Atrial natriuretic peptide promotes cardiomyocyte survival by cGMP-dependent nuclear accumulation of zyxin and Akt
J. Clin. Invest. Takahiro Kato, et al. 115:2716 doi:10.1172/JCI24280 [Go to this article.]

Figure 4
Nuclear accumulation of activated Akt kinase in response to ANP treatment or expression of nuclear-targeted zyxin. Cultured cardiomyocytes (A–Z) or myocardial sections (AA–JJ) showing nuclear accumulation of Akt. Confocal micrographs (A) and immunoblot analysis (B) of myocytes treated with vehicle (control) or ANP (10^–6 M) show nuclear accumulation of both zyxin and phospho-Akt⁴⁷³ (p-Akt) in response to ANP exposure. Overlay channels in A correspond to zyxin (red), phospho-Akt (green), and actin filaments to show myofibril organization (blue). In B, immunoblots for GAPDH, histone H3, and connexin 43 demonstrate subcellular fractionation of the cytosolic, nuclear, and membrane fractions, respectively. Also shown are confocal micrographs of cardiomyocytes labeled with antibodies (shown in red) to either total Akt (C–N) or phospho-Akt⁴⁷³ (O–Z). Cells were infected with adenovirus (shown in green) expressing GFP (K–N and W–Z), nuclear-targeted zyxin (C–F and O–R), or full-length zyxin (G–J and S–V). Myofibrillar organization was observed with phalloidin to label actin filaments (shown in blue). (C–N) Akt immunoreactivity was mainly observed in cytoplasm in cultures expressing full-length zyxin or GFP but accumulated in the nucleus following expression of nuclear-targeted zyxin virus. (O–Z) Phospho-Akt⁴⁷³ immunoreactivity was observed at low levels in cells expressing either full-length zyxin or GFP but accumulated in the nucleus following expression of nuclear-targeted zyxin. Myocardial sections (AA–JJ) show nuclear accumulation of phospho-Akt⁴⁷³ in transgenic mice expressing cardiac-specific nuclear-targeted zyxin (zyxin-n.t. Tg) but not in nontransgenic control samples. Arrows indicate nuclei positive for both zyxin and phospho-Akt⁴⁷³. Transgenic zyxin was visualized using antibody to myc-tag (tag) and desmin to show myofibrillar structure. Overlay colors correspond to phospho-Akt⁴⁷³ (red), myc-tag (green), nuclei (pink), and desmin (blue) with coincidence of red and blue labeling appearing yellow. Scale bars: 20 μm (A and AA–JJ); 30 μm (C–Z).