PALMITATE MEDIATED REPRESSION OF PPAR ACTIVITY AND SERCA2A EXPRESSION IS ASSOCIATED WITH CYTOKINE ACTIVATION IN PRIMARY CARDIOMYOCYTES

Abstract

Patients with diabetes and the metabolic syndrome frequently exhibit hyperlipidemia, which is believed to contribute to cardiac inflammation. Palmitate, one of the most abundant fatty acids in human diets has been shown to induce the expression of inflammatory cytokines in skeletal muscle cells, which may cause skeletal muscle insulin resistance. In addition, it has been proposed that palmitate mediated cytotoxicity is due in part to its inhibitory effects on β-oxidation. Here we were interested in determining if palmitate induces inflammation in primary cardiomyocytes and whether this has any effect on PPAR activity and hence β-oxidation. Rat neonatal cardiomyocytes were treated with palmitate for various time points and at various concentrations. We found that palmitate induced cardiomyocyte inflammation in a dose and time dependent manner as measured by TNF-α and IL-6 mRNA expression by qRT-PCR. PPAR activity was determined by assessing the expression of PPAR target genes (i.e. Acsl, Acadl, Cpt1b, Ucp2). We found that palmitate caused an initial induction of PPAR activity (at 4-8 hours) as evidenced by a significant increase in PPAR target gene expression. However, this was followed by a later repression of PPAR activity with a significant decrease in PPAR target gene expression after 24 hour palmitate exposure (Figure). In contrast, oleate, a non-toxic monounsaturated fatty acid inhibited the expression of pro-inflammatory cytokines. Furthermore oleate also induced PPAR target gene expression at early time points, but in contrast to palmitate, oleate’s effect on PPAR induction persisted after 24 hours and hence showed no signs of later repression. Because previous studies have shown a link between inflammation, PPAR activity, and Serca2a expression and because of the importance of Serca2a in cardiomyocyte physiology and health we evaluated Serca2a mRNA and protein expression. Palmitate induced a significant decrease in both mRNA and protein levels of Serca2a, while oleate had no effect on Serca2 mRNA levels. To evaluate if this translated into changes in Serca2 activity we assessed calcium transients in Fluo4 loaded cardiomyocytes. We found that calcium uptake, an indicator of Serca2a activity, was significantly prolonged in palmitate treated cardiomyocytes. This is the first study to demonstrate palmitate induced inflammation in primary mammalian cardiomyocytes. The resulting inflammation was associated with a late stage repression of PPAR activity and progressive repression of Serca2a expression which can lead to attenuated β-oxidation and calcium uptake, respectively.