Histological assessment of myocardium in lethal ethanol intoxication

Elżbieta Litwiejko-Pietryńczak, Magdalena Szkudlarek, Anna Niemcunowicz-Janica, Andrzej Namiot, Beata Klim, Paweł Łaskowski, Janusz Dzięcioł

Medical University of Bialystok, Białystok, Poland

Address for correspondence:
Elżbieta Litwiejko-Pietryńczak, MD, PhD, Department of Human Anatomy, Medical University of Bialystok, ul. Mickiewicza 2A, 15–089 Białystok, Poland,
e-mail: litwiejko@tlen.pl
Received: 05.06.2014 Accepted: 22.01.2015 Available as AoP: 25.03.2015

INTRODUCTION

Diseases of the cardiovascular system constitute some of the most frequent causes of death in the adult population of economically developed countries, including Poland [1]. The steadily growing rate of alcohol consumption goes hand in hand with an increased number of sudden deaths of apparently healthy persons [2]. The majority of studies have so far focused on the evaluation of the impact of chronic alcohol consumption on the viscera, with special attention paid to the liver. However, the influence of acute incidental alcohol intoxication on the myocardium and, in particular, of the consumption of high-percentage alcohol in large quantities within a relatively short period of time have not yet been scrutinised. Therefore, the pathological mechanism leading to the sudden death which sometimes occurs after an incidental ethanol intoxication is not precisely known [3–5]. The dysfunction of the circulatory and respiratory centres in the brainstem is believed to be one of the causes of sudden death. It is also supposed that some typical cardiogenic causes (the pathology of cardiomyocytes, cell skeleton and extracellular matrix, as well as alterations in the coronary vessels and coagulation system) may significantly participate in the mechanism of death [6–8]. Also, the electrolytic disturbances accompanying hypoxia of the myocardium may play an important role because they lead to electric instability of the heart, which may result in death caused by ventricular tachycardia or ventricular fibrillation [9]. The aim of the study was a histological assessment of the myocardium in lethal ethanol intoxication.

METHODS

The study, approved by the Bioethical Commission of the Medical University of Bialystok (No. R-I-002/129/2008), was based on a histological assessment of specimens of the myocardium obtained from the hearts of 30 deceased males within the age range 29–45 years, who died after an incidental ethanol intoxication (hereafter referred to as the alcohol-exposed group). Autopsies were carried out in the Department of the Forensic Medicine at the Medical University of Bialystok. The results of the post-mortem autopsies conducted in the studied group of males did not reveal other causes of death. The subjects had not been previously treated. The medical history provided by their relatives excluded the occurrence of the following illnesses: chronic ischaemic cardiac disease, cardiac infarction, cardiomyopathy, arterial hypertension, cerebral stroke, diabetes, chronic renal disease, and alcohol and drug abuse. Before death, the studied males had occasionally (2–3 times per month) consumed moderate amounts of alcohol. The only explicable factor triggering their sudden death was an alcohol intake confirmed by its concentration in the blood and urine samples. The control group (n = 10) comprised the myocardium collected from deceased males within the age range 20–30 years, victims of a violent death, whose blood and urine did not reveal the presence of alcohol (hereafter referred to as the control group). The direct cause of their death was a post-traumatic intracranial haemorrhage. Prior to the lethal traumatic injury, the males in the control group had been healthy. Their post-mortem examination did not reveal any significant lesions in the other viscera.

