Hypoplastic left heart syndrome (HLHS) is a congenital heart disease with a high mortality, especially during the early neonatal period and surgical treatment [1]. HLHS is mainly characterized by severe hypoplasia of the mitral valve, aortic valve, left ventricle, and ascending aorta. Children born with HLHS require staged surgery, which, however, only works to reconfigure the structure and blood flow of the heart. Even after surgical treatment, children with HLHS often have poor exercise capacity and might develop right ventricular failure [2]. Currently, a broad spectrum of variations and molecular pathways correlated with the etiology of HLHS have been discovered [3], but the intricate cellular interactions that result in HLHS remain to be comprehensively studied.

Macrophages as an important component of the innate and adaptive immune responses play crucial roles in maintaining the normal functions of the cardiovascular system. Normally, macrophages eliminate apoptotic cells to protect the organism from pathogens, participating in electrical conduction in arterial tone regulation, and heart, and vessel patrolling [4,5]. The predominant cell population in a healthy heart consists of resident macrophages with only a limited proportion of macrophages derived from monocytes [6]. However, when the heart is injured, cardiomyocytes experience necrosis to attract circulating monocytes expressing the CCR2 receptor, and these monocytes, along with the monocyte-derived macrophages, play a crucial role in eliminating dead cells and debris from the injured site. Moreover, they release pro-inflammatory cytokines to promote the degradation of extracellular matrix during the initial phases of cardiac injury, and then a specific subgroup of resident macrophages will secrete substantial quantities of anti-inflammatory cytokines to enhance tissue repair at the inflammation subsides [7–9]. The highly plastic nature of macrophages often blurs the boundaries of different phenotypes, which is largely correlated with the tissue microenvironment and macrophage-specific functions [5]. At present, the molecular mechanisms through which macrophages influence the progression of HLHS remain unclear, which requires a comprehensive analysis of macrophage functions in HLHS to help discover effective therapeutic targets.

Advances in single-cell and single-nucleus RNA sequencing (snRNA-seq) technologies allow for a better understanding of the underlying transcriptional profiles of healthy and diseased human hearts [10]. In this study, snRNA-seq data were employed to analyze the macrophage landscape in HLHS and to explore its molecular mechanisms. The workflow diagram is shown in Fig. S1. The snRNA-seq data of HLHS were collected from public databases to define macrophage subtypes by performing clustering analysis. Trajectory analysis of macrophage subpopulations and transcription factor regulatory networks were used to characterize the dynamic changes of macrophage from healthy state to HLHS. In conclusion, our study confirmed that macrophages played a key role in the development of HLHS, providing a new direction for the treatment of HLHS patients.

HLHS refers to an anatomical and functional inadequacy of the left side of the heart and an inability of the left ventricle to perform systemic perfusion [18]. Macrophages have prominent immune-independent tissue-specific functions in the heart. In particular, for example, tissue-resident macrophages could prevent fibrosis, increase electrical conduction in the heart, and facilitate wound healing [4,16]. However, the extreme plasticity of macrophages often blurs the boundaries between their different phenotypes. Macrophage expression in different tissues can be distinguished by different gene expression profiles and protein levels [5,19–21]. Therefore, we used snRNA-seq data to further analyze the molecular mechanisms of macrophages in HLHS.

