Amphiregulin is overexpressed in human cardiac tissue in hypothermia deaths; associations between the transcript and stress hormone levels in cardiac deaths

Abstract

Background: Amphiregulin (AREG) is a growth factor linked to cardioprotection and heart pathology during myocardial stress. Our aim was to investigate cardiac AREG expression, its potential as a postmortem hypothermia marker and its possible stress hormone dependency in different types of deaths.

Materials and methods: Heart RNA was isolated from hypothermic, cardiac and non-cardiac deaths. Relative AREG mRNA levels and urine stress hormone concentrations were measured by qPCR and enzyme-linked immunosorbent assays from eight different death cause groups. Receiver operating characteristic curve was used to evaluate a cut-off point for AREG expression as a hypothermia marker. Regulatory elements were predicted by PROMO.

Results: The AREG mRNA levels were significantly higher in hypothermic deaths than in most cardiac and non-cardiac deaths. AREG expression indicated hypothermic deaths with nearly 70% sensitivity and specificity. However, high expression levels were also detected in non-ischaemic deaths. The highest concentrations of adrenaline and cortisol were detected in hypothermic deaths, while the highest noradrenaline concentrations associated with atherosclerotic heart disease (AHD) deaths with acute myocardial infarction and trauma deaths. There were no significant correlations between stress hormones and AREG mRNA in hypothermic and non-cardiac deaths, whereas moderate-to-high associations were detected in cardiac deaths. Putative response elements for cortisol and catecholamines were found in AREG.

Conclusions: Severe hypothermia activates cardiac AREG expression practicable as a postmortem hypothermia marker. Cortisol and catecholamines may act as transcriptional modifiers of this gene, especially in long-term ischaemic heart disease. However, the exact role of these hormones in upregulation of AREG during hypothermia remains unclear.

Keywords: Amphiregulin; cardiac stress; catecholamines; cortisol; hypothermia and cardiovascular deaths; ischaemia; postmortem hypothermia marker.

Figure 1.

Amphiregulin expression in human heart tissue in different types of deaths. Mean relative AREG mRNA level is shown with standard error and including individual data points on logarithmic scale. COD, cause of death; CVD, cardiovascular disease; AHD, atherosclerotic heart disease; AMI, acute myocardial infarction; HTA, arterial hypertension. *p < 0.05, ***p < 0.001 vs. hypothermia main COD; ^Ɨp < 0.05, ^Ɨ Ɨp < 0.01 vs. hypothermia contributory COD.

Figure 2.

Localization of amphiregulin in the human heart from hypothermia victim. (A,C) AREG transcripts (red dots) are present in cytoplasm and in some nuclei of cardiomyocytes. (B,D) Signal for AREG protein is detected in cardiomyocytes with cytoplasmic staining and membranous staining of nuclei.

Figure 3.

Urine concentrations of the stress hormones in different death cause groups. Median level (ng/ml) with standard error for A and NA (A) and cortisol (B). COD, cause of death; CVD, cardiovascular disease; AHD, atherosclerotic heart disease; AMI, acute myocardial infarction; HTA, arterial hypertension. *p < 0.05, **p < 0.01, ***p < 0.001 vs. hypothermia main COD; ^Ɨp < 0.05, ^Ɨ Ɨp < 0.01 vs. hypothermia contributory COD; ⁺p < 0.05 vs. ischaemic CVD/AHD; ^oop < 0.01 vs. CVD/AHD + AMI; ^¤p < 0.05 vs. CVD/AHD + HTA.

Figure 4.

Visualization of correlations between amphiregulin expression and stress hormones. The non-parametric Spearman’s rho correlation tests were carried out and presented as scatterplots using the GraphPad Prism. FC, fold change; CI, confidence interval; A/NA, adrenaline to noradrenaline ratio.

Figure 5.

Visualization of correlations between catecholamines and cortisol. The non-parametric spearman’s rho correlation tests were carried out and presented as scatterplots using the GraphPad Prism. CI, confidence interval; A/NA, adrenaline to noradrenaline ratio.

Figure 6.

Potential regulatory elements for catecholamines and cortisol in AREG proximal promoter. Selected transcription factor binding sites were predicted by PROMO using TRANSFAC database. Putative GR binding sites and sites for CREB are shown along the AREG sequence 500 bp upstream from ATG-codon. Blocks marked with 0, 1 and 2 represent putative GR, GR-alpha and GR-beta binding sites, respectively. Blocks marked with 3 represent CREB binding sites. Sites marked with * have dissimilarity margins ≤0.21% and others ≤10%.