The human CYP2J2 cDNA has been cloned, expressed in Sf9 insect cells, and extensively characterized with respect to its tissue distribution, cellular localization and function. This enzyme appears to be the major human P450 expressed in heart, where it is highly localized to cardiac myocytes and endothelial cells, and highly active in the metabolism of arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs) and 19-hydroxyeicosatetraenoic acid (19-HETE). Interestingly, one of the EETs (11,12-EET) improves cardiac contractility following prolonged global ischemia, causes hyperpolarization of the resting membrane potential and shortening of the cardiac action potential, and markedly inhibits cardiac L-type Ca++ channel activity. The human CYP2J2 gene has been cloned, characterized with respect to its intron/exon organization, and mapped to Chromosome 1p31.3-31.2 in close proximity to the CYP4A and CYP4B loci. The CYP2J2 gene appears to be regulated, in part, by alternative splicing at the exon 1/intron 1 junction. Two alternative splice variants (CYP2J2-H2 and CYP2J2-H5) have been cloned and shown to be expressed in multiple tissues including the heart by RT-PCR and protein immunoblotting. We have also identified a polymorphic variant of the CYP2J2 gene that results in a T to A substitution at position 143 within the C-helix in close proximity to one of the putative P450 substrate recognition sites. These and other CYP2J2 variants will be identified and functionally characterized as part of the NIEHS Environmental Genome Project. Efforts are currently underway to: (a) construct endothelial and vascular smooth muscle cell lines that stably overexpress CYP2J2 to examine the effects of CYP2J2 products on Ca++ signaling; (b) co-express human CYP2J2 and green fluorescent protein in cultured neonatal rat heart myocytes to examine the role of this enzyme and its products on cardiac myocyte function under basal conditions and during metabolic stress; and (c) construct transgenic mice that overexpress CYP2J2 in heart muscle to examine the effects of CYP2J2-derived eicosanoids on cardiac function in vivo. In related studies, we have identified five novel mouse Cyp2j genes, at least two of which (Cyp2j6 and Cyp2j8) appear to be expressed in mouse heart by RT-PCR analysis. We have cloned the corresponding cDNAs, and are in the process of expressing the recombinant proteins in Sf9 insect cells to examine their AA metabolic activity. Preliminary studies suggest that the CYP2J6 heme-thiolate protein is unstable. This has precluded enzymatic studies and analysis of the distribution of CYP2J6 protein using a peptide-based antibody. We are currently investigating ways to stabilize the recombinant CYP2J6 so that these studies will be possible.
