Abstract 3296: shRNA Knockdown of Diacylglycerol Kinase Zeta Abolishes the Anti-Hypertrophic Actions of PPAR Ligands on Cardiac Myocytes
Activation of peroxisome proliferator-activated receptors (PPARs) prevents cardiac hypertrophy, but by unknown signaling pathways. Three isoforms of diacylglycerol kinase (DGK), which phosphorylates diacylglycerol to modulate protein kinase C and G-protein signaling, are found in cardiac myocytes: α, ϵ, and ζ. The predominant isoform in myocytes, DGKζ, negatively regulates hypertrophy. Since we found that
a non-selective inhibitor of DGK blocked the antihypertrophic actions of PPARs, and
PPAR ligands upregulate DGKζ levels, we hypothesized that specifically the DGKζ isoform mediates the antihypertrophic actions of PPARs.
Using neonatal rat cardiac myocytes stimulated with endothelin-1 (ET1), we examined 2 parameters of hypertrophy: myocyte size and hypertrophic gene activation. The ability of ET1 (0.1 μM) to increase myocyte size (130±4% vs control; n=6, p<0.01) was blocked by ligands of PPARγ (troglitazone, 1 μM; 112±7% vs control; p=NS) or PPARα (fenofibrate, 10 μM; 115±4% vs control; p=NS). shRNA knockdown of DGKζ abolished the inhibitory effects of PPARs to restore ET1-induced hypertrophy, even in the presence of troglitazone (169±9% vs control; p<0.01) and fenofibrate (161±9% vs control; p<0.01) (n=3). We next queried the role of DGKζ in PPAR-mediated suppression of hypertrophic genes by real-time PCR. Troglitazone and fenofibrate blocked ET1-induced activation of 3 fetal genes (brain natriuretic peptide, β-myosin heavy chain and skeletal muscle α-actin), and cardiac muscle α-actin (see table⇓). DGKζ knockdown restored hypertrophic gene expression, even in the presence of the PPAR ligands. Since the inhibitory effects of PPAR ligands on myocyte growth and hypertrophic gene expression are abolished by shRNA knockdown of DGKζ, we conclude that the ability of PPARs to prevent hypertrophy requires DGKζ..