Original Research Paper
mRNA expression patterns in human myocardial tissue, pericardial fluid and blood, and its contribution to the diagnosis of cause of death

https://doi.org/10.1016/j.forsciint.2019.109876Get rights and content

Highlights

Abstract

Gene expression has become an interesting research area in forensic pathology to investigate the process of death at the molecular level. The aims of this study were to analyze changes in gene expression patterns in relation to the cause of death, and to propose new molecular markers of myocardial ischemia of potential use for the postmortem diagnosis of early ischemic heart damage in cases of sudden cardiac death (SCD). We determined mRNA levels of five proteins related with ischemic myocardial damage and repair – TNNI3, MYL3, TGFB1, MMP9 and VEGFA – in specific sites of the myocardium, blood and pericardial fluid in samples from 30 cadavers with different causes of death (SCD, multiple trauma, mechanical asphyxia, and other natural deaths). TNNI3 expression in blood, and MMP9 expression in pericardial fluid, were significantly higher when the cause of death was mechanical asphyxia, probably because of the more sensitive response of these proteins to acute systemic hypoxia/ischemia. Specifically, among SCD cases, increased MYL3, VEGFA and MMP9 values in the anterior wall of the right ventricle were found when the confirmed cause of death was acute myocardial infarction (AMI). Higher TGFB1 expression was found in the interventricular septum when AMI was not the cause of death, most likely as a reflection of the short duration of ischemia. Molecular biology techniques can provide complementary tools for the forensic diagnosis of early ischemic myocardial damage and AMI, and may make it possible to determine the duration and severity of myocardial ischemia.

Introduction

Determining the cause of death can be difficult in forensic practice, especially in cases of sudden death. In adults, sudden death is defined as natural unexpected death occurring within 1 h of the beginning of symptoms. Most cases of sudden death are related with cardiac disease, especially ischemic heart disease [1], with myocardial infarction (MI) being a common cause of sudden cardiac death (SCD). However, the postmortem diagnosis of early ischemic lesions in the heart is a challenging problem in practical forensic medicine, since the typical morphological changes that occur in myocardial tissue because of ischemia require a minimum survival time to be detectable after death. Thus, it is often difficult to verify acute ischemic heart disease on autopsy. This makes it necessary to develop diagnostic tests that can ensure the accurate diagnosis of acute cardiac ischemia as the cause of SCD.

Many different approaches have been tried to find reliable markers of early myocardial ischemic damage. Morphological, histochemical, immunohistochemical and biochemical methods have all been tried for the diagnosis of myocardial injury, especially to diagnose early MI [2], [3], [4], [5], [6], [7], [8], [9], [10]. The use of conventional histological methods is limited because myocardial damage is usually manifested at least 4–6 h after MI occurs [3], [11], [12]. In contrast, myocardial injury can be detected earlier with immunohistochemical techniques, i.e. about 1 or 2 h after the beginning of ischemia, although these techniques are difficult to interpret due to artifacts caused by autolysis, and are not easy to use in routine forensic practice [3], [11], [13]. Biochemical methods include those based on the study of cardiac structural molecules such as cardiac troponin I and T in blood and pericardial fluid as postmortem markers of myocardial damage. In this case, however, it should be recalled that elevated levels of troponin in blood and pericardial fluid are also found in other causes of death such as hyperthermia, methamphetamine abuse and carbon monoxide poisoning, and the postmortem interval may also affect the detectability of these molecules. In addition, it is important to note that variability has been reported in the levels of cardiac markers in blood from different body locations in the same individual [2], [9], [11], [14], [15]. Thus, some authors have used different biochemical methods for cardiac tissues, such as the potassium/sodium ratio and formazan test [5], [6], [16]. Despite these limitations, biochemical methods have been shown to be more useful than histological methods in detecting myocardial damage at earlier stages [12], [17].

Aside from the approaches summarized above, postmortem gene expression studies are becoming an interesting field of research in forensic pathology to investigate the cause and process of death at the molecular level. In the context of postmortem diagnosis of the cause of death, gene expression studies in cardiac tissues and fluids from cadavers are needed to reach a better understanding of the underlying mechanisms of myocardial ischemia and its repair [10], [18], and to make it possible to identify early molecular markers of myocardial ischemia for the postmortem diagnosis of SCD.

In previous work we investigated the mRNA integrity, mRNA expression levels and postmortem stability of five genes related to ischemic myocardial injury and its repair – cardiac troponin I (TNNI3), myosin light chain 3 (MYL3), transforming growth factor beta 1 (TGFB1), matrix metalloprotease 9 (MMP9), and vascular endothelial growth factor A (VEGFA) – in postmortem samples from the heart (five myocardial sites) and body fluids (femoral vein blood and pericardial fluid). Our results showed that RNA extracted in all samples showed good integrity and remained stable for up to 24 h after death [19].

