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. 2022 Mar 24;11(7):e023401. doi: 10.1161/JAHA.121.023401

Figure 6. Purified chondroitin sulfate A (CS‐A) shows cardioprotective effects.

Figure 6

A, Immunoblotting for caspase‐3, cleaved caspase‐3 (c.caspase 3), phospho‐AKT (p‐AKT), total‐AKT (t‐AKT), and GAPDH in H9C2 cells treated with either vehicle or CS‐A in the presence or absence of doxorubicin. Molecular weight (MW) for protein ladders are shown. B, Apoptosis was assessed by the cleaved caspase‐3 (c.caspase 3) expression levels compared with caspase 3 (n=9 for each group). Quantitative data show cleaved caspase 3/total caspase 3 value relative to that in vehicle group. C, Akt activity was assessed by quantification of p‐AKT compared with t‐AKT (n=9 for each group). Quantitative data show p‐AKT/t‐AKT value relative to that in vehicle group. D, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining in neonatal rat cardiomyocytes treated with either vehicle or CS‐A in the presence of doxorubicin. TUNEL‐positive apoptotic cells were quantified (n=4 for each group); bars: 100 μm. TUNEL, green; and Hoechst, blue. E, Immunoblotting for caspase‐3, cleaved caspase‐3, p‐AKT, t‐AKT, and GAPDH in H9C2 cells treated with either vehicle or CS‐A in the presence of doxorubicin. Molecular weight (MW) for protein ladders are shown. Some cells were treated with phosphoinositide 3‐kinases inhibitor, LY294002 (LY). F, Apoptosis was assessed by the cleaved caspase‐3 expression levels compared with caspase 3 (n=9 for each group). Quantitative data show cleaved caspase 3/total caspase 3 value relative to that in vehicle + doxorubicin group. G, Akt activity was assessed by quantification of p‐AKT compared with t‐AKT (n=9 for each group). Quantitative data show p‐AKT/t‐AKT value relative to that in vehicle + doxorubicin group. H, Immunoblotting for caspase‐3, cleaved caspase‐3, p‐AKT t‐AKT, and GAPDH in H9C2 cells treated with either vehicle or CS‐A in the presence of doxorubicin. Molecular weight (MW) for protein ladders are shown. Some cells were treated with anti‐CD44 antibody (CD44ab). I, Apoptosis was assessed by the cleaved caspase‐3 expression levels compared with caspase 3 (n=9 for each group). Quantitative data show cleaved caspase 3/total caspase 3 value relative to that in vehicle + doxorubicin group. J, Akt activity was assessed by quantification of p‐AKT compared with t‐AKT (n=9 for each group). Quantitative data show p‐AKT/t‐AKT value relative to that in vehicle + doxorubicin group. K, TUNEL staining in neonatal rat cardiomyocytes treated with either vehicle or CS‐A in the presence of doxorubicin. Some cells were treated with anti‐CD44 antibody (CD44ab). TUNEL‐positive apoptotic cells were quantified (n=3 for each group). Bars: 100 μm. TUNEL, green; and Hoechst, blue. CS‐A at 500 μg/mL; LY294002 at 25 μmol/L; CD44ab at 1: 200 dilution; doxorubicin at 1 μmol/L were used for experiments unless otherwise mentioned. Data represent median and interquartile range. *P<0.05, **P<0.01, and ***P<0.001. n.s., not significant. Statistical analyses were performed using Kruskal‒Wallis test followed by Wilcoxon rank‐sum test (B, C, D, F, G, I, J, and K). CS‐A indicates chondroitin sulfate A; LY, LY294002; pAKT, phospho‐AKT; tAKT, total‐AKT; and TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.