ChGn-2 Plays a Cardioprotective Role in Heart Failure Caused by Acute Pressure Overload

Abstract

Background Cardiac extracellular matrix is critically involved in cardiac homeostasis, and accumulation of chondroitin sulfate glycosaminoglycans (CS-GAGs) was previously shown to exacerbate heart failure by augmenting inflammation and fibrosis at the chronic phase. However, the mechanism by which CS-GAGs affect cardiac functions remains unclear, especially at the acute phase. Methods and Results We explored a role of CS-GAG in heart failure using mice with target deletion of ChGn-2 (chondroitin sulfate N-acetylgalactosaminyltransferase-2) that elongates CS chains of glycosaminoglycans. Heart failure was induced by transverse aortic constriction in mice. The role of CS-GAG derived from cardiac fibroblasts in cardiomyocyte death was analyzed. Cardiac fibroblasts were subjected to cyclic mechanical stretch that mimics increased workload in the heart. Significant CS-GAGs accumulation was detected in the heart of wild-type mice after transverse aortic constriction, which was substantially reduced in ChGn-2^-/- mice. Loss of ChGn-2 deteriorated the cardiac dysfunction caused by pressure overload, accompanied by augmented cardiac hypertrophy and increased cardiomyocyte apoptosis. Cyclic mechanical stretch increased ChGn-2 expression and enhanced glycosaminoglycan production in cardiac fibroblasts. Conditioned medium derived from the stretched cardiac fibroblasts showed cardioprotective effects, which was abolished by CS-GAGs degradation. We found that CS-GAGs elicits cardioprotective effects via dual pathway; direct pathway through interaction with CD44, and indirect pathway through binding to and activating insulin-like growth factor-1. Conclusions Our data revealed the cardioprotective effects of CS-GAGs; therefore, CS-GAGs may play biphasic role in the development of heart failure; cardioprotective role at acute phase despite its possible unfavorable role in the advanced phase.

Keywords: cardiac fibroblasts; cardiomyocytes; chondroitin sulfate glycosaminoglycan; extracellular matrix; heart failure; mechanical stretch.

Figure 1. Accumulation of chondroitin sulfate glycosaminoglycans in the heart after acute pressure overload is reduced in ChGn‐2^‐/‐ (chondroitin sulfate N‐acetylgalactosaminyltransferase‐2) mice.

A, Quantitative real time polymerase chain reaction (n=8 for each group) and immunoblotting (n=3 for each group) analysis for ChGn‐2 in the heart of wild‐type (WT) and ChGn‐2^‐/‐ mice. Molecular weight (MW) for protein ladders are shown. B, Quantification of extracted total glycosaminoglycans using 1,9‐dimethylmethylene blue assay in the heart of WT and ChGn‐2^‐/‐ mice in either sham or transverse aortic constriction (TAC) condition (n=7 for sham WT, n=5 for sham ChGn‐2^‐/‐, n=12 for TAC WT, n=9 for TAC ChGn‐2^‐/‐). C, Representative images for Alcian blue staining in the heart of WT and ChGn‐2^‐/‐ mice in either sham or TAC condition; bars: 100 μm. D, Representative images of immmunofluorescence for chondroitin‐4‐sulfate (C4S) in the heart of WT and ChGn‐2^‐/‐ in either sham or TAC condition. C4S fluorescence intensity was quantified (n=5 for sham WT, n=5 for sham ChGn‐2^‐/‐, n=7 for TAC WT, n=7 for TAC ChGn‐2^‐/‐); bars: 100 μm. C4S, red; and 4’,6‐diamidino‐2‐28 phenylindole dihydrochloride, blue. E, Dot blot analysis for C4S in the heart of WT and ChGn‐2^‐/‐ mice in either sham or TAC conditions. C4S expression was quantified and normalized to total protein assessed by the ponceau staining (n=6 for each group). F, Immunoblotting for decorin in heart of WT and ChGn‐2^‐/‐ mice in either sham or TAC conditions. High molecular weight decorin protein expression was quantified and normalized to GAPDH expression (n=6 for each group). Bottom blot shows the results after degradation of glycosaminoglycans by chondroitinase ABC treatment. Molecular weight (MW) for protein ladders are shown. Data represent median and interquartile range. *P<0.05, **P<0.01, and ***P<0.001. Statistical analyses were performed using Wilcoxon rank‐sum test (A) or Kruskal‒Wallis test followed by Wilcoxon rank‐sum test (B, D, E, and F). 18s indicates 18s ribosomal RNA; A.U., arbitrary unit; C4S, chondroitin‐4‐sulfate; ChABC, chondroitinase ABC; ChGn‐2, chondroitin sulfate N‐acetylgalactosaminyltransferase‐2; DMMB, 1,9‐dimethylmethylene blue; TAC, transverse aortic constriction; and WT, wild‐type.

