Abstract 198: Endothelin-1 and Phenylephrine Activate Global Protein Kinase D Similarly in Adult Cardiac Myocytes, but Differ Greatly in Nuclear versus Sarcolemmal Targeting

Abstract

Endothelin-1(ET-1) and phenylephrine (PE) both activate hypertrophic signaling in cardiac myocytes via nuclear export of class II histone deacetylase (HDAC), and protein kinase D (PKD) is critical in mediating this for HDAC5. However, we now find that in adult ventricular myocytes PE-induced HDAC5 nuclear export is more critically PKD-dependent than for ET-1. Here, we use novel FRET-based PKD activity reporter (DKAR) and GFP-tagged PKD (both in adenoviral vectors) expressed in adult rabbit cardiac myocytes, along with confocal and TIRF microcsopy to localize ET-1 vs . PE-induced PKD activation and translocation. On-line PKD activity (sensed by DKAR FRET) was similarly activated by 100 nM ET-1, 10 μM PE or 300 nM PDBu (4.6 ± 0.4, 5.9 ± 0.7 or 5.6 ± 0.6 units) in adult myocytes overexpressing PKD. PKD-GFP expressed in myocytes is relatively uniform and cytosolic at rest (slightly higher at Z-lines), and largely non-nuclear (F nuc /F cyto 0.5 ± 0.03). ET-1 caused rapid sustained PKD recruitment to the plasma membrane (46 ± 3% increase) and also at Z-lines, but only slow and modest PKD nuclear import (12 ± 8% rise in F nuc /F cyto ). In contrast, PE more modestly increased membrane and Z-line recruitment (12 ± 0.9%), but caused more dramatic nuclear translocation (47 ± 4% rise in F nuc /F cyto ). PDBu caused both membrane (120 ± 15% increase) and nuclear localization of PKD (44 ± 4% increase in F nuc /F cyto ). Interestingly, SR Ca depletion by thapsigargin caused an ET-1-like effect, whereas strophantidin did not elicit PKD mobilization. We conclude that ET-1 and PE differ in adult myocyte PKD activation, not in extent, but in cellular localization or translocation. These results are consistent with a more prominent nuclear action of PKD in response to PE vs. ET-1, and with the more critical role of PKD in PE-induced HDAC5 nuclear export.