Exercise-derived peptide protects against pathological cardiac remodeling

Figures

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  Figure 1. CCDC80 is a potential exerkine involved in cardiovascular pathophysiology. a. Circos plot visualizes the overlap between upregulated gene lists from three databases, and the blue curves link genes that belong to the same enriched GO term. b. Pie charts represent the network of enriched terms in upregulated genes. Each circle node represents an enriched term and is clustered by its identity, where the size of a slice represents the percentage of genes under the term that originated from the corresponding database. c. Heatmap of enriched terms across the three upregulated gene lists, colored by p-values. d. Heatmap of enrichment analysis in DisGeNET, colored by p-values. e. Circos plot displays the overlap between upregulated gene lists, and the purple curves link identical genes. The five genes within the black box represent the genes with significant upregulation throughout the three databases. f. RT-PCR analysis of the expression levels of CCDC80 in various tissues of mice. g, h. After overexpression of PGC-1α in C2C12 cells, the expressions of Pgc-1α (g) and Ccdc80 (h) was determined by qPCR. i, j and k. Eight-week-old male mice were forced to swim 5 days per week for 3 months.…

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  Figure 2. CCDC80 generates a peptide encapsulated in EVs in response to PGC-1α overexpression. a. The cell lysates and culture supernatants collected from vehicle- or Flag-PGC-1α-overexpressing C2C12 cells (treated or not with BFA, 1 μg/mL, 16 h) were prepared and analyzed for CCDC80 protein levels by WB. N=3 b. The supernatants of CCDC80-EGFP-overexpressing C2C12 cells were harvested and filtered through a sterile 0.22-μm filter and then added to H9c2 cells. After 12-h incubation, the cell fluorescence was detected. The fluorescent spots indicated by white arrows were supposedly EVs. N=3 c, d, e. The supernatants of CCDC80-overexpressing C2C12 cells were collected, and EVs were extracted for TEM (c), NTA (d), and EVs markers (e) determinations. N=3. WCL: whole cell lysates. f. The cell lysates and EVs isolated from the supernatants of vehicle- or CCDC80-HA-overexpressing C2C12 cells were prepared and analyzed for CCDC80 protein levels by WB. g. The schematic diagram of CCDC80tide in CCDC80.

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  Figure 3. Acute exercise stimulates a rapid release of CCDC80tide into the circulation. a. Schematic illustration of the experimental design for acute exercise. Twenty subjects (n = 20, males (n=10) and female (n=10)) were included, and the plasma were obtained before, immediately after, and 2 h after exercise; then, EVs were purified. b, c. Plasma EVs were extracted for NTA (b) and TEM (c) determinations. d. The EVs proteins were extracted and analyzed for CCDC80tide and EVs markers by WB. e, f. The EVs-derived CCDC80tide level was measured by ELISA and represented separately by gender. Data are presented as mean ± SD, n = 20. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test, *p < 0.05.

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  Figure 4. CCDC80tide protects against angiotensin II (Ang II)-induced cardiac remodeling. AAV9-cTNT CCDC80tide-HA-EGFP was intravenously injected into mice, and the equal amount of AAV9-HA-EGFP vehicle was injected as a control, n = 9 mice per group. Eighteen days after the injection, mice were chronically infused with Ang II (1.5 mg/kg/day) for 4 weeks. Mice were subjected to the following detections upon the termination of the experiment. a. Left panel, frozen heart tissue sections from AAV9-treated mice were prepared for the detection of EGFP fluorescence. Right panel, the relative quantification of fluorescence intensity. Statistical significance: *** p < 0.001 Student t-test. b. Mice were sacrificed and hearts were dissected, weighed, and photographed. c. Heart weight (HW), body weight (BW), and heart and body weight ratio (HW/BW). Each dot represents a single animal, the same as below. d. Before sacrifice, systolic blood pressure was determined by tail-cuff plethysmography. e, f, g, h. Echocardiography (e) was performed to calculate left ventricular (LV) mass (f), LV posterior wall diastolic (LVPWd) (g), and LV wall thickness (LVAW) (h). i. HE-stained heart transverse sections of…

