Proteomic signatures of mitochondrial dysfunction associated with atrial fibrillation in goats

Figures

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  Fig. 1. Gene Ontology (GO) Biological Process (BP) enrichment analysis reveals mitochondrial ATP synthesis and respiratory dysfunction in AF samples. (A) Dot plot showing significantly enriched GO BP terms from over-representation analysis of differentially abundant proteins. The x-axis represents enrichment z-score, dot size indicates gene count, and colour intensity represents adjusted p-value (Benjamini-Hochberg correction). Terms are ranked by significance, with proton motive force-driven mitochondrial ATP synthesis showing the highest enrichment. (B) Concept Network Enrichment (CNE) plot displaying gene-concept relationships for top enriched BP. Genes (outer nodes) are connected to their associated GO terms (inner nodes), with node size proportional to gene count or term size. Gene nodes are coloured by log2-fold change (red = upregulated, blue = downregulated in AF). (C) Enrichment Map Analysis (EMA) plot showing functional relationships between enriched biological process terms. Nodes represent GO terms sized by gene count, and edges connect terms sharing genes. Terms cluster by functional similarity, revealing coordinated enrichment of mitochondrial ATP synthesis, electron…

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  Fig. 2. Gene Ontology (GO) Molecular Function (MF) enrichment analysis highlights altered ATP synthase and cytoskeletal binding activities in AF samples. (A) Dot-plot showing significantly enriched GO Molecular Function terms from over-representation analysis of differentially abundant mitochondrial proteins. The x-axis represents enrichment z-score, dot size indicates gene count, and colour intensity represents adjusted p-value (Benjamini-Hochberg correction). Proton-transporting ATP synthase activity and actin filament binding show the most significant enrichment. (B) Concept Network Enrichment (CNE) plot displaying gene-concept relationships for top enriched molecular functions. Genes (outer nodes) are connected to their associated GO terms (inner nodes), with node size proportional to gene count or term size. Gene nodes are coloured by log2 fold change (red = upregulated, blue = downregulated in AF). (C) Enrichment Map Analysis (EMA) plot showing functional relationships between enriched molecular function terms. Nodes represent GO terms sized by gene count, and edges connect terms sharing genes. Terms cluster by functional similarity, with prominent groupings of structural…

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  Fig. 3. Gene Ontology (GO) Cellular Component (CC) enrichment analysis reveals reorganization of focal adhesions and mitochondrial complexes in AF samples. (A) Dot-plot showing significantly enriched GO Cellular Component terms from over-representation analysis of differentially abundant mitochondrial proteins. The x-axis represents enrichment z-score, dot size indicates gene count, and colour intensity represents adjusted p-value (Benjamini-Hochberg correction). Focal adhesion, Z disc, and actin cytoskeleton components show the highest enrichment scores. (B) Concept Network Enrichment (CNE) plot displaying gene-concept relationships for top enriched cellular components. Genes (outer nodes) are connected to their associated GO terms (inner nodes), with node size proportional to gene count or term size. Gene nodes are coloured by log2 fold change (red = upregulated, blue = downregulated in AF). (C) Enrichment Map Analysis (EMA) plot showing functional relationships between enriched cellular component terms. Nodes represent GO terms sized by gene count, and edges connect terms sharing genes. Terms cluster into distinct functional modules including focal adhesion/Z disc/actin cytoskeleton,…

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  Fig. 4. WikiPathways enrichment analysis identifies mitochondrial electron transport chain dysfunction in AF samples. (A) Dot-plot showing significantly enriched WikiPathways terms from over-representation analysis of differentially abundant proteins from AF-induced left atrial tissue versus sham-operated controls. The x-axis represents enrichment z-score, dot size indicates gene count, and colour intensity represents adjusted p-value (Benjamini-Hochberg correction). Electron transport chain OXPHOS system in mitochondria shows the most significant enrichment. (B) Concept Network Enrichment (CNE) plot displaying gene-concept relationships for top enriched pathways. Genes (outer nodes) are connected to their associated pathway terms (inner nodes), with node size proportional to gene count or term size. Gene nodes are coloured by log2 fold change (red = upregulated, blue = downregulated in AF). Electron transport chain and oxidative phosphorylation pathways cluster together with multiple NADH dehydrogenase (NDUF) subunits and ATP synthase components. All enrichments shown have adjusted p-value < 0.05.

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  Fig. 5. KEGG pathway enrichment analysis reveals muscle-specific and metabolic adaptations in AF samples. (A) Dot-plot showing significantly enriched KEGG pathways from over-representation analysis of differentially abundant proteins from AF-induced left atrial tissue versus sham-operated controls. The x-axis represents enrichment z-score, dot size indicates gene count, and colour intensity represents adjusted p-value (Benjamini-Hochberg correction). Cytoskeleton in muscle cells and oxidative phosphorylation show the highest enrichment scores. All enrichments shown have adjusted p-value < 0.05.