The present study was designed to analyze changes in gene expression patterns in relation to the cause of death. The aim of this study was to identify differences in the expression levels of five proteins related with ischemic myocardial damage and repair (TNNI3, MYL3, TGFB1, MMP9, and VEGFA) in myocardial tissue, blood and pericardial fluid, in cadavers with different known causes of death. Our ultimate goal was to propose new molecular markers of myocardial ischemia of potential use for the postmortem diagnosis of early ischemic heart damage in cases of SCD.

Section snippets

Samples

Samples were obtained from a total of 30 cadavers (5 females and 25 males; mean age 65.03 ± 16.50 years; range 36–90 years) autopsied at the Institute of Forensic Medicine of Malaga (Spain) in accordance with the principles of the Declaration of Helsinki. The research protocol was approved by the Ethics Committee for Human Research of the University of Granada (Spain). All cadavers were kept at 4 °C until autopsy was performed at a known postmortem interval (mean PMI: 15.36 ± 5.67 h, range 5–24 h).

Results

Postmortem mRNA levels of five proteins related with ischemic myocardial injury and repair (TNNI3, MYL3, TGFB1, MMP9 and VEGFA) were studied in myocardial tissue, blood and pericardial fluid from cadavers with different known causes of death (SCD, multiple trauma, mechanical asphyxia, and other natural deaths). When gene expression levels were compared across each of the four cause-of-death groups, significant differences in TNNI3 mRNA expression in blood samples were found between mechanical

Discussion

There is no ideal method for the postmortem diagnosis of early myocardial ischemic injury, but mRNA expression profiling has become an interesting and potentially fruitful field of research in forensic pathology to investigate the cause and process of death at the molecular level. Studies of gene expression in different human tissues and fluids are important to elucidate the molecular mechanisms of pathological situations, because they offer insights into gene expression patterns in specific

Conflict of interest

The authors declare that they have no conflict of interest.

Funding

This work was supported by funding from the Centro para la Excelencia Forense en Andalucía (CEIFA-01/2008).

Acknowledgments

The authors gratefully acknowledge the scientific advice, guidance and support of Dr. Luis Javier Martinez from the Centro Pfizer – Universidad de Granada – Junta de Andalucía de Genómica e Investigación Oncológica (GENYO), and thank K. Shashok for improving the use of English in the manuscript.

References (41)

  • H. Maeda et al.

    Forensic molecular pathology: its impacts on routine work, education and training

    Leg. Med. (Tokyo)

    (2014)
  • X. Xu et al.

    Expression of vascular endothelial growth factor and its receptors is increased, but microvascular relaxation is impaired in patients after acute myocardial ischemia

    J. Thorac. Cardiovasc. Surg.

    (2001)
  • D. Zhao et al.

    Real-time RT-PCR quantitative assays and postmortem degradation profiles of erythropoietin, vascular endothelial growth factor and hypoxia-inducible factor 1 alpha mRNA transcripts in forensic autopsy materials

    Leg. Med. (Tokyo)

    (2006)
  • J. Dabek et al.

    Transcriptional activity of genes encoding transforming growth factor beta and its receptors in peripheral blood mononuclear cells from patients with acute coronary syndromes

    Int. J. Cardiol.

    (2006)
  • J.H. Chen et al.

    Cardiac biomarkers in blood, and pericardial and cerebrospinal fluids of forensic autopsy cases: a reassessment with special regard to postmortem interval

    Leg. Med. (Tokyo)

    (2015)
  • N.I. Batalis et al.

    The role of postmortem cardiac markers in the diagnosis of acute myocardial infarction

    J. Forensic Sci.

    (2010)
  • C.P. Campobasso et al.

    Sudden cardiac death and myocardial ischemia indicators: a comparative study of four immunohistochemical markers

    Am. J. Forensic Med. Pathol.

    (2008)
  • K. He et al.

    Research evolvement of postmortem diagnosis of early myocardial infarction in forensic pathology

    Fa Yi Xue Za Zhi

    (2006)
  • R.M. Mao et al.

    The application of biochemical indexes detecting in sudden cardiac death in forensic autopsy

    Fa Yi Xue Za Zhi

    (2009)
  • E. Tomaskova et al.

    Some possibilities in the diagnosis of early acute ischaemic changes in the heart muscle in sudden death

    Soud. Lek.

    (2010)
  • Cited by (0)

    View full text