Cardiac CaMKII δ and Wenxin Keli Prevents Ang II-Induced Cardiomyocyte Hypertrophy by Modulating CnA-NFATc4 and Inflammatory Signaling Pathways in H9c2 Cells

Abstract

Previous studies have demonstrated that calcium-/calmodulin-dependent protein kinase II (CaMKII) and calcineurin A-nuclear factor of activated T-cell (CnA-NFAT) signaling pathways play key roles in cardiac hypertrophy (CH). However, the interaction between CaMKII and CnA-NFAT signaling remains unclear. H9c2 cells were cultured and treated with angiotensin II (Ang II) with or without silenced CaMKIIδ (siCaMKII) and cyclosporine A (CsA, a calcineurin inhibitor) and subsequently treated with Wenxin Keli (WXKL). Patch clamp recording was conducted to assess L-type Ca²⁺ current (I_Ca-L), and the expression of proteins involved in signaling pathways was measured by western blotting. Myocardial cytoskeletal protein and nuclear translocation of target proteins were assessed by immunofluorescence. The results indicated that siCaMKII suppressed Ang II-induced CH, as evidenced by reduced cell surface area and I_Ca-L. Notably, siCaMKII inhibited Ang II-induced activation of CnA and NFATc4 nuclear transfer. Inflammatory signaling was inhibited by siCaMKII and WXKL. Interestingly, CsA inhibited CnA-NFAT pathway expression but activated CaMKII signaling. In conclusion, siCaMKII may improve CH, possibly by blocking CnA-NFAT and MyD88 signaling, and WXKL has a similar effect. These data suggest that inhibiting CaMKII, but not CnA, may be a promising approach to attenuate CH and arrhythmia progression.

Figure 1

The HPLC chromatograms of WXKL. (a) Chemical reference substances, (b) WXKL, (c) negative samples of Panax notoginseng, and (d) Codouopsis pilosula. (1) notoginsenoside R1; (2) ginsenoside Rg1; (3) obetyolin; (4) ginsenoside Rb1; (5) ginsenoside Rd.

Figure 2

SiCaMKII decreases Ang II-induced cell surface area enlargement in H9c2 cells. H9c2 cells were treated with Ang II (10⁻⁷ M for 48 h). CaMKII was silenced or CsA (10⁻⁶ M) was added to the culture medium prior to Ang II administration. After administering Ang II, WXKL (5 g/L) was added, and the culture medium was incubated for 24 h. The control cells received no treatment. (a) Representative images of immunofluorescence staining for phalloidin following treatment (n = 10 cells per group). Scale bar: 30 μm. (b) Mean cell length measurement (n = 10 cells per group). (c) Mean cell width measurement (n = 10 cells per group). Data are presented as the mean ± SD. Statistical significance was determined by one-way ANOVA. ^∗P < 0.05 and ^∗∗P < 0.01 vs. the control group. ^#P < 0.05 and ^##P < 0.01 vs. the Ang II group. ^▲P < 0.05 and ^▲▲P < 0.01; the control group in SiCaMKII or CsA vs. the control group in normal. ^△P < 0.05 and ^△△P < 0.01; the Ang II group in SiCaMKII or CsA vs. the Ang II group in normal.

Figure 3

Changes in patch clamp inspection. (a) I_Ca-L current in the control, Ang II, Ang II + WXKL, SiCaMKII, and CsA groups. (b) Effects of siCaMKII and CsA on the I_Ca-L current-voltage (I–V) curve in H9c2 cells. I–V curves and peak current density (PA/PF) of each group (n = 10 cells per group). (c) Effects of siCaMKII and CsA on I_Ca-L steady-state activation (SSA) in H9c2 cells. I_Ca-L steady-state activation curves (original data point diagram and curve fit by Boltzmann equation), comparison of semiactivation voltage (V_{1/2, act}), and comparison of slope factors (K_,act) in each group (n = 10 cells per group). (d) Effects of siCaMKII and CsA on I_Ca-L steady-state inactivation in H9c2 cells. I_Ca-L steady-state inactivation curves (original data point diagram and curve fitting by Boltzmann equation); comparison of semi-inactivation voltage (V_{1/2, inact}); comparison of slope factors (K_,inact) in each group (n = 10 cells per group). Values are presented as mean ± SD; Statistical significance was determined by one-way ANOVA. ^∗P < 0.05 and ^∗∗P < 0.01 vs. the control group. ^#P < 0.05 and ^##P < 0.01 vs. the Ang II group.

Figure 4

Effects of siCaMKII and CsA on the CaMKII signaling cascade. (a) Double immunofluorescence staining to observe the effects of siCaMKII, CsA, and WXKL on CaMKII expression in each group. Scale bar: 50 μm. (b) Mean density of CaMKII in each group. Data are presented as the mean ± SD. Statistical significance was determined by one-way ANOVA. ^∗P < 0.05 and ^∗∗P < 0.01 vs. the control group. ^#P < 0.05 and ^##P < 0.01 vs. the Ang II group. ^▲P < 0.05 and ^▲▲P < 0.01; the control group in SiCaMKII or CsA vs. the control group in normal. ^△P < 0.05 and ^△△P < 0.01; the Ang II group in SiCaMKII or CsA vs. the Ang II group in normal.

