Rom J Leg Med [25] 8-13 [2017]
DOI: 10.4323/rjlm.2017.8
Fundamental research
© 2017 Romanian Society of Legal Medicine Heat shock protein expression in cardiac tissue in amphetamine-related
deaths
Burkhard Madea1,*, Rebecca Wagner1, Philipp Markwerth1, Elke Doberentz1
_________________________________________________________________________________________
Abstract: Both amphetamines and cocaine promote perivascular and interstitial fibrosis and myocyte hypertrophy, but
acute myocardial infarction is much more common in cocaine abusers than in amphetamine-class fatalities. As a hypothesis was
speculated that amphetamine-induced hyperthermia may promote the expression of heat shock proteins, which may in turn
increase myocardial resistance to infarction. We examined the expression of heat shock proteins 27, 60, and 70 in a random
sample of 19 amphetamine-class fatalities with different causes of death using immunohistochemistry. Five cases demonstrated
strong heat shock protein-positive reactions in myocytes. This result may support the hypothesis of Karch. The lack of a positive
reaction in the remaining cases may be accounted for by the different causes of death and agonal events.
Key Words: amphetamine, class fatality, heart, heat shock protein expression, myocyte.
A
few years ago a total of 169 cases were
reviewed at the Victorian institute of Forensic
Medicine where an involvement of amphetamines in
sudden and unexpected death was proven [1]. Cause of
death was classified according to table 1. Often not the
drug alone is the cause of death but the association with
amphetamine related heart disease [1-9].
Methamphetamine (MA) was the principal
class of abused amphetamines in the state of Victoria,
followed
by
methylenedioxymethamphetamine
(MDMA, Ecstasy). The review identified six cases of
death due to cerebral hemorrhage, and three in which
serotonin syndrome was caused by the interaction
between MDMA and moclobemide. Long-term use of
amphetamines was associated with heart diseases in 19
cases, and amphetamine-class drugs alone were regarded
as the cause of death in only three cases, who exhibited
high levels of MDMA and lower levels of MA and/or
amphetamine. There were no cases without significant
natural disease in whom MA was regarded as the cause
of death [1].
Amphetamine-class drugs dramatically increase
sympathetic stimulation and circulating levels of
neurotransmitters in the periphery. Ecstasy (MDMA)
has frequently been reported to cause death, often
through the development of malignant hyperthermia or
liver damage [1, 4, 8, 9].
Well-recognized side effects of amphetamine
and MA abuse identified at autopsy include fatty liver,
moderate coronary artery disease, cirrhosis, pneumonia,
myocardial fibrosis, triaditis, severe coronary artery
disease, emphysema, and hepatitis [2, 3]. These side effects
have been confirmed in several studies [4-9]. Hearts
demonstrate similar microscopic features at autopsy
in amphetamine/MA and cocaine users, including
hypertrophy, interstitial fibrosis, and microvascular
disease, and both amphetamines and cocaine promote
perivascular and interstitial fibrosis, myocyte hypertrophy,
and intimal and medial hyperplasia. However, acute
myocardial infarction is much more common in cocaine
abusers than in MA abusers, despite apparently similar
levels of disease [2, 3].
1) University of Bonn, Institute of Forensic Medicine, Bonn, Germany
* Corresponding author: E-mail: [email protected]
8
Romanian Journal of Legal Medicine Karch [3] suggested that amphetamines
possess several properties that may reduce the risk of
myocardial infarction in amphetamine users compared
with cocaine users. Notably, MA induces the production
of heat shock proteins (HSPs), while cocaine does not.
According to Karch [3] it has been shown for decades
that HSP production is an adaptive myocardial response
that occurs within 24 h after short episodes of cardiac
ischemia and that the production of HSP proteins (it is
not clear which of the proteins predominate) increases
myocardial resistance to infarction. The production
of HSPs may provide a logical explanation for the
recognized ability of MA and most other amphetamines
to cause hyperthermia [10-14].
The present study aimed to investigate the
expression of HSPs in cardiac tissue from individuals
with amphetamine-class drug-related deaths.
