Advertisement

International Journal of Legal Medicine

, Volume 133, Issue 2, pp 335–345 | Cite as

Identification of potential markers of fatal hypothermia by a body temperature-dependent gene expression assay

  • Takahiro UmeharaEmail author
  • Takehiko Murase
  • Yuki Abe
  • Hiromi Yamashita
  • Yoshinori Shibaike
  • Shinichiro Kagawa
  • Takuma Yamamoto
  • Kazuya Ikematsu
Original Article

Abstract

Diagnosis of fatal hypothermia is considered to be difficult in forensic practice and even if findings due to cold exposure are evident, cold exposure is not necessarily a direct cause of death. Identification of useful molecular markers for the diagnosis of fatal hypothermia has not been successful. In this study, to identify novel molecular markers that inform the diagnosis of fatal hypothermia, we focused on skeletal muscle, which plays a role in cold-induced thermogenesis in mammals. We made rat models of mild, moderate, and severe hypothermia and performed body temperature-dependent gene expression analysis in the iliopsoas muscle using next-generation sequencing (NGS). NGS showed that after severe hypothermia, the expression levels of 91 mRNAs were more than double those in mild and moderate hypothermia and control animals. Gene ontology (GO) analysis indicated that these mRNAs are involved in a number of biological processes, including response to stress and lipids, and cellular response to hypoxia. The expression of four genes [connective tissue growth factor (Ctgf), JunB proto-oncogene, AP-1 transcription factor subunit (Junb), nuclear receptor subfamily 4, group A, member 1 (Nr4a1), and Syndecan 4 (Sdc4)] and the level of one protein (CTGF) were induced only by severe hypothermia. These genes and protein are involved in muscle regeneration, tissue repair, and lipid metabolism. These results indicate that heat production to maintain body temperature in a process leading to fatal hypothermia might be performed by the iliopsoas muscle, and that Ctgf, Junb, Nr4a1, and Sdc4 genes are potential diagnostic markers for fatal hypothermia.

Keywords

Iliopsoas muscle Body temperature-dependent gene expression analysis Thermogenesis Marker gene 

Notes

Acknowledgements

We thank Jeremy Allen, PhD, from Edanz Group (www.edanzediting.com/ac) for editing the draft of this manuscript.

Funding information

This work was supported in part by the Japan Society for the Promotion of Science (Grant-in-Aid for Scientific Research C, 16K09210).

Compliance with ethical standards

The Animal Care Committee of Nagasaki University approved this research protocol (approval number 1606081312).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

414_2018_1888_Fig8_ESM.png (681 kb)
Figure. 1

In the severe hypothermia group, the expression levels of 77 mRNAs were less than half those in control, mild and moderate hypothermia groups. The heatmap illustrates profiles of 77 mRNAs from the iliopsoas muscle of control, mild, moderate and severe hypothermia rats. Quantification and log2 calculations were performed using Deseq, and normalization for each gene was performed using the median of all samples. (PNG 680 kb)

414_2018_1888_MOESM1_ESM.tiff (1.5 mb)
High resolution image (TIFF 1521 kb)
414_2018_1888_Fig9_ESM.png (826 kb)
Figure. 2

A. In the moderate hypothermia group, 67 mRNAs showed a more than twofold difference in expression level compared with levels in control, mild and severe hypothermia groups. B. In the mild hypothermia group, 28 mRNAs showed a more than twofold difference in expression level compared with levels in control, moderate and severe hypothermia groups. The heatmap illustrates profiles of 67 and 28 mRNAs from the iliopsoas muscle of control, mild, moderate and severe hypothermia rats. Quantification and log2 calculations were performed using Deseq, and normalization for each gene was performed using the median of all samples. (PNG 826 kb)

414_2018_1888_MOESM2_ESM.tiff (1.5 mb)
High resolution image (TIFF 1521 kb)

