Sex differences in mitochondrial gene expression during viral myocarditis

Fig. 1. Myocardial inflammation is increased in males compared to females. a, Myocarditis severity (% inflammation) between female controls (F-CON, n = 27), females with myocarditis (F-MYO, n = 41), male controls (M-CON, n = 30), and males with myocarditis (M-MYO, n = 40); white symbols indicate the samples chosen for RNA sequencing (n = 3); b, representative heart sections (scale bars = 80 μm); c-h, relative gene expression (RGE) for F-CON (n = 15–16), F-MYO (n = 15–21), M-CON (n = 18), and M-MYO (n = 15–20)

Fig. 2. RNA sequencing reveals sex differences in immune and mitochondrial genes. a, RNA-sequencing experimental pipeline; b, principal component analysis plot showing female controls (F-CON), females with myocarditis (F-MYO), male controls (M-CON), and males with myocarditis (M-MYO); c, results from GSEA pre-ranked plotted on Cytoscape with Enrichment Map and AutoAnnotate, pink = F-MYO and blue = M-MYO, nodes circled in black are mitochondrial-related pathways; heat map for d, the top 273 most differentially expressed genes and e, mitochondrial genes between all four groups

Fig. 3. Sex-specific gene expression pathways during myocarditis. The top ten most enriched pathways from GSEA ranked by normalized enrichment score comparing a, female controls (F-CON) to females with myocarditis (F-MYO), b, male controls (M-CON) to males with myocarditis (M-MYO) (pathway text marked with astricts indicate abbreviated pathway names, see supplement for abbreviations and full pathway names) c, Row normalized heatmaps for the most enriched pathways from F-CON vs. F-MYO and M-CON vs. M-MYO, respective to colors highlighting pathways in (a) and (b). NES = normalized enrichment score, Extracellular Matrix Structural (Struct.) Constituent (Const.). * FDRq < 0.05, ** FDRq < 0.01, *** FDRq < 0.001, **** FDRq < 0.0001

Fig. 4. Sex differences in differential gene expression pathways during myocarditis. The top ten most enriched pathways from GSEA ranked by normalized enrichment score comparing a, females with myocarditis (F-MYO) and males with myocarditis (M-MYO) and heatmap of auto-annotated cluster of pathways/nodes describing the mitochondrial respiratory complex, b, Row normalized heatmaps for pathways highlighted in pink for F-MYO (from F-MYO vs. M-MYO comparison; other 2 pathways share common genes with those in (b) and are available in Additional File 1: Figure S3). * FDRq < 0.05, ** FDRq < 0.01, *** FDRq < 0.001, **** FDRq < 0.0001

Fig. 5. Females with myocarditis are enriched for pathways related to mitochondrial respiration compared to males with myocarditis. Metascape enrichment results for females with myocarditis (comparing females and males with myocarditis) a, the top enriched pathways colored by cluster/pathway and b, by p-value. Top enriched pathways c, Protein-protein interaction analysis clustered by interaction outside of pathways (i) and interaction inside pathways (ii). Log10(P) vals are derived by averaging the Log10(P) vals for the 3 MCODE annotations, rounded to whole number with colors indicating respective pathways. Images in (i) are cropped to show the bulk of pathways and interactors and the top 3 pathways only are shown in (ii)

Fig. 6. Males with myocarditis are enriched for pathways related immune activation compared to females with myocarditis. Metascape enrichment results for males with myocarditis (comparing females and males with myocarditis) a, the top enriched pathways colored by cluster/pathway and b, by p-value. Top enriched pathways c, Protein-protein interaction analysis clustered by interaction outside of pathways (i) and interaction inside pathways (ii). Log10(P) vals are derived by averaging the Log10(P) vals for the 3 MCODE annotations, rounded to whole number with colors indicating respective pathways. Images in (i) are cropped to show the bulk of pathways and interactors and the top 3 pathways only are shown in (ii)