Alcohol concentration in the blood and urine of the deceased patients was evaluated by means of the gas chromatography method and the use of a flame-ionisation detector. The hearts were opened according to the standard procedure, blood and blood clots were washed out, and approximately 1 cm of the ascending aorta and the pulmonary trunk were included. The hearts were weighed during the autopsy with a precision to the nearest tenth of a gram. The material for the histological examination was obtained from the myocardium of the anterior wall of the left ventricle and interventricular septum of the researched hearts. Three specimens of the myocardium of the left ventricle were collected from each heart: from the superior, middle, and inferior part of the anterior wall of the left ventricle at a distance of 1 cm from the anterior interventricular sulcus, which is the place of the course of the anterior interventricular branch of the left coronary artery. Specimens were also collected from the muscular part of the interventricular septum in its superior, middle, and inferior part. All the specimens were of the size 10 × 10 × 1 mm. The obtained material was fixed in 10% buffered formalin. After 24-h fixation, the material was embedded in paraffin blocks and cut on the microtome into 6-mm paraffin sections that were subsequently stained with haematoxylin and eosin according to the standard histological procedure. Afterwards, the material was subjected to a histomorphometric analysis performed in the Department of Human Anatomy of the Medical University of Bialystok with the use of an Olympus B×41 microscope equipped with an Olympus DP20 camera, and computer software for the histomorphometric analysis — Olympus Cell D Imaging Software for Life Science Microscopy. The histomorphometric examination in the alcohol-exposed group and control group was carried out at a magnification of ×400. Transverse sections of three randomly chosen microscopic fields were examined focusing on the following morphometric parameters of cardiomyocytes: surface area, circumference of myocardium fibres, and the number of myocardium fibres. The shape of cardiomyocytes (circular deviation — CD) was assessed by applying the formula CD = 4 µA/C2. Mean values for the examined parameters were defined in the alcohol-exposed group and control group. The results were then analysed and elaborated by means of the computer programme Statistica 10PL. Statistical analysis was performed with the t-test (statistical significance) and the U Mann-Whitney test. Statistical significance was accepted for p < 0.05.

RESULTS

As far as the alcohol-exposed group was concerned, the concentration of ethanol in the analysed blood samples stood at between 2.5 and 4.4‰ [3.73 ± 0.33], whereas in the analysed samples of urine it amounted to between 2.6 and 5.4‰ [4.19 ± 0.72]. Nothing more than trace amounts of endogenous alcohol (less than 0.5‰) were revealed in the control group. Table 1 depicts the statistical differences in alcohol concentration in the alcohol-exposed group and control group. No significant difference in the heart weight was seen between the examined groups. The mean heart weight in the alcohol-exposed group was 352 ± 52 g, whereas in the control group it stood at 348 ± 58 g. Statistically significant differences between the groups concerned the number, area, and circumference of their cardiomyocytes (Fig. 1). A lower number of cardiomyocytes in the microscopic field in transverse sections and a hypertrophy of individual cardiomyocytes with a significant enlargement of cell nuclei (Fig. 2) was observed in the alcohol-exposed group. The said cardiomyocytes exhibited also an increased area and circumference of fibres. Additionally, fragmentation and segmentation of individual cardiomyocytes with a significant enlargement of cell nuclei was observed in 26 cases. The number of cardiomyocytes was higher in the control group (Fig. 3). There were no statistically significant differences between the alcohol-exposed group and the control group with regard to the parameter of CD. The presence of a small number (1–3) of lymphocytic cells was noticed in several cases of the microscopic fields. No morphological features typical of cardiomyopathy were seen in either group.

Table 1. Concentration of ethanol in urine and blood and heart weight

Figure 1. Values of the analysed morphometric parameters in the alcohol-exposed group (GI) and control group (G II); A. Number of cardiomyocytes in the microscopic field; B. Area of cardiomyocytes in the transverse section [μm2]; C. Circumference of cardiomyocytes in the transverse section [μm]; D. Circular deviation

Figure 2. Transverse section of cardiomyocytes in the alcohol-exposed group (HE, ×400). Visibly lower number of cardiomyocytes in the microscopic field and hypertrophy of individual cardiomyocytes with a significant enlargement of cell nuclei

Figure 3. The transverse section of normal cardiomyocytes in the control group (HE, ×400)