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  Fig. 6. Reactome pathway enrichment analysis highlights immune signalling and translational control alterations in AF samples. (A) Dot-plot showing significantly enriched Reactome pathways from over-representation analysis of differentially abundant proteins from AF-induced left atrial tissue versus sham-operated controls. The x-axis represents enrichment z-score, dot size indicates gene count, and colour intensity represents adjusted p-value (Benjamini-Hochberg correction). Gene and protein expression by JAK-STAT signalling and interleukin-12 family signalling pathways show the most significant enrichment. (B) Concept Network Enrichment (CNE) plot displaying gene-concept relationships for top enriched Reactome pathways. Genes (outer nodes) are connected to their associated pathway terms (inner nodes), with node size proportional to gene count or term size. Gene nodes are coloured by log2-fold change (red = upregulated, blue = downregulated in AF). (C) Enrichment Map Analysis (EMA) plot showing functional relationships between enriched Reactome pathways. Nodes represent pathway terms sized by gene count, and edges connect pathways sharing genes. Terms cluster into distinct functional…

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  Fig. 7. Multi-database pathway enrichment analysis reveals consensus on the Oxidative Phosphorylation pathway. (A) UpSet plot displaying the distribution of enriched pathways across Gene Ontology (GO), KEGG, and WikiPathways databases. Vertical bars indicate the number of pathways found in each database combination (intersections), while horizontal bars show the total number of enriched pathways per database. Pathways were considered significantly enriched at adjusted p-value < 0.01. Note only the Oxidative Phosphorylation pathway is enriched in all three databases. (B) Heatmap of pathway enrichment significance across databases. Colour intensity represents -log10 (adjusted p-value), with darker red indicating higher statistical significance. Each row represents a unique pathway description (lowercase-matched across databases). The top consensus pathway, appearing in multiple databases with the strongest median significance, is labelled (Oxidative Phosphorylation). Columns represent individual enrichment databases; rows are unsupervised hierarchically clustered.

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  Fig. 8. Mitochondrial-specific protein set enrichment analysis reveals upregulation of oxidative phosphorylation subunits in AF samples. (A) Gene Set Enrichment Analysis (GSEA) plot for the OXPHOS subunits pathway from the MitoCarta database. The top panel shows the running enrichment score (green line) across the ranked protein list, with vertical black lines indicating the position of genes within the OXPHOS subunits protein set. The bottom panel displays the ranked list metric (log2-fold change weighted p-value), with red-to-blue colouring indicating upregulated to downregulated genes in AF versus sham-operated samples. The enrichment score peaks early in the ranked list, indicating significant upregulation of OXPHOS subunits (NES = 1.76, p-value = 0.00025, adjusted p-value = 0.016). (B) Concept Network Enrichment (CNE) plot showing gene-concept relationships for the OXPHOS subunits pathway. Individual proteins (outer nodes) are connected to the central OXPHOS subunits pathway node, with node size proportional to gene count (n = 12). Protein nodes are coloured by rank score, with darker red indicating higher-ranked (more significantly upregulated) genes in AF samples. Key NADH…

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  Fig. 9. Comprehensive mitochondrial pathway analysis reveals functional remodelling across major mitochondrial processes in AF samples. Heatmap displaying single-sample Gene Set Enrichment Analysis (ssGSEA) scores for hierarchically organized mitochondrial pathways from the MitoCarta database. The central phylogenetic tree represents the hierarchical relationships between mitochondrial gene sets, organized into seven major functional categories (indicated by purple arc labels): OXPHOS, Protein import sorting and homeostasis, Mitochondrial dynamics and surveillance, Signalling, Small molecule transport, Mitochondrial central dogma, and Metabolism. Each concentric ring represents an individual sample (AF1, AF2, AF3 = AF-induced left atrial tissue; Con1, Con2, Con3 = sham-operated controls). Colour intensity represents ssGSEA enrichment scores, with red indicating pathway downregulation, purple indicating pathway upregulation, and white indicating no enrichment (score range: −0.50 to + 0.50). Each radial segment corresponds to a specific mitochondrial pathway or gene set, with related pathways clustering together based on functional similarity. The visualization reveals coordinated patterns…

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  Fig. 10. Inferred regulatory network reveals HSPA9 as a master coordinator of OXPHOS complex assembly in AF samples. Directed network graph displaying multi-step causal cascades within the OXPHOS pathway showing high concordance between regulator and target protein expression changes. The network was constructed by overlaying differential abundance data onto directional regulatory relationships from the Reactome “Aerobic respiration and respiratory electron transport” pathway. Nodes represent proteins identified in AF-induced left atrial tissue versus sham-operated controls, with all proteins shown in orange indicating upregulation in AF samples. Node size reflects the protein’s hub centrality in the network architecture. Directed edges (arrows) represent activation relationships derived from Reactome regulatory annotations. HSPA9 (mitochondrial heat shock protein 70, mt-Hsp70) emerges as the primary regulatory hub, participating in 7 of 22 identified three-step directional cascades. HSPA9 forms a reciprocal activation loop with UQCRFS1 (Rieske iron-sulphur protein, Complex III) and coordinates the expression of multiple NADH dehydrogenase subunits (NDUFV1, NDUFV2, NDUFS1, NDUFS2, NDUFS7,…