Figure 2. ChGn‐2^‐/‐ (chondroitin sulfate N‐acetylgalactosaminyltransferase‐2) mice exhibit severe left ventricle dysfunction after acute pressure overload.

A, Representative echocardiographic images in wild‐type (WT) and ChGn‐2^‐/‐ mice in either sham or transverse aortic contriction (TAC) group 2 weeks after surgery. White bars: 200 msec; red bars: 2 mm. B, Left ventricular systolic function was analyzed by the fractional shortening at day 0, 3, 7, 10, and 14 days after TAC (n=6 for sham WT, n=5 for sham ChGn‐2^‐/‐, n=8 for TAC WT, n=8 for TAC ChGn‐2^‐/‐). Kruskal‒Wallis test followed by Wilcoxon rank‐sum test was used to analyze the difference of fractional shortening between the groups at each time point. ^$$ P<0.01 between sham WT and TAC WT. ⁺⁺ P<0.01 between sham ChGn‐2^‐/‐ and TAC ChGn‐2^‐/‐. ^%% P<0.01 and ^%%% P<0.001 between TAC WT and TAC ChGn‐2^‐/‐. C, Left ventricular systolic function was analyzed by the ejection fraction at day 0, 3, 7, 10, and 14 days after TAC (n=6 for sham WT, n=5 for sham ChGn‐2^‐/‐, n=8 for TAC WT, n=8 for TAC ChGn‐2^‐/‐). Kruskal‒Wallis test followed by Wilcoxon rank‐sum test was used to analyze the difference of ejection fraction between the groups. ^$$ P<0.01 between sham WT and TAC WT. ⁺⁺ P<0.01 between sham ChGn‐2^‐/‐ and TAC ChGn‐2^‐/‐. ^%% P<0.01 and ^%%% P<0.001 between TAC WT and TAC ChGn‐2^‐/‐. D, Representative images for transversal section of the heart of WT and ChGn‐2^‐/‐ mice in either sham or TAC group 2 weeks after surgery; bars: 500 μm. E, Representative images for Masson trichome staining of the heart in WT and ChGn‐2^‐/‐ mice in either sham or TAC condition; bars: 200 μm. F, Quantification of heart weight normalized to body weight in WT and ChGn‐2^‐/‐ mice in either sham or TAC condition (n=13 for sham WT, n=10 for sham ChGn‐2^‐/‐, n=17 for TAC WT, n=15 for TAC ChGn‐2^‐/‐). G, Quantification of heart weight normalized to tibia length in WT and ChGn‐2^‐/‐ mice in either sham or TAC condition (n=13 for sham WT, n=10 for sham ChGn‐2^‐/‐, n=17 for TAC WT, n=15 for TAC ChGn‐2^‐/‐). H, Quantification of collagen fibrosis area (%) in the heart of WT and ChGn‐2^‐/‐ mice in either sham or TAC group 2 weeks after surgery (n=6 for sham WT, n=5 for sham ChGn‐2^‐/‐, n=8 for TAC WT, n=8 for TAC ChGn‐2^‐/‐). I, Quantitative real time polymerase chain reaction analysis for collagen‐1a (Col1a1) in the heart of WT and ChGn‐2^‐/‐ mice in either sham or TAC group 2 weeks after surgery (n=6 for sham WT, n=5 for sham ChGn‐2^‐/‐, n=8 for TAC WT, n=8 for TAC ChGn‐2^‐/‐). J, Representative images for TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) and cTnT (cardiac troponin T) double immunofluorescence staining in the heart of WT and ChGn‐2^‐/‐ mice in either sham or TAC group 2 weeks after surgery. TUNEL and cTnT double‐positive apoptotic cells was quantified (n=5 for each group); bars: 50 μm. TUNEL, green; cTnT, red; and 4’,6‐diamidino‐2‐28 phenylindole dihydrochloride, blue. K, Immunoblotting for caspase‐3, cleaved caspase‐3 (c.caspase 3), and GAPDH in the heart of WT and ChGn‐2^‐/‐ mice in either sham or 2 weeks after TAC. Molecular weight (MW) for protein ladders are shown. Apoptosis was assessed by the cleaved caspase‐3 expression levels (n=6 for each group). Quantitative data show cleaved caspase 3/total caspase 3 value relative to that in sham WT group. Data represent median and interquartile range. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. Statistical analyses were performed using Kruskal‒Wallis test followed by Wilcoxon rank‐sum test (F, G, H, I, J, and K). 18s indicates 18s ribosomal RNA; BW, body weight; ChGn‐2, chondroitin sulfate N‐acetylgalactosaminyltransferase‐2; cTnT, cardiac troponin T; DAPI, 4’,6‐diamidino‐2‐28 phenylindole dihydrochloride; ChGn‐2, chondroitin sulfate N‐acetylgalactosaminyltransferase‐2; EF, ejection fraction; HW, heart weight; TAC, transverse aortic constriction; TL, tibia length; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; and WT, wild‐type.

