Elsevier

Vascular Pharmacology

Volume 58, Issue 4, April 2013, Pages 326-333
Vascular Pharmacology

Murine atrial HL-1 cell line is a reliable model to study drug metabolizing enzymes in the heart

https://doi.org/10.1016/j.vph.2012.12.002Get rights and content

Abstract

HL-1 cells are currently the only cells that spontaneously contract while maintaining a differentiated cardiac phenotype. Thus, our objective was to examine murine HL-1 cells as a new in vitro model to study drug metabolizing enzymes. We examined the expression of cytochrome P450s (Cyps), phase II enzymes, and nuclear receptors and compared their levels to mice hearts. Our results demonstrated that except for Cyp4a12 and Cyp4a14 all Cyps, phase II enzymes: glutathione-S-transferases (Gsts), heme oxygenase-1 (HO-1), and NAD(P)H: quinone oxidoreductase (Nqo1), nuclear receptors: aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), and peroxisome proliferator activated receptor (PPAR-alpha) were all constitutively expressed in HL-1 cells. Cyp2b19, Cyp2c29, Cyp2c38, Cyp2c40, and Cyp4f16 mRNA levels were higher in HL-1 cells compared to mice hearts. Cyp2b9, Cyp2c44, Cyp2j9, Cyp2j11, Cyp2j13, Cyp4f13, Cyp4f15 mRNA levels were expressed to the same extent to that of mice hearts. Cyp1a1, Cyp1a2, Cyp1b1, Cyp2b10, Cyp2d10, Cyp2d22, Cyp2e1, Cyp2j5, Cyp2j6, Cyp3a11, Cyp4a10, and Cyp4f18 mRNA levels were lower in HL-1 cells compared to mice hearts. Moreover, 3-methylcholanthrene induced Cyp1a1 while fenofibrate induced Cyp2j9 and Cyp4f13 mRNA levels in HL-1 cells. Examining the metabolism of arachidonic acid (AA) by HL-1 cells, our results demonstrated that HL-1 cells metabolize AA to epoxyeicosatrienoic acids, dihydroxyeicosatrienoic acids, and 20-hydroxyeicosatetraenoic acids. In conclusion, HL-1 cells provide a valuable in vitro model to study the role of Cyps and their associated AA metabolites in addition to phase II enzymes in cardiovascular disease states.

Introduction

Cardiovascular diseases (CVD) are the leading cause of death worldwide; every 36 seconds 1 person dies from CVD in the United States. Moreover, CVD exceeds the next four leading causes of death (cancer, lung disease, accidents and diabetes) combined (DiPiro, 2008). Recently, it was reported that there is a strong correlation between arachidonic acid (AA) metabolism and the pathogenesis of many CVD (Roman, 2002). It has long been recognized that cyclooxygenase (COX) and lipoxygenase (LOX) enzymes metabolize AA; however, a third pathway has been identified for AA metabolism, this pathway is the cytochrome P450 (Cyp) pathway (Capdevila et al., 1981). The Cyp pathway is further divided into Cyp epoxygenases and Cyp hydroxylases sub-pathways (Roman, 2002), Cyp epoxygenases metabolize AA to form epoxyeicosatrienoic acids (EETs), while Cyp hydroxylases form hydroxyeicosatetraenoic acids (HETEs). Now it is well established that, both EETs and HETEs play critical roles in cardiovascular system (Roman, 2002); EETs produce important biological effects such as regulating vascular tone in the coronary, cerebral, mesenteric, renal, pulmonary, and peripheral circulations, besides its anti-inflammatory activities (Anwar-mohamed et al., 2010). In contrast to EETs, 20-HETE is involved in many cardiovascular diseases including cardiac hypertrophy and heart failure (Anwar-mohamed et al., 2010).

