P2X7 receptors promote atrial remodeling and atrial fibrillation susceptibility via reactive oxygen species‐mediated mitogen‐activated protein kinase signaling activation

Figures

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  FIGURE 1. Transcriptomic profiling of HL‐1 atrial cardiomyocytes under rapid electrical stimulation. (A) Experimental workflow: HL‐1 atrial cardiomyocytes were subjected to either rapid electrical stimulation or left untreated. RNA was extracted for microarray analysis and identification of DEGs. (B) Distribution of normalized signal intensities across RNA‐seq samples. (C) Principal component analysis reveals clear separation between ref and test groups along PC1 and PC2 dimensions, indicating distinct transcriptomic profiles. (D) Volcano plot displaying the distribution of DEGs—red denotes upregulated genes and blue denotes downregulated genes. (E) Heat map displaying the expression patterns of the top 20 upregulated genes. (F) Gene ontology and and Kyoto Encyclopedia of Genes and Genomes enrichment analysis bubble plots, showing that differentially expressed genes are significantly enriched in pathways such as mitogen‐activated protein kinase signaling, apoptotic signaling, and protein folding chaperone‐related pathways. (G) Bar graph showing a significant upregulation of P2rx7 expression in the test group. “Ref” refers to spontaneously beating HL‐1 cardiomyocytes (n = 2), and “test”…

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  FIGURE 2. Shortening of action potential duration in HL‐1 cells following rapid electrical stimulation. (A) Representative action potential traces recorded from HL‐1 cells under control conditions and after 2 days of rapid pacing at 300 bpm; the recording frequency was 1 Hz. (B) Summary of APD90 measurements at different stimulation frequencies, comparing control and paced groups. (C) Representative ICa, L traces recorded via patch clamp, with voltage steps initiated from −90 mV (left) and −40 mV (right). (D) Dose–response curves of L‐type calcium current inhibition by varying concentrations of nimodipine. (E) Current–voltage (I–V) relationships under different holding potentials (−90 mV and −40 mV), illustrating voltage dependence. All experiments were repeated in triplicate (n = 3 biological replicates). *p < 0.05.

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  FIGURE 3. Effects of rapid electrical stimulation on ion channel expression, oxidative stress, and mitogen‐activated protein kinase (MAPK) pathway activation in HL‐1 cells. (A) Experimental grouping schematic. (B) WB analysis of Cav1.2 and Kv1.5 protein expression in control (control) and rapid stimulation (paced) groups. (C) Fluorescence microscopy images and quantitative analysis of reactive oxygen species accumulation using the DCFH‐DA fluorescent probe (scale bar: 50 μm). (D–E) WB analysis of ROS‐related proteins NOX2 and NOX4. (F) WB analysis of MAPK pathway proteins including phosphorylated and total ERK, p38, and JNK. (G) Quantification of relative expression levels of phosphorylated MAPK proteins (p‐ERK, p‐p38, and p‐JNK). (H) ELISA results showing concentrations of inflammatory cytokines IL‐6, IL‐1β, TNF‐α, and IL‐18 in cell culture supernatants. All experiments were performed in triplicate (n = 3 biological replicates). *p < 0.05, **p < 0.01, and ***p < 0.001.

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  FIGURE 4. Inhibition of P2X7 confirms its central role in the ROS‐MAPK inflammation pathway. (A) Schematic overview of the experimental design, (B) representative fluorescence images and quantitative analysis of intracellular reactive oxygen species levels in HL‐1 cells from control, paced, and paced + A‐438079 groups using DCFH‐DA staining (scale bar: 50 μm), (C–F) WB analysis and corresponding densitometry of ROS‐generating enzymes and mitogen‐activated protein kinase pathway proteins, and (G) ELISA quantification of IL‐6, IL‐1β, TNF‐α, and IL‐18 levels in cell culture supernatants. All cell experiments were performed in triplicate (n = 3 biological replicates). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

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  FIGURE 5. Reactive oxygen species (ROS) as a critical mediator of P2X7‐induced mitogen‐activated protein kinase (MAPK) activation and inflammatory cytokine expression. (A) Experimental workflow illustrating four groups, control, paced, paced + NAC, and paced + NAC + P2X7 overexpression (OE), to assess whether MAPK signaling and inflammatory responses depend on ROS accumulation, (B) DCFH‐DA staining of cytosolic ROS in HL‐1 cells across groups, with quantification of fluorescence intensity (scale bar: 50 μm), (C) MitoSOX Red staining to visualize mitochondrial ROS levels (scale bar: 50 μm), (D and E) WB detection of phosphorylated MAPK proteins (p‐ERK, p‐p38, and p‐JNK) and ROS‐associated enzymes (NOX2 and NOX4), with corresponding densitometry, and (F) ELISA measurement of IL‐6, IL‐1β, TNF‐α, and IL‐18 levels in supernatants. All cell experiments were conducted in triplicate (n = 3 biological replicates). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant.

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  FIGURE 6. Inhibition of the P2X7 receptor improves atrial conduction and structural remodeling in PAF rats. (A) Schematic of experimental grouping and treatment protocol, (B) representative ECG recordings from each group, (C and D) atrial conduction dispersion assessed using the MappingLab system, (E and F) echocardiographic measurement of the left atrial systolic diameter and the area, (G and H) Masson's trichrome staining to evaluate atrial collagen deposition (scale bar: 50 μm), (I and J) immunohistochemical detection of tyrosine hydroxylase protein expression in atrial tissue (scale bar: 50 μm), and (K) representative hematoxylin and eosin (H&E) staining images showing pathological alterations in each group. n = 6 animals per group. *p < 0.05, **p < 0.01, and ****p < 0.0001; ns indicates no significant difference.

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  FIGURE 7. In vivo validation showing that P2X7 receptor activation promotes mitogen‐activated protein kinase signaling and inflammation via reactive oxygen species (ROS) in atrial tissue. (A) Experimental workflow diagram, (B) DCFH‐DA fluorescence staining of ROS levels in left atrial tissue (scale bar: 50 μm), (C–F) WB analysis of NOX2, NOX4, phosphorylated and total ERK, p38, and JNK proteins, and (G) ELISA quantification of IL‐6, IL‐1β, TNF‐α, and IL‐18 in left atrial tissue. Sample size per group: n = 6. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.