Figure 3. Mechanical stretch increases glycosaminoglycans production and enhances cardioprotective effects in human cardiac fibroblasts.

A, Quantitative real time polymerase chain reaction analysis for ChGn‐2 (chondroitin sulfate N‐acetylgalactosaminyltransferase‐2) in human cardiac fibroblasts (HCFs) in either control and stretch condition (n=6 for each group). B, Immunoblotting for ChGn‐2 in HCFs in either control and stretch condition. Molecular weight (MW) for protein ladders are shown. ChGn‐2 protein levels were quantified and normalized to GAPDH expression (n=3 for each group). C, Dot blot analysis for chondroitin‐4‐sulfate (C4S) in conditioned medium (CM) derived from HCFs in either control (C‐CM) and stretch (S‐CM) condition. D, Quantification of extracted total glycosaminoglycans in CM derived from HCFs in either C‐CM or S‐CM (n=3 for each group). E, Immunoblotting for caspase‐3, cleaved caspase‐3 (c.caspase 3), and GAPDH in H9C2 cells treated with CM derived from HCFs in either C‐CM or S‐CM in the presence or absence of doxorubicin. Molecular weight (MW) for protein ladders are shown. Apoptosis was assessed by the cleaved caspase‐3 expression levels normalized to caspase 3 (n=9 for each group). Quantitative data show cleaved caspase 3/total caspase 3 value relative to that in HCF C‐CM group. F, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining in neonatal rat cardiomyocytes treated with CM derived from HCFs in either C‐CM or S‐CM in the presence of doxorubicin. TUNEL‐positive apoptotic cells were quantified (n=4 for each group); bars: 100 μm. TUNEL, green; and Hoechst, blue. G, Immunoblotting for caspase‐3, cleaved caspase‐3, and GAPDH in H9C2 cells treated with CM derived from HCFs in either C‐CM or S‐CM in the presence of doxorubicin. Molecular weight (MW) for protein ladders are shown. Some cells were cultured in the CM pretreated with chondroitinase ABC. 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 HCF C‐CM + doxorubicin group. H, TUNEL staining in neonatal rat cardiomyocytes treated with CM derived from HCFs in either C‐CM or S‐CM in the presence of doxorubicin. Cells were cultured in the CM pretreated with chondroitinase ABC. TUNEL‐positive apoptotic cells were quantified (n=4 for each group); bars: 100 μm. TUNEL, green; and Hoechst, blue. I, Immunoblotting for ChGn‐2 in HCFs infected with retroviruses carrying either green fluorescent protein or ChGn‐2 (ChGn‐2 overexpression). Molecular weight (MW) for protein ladders are shown. ChGn‐2 protein levels were quantified and normalized to GAPDH expression (n=3 for each group). J, Immunoblotting for caspase‐3, cleaved caspase‐3, and GAPDH in H9C2 cells treated with CM derived from HCFs in either C‐CM or S‐CM in the presence of doxorubicin. Molecular weight (MW) for protein ladders are shown. HCFs infected with retroviruses carrying either green fluorescent protein or ChGn‐2 were used. 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 green fluorescent protein HCF C‐CM + doxorubicin group. Chondroitinase ABC at 10 mIU/mL; doxorubicin at 1 μmol/L were used unless otherwise mentioned. Data represent median and interquartile range. *P<0.05, **P<0.01, and ***P<0.001. Statistical analyses were performed using Wilcoxon rank‐sum test (A, B, D, and I) or Kruskal‒Wallis test followed by Wilcoxon rank‐sum test (E, F, G, H, and J). 18s indicates 18s ribosomal RNA; C‐CM, control conditioned medium; ChABC, chondroitinase ABC; ChGn‐2, chondroitin sulfate N‐acetylgalactosaminyltransferase‐2; DMMB, 1,9‐dimethylmethylene blue; GFP, green fluorescent protein; HCF, human cardiac fibroblasts; and S‐CM, stretch conditioned medium.

