Pharmacological regulation of cytochrome P450 metabolites of arachidonic acid attenuates cardiac injury in diabetic rats.

Diabetic cardiomyopathy (DCM) is a well-established complication of type 1 and type 2 diabetes associated with a high rate of morbidity and mortality. DCM is diagnosed at advanced and irreversible stages. Therefore, it is of utmost need to identify novel mechanistic pathways involved at early stages to prevent or reverse the development of DCM. In vivo experiments were performed on type 1 diabetic rats (T1DM). Functional and structural studies of the heart were executed and correlated with mechanistic assessments exploring the role of cytochromes P450 metabolites, the 20-hydroxyeicosatetraenoic acids (20-HETEs) and epoxyeicosatrienoic acids (EETs), and their crosstalk with other homeostatic signaling molecules. Our data displays that hyperglycemia results in CYP4A upregulation and CYP2C11 downregulation in the left ventricles (LV) of T1DM rats, paralleled by a differential alteration in their metabolites 20-HETEs (increased) and EETs (decreased). These changes are concomitant with reductions in cardiac outputs, LV hypertrophy, fibrosis, and increased activation of cardiac fetal and hypertrophic genes. Besides, pro-fibrotic cytokine TGF-ß overexpression and NADPH (Nox4) dependent-ROS overproduction are also correlated with the observed cardiac functional and structural modifications. Of interest, these observations are attenuated when T1DM rats are treated with 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA), which blocks EETs metabolism, or N-hydroxy-N'-(4-butyl-2-methylphenol)Formamidine (HET0016), which inhibits 20-HETEs formation. Taken together, our findings confer pioneering evidence about a potential interplay between CYP450-derived metabolites and Nox4/TGF-ß axis leading to DCM. Pharmacologic interventions targeting the inhibition of 20-HETEs synthesis or the activation of EETs synthesis may offer novel therapeutic approaches to treat DCM.

Region of interest methylation analysis: a comparison of MSP with MS-HRM and direct BSP.

The aim of this study was to compare and contrast three DNA methylation methods of a specific region of interest (ROI): methylation-specific PCR (MSP), methylation-sensitive high resolution melting (MS-HRM) and direct bisulfite sequencing (BSP). The methylation of a CpG area in the promoter region of Estrogen receptor alpha (ESR1) was evaluated by these three methods with samples and standards of different methylation percentages. MSP data were neither reproducible nor sensitive, and the assay was not specific due to non-specific binding of primers. MS-HRM was highly reproducible and a step forward into categorizing the methylation status of the samples as percent ranges. Direct BSP was the most informative method regarding methylation percentage of each CpG site. Though not perfect, it was reproducible and sensitive. We recommend the use of either method depending on the research question and target amplicon, and provided that the designed primers and expected amplicons are within recommendations. If the research question targets a limited number of CpG sites and simple yes/no results are enough, MSP may be attempted. For short amplicons that are crowded with CpG sites and of single melting domain, MS-HRM may be the method of choice though it only indicates the overall methylation percentage of the entire amplicon. Although the assay is highly reproducible, being semi-quantitative makes it of lesser interest to study ROI methylation of samples with little methylation differences. Direct BSP is a step forward as it gives information about the methylation percentage at each CpG site.