycle re-entry on its own or in combination with other stimuli has not been addressed.

Translational opportunities of cardiac single-cell RNA sequencing

Although the leap from bench to bedside is still premature, certain trends for translation are clearly visible as we discussed ahead. First, immuno-inflammatory mechanisms play an important role in mediating or modulating cardiovascular homeostasis and disorders.[35] Existing single-cell studies thus support immune cell-targeted approaches in cardiovascular disorders. Second, dynamic cell crosstalk during disease progression can serve as a viable target for intervention. In these models, changes in non-myocytes precede cardiomyocyte (effector cell type) deterioration, and paracrine-induced cell signaling diminishes the effect of pathogenic stimuli. Determining the exact target and therapeutic window may be critical in this setting because disease progression is complex, dynamic, and patient-specific. Third, strategies directed directly against cardiomyocytes are still lacking despite their preponderant roles in cardiac abnormalities. However, the reason for this phenomenon remains unclear. If cardiomyocytes function predominantly as mechanical mini-pumps, primarily influenced by their microenvironment, targeting the final effector may not prove efficient. On the other hand, current biological and biochemical approaches are probably not particularly well suited to impact cardiomyocytes (which may be more amenable to electro-mechanical modulation). Finally, when converged with trends in precision medicine, scRNA-seq and related techniques are expected to play an encouraging role. In cancer treatment, scRNA-seq has been proposed to be integrated into tumor surveillance and identification of tumor clonality.[36] In cardiovascular diseases, clinical observations suggest patient-specific differences in drug response,[37] and therefore precision medicine has become a popular goal.[38–40] In an ideal scenario, profiling cells in patients’ blood or cardiac biopsy samples may be used to monitor disease progression or reveal underlying complications that can assist decision-making in the clinic.


This work was supported by the National Key R&D Program of China (2017YFA0103700), CAMS Innovation Fund for Medical Sciences (CIFMS, 2018-I2M-3-002, 2016-I2M-1-015), grants from the National Natural Science Foundation of China (82070287 to BYZ), and the Beijing Natural Science Foundation (Z200026).