Figure 4. ChGn‐2 (chondroitin sulfate N‐acetylgalactosaminyltransferase‐2) is inevitable for the stretch‐mediated cardioprotective effects in mouse cardiac fibroblasts (MCFs).

A, Immunoblotting for ChGn‐2 in mouse cardiac fibroblasts (MCFs) in control and stretch conditions. MCFs were isolated from wild‐type (WT) mice. ChGn‐2 protein levels were quantified and normalized to GAPDH expression (n=3 for each group). B, Dot blot analysis for chondroitin‐4‐sulfate (C4S) in conditioned medium (CM) derived from MCFs in either control (C‐CM) or stretch (S‐CM) condition. MCFs were isolated from either WT or ChGn‐2^‐/‐ mice. C, Quantification of extracted total glycosaminoglycans in CM derived from MCFs in either control (C‐CM) or stretch (S‐CM) condition (n=3 for each group). MCFs were isolated from either WT or ChGn‐2^‐/‐ mice. D, Immunoblotting for caspase‐3, cleaved caspase‐3 (c.caspase 3), and GAPDH in H9C2 cells treated with CM derived from WT‐MCFs in either C‐CM or S‐CM in the presence or absence of doxorubicin. Molecular weight (MW) for protein ladders are shown. 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 MCF C‐CM group. E, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining in neonatal rat cardiomyocytes treated with CM derived from WT‐MCFs in either C‐CM or S‐CM in the presence of doxorubicin. TUNEL‐positive apoptotic cells were quantified (n=4 for each group); bars: 100 μm. TUNEL, green; and Hoechst, blue. F, Immunoblotting for caspase‐3, cleaved caspase‐3, and GAPDH in H9C2 cells treated with CM derived from WT‐MCFs in either C‐CM or S‐CM in the presence of doxorubicin. Molecular weight (MW) for protein ladders are shown. Some cells were cultured in the CM pretreated with chondroitinase ABC. 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 MCF C‐CM + doxorubicin group. G, TUNEL staining in neonatal rat cardiomyocytes treated with CM derived from WT‐MCFs in either C‐CM or S‐CM in the presence of doxorubicin. Cells were cultured in the CM pretreated with chondroitinase ABC. TUNEL‐positive apoptotic cells were quantified (n=4 for each group); bars: 100 μm. TUNEL, green; and Hoechst, blue. H, Immunoblotting for caspase‐3, cleaved caspase‐3, and GAPDH in H9C2 cells treated with CM derived from MCFs in either C‐CM or S‐CM condition in the presence of doxorubicin. Molecular weight (MW) for protein ladders are shown. MCFs were isolated from either WT or ChGn‐2^‐/‐ mice. 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 MCF C‐CM +doxorubicin group. I, TUNEL staining in neonatal rat cardiomyocytes treated with CM derived from ChGn‐2^‐/‐ MCFs in either C‐CM or S‐CM in the presence of doxorubicin. TUNEL‐positive apoptotic cells were quantified (n=4 for each group); bars: 100 μm. TUNEL, green; and Hoechst, blue. Chondroitinase ABC at 10 mIU/mL; 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. Statistical analyses were performed using Wilcoxon rank‐sum test (A) or Kruskal‒Wallis test followed by Wilcoxon rank‐sum test (C, D, E, F, G, H, and I). C‐CM indicates control conditioned medium; ChABC, chondroitinase ABC; ChGn‐2, chondroitin sulfate N‐acetylgalactosaminyltransferase‐2; DMMB, 1,9‐dimethylmethylene blue; MCFs, mouse cardiac fibroblasts; S‐CM, stretch conditioned medium; and WT, wild‐type.

