Human fetal ventricular cardiomyocyte, RL-14 cell line, is a promising model to study drug metabolizing enzymes and their associated arachidonic acid metabolites

Abstract

Introduction

RL-14 cells, human fetal ventricular cardiomyocytes, are a commercially available cell line that has been established from non-proliferating primary cultures derived from human fetal heart tissue. However, the expression of different drug metabolizing enzymes (DMEs) in RL-14 cells has not been elucidated yet. Therefore, the main objectives of the current work were to investigate the capacity of RL-14 cells to express different cytochrome P450 (CYP) isoenzymes and correlate this expression to primary cardiomyocytes.

Methods

The expression of CYP isoenzymes was determined at mRNA, protein and catalytic activity levels using real time-PCR, Western blot analysis and liquid chromatography-electron spray ionization-mass spectrometry (LC-ESI-MS), respectively.

Results

Our results showed that RL-14 cells constitutively express CYP ω-hydroxylases, CYP1A, 1B, 4A and 4F; CYP epoxygenases, CYP2B, 2C and 2J; in addition to soluble epoxide hydrolayse (EPHX2) at mRNA and protein levels. The basal expression of CYP ω-hydroxylases, epoxygenases and EPHX2 was supported by the ability of RL-14 cells to convert arachidonic acid to its biologically active metabolites, 20-hydroxyeicosatetraenoic acids (20-HETEs), 14,15-epoxyeicosatrienoic acids (14,15-EET), 11,12-EET, 8,9-EET, 5,6-EET, 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), 11,12-DHET, 8,9-DHET and 5,6-DHET. Furthermore, RL-14 cells express CYP epoxygenases and ω-hydroxylase at comparable levels to those expressed in adult and fetal human primary cardiomyocytes cells implying the importance of RL-14 cells as a model for studying DMEs in vitro. Lastly, different CYP families were induced in RL-14 cells using 2,3,7,8-tetrachlorodibenzo-p-dioxin and fenofibrate at mRNA and protein levels.

Discussion

The current study provides the first evidence that RL-14 cells express CYP isoenzymes at comparable levels to those expressed in the primary cells and thus offers a unique in vitro model to study DMEs in the heart.

Introduction

Mounting evidence is shedding the light on the role of cytochrome P450 (CYP) in the pathogenesis of cardiovascular disease (CVD) (Roman, 2002). The CYP are superfamily of cysteinato-heme mixed function mono-oxygenases that are key mediators of the oxidative metabolism of xenobiotics as well as endogenous substances. Although CYP are mainly expressed in the liver, many CYP enzyme families have been extensively reviewed in the extrahepatic tissues such as heart, endothelium and smooth muscle of blood vessels (Elbekai & El-Kadi, 2006).

One of the important physiological roles of CYP enzymes is the metabolism of arachidonic acid (AA) into epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs) which are known to play an important role in the maintenance of cardiovascular health (Zordoky & El-Kadi, 2010). CYP ω-hydroxylases, namely CYP1 and CYP4 families, metabolize AA into 20-HETE whereas, CYP epoxygenases, mainly CYP2B, CYP2C and CYP2J subfamilies, metabolize AA into four regioisomers of EETs, 14,15-EET, 11,12-EET, 8,9-EET and 5,6-EET metabolites (Roman, 2002). EETs are further metabolized by soluble epoxide hydrolase (sEH) into their corresponding degradation products dihydroxyeicosatrienoic acid (DHETs) (Imig, Zhao, Capdevila, Morisseau, & Hammock, 2002).

EETs are the major anti-inflammatory and cardioprotective products of AA metabolism by CYP enzymes. EETs have been identified as potential endothelium-derived hyperpolarizing factors (EDHFs). It has been proposed that diminished production or concentration of EETs contributes to several cardiovascular disorders such hypertension and cardiac hypertrophy (Althurwi et al., 2013, Imig et al., 2002). On the other hand, perturbations in the levels of 20-HETE in tissues and biological fluids have been observed in multiple pathological states, including hypertension, ischemic cerebrovascular diseases, cardiac ischemia–reperfusion injury and isoproterenol and doxorubicin-induced cardiac hypertrophy (Althurwi et al., 2013, Elshenawy, Anwar-Mohamed and El-Kadi, 2013, Fava et al., 2012, Gross et al., 2005, Schwartzman et al., 1996, Wu and Schwartzman, 2011, Yousif et al., 2009, Zordoky et al., 2008, Zordoky et al., 2010).

The role of drug metabolizing enzymes was studied in the heart using either in vivo models such as mice or rats (Imaoka et al., 2005, Zordoky et al., 2008), or in vitro systems such as isolated primary cardiomyocytes (Lee et al., 2004, Thum and Borlak, 2000) or immortalized cell lines such as H9C2 and HL-1 cells (Elshenawy, Anwar-Mohamed, Abdelhamid and El-Kadi, 2013, Zordoky and El-Kadi, 2007). However, each model has its limitations and drawbacks. For example, in vivo experiments are expensive and time consuming. In addition, animal models may not be readily suited to detailed investigations at a cellular and molecular level. Isolation of primary cardiomyocytes is a complicated technique as heart muscle cells are firmly connected to each other and it is hard to cleave these connections without injuring the cells (Schluter & Schreiber, 2005). Moreover, it is difficult to cultivate and maintain these cells in vitro, because of their low yield and limited viability. Rat cardiomyoblast cells, H9c2, and a mouse atrial cardiomyocyte cell line, HL-1, are useful tools for cardiovascular research as they can be passaged serially, differentiate, and maintain the characteristics of rat and mouse cardiomyocytes (Claycomb et al., 1998, Kimes and Brandt, 1976). However, a mouse and rat cell lines cannot answer questions that are specific to the human system.

Therefore, there is an urgent need for a reliable human in vitro cell line model to study the role of drug metabolizing enzymes in the heart. Currently, the human fetal ventricular cardiomyocyte RL-14 cells are a commercially available cell line that has been established from non-proliferating primary cultures derived from human fetal heart tissues (Zhang, Nuglozeh, Toure, Schmidt, & Vunjak-Novakovic, 2009). RL-14 cells proliferate and differentiate in culture and can express the human cardiomyocyte phenotype when culture conditions are manipulated. However, the expression of different CYP isoenzymes in RL-14 cells has not been fully elucidated yet. Accordingly, we hypothesize that RL-14 cell line may express different CYP isoenzymes and offers a unique in vitro model to study drug metabolizing enzymes in the heart. For this purpose, the present study was designed to investigate the capacity of RL-14 cells to express different CYP isoenzymes and correlate this expression to primary cardiomyocyte. Our study provides the first evidence that RL-14 cells express CYP isoenzymes at comparable levels to those expressed in the primary cell line and thus validate RL-14 cells as a reliable model for studying drug metabolizing enzymes in the heart.