Conflicts of Interest



[1]. DeLaughter DM, Bick AG, Wakimoto H, et al. Single-cell resolution of temporal gene expression during heart development. Dev Cell 2016;39(4):480–490. doi: 10.1016/j.devcel.2016.10.001.
[2]. Li G, Xu A, Sim S, et al. Transcriptomic profiling maps anatomically patterned subpopulations among single embryonic cardiac cells. Dev Cell 2016;39(4):491–507. doi: 10.1016/j.devcel.2016.10.014.
[3]. Lescroart F, Wang X, Lin X, et al. Defining the earliest step of cardiovascular lineage segregation by single-cell RNA-seq. Science 2018;359(6380):1177–1181. doi: 10.1126/science.aao4174.
[4]. Jia G, Preussner J, Chen X, et al. Single cell RNA-seq and ATAC-seq analysis of cardiac progenitor cell transition states and lineage settlement. Nat Commun 2018;9(1):4877. doi: 10.1038/s41467-018-07307-6.
[5]. Xiong H, Luo Y, Yue Y, et al. Single-cell transcriptomics reveals chemotaxis-mediated intraorgan crosstalk during cardiogenesis. Circ Res 2019;125(4):398–410. doi: 10.1161/CIRCRESAHA.119.315243.
[6]. de Soysa TY, Ranade SS, Okawa S, et al. Single-cell analysis of cardiogenesis reveals basis for organ-level developmental defects. Nature 2019;572(7767):120–124. doi: 10.1038/s41586-019-1414-x.
[7]. Goodyer WR, Beyersdorf BM, Paik DT, et al. Transcriptomic profiling of the developing cardiac conduction system at single-cell resolution. Circ Res 2019;125(4):379–397. doi: 10.1161/CIRCRESAHA.118.314578.
[8]. Xiao Y, Hill MC, Zhang M, et al. Hippo signaling plays an essential role in cell state transitions during cardiac fibroblast development. Dev Cell 2018;45(2):153–169. e6. doi: 10.1016/j.devcel.2018.03.019.
[9]. Cui Y, Zheng Y, Liu X, et al. Single-cell transcriptome analysis maps the developmental track of the human heart. Cell Rep 2019;26(7):1934–1950. e5. doi: 10.1016/j.celrep.2019.01.079.
[10]. Asp M, Giacomello S, Larsson L, et al. A Spatiotemporal organ-wide gene expression and cell atlas of the developing human heart. Cell 2019;179(7):1647–1660. e19. doi: 10.1016/j.cell.2019.11.025.
[11]. Nomura S, Satoh M, Fujita T, et al. Cardiomyocyte gene programs encoding morphological and functional signatures in cardiac hypertrophy and failure. Nat Commun 2018;9(1):4435. doi: 10.1038/s41467-018-06639-7.
[12]. Satoh M, Nomura S, Harada M, et al. High-throughput single-molecule RNA imaging analysis reveals heterogeneous responses of cardiomyocytes to hemodynamic overload. J Mol Cell Cardiol 2019;128:77–89. doi: 10.1016/j.yjmcc.2018.12.018.
[13]. Hu P, Liu J, Zhao J, et al. Single-nucleus transcriptomic survey of cell diversity and functional maturation in postnatal mammalian hearts. Genes Dev 2018;32(19–20):1344–1357. doi: 10.1101/gad.316802.118.
[14]. Linscheid N, Logantha S, Poulsen PC, et al. Quantitative proteomics and single-nucleus transcriptomics of the sinus node elucidates the foundation of cardiac pacemaking. Nat Commun 2019;10(1):2889. doi: 10.1038/s41467-019-10709-9.
[15]. Wolfien M, Galow AM, Müller P, et al. Single-nucleus sequencing of an entire mammalian heart: cell type composition and velocity. Cells 2020;9:2. doi: 10.3390/cells9020318.
[16]. Selewa A, Dohn R, Eckart H, et al. Systematic comparison of high-throughput single-cell and single-nucleus transcriptomes during cardiomyocyte differentiation. Sci Rep 2020;10(1):1535. doi: 10.1038/s41598-020-58327-6.
[17]. Tucker NR, Chaffin M, Fleming SJ, et al. Transcriptional and cellular diversity of the human heart. Circulation 2020;142(5):466–482. doi: 10.1161/CIRCULATIONAHA.119.045401.
[18]. Litviňuková M, Talavera-López C, Maatz H, et al. Cells of the adult human heart. Nature 2020;588(7838):466–472. doi: 10.1038/s41586-020-2797-4.
[19]. Skelly DA, Squiers GT, McLellan MA, et al. Single-cell transcriptional profiling reveals cellular diversity and intercommunication in the mouse heart. Cell Rep 2018;22(3):600–610. doi: 10.1016/j.celrep.2017.12.072.
[20]. King KR, Aguirre AD, Ye YX, et al. IRF3 and type I interferons fuel a fatal response to myocardial infarction. Nat Med 2017;23(12):1481–1487. doi: 10.1038/nm.4428.