Figure 5. Overexpression of ChGn‐2 augments the cardioprotective effects in mouse cardiac fibroblasts (MCFs).

A, Immunoblotting for ChGn‐2 (chondroitin sulfate N‐acetylgalactosaminyltransferase‐2) in wild‐type (WT) mouse cardiac fibroblasts (MCFs) infected with retroviruses carrying either green fluorescent protein (GFP) or ChGn‐2 (ChGn‐2 overexpression). Molecular weight (MW) for protein ladders are shown. ChGn‐2 protein levels were quantified and normalized to GAPDH expression (n=3 for each group). B, Immunoblotting for caspase‐3, cleaved caspase‐3 (c.caspase 3), and GAPDH in H9C2 cells treated with conditioned medium (CM) derived from WT MCFs in either control (C‐CM) or stretch (S‐CM) condition in the presence of doxorubicin. Molecular weight (MW) for protein ladders are shown. WT MCFs infected with retroviruses carrying either GFP or ChGn‐2 (ChGn‐2 overexpression) were used. Apoptosis was assessed by the cleaved caspase‐3 expression levels compared with caspase 3 (n=6 for each group). Quantitative data show cleaved caspase 3/total caspase 3 value relative to that in GFP MCF C‐CM + doxorubicin group. C, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining in in H9C2 cells treated with CM derived from WT MCFs in either C‐CM or S‐CM in the presence of doxorubicin. WT MCFs infected with retroviruses carrying either GFP or ChGn‐2 (ChGn‐2 overexpression) were used. TUNEL‐positive apoptotic cells were quantified (n=6 for each group); bars: 100 μm. TUNEL, green; and Hoechst, blue. D, Immunoblotting for ChGn‐2 in ChGn‐2^‐/‐ MCFs infected with retroviruses carrying either GFP or ChGn‐2 (ChGn‐2 overexpression). Molecular weight (MW) for protein ladders are shown. E, Immunoblotting for caspase‐3, cleaved caspase‐3, and GAPDH in H9C2 cells treated with CM derived from ChGn‐2^‐/‐ MCFs in either C‐CM or S‐CM in the presence of doxorubicin. Molecular weight (MW) for protein ladders are shown. ChGn‐2^‐/‐ MCFs infected with retroviruses carrying either GFP or ChGn‐2 (ChGn‐2 overexpression) were used. Apoptosis was assessed by the cleaved caspase‐3 expression levels (n=9 for each group). Quantitative data show cleaved caspase 3/total caspase 3 value relative to that in GFP ChGn‐2^‐/‐ MCF C‐CM + doxorubicin group. F, TUNEL staining in H9C2 cells treated with CM derived from ChGn‐2^‐/‐ MCFs in either C‐CM or S‐CM in the presence of doxorubicin. ChGn‐2^‐/‐ MCFs infected with retroviruses carrying either GFP or ChGn‐2 (ChGn‐2 OE) were used. TUNEL‐positive apoptotic cells were quantified (n=6 for each group); bars: 100 μm. TUNEL, green; and Hoechst, blue. Doxorubicin was used at 1 μmol/L. Data represent median and interquartile range. *P<0.05, **P<0.01, and ***P<0.001. Statistical analyses were performed using Wilcoxon rank‐sum test (A) or Kruskal‒Wallis test followed by Wilcoxon rank‐sum test (B, C, E, and F). C‐CM indicates control conditioned medium; ChABC, chondroitinase ABC; ChGn‐2, chondroitin sulfate N‐acetylgalactosaminyltransferase‐2; GFP, green fluorescent protein; MCFs, mouse cardiac fibroblasts; S‐CM, stretch conditioned medium; and WT, wild‐type.

