Abstract
Left ventricular diastolic dysfunction (LVDD) is characterized by the disturbance of ventricle’s performance due to its abnormal relaxation or to its increased stiffness during the diastolic phase. The molecular mechanisms underlying LVDD remain unknown. We aimed to identify normalization genes for accurate gene-expression analysis of LVDD using quantitative real-time PCR (RT-PCR) in a new rabbit model of LVDD. Eighteen rabbits were fed with a normal diet (n = 7) or a 0.5% cholesterol-enriched diet supplemented with vitamin D2 (n = 11) for an average of 14.5 weeks. We validated the presence of LVDD in this model using echocardiography for diastolic function assessment. RT-PCR was performed using cDNA derived from left ventricle samples to measure the stability of 10 genes as candidate reference genes (Gapdh, Hprt1, Ppia, Sdha, Rpl5, Actb, Eef1e1, Ywhaz, Pgk1, and G6pd). Using geNorm analysis, we report that Sdha, Gapdh and Hprt1 genes had the highest stability (M <0.2). By contrast, Hprt1 and Rpl5 genes were found to represent the best combination for normalization when using the Normfinder algorithm (stability value of 0.042). Comparison of both normalization strategies highlighted an increase of natriuretic peptides (Bnp and Anp), monocytes chemotactic protein-1 (Mcp-1) and NADPH oxidase subunit (Nox-2) mRNA expressions in ventricle samples of the hypercholesterolemic rabbits compared to controls (P<0.05). This increase correlates with LVDD echocardiographic parameters and most importantly it molecularly validates the presence of the disease in our model. This is the first study emphasizing the selection of stable reference genes for RT-PCR normalization in a rabbit model of LVDD.
Highlights
Left ventricular diastolic dysfunction (LVDD) accounts for almost half of the cases of heart failure in clinical practice [1]
Given our prior experience with a hypercholesterolemic rabbit model that develops aortic valve stenosis [26], and since LVDD is often associated with aortic valve stenosis, we performed a detailed characterization of diastolic heart function in this animal model using echocardiography
We report a significant decrease in E wave deceleration time and decreased values of left and right pulmonary vein A-atrial flow (Ar) durations in the hypercholesterolemic group at these two time-points compared with the normal group (Figure S1 in File S1)
Summary
Left ventricular diastolic dysfunction (LVDD) accounts for almost half of the cases of heart failure in clinical practice [1] It is characterized by abnormal left ventricle (LV) relaxation or reduced ventricular compliance, which leads to increased filling pressures [2]. Large animal models have been described for LVDD [16], only a limited number of models of experimental LVDD have been described in rabbits Among these models, the development of LVDD with age or hypercholesterolemia in rabbits has been shown to be similar to the changes seen in PLOS ONE | www.plosone.org qPCR Reference Genes for a New Rabbit LVDD Model humans [4,5,17], making rabbits a valid model to study the mechanisms underlying this pathology
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
