DR region of Na+-K+-ATPase is a new target to protect heart against oxidative injury

Figures

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  Figure 1. Properties of DR-Ab and its therapeutic effects in an ISO-induced mouse cardiac hypertrophy model. (a) Titer of DR-Ab purified from immunized rat sera and IgGs from normal rat sera (NS). (b) Western blots showing purified pig kidney NKA protein detected by DR-Ab and positive control (anti-NKA α, Santa Cruz, H3) at 1:1000 dilutions. IB: immunoblotting. (c) Immunoprecipitation assay showing that DR-Ab, but not control IgG, immunoprecipitated NKA from mouse heart lysate. IP: immunoprecipitation. (d,e) Quantitative results of ejection fraction (d) and fractional shortening (e) in each group (n = 7–8); (f–i) Statistical results for the body weight (f), heart weight (g), and the ratios of heart weight/body weight (HW/BW, h), HW/tibia length (HW/TL, i) at the end of 2 weeks post the ISO injection. n = 4–6. Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs control; #p < 0.05, ##p < 0.01, ###p < 0.001 vs ISO.

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  Figure 2. Protective effects of DR-Ab against ISO-induced hypertrophy. (a) Representative images of FITC-conjugated wheat germ agglutinin (WGA) staining of hearts from each group; scale bar, 50 μm; (b) Quantitative results of the cardiomyocyte cross sectional area in each group (n = 150 cells per group); (c,d) The relative mRNA levels of hypertrophic marker ANP (c) and β-MHC (d) over GAPDH (n = 4–5); (e,f) Representative Western blots (e) and quantification (f) of ANP to HPRT in each group (n = 5–8). (g) Representative images of picrosirius red staining of hearts from each group; scale bar, 50 μm; (h) Quantitative results of the left ventricular (LV) collagen volume in each group (n = 10 fields per group); Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs control; #p < 0.05, ##p < 0.01, ###p < 0.001 vs ISO.

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  Figure 3. DR-Ab alleviates Isoproterenol (ISO)-induced oxidative stress. (a) DCF florescence of heart lysates in each group after incubation with H2DCF-DA for 45 min (n = 4); (b) Representative dot blots and quantification of total 3-nitrotyrosine levels (n = 4); (c) Representative images of DHE staining on the whole heart slice and enlarged view in each group. Fluorescent images of whole heart section were obtained via tile scanning with Leica DMi8 automated system. Right, quantitative results of the DHE staining in each group (3 hearts per group and 6 areas per heart). Scare bar, 1 mm for whole heart slice and 50 μm for enlarged view. Mean ± SEM. *p < 0.05, ***p < 0.001 vs control; #p < 0.05, ###p < 0.001 vs ISO.

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  Figure 4. Effect of DR-Ab on ROS-induced injury in AC16 human cardiac myocytes or H9C2 rat myoblast cells. (a) Cytotoxicity induced by exposure to various concentrations of H2O2 for 4 hours, as measured by MTT. n = 8; (b) Pretreatment with DR-Ab for 1 hr inhibits cell death caused by various concentrations of H2O2. n = 8; (c) Time-course study showing the protective effect lasted for at least 24 hr. n = 4–8; (d) H2O2 increased LDH level in the culture media of H9C2 cells and DR-Ab pretreatment reduced it. n = 3. (e) Morphological changes (shrinking and detachment) in H2O2-treated H9C2 cells were absent in DR-Ab pretreated cells from three independent experiments; scale bar, 10 μm; (f) Effect of NKA α1 silencing on cell viability of AC16 cells under oxidative stress. n = 6; (g) Premixing DR-Ab with DR-peptide for 1 hour at room temperature under agitation abolished the protective effect of DR-Ab on AC16 cells. n = 8, Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs control, #p < 0.05, ##p < 0.01, ###p < 0.001 vs corresponding NS group. +++p < 0.001 WT vs SiRNA; ^^^p < 0.001 vs DR-Ab group. Con: control; Veh: Vehicle (PBS); NS: Rat normal antibody, as negative control.

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  Figure 5. Effect of DR-Ab on H2O2-induced apoptosis in H9C2 cells. (a) DR-Ab reduced the number of apoptotic cells induced by overnight incubation with 100 μM H2O2, as measured by the condensed and bright nuclei in Hoechst33342 staining. From 3 independent experiments; (b) Representative Western blots and group analysis showing protein expression of Bcl-2, Bax. n = 4. Mean ± SEM. *p < 0.05, ***p < 0.001 vs control, #p < 0.05, vs corresponding NS group. NS: Rat normal antibody, as negative control.

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  Figure 6. Effect of DR-Ab on the reduced NKA activity and membrane NKA abundance in H2O2 treated AC16 cells and Isoproterenol (ISO)-challenged rat hearts. (a) Pretreatment with DR-Ab for 1 hr rescued the inhibition of NKA activity caused by H2O2 (100 μM, 1 hr). n = 4; (b) Representative Western blots and group analysis showing that DR-Ab treatment reversed the plasma membrane (PM) NKA loss in AC 16 cells treated with H2O2 (100 μM, 1 hr). n = 6; (c) Representative Western blots and group analysis showing the effect of DR-peptide immunization on NKA abundance in the rat hearts at the end of 10 weeks post the ISO injection. n = 5; (d) Representative confocal immunostaining images showing reduced NKA density on the plasma membrane of isolated rat cardiomyocytes subjected to metabolic inhibition/acidosis (15 min) and normal culture medium (15 min). The lower left panel shows the fluorescent signal intensities of NKA protein collected from the lines across the cells as indicated in the green channel (NKA). The lower right histogram shows a group analysis of plasma membrane NKA staining/peak intensity. Values were the averages from 10 cells in each group. Membrane NKA abundance was maintained in…

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  Figure 7. Immunofluorescence staining showing the endocytosis of NKA in AC16 cells. Representative immunofluorescence staining and co-localization analysis showing the increased co-localization of NKA (Green) with late endosome marker, rab7 (Red) in AC16 cells treated with 100 µM H2O2 for 1 hr. n = 17. Scale bar, 10 μm.

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  Figure 8. Involvement of PP2A in H2O2-induced NKA endocytosis and cardioprotective effect of DR-Ab. (a) Representative Western blots and group analysis showing NKA in endosomes isolated from cells of different treatment groups. Incubation with 100 µM H2O2 for 1 hr induced enrichment of NKA in the endosomes. n = 6; (b) Representative Western blots and group analysis of McK1 showing the unphosphorylated NKA at Ser18 in H9C2 rat myoblast cell line exposed to 100 µM H2O2 for 30 min. n = 3; (c) Representative Western blots and group analysis of PKCζ in the membrane and cytosolic fractions of cell lysate. n = 4; (d) Effect of H2O2 (100 µM, 30 min) and DR-Ab (160 ug/ml, 1 h pretreatment) on PP2A activity in AC16 cells. n = 3; (e) Effect of PP2A inhibitors, okadaic acid (OK, 100 nM) and cantharidin (Cant, 1 µM), on the DR-Ab conferred antioxidative protection. n = 8. (f) Effect of PP2A silencing on cell viability of AC16 cells under oxidative stress. Insert, Western blots showing protein expression of PP2A. n = 8. Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs control, #p < 0.05, ##p < 0.01, ###p < 0.001 vs H2O2 treatment group, ^^^p < 0.001 vs DR-Ab + H2O2 group. Veh: PBS. NS: Normal rat…