# Single-Nucleus RNA Sequencing Reveals SPP1+ Macrophages Induce Cardiomyocyte Apoptosis to Promote Atrial Fibrillation Susceptibility

## TL;DR

This study shows that SPP1+ macrophages in heart tissue cause heart cell death, increasing the risk of atrial fibrillation.

## Contribution

The novel finding is that SPP1+ macrophages induce cardiomyocyte apoptosis via PI3K/AKT pathway downregulation, promoting atrial fibrillation.

## Key findings

- SPP1+ macrophages are significantly increased in atrial tissues of AF patients and communicate strongly with cardiomyocytes.
- SPP1+ macrophages induce cardiomyocyte apoptosis by downregulating the PI3K/AKT pathway.
- Activating the PI3K/AKT pathway reverses apoptosis caused by SPP1+ macrophages in co-culture experiments.

## Abstract

Atrial fibrillation (AF) is closely linked to atrial remodeling, while its underlying immune mechanisms remain elusive. This study sought to investigate the role of SPP1+ macrophages in the development and progression of AF and further elucidate the underlying mechanisms. Single-nucleus RNA sequencing was performed on right atrial tissues from 3 patients with persistent AF and 3 with sinus rhythm (all with rheumatic valvular heart disease). The results revealed significant immune cell infiltration in AF atrial tissues, with a marked increase in the proportion of SPP1+ macrophages, which exhibited the strongest intercellular communication with cardiomyocytes. Phenotypic scoring indicated that apoptosis was the dominant mode of cardiomyocyte death in AF. Immunohistochemical and Western blot analyses confirmed elevated levels of pro-apoptotic proteins (Bax, Cleaved-Caspase3) and reduced levels of the anti-apoptotic protein Bcl2 in AF tissues. In a mouse model with macrophage-specific SPP1 overexpression, increased AF inducibility and duration were observed, accompanied by enhanced cardiomyocyte apoptosis. In vitro co-culture experiments using SPP1-overexpressing RAW264.7 macrophages and HL-1 cardiomyocytes confirmed that SPP1+ macrophages could induce cardiomyocyte apoptosis. Mechanistically, KEGG and GSEA analyses identified downregulation of the PI3K/AKT pathway in AF. Treatment with the PI3K/AKT activator Recilisib reversed apoptosis and restored p-PI3K/p-AKT levels in HL-1 cells co-cultured with SPP1-overexpressing RAW264.7 macrophages. These findings demonstrate that SPP1+ macrophages accumulate in atrial tissues of AF patients and induce cardiomyocyte apoptosis by downregulating the PI3K/AKT pathway, thereby increasing AF susceptibility.