The RT-qPCR is a powerful tool to study gene expression at mRNA level. As many factors can influence the accuracy of results obtained by this method (RNA quality, efficiency of reverse transcription, primer design), a correct normalization of primary data is necessary to reduce the variations caused by experimental and analytical procedures. Accordingly, the selection of proper reference genes is crucial, as these genes directly influence the interpretation of RT-qPCR results. Furthermore, in clinical HF samples, the variations in gene expression can be also affected by the number of samples analyzed, specific pathophysiology of the samples and pharmacological treatment, as well as the normalization strategy. Therefore, it is not surprising that the final choice of RGs selected for normalization in various studies of LV from normal and failing heart tissues differ significantly [10,18-23,31,32].
In this study, we selected 15 most commonly used CRGs and compared their stability in both the healthy and diseased samples. First, we employed an ANOVA-based approach [15] and F-statistics to calculate the stability index for each CRG (Table 2). Based on these calculations, IPO8, PGK1 and HPRT1 were considered the most stable genes, with nonsignificant p-values and the lowest intergroup variance. This analysis also revealed that 5 genes (TBP, YWHAZ, ACTB, B2M, RPLP0) were expressed variantly (p ˂0.05) and thus were excluded from further analyses. Among these genes, TBP was previously reported as stably expressed only in post mortem human cardiac muscle tissue [21], while in other studies where it was analyzed, it was never ranked among the most stably expressed genes [18,31]. On the other hand, YWHAZ was a top–ranked RG when samples from left and right ventricles of patients with LVAD (left ventricular assist device) or HT (heart transplantation without previously implanted LVAD) were tested [22]. Moreover, along with GAPDH, IPO8, POLR2A and PPIA, YWHAZ was identified as a suitable RG in several heart cavities and disease conditions [18]. In contrast, the identification of endogenous controls in LV of hearts from organ donors ranked YWHAZ among the less stable genes [31], which is in concert with our analysis and questions its role as RG in studies aimed at the differential gene expression in LV of failing hearts. The RPLP0 was described as suitable RG in study of hearts from human donors [32], but not in the study concerning normal and diseased hearts [18]. Regarding these genes, however, we would like to note that no data have been reported as to whether they had been tested for systematic variation or not. Therefore, we cannot exclude the possibility that in other clinical studies on LV of failing human hearts including different sets of cardiomyopathies and/or different sizes of both control and diseased groups, no statistical significant difference between the tested groups will be observed. Hence, we propose to include these genes in the CRG sets used for the identification of the most stably expressed genes in LV of normal and failing hearts in the future. Importantly, this does not apply to the last two genes, ACTB and B2M (see below).
Afterwards, the 10 stably expressed CRGs were further analyzed with geNorm, NormFinder and BestKeeper algorithms, as well as the Delta Cq method. Since geNorm, NormFinder or a combination of both is used in the majority of studies searching for suitable RGs, we used them as the primary tools and compared the resulting ranking lists with those generated by BestKeeper and Delta Cq (Fig. 1). Both of these algorithms provide reliable results if none of the evaluated genes shows a systematic variation in the expression profile during experiment. Unfortunately, there is no way to predict whether a particular CRG is expressed invariantly before performing the experiment and the preliminary analyses of CRGs are frequently based on publicly unavailable microarray or RNAseq data. One way to overcome this problem is to use the statistical approach, which evaluates each CRG individually and identifies genes with systematic variation in gene expression. Using all 5 aforementioned methods, we generated individual ranking lists for each algorithm and eventually combined them into the final ranking. This analysis identified IPO8, PGK1 and POLR2A as the most stably expressed genes, suggesting they might serve as reliable RGs in RT-qPCR analyses (Table 3).
Among the other seven genes (ranked 4.-10.), HPRT1 has been previously selected for normalization of gene expression profiles in cardiac tissue and blood cells in the study of myocarditis [10,19], PPIA was shown to be a functional internal control in study of healthy organ donors [31] and cytokine expression in patients with LVAD [22]. Interestingly, the results on GAPDH are contradictory, as it was reported as reliable RG in three separate studies [19, 20, 31], while in one report it was found to be the least stable gene [32]. In our study, GAPDH was scored as a less stable gene, ranked sixth in the final calculation. This is in agreement with results of [18], where GAPDH was ranked as a less stable gene in LV of control and failing hearts. On the other hand, the same study showed that GAPDH is a stable reference gene across different heart cavities and disease conditions [18]. GUSB has been analyzed only in one study, where it was described as the second most stable gene in LV of human controls and failing heart tissues [18]. This is inconsistent with the results of our analysis, where GUSB was ranked among the less stable genes. The UBC gene was analyzed in three studies [18,21,31], always found as a less stable, whereas the HMBS gene was reported as a suitable RG in postmortem human cardiac muscle tissue, but not in sudden cardiac death (SDC) analysis [21,23]. Together with TFRC gene, HMBS was found as the least stable CRG in our analysis, occupying the last two positions of the final ranking.