Fig. 7. Metascape Enrichment QC shows cell specific signals and identifies potential transcriptional regulators of sex differences in myocarditis. a, Enrichment quality control (QC) for males with myocarditis (M-MYO) shows cell specific enrichment signals from Pattern Gene Database (PaGenBase) and suggested transcription factors from Transcriptional Regulatory Relationships Unraveled by Sentence-based Text mining (TRRUST) and for b, females with myocarditis (F-MYO)

Fig. 8. Sex differences in mitochondrial electron transport genes during myocarditis. Row normalized RPKM comparing female controls (FC), females with myocarditis (FM), male controls (MC) and males with myocarditis (MM) for nuclear encoded genes for a, complex I, b, color-coded illustration of the mitochondrial electron transport chain; c, complex II, d, complex III, e, complex IV, and f, ATP synthase. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Fig. 9. Isolated cardiac mitochondria from male mice with myocarditis have reduced oxygen consumption. Mitochondria were isolated from the heart of male (M) and female (F) BALB/c mice treated with saline (CON) or CVB3 (MYO) and harvested at day 10 pi. a, Representative examples of one sample from each group show mitochondrial oxygen consumption over time measured using a Clark electrode (OxyTherm). b, Representative examples of one sample from each group of mitochondrial oxygen consumption/concentration for State 3 are shown highlighting the slope of the solid line, which is less steep in males with myocarditis. Rate is represented by a dashed line

Fig. 10. Isolated cardiac mitochondria from male mice with myocarditis have reduced oxygen consumption rate for State 2–4. Mitochondria were isolated from the heart of male (M) and female (F) BALB/c mice treated with saline (CON) or CVB3 (MYO) and harvested at day 10 pi. Mitochondrial oxygen consumption over time was measured using a Clark electrode (OxyTherm) after administration of respiratory substrate. a, Graphs show when mitochondria were added (Mito) followed by the administration of glutamate, malate and succinate which stimulates the tricarboxylic acid (TCA) cycle, ADP, oligomycin (Omy), and antimycin A (AA). Respiratory states were calculated after administration of the substrate and are represented by two perpendicular dotted lines with the state name inside. Background oxygen consumption rate is shown in Additional File 1: Figure S9. Solid line depicts oxygen concentration and dotted line the rate. b, Graphs represent the slope of the oxygen consumption rate (OCR) during the states shown in a. Baseline represents the OCR before addition of substrates to stimulate the TCA cycle. Oxidative phosphorylation (OxPhos) occurs after induction with ADP. Leakage represents leakage of…

Fig. 11. TRANSFAC analysis identifies interferon regulatory factors and estrogen-related receptors as potential mediators of sex difference during myocarditis. a, TRANSFAC results comparing females with myocarditis (F-MYO) and males with myocarditis (M-MYO); RPKM (reads per kilobase per million) using false discovery rate to compare F-MYO and M-MYO for b, interferon regulatory factors (IRFs) and c, estrogen-related receptors (ERRs); d, predicted binding capacity of ERRs for electron transport chain transcripts; e, significantly different transcripts by sex in electron transport genes are indicated by bold blue lettering, green boxes indicate genes that ERRα predicted to bind to

Fig. 12. Females with myocarditis express higher levels of cardiac ERRα transcript and protein compared to males. a, b, Relative gene expression (RGE) of ESRRA/ ERRα relative to the housekeeping gene HPRT for females (F) or males and (M) controls (CON) or myocarditis (MYO): F-CON (n = 15), F-MYO (n = 20–21), M-CON (n = 17–18), and M-MYO (n = 19–20). c, Representative heart sections of IHC for ERRα, Scale bars = 200 μm. d, Quantification of IHC using Image J. e, f, g, ERRα ELISA from whole heart homogenate supernatant comparing e, CON (n = 14) to MYO (n = 42); f, all females (F, n = 27) to all males (M, n = 29) regardless of disease state; g, two-way ANOVA of F-CON (n = 7), F-MYO (n = 20), M-CON (n = 7), and M-MYO (n = 22) found effect of sex (p = 0.006) and myocarditis (p = 0.017). *p < 0.05, ** p < 0.01, **** p < 0.0001