All body cells and cell compartments contain
molecular chaperones, which help to manage protein
biosynthesis [15-17] by supporting the folding of
newly synthesized, unfolded proteins into their threedimensional structure, and their subsequent transport to
the place of action [18, 19]. HSPs belong to this group
of chaperones. They are highly conserved proteins that
were discovered in relation to their high expression levels
after exposure to heat [20]. Ritossa initially detected
upregulated genes after heat exposure [21], while
Tissieres et al. subsequently described the corresponding
proteins as HSPs [22]. HSPs are located in various
cellular compartments (Table 2). They are classified into
families according to their molecular size and can also be
separated into non-ATP-dependent and ATP-dependent
chaperones, according to their energy dependence [2327]. Chaperones of small molecular size are called small
HSPs, which work without an energy source, whereas
larger chaperones, such as HSP70, HSP90, and HSP100,
depend on the availability of ATP.
Vol. XXV, No 1(2017)
HSPs play an important role in maintaining
protein homeostasis in cells and preventing apoptotic
cell death [28, 29]. They are responsible for the repair or
degradation of wrongly folded or degraded proteins [3033]. Proteins may unfold and even aggregate during cell
stress, such as at non-optimal physiological temperatures
[19]. The expression of protective HSPs increases under
these conditions as part of the stress or heat shock
response, thus supporting cell survival under stressful
conditions. They are involved both in local cellular
reactions, and also in systemic reactions [34], such as
inducing fever [35, 36].
Rapid HSP expression has been demonstrated in
pulmonary tissues in fire-related deaths [37, 38], as well
as in renal tissue following hypothermia [39]. However, to
the best of our knowledge, immunohistochemical studies
examining HSP expression in cardiac tissue are relatively
scarce [37, 38, 40, 41]. The HSP content of cardiac tissue
can be increased by either ischemic or thermal stress,
as shown by western blotting. HSP60 was preferentially
elevated by ischemic pretreatment [13], while myocardial
induction of HSP72 occurred after increasing the rectal
temperature in heat-stressed anaesthetized rabbits to at
least 42°C for 15 minutes [13].
HSPs are present in body cells at low or very low
levels, especially in the cytoplasm, even in the absence
of stress. Under normal physiological conditions,
HSPs bind to heat shock factor (HSF), which is a
transcriptional regulator of HSPs, leading to increased
HSP synthesis. HSF-1 regulates the response to different
kinds of stress and increases the expression of heat
shock genes following heat shock. HSP expression in
body cells increases in the event of exposure to various
stress stimuli, including hypo- or hyperthermia, as well
as other external and internal physiological stresses such
as lack of energy (ATP), oxidative stress, heavy metals,
ischemia, UV-light, chemicals, injuries, mechanical
Table 1. Categorization of amphetamine group cases [1]
Group
A1
A2
A3
A4
B
C
Cause of death
Hemorrhage and presence of amphetamines
Amphetamine toxicity and heart disease
Amphetamine toxicity leading to serotonine syndrome
Amphetamine toxicity
Drug overdose
Mechanical injury
Drugs involved
Amphetamines and no other significant drug
Amphetamines and no other significant drug
Amphetamines and other significant serotonine active drugs
Amphetamines only
Death caused predominantly by drugs other than amphetamines
Amphetamines and other drugs
Table 2. Most important hsp families with localisation and the main function [9]
Hsp family
Hsp27
Hsp60
Hsp70
Hsp72
Hsp73
Hsp75
Hsp78
Hsp90
Hsp100-104
Localization
Cytoplasm and nucleus
Mitochondria
Cytoplasm and nucleus
Cytoplasm and nucleus
Cytoplasm and nucleus
Mitochondria
Endoplasmic reticulum
Cytoplasm
Cytoplasm
Main function
Stabilization of microfilaments
Protection of proteins and reparation of proteins
Protein folding and cytoprotection
Protein folding and cytoprotection
Protein translocation
Protein translocation, cytoprotection against apoptosis
Cytoprotection against apoptosis and protein translocation
Refolding of protein aggregates
9
Madea B. et al.
Heat shock protein expression in cardiac tissue in amphetamine-related deaths
stress, and chemotherapeutic substances or cytokines.