Figure 5

Effects of siCaMKII and CsA on Ang II-induced activation of CaMKII signaling in H9c2 cells. (a) Representative western blotting images. Densitometric analysis of (b) CaMKII, (c) p-CaMKII, (d) RyR2, (e) p-RyR2, (f) PLB, and (g) p-PLB expression levels (n = 3 cells per group). Data are presented as the mean ± SD. Statistical significance was determined by one-way ANOVA. ^∗P < 0.05 and ^∗∗P < 0.01 vs. the control group. ^#P < 0.05 and ^##P < 0.01 vs. the Ang II group. ^▲P < 0.05 and ^▲▲P < 0.01; the control group in SiCaMKII or CsA vs. the control group in normal. ^△P < 0.05 and ^△△P < 0.01; the Ang II group in SiCaMKII or CsA vs. the Ang II group in normal.

Figure 6

Effects of siCaMKII and CsA on Ang II-induced activation of CnA-NAFT signaling in H9c2 cells. (a) Representative western blotting images. Densitometric analysis of (b) CnA, (c) p-CnA, (d) NFATc4, (e) p-NFATc4, (f) GATA4, (g) p-GATA4, (h) ANP, and (i) BNP expression levels (n = 3 cells per group). Data are presented as the mean ± SD. Statistical significance was determined by one-way ANOVA. ^∗P < 0.05 and ^∗∗P < 0.01 vs. the control group. ^#P < 0.05 and ^##P < 0.01 vs. the Ang II group. ^▲P < 0.05 and ^▲▲P < 0.01; the control group in SiCaMKII or CsA vs. the control group in normal. ^△P < 0.05 and ^△△P < 0.01; the Ang II group in SiCaMKII or CsA vs. the Ang II group in normal.

Figure 7

Ang II-induced NFATc4 nuclear translocation is inhibited by siCaMKII. H9c2 cells were treated with Ang II (10⁻⁷ M) for 48 h. CaMKII was silenced or CsA (10⁻⁶ M) was added to the culture medium prior to Ang II administration. WXKL (5 g/L) was added for 24 h after Ang II administration. The control cells received no treatment. (a) Representative images of immunofluorescence staining of NFATc4. Blue: DAPI staining; red: NFATc4. Scale bar: 50 μm. Yellow arrow: nuclear translocation; white arrow: no nuclear translocation or nuclear translocation was decreased. (b) Semiquantitative analysis of NFATc4 nuclear translocation. Data are presented as the mean ± SD. Statistical significance was determined by one-way ANOVA. ^∗P < 0.05 and ^∗∗P < 0.01 vs. the control group. ^#P < 0.05 and ^##P < 0.01 vs. the Ang II group. ^▲P < 0.05 and ^▲▲P < 0.01; the control group in SiCaMKII or CsA vs. the control group in normal. ^△P < 0.05 and ^△△P < 0.01; the Ang II group in SiCaMKII or CsA vs. the Ang II group in normal.

Figure 8

Effects of siCaMKII and CsA on Ang II-induced activation of the inflammatory signal transduction pathway in H9c2 cells. (a) Representative western blotting images. Densitometric analysis of (b) MyD88, (c) NF-κB, (d) p-NF-κB, (e) TLR2, and (f) TLR4 expression levels (n = 3 cells per group). Data are presented as the mean ± SD. Statistical significance was determined by one-way ANOVA. ^∗P < 0.05 and ^∗∗P < 0.01 vs. the control group. ^#P < 0.05 and ^##P < 0.01 vs. the Ang II group. ^▲P < 0.05 and ^▲▲P < 0.01; the control group in SiCaMKII or CsA vs. the control group in normal. ^△P < 0.05 and ^△△P < 0.01; the Ang II group in SiCaMKII or CsA vs. the Ang II group in normal.

Figure 9

Ca²⁺-dependent signaling pathways in cardiac hypertrophy. Upon activation by CaM, CaN dephosphorylates cytoplasmic NFAT, a known hypertrophic transcription factor, and promotes its translocation to the nucleus and subsequent transcriptional activity. After silencing CaMKII, protein expression in the CaMKII and CaN-NFAT signaling pathways was inhibited, and the nuclear transfer of NFATc4 was decreased. CsA, an inhibitor of CaN, inhibits the expression in the CaN-NFAT pathway but activates the CaMKII signaling pathway, MyD88 inflammatory pathway, and I_Ca-L. WXKL improves CH through the CaMKII and CaN-NFAT signaling pathways. CaM: calmodulin; CaN: calcineurin; NFAT: nuclear factor of activated T cells; CaMKII: Ca²⁺/calmodulin-dependent protein kinase II; L-type Ca²⁺ current: I_Ca-L; CsA: cyclosporine A; WXKL: Wenxin Keli; CH: cardiac hypertrophy.