References

  1. 1.
    Hirvonen J (1976) Necropsy findings in fatal hypothermia cases. Forensic Sci 8:155–164CrossRefGoogle Scholar
  2. 2.
    Mizukami H, Shimizu K, Shiono H, Uezono T, Sasaki M (1999) Forensic diagnosis of death from cold. Legal Med 1:204–209CrossRefGoogle Scholar
  3. 3.
    Turk EE (2010) Hypothermia. Forensic Sci Med Pathol 6:106–115CrossRefGoogle Scholar
  4. 4.
    Brändström H, Eriksson A, Giesbrecht G, Angquist KA, Haney M (2012) Fatal hypothermia: an analysis from a sub-arctic region. Int J Circumpolar Health 71:1–7CrossRefGoogle Scholar
  5. 5.
    Ogata M, Ago K, Ago M, Kondo T, Kasai K, Ishikawa T, Mizukami H (2007) A fatal case of hypothermia associated with hemorrhages of the pectoralis minor, intercostal, and iliopsoas muscles. Am J Forensic Med Pathol 28:348–352CrossRefGoogle Scholar
  6. 6.
    Preuss J, Lignitz E, Dettmeyer R, Madea B (2007) Pancreatic changes in cases of death due to hypothermia. Forensic Sci Int 166:194–198CrossRefGoogle Scholar
  7. 7.
    Hejna P, Zátopková L, Tsokos M (2012) The diagnostic value of synovial membrane hemorrhage and bloody discoloration of synovial fluid (“inner knee sign”) in autopsy cases of fatal hypothermia. Int J Legal Med 126:415–419CrossRefGoogle Scholar
  8. 8.
    Ishikawa T, Yoshida C, Michiue T, Perdekamp MG, Pollak S, Maeda H (2010) Immunohistochemistry of catecholamines in the hypothalamic-pituitary-adrenal system with special regard to fatal hypothermia and hyperthermia. Legal Med 12:121–127CrossRefGoogle Scholar
  9. 9.
    Kitamura O, Gotohda T, Ishigami A, Tokunaga I, Kubo S, Nakasono I (2005) Effect of hypothermia on postmortem alterations in MAP2 immunostaining in the human hippocampus. Legal Med 7:340–344CrossRefGoogle Scholar
  10. 10.
    Umehara T, Usumoto Y, Tsuji A, Kudo K, Ikeda N (2011) Expression of material mRNA in the hypothalamus and frontal cortex in a rat model of fatal hypothermia. Legal Med 13:165–170CrossRefGoogle Scholar
  11. 11.
    Takamiya M, Saigusa K, Dewa K (2013) DNA microarray analysis of the mouse adrenal gland for the detection of hypothermia biomarkers: potential usefulness for forensic investigation. Ther Hypothermia Temp Manag 3:63–73CrossRefGoogle Scholar
  12. 12.
    Hirvonen J, Penttinen J, Huttunen P, Saukko P (1980) Changes in the myocardium and skeletal muscle in Guinea pigs in cold exposure with and without ethanol. Z Rechtsmed 84:195–207CrossRefGoogle Scholar
  13. 13.
    Sadler DW, Pounder DJ (1995) Urinary catecholamines as markers of hypothermia. Forensic Sci Int 76:227–230CrossRefGoogle Scholar
  14. 14.
    Hirvonen J, Huttunen P (1995) Hypothermia markers: serum, urine and adrenal gland catecholamines in hypothermic rats given ethanol. Forensic Sci Int 72:125–133CrossRefGoogle Scholar
  15. 15.
    Ishikawa T, Miyaishi S, Tachibana T, Ishizu H, Zhu BL, Maeda H (2004) Fatal hypothermia related vacuolation of hormone-producing cells in the anterior pituitary. Legal Med 6:157–163CrossRefGoogle Scholar
  16. 16.
    Rada A, Tonino P, Anselmi G, Strauss M (2005) Is hypothermia a stress condition in HepG2 cells? Expression and localization of Hsp70 in human hepatoma cell line. Tissue Cell 37:59–65CrossRefGoogle Scholar
  17. 17.