Stressful conditions increase the incidence of damaged
or misfolded proteins. HSPs then bind to these misfolded
proteins and dissociate from HSF, and the free HSF then
induces the expression of new HSPs. This mechanism
provides cell protection by increasing HSP expression
during cellular damage. HSP induction occurs within
minutes of exposure to the stressor, with the increase
correlating with the severity of heat stress. HSPs can then
be detected by immunohistochemical staining.
Materials and methods
We investigated 19 amphetamine-associated
fatalities in whom the causes of death were amphetamine
intoxication alone, mixed intoxication, or intoxication
together with preexisting amphetamine-related diseases.
The subjects included three women and 16 men, aged
22–58 years (mean age 34.5 years) (Table 3).
Heart, lung, and kidney tissue samples were
taken during forensic autopsies for histological and
immunohistochemical examination. The samples were
fixed in 8%–10% formalin, embedded in paraffin wax,
sliced at 3–4 µm, and stained with antibodies to HSP27
(mouse anti-HSP27 monoclonal antibody; Novocastra
Laboratories Ltd., Newcastle upon Tyne, UK), HSP60
(mouse monoclonal anti-HSP60 clone LK 1; SigmaAldrich, St. Louis, MO, USA), and HSP70 (HSP70
mouse monoclonal antibody; Novocastra), and with
hematoxylin-eosin. Tonsillar tissue was used as a positive
control in every staining procedure. One sample of
tonsillar tissue was treated without primary antibody and
one without secondary antibody as negative controls.
Thirty visual fields from each slide were examined
under a light microscope at 400× magnification, and the
structures of each organ were evaluated (Table 4) [37-41].
The immunohistochemical reaction of the tissue was
measured semi-quantitatively on a four-point scale,
according to Preuss et al. [39]. The number of reddishstained structures/cells in relation to all studied
structures/cells visible in each visual field was estimated
as a percentage. A mean value over all 30 visual fields was
calculated for each analyzed organ structure and case,
and ranked as shown in Table 5 and Figures 1–3.
Table 3. Cause of death in amphetamine/methamphetamine related deaths. Cause of death mostly intoxication or intoxication
together with preexisting diseases. The Hsp positive cases are highlighted (grade in brackets). Concentration in ng/mL.
A/MDMA Amph
Amph
MDMA HSP / Grade of
contribution heart
Femoral Femoral HSP expression
to death
blood vein blood vein blood in brackets
negative
Amphetamine and preexisting cardiac disease
Yes
246.5
negative
Mixed intoxication
No
35.01
negative
Ampehtamine and preexisting cardiac disease
Yes
47.73
negative
Mixed intoxication / heroin
No
52.3
negative
Mixed intoxication
?
165.71
negative
Amphetamine and preexisting cardiac disease
Yes
360.95
negative
Mixed intoxication MDMA
Yes
519
1353
negative
Mixed intoxication amphetamine
Yes
147.69
MDMA intoxication
Yes
249.5
1300
negative
Amphetamine intoxication
Yes
1084
27 (heart) [2].
Mixed intoxication / heroin
No
89.34
70 (heart) [1]
Mixed intoxciation Amphetamine and
Negative
?
67.9
preexisting cardiac disease
Negative
Mixed intoxication
Yes
222.51
27 (heart) [3].
Amphetamine intoxication
Yes
pos.
304
27 (kidney) [1]
27 (hear) [1]
Mixed intoxication Amphetamine / heroine
Yes
93.6
Amphetamine and preexisting cardiac
Negative
Yes
255
disease
27 (heart) [2]
Mixed intoxication MDMA
Yes
238
8142
Amphetamine and preexisting cardiac
27 (heart) [1]
Yes
pos.
170
disease
Negative
Amphetamine intoxication
Yes
107
Case Autopsy
Cause of death
number number
1
2
3
4
5
6
7
8
9
10
168/09
284/09
137/10
139/11
190/11
291/11
321/11
147/12
240/12
275/13
11
373/13
12
453/13
13
174/14
14
412/14
15
004/15
16
022/15
17
288/15
18
066/16
19
076/16
Table 4. Studied structures in the different organs
Organs
Heart
Lung
Kidney
10
Structures
Myocytes, fibrocytes, vessels
Peripheral and central bronchial tubes, vessels (endothelium, lumen), inter-alveolar septa, pleura, peribronchoal
glands, peribronchial connective tissue, ciliated epithelium
Glomerula, tubuli, vessels, connective tissue
Romanian Journal of Legal Medicine Vol. XXV, No 1(2017)
Table 5. Table of graduation according to insensitivity of stained organ structures
Percentage of reddish stained
structures in total
0
>0 to 29.99 %
30 to 59.99 %
60 to 100 %
Graduation Explanation
Grade 0
Grade 1
Grade 2
Grade 3
Grade 4
No reaction
Weak reaction
Moderate staining
Intensive staining
Analyzed structures were not present in the section of the tissue sample
Figure 1. Amphetamine associated death, cardiac tissue, HSP
27, grade 2, x 200.
