Fig. 4: Deciphering intra- and intercluster heterogeneity by repurposing pseudotime trajectories and employing spatial trajectories towards fibrosis.
a UMAP projection of spatially-unaware of Raman data with overlayed pseudotime trajectories. Branches denote crucial differences from pixels along the main trajectory and are predominantly found in clusters assigned to myocardium (dashed box). b DDRTree based pseudotime trajectory of pixels from center of Raman scan (area highlighted in red). c Branches almost exclusively occur with pixels assigned to cluster 1 (remodeling myocardium, dark blue), while spectra derived from healthy myocardium (light blue) appear homogenously. d Spatial trajectory along red arrow in section of subendocardial fibrosis. Heatmap of log2 normalized intensity changes in Raman fingerprint spectrum along this trajectory. Corresponding clusters along trajectory are plotted on bottom by their color code. e, f Selected wavenumbers with intensity shifts along the spatial trajectory. Blue lines are mean, grey ribbons are 0.95 confidence intervals. 858 cm−1 band corresponds to hydroxyproline from collagen, 1314 cm−1 to cytochrome c. g A similar myocardial pattern with a distinct healthy (light blue) and remodeling (dark blue) cluster when approaching fibrotic regions (pink) was reproduced in 4 individual hearts. Scale bar 50 µm. h Reproduction of intensity shifts of collagen and cytochrome c (n = 4). i Log2 normalized intensity changes filtered to pixels assigned to cluster 0 and 1 along the spatial trajectory (red arrow). j Representative wavenumber demonstrating intensity dynamics within cluster 0 and 1. 1569 cm−1 corresponds to NH bending especially found in in α-helical protein structures. Grey ribbons are 0.95 confidence intervals. k Violin plots showing significantly lower dynamics in all spectra from cluster 0 (light blue, healthy myocardium) in comparison to cluster 1 (dark blue), implying molecular dynamics of myocardium under remodeling. Quantification by calculating the maximum variability of the local polynomial regression fitting (loess) curve (left, p = 5.02 × 10−4) and thresholding of the derivation from loess curve (right, p < 2.2*10−16). Welch two-sided, paired t-test. n = 1 (sample from a). l Reproduction of spectral dynamics in the remodeling subcluster in n = 4 individual hearts. Welch two-sided, paired t-test. Bars display mean ± SEM.