    Zhu BL, Ishikawa T, Michiue T, Li DR, Zhao D, Quan L, Oritani S, Bessho Y, Maeda H (2007) Postmortem serum catecholamine levels in relation to the cause of death. Forensic Sci Int 173:122–129CrossRefGoogle Scholar
  18. 18.
    Maeda H, Zhu BL, Bessho Y, Ishikawa T, Quan L, Michiue T, Zhao D, Li DR, Komatsu A (2008) Postmortem serum nitrogen compounds and C-reactive protein levels with special regard to investigation of fatal hyperthermia. Forensic Sci Med Pathol 4:175–180CrossRefGoogle Scholar
  19. 19.
    Preuss J, Dettmeyer R, Poster S, Lignitz E, Madea B (2008) The expression of heat shock protein 70 in kidneys in cases of death due to hypothermia. Forensic Sci Int 176:248–252CrossRefGoogle Scholar
  20. 20.
    Ishikawa T, Quan L, Li DR, Zhao D, Michiue T, Hamel M, Maeda H (2008) Postmortem biochemistry and immunohistochemistry of adrenocorticotropic hormone with special regard to fatal hypothermia. Forensic Sci Int 179:147–151CrossRefGoogle Scholar
  21. 21.
    Li DR, Michiue T, Zhu BL, Ishikawa T, Quan L, Zhao D, Yoshida C, Chen JH, Wang Q, Komatsu A, Azuma Y, Maeda H (2009) Evaluation of postmortem S100B levels in the cerebrospinal fluid with regard to the cause of death in medicolegal autopsy. Legal Med 11(Suppl 1):S273–S275CrossRefGoogle Scholar
  22. 22.
    Jakubeniene M, Chaker GA, Becelis A, Malakiene D, Raudys R (2009) Investigation of calcium and sodium in postmortem material as biochemical markers defining the cause of death from hypothermia. Legal Med 11(Suppl 1):S304–S306CrossRefGoogle Scholar
  23. 23.
    Ishikawa T, Michiue T, Zhao D, Komatsu A, Azuma Y, Quan L, Hamel M, Maeda H (2009) Evaluation of postmortem serum and cerebrospinal fluid levels of thyroid-stimulating hormone with special regard to fatal hypothermia. Legal Med 11(Suppl 1):S228–S230CrossRefGoogle Scholar
  24. 24.
    Zhou C, Byard RW (2011) Armanni-Ebstein phenomenon and hypothermia. Forensic Sci Int 206:e82–e84CrossRefGoogle Scholar
  25. 25.
    Yoshida C, Ishikawa T, Michiue T, Quan L, Maeda H (2011) Postmortem biochemistry and immunohistochemistry of chromogranin A as a stress marker with special regard to fatal hypothermia and hyperthermia. Int J Legal Med 125:11–20CrossRefGoogle Scholar
  26. 26.
    Palmiere C, Sporkert F, Werner D, Bardy D, Augsburger M, Mangin P (2012) Blood, urine and vitreous isopropyl alcohol as biochemical markers in forensic investigations. Legal Med 14:17–20CrossRefGoogle Scholar
  27. 27.
    Palmiere C, Lesta MM, Sabatasso S, Mangin P, Augsburger M, Sporkert F (2012) Usefulness of postmortem biochemistry in forensic pathology: illustrative case reports. Legal Med 14:27–35CrossRefGoogle Scholar
  28. 28.
    Palmiere C, Bardy D, Letovanec I, Mangin P, Augsburger M, Ventura F, Iglesias K, Werner D (2013) Biochemical markers of fatal hypothermia. Forensic Sci Int 226:54–61CrossRefGoogle Scholar
  29. 29.
    Quan L, Ishikawa T, Michiue T, Li DR, Zhao D, Zhu BL, Maeda H (2005) Quantitative analysis of ubiquitin-immunoreactivity in the midbrain periaqueductal gray matter with regard to the causes of death in forensic autopsy. Legal Med 7:151–156CrossRefGoogle Scholar
  30. 30.
    Doberentz E, Preuss-Wössner J, Kuchelmeister K, Madea B (2011) Histological examination of the pituitary glands in cases of fatal hypothermia. Forensic Sci Int 207:46–49CrossRefGoogle Scholar
  31. 31.
