Myocardial disarray drives metabolic inefficiency in human cardiomyocytes

Figures

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  Fig. 1. Linear micropatterning of human cardiomyocytes improves morphological features and sarcomere organization. A Schematic overview of the study. On day 35 of differentiation, hiPSC-CMs were seeded onto micropatterned or non-patterned slides for 7 days before functional phenotyping. Created in BioRender. B Schematic representation of the profile (left) and top (right) view of the adhesive micropatterns. Created with Biorender.com. C Representative phase-contrast images of non-patterned cells (left) and micropatterned-cells (right), scale bar 400 µm. D Representative immunofluorescence images of non-patterned and micropatterned hiPSC-CMs, for alpha-sarcomeric actinin (green), cardiac Troponin T2 (red) at day 7 after seeding. DAPI (blue) was used to stain nuclei. The insert represents a zoomed view of sarcomeres. Global scale bar 100 µm, insert scale bar 10 µm. E Automated measures of sarcomere organization and sarcomere length. (Left) Immunofluorescence images of non-patterned (top) and micropatterned (bottom) hiPSC-CMS. Scale bar 25 µm. (Right) Haralick correlation metric computed at multiple offset distances and angles to determine sarcomere organization and sarcomere length.…

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  Fig. 2. Transcriptomics and pathway analyses reveal changes in metabolic genes. A Pie chart summarizing the classification of all the significantly changed pathways (FDR < 0.01) in micropatterned vs. non-patterned hiPSC-CMs as found with ReactomeGSA. B Voronoi diagram displaying the significantly altered lipid metabolism pathways (FDR < 0.01) in micropatterned vs. non-patterned hiPSC-CMs (generated by ReactomeGSA). The upregulated pathways are colored yellow, while the downregulated pathways are colored blue. C Upregulated energy metabolism pathways identified by ReactomeGSA comparing micropatterned vs non-patterned hiPSC-CMs showing their corresponding p-value as -Log10(p-value). D Downregulated energy metabolism pathways identified by ReactomeGSA comparing micropatterned vs non-patterned hiPSC-CMs showing their corresponding p-value as -Log10(p-value). E Representative Western blot images showing the expression of ACC (total and phosphorylated at Ser79), AMPK (total and phosphorylated at Thr172), and CPT1 on non-patterned (NP) and micropatterned (M) hiPSC-CMs. Vinculin was used as loading control. F Quantification of total proteins ACC, AMPK, and CPT1 in micropatterned hiPSC-CMs (n =…

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  Fig. 3. Mitochondrial respiration is increased in micropatterned hiPSC-CMs. A Real-time oxygen consumption rate (OCR) measurements of micropatterned and non-patterned hiPSC-CMs by Seahorse extracellular flux analyzer. Cells were treated with the ATP synthase inhibitor oligomycin, the respiratory uncoupler FCCP, and the respiratory chain blockers rotenone and antimycin A. B Scatter plots show basal respiration, production of ATP, and maximal respiration. N = 8–11 differentiations. C Real-time extracellular acidification rate (ECAR) measurements of hiPSC-CMs non-patterned and micropatterned by Seahorse extracellular flux analyzer. Cells were treated with glucose, ATP synthase inhibitor oligomycin, and glucose analog 2-DG. D Scatter plots show glycolysis, non-glycolytic acidification, and glycolytic capacity. N = 4–12 differentiations. E Cell energy diagram showing normalized oxygen consumption rate (OCR) and normalized extracellular acidification rate (ECAR) of micropatterned and non-patterned hiPSC-CMS at the basal (circle points) and stressed (FCCP exposed, square points) conditions. F Representative profiles of oxygen consumption of non-patterned (top) and micropatterned (bottom)…

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  Fig. 4. Micropatterning impacts mitochondrial distribution independently of mitochondrial mass. A Mitochondrial distribution stained by MitoTrackerTM Orange (red) and DAPI (blue) and analysed by confocal microscopy. The insert represents a zoomed view of mitochondrial network. Global scale bar 50 µm, insert scale bar 10 µm. B Quantitative analysis of mitochondrial direction measured with Fiji software. n = 18 non-patterned cells, n = 16 micropatterned cells; N = 2 differentiations. C Representative pictures obtained with MitoTrackerTM Orange and IMARIS software. Each mitochondrion is identified by a color in accordance with its volume (the smallest mitochondria are blue and the largest are red). D Total mitochondrial volume quantified by IMARIS. n = 28,675 non-patterned cells, n = 64,919 micropatterned cells; N = 3 differentiations. E Scatter plots show the number of mitochondria per cell. n = 18 non-patterned cells, n = 20 micropatterned cells; N = 3 differentiations. F Mitochondrial DNA (mtDNA) content in non-patterned (n = 17) and micropatterned (n = 11) hiPSC-CMs, quantified by amplification of the mitochondrial Mttl1 gene relative to nuclear gene b-Actin. N = 3 differentiations. G…

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  Fig. 5. Increase of mitochondria-SR contact sites by structural reorganization with micropatterns. A Transmission electron microscopy (TEM) images of the cellular structure of non-patterned (top) and micropatterned (bottom) hiPSC-CMS in transverse cross-sections; scale=1 µm. Mf indicates myofibril; Mt mitochondria; and SR sarcoplasmic reticulum. B Quantitative measurement of mitochondria surface (left) and circularity (right) on the TEM images. n = 10 fields per conditions with 5 to 15 mitochondria per field, N = 3 differentiations. C Schematic representation of the VDAC1/GRP75/IP3R1 complex at MAM interface. Created with Biorender.com. D Left: representative PLA images. The insert represents a zoomed view of PLA signal. Each white dot corresponds to a positive PLA signal. Right: quantitative analysis of VDAC1-IP3R1, GRP75-IP3R1 and VDAC1-GRP75 interactions signal in non-micropatterned and micropatterned hiPSC-CMs. n = 10 images/conditions, N = 3 differentiations. Scale bar 100 µm. E Representative images of Rhod-2 AM (green) and MitoTrackerTM Deep Red (red) staining in non-patterned and micropatterned hiPSC-CMs. Scale bar 10 µm. F Quantitative analysis of Rhod-2 AM intensity of loaded…