Ultrastructure analysis of mitochondria, lipid droplet and sarcoplasmic reticulum apposition in human heart failure

Figures

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  Fig. 1. Mitochondrial dynamics are altered in failing human cardiomyocytes (A) Schematic for mitochondrial ultrastructure analysis. (B) Representative EM Images. Cardiac tissue from NF, DCM, HCM, ICM samples were sectioned and imaged by TEM. TEM images were analyzed and quantified using ImageJ Fiji (Scale bars: 800 nm). Ultrastructure measurements include (C) percent mitochondria area, (D) number of mitochondria/cardiomyocyte (CM) area, (E) mitochondria feret diameter, (F) mitochondria perimeter, (G) frequency of mitochondrial perimeter in HF etiologies, and (H) mitochondrial circularity. Number of patients for EM Analysis = 8 NF, 9 DCM, 3 ICM, 5 HCM. Number of mito = 2760 NF, 4546 DCM, 1368 ICM, 2350 HCM. qPCR analysis of genes involved in mitochondrial dynamics including (I) DRP1, (J) OPA1, (K) FIS1, (L) ERMIT2, (M) and ERMIN2. mRNA expression of genes involved in mitochondrial dynamics depicted as fold change corrected to RPS13. Number of patients for qPCR analysis = 6 NF, 6 DCM, 6 ICM, 6 HCM. Data is presented as mean ± SEM. ****p < 0.0001, **p < 0.01, *p < 0.05; One-way ANOVA with Dunnett's Post Hoc Test. Abbreviations: EM: Electron Microscopy, HF- Heart Failure, NF-Nonfailing, DCM-…

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  Fig. 2. Failing cardiomyocytes have altered sarcoplasmic reticulum- mitochondria apposition (A) Schematic of mitochondria-sarcoplasmic reticulum apposition analysis. (B) Representative EM Images. Cardiac tissue from NF, DCM, HCM, ICM samples were sectioned and imaged by TEM. TEM images were analyzed and quantified using ImageJ Fiji (Scale bars: 800 nm). Examples of mitochondria-SR interactions are highlighted in orange and green respectively. Number of patients for EM analysis = 8 NF, 9 DCM, 3 ICM, 5 HCM. Number of contacts = 405 NF, 526 DCM, 163 ICM, 237 HCM. Ultrastructure measurements include (C) mean SR-mitochondria distance, (D) minimum SR-mitochondria distance, (E) total length of SR <50 nm from mitochondria, (F) mitochondrial perimeter in association with SR, (G) binning of the length of OMM <50 nm from SR, (H) frequency of SR-mito contacts, (I) and total number of SR-mitochondrial contracts per image area. (J) Heatmap representation of mRNA levels of tethers in HF depicted as fold change corrected to calnexin. Number of patients for qPCR analysis = 6 NF, 6 DCM, 6 ICM, 6 HCM. Data is presented as mean ± SEM. ****p < 0.0001, **p < 0.01, *p < 0.05; One-way ANOVA with Dunnett's Post…

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  Fig. 3. LD ultrastructure is altered in failing cardiomyocytes (A) Schematic of lipid droplet (LD) ultrastructure analysis. (B) Representative EM Images. Cardiac tissue from NF, DCM, HCM, ICM samples were sectioned and imaged by TEM. TEM images were analyzed and quantified using ImageJ Fiji (Scale bars: 800 nm). Number of patients for EM analysis = 8 NF, 9 DCM, 3 ICM, 5 HCM. Number of LD = 178 NF, 60 DCM, 12 ICM, 29 HCM. Ultrastructure measurements include, (C) LD area/total cardiomyocyte area, (D) number of LD/CM area, (E) LD perimeter, (F) Percentage of LD perimeter sizes per HF etiology, (G) LD Feret diameter, (H) and LD Circularity. Data is presented as mean ± SEM. **p < 0.01, *p < 0.05; One-way ANOVA with Dunnett's Post Hoc Test. Abbreviations: EM: Electron Microscopy, HF- Heart Failure, NF-Nonfailing, DCM- Dilated Cardiomyopathy, ICM- Ischemic Cardiomyopathy, HCM- Hypertrophic Cardiomyopathy, TEM-Transmission Electron Microscopy, CM- Cardiomyocyte.

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  Fig. 4. LD-Mitochondria Association is diminished in failing cardiomyocytes (A). Schematic of LD-mitochondria apposition analysis. (B) Representative EM Images. Cardiac tissue from NF, DCM, HCM, ICM samples were sectioned and imaged by TEM. TEM images were analyzed and quantified using ImageJ Fiji (Scale bars: 800 nm). Number of patients for EM analysis = 8 NF, 9 DCM, 3 ICM, 5 HCM. Number of contacts = 248 NF, 116 DCM, 15 ICM, 25 HCM. Ultrastructure measurements include (C) mean LD-mitochondria distance, (D) minimum LD-Mito distance, (E) LD perimeter within 100 nm of OMM, (F) OMM perimeter in contact with LD, (G) Binning of different LD perimeter sizes within 100 nm of OMM in different HF etiologies, (H) Total LD-Mito contacts within image area, (I) mRNA expression of proposed LD-Mito tethers including PLIN3, (J) MIGA2, (K) FACL2, (L) and SNAP23. mRNA levels of genes involved in mitochondrial dynamics depicted as fold change corrected to RPS13. Number of patients for qPCR analysis = 6 NF, 6 DCM, 6 ICM, 6 HCM. Data is presented as mean ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05; One-way ANOVA with Dunnett's Post Hoc Test unless stated otherwise. 4G Two-Way ANOVA with Dunnett's Post Hoc…