    Park JJ, Lee HK, Shin MW, Kim SJ, Noh SY, Shin J, Yu WS (2007) Short-term cold exposure may cause a local decrease of neuropeptide Y in the rat hypothalamus. Mol Cells 23:88–93Google Scholar
  32. 32.
    Jedema HP, Gold SJ, Gonzalez-Burgos G, Sved AF, Tobe BJ, Wensel T, Grace AA (2008) Chronic cold exposure increases RGS7 expression and decreases alpha(2)-autoreceptor-mediated inhibition of noradrenergic locus coeruleus neurons. Eur J Neurosci 27:2433–2443CrossRefGoogle Scholar
  33. 33.
    Sánchez E, Uribe RM, Corkidi G, Zoeller RT, Cisneros M, Zacarias M, Morales-Chapa C, Charli JL, Joseph-Bravo P (2001) Differential responses of thyrotropin-releasing hormone (TRH) neurons to cold exposure or suckling indicate functional heterogeneity of the TRH system in the paraventricular nucleus of the rat hypothalamus. Neuroendocrinology 74:407–422CrossRefGoogle Scholar
  34. 34.
    Mollica MP, Lionetti L, Crescenzo R, Tasso R, Barletta A, Liverini G, Iossa S (2005) Cold exposure differently influences mitochondrial energy efficiency in rat liver and skeletal muscle. FEBS Lett 579:1978–1982CrossRefGoogle Scholar
  35. 35.
    Wijers SL, Schrauwen P, Saris WH, van Marken Lichtenbelt WD (2008) Human skeletal muscle mitochondrial uncoupling is associated with cold induced adaptive thermogenesis. PLoS One 3:e1777CrossRefGoogle Scholar
  36. 36.
    Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M, Lightfoot S, Menzel W, Granzow M, Ragg T (2006) The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol 7:3CrossRefGoogle Scholar
  37. 37.
    Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3:1101–1108CrossRefGoogle Scholar
  38. 38.
    Ouellet V, Labbé SM, Blondin DP, Phoenix S, Guérin B, Haman F, Turcotte EE, Richard D, Carpentier AC (2012) Brown adipose tissue oxidative metabolism contributes to energy expenditure during acute cold exposure in humans. J Clin Invest 122:545–552CrossRefGoogle Scholar
  39. 39.
    Brigstock DR, Goldschmeding R, Katsube KI, Lam SC, Lau LF, Lyons K, Naus C, Perbal B, Riser B, Takigawa M, Yeger H (2003) Proposal for a unified CCN nomenclature. Mol Pathol 56:127–128CrossRefGoogle Scholar
  40. 40.
    Vial C, Zúñiga LM, Cabello-Verrugio C, Cañón P, Fadic R, Brandan E (2008) Skeletal muscle cells express the profibrotic cytokine connective tissue growth factor (CTGF/CCN2), which induces their dedifferentiation. J Cell Physiol 215:410–421CrossRefGoogle Scholar
  41. 41.
    Cabello-Verrugio C, Morales MG, Cabrera D, Vio CP, Brandan E (2012) Angiotensin II receptor type 1 blockade decreases CTGF/CCN2-mediated damage and fibrosis in normal and dystrophic skeletal muscles. J Cell Mol Med 16:752–764CrossRefGoogle Scholar
  42. 42.
    Morales MG, Cabello-Verrugio C, Santander C, Cabrera D, Goldschmeding R, Brandan E (2011) CTGF/CCN-2 over-expression can directly induce features of skeletal muscle dystrophy. J Pathol 225:490–501CrossRefGoogle Scholar
  43. 43.
    Brigstock DR (2002) Regulation of angiogenesis and endothelial cell function by connective tissue growth factor (CTGF) and cysteine-rich 61 (CYR61). Angiogenesis 5:153–165CrossRefGoogle Scholar
  44. 44.
    Nishida T, Kubota S, Aoyama E, Janune D, Lyons KM, Takigawa M (2015) CCN family protein 2 (CCN2) promotes the early differentiation, but inhibits the terminal differentiation of skeletal myoblasts. J Biochem 157:91–100CrossRefGoogle Scholar
