ANP exposure exerts concentration-dependent antiapoptotic effects upon cardiomyocytes and is protective in vivo. (A) Cardiomyocytes were plated onto chamber slides and preincubated with or without ANP (concentration range from 10^–10.5 M to 10^–6 M as indicated for 1 hour) followed by 2 hours of vehicle only (–) or staurosporine treatment (+; 1 μM). Apoptosis was assessed by TUNEL assay. (B) Apoptotic signaling in cultured cardiomyocytes evaluated by cleaved caspase-3. Quantitation of caspase-3 immunoblot was performed by densitometric analysis using GAPDH as a loading standard to correct for slight differences in protein loading. ANP treatment protocol was the same as in A. (C) Cardiomyocytes were treated with ANP for 24 hours prior to 2 hours apoptotic stimulus with staurosporine. Apoptosis was assessed by TUNEL assay. (D) Circulating serum levels of ANP from mice implanted with osmotic pumps as determined by ELISA assay (see Methods; n = 4 for each group). (E–H) Analyses of hearts from mice implanted with osmotic pumps (saline control versus ANP treated) or genetically engineered to express nuclear-targeted zyxin (nontransgenic [NTg] control versus nuclear-targeted zyxin [zyxin-n.t.]) as described in Methods. (E) Representative confocal micrographs of TUNEL assays performed on myocardial sections of hearts subjected to ischemia/reperfusion damage. Scale bars: 10 μm. (F) Quantitation of TUNEL-positive nuclei from myocardial sections of mice as indicated. (G–H) Recovery of hemodynamic function during reperfusion phase for mouse groups receiving osmotic pump implants (G) or genetically engineered to express nuclear targeted zyxin (H). *P < 0.05 or **P < 0.01 for each indicated comparison in A–H. n = 3 for experiments in A–C; n = 4 for experiments in D–H. LVDP, left ventricular developed pressure.