Figure 2. Amphetamine associated death, cardiac tissue, HSP
27, grade 3, x 400.
was heterogeneous in terms of the cause of death and
agonal period.
Positive HSP expression in cardiac tissue was only
accompanied by positive expression in other organs, i.e.,
the kidney glomeruli and tubules, in one case. Notably,
strong positive HSP expression in the kidney was previously
observed in cases of fatal hypothermia [39].
Discussion
Figure 3. Fatal hypothermia in amphetamine intoxication,
strongly positive Hsp 27 expression (see also Fig. 4).
Results
The various grades of HSP expression are shown
in Figs 1–3 and a case example is given in Fig. 4. Positive
HSP expression was especially notable in myocytes.
Although the intensity of staining varied considerably, it
was strong in some cases, as shown in Figs 2 and 3.
The results of this pilot study verified positive
HSP expression in five of the 19 cases of amphetaminerelated deaths, though most cases were negative. All cases
were negative for HSP60, five were positive for HSP27,
and one was also positive for HSP70 (Table 3). The
positive cases are highlighted. However, the case material
The causes of death varied considerably among
the amphetamine-related fatalities (Table 3). Monointoxication was rare, and the predominant causes of
death were mixed intoxications and long-term use of
amphetamines associated with heart diseases. In line
with the different causes of death, the agonal periods may
also have differed considerably, and a dramatic increase
in sympathetic stimulation and the development of
malignant hyperthermia may have occurred in some but
not all cases.
The positive HSP expression in some cases in
the current study may support the hypothesis of Karch
[3], whereby hyperthermia-induced HSP expression [42,
43] may increase myocardial resistance to infarction. The
high levels of HSP expression observed in cardiac tissue
in some of the current cases were not seen in cases of
death due to fire or hypothermia [37].
All the current cases were negative for HSP60,
while five were positive for HSP27 and one for HSP70.
The reasons for this expression pattern remain
unclear. However, HSPs are located in various cellular
11
Madea B. et al.
Heat shock protein expression in cardiac tissue in amphetamine-related deaths
A
B
Figure 4. (A) A 24 year old man was found undressed in December in a river. Mouth and nose above the water level. Body
weight 102,7 kg, body length 185 cm. Some meters away the clothing of the deceased was found. (B) Main autopsy findings: Frost
erythema above the knee joints , the elbow joints on both sides. Hemorrhagic gastric erosions. No further preexisting diseases.
Cause of death: Death due to hypothermia. Blood alcohol concentration: negative. Urine: positive immunological test for
amphetamine, metamphetamine and ecstasy. Amphetamine about 84965 ng/mL, metamphetamine 22.0 ng/mL. Femoral blood:
amphetamine 304 ng/mL, olanzapine 56.0 ng/mL, BAC negative.
compartments and have different functions, and an
analysis of HSPs in pulmonary tissue from fire-related
fatalities indicated that HSP27 was the predominant HSP
in short-term survivors, compared with HSP70 in longterm survivors [37-38].
Further studies are needed to determine the
patterns of HSP expression in amphetamine-related
fatalities, especially in cases where amphetamines are
the leading cause of death, and in cases for which details
of the terminal period and body core temperature are
available.
Key points
Amphetamines and cocaine both promote
perivascular and interstitial fibrosis, myocyte hypertrophy,
and intimal and medial hyperplasia.
Acute myocardial infarction is more common
in cocaine abusers than in amphetamine-class fatalities,
despite similar extents of cardiac disease.
Karch speculated that amphetamine-induced
hyperthermia may induce HSP expression, which may
increase myocardial resistance to infarction.
