Exogenous L6 myotube mitochondrial transplantation attenuates hypertrophy and elicits a unique proteomic signature in phenylephrine-treated H9C2 cardiomyocytes

Figures

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  FIGURE 1.. AIM 1 results demonstrating successful L6 mitochondrial transplantation in H9C2 cardiomyoctes A) Internalization of exogenous L6 myotube mitochondria (pre-labelled with Mito-Tracker Red FM) into non-perturbed H9C2 cardiomyocytes. Control cells received a vehicle treatment of PBS. Fluorescent microscopy revealed perinuclear localization of the stained exogenous mitochondria following coincubation. The brightfield and associated fluorometric images were taken after 24 hours of coincubation using 10x magnification (white bar = 100 μM). B) Respirometry data on n=3 replicates per condition indicates that mitochondrial transplantation increased complex I state 3 (complex I-linked OXPHOS) and maximum capacity of the electron transfer system (ET capacity) without affecting cytochrome c flux. Values are normalized to cell count. Abbreviations: CTL, control cells with no mitochondrial transplantation; MitoTx, cardiomyocytes were coincubated with L6 myotube mitochondrial isolates for 24 hours.

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  FIGURE 2.. Phenylephrine pilot experiment to ensure cardiomyocyte hypertrophy A) Cell size differences presented as means ± standard deviation values between Control and different phenylephrine doses (expressed as fold-change from control treatments). Two replicate wells were used for each group, and 5 images were taken randomly per well. ~20–30 cardiomyocytes were analyzed for morphology per image under 10x magnification (white bar = 100 μM), B) Representative 10x image of each treatment condition. C) Respirometry data on n=3 replicates per condition indicates that 100 μM of phenylephrine did not affect complex I state 3 (complex I-linked OXPHOS), complex IV state 3 (complex IV-linked OXPHOS), maximum capacity of the electron transfer system (ET capacity), or cytochrome c flux. Values are normalized to cell count. Abbreviations: CTL, control cells with no phenylephrine; Phe, 25–100 μM phenylephrine was applied to cells for 24 hours.

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  FIGURE 3.. AIM 2 results examining the effects of mitochondrial transplantation on cell size A) Cell size differences presented as means ± standard deviation values between Control, Phe-only, and Phe+MitoTx (expressed as fold-change from control treatments). Three replicate wells were used for each group and ~20–30 cardiomyocytes per treatment were analyzed for morphology per image under 10x magnification (white bar = 100 μM). B) Phalloidin+DAPI representative 10x images of Control, Phe-only, and Phe+MitoTx. C) Bottom micrograph demonstrates internalization of exogenous L6 myotube mitochondria (pre-labelled with Mito-Tracker Red FM) into H9C2 cardiomyocytes following coincubation with 100 μM Phe relative to the other two images whereby mitochondrial transplantation was not applied. Abbreviations: CTL, control cells with no phenylephrine and no mitochondrial transplantation; Phe-only, 100 μM phenylephrine was applied to cells for 48 hours; Phe+MitoTx, cardiomyocytes were treated with 100 μM phenylephrine for 48 hours and transplanted with L6 myotube mitochondrial isolates for the last 24 of the 48 hours.

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  Figure 4.. AIM 2 proteomic contrasts between treatments A) PCA on the missForest-imputed matrix (24 samples (n=6 per treatment) x 4,806 proteins, 80% confidence ellipses; PC1 23.8%, PC2 12.2%). B) Differentially abundant proteins per contrast as % of proteome, split Up versus Down, at three cutoffs (p < 0.05, BH-FDR < 0.10, Π < 0.05); Π < 0.05 is reported throughout. C) log2FC distribution per contrast. D) Area-proportional Euler diagram of Π < 0.05 proteins across Disease, Transplant, and Rescue. E) Up/Down direction strip for the same proteins. F) Significant-pathway counts per contrast and direction before deduplication (five-database, fgsea BH-FDR < 0.05); deduplicated representative counts are given in Fig. 5 and supplemental data S2 Table. Abbreviations: CTL, control cells with no phenylephrine and no mitochondrial transplantation; Phe-only, 100 μM phenylephrine was applied to cells for 48 hours; MitoTx, cardiomyocytes were transplanted with L6 myotube mitochondrial isolates for the 24 hours; Phe+MitoTx, cardiomyocytes were treated with 100 μM phenylephrine for 48 hours and transplanted with L6 myotube mitochondrial isolates for the last 24 of the 48 hours.

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  Figure 5.. AIM 2 pathway analysis Per-contrast enrichment volcano-in-ring panels for: A) Disease contrast (Phe-only vs. CTL), B) Transplant contrast (MitoTx vs. CTL), C) Rescue contrast (Phe+MitoTx vs. Phe-only), and D) Interaction contrast ((Phe+MitoTx – MitoTx) – (Phe-only – CTL)). Note that each panel pairs a central per protein volcano (log2FC against significance, with DEP counts) with a surrounding ring of pathways drawn from a pooled five-database lens (Hallmark, Reactome, KEGG, GO-Slim, MitoCarta) at fgsea BH-FDR < 0.05 and collapsed by EnrichmentMap overlap/Jaccard deduplication. Each panel subtitle gives the number of representative programs displayed out of the total surviving deduplication (Disease 10/10, Transplant 12/14, Rescue 12/51, Interaction 7/7); arc height is −log10 FDR and arc colour is NES. Abbreviations: CTL, control cells with no phenylephrine and no mitochondrial transplantation; Phe-only, 100 μM phenylephrine was applied to cells for 48 hours; MitoTx, cardiomyocytes were transplanted with L6 myotube mitochondrial isolates for 24 hours; Phe+MitoTx, cardiomyocytes were treated with 100 μM phenylephrine for 48 hours and transplanted with L6 myotube mitochondrial…

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  Figure 6.. AIM 2 co-expression module analysis Co-expression modules responsive to treatment. A) Module size in proteins. B) Module eigengene across the four treatment groups (group mean ± SE, n = 6); the omnibus moderated F FDR is printed per module and starred at FDR < 0.05. C) Top five over-representation terms per module (per-database BH-FDR; bars at full color are FDR < 0.05, washed-out bars are not significant). D) Module normalized enrichment score across the Disease, Transplant, Rescue and Interaction contrasts, shown as descriptive direction and magnitude only; a ring outline marks cells clearing the fry self-contained rotation gate at FDR < 0.05. The full module × contrast fry grid, including the Recovery and disease-after-transplant contrasts, is in supplemental data S3 Table. Abbreviations: CTL, control cells with no phenylephrine and no mitochondrial transplantation; MitoTx, cardiomyocytes transplanted with L6 myotube mitochondrial isolates for 24 hours; Phe-only, 100 μM phenylephrine applied to cells for 48 hours; Phe+MitoTx, cardiomyocytes treated with 100 μM phenylephrine for 48 hours and transplanted with L6 myotube mitochondrial isolates for the last 24 of the 48 hours;…