Feb 14, 2022 / Immunology

Single cell tools open the heart-shaped black box of cardiovascular disease

Jeanene Swanson

As we acknowledge American Heart Month in February and celebrate Valentine’s Day mid-month, it’s a good reminder that, even though we “heart” our loved ones, disease of this vital, life-giving organ is the leading cause of death in the US and worldwide (1). Initiated by President Lyndon B. Johnson in 1964, American Heart Month was designed to shine a light on the epidemic of heart disease and stroke. Recognition is even more relevant in recent years as we’ve begun to learn more about the ways in which the coronavirus can potentially harm the heart and cardiovascular system (2).

To honor groundbreaking discoveries in cardiovascular research that single cell technologies have enabled, we feature two publications that delve into the mechanisms behind heart disease at the single cell level. Armed with this newfound ability to probe deeper than ever before, scientists are making inroads into how we can improve both the prevention and treatment of this silent killer in the future.

Heart disease is the leading cause of death worldwide according to the American Heart Association. A recent report revealed that almost 18.6 million people died of cardiovascular disease in 2019, an increase of 17.1% over the past decade, and there were 523.2 million reported cases, a 26.6% increase over 2010 (3).

These jarring statistics underscore the urgency of advancing cardiovascular research to come up with more actionable insights into prevention and treatments. To that end, scientists are using single cell tools to dig deeper into studying the fibrosis that leads to heart failure. In two recent publications, they’ve made discoveries around fibroblast phenotypic switching and have created a single cell transcriptomic map of the failing heart. Both works contribute essential knowledge to a foundational understanding of the cellular pathways that drive cardiac disease.

Exploring how fibroblast phenotypic switching contributes to heart disease

In the first publication (4), A transcriptional switch governs fibroblast activation in heart disease, researchers in the lab of Deepak Srivastava, MD, at the Gladstone Institutes in San Francisco used a combination of Chromium Single Cell Gene Expression and Single Cell ATAC for chromatin accessibility profiling to study fibroblast activation in the mouse heart. When activated, cardiac fibroblasts initiate a stress response that leads to heart disease. It is known that treating cardiac disease with the molecule, JQ1, which inhibits BET proteins, improves heart function (5). In their study, they used single cell RNA sequencing (scRNA-seq) to learn what happens to different cell types when this treatment is started and stopped. They performed scRNA-seq on various groups, including control mice, mice with untreated heart failure, mice being treated with JQ1, and mice that had been recently taken off treatment. Surprisingly, they found a “reversible transcriptional switch,” in that fibroblasts’ transcriptomes alternated between quiescent and activated states depending on whether or not they were being treated.

Next, they used Single Cell ATAC to perform single cell assay for transposase-accessible chromatin sequencing (scATAC-seq) on the same hearts, with the aim of focusing on enhancers (DNA sequences located anywhere in the genome that jumpstart transcription of a given gene) where increased accessibility is correlated with worse heart function. They discovered an enhancer showing a large negative correlation to heart function located downstream of transcription factor MEOX1. Functional studies revealed that this element is a necessary component of MEOX1 expression in TGFβ-induced fibroblast activation. In essence, MEOX1 may serve as the “switch” that turns on cardiac fibroblasts and induces fibrosis in the heart. Further research into how cell state plasticity contributes to disease mechanisms in cardiac failure has the potential to provide new avenues for developing new treatments.

Uncoupling inflammation and fibrosis to understand their fundamental involvement in heart failure

In the second publication (6), Resolving the intertwining of inflammation and fibrosis in human heart failure at single-cell level, the lab of Jiangping Song, MD, PhD, of Fuwai Hospital in Beijing created the first single cell transcriptomic atlas of leukocytes and non-myocytes in the adult human failing heart by blending Chromium Single Cell Gene Expression and Single Cell Immune Profiling. Recent research suggests that inflammation in the heart is essential to the process of cardiac fibrosis, effectively leading to heart failure (7). Ischemic cardiomyopathy (ICM) and dilated cardiomyopathy (DCM) are both prevalent conditions, and the team chose to sample both states in order to provide the most comprehensive map. Using scRNA-seq, they profiled the transcriptomes of three DCM hearts, three ICM hearts, and two non-failing donor hearts to reveal 13 major cell types, including fibroblasts, various immune cell types, and others. Within this heterogeneity that showed extensive phenotypic changes, they used pseudo-time trajectory analysis to discover that transcription factor AEBP1 was increasingly expressed in POSTN+ fibroblasts and ACTA2+ myofibroblasts, making it a possibly novel regulator of cardiac fibrosis.

Considering that they found T cells within the failing hearts, they used Chromium Single Cell Immune Profiling to perform single cell immune repertoire profiling on T cells collected from two additional DCM patients and one ICM patient. They found that cytotoxic CD8+ T cells infiltrate the fibrotic heart and CD4+ T cells transition to a pro-inflammatory state, leading to the inflammation seen in cardiac failure. Further transcriptome analysis of myeloid cells in their scRNA-seq data showed that macrophages could be divided by CCR2 and MHC class II molecule expression (CCR2-HLA-DRhi and CCR2+HLA-DRhi macrophages) and that the pro-angiogenic chemokine CXCL8 was upregulated in some cells. Further, a subset of CXCL8hiCCR2+HLA-DRhi macrophages move specifically to the myocardium in severe cardiac fibrosis and spur the recruitment of leukocytes and inflammation. Finally, they mapped receptor–ligand pairs using CellPhoneDB to look at cell–cell interactions, finding that there is crosstalk between DARC+ endothelial cells and CXCL8hiCCR2+HLA-DRhi macrophages, which could potentially lead to more leukocyte infiltration and increased inflammation. This single cell map of the heart immune microenvironment will go a long way towards discovering novel pathways and crosstalk, helping to increase understanding of the mechanisms behind heart disease, and may potentially lead to new therapeutic targets.


  4. Alexanian M, et al. A transcriptional switch governs fibroblast activation in heart disease. Nature 595: 438–443 (2021).
  5. Antolic A, et al. BET bromodomain proteins regulate transcriptional reprogramming in genetic dilated cardiomyopathy. JCI Insight 5: e138687 (2020).
  6. Rao M, et al. Resolving the intertwining of inflammation and fibrosis in human heart failure at single-cell level. Basic Res Cardiol 116: 55 (2021).
  7. Bansal SS, et al. Dysfunctional and proinflammatory regulatory T-lymphocytes are essential for adverse cardiac remodeling in ischemic cardiomyopathy. Circulation 139: 206–221 (2019).