Abstract 1360: A Potential Role for Nuclear-Delimited Signaling via Nuclear-Envelope Angiotensin Receptors in the Control of Cardiac Gene Expression
Angiotensin-II (A-II) regulates cardiac homeostasis via AT1 (AT1R) and AT2 (AT2R) receptors. A-II mitogenic and growth promoting effects contribute to various cardiac remodeling paradigms, largely by altering gene expression. Here, we assessed the possibility that A-II effects on gene expression may be mediated by intracellular A-II acting on AT1Rs or AT2Rs in the nuclear envelope. Western blots of nucleus-enriched rat heart fractions indicated the presence of AT1R and AT2R proteins that preferentially copurified with a nuclear-membrane marker (Nup 62) but not markers of plasma (Calpactin I), Golgi (GM130) or endoplasmic-reticulum (GRP78) membranes. Confocal immunoflorescent microscopy of adult rat and dog cardiomyocytes revealed AT1R and AT2R proteins on nuclear membranes and T tubules. Microinjection of AngII-Alexa488 into single cardiomyocytes indicated preferential binding of A-II to nuclear vs cytoplasmic sites (197 ± 7* vs 110 ± 7 respectively; *p<0.05). Ca2+ i-recordings on nuclear preparations (Fura-2 AM) revealed AT1R-mediated Ca2+ release (e.g. 17 ± 2 nM control, 264 ± 13 nM* Ang-II and 177±6 nM* for the AT1-selective agonist L-162313). AT1R ligands enhanced de novo RNA synthesis in isolated cardiac nuclei incubated with [α32P]-UTP (eg 45±1 CPM/ng DNA control vs 244±3 CPM/ng DNA* with A-II, N=6/group, P<0.001). NFκB mRNA expression (RT-qPCR, relative to β-actin) was enhanced by A-II or L-162313 applied to isolated cardiac nuclei, a response that was suppressed by co-administration of valsartan (AT1-selective antagonist). Dose-response experiments with A-II applied to purified nuclei vs whole myocytes showed a 1.5-fold* greater increase in NFκB mRNA levels at saturating concentrations with similar EC50 values, suggesting preferential nuclear signaling. Cardiac nuclear membranes possess AT2Rs and AT1Rs that are coupled to nuclear signaling pathways and regulate transcription. Intracellularly synthesized A-II signaling through cardiac nuclear envelopes may be particularly important for A-II regulation of cardiac gene expression, suggesting potential differential actions of A-II from intra- vs extracellular sources, with important mechanistic and therapeutic implications.