Pro-regenerative fingerprints identified in a sub-population of adult mouse cardiomyocytes by integrative single-cell proteomics

Figures

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  Fig. 1. General overview of our single-cardiomyocyte proteomics workflow. a Scheme of the methodological procedure followed for single-cardiomyocyte isolation and sample digestion. b Diagram of the steps for TMT labelling and data analysis of samples after digestion. TMT-isobaric labelled samples are multiplexed prior sample desalting and data acquisition by LC–MS. c Design A and B for TMT labelling of single-cardiomyocytes and booster channel. In the pilot analysis, 12 different 10-plex TMTs were used. d Median values of reporter ion intensities for single-cardiomyocytes in two experimental examples of Design A and Design B. Each bar represents the median values of reporter ion intensities of one cell. e Optical images of tissue sections from control and Myc-overexpressing hearts, along with quantification of cardiomyocyte cell area [25]

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  Fig. 2. Normalizing strategies for analyzing mass spectrometry-based single-cell proteomics data. a Violin plots showing the log10 transformed median values of proteins in control and Myc-overexpressing cardiomyocytes with non-normalized data. b PCA distribution of cells based on non-normalized protein expression values highlighting the cell type as control and Myc-overexpressing cardiomyocytes. c PCA distribution of cells based on non-normalized protein expression values highlighting the TMT where each cell belongs to. d Violin plot showing the Zqall median values, where Zqall represents the relative abundance changes of proteins, comparing control and Myc-overexpressing cardiomyocytes. e PCA representing the separation of control and Myc-overexpressing cardiomyocytes based on Zqall values. f PCA representing the separation of cardiomyocytes based on Zqall values and indicating the TMT in which each cardiomyocyte was analyzed. In every section, violin representations include the median values of 503 proteins out of 48 control cardiomyocytes and 48 Myc-overexpressing cardiomyocytes. PCA representations include the 48 control cardiomyocytes and 48 Myc-overexpressing cardiomyocytes used in…

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  Fig. 3. Comparison of two strategies for isolating single-cardiomyocytes. a PCA representing the separation of control and Myc-overexpressing cardiomyocytes isolated by FACS-sorter. b PCA representing the separation of control and Myc-overexpressing cardiomyocytes manually isolated. 948 common proteins to both separation techniques have been compared considering their identification in at least 16 cells, that would include proteins present in at least 1 TMT per condition. Metaboanalyst filters out proteins with constant imputed values under standard deviation and mean filtering criteria. Each PCA representation includes 56 control and 56 Myc-overexpressing cardiomyocytes. A total of 14 different 10-plex TMTs were used in each of the analyses. The 10-plex TMTs used in the manual isolation were included in the final analytical dataset. PERMANOVA test results: In Fig. 3a, F-value = 1.72 and p-value = 0.184. In Fig. 3b, F-value = 31.741 and p-value = 0.001. c and d Violin plots showing the distribution of Zqall values of proteins related to fatty acid oxidation that show to be discriminant in the differential analysis when manual isolation of cardiomyocytes is conducted. c Represents the…

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  Fig. 4. Pseudobulk analysis based on Zqall values, comparing control and Myc-overexpressing cardiomyocytes in the analytical dataset. a Violin plot representing the Zqall median values of the proteins in control and Myc-overexpressing cardiomyocytes in the analytical dataset when integrating data to all experiments in iSanXoT application. Comparison between groups was performed by Student’s t-test. b PCA distribution of control and Myc-overexpressing cardiomyocytes based on Zqall values. PERMANOVA test results: F-value = 9.81and p-value = 0.001. c PLS-DA distribution of control and Myc-overexpressing cardiomyocytes based on Zqall values. d Functional protein interaction network analysis using STRING. The represented proteins are the ones extracted out of the five first components of the PLS-DA analysis with a VIP score higher or equal to 1.5. Fatty acid beta- oxidation-related proteins are highlighted in red. e Box plots representing the Zqall values of the fatty acid beta- oxidation-related proteins that contributes to the separation of control and Myc-overexpressing cardiomyocytes and highlighted in red in 4d. Each dot in the box plots represents the correspondent Zqall value in one…

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  Fig. 5. Pseudobulk analysis based on Zqcc values, comparing control and Myc-overexpressing cardiomyocytes in the analytical dataset. a Violin plot representing the Zqcc median values of the proteins in control and Myc-overexpressing cardiomyocytes in the analytical dataset when integrating data to the cellular compartment where they canonically belong to, using the iSanXoT application. Comparison between groups was performed by Student’s t-test. b PCA distribution of control and Myc-overexpressing cardiomyocytes based on Zqcc values. PERMANOVA test results: F-value = 11.78 and p-value = 0.001. c PLS-DA distribution of control and Myc-overexpressing cardiomyocytes based on Zqcc values. d Functional protein interaction network analysis using STRING of the top proteins extracted out of the five first components of the PLS-DA analysis. The selected proteins are the ones with a VIP score higher or equal to 1.5. Fatty acid beta-oxidation-related proteins are highlighted in red. Canonical glycolysis related proteins are highlighted in blue. e Box plots representing the Zqcc values of the fatty acid-beta oxidation-related proteins that contributes to the separation of control and…

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  Fig. 6. Comparison of cellular compartment behaviour based on Zcca values. Violin plots representing the Zcca values per cell of each cellular compartment in control and Myc-overexpressing cardiomyocytes. Zcca value is the result of integrating cellular compartment values to the grand mean (all) using the SBT algorithm, performed with iSanXoT. The analysis includes 323 control cardiomyocytes and 324 Myc-overexpressing cardiomyocytes in each violin plot. A total of 81 different 10-plex TMTs were used for this analysis (analytical dataset). Comparison between groups was performed by Student’s t-test. (ECM = Extracellular matrix; ER = Endoplasmic reticulum). (**: p < 0.01; ***: p < 0.001; ****: p < 0.0001). See also Additional file 1: Fig. S6

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  Fig. 7. Clustering analysis of control and Myc-overexpressing cardiomyocytes. a Results of UMAP analysis using Zqcc and Zcca values, where control and Myc-overexpressing cardiomyocytes are represented distributed in five different clusters. b UMAP diagrams showing control and Myc-overexpressing cardiomyocytes separately. c Representation of the proportion of control and Myc-overexpressing cardiomyocytes in each of the five clusters shown in Fig. 7a. (CTRL = Control cardiomyocytes; MYC = Myc-overexpressing cardiomyocytes). For this representation, the amount of each type of cardiomyocytes per cluster is normalized by the total amount of cells of each cluster. d Heat map showing the differential expression of the top 100 proteins contributing to the clustering of cardiomyocytes in the five clusters in UMAP shown in Fig. 7a (see also Additional file 1: Fig. S7). e Heat map showing the differential expression of the subcellular compartments analyzed per cluster. The analyses of all the items include 323 control cardiomyocytes and 324 Myc-overexpressing cardiomyocytes. A total of 81 different 10-plex TMTs were used for this analysis (analytical dataset). See also Additional file 1: Figs. S7,…