ANP induces nuclear accumulation of zyxin and phospho-Akt in the myocardium. Myocardial sections (A–JJ) and immunoblot (KK) demonstrating nuclear accumulation of zyxin or phospho-Akt⁴⁷³. All ANP treatments were performed for 24 hours using an implanted osmotic pump with 200 μl of 10^–4 M ANP. For all cGMP treatments, agonist at a concentration of 10–² M was administered intravenously in 100 μl volume, and hearts were removed 5 hours later. (A–P) Control (A–D and I–L) or experimental mice (E–H and M–P) labeled with antibody to zyxin (A, E, I, and M; green in overlay). Cardiomyocytes were identified with antibodies to α-actinin (C and G) or desmin (K and O) (both blue in overlay). Nuclei were stained with TOPRO (B, F, J, and N; red in overlay). Nuclear accumulation of zyxin was observed either following ANP treatment or in transgenic mice expressing cardiac-specific GC (GC Tg). (Q–JJ) Control (Q–T and CC–FF) or experimental (U–BB and GG–JJ) mouse hearts labeled with antibody to phospho-Akt⁴⁷³ (Q, U, Y, CC, GG; yellow in overlay in panels X, BB, and JJ is due to coincident nuclear staining). Cardiomyocytes were identified with staining for desmin (S, W, AA, EE, and II; blue in overlays), and nuclei were identified by staining with TOPRO (R, V, Z, DD, and HH; red in overlays). Nuclear accumulation of phospho-Akt⁴⁷³ was observed following cGMP treatment (U–X; 5 hr intravenous), ANP treatment (Y–BB), andn in the cardiac-specific GC Tg mouse hearts (GG–JJ). (KK) Immunoblot of cardiac protein lysates from fractions of control or ANP-treated mice partitioned into nuclear and cytoplasmic/membrane fractions. Enrichment of zyxin or phospho-Akt⁴⁷³ was observed in samples from ANP-treated mice. Enrichment for nuclei was demonstrated by partitioning of GAPDH (cytoplasmic), histone H3 (nuclear), and connexin 43 (membrane associated) to appropriate fractions. Appropriate mobilities of bands on blots were confirmed using molecular weight standards in combination with labeling of unfractionated lysate (not shown). Scale bars: 10 μm.