A Study of Gene Expression, Structure, and Contractility of iPSC-Derived Cardiac Myocytes from a Family with Heart Disease due to LMNA Mutation

Figures

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  Figure 1. iPSC-derived cardiomyocytes. (a) Confocal images of cardiac tissues for one of the individuals. Stained for actin fibrils (phalloidin: green), nuclei (DAPI: blue), and sarcomeric z-lines (α-actinin: red). Scale bar 20 μm. (b) Single cell RNA sequencing of PA1 and CA1 cell lines represented with a heat map for the top 90 genes shows a varied expression profile for the two cells lines. (c) Feature maps for the two cells lines show mostly matching clusters for both cell lines, and the two cardiac-specific markers indicate that 99% of the cells are considered cardiomyocytes.

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  Figure 2. Nuclei analysis. (a) Nuclei related gene expressions from single cell RNA-sequencing of two cell lines—PA1 (cell number = 13,028) and CA1 (cell number = 12,591). (b) Example images of normal and defective nuclei, stained for DAPI, for a patient and control cell line. Scale bar 5 μm. (c) Quantitative analysis of nuclear morphology for all five cell lines (error bars are standard deviations; black horizontal lines indicate significance of p < 0.05). Unless otherwise indicated sample sizes (n) is the number of nuclei analyzed for each condition: (i) percent dysmorphic nuclei grouped by mutation status—controls (CTRL) and patients (PT) (sample sizes, n based on the number of coverslips). (ii) Mean negative curvature, (iii) relative concavity, (iv) maximum negative curvature, and (v) area for all controls and for each individual patient line. (vi) Comparison of dysmorphic nuclei percentage for both cardiomyocytes and fibroblasts in all individual cell lines (sample sizes, n based on the number of coverslips). (vii) The increase in dysmorphic nuclei percentage from fibroblasts to cardiomyocytes for each individual [errors were propagated from part (vi) and smaller sample size was used…

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  Figure 3. Extracellular matrix. Extracellular matrix gene expression levels for CA1 (cell number = 12,591) and PA1 (cell number = 13,028) cell lines for (a) Laminin subunit Alpha 4 (LAMA4), (b) Collagen type IV Alpha 4 (COL4A1), and (c) Collagen type VI Alpha 1 (COL6A1).

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  Figure 4. Cardiomyocyte cytoskeleton. (a) Expression levels for cytoskeleton related genes that were differentially expressed in CA1 (cell number = 12,591) and PA1 (cell number = 13,028) lines. (b) Example analysis of actin orientation (left image, green arrows) and sarcomere identification [right image sarcomeres identified with purple (longer) and blue (shorter)]. (c) The density of sarcomeres averaged for three control lines (CTRL) and two patient lines (PT). (d) Quantification of myofibril architecture (error bars are standard deviations; black horizontal lines indicate significance of p < 0.05): (i) average sarcomere length for each individual cell line; orientational order parameter (OOP) for the (ii) sarcomeres and (iii) actin. Sample sizes (n) are based on the number of coverslips analyzed for each condition.

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  Figure 5. Contractility of cardiac tissues. (a) Expression levels for genes, which contribute to contractile properties, differentially expressed for PA1 (cell number = 13,028) and CA1 (cell number = 12,591). (b) An image of a “heart-on-a-chip” device for systole and diastole. Blue outlines the length of the film when it lies flat, and red bars track the horizontal projection of the films that is used to calculate stress as a function of time. Scale bar 1 mm. Measured beating frequency vs. pacing frequency with a linear regression for (c) control tissues (sample size Table S1) and (d) patient tissues (sample size Table S1). (e) Log normalized Diastolic (i), Systolic (ii), and Active (iii) stresses averaged for the three control and two patient lines shows a statistically significant difference for systolic and active stresses.