  45. 45.
    Morales MG, Acuña MJ, Cabrera D, Goldschmeding R, Brandan E (2018) The pro-fibrotic connective tissue growth factor (CTGF/CCN2) correlates with the number of necrotic-regenerative foci in dystrophic muscle. J Cell Commun Signal 12:413–421CrossRefGoogle Scholar
  46. 46.
    Bentzinger CF, Wang YX, von Maltzahn J, Soleimani VD, Yin H, Rudnicki MA (2013) Fibronectin regulates Wnt7a signaling and satellite cell expansion. Cell Stem Cell 12:75–87CrossRefGoogle Scholar
  47. 47.
    Le Grand F, Jones AE, Seale V, Scimè A, Rudnicki MA (2009) Wnt7a activates the planar cell polarity pathway to drive the symmetric expansion of satellite stem cells. Cell Stem Cell 4:535–547CrossRefGoogle Scholar
  48. 48.
    Pinent M, Prokesch A, Hackl H, Voshol PJ, Klatzer A, Walenta E, Panzenboeck U, Kenner L, Trajanoski Z, Hoefler G, Bogner-Strauss JG (2011) Adipose triglyceride lipase and hormone-sensitive lipase are involved in fat loss in JunB-deficient mice. Endocrinology 152:2678–2689CrossRefGoogle Scholar
  49. 49.
    Maxwell MA, Cleasby ME, Harding A, Stark A, Cooney GJ, Muscat GE (2005) Nur77 regulates lipolysis in skeletal muscle cells. Evidence for cross-talk between the beta-adrenergic and an orphan nuclear hormone receptor pathway. J Biol Chem 280:12573–12584CrossRefGoogle Scholar
  50. 50.
    Pei L, Waki H, Vaitheesvaran B, Wilpitz DC, Kurland IJ, Tontonoz P (2006) NR4A orphan nuclear receptors are transcriptional regulators of hepatic glucose metabolism. Nat Med 12:1048–1055CrossRefGoogle Scholar
  51. 51.
    Fassett MS, Jiang W, D'Alise AM, Mathis D, Benoist C (2012) Nuclear receptor Nr4a1 modulates both regulatory T-cell (Treg) differentiation and clonal deletion. Proc Natl Acad Sci 109:3891–3896CrossRefGoogle Scholar
  52. 52.
    Hanna RN, Carlin LM, Hubbeling HG, Nackiewicz D, Green AM, Punt JA, Geissmann F, Hedrick CC (2011) The transcription factor NR4A1 (Nur77) controls bone marrow differentiation and the survival of Ly6C- monocytes. Nat Immunol 12:778–785CrossRefGoogle Scholar
  53. 53.
    Zeng H, Qin L, Zhao D, Tan X, Manseau EJ, Van Hoang M, Senger DR, Brown LF, Nagy JA, Dvorak HF (2006) Orphan nuclear receptor TR3/Nur77 regulates VEGF-A-induced angiogenesis through its transcriptional activity. J Exp Med 203:719–729CrossRefGoogle Scholar
  54. 54.
    Arkenbout EK, de Waard V, van Bragt M, van Achterberg TA, Grimbergen JM, Pichon B, Pannekoek H, de Vries CJ (2002) Protective function of transcription factor TR3 orphan receptor in atherogenesis: decreased lesion formation in carotid artery ligation model in TR3 transgenic mice. Circulation 106:1530–1535CrossRefGoogle Scholar
  55. 55.
    Cortez-Toledo O, Schnair C, Sangngern P, Metzger D, Chao LC (2017) Nur77 deletion impairs muscle growth during developmental myogenesis and muscle regeneration in mice. PLoS One 12:e0171268CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Division of Forensic Pathology and Science, Unit of Social Medicine, Course of Medical and Dental Sciences, Graduate School of Biomedical SciencesNagasaki University School of MedicineNagasakiJapan
  2. 2.Center for Forensic Pathology and ScienceNagasaki University Graduate School of Biomedical SciencesNagasakiJapan

Personalised recommendations