Strong HSP expression in cardiac myocytes
was detected in five of 19 cases of amphetamine-related
fatalities, potentially supporting Karch’s hypothesis.
Positive HSP expression in amphetaminerelated fatalities was only observed in the heart and not
in the kidney, which has shown a strong reaction to
hypothermia.
The lack of HSP-positivity in most cases of
amphetamine-related deaths may be attributable to the
different causes of death and agonal events.
Further studies are needed focusing on cases in
which amphetamines were the leading cause of death.
Conflict of interest. The authors declare that
they have no conflict of interest concerning this article.
References
1.
Pilgrim JL, Gerostamoulos D, Drummer OH, Bollmann M. Insolvement of amphetamines in sudden and unexpected death. J Forensic Sci
2008; 54: 478-485.
2. Karch SB, Chih-Hsieng H. Medamphetamine-related deaths in San Francisco: demographic, pathologic, and toxicologic profiles. J Forensic
Sci 1999; 44: 359-368.
3. Karch SB, Drummer OH. Karch’s Pathology of Drug Abuse. 5th Edition CRC Press 2016; Boca Raton London New York.
4. Milroy CM. “Ecstasy” associated deaths: what is a fatal concentration? Analysis of a case series. Forensic Sci Med Pathol 2011; 7: 248-252.
5. Püschel K, Schmoldt A (2004) Drogennot- und -todesfälle. In: Madea B, Brinkmann B (Hrsg.) Handbuch gerichtliche Medizin. Band 2.
Springer Verlag Berlin Heidelberg. 689-735.
6. von Schrenck T (1999) Internistische Komplikationen nach Ecstasy. Deutsches Ärzteblatt (96)6:347-352.
7. Verschraagen M, Maes A, Ruiter B, Bosman IJ, Smink BE, Lusthof KJ. Post-mortem cases involving amphetamine-based drugs in the
Netherlands. Comparison with driving under the influence cases. Forensic Sci Int 2007; 170: 163-170.
8. Wöllner K, Stockhausen S, Musshoff F, Madea B. Todesfälle nach der Einnahme von Amphetamin/Ecstasy: zwei Fallvorstellungen. Arch
Kriminol 2015; 235:53-61.
9. Wöllner K, Mußhoff F, Heß C, Madea B. Retrospektive Analyse von Todesfällen durch Amphetamin- und Ecstasy-Konsum. Arch Kriminol
2015; 236: 199-207.
10. Kiyatkin EA. Brain temperature homeostasis: Physiological fluctuations and pathological shifts. Front Biosci 2010; 15: 73-92.
11. Sanchez Y, Lindquist SL. HSP104 required for induced thermotolerance. Science 1990; 248 (4959): 1112-1115.
12
Romanian Journal of Legal Medicine Vol. XXV, No 1(2017)
12. Srivastava P. Heat shock proteins and immune response: Methods to madness. Methods 2004; 32(1): 1-2.
13. Marber MS, Latchman DS et al. Cardiac stress protein elevation 24 hours after brief ischemia or heat stress in associated with resistance to
myocardial infarction. Circulation 1993; 88(3): 1264-1272.
14. DeMaio A. Heat shock proteins, oxygen radiacs, and apoptosis: The conflict between protection and destruction. Crit Care Med 2000;
28(5): 1679-1681.
15. Macario AJL, Conway de Macario E (2005) Sick chaperones, cellular stress, and disease. N Engl J Med 353(14):1489-1501.
16. Walter S, Buchner J (2002) Molecular chaperones--cellular machines for protein folding. Angew Chem Int Ed Engl 41(7):1098-1113.
17. Wilson J, Sukyeong L, Nuri S, Jungsoon L, Francis TFT (2015) Molecular chaperones: guardians of the proteome in normal and disease
states. F1000 Facult Rev 4:1448.
18. Deshaies RJ, Koch BD, Werner-Washburne M, Craig EA, Schekman R (1988) A subfamily of stress proteins facilitates translocation of
secretory and mitochondrial precursor polypeptides. Nature 332(6167):800-805.
19. Walter S, Buchner J (2002) Molecular chaperones--cellular machines for protein folding. Angew Chem Int Ed Engl 41(7):1098-1113.