Finally, when comparing the top three RGs from our study (IPO8, PGK1 and POLR2A) with previously published reports, IPO8 and POLR2A were described as suitable RGs in left ventricles of failing hearts and also in all heart chambers and disease conditions [18]. PGK1 has been also reported as appropriate internal control in two analyses of human heart failure [10,18], suggesting all of these genes might serve as reliable RGs.
Analysis of MYH6 and MYH7 genes expression using top three CRGs
To experimentally test these three RGs, we analyzed the expression of MYH6 (downregulated in LV of failing hearts) and MYH7 (upregulated in LV of failing hearts) genes by RT-qPCR. Even though the control samples showed a slight variation in expression of MYH6, the expression of both MYH6 and MYH7 was significantly altered in LV of failing hearts, showing the expected patterns of differential regulation (Fig. 2). These data indicate that selected RGs are appropriate for examining the differential gene expression in proposed experimental set up and might be used in RT-qPCR analyses aimed at the identification of both upregulated and downregulated genes of interest.
Analysis of ACTB and B2M genes expression using top three CRGs
ACTB and B2M genes are very frequently used as RGs in gene expression studies. In our analysis, however, both showed a highly variant expression and were excluded from the dataset in the first step due to low p-values and high ANOVA stability indexes (Table 2). Similarly, these genes were also found as less stably expressed in other studies focusing on the identification of RGs in LV of failing human hearts, regardless of their clinical background [10,18,20-21,31]. Interestingly, the significant decrease in expression of these two genes might be directly associated with the pathophysiology of the LV of failing heart tissue.
In case of B2M gene, it has been recently shown that high levels of B2M protein in serum are associated with severity of coronary artery disease without renal dysfunction [33]. In addition, B2M levels are higher in plasma of patients hospitalized with chronic heart failure and correlate with the severity of cardiorenal failure [34]. Recently, the increased expression of B2M in stressed mouse cardiomyocytes was reported to stimulate a wound healing response in mouse fibroblasts with induced ischemia-reperfusion injury [35]. In contrast to these observations, we found the B2M mRNA levels decreased in failed hearts. One possible explanation of this discrepancy is that decreased expression of B2M gene could lead to a decreasing ability of wound healing in end-stage human hearts. However, the exact role of B2M gene in pathophysiology of human hearts remain to be investigated.
The role of ACTB gene in failing human hearts is also not yet fully understood. ACTB is one of the cytoskeletal proteins and participates in many important cellular processes, including muscle contraction, cell motility and division, organelle movement, the establishment and maintenance of cell junctions and cell shape [36]. The disorganization of cytoskeletal proteins and loss of sarcomeric structures contribute to the pathogenesis of contractile dysfunction in mammalian hearts and the decreased expression of this gene might lead to the loss of contractile force in end-stage hearts and subsequent heart failure [37]. Moreover, in many eukaryotic cells, β-actin (protein product of ACTB gene) is also present in nucleus, where it regulates the expression of specific genes and processes connected to cell division, and proliferation [38,39]. One of the best-characterized examples of β-actin role in the regulation of nuclear complexes is its involvement in regulation of MRTF (myocardin-related transcription factor) system, an activator of SRF (serum response factor). SFR is a regulator of cardiac gene expression in response to hypertrophic stimuli and several processes which reduces the monomer-actin (G-actin) lead to the retention of MRTF in nucleus and SRF activation. This in turn could lead to the expression of genes responsible for cardiac remodeling [40,41]. Interestingly, the cardiac remodeling might be also regulated via interaction of β-actin with endothelial nitric oxide synthase (eNOS). The association of eNOS with β-actin increases eNOS activity and the nitric oxide (NO) production [41]. The following activation of cGMP-dependent protein kinase (PKG) by nitric oxide (NO) might inhibit the hypertrophic signaling pathways and act as pro-hypertrophic brake [5,42].