Fig 1: ROS production was involved in IS-induced reductions of Ito,f-related proteins.(A and D) Measurements of ROS productions based on DHE fluorescence in rat hearts. (A) Representative images of DHE immunofluorescence. Scale bar: 200 μm. (D) Relative ROS fluorescence intensities in sham, CKD, and CKD plus BB536 groups (n = 5 per group), and in vehicle and IS treatment groups (n = 6 per group). (B and E) Measurements of ROS productions based on flow cytometry in NRVMs treated with different concentrations of IS. (B) Representative flow cytometric histograms in NRVMs. (E) Relative ROS fluorescence intensities in NRVMs (n = 5 per group). (C and F) NAC reversed ROS production induced by IS in NRVMs. (C) Representative flow cytometric histograms in 4 groups. (F) Relative ROS fluorescence intensities detected by flow cytometry in 4 groups (n = 5 per group). (G and H) Representative immunoblots of NOX2 proteins in sham, CKD, and CKD plus BB536 groups (n = 5 per group) (G), and in vehicle and IS treatment groups (n = 6 per group) (H). (I) Average immunoblots data of NOX2 proteins in the hearts of rats. (J and M) Representative immunoblots (J) and average data (M) of NOX2 proteins in NRVMs treated with different concentrations of IS (n = 3 per group). (K and N) Representative immunoblots (K) and average data (N) of NOX2 proteins in control, DPI, IS, and IS plus DPI groups (n = 3 per group). (L and O) Representative immunoblots (L) and average data (O) of NOX2 proteins in control, APO, IS, and IS plus APO groups (n = 3 per group). (P and Q) Representative immunoblots (P) and average data (Q) of Kv4.2, Kv4.3, and KChIP2 proteins in control, NAC, IS, and IS plus NAC groups (n = 3 per group). (R) Relative mRNA expressions for Kv4.2, Kv4.3, and KChIP2 in control, NAC, IS, and IS plus NAC groups (n = 3 per group). NRVMs in IS and IS plus NAC groups were treated with 10 μM IS. Data are presented as mean ± SEM. Statistical analysis was performed using 2-tailed Student’s t test (D and I) and 1-way ANOVA followed by Bonferroni post hoc test (D–F, I, M–O, Q, and R). *P < 0.05, **P < 0.01.
Fig 2: Activation of p38 MAPK, p44/42 MAPK, and NF-κB signaling pathways downregulated Ito,f-related protein and mRNA.(A and B) Representative immunoblots (A) and average data (B) of Kv4.2, Kv4.3, KChIP2, and P-p38 MAPK in control, SB, IS, and IS plus SB groups (n = 3 per group). (C) Relative mRNA expressions for Kv4.2, Kv4.3, and KChIP2 in control, SB, IS, and IS plus SB groups (n = 3 per group). (D and E) Representative immunoblots (D) and average data (E) of Kv4.2, Kv4.3, KChIP2, and P-p44/42 MAPK in control, U0126, IS, and IS plus U0126 groups (n = 3 per group). (F) Relative mRNA expressions for Kv4.2, Kv4.3, and KChIP2 in control, U0126, IS, and IS plus U0126 groups (n = 3 per group). (G and H) Representative immunoblots (G) and average data (H) of Kv4.2, Kv4.3, KChIP2, and P-NF-κB in control, BAY, IS, and IS plus BAY groups (n = 3 per group). (I) Relative mRNA expressions for Kv4.2, Kv4.3, and KChIP2 in control, BAY, IS, and IS plus BAY groups (n = 3 per group). NRVMs in IS, IS plus SB, IS plus U0126, and IS plus BAY groups were treated with 10 μM IS. Data are presented as mean ± SEM. Statistical analysis was performed using 1-way ANOVA, followed by Bonferroni post hoc test. *P < 0.05, **P < 0.01.
Fig 3: IS reduced Ito,f-related protein expression levels and current densities in vitro.(A and B) Representative immunoblots (A) and average data (B) of Kv4.2, Kv4.3, and KChIP2 proteins in NRVMs treated with different concentrations of IS (n =3 per group). (C) Relative mRNA expressions for Kv4.2, Kv4.3, and KChIP2 in NRVMs treated with different concentrations of IS (n = 3 per group). (D and E) Representative immunofluorescence images of Kv4.2 (D) and KChIP2 (E) proteins in NRVMs. Scale bar: 50 μm. (F and G) Representative Ito,f traces (F) and average Ito,f densities (peak minus steady state) versus membrane potentials (G) in NRVMs treated with different concentrations of IS (n = 5 per group).The inset in F shows the voltage-clamp protocol. (H) Average time constants (τ) of decay of Ito,f at +60 mV in NRVMs (n = 5 per group). (I and J) Average values of constants (k) of activation (I) and half-maximal voltage of activation(V0.5, act) (J) in NRVMs (n = 5 per group). (K) Voltage-dependent activation curves of Ito,f in NRVMs (n = 5 per group). Data are presented as mean ± SEM. Statistical analysis was performed using 1-way ANOVA followed by Bonferroni post hoc test. *P < 0.05 versus control, **P < 0.01 versus control.
Fig 4: Ito,f-related proteins were downregulated in CKD rats with a high levels of IS.(A) Flow chart of animal experiments. (B and D) Determinations of IS levels in serum (B) and heart tissues (D) at 8 weeks after right nephrectomy (n = 5 per group). (C and E) Measurements of IS levels in serum (C) and heart tissues (E) after 8 weeks of IS treatment (n = 6 per group). (F and I) Representative immunoblots (F) and average data (I) of Kv4.2, Kv4.3, and KChIP2 proteins in vehicle and IS treatment groups (n = 6 per group). (G and H) Representative immunoblots (G) and average data (H) of Kv4.2, Kv4.3, and KChIP2 proteins in sham, CKD, and CKD plus BB536 groups (n = 5 per group). (J) Representative IHC images of Kv4.2 and Kv4.3 proteins in rats. Scale bar: 100 μm. Data are presented as mean ± SEM. Statistical analysis was performed using 2-tailed Student’s t test (C, E, and I) and 1-way ANOVA followed by Bonferroni post hoc test (B, D, and H). *P < 0.05, **P < 0.01.
Supplier Page from Abcam for Anti-KChIP2 antibody