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Atrial fibrillation (AF) is the most common sustained arrhythmia in humans, yet the molecular basis of AF remains incompletely understood. To determine the cell type-specific transcriptional changes underlying AF, we perform single-nucleus RNA-seq (snRNA-seq) on left atrial (LA) samples from patients with AF and controls. From more than 175,000 nuclei we find that only cardiomyocytes (CMs) and macrophages (MΦs) have a significant number of differentially expressed genes in patients with AF. Attractin Like 1 (ATRNL1) was overexpressed in CMs among patients with AF and localized to the intercalated disks. Further, in both knockdown and overexpression experiments we identify a potent role for ATRNL1 in cell stress response, and in the modulation of the cardiac action potential. Finally, we detect an unexpected expression pattern for a leading AF candidate gene, KCNN3. In sum, we uncover a role for ATRNL1 which may serve as potential therapeutic target for this common arrhythmia., Competing Interests: Competing interests C.A.K. is an employee of Bayer US LLC (a subsidiary of Bayer AG) and may own stock in Bayer. The Precision Cardiology Laboratory is joint effort between the Broad Institute and Bayer AG. P.T.E. has received sponsored research support from Bayer AG, Novo-Nordisk, Bristol Myers Squibb and Pfizer; he has also served on advisory boards or consulted for Bayer AG. C.R. is a full-time employee at GSK as of July 2024. All remaining authors declare no competing interests., (© 2024. The Author(s).)

The adult mammalian heart's potential for regeneration is very inefficient. Importantly, adult mammalian cardiomyocytes (CMs) are characterized as a cell population with very limited mitotic potential. Conversely, the neonatal mouse heart possesses a brief, yet robust, regenerative capacity within the first week of life. Cell type-specific enrichment procedures are essential for characterizing the full spectrum of epigenomic landscapes and gene regulatory networks deployed by mammalian CMs. In this chapter, we describe a protocol useful for purifying CM nuclei from mammalian cardiac tissue. Furthermore, we detail a low-input procedure suitable for the parallel genome-wide profiling of chromatin accessibility, histone modifications, and transcription factor-binding sites. The CM nuclei purified using this process are suitable for multi-omic profiling approaches.

Background: Surviving cells in the postinfarction border zone are subjected to intense fluctuations of their microenvironment. Recently, border zone cardiomyocytes have been specifically implicated in cardiac regeneration. Here, we defined their unique transcriptional and regulatory properties, and comprehensively validated new molecular markers, including Nppb, encoding B-type natriuretic peptide, after infarction., Methods: Transgenic reporter mice were used to identify the Nppb-positive border zone after myocardial infarction. Transcriptome analysis of remote, border, and infarct zones and of purified cardiomyocyte nuclei was performed using RNA-sequencing. Top candidate genes displaying border zone spatial specificity were histologically validated in ischemic human hearts. Mice in which Nppb was deleted by genome editing were subjected to myocardial infarction. Chromatin accessibility landscapes of border zone and control cardiomyocyte nuclei were assessed by using assay for transposase-accessible chromatin using sequencing., Results: We identified the border zone as a spatially confined region transcriptionally distinct from the remote myocardium. The transcriptional response of the border zone was much stronger than that of the remote ventricular wall, involving acute downregulation of mitochondrial oxidative phosphorylation, fatty acid metabolism, calcium handling, and sarcomere function, and the activation of a stress-response program. Analysis of infarcted human hearts revealed that the transcriptionally discrete border zone is conserved in humans, and led to the identification of novel conserved border zone markers including NPPB, ANKRD1, DES, UCHL1, JUN, and FOXP1. Homozygous Nppb mutant mice developed acute and lethal heart failure after myocardial infarction, indicating that B-type natriuretic peptide is required to preserve postinfarct heart function. Assay for transposase-accessible chromatin using sequencing revealed thousands of cardiomyocyte lineage-specific MEF2-occupied regulatory elements that lost accessibility in the border zone. Putative injury-responsive enhancers that gained accessibility were highly associated with AP-1 (activator protein 1) binding sites. Nuclear c-Jun, a component of AP-1, was observed specifically in border zone cardiomyocytes., Conclusions: Cardiomyocytes in a discrete zone bordering the infarct switch from a MEF2-driven homeostatic lineage-specific to an AP-1-driven injury-induced gene expression program. This program is conserved between mouse and human, and includes Nppb expression, which is required to prevent acute heart failure after infarction.

The Pitx2 gene encodes a homeobox transcription factor that is required for mammalian development. Disruption of PITX2 expression in humans causes congenital heart diseases and is associated with atrial fibrillation; however, the cellular and molecular processes dictated by Pitx2 during cardiac ontogeny remain unclear. To characterize the role of Pitx2 during murine heart development we sequenced over 75,000 single cardiac cell transcriptomes between two key developmental timepoints in control and Pitx2 null embryos. We found that cardiac cell composition was dramatically altered in mutants at both E10.5 and E13.5. Interestingly, the differentiation dynamics of both anterior and posterior second heart field-derived progenitor cells were disrupted in Pitx2 mutants. We also uncovered evidence for defects in left-right asymmetry within atrial cardiomyocyte populations. Furthermore, we were able to detail defects in cardiac outflow tract and valve development associated with Pitx2 Our findings offer insight into Pitx2 function and provide a compilation of gene expression signatures for further detailing the complexities of heart development that will serve as the foundation for future studies of cardiac morphogenesis, congenital heart disease and arrhythmogenesis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Loss of the paired-like homeodomain transcription factor 2 ( Pitx2 ) in cardiomyocytes predisposes mice to atrial fibrillation and compromises neonatal regenerative capacity. In addition, Pitx2 gain-of-function protects mature cardiomyocytes from ischemic injury and promotes heart repair. Here, we characterized the long-term myocardial phenotype following myocardial infarction (MI) in Pitx2 conditional-knockout ( Pitx2 CKO) mice. We found adipose-like tissue in Pitx2 CKO hearts 60 days after MI induced surgically at postnatal day 2 but not at day 8. Molecular and cellular analyses showed the onset of adipogenic signaling in mutant hearts after MI. Lineage tracing experiments showed a non-cardiomyocyte origin of the de novo adipose-like tissue. Interestingly, we found that Pitx2 promotes mitochondrial function through its gene regulatory network, and that the knockdown of a key mitochondrial Pitx2 target gene, Cox7c , also leads to the accumulation of myocardial fat tissue. Single-nuclei RNA-seq revealed that Pitx2 -deficient hearts were oxidatively stressed. Our findings reveal a role for Pitx2 in maintaining proper cardiac cellular composition during heart regeneration via the maintenance of proper mitochondrial structure and function., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)