Abstract 19320: Protein Kinase G Oxidation Blunts Its Suppression of Cardiac Hypertrophy by Diminishing Phosphorylation of Tuberin and Consequent Autophagy
Stimulation of protein kinase G (PKG) suppresses maladaptive cardiac hypertrophy in part by blunting signaling cascades such as calcineurin/NFAT (Cn) and calcium-calmodulin dependent kinase II (CamKII). Here we report PKG stimulation also enhances autophagy as an anti-hypertrophic mechanism, identify a novel phosphorylation target for this effect, and show this signaling is blunted when PKG becomes oxidized at cysteine 42 (C42). Mice with knock-in expression of mutant PKG (C42S) or littermate controls were subjected to pressure-overload. C42S mice were protected, with reduced hypertrophy and fibrosis, improved function, and blunted Cn, CamKII, and hypertrophic gene activation. Neonatal rat ventricular myocytes (NRVMs) expressing C42S-PKG exposed to 48hr endothelin-1 (ET1) also displayed less hypertrophic responses. In both C42S mice and NRVMs expressing C42S, autophagic flux was increased over WT, determined by autophagic flux assays, which was associated with inhibited mammalian target of rapamycin (mTOR) signaling. Control mice improved responses similar to C42S mice if autophagic flux was stimulated by inhibiting mTOR with everolimus. Analogous findings were obtained by mTOR suppression in NRVMs. To determine how PKG activation influences mTOR-regulated autophagy, we performed unbiased phospho-proteome analysis using mass spectrometry that revealed a unique PKG targeted site on tuberin (TSC2) at S1343. To test the functional importance of this site, we expressed a mutated TSC2, serine to glutamic acid (phospho-mimetic, S1343E), which depressed mTOR signaling, increased autophagic flux, and blunted hypertrophic signaling and cell enlargement stimulated by ET1 in NRVMs. By contrast, the cardio-protection elicited by PKG C42S was lost when TSC2 or a downstream critical autophagy protein, ATG5 were silenced. Taken together, these results provide the first evidence that PKG activation augments autophagy via targeting of TSC2 as a mechanism of anti-hypertrophic signaling, and that this occurs much more when PKG is in a reduced state (e.g. no C42-disulfide). Loss of this mechanism in myocardium under oxidative stress may play a role in the decline in protection from PKG in cardiac disease which often involves an oxidative environment.
Author Disclosures: M.J. Ranek: None. T. Nakamura: None. R. Holewinski: None. D. Lee: None. G. Zhu: None. K. Chakir: None. P. Eaton: None. J. Van Eyk: None. D.A. Kass: None.
This research has received full or partial funding support from the American Heart Association.
- © 2014 by American Heart Association, Inc.