Table 1.
Charasteristics of DS rats at 12 and 16 weeks.
Fig 1.
Upregulation of glycogene expression in the LV of DS hypertensive rats.
(A) Relative expression levels of glycogenes selected after qPCR array in the LV of DS rats fed HS and LS diets were quantified by qPCR. Rps18 was used as an internal control. The numbers of examined rats were n = 12 and n = 15 for the HS groups at 12 and 16 weeks, respectively; n = 6 for LS groups at each period. Expression levels were normalized to that of TATA box-binding protein (Tbp). (B) Protein levels of glycogenes in LV extracts were analyzed by western blotting. β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as internal controls. Representative results from three rats per group are indicated. (C) Densitometry analysis of immunoblots shown in (B). Intensity of each band was normalized to that of GAPDH. Data are presented as the fold change compared with LS rats at 12 weeks (n = 6). (A,C) *, p < 0.05 (Tukey-HSD).
Fig 2.
Lectin microarray analysis of LV extracts and plasma.
Reactivity of fucose-binding lectins AOL and AAL, and mucin-type O-glycan-binding lectin ACA was analyzed in LV extracts (A) and plasma depleted of high-abundance proteins (B) of DS rats (n = 3). Entire lectin microarray datasets are shown in the S3 and S4 Tables. Data are presented as normalized intensity. *, p < 0.05 (Tukey-HSD).
Table 2.
Summary of the lectin microarray results of LV extracts and plasma in DS rats.
Fig 3.
Altered mucin-type O-glycosylation in the LV of DS hypertensive rats.
(A) T-synthase activity in LV extracts. Data were normalized to protein content. (B) Correlation of T-synthase activity with ANP gene expression. ANP gene expression level was quantified by qPCR and normalized to that of Tbp. Data are presented as the fold change compared with LS rats at 12 weeks. (C) Correlation of T-synthase activity with ejection fraction. (D) Relative expression levels of glycogenes involved in the early stage of mucin-type O-glycosylation in the LV tissues of DS rats were analyzed by qPCR and normalized to that of Tbp. Data are presented as the fold change compared with LS rats at 12 weeks. (E) Schematic summary of gene expression analysis data shown in (D). Examined glycosyltransferases in the mucin-type O-glycosylation pathway are shown in red (upregulated), blue (downregulated), or black (no change) letters. Relatively rare core structures (core 5, 6, 7, and 8) synthesized from Tn are omitted. The biosynthetic pathway of disialyl-T is upregulated, as indicated with bold arrows. GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; Gal, galactose; NeuAc, N-acetylneuraminic acid. (F) Lectin blot analysis of sialidase-treated LV extracts using ACA. Representative images demonstrate ACA-reactive glycoproteins and SYPRO Ruby-stained total proteins of three individual rats in each group. Lower panel shows densitometry analysis; intensity of each band was normalized to total protein amount. Data are presented as the fold change (n = 6) compared with sialidase-untreated LV extracts of HS rats at 12 weeks. (A,D) The numbers of examined rats were n = 12 and n = 15 for the HS groups at 12 and 16 weeks, respectively; n = 6 for LS groups at each period. (A,D,F) *, p < 0.05 (Tukey-HSD).
Fig 4.
Decrease of core fucosylation on N-glycans in DS hypertensive rats.
(A) Lectin blot analysis of LV extracts using AOL. Representative images demonstrate AOL-reactive glycoproteins and SYPRO Ruby-stained proteins of three individual rats in each group. Right panel shows densitometry analysis data; intensity of each band was normalized to total protein. Data are presented as the fold change (n = 6) compared with LS rats at 12 weeks. (B) Relative expression levels of the genes responsible for core fucosylation (Fut8) and defucosylation (Fuca1 and Fuca2) on N-glycans were examined by qPCR; levels in the LV and liver were normalized to that of Tbp and Actb, respectively. Data are presented as the fold change compared with the LS group at 12 weeks. (C) AFU activity in the LV and liver extracts, and plasma. Data were normalized to protein content. (D) Plasma levels of FUCA1 and FUCA2. (E) Correlation of AFU activity in LV extracts shown in (C) with relative ANP expression shown in Fig 3B. (F,G) Correlation of plasma AFU activity shown in (C) with plasma NT-proANP concentration (F) and LV anterior wall thickness during diastole (LVAWd) (G). (B-G) The numbers of examined rats were n = 12 and n = 15 for HS groups at 12 and 16 weeks, respectively; n = 6 for LS groups at each period. (A-D) *, p < 0.05 (Tukey-HSD).
Fig 5.
Altered O-glycosylation on CSRP3 in the LV of DS hypertensive rats.
(A) ACA lectin blot analysis and SYPRO Ruby staining of fractions from sialidase-treated LV extracts. Arrow indicates the ACA-positive band, which is observed strongly in fraction 3 of HS (H) but weakly in that of the LS (L) group. (B) Two-dimensional PAGE images of sialidase-treated LV fraction 3. Proteins transferred to membranes were subjected to SYPRO Ruby staining, and then to ACA lectin blotting. Insets show magnified images of two spots used for protein identification. (C) Western blot (WB) and ACA lectin blot (LB) analyses of recombinant human CSRP3. Recombinant proteins expressed in E. coli (unglycosylated negative control) and in HEK293 cells (potentially glycosylated reference) were analyzed after treatment with sialidase and O-glycosidase. (D) Relative expression levels of Csrp3 in the LV tissues. qPCR data were normalized to Tbp expression levels. The numbers of examined rats were n = 12 and n = 15 for HS groups at 12 and 16 weeks, respectively; n = 6 for LS groups at each period. (E) Protein levels of CSRP3 in LV extracts. Densitometry analysis data of western blotting are shown (n = 6). (D,E) The data are presented as the fold change compared with LS rats at 12 weeks. (F) Western blot (WB) and ACA lectin blot (LB) analyses of CSRP3 from LV extracts of DS rats. CSRP3 in LV extracts was immunoprecipitated, denatured, separated by SDS-PAGE, and analyzed. Recombinant human CSRP3 was used as an experimental control of immunoprecipitation with anti-CSRP3 antibody (+) or normal IgG (-). Lower panel shows densitometry analysis data; the intensity of each band in LB was normalized to that in WB (n = 6). (G) Effects of glycosidases on CSRP3 dimerization. LV extracts from three HS (H) or LS (L) rats at 16 weeks were treated with three glycosidases as indicated and then analyzed by western blotting for CSRP3. Arrows indicate the bands corresponding to monomers and dimers. Lower panels show densitometry analysis from five experiments; dimer/monomer ratios are presented as the fold change compared with LS rats without glycosidase treatment. (D-G) *, p < 0.05 (Tukey-HSD). In (G), statistical comparison of HS and LS groups in the same condition and that of HS groups in five conditions are indicated.