Lamin B1 loss in human cardiomyocytes is associated with nuclear shape deformation in dilated cardiomyopathy

Figures

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  Fig. 1. Lamin B1 quantification in human heart homogenates. Human myocardium tissue homogenates were analysed by western blot using lamin B1 and emerin antibodies and actin was visualised by ponceau red staining. A) Results expressed as the densitometric ratio of Lamin B1/Actin or Lamin B1/Emerin of n = 3 control and n = 3 DCM samples are shown. Each n represents a human myocardium tissue sample from a single donor. Bars represent the mean and standard deviation. Data was analysed by unpaired non-parametric t-test with Mann-Whitney corrections applied, ns non-significant. B) Lamin B1, emerin and actin western blot and ponceau red staining images used for the quantification in A. Control and donor IDs and diagnostic data are reported in Supplementary File 1. Human myocardium tissue homogenates were analysed by western blot using lamin B1 and emerin antibodies and actin was visualised by ponceau red staining. A) Results expressed as the densitometric ratio of Lamin B1/Actin or Lamin B1/Emerin of n = 3 control and n = 3 DCM samples are shown. Each n represents a human myocardium tissue sample from a single donor. Bars represent the mean and standard deviation. Data was analysed by unpaired…

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  Fig. 2. Lamin B1 quantification in human heart nuclei. Representative confocal images of immunofluorescence-stained sections of human myocardium: lamin B1 (green), DAPI (blue), and sarcomeric alpha-actinin (red). A) cardiomyocyte nuclei B) non-cardiomyocyte nuclei C) human heart nuclei lamin B1 quantification from immunofluorescence staining of myocardium sections from control non-failing (n = 3) and DCM (n = 8) donors. Small dots represent individual nuclei (cardiomyocyte: 95 control, 263 DCM; non-cardiomyocyte: 416 control, 1390 DCM; 2164 nuclei total from 79 fields of view). Large, white-outlined circles represent per-donor means. Boxes show the median and interquartile range. Significance was assessed by nested linear mixed-effects models fitted to nucleus-level data with donor and image (nested within donor) as random intercepts. p-Values were Holm–Bonferroni corrected across the four comparisons: cardiomyocyte control vs. DCM p = 0.0013; non-cardiomyocyte control vs. DCM p = 0.62; cardiomyocyte vs. non-cardiomyocyte in control p = 0.66; cardiomyocyte vs. non-cardiomyocyte in DCM p = 1.1 × 10−4. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. (For interpretation of the…

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  Fig. 3. Dimensionality reduction and unsupervised clustering of cardiomyocytes features. A) Principal Component Analysis (PCA) of cardiomyocyte nuclei features. B) Silhouette score Cluster over number of clusters. C) Silhouette plot for 2 clusters. The dashed red line indicates the overall mean silhouette score of 0.157. D) UMAP projection coloured by condition. Small dots represent individual cells coloured by condition. Large dots indicate calculated sample centroids for individual replicates. E) UMAP projection coloured by cluster. Same UMAP embedding shown in (D) but coloured by cluster assignment. F) Cluster distribution by condition and biological sample. Stacked bar charts showing the percentage composition of cells assigned to Cluster 0 (blue) and Cluster 1 (orange). Left panel: proportional distribution aggregated by experimental condition (Control vs. DCM). Right panel: Proportional distribution by individual biological samples, with samples coloured by their respective cluster assignment. Control and donor IDs and diagnostic data are reported in Supplementary File 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web…

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  Fig. 4. UMAP plot coloured by K-means cluster showing ten randomly samples images. UMAP projection coloured by cluster assignment (Cluster 0: blue Cluster 1: orange). Randomly selected images are shown at their corresponding location. The colour of images border indicates the sample origin (Blue: control and Orange: DCM). Displayed nuclei were randomly selected by setting np.random.default_rng(). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Supplementary Fig. 1. Representative confocal images of immunoflourescencestained sections of sections of human myocardium.

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  Supplementary Fig. 2. Representative confocal images of immunoflourescencestained sections of sections of human myocardium.