Atrial natriuretic peptide promotes cardiomyocyte survival by cGMP-dependent nuclear accumulation of zyxin and Akt
Takahiro Kato, John Muraski, Yan Chen, Yasuyuki Tsujita, Jason Wall, Christopher C. Glembotski, Erik Schaefer, Mary Beckerle, Mark A. Sussman
Takahiro Kato, John Muraski, Yan Chen, Yasuyuki Tsujita, Jason Wall, Christopher C. Glembotski, Erik Schaefer, Mary Beckerle, Mark A. Sussman
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Research Article Immunology

Atrial natriuretic peptide promotes cardiomyocyte survival by cGMP-dependent nuclear accumulation of zyxin and Akt

Abstract

This study delineates a mechanism for antiapoptotic signaling initiated by atrial natriuretic peptide (ANP) stimulation leading to elevation of cGMP levels and subsequent nuclear accumulation of Akt kinase associated with zyxin, a cytoskeletal LIM-domain protein. Nuclear targeting of zyxin induces resistance to cell death coincident with nuclear accumulation of activated Akt. Nuclear translocation of zyxin triggered by cGMP also promotes nuclear Akt accumulation. Additional supportive evidence for nuclear accumulation of zyxin-enhancing cardiomyocyte survival includes the following: (a) promotion of zyxin nuclear localization by cardioprotective stimuli; (b) zyxin association with phospho-Akt473 induced by cardioprotective stimuli; and (c) recruitment of zyxin to the nucleus by activated nuclear-targeted Akt as well as recruitment of Akt by nuclear-targeted zyxin. Nuclear accumulation of zyxin requires both Akt activation and nuclear localization. Potentiation of cell survival is sensitive to stimulation intensity with high-level induction by ANP or cGMP signaling leading to apoptotic cell death rather than enhancing resistance to apoptotic stimuli. Myocardial nuclear accumulation of zyxin and Akt responds similarly in vivo following treatment of mice with ANP or cGMP. Thus, zyxin and activated Akt participate in a cGMP-dependent signaling cascade leading from ANP receptors to nuclear accumulation of both molecules. Nuclear accumulation of zyxin and activated Akt may represent a fundamental mechanism that facilitates nuclear-signal transduction and potentiates cell survival.

Authors

Takahiro Kato, John Muraski, Yan Chen, Yasuyuki Tsujita, Jason Wall, Christopher C. Glembotski, Erik Schaefer, Mary Beckerle, Mark A. Sussman

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Nuclear accumulation of activated Akt kinase in response to ANP treatmen...
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).