Consiglio Nazionale delle Ricerche CNR, Institute of Clinical Physiology, Laboratory of Cardiovascular Biochemistry, Pisa, Italy.
Cytokine. 2013 Aug;63(2):113-22. doi: 10.1016/j.cyto.2013.04.021. Epub 2013 May 10.
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.
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-α, whose protein levels were measured by immunometric assay.
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.
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.
新的设备治疗方法已经扩大了治疗心力衰竭（HF）患者的策略。使用左心室辅助装置（LVAD）减轻心脏负担可以导致许多重构变化的逆转，其潜在机制尚未完全清楚。分子分析可能在进一步深入了解该过程的调节机制方面发挥作用。RT-PCR 研究的关键步骤是选择用于数据归一化的参考基因。本研究旨在确定人类心脏不同区域稳定表达的参考基因的最佳组合，以研究 LVAD 植入对心脏重构的影响，特别是检查它们在评估促炎细胞因子表达方面的可靠性。
我们在从接受 LVAD 植入（共 6 例患者，n=30）和心脏移植（共 7 例患者，n=35）的患者心脏移植中获得的人心肌样本中验证了最常用的 9 种参考基因中的 9 种，作为对照。分析了来自左心室（LV）和右心室（RV）的样本。通过分析白细胞介素-6（IL-6）、白细胞介素-8（IL-8）和肿瘤坏死因子-α（TNF-α）的 mRNA 表达来测试归一化策略，其蛋白水平通过免疫计量测定法测量。
根据实验分组（LVAD 和 HT 组以及不同的心肌区域），最稳定的基因组合发生了变化。考虑到所有心脏样本作为一个整体，表达最稳定的三个基因是 PPIA、RPL13A 和 YWHAZ（M=0.70）。使用最佳归一化策略，与 HT 相比，LVAD 样本中 IL-6、IL-8 mRNA 表达显著增加（p<0.0001）。蛋白质分析也得到了类似的结果。
我们的结果强调了为特定研究系统选择参考基因的重要性。对于理解与心力衰竭病理生理学相关的分子机制（如炎症），最合适的归一化策略至关重要。