It was found that the proportion of macrophage clusters was reduced in HLHS patients compared to donors. To further explore the heterogeneity of macrophages in HLHS, macrophages were divided into 3 cellular subpopulations (Macrophages 1, 2, and 3). Macrophages 1 were largely involved in nervous system developmental, angiogenic, and apoptotic pathways. The heart is highly innervated by autonomic nerve cells, and dynamic autoregulation of the heart and blood vessels is essential for maintaining normal life activities in mammals [22]. Fujiu et al. showed that in mice with cardiac pressure overload, sympathetic activation activates cells within the kidney in which cytokine CSF2 causes secretion between tissue macrophages and endothelial cells and then stimulates cardiac-resident macrophages. This dynamic interaction also highlights the homeostatic function of tissue macrophages and the sympathetic nervous system in the context of cardiovascular disease [23]. HLHS exhibits pathological changes such as empty areas within the heart muscle, formation of vascular channels, and infiltration of mononuclear cells along the septum of the ventricle [24]. Analysis of the communication between Macrophages 1 and cardiomyocytes showed that some ligand-acceptors were absent in the HLHS group but not in donors, which suggested that these ligands and receptors functioned critically in the development of HLHS. For example, AXL/GAS6 is essential for the secretion of gonadotropin-releasing hormone (GnRH) to promote survival and migration of hypothalamic neurons. AXL enhances this process by facilitating Rac1-mediated chemotactic migration of the developing GnRH neurons [25,26]. IL6, a pleiotropic cytokine, plays a key role in tissue regeneration and cell differentiation [27–30]. Hunter and his colleagues found that the functions of IL6 and its receptors vary when IL6 is expressed in different cells or activated at different times or modes [31]. Although IGF1 is structurally and functionally related to insulin, it has a high growth-promoting activity. By stimulating glucose transport in bone-derived osteoblasts, IGF1 acts at a much lower concentration than insulin, which contributes to synapse maturation [32,33]. In addition, in cell-cell contact, THY1 also appeared only in the donor group and was absent in the HLHS group. During synaptogenesis and other events in the brain, THY1 also plays an important role in cell-ligand or cell-cell interactions [34]. These results tentatively confirmed that the Macrophages 1 subpopulation identified in this study can promote nervous system development, tissue regeneration, and angiogenesis, which were similar to the functions of cardiac tissue-resident macrophages [17,35]. Hence, we speculated that Macrophages 1 may be a cardiac tissue-resident macrophages.

Our pseudotime-based analyses demonstrated that Macrophages 1 had the same potential to promote the nervous system and cardiac development and the growth of multiple tissues. In addition, cardiac and neurodevelopment-related genes progressively reduced in Macrophages 1 along the pseudotime trajectories. In particular, we observed that two TFs (FOXO3 and ELF2) were significantly negatively correlated with sham time, and that the target genes associated with pathways such as cardiac development, WNT, cell cycle, and signaling were all significantly reduced in the HLHS group. FOXO3 could promote gene-gene interactions, changes in chromatin conformation, and interactions of topologically related structural domains, which are involved in the regulation of several genes in cellular resilience processes such as stress response, autophagy, cell proliferation, and apoptosis [36,37]. Hill et al. observed a state of congenital heart disease-specific cells in cardiomyocytes with increased signaling of FOXO [38]. FOXO3 has been shown to have a decisive effect on cardiomyocytes, and some researchers indicated that microRNAs (miR-122) can facilitate cardiomyocyte hypertrophy progression partially by directly regulating the FoxO3-calcineurin pathway [39]. In addition, Willcox et al. showed that the main reason for the protective allele of FOXO3 to extend human lifespan is its protection against coronary death [40]. Similarly, Chen et al. demonstrated that the FOXO3-associated longevity genotype could also extend the lifespan of high-risk patients with cardiometabolic diseases by protecting the heart under metabolic stress [41]. As a member of the Ets family of transcription factors, ELF2 participates in the regulatory regions of lymphocyte development and functional genes [42]. Guan et al. identified ELF2 isoforms as important regulators of cell proliferation, cell cycle, and apoptosis [43]. Consistently, we found that the expression levels of the relevant cell cycle target genes of ELF2 in Macrophages 1 were downregulated in patients with HLHS. These results reconfirmed that the critical role of the Macrophages 1 subpopulation in promoting homeostasis of cardiac function may be achieved by targeting FOXO3 and ELF2.

However, there were some limitations in our study. Firstly, the data in this paper were obtained mainly from public databases, and due to the rarity of cardiac explants in children, we were unable to obtain snRNA-seq derived from fresh tissues of healthy controls. Hence, our future study will increase the diversity of datasets in order to provide a more comprehensive analysis of biosignatures and disease mechanisms. In addition, an animal model of HLHS and fresh tissue samples will be used to verify the reliability and accuracy of the current results.

In summary, we analyzed snRNA-seq data to classify macrophage subtypes that maintained the cardiac functions in HLHS and revealed the differentiation status of Macrophages 1 and the activity of TFs. Our study confirmed that the role of Macrophages 1 in maintaining functional homeostasis of cardiomyocytes was possibly realized through regulating FOXO3 and ELF2 or targeting their genes. The present findings provided novel insights into the heterogeneity of macrophages in HLHS and the molecular mechanisms of macrophages in the development of HLHS.