Post-transcriptional Rac1 activation driven by lipid modification enzymes in atrial fibrillation: a multi-dataset bioinformatics study with experimental validation
Rac1 activation in atrial fibrillation may be more influenced by post-translational modifications than by changes in gene transcription, but further validation is necessary.
Where it sits
this study against the rest of the glutathione (gsh) corpusSummary and findings
This study examined the role of post-translational modifications in Rac1 activation in atrial fibrillation (AF) using a multi-dataset bioinformatics approach. The GSH metabolism arm was significantly downregulated in AF, with a p-value of 1.57 × 10⁻⁴. Rac1 protein activation showed a trend toward higher intensity in AF tissue, but this was not statistically significant.
Abstract
<title>Abstract</title> <p>Background Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, but the post-translational mechanisms linking mitochondrial dysfunction to arrhythmogenic signaling remain poorly understood. Whether the small GTPase Rac1—central to cardiomyocyte redox signaling—is activated through transcriptional upregulation or post-translational modification (PTM) has not been dissected. Methods We constructed a 77-gene Mitoxyperilysis Gene Set (MGS) and performed differential expression analysis in the discovery cohort GSE115574 (AF = 26, SR = 31; Affymetrix GPL570). ssGSEA scored four mechanistic arms: glutathione (GSH) metabolism, mitochondrial damage, RhoA axis, and mTORC2 axis. A 44-gene PTM enzyme panel was assessed across three datasets (GSE115574, GSE41177, GSE79768; AF = 29, SR = 5 and AF = 11, SR = 9 in the validation cohorts). Rac1 protein activation was examined via immunofluorescence (IF) in human right atrial tissue (AF = 3, SR = 3; TOM20/RAC1 dual staining, eight fields per sample). Results Among 170 differentially expressed genes, 3 MGS genes were significantly altered. The GSH metabolism arm was markedly downregulated in the discovery cohort (P = 1.57 × 10⁻⁴, FDR = 0.0012). The PTM enzyme panel revealed 30/44 genes significantly altered in ≥ 1 dataset, with 5 showing cross-dataset concordant upregulation (GGPS1, ZDHHC4, ZDHHC5, ZDHHC20, AKT1). RAC1 mRNA was not significantly different (log₂FC = 0.12, P = 0.34), and the transcriptional RAC1/RHOA ratio was consistently lower in AF across all three datasets (a negative finding). In human atrial tissue, Rac1 immunofluorescence showed a trend toward higher intensity (8.51 ± 2.95 vs 7.08 ± 2.00, P = 0.53) and membrane-to-cytoplasm ratio (0.93 ± 0.07 vs 0.84 ± 0.13, P = 0.40) in AF (pilot validation, n = 3/group); trimmed-mean and median analyses confirmed that these trends were not driven by bright spots. Conclusions AF atrial tissue exhibits concurrent "transcriptional RhoA relative dominance" and "post-translational Rac1 activation trend," which are not contradictory. Rac1 activation in AF appears to be modification-driven (geranylgeranylation/palmitoylation) rather than transcription-driven, warranting further validation by site-specific PTM assays.</p>
Background
Atrial fibrillation (AF) is a prevalent cardiac arrhythmia, yet the mechanisms linking mitochondrial dysfunction to arrhythmogenic signaling are not well defined. Previous studies have suggested that Rac1, a small GTPase involved in redox signaling, may play a role in AF. This study aims to clarify whether Rac1 activation is due to transcriptional upregulation or post-translational modification, which is crucial for understanding AF pathology.
Methods
The study utilized a multi-dataset approach, analyzing gene expression data from GSE115574 (AF = 26, SR = 31) and two additional datasets (AF = 29, SR = 5; AF = 11, SR = 9). A 77-gene Mitoxyperilysis Gene Set was constructed, and differential expression analysis was performed. Rac1 protein activation was assessed via immunofluorescence in human atrial tissue (AF = 3, SR = 3).
Results
Among 170 differentially expressed genes, 3 genes from the Mitoxyperilysis Gene Set were significantly altered. The glutathione metabolism arm was downregulated with a p-value of 1.57 × 10⁻⁴ and an FDR of 0.0012. The PTM enzyme panel showed that 30 out of 44 genes were significantly altered in at least one dataset. The RAC1 mRNA levels did not show significant differences (log₂FC = 0.12, P = 0.34). Immunofluorescence results indicated a trend toward higher Rac1 intensity and membrane-to-cytoplasm ratio in AF, although these findings were not statistically significant.
Interpretation
The findings indicate that Rac1 activation in AF may be driven more by post-translational modifications than by transcriptional changes, which contrasts with previous assumptions. However, the effect sizes observed are not clinically significant, and the small sample sizes in the validation cohorts limit the robustness of these conclusions. This suggests that further research is necessary to validate these findings and understand their implications for AF management.
Key findings
- 3 MGS genes significantly altered among 170 differentially expressed genes.
- GSH metabolism arm downregulated (P = 1.57 × 10⁻⁴, FDR = 0.0012).
- 30/44 PTM enzyme genes significantly altered in ≥ 1 dataset.
- RAC1 mRNA not significantly different (log₂FC = 0.12, P = 0.34).
- Rac1 immunofluorescence showed a trend toward higher intensity (8.51 ± 2.95 vs 7.08 ± 2.00, P = 0.53) in AF.
Limitations
- small sample sizes in validation cohorts
- pilot nature of immunofluorescence analysis
- no significant changes in RAC1 mRNA levels
- not all findings reached statistical significance