Available online 16 May 2025, 107743

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Comprehensive single-nucleus RNA sequencing reveals cellular alterations in LACM.

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Identified key cardiomyocyte, fibroblast, and adipocyte subclusters driving pathology.

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CM-to-adipocyte transdifferentiation and PI3K-AKT signaling pathway highlighted.

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ANKRD1, SLC8A1, and LTBP1 validated as markers of pathological remodeling.

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Insights provide novel therapeutic targets for left-dominant arrhythmogenic cardiomyopathy.

AbstractBackground

Arrhythmogenic cardiomyopathy (ACM) is a myocardial disorder characterized by arrhythmias and an increased risk of sudden cardiac death, particularly in left-dominant arrhythmogenic cardiomyopathy (LACM), which primarily affects the left ventricle. This study aims to elucidate the cellular and molecular mechanisms underlying LACM by performing an in-depth single-nucleus RNA sequencing (snRNA-seq) analysis to identify key transcriptional signatures and pathways involved in the disease's pathogenesis.

Method

Human heart samples were collected from five patients undergoing heart transplantation due to ACM and from four healthy donors. Single nuclei were isolated from myocardial tissues and subjected to snRNA-seq using the 10 × Genomics Chromium platform. Data were processed and analyzed to identify distinct cell populations and their differentially expressed genes. Immunofluorescence staining was used to validate key findings.

Result

The snRNA-seq analysis revealed an increased proportion of fibroblasts and adipocytes in the left ventricles of LACM patients, suggesting a cellular basis for the fibrofatty remodeling observed in the disease.Key cell populations, including cardiomyocytes (CMs), fibroblasts (Fbs), and adipocytes, were identified with distinct transcriptional profiles. We identified a disease-associated cardiomyocyte subpopulation (CM1) characterized by upregulation of fibrosis-, metabolism-, and stress-related markers, indicating transcriptional remodeling processes involved in LACM. The Fb subgroup Fb1 was characterized by genes involved in the PI3K-AKT signaling pathway. Adipocyte subpopulations exhibited gene expression features reflecting adaptation to the cardiac pathological environment, including markers associated with extracellular matrix remodeling and metabolic stress.

Immunofluorescence staining validated the high expression of key markers of LACM patients.

Conclusion

This study provides a cellular and molecular characterization of LACM, identifying key pathways and transcriptional signatures that contribute to the disease's pathogenesis. These findings enhance our understanding of LACM and offer potential targets for therapeutic intervention.

Graphical abstractDownload: Download high-res image (128KB)Download: Download full-size imageIntroduction

Arrhythmogenic cardiomyopathy (ACM) encompasses a heterogeneous group of myocardial disorders with a predilection for arrhythmias and an elevated risk of sudden cardiac death[1]. Within this spectrum, left-dominant ACM (LACM) is distinguished by its primary affliction of the left ventricle (LV), posing distinct diagnostic and therapeutic challenges[2]. The intricacies of LACM have hitherto been veiled, largely due to the variable presentation and the complex molecular milieu of the affected myocardium.

Despite its lower prevalence compared to classical right-dominant ACM associated with pathogenic variants such as those in DSP and LMNA, LACM carries a significantly worse prognosis due to the essential role of the left ventricle in systemic circulation and pump function. Importantly, according to our previous findings1, a distinct subtype of LACM, termed Type IV in the Fuwai classification, is characterized by extensive myocardial fibrosis and fatty infiltration without detectable pathogenic genetic variants. Given the high-risk clinical implications and the relative paucity of molecular data on this subgroup, we prioritized the investigation of these rare yet clinically consequential cases. Our previous study leverages the foundational insights provided by the fuwai classification to dissect LACM at an cellular resolution. By integrating single-nucleus RNA sequencing (snRNA-seq) data with the robust classification framework, we have identified key transcriptional signatures and pathways that reveal the potential mechanistic trajectories from early disease states to advanced cardiomyopathy. By cataloging the single-cell transcriptomic alterations within the LV, we have illuminated the cellular heterogeneity and pivotal molecular pathways that drive LACM pathogenesis. The data acquired from LACM tissue, interpreted through the lens of our classification, elucidate novel aspects of disease progression and myocardial deterioration.

The research provides an resource for the identification of biomarkers and therapeutic targets, offering a transformative perspective on LACM's molecular landscape.

Section snippetsHuman heart sample collection and single nucleus acquisition

The Human Ethics Committee of Fuwai Hospital, Chinese Academy of Medical Sciences approved human tissue use in the present study, with the approval number of FW-2022–0043. Written informed consent was obtained from each patient.1g of the left anterior ventricular samples of ACM patients were obtained at the time of HTx. All patients were negative for the gene mutation associated with ACM. The identical regions from the NC samples were obtained from brain-death donors with normal circulatory

Fibrofatty infiltration of LACM

5 HTx patients diagnosed as LACM were enrolled in this study. The clinical characterization of the LACM patients was presented in Table S1 and S2. The late gadolinium enhancement-cardiac magnetic resonance identified that the LV but not the right ventricle (RV) of LACM patients underwent significantly fibrofatty remodeling (Figure 1A). In addition, Masson's trichrome staining was used to further quantify the fibrofatty infiltration of the LV of the LACM. The fat replacement level of LV in LACM

Discussion

In this study, we constructed the first single-cell atlas of LACM, providing an view of the cellular and molecular alterations underlying this disease. Our snRNA-seq analysis identified distinct cell populations in the LV of LACM patients, including CMs, fibroblasts, and adipocytes, each exhibiting unique transcriptional profiles. Given the variability inherent in snRNA-seq data, we interpreted cardiomyocyte subcluster changes based on overall trends. While CM2 was reduced in disease groups,

Funding

This study was supported by National Science Fund for Distinguished Young Scholars (grant no.82125004; to JPS), Shenzhen Science and Technology Innovation Commission (grant no.JCYJ20220818103414030), Independent Programs of Shenzhen Key Laboratory of Cardiovascular Disease, Fuwai Shenzhen Hospital, Chinese Academy of Medical Sciences [Grant No. GSP-ZDSYS-004, GSP-ZDSYS-008, GSP-ZDSYS-019, GSP-ZDSYS-023]

CRediT authorship contribution statement

Yiqi Zhao: Writing – original draft, Methodology, Investigation, Formal analysis. Mengda Xu: Writing – original draft, Visualization, Methodology, Formal analysis. Xiumeng Hua: Supervision, Investigation, Data curation. Yuan Chang: Methodology, Data curation. Yixuan Sheng: Resources, Methodology. Dan Shan: Resources, Methodology. Ningning Zhang: Resources, Methodology. Xiao Chen: Writing – review & editing, Data curation, Conceptualization. Jiangping Song: Supervision, Funding acquisition,

Declaration of competing interest

The authors reported no conflicts of interest.

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