Secretome Signature of Cardiopoietic Cells Echoed in Rescued infarcted Heart Proteome

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  FIGURE 1. Cardiopoietic secretome harbors pro‐cardiovasculogenic traits. A, Conditioned media was assessed by biotin labeling on a 507 protein streptavidin‐conjugated fluorescent chip array, indicating that effective cardiopoietic cells secreted 155 differentially expressed proteins at >2‐fold change (107 upregulated—orange; 48 downregulated—blue). B, Ingenuity Pathway Analysis (IPA) for enriched cardiovascular developmental functions revealed vascular development, angiogenesis, vasculogenesis, and multiple endothelial cell (EC) functions as prioritized annotations associated with the differential secretome, with positive z‐scores representing increased likelihood of activation and negative scores increased likelihood of inhibition. C, IPA integration of the differential secretome generated an interaction network comprising 520 nodes, clustered along matrix axes by inclusion basis (colored bars), with 8977 pairwise connections (edges) denoted as black squares. Node names and topological properties are listed in supporting Table S1. The top 10 IPA cardiovascular system development functional annotations associated with the network were angiogenesis, vascular development, vasculogenesis, EC…

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  FIGURE 2. Cardiopoietic secretome miRNA regulators. A, Differential secretome interrogation by Ingenuity Pathway Analysis identified 65 miRNAs as upstream candidate regulators induced by the cardiopoietic process, with individual identities rank ordered by corresponding enrichment P‐value. B, Independently, the miRNA makeup of high vs low response cardiopoietic cells was evaluated by miRNA‐Seq detecting 447 discrete miRNAs, of which 17 were differentially expressed at >1.5‐fold change, P < .05, as visualized by volcano plot (B, upper, with 11 upregulated—orange; 6 downregulated—blue). Differential miRNA clustering segregated high vs low response cardiopoietic cells (hrCP and lrCP, respectively), as visualized by agglomerative hierarchical heatmap (B, lower, with differentially expressed miRNAs listed). For each miRNA, z‐scores represent intensity for individual samples normalized to mean intensity. C, Overlap between the 65 candidate miRNA regulators of the secretome (blue) and the 17 differentially expressed miRNAs of hrCP (red) revealed a subset of 4 shared miRNAs, visualized by Venn diagram. miR‐146, 146a‐5p/146b‐5p (black ellipse) exhibited consistent directionality, with observed…

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  FIGURE 3. Secretome functionality concentrated within the miR‐146 dependent sub‐secretome. (A) Pathway interrogation pinpointed miR‐146 at the apex of a 14‐protein network upstream of the secretome (A, upper) comprising interdependent transcriptional regulators (TNFα; CREB1; PPARγ; CCL5; IRF8; STAT1; STAT3; STAT6; and the NFκB complex, including NFκB1, NFκBIA, NFκBIB, and RELA) predicted to be activated or inhibited (orange or blue, respectively). Each node of the miR‐146 directed network regulates one or more downstream components of a cardiopoietic sub‐secretome (A, lower), comprising 65% of the overall differential secretome (101 of 155 proteins), with protein increase or decrease shown (red or green, respectively). B, Integrating the 101 protein miR‐146 dependent sub‐secretome into an expanded interaction neighborhood generated a 430 node network, clustered along matrix axes by inclusion basis (colored bars), connected by 6755 interactions (edges) shown as black squares. Network node names and topological parameters are listed in supporting Table S3. C, BiNGO analysis of the miR‐146 dependent secretome network yielded 558 significantly enriched terms (P < .001; with the top 100 listed…

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  FIGURE 4. Subproteome linked to cardiopoietic cell therapeutic response. A, At 2‐months postinfarction, 13.7% of the murine ventricular proteome differed (6.4% up, 7.3% down ≥2‐fold, P < .05) compared with non‐infarction, measured by label‐free quantitative proteomics and shown by volcano plot. B, Ejection fraction on average decreased −7 ± 2% in untreated infarction (n = 28) but improved +8 ± 1% (n = 34; P < .0001) with human cardiopoietic cell (600 000 cells/heart delivered epicardially) treatment. C, Compared with untreated infarcted hearts, high and low response proteomes (hrCP and lrCP) exhibited a common core of 280 changes (purple), with a greater number of additional proteins altered in hrCP (blue) vs lrCP (red). D, Ingenuity Pathway Analysis prioritized infarction associated (top ranked shown, gray) cardiac adverse effects (P < .05) predicted to be nullified through full or partial subproteome reversal in both lrCP and hrCP cohorts. E, hrCP hearts were enriched across cardiac function, structure, conduction, muscularization and angiogenesis, while lrCP hearts displayed limited overlap in the predicted systems spectrum. hrCP achieved superior pump function (F), reversal of left…