All studies examining the role of drug metabolizing enzymes in the heart use either in vivo models such as mice or rats (Imaoka et al., 2005, Zordoky et al., 2008), or in vitro systems such as isolated neonatal and adult cardiomyocytes (Lee et al., 2004, Thum and Borlak, 2000a) or immortalized cell lines such as H9C2 cells (Zordoky and El-Kadi, 2007). However, each model has its limitations and drawbacks. For example, in vivo experiments are expensive and require multiple approvals due to ethical concerns (Festing and Wilkinson, 2007). Isolation of adult cardiomyocytes is a difficult technique as heart muscle cells are firmly connected to each other and it is difficult to cleave these connections without injuring the cells (Schlüter and Piper, 2005). Neonatal cardiomyocytes lack many characteristics of adult cardiomyocytes and are usually overgrown by nonmyocytes after a few days in culture (White et al., 2004). Finally the H9C2 cell line was originally derived from embryonic rat ventricular tissue which is no longer able to beat (Watkins et al., 2011).

Therefore, there is an urgent need for a reliable in vitro cell line model to study the role of drug metabolizing enzymes in the heart. Currently, the murine HL-1 cell line, from C57BL/6 mouse, is the only established immortalized cardiac cells that continuously divide and spontaneously contracts while maintaining a differentiated cardiac phenotype (Claycomb et al., 1998). However, to the best of our knowledge, there has been no previous attempt to investigate the expression of different Cyp isoenzymes in HL-1 cells. Therefore, we hypothesize that HL-1 cell line may express different Cyp isoenzymes and therefore could be a good in vitro model to study drug metabolizing enzymes and their role in cardiac diseases. Thus, the present study aims to investigate the expression of Cyp isoenzymes in HL-1 cells and correlate this expression to in vivo model.

Section snippets

Cell culture

HL-1 cells were cultured as previously described (Andersen et al., 2009, Ikeda et al., 2009) with slight modification. Briefly, cells were plated in 6-well culture plates pre-coated with 0.02% Bacto gelatin (Becton, Dickinson, and Company, Franklin Lakes, NJ, USA) and 5  μg/ml fibronectin (Sigma–Aldrich Inc., St Louis, MO, USA) and cultured in Claycomb Medium (Sigma–Aldrich Inc.) supplemented with 10% fetal bovine serum (FBS, Sigma–Aldrich Inc.), 0.1  mM norepinephrine (Sigma–Aldrich Inc.), 2 mM l

Expression level of Cyps, phase II metabolizing enzymes, nuclear receptors, and cardiac hypertrophic markers mRNA levels in HL-1 cells and C57BL/6 mice hearts

In the present study we showed that Cyp1, 2, 3 and 4 families are constitutively expressed in HL-1 cells as well as in C57BL/6 mice hearts. Levels of expression were determined based on criteria mentioned in Table 2. Cyp2d22, Cyp2j6, Cyp2j9, Cyp2j13, Cyp4f13, and Cyp4f16 were found to be highly expressed in both HL-1 cells and C57BL/6 mice hearts. Cyp2c29 was highly expressed in HL-1 cells, whereas, Cyp1a1, Cyp2b10, Cyp2e1, Cyp2j5, Cyp4a10, and Cyp4f18 were highly expressed in C57BL/6 mice

Discussion

HL-1 cell line was employed to determine whether it will serve as a reliable in vitro model to study the drug metabolizing enzymes in the heart. Biotransformation of endogenous and exogenous substances is performed via phases I and phase II metabolizing enzymes (Nebert and Dalton, 2006). In general, transcriptional activation of most Cyps occurs through four main nuclear receptor mechanisms: the AhR for Cyp1 family, the CAR for the Cyp2 family; the PXR for the Cyp3 family; and PPAR for Cyp4

Acknowledgments

This work was supported by a grant from the Canadian Institutes of Health Research [Grant 106665] to A.O.S.E. O.H.E. is the recipient of University of Alberta Doctoral Recruitment Scholarship. A.A-M. is the recipient of Alberta Innovates Technology Futures Scholarship and Izaak Walton Killam Memorial Graduate Scholarship. We are grateful to Dr. John M. Seubert and Dr. Victor Samokhvalov for helping us with HL-1 cell culture.

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