Figure 6.
Role of NMT2 in Ang II–Induced Pathological Hypertrophy
(A) Representative immunofluorescence images for localization of MARCKS in H9c2 myocytes. Twenty-four hours after transfection with siCTRL or siNMT2, HA-MARCKSWT was transfected and stained for anti-Na, K-ATPase (red), and anti-HA (green) antibodies with DAPI (blue). Images in boxed areas at higher magnification are shown in lower panels. Scale bars = 5 μm. (B) Immunofluorescence images for the assessment of myocyte hypertrophy altered by the inhibition of NMT2. Transfected H9c2 myocytes with siCTRL or siNMT2 were stimulated with vehicle or Ang II (1 μmol/L) for 24 hours and stained with phalloidin (green) and DAPI (blue). Scale bars = 5 μm. (C) Quantitative analysis of the cell surface area determined by phalloidin staining. More than 100 cells were counted. Data are expressed as a relative ratio over siCTRL with vehicle from 3 independent experiments. (D) Messenger RNA expression levels in Nppa, Nppb, and Myh7. The data were normalized to Actb levels (n = 4 in each). (E) Western blot analysis on HDAC4 phosphorylation and histone H3 acetylation. Quantitative analyses of the ratios of phosphorylated HDAC4 (p-HDAC4) to total HDAC4 and acetylated H3 (ac-H3) to total H3 are shown in the graphs (n = 3 in each). (F) Involvement of CaMKII-related signaling in Ang II–induced hypertrophic response in NMT2-knockdown H9c2 myocytes. siRNA-transfected cells were incubated with CaMKII inhibitor (KN-93, 5 μmol/L) or vehicle for 1 hour before Ang II (1 μmol/L) stimulation. Twenty-four hours after Ang II, cell surface area was assessed by phalloidin (green) and DAPI (blue) staining. Scale bar = 5 μm. (G) Quantitative analysis of the cell surface area determined by phalloidin staining. Data are expressed as a relative ratio over siCTRL with vehicle from 5 independent experiments. (H) mRNA expression levels in Nppa, Nppb, and Myh7. The data were normalized to Actb levels (n = 9 in each). (I) Immunoblot analysis for phosphorylation of CaMKII and HDAC4 and acetylation of histone H3 (n = 6 in each). Data are expressed as a relative ratio over vehicle-treated siCTRL group. (J) Assessment of hypertrophic responses with overexpression of NMT2 by immunofluorescence. Transfected H9c2 myocytes with empty vector or FLAG-NMT2 were stimulated with vehicle or Ang II (1 μmol/L) for 24 hours and stained with phalloidin (green) and DAPI (blue). Scale bar = 5 μm. (K) Quantitative analysis of the cell surface area determined by phalloidin staining. Data are expressed as a relative ratio over empty vector with vehicle from 3 independent experiments. (L) Messenger RNA expression levels in Nppa, Nppb, and Myh7. The data were normalized to Actb levels (n = 4-5 in each). (M) Western blot analysis on HDAC4 phosphorylation and histone H3 acetylation (n = 3 in each). All data are presented as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 by 1-way analysis of variance with Tukey’s post hoc analysis. Abbreviations as in Figures 1, 2, 3, 4, and 5.