Abstract 12704: Activation of the Valosin-Containing Protein by H11 Kinase/Hsp22 Promotes Cardiac Cell Survival
The small heat shock protein H11 kinase/Hsp22 (Hsp22) stimulates cardiac cell survival through activation of the Akt pathway and expression of the inducible nitric oxide (NO) synthase (iNOS), resulting in cardioprotection equivalent to that provided by ischemic preconditioning. Our goal was to elucidate the downstream effector by which Hsp22 and Akt increase iNOS expression. We tested the hypothesis that such molecular link is the valosin-containing protein (VCP), an Akt substrate, which activates the transcription factor NF-κB by promoting the poly-ubiquitination of the NF-κB inhibitor, IκBα. Using two-dimensional gel electrophoresis and mass spectrometry, we found that VCP abundance is increased by 3-fold (P<0.05) in hearts from transgenic mice with cardiac-specific over-expression (5-fold) of Hsp22 as compared to wild type. VCP expression predominated in the nuclear fraction of adult heart, where it co-precipitated with both Hsp22 and Akt. To reproduce this increased abundance, adeno-mediated over-expression of VCP (up to 6-fold) was performed in isolated cardiac myocytes. VCP dose-dependently increased the NF-κB DNA binding activity by up to 4-fold (P<0.01), the nuclear translocation of NF-κB, and the expression of iNOS by up to 10-fold (P<0.01), which was abolished by the NF-κB inhibitor SN50. Over-expression (10-fold) of a dominant-negative mutant of VCP (VCP DN) lacking the N terminal domain of poly-ubiquitination did not activate NF-κB and did not increase iNOS expression. Addition of chelerythrine to cardiomyocytes increased apoptosis (as measured by TUNEL) by 3-fold in β-Gal control (P<0.05 vs vehicle), but such increase was totally suppressed upon over-expression of VCP. This protective effect of VCP was abolished upon addition of SN50 (2.7-fold increase in apoptosis with chelerythrine compared to vehicle, NS vs β-Gal), or of the NOS inhibitor L-NMMA (2.8-fold increase, NS vs β-Gal). Chelerythrine-mediated apoptosis was not significantly decreased upon over-expression of VCP DN compared to control (2.8-fold increase, NS vs β-Gal). We conclude that Hsp22 increases the expression of VCP, which mediates iNOS induction through an NF-κB-dependent mechanism, resulting in improved cardiac cell defense against apoptosis.
- © 2010 by American Heart Association, Inc.