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

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.

Figure 7. Chondroiton sulfate A (CS‐A) binds to insulin‐like growth factor 1 (IGF‐1) and augments its cardioprotective effects.

A, Quantitative real time polymerase chain reaction analysis for insulin‐like growth factor 1, basic fibroblast growth factor, platelet derived growth factor subunit A, and granulocyte colony‐stimulating factor in human cardiac fibroblasts in either control or stretch condition (n=6 for each group). Cells were given stretch stimuli for 3h S, 6h S, 12h S, and 24h S, respectively. B, Culture plates were coated with CS‐A in the presence or absence of chondroitinase ABC. Negative control plates were prepared by incubating with PBS. Subsequently, plates were incubated with 100 ng/mL IGF‐1 for 2 hours at 37 °C, followed by washing with PBS. H9C2 cells were then seeded onto the plates, and incubated for 24 hours. RNAs were extracted from cells, and atrial natriuretic peptide and brain natriuretic peptide mRNA expression was quantitatively analyzed with GAPDH as normalization (n=3 each). As a positive control, cells were seeded on non‐coated plate (without incubation with IGF‐1), and 100 ng/mL IGF‐1 was added in the culture medium at the time of cell seeding. For detailed experimental procedures, see Figure S8. C, Culture plates were coated with CS‐A in the presence or absence of chondroitinase ABC. Negative control plates were prepared by incubating with PBS. Subsequently, plates were incubated with 100 ng/mL IGF‐1 for 2 hours at 37 °C, followed by washing with PBS. H9C2 cells were then seeded onto the plates, and incubated for 24 hours. Then, the cells were treated with doxorubicin for 3 hours (see Figure S8 for detailed experimental procedures). Caspase‐3, cleaved caspase‐3 (c.caspase 3), phospho‐AKT, total‐AKT, and GAPDH expressions were analyzed by immunoblotting. Molecular weight (MW) for protein ladders are shown. As a positive control, cells were seeded on non‐coated plate (without incubation with IGF‐1), and 100 ng/mL IGF‐1 was added in the culture medium at the time of cell seeding. D, Immunoblotting for caspase‐3, cleaved caspase‐3, phospho‐AKT, total‐AKT, and GAPDH in H9C2 cells treated with either vehicle, IGF‐1, CS‐A, or IGF‐1+CS‐A in the presence of doxorubicin. Apoptosis was assessed by the cleaved caspase‐3 expression levels compared with caspase 3 (n=6 for each group). AKT activity was also assessed by quantification of phospho‐AKT compared with total‐AKT (n=6 for each group). Quantitative data show cleaved caspase 3/total caspase 3 and phospho‐AKT/total‐AKT values relative to those in vehicle + doxorubicin group. E, Immunoblotting for caspase‐3, cleaved caspase‐3, and GAPDH in H9C2 cells treated with conditioned medium (CM) derived from wild‐type (WT) mouse cardiac fibroblasts in either control‐CM or stretch‐CM in the presence of doxorubicin. Molecular weight (MW) for protein ladders are shown. Some cells were treated with anti‐CD44 antibody (CD44ab). 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 mouse cardiac fibroblasts control‐CM + doxorubicin group. F, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining in neonatal rat cardiomyocytes treated with CM derived from WT‐ mouse cardiac fibroblasts in either control‐CM or stretch‐CM condition 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; IGF‐1 at 10 ng/mL; CD44ab at 1: 200 dilution; doxorubicin at 1 μmol/L (D through F) were used for experiments. 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 (D through F). 18s indicates 18s ribosomal RNA; ANP, atrial natriuretic peptide; bFGF, basic fibroblast growth factor; BNP, brain natriuretic peptide; C‐CM, control conditioned medium; ChABC, chondroitinase ABC; CS‐A, chondroitin sulfate A; GCSF, granulocyte colony‐stimulating factor; IGF‐1, insulin‐like growth factor 1; MCFs, mouse cardiac fibroblasts; pAKT, phospho‐AKT; PDGFA, platelet derived growth factor subunit A; S‐CM, stretch conditioned medium; and tAKT, total‐AKT.