[21]. Dick SA, Macklin JA, Nejat S, et al. Self-renewing resident cardiac macrophages limit adverse remodeling following myocardial infarction. Nat Immunol 2019;20(1):29–39. doi: 10.1038/s41590-018-0272-2.
[22]. Martini E, Kunderfranco P, Peano C, et al. Single-cell sequencing of mouse heart immune infiltrate in pressure overload-driven heart failure reveals extent of immune activation. Circulation 2019;140(25):2089–2107. doi: 10.1161/CIRCULATIONAHA.119.041694.
[23]. Gladka MM, Molenaar B, de Ruiter H, et al. Single-cell sequencing of the healthy and diseased heart reveals cytoskeleton-associated protein 4 as a new modulator of fibroblasts activation. Circulation 2018;138(2):166–180. doi: 10.1161/CIRCULATIONAHA.117.030742.
[24]. Kretzschmar K, Post Y, Bannier-Hélaouët M, et al. Profiling proliferative cells and their progeny in damaged murine hearts. Proc Natl Acad Sci U S A 2018;115(52):E12245–E12254. doi: 10.1073/pnas.1805829115.
[25]. Wang L, Yu P, Zhou B, et al. Single-cell reconstruction of the adult human heart during heart failure and recovery reveals the cellular landscape underlying cardiac function. Nat Cell Biol 2020;22(1):108–119. doi: 10.1038/s41556-019-0446-7.
[26]. Ren Z, Yu P, Li D, et al. Single-cell reconstruction of progression trajectory reveals intervention principles in pathological cardiac hypertrophy. Circulation 2020;141(21):1704–1719. doi: 10.1161/CIRCULATIONAHA.119.043053.
[27]. Yekelchyk M, Guenther S, Preussner J, et al. Mono- and multi-nucleated ventricular cardiomyocytes constitute a transcriptionally homogenous cell population. Basic Res Cardiol 2019;114(5):36. doi: 10.1007/s00395-019-0744-z.
[28]. Zhou B, Wang L. Reading the heart at single-cell resolution. J Mol Cell Cardiol 2020;148:34–45. doi: 10.1016/j.yjmcc.2020.08.010.
[29]. Guo GR, Chen L, Rao M, et al. A modified method for isolation of human cardiomyocytes to model cardiac diseases. J Transl Med 2018;16(1):288. doi: 10.1186/s12967-018-1649-6.
[30]. Winkels H, Ehinger E, Vassallo M, et al. Atlas of the immune cell repertoire in mouse atherosclerosis defined by single-cell RNA-sequencing and mass cytometry. Circ Res 2018;122(12):1675–1688. doi: 10.1161/CIRCRESAHA.117.312513.
[31]. Cochain C, Vafadarnejad E, Arampatzi P, et al. Single-cell RNA-Seq reveals the transcriptional landscape and heterogeneity of aortic macrophages in murine atherosclerosis. Circ Res 2018;122(12):1661–1674. doi: 10.1161/CIRCRESAHA.117.312509.
[32]. Fernandez DM, Rahman AH, Fernandez NF, et al. Single-cell immune landscape of human atherosclerotic plaques. Nat Med 2019;25(10):1576–1588. doi: 10.1038/s41591-019-0590-4.
[33]. Hua X, Hu G, Hu Q, et al. Single-cell RNA sequencing to dissect the immunological network of autoimmune myocarditis. Circulation 2020;142(4):384–400. doi: 10.1161/CIRCULATIONAHA.119.043545.
[34]. Honkoop H, de Bakker DE, Aharonov A, et al. Single-cell analysis uncovers that metabolic reprogramming by ErbB2 signaling is essential for cardiomyocyte proliferation in the regenerating heart. Elife 2019;8. doi: 10.7554/eLife.50163.
[35]. Swirski FK, Nahrendorf M. Cardioimmunology: the immune system in cardiac homeostasis and disease. Nat Rev Immunol 2018;18(12):733–744. doi: 10.1038/s41577-018-0065-8.
[36]. Bagger FO, Probst V. Single cell sequencing in cancer diagnostics. Adv Exp Med Biol 2020;1255:175–193. doi: 10.1007/978-981-15-4494-1_15.
[37]. Kolandaivelu K, Leiden BB, O’Gara PT, et al. Non-adherence to cardiovascular medications. Eur Heart J 2014;35(46):3267–3276. doi: 10.1093/eurheartj/ehu364.
[38]. Leopold JA, Loscalzo J. Emerging role of precision medicine in cardiovascular disease. Circ Res 2018;122(9):1302–1315. doi: 10.1161/CIRCRESAHA.117.310782.
[39]. Currie G, Delles C. Precision medicine and personalized medicine in cardiovascular disease. Adv Exp Med Biol 2018;1065:589–605. doi: 10.1007/978-3-319-77932-4_36.
[40]. Antman EM, Loscalzo J. Precision medicine in cardiology. Nat Rev Cardiol 2016;13(10):591–602. doi: 10.1038/nrcardio.2016.101.
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