S-Enantiomer of 19-Hydroxyeicosatetraenoic Acid Preferentially Protects Against Angiotensin II-Induced Cardiac Hypertrophy

Abstract

We had recently demonstrated that the racemic mixture of 19-hydroxyeicosatetraenoic acid (19-HETE) protects against angiotensin II (Ang II)-induced cardiac hypertrophy. Therefore, the purpose of this study was to investigate whether the orenantiomer of 19-HETE confers cardioprotection against Ang II-induced cellular hypertrophy in RL-14 and H9c2 cells. Both cell lines were treated with vehicle or 10 M Ang II in the absence and presence of 20M 19()HETE or 19()HETE for 24 hours. Thereafter, the level of midchain HETEs was determined using liquid chromatography-mass spectrometry. Gene- and protein-expression levels were measured using real-time polymerase chain reaction and Western blot analysis, respectively. The results showed that both 19()HETE and 19()HETE significantly decreased the metabolite formation rate of midchain HETEs, namely 8-, 9-, 12-, and 15-HETE, compared with control group, whereas the level of 5-HETE was selectively decreased by enantiomer. Moreover, both 19()HETE and 19()HETE significantly inhibited the catalytic activity of CYP1B1 and decreased the protein expression of 5- and 12-lipoxygenase (LOX) as well as cyclo-oxygenase-2 (COX-2). Notably, the decrease in 15-LOX protein expression was only mediated by 19()HETE. Interestingly, both enantiomers protected against Ang II-induced cellular hypertrophy, as evidenced by a significant decrease in mRNA expression of /-myosin heavy chain ratio, atrial natriuretic peptide, and interleukins 6 and 8. Our data demonstrated that enantiomer of 19-HETE preferentially protected against Ang II-induced cellular hypertrophy by decreasing the level of midchain HETEs, inhibiting catalytic activity of CYP1B1, decreasing protein expression of LOX and COX-2 enzymes, and decreasing mRNA expression of IL-6 and IL-8.

Identification of 19-(S/R)Hydroxyeicosatetraenoic Acid as the First Endogenous Noncompetitive Inhibitor of Cytochrome P450 1B1 with Enantioselective Activity

“…The K i values of both enantiomers showed that the S-enantiomer is more potent than the R-enantiomer by approximately 2.4-fold. This comes in agreement with our previous work demonstrating that the S-enantiomer of 19-HETE has a preferential effect over the R-enantiomer in inhibiting the formation of midchain HETEs (Shoieb and El-Kadi, 2018). In accordance with being noncompetitive inhibitors, the in vitro stability study showed that both enantiomers of 19-HETE are metabolically stable in solution during the 30-minute time course of the kinetic experiment as there was no significant difference in the level of both enantiomers in the duration of the experiment (Supplemental Figs.…”

“…1) protect against angiotensin II (Ang II)-induced cardiac hypertrophy with preferential effect of 19(S)-HETE. One important finding of our study was that both enantiomers of 19-HETE inhibited the formation of midchain HETEs and the catalytic activity of CYP1B1 in two different cell lines, human ventricular cardiomyocytes RL-14 cells and rat cardiomyoblasts H9c2 cells (Shoieb and El-Kadi, 2018). To obtain a comprehensive understanding of the protective role of R-and S-enantiomers of 19-HETE in the case of cardiac hypertrophy, we undertook the current study to examine, using 7-ethoxyresorufin O-deethylation (EROD) assay, the inhibitory effect of both enantiomers on human recombinant CYP1B1 enzyme.…”

“…Inhibition of CYP1B1 is proposed as one of the strategies in the treatment of cardiovascular diseases and cancer chemoprevention (Mikstacka et al, 2007). In this regard, we tested the capacity of both enantiomers of 19-HETE to inhibit the catalytic activity of human recombinant CYP1B1, on the basis of our previous finding that 19(R)and 19(S)-HETE protected against Ang II-induced cellular hypertrophy via decreasing the level of cardiotoxic midchain HETEs, the major metabolites of CYP1B1 (Shoieb and El-Kadi, 2018). For that purpose, the inhibition kinetics of both enantiomers of 19-HETE on CYP1B1 activity were carried out using CYP1B1 substrate 7-ER, a compound that is metabolized via oxidative reaction by this enzyme (Shimada et al, 1997).…”

“…The reaction was stopped using 400 ml icecold acetonitrile and the extraction of 19-HETE enantiomers was performed using ethyl acetate and dried using speed vacuum (Savant, Farmingdale, NY). The analysis of 19-HETE enantiomers was performed using liquid chromatographyelectrospray ionization mass spectrometry (LC-ESI-MS) as previously described (Shoieb and El-Kadi, 2018).…”

Overview of the Components of Cardiac Metabolism

“…Neither enantiomer of 19-HETE produced significant effects on mRNA or protein for CYP2B6, CYP2C8, or CYP2J2. The S-enantiomer increased CYP4F2 and CYP4F11 mRNA and protein expression levels (Shoieb and El-Kadi, 2018). The opposite roles of the EETs and HETEs suggest that the ratios between the two classes are important.…”