P5437The BNP reporter mouse by knock-in technology is useful for the analysis of mechanism in reactivation of BNP in adult heart

Abstract Background It has been thought BNP is induced by undetermined stretch-activated receptors, however, which receptor is associated remains unknown. The stretch-activated receptors include mechanically gated channels, which can be activated by a mechanical stimulus alone, and mechanically modulated channels, which require nonmechanical stimuli such as agonists. It has been recently shown that 1.1kb segment of mouse NPPB promoter dose not reproduce the pattern of reactivation of BNP in adult heart, although it could monitor the expression of BNP in neonatal cardiomyocytes. Purpose Our aim is to develop a true BNP reporter mouse and examine whether this mouse is useful or not for the investigation of BNP reactivation mechanism in adult heart and for the measurement of serum-induced BNP expression in patients with heart failure. Methods We generated the BNP reporter mice by knocking luciferase cDNA in the initiation site of NPPB. In vivo imaging of luciferase was performed in the BNP reporter mice after the intraperitoneal injection of luciferin. The luciferase activity was examined in neonatal cardiomyocyte, isolated adult cardiomyocytes, adult cardiac dissected tissue with or without 120–150% stretch or angiotensin II stimulation. Left anterior descending (LAD) coronary artery was ligated to study myocardial infarction. Cardiac dissected tissue segments from the BNP reporter mouse were incubated for 8 hours with 20% serum from patients with or without heart failure and the luciferase activity was measured after homogenization. Results The in vivo imaging system showed the activity of BNP was high in 1 day-old neonates and the reactivation of BNP in the adult heart after LAD ligation could be monitored by the luciferase activity (figure). The treatment of Ang II could increase the activity of pBNP more than ten folds in heart tissue from adult mice. On the other hand, the 120–150% stretch did not show any effect on the activity of pBNP in this system. We could not observe any activation of pBNP in cultured neonatal or adult cardiomyocytes demonstrated by immunostain with antibodies against luciferase after 120–150% stretch. Interestingly, the luciferase activity was extensively higher in cultured heart tissue segments from the BNP reporter mice after the treatment of serum from patients with heart failure than without heart failure. In vivo imaging of BNP reporter mice Conclusion These results indicate the BNP reporter mouse by knock-in technology is u