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  Figure 5. CCDC80tide ameliorates Ang II-induced hypertrophy in cultured cardiomyocytes. a, b, c. Rat neonatal primary cardiomyocytes were infected with increasing levels of CCDC80tide-overexpressed lentivirus or empty lentivirus for 36 h, followed by 1-μM Ang II treatment for 6 h. Total RNA was extracted for qPCR analysis of Myh7 (a), Col1a1 (b), and Tgfb1 (c) mRNA levels. The results are a representative of four biological repetitions. d. Left panel, primary cardiomyocytes were infected with indicated lentivirus for 36 h and treated with 1 μM Ang II for 24 h. The total proteins were extracted and subjected to analysis of above mentioned three protein levels by WB. Right panel, densitometric quantification of WB. N=3 e. Left panel, representative images showing cultured primary cardiomyocytes stained with rhodamine phalloidin. Right panel, quantification of cardiomyocyte area. The results are representative of three biological repetitions (approximately 100 cells were randomly selected for calculation of mean value in each biological replicate, the same as below). f. Left panel, AC16 cells were infected with increasing levels of CCDC80tide-overexpressed lentivirus or empty lentivirus for…

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  Figure 6. CCDC80tide ameliorated Ang II-induced hypertrophy through the regulation of STAT3 signaling. Rat neonatal primary cardiomyocytes were infected with CCDC80tide-overexpressed lentivirus or empty lentivirus for 12 h; then, total RNA was extracted and subjected for RNA-seq analysis. Three biological replicates were used for RNA-seq analysis. a. Volcano plot representing significant DEGs, p < 0.01 (CCDC80tide-overexpression group versus vehicle-overexpression group, upregulated in red and downregulated in blue). The top 10 ranked genes were labeled. b. Establishing DEG network using a text mining approach based on GenCLiP 3. Red, the upregulated genes. Green, downregulated genes. c. GSEA showing negative correlation between CCDC80tide overexpression and STAT3 signaling pathway. d. Left panel, primary cardiomyocytes were infected with CCDC80tide-overexpressed lentivirus or empty lentivirus for 36 h, followed by 1-μM Ang II treatment for 24 h. The total proteins were extracted and subjected to analysis of STAT3 and p-STAT3 (Tyr705) protein levels by WB. Right panel, densitometric quantification of WB. e. Left panel, mice were treated as described in Figure 5. The heart tissue protein…

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  Figure 7. CCDC80tide interacts with kinase-active JAK2 dependent on DUDES domain. a, b. 293T cells were co-transfected with HA-tagged CCDC80tide and Flag-tagged JAK2. Cell lysates were collected 48 h after transfection and immunoprecipitated with anti-HA magnetic beads (a) or anti-Flag agarose beads (b). The immunoprecipitates were probed by WB. c. 293T cells were co-transfected with HA-tagged CCDC80tide and Flag-tagged JAK2 or catalytically inactive JAK2 (K882E). Cell lysates were collected 48 h after transfection and immunoprecipitated with anti-HA magnetic beads and analyzed by WB. d. Schematic diagram of the DUDES domains of CCDC80tide. e. 293T cells were co-transfected with Flag-tagged JAK2 and HA-tagged CCDC80tide or DUDES domain-deleted CCDC80tide (CCDC80tide/△DUDES). Cell lysates were collected 48 h after transfection and immunoprecipitated with anti-HA magnetic beads and analyzed by WB. f. Left panel, rat primary cardiomyocytes were infected with CCDC80tide-overexpressed lentivirus or CCDC80tide/△DUDES lentivirus for 36 h and then treated with 1 μM Ang II for 24 h. The total proteins were extracted and subjected to analysis of indicated protein levels by WB. Right panel,…