Impact of normalization strategy on cardiac expression of pro-inflammatory cytokines: Evaluation of reference genes in different human myocardial regions after Left Ventricular Assist Device support

Objective: New device therapies have expanded the strategies for treating heart failure (HF) patients. Unloading of the heart with a left ventricular assist device (LVAD) can lead to the reversal of many remodeling changes whose underlying mechanisms are not yet completely known. Molecular analysis might play a role in obtaining further insight into the regulatory mechanisms of this process. A critical step in an RT-PCR study is the selection of reference genes for data normalization. This study aimed to determine an optimal combination of stably expressed reference genes in different regions of the human heart in order to study the effects of LVAD implants on cardiac remodeling, and in particular to check their reliability on the evaluation of pro-inflammatory cytokine expression. Design and methods: We validated nine of the most commonly used reference genes in human myocardium samples obtained at heart transplantation from patients with LVAD implant (n = 30 from a total of six patients) and from heart transplant (HT from a total of seven patients) recipients as controls (n = 35). Samples from both left (LV) and right (RV) ventricles were analyzed. The normalization strategy was tested by analyzing mRNA expression of IL-6, IL-8 and TNF-alpha, whose protein levels were measured by immunometric assay. Results: The most stable gene combinations changed according to the experimental groups (the LVAD and HT groups and the different myocardial regions). Considering all the cardiac samples as a whole, the three most stably expressed genes were PPIA, RPL13A, and YWHAZ (M = 0.70). Using the best normalization strategy, a significant increase in IL-6, IL-8 mRNA expression was observed in LVAD samples compared to HT (p < 0.0001). Similar results were obtained by protein analysis. Conclusions: Our results underline the importance of always selecting reference genes for the specific system studied. The most appropriate normalization strategy is of pivotal importance for understanding the molecular mechanisms associated with the pathophysiology of HF, such as inflammation. (c) 2013 Elsevier Ltd. All rights reserved.