Cardiopoietic stem cell therapy restores infarction-altered cardiac proteome

Figures

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  Fig. 1. Cardiopoietic cell therapy reduces infarction altered cardiac proteome. a Of the 3987 identified ventricular proteins, 450 (11.3%) differed significantly between Ctrl and MI (two-fold change, p < 0.01), with z-score transformed agglomerative heatmap showing 243 downregulated (blue) and 207 upregulated (red) proteins in infarcted hearts. b CP cell treatment reduced infarction-induced change to 283 differences from Ctrl (7.1% of total), with heatmap detailing 130 down (blue) and 153 up (red). Ctrl control, MI myocardial infarction, MI + CP MI plus cardiopoietic cell therapy.

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  Fig. 2. Reversal of infarcted proteome and predicted upstream regulators. a A majority of proteins altered by MI responded to cardiopoietic (CP) cell therapy (n = 292, 64.9%), either by full (n = 64, 14.2%, blue) or partial (n = 228, 50.7%, red) reversal, whereas 158 proteins (35.1%, green) did not. A larger proportion of downregulated (70%) than upregulated (59%) proteins were responsive to therapy. b Full reversal, following cell treatment, occurred for 32 proteins upregulated and 32 downregulated by infarction. Proteins altered in MI vs. Ctrl (gray bars) were considered fully reversed when altered in the opposite direction in MI + CP vs. MI (red bars) such that MI + CP did not differ from Ctrl. c Partial reversal occurred for 91 proteins upregulated and 137 downregulated by infarction. Proteins considered partially reversed were altered in the opposite direction in MI + CP (versus MI), and either without reaching significance versus MI and not differing from Ctrl; or significantly differing from both Ctrl and MI. d Reversed subproteome (n = 292 proteins) interpretation by pathway analysis identified 78 upstream regulators possessing z-scores for both infarction-induced (MI versus…

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  Fig. 3. Infarction susceptible protein classes respond to cell therapy. Categorical impact was assessed for 33 functional classes into which proteins were allocated, with radial graphs plotting changes induced by myocardial infarction without (MI vs. Ctrl; upregulated in a, downregulated in b) or with cardiopoietic cell therapy (MI + CP vs. Ctrl; upregulated in c, downregulated in d). Radial displacement (red projection) indicates intra-class percentage differing from controls (Categorical Δ, %). Blue circles represent total proteome change (Total Δ, %). Proteins upregulated by infarction were reduced for most categories (26% overall) by cell therapy (a versus c), and likewise for categories downregulated by infarction (b versus d), which reduced 46.5% overall. For each graph a–d, n indicates number of proteins changed (>2-fold, p < 0.01). Functional categories are arrayed alphabetically, with identities and number of proteins listed.

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  Fig. 4. Functional enrichment at proteome and network levels indicative of cell therapy impact. a Screening of proteins differentially expressed by myocardial infarction (–CP) for Ingenuity Pathway Analysis (IPA) enrichment (p < 0.001) of cardiovascular system development and physiological functions revealed prioritization of cardiovascular disease and abnormal morphology. b IPA screening of the cell therapy differential proteome (+CP) indicated a transition from infarction, with numerous enriched functions prioritizing vasculogenesis and associated vascular properties. Asterisks indicate annotations common to a and b, shown in blue or orange if reduced or increased in significance, respectively, following cell therapy (+CP versus –CP). c Venn diagram indicates enriched (Benjamini–Hochberg corrected p < 0.001) Gene Ontology (GO) Biological Processes shared between or unique to the –CP and +CP networks from Supplementary Data Fig. 2. Bubble plots indicate processes unique to each network, with circle diameter proportional to intra-cluster number of annotations, and −log(p-value) for each circle representing the mean of all annotations represented within the cluster. Clusters present in…

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  Fig. 5. Cell therapy-reduced disease associations and -nullified adverse effects validated in vivo. a Ingenuity Pathway Analysis (IPA) interrogation of the infarction altered proteome for cardiovascular disease annotations revealed enriched functions (57 gray bars, p < 0.001), of which only 15 remained in the cell therapy altered proteome (red asterisks). b In IPA Tox Function analysis, hypergeometric overrepresentation predicted 24 cardiac adverse effects associated with the infarcted proteome (gray), 20 of which were significant (p < 0.05). Following interpretation of the reversed subproteome, nearly all adverse effects were predicted to be nullified by cell therapy (red). c–e Infarcted animals were randomized to those without (MI) versus those receiving cardiopoietic (CP) cell therapy (MI + CP), and assessed by echocardiography and electrocardiography for cardiac structure c, electrophysiology d and contractility e. Ischemic cardiomyopathy (MI, n = 8) was characterized by left ventricular dilation and pump failure, quantified by increased end-systolic volume c and decreased ejection fraction e, respectively, 4 weeks post- relative to pre-randomization. In contrast, CP cell therapy (MI…

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  Fig. 6. Cardiopoietic cell therapy redirects infarction modified proteome toward pre-disease state. a Principal component (PC) analysis of differential protein expression segregated myocardial infarction hearts (MI, blue) away from pre-disease controls (Ctrl, red), with cardiopoietic cell therapy (MI + CP, green) counterparts positioned in-between. PC1 represents 25%, PC2 13%, and PC3 11% of total data. b Euclidean squared distance between cohorts was maximal for MI versus Ctrl (2.28 × 105) representing the full extent of disease induced transition, i.e., 100% displacement. Distance between cardiopoietic cell therapy cohort and control (MI + CP versus Ctrl) was substantially lower (1.18 × 105), i.e., 52% of disease-induced displacement. Moreover, the MI + CP versus MI distance was 1.59 × 105, estimating that treatment outcome (MI + CP) response displacement returned ≈57% of the way [1.18 × 105/(1.18 × 105 + 1.59 × 105)] toward pre-disease.