20. Brinkmeier H, Ohlendieck K (2014) Chaperoning heat shock proteins: proteomic analysis and relevance for normal and dystrophindeficient muscle. Proteomics Clin Appl 8(11-12):875-895.
21. Ritossa F (1962) A new puffing pattern induced by temperature shock and DNP in drosophila. Experientia 18(12):571-573.
22. Tissières A, Mitchell HK, Tracy UM (1974) Protein synthesis in salivary glands of Drosophila melanogaster: relation to chromosome puffs.
J Mol Biol 84(3):389-398.
23. Jee H (2016) Size dependent classification of heat shock proteins: a mini-review. J Exerc Rehabil 12(4):255-259.
24. Hartl FU, Bracher A, Hayer-Hartl M (2011) Molecular chaperones in protein folding and proteostasis. Nature 475(7356):324-332.
25. Mayer MP (2013) HSP70 chaperone dynamics and molecular mechanism. Trends Biochem Sci 38(10):507-514.
26. Wang X, Chen M, Zhou J, Zhang X (2014) HSP27, 70 and 90, anti-apoptotic proteins, in clinical cancer therapy (Review). Int J Oncol
45(1):18-30.
27. Morrow G, Hightower LE, Tanguay RM (2015) Small heat shock proteins: big folding machines. Cell Stress Chaperones 20(2):207-212.
28. Garrido C, Gurbuxani S, Ravagnan L, Kroemer G (2001) Heat shock proteins: endogenous modulators of apoptotic cell death. Biochem
Biophys Res Commun 286(3):433-442.
29. Joly AL, Wettstein G, Mignot G, Ghiringhelli F, Garrido C (2010) Dual role of heat shock proteins as regulators of apoptosis and innate
immunity. J Innate Immun 2(3):238-247.
30. Javid B, MacARy PA, Lehner PJ (2007) Structure and function: heat shock proteins and adaptive immunity. J Immunol 179(4):2035-2040.
31. Jee H (2016) Size dependent classification of heat shock proteins: a mini-review. J Exerc Rehabil 12(4):255-259.
32. Sreedharan R, Van Why SK (2016) Heat shock proteins in the kidney. Pediatr Nephrol 31(10):1561-1570.
33. Kennedy D, Jäger R, Mosser DD, Samali A (2014) Regulation of apoptosis by heat shock proteins. IUBMB Life 66(5):327-338.
34. Welch WJ, Feramisco JR (1984) Nuclear and nucleolar localization of the 72,000-dalton heat shock protein in heat-shocked mammalian
cells. J Biol Chem 259(7):4501-4513.
35. Hasday JD, Singh IS (2000) Fever and the heat shock response: distinct, partially overlapping processes. Cell Stress Chaperones 5(5):471480.
36. Feder ME, Hofmann GE (1999) Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological
physiology. Annu Rev Physiol 61:243-282.
37. Doberentz E, Genneper L, Böker D, Lignitz E, Madea B (2014) Expression of heat shock proteins (HSP) 27 and 70 in various organ systems
in cases of death due to fire. Int J Legal Med 128(6):967-978.
38. Doberentz E, Genneper L, Wagner R, Madea B. Expression times for hsp27 and hsp70 as an indicator of thermal stress during death due to
fire. Int J Legal Med. 2017. doi: 10.1007/s00414-017-1566-x.
39. Preuss J, Dettmeyer R, Poster S, Lignitz E, Madea B. The expression of heat shock protein 70 in kindneys in case of death due to hypothermia.
Forensic Sci Int 2008; 176:248-252.
40. Doberentz E, Führing S, Madea B. HSP27 and 70 expression in heart, lung and kidney in SIDS. Rom J Legal Med 24:248-252.
41. Doberentz E, Führing S, Madea B. Sudden infant death syndrome: no significant expression of heat-shock proteins (HSP27, HSP70).
Forensic Sci Med Pathol 2016; 12:33-39.
42. Dias da Silva D, Silva E, Carmo H. Combination effects of amphetamines under hyperthermia – the role played by oxidative stress. J. Appl.
Toxicol 2014; 34:637-650.
43. Felgenhauer N, Zilker T. Intoxikation mit Amphetaminen und Designer-Drogen. Der Internist 1999; 40:617-623.
13