DISCUSSION

The most frequent causes of sudden cardiac death (SCD) in the general population are either ventricular arrhythmias or electromechanical dissociation of the heart. It is estimated that ventricular arrhythmias such as ventricular fibrillation or stable ventricular tachycardia are responsible for 60–80% of SCD cases, whereas electromechanical dissociation, atrioventricular block and sinusal arrest account for 20–40% of them. In 4% of cases, SCD may be the first symptom of post-alcoholic dilated cardiomyopathy [9, 10]. Despite the enlargement of cardiomyocytes observed in our study, no morphological features of cardiomyopathy were found in the examined heart structures. Among the factors inducing an arrhythmogenic environment in the myocardium the following are usually noted: structural alteration resulting from hypoxia, inflammatory process, toxic action of various chemical substances including ethanol and haemodynamic factors, disturbance of the ionic balance, and disturbance of the regulation of the autonomic nervous system [11–14]. The results of studies carried out in vitro and on animals suggest that alcohol may activate the apoptosis of cardiomyocytes, severely impair the function of mitochondria and the sarcoplasmatic reticulum, affect the expression of sarcomeric proteins, and result in disturbances of calcium metabolism in cardiomyocytes, which may produce serious consequences. In the group of males who died after an incidental ethanol intoxication we observed the following types of significant alterations in the myocardium: hypertrophy of individual cardiomyocytes with a significant enlargement of cell nuclei, and a lower number of cardiomyocytes in the microscopic field in transverse sections under ×400 magnification. It is worth underlining that Figures 2 and 3 convey a false visual illusion of the two different magnifications owing to the fact that the cardiomyocytes examined in the alcohol-exposed group had an increased area and circumference of fibres (Fig. 1) compared to the cardiomyocytes in the control group (Fig. 3). No statistically significant differences were found with regard to CD. Fragmentation and segmentation of individual cardiomyocytes with a significant enlargement of cell nuclei was observed in 26 subjects from the alcohol-exposed group. In seven subjects from this group we observed the presence of a small number (1–3) of lymphocytic cells in the microscopic fields. The higher the ethanol concentration found in the blood and urine of a deceased person, the more intensive the disturbances in the examined histopathological specimens. In the control group, no significant pathology of the histomorphological structure of the examined samples of the myocardium was observed. Our study suggests that the toxic influence of alcohol depends on its dose, but according to other authors it is not a linear dependence [15]. In addition, as we examined only a group of males, we cannot refer to another interesting effect that is widely known, i.e. an increased sensitivity to the toxic influence of alcohol in females. Furthermore, previous studies concerning the influence of alcohol on the human organism focused primarily on persons addicted to alcoholic beverages [16–18], which makes any comparison between their results and ours difficult to make.

CONCLUSIONS

The obtained results indicate a harmful influence of alcohol on the structure of myocardium. The observed destructive alterations in the myocardium may induce circumstances conducive to an electric destabilisation of the heart, which may result in sudden death in the mechanism of arrhythmia.

Conflict of interest: none declared

References

1. 1. Dalen JE, Alpert JS, Golgberg RJ, Weinstein RS. The epidemic of the 20th century: coronary heart disease. Am J Med, 2014; 127: 807–812. doi: 10.1016/j.amjmed.2014.04.015.
2. 2. O’Keefe JH, Bhatti SK, Bajwa A et al. Alcohol and cardiovascular health: the dose makes the poison…or the remedy. Mayo Clin Proc, 2014; 89: 382–393. doi: 10.1016/j.mayocp.2013.11.005.
3. 3. Kelly P, Coats A. Variation in mode of sudden cardiac death in patients with dilated cardiomyopathy. Eur Heart J, 2014; 18: 879–880.
4. 4. Bansch D, Antz M, Boczor S et al. Primary prevention of sudden cardiac death in idiopathic dilated cardiomyopathy. The Cardiomyopathy Trial. Circulation, 2002; 105: 1453–1458. doi: 10.1161/01.CIR.0000012350.99718.AD.
5. 5. Fauchier L, Babuty D, Poret P et al. Comparison of long-term outcome of alcoholic and idiopathic dilated cardiomyopathy. Eur Heart J, 2000; 21: 306–314.
6. 6. Janica J. Morfologiczne aspekty ostrego zatrucia etanolem. Arch Med Sąd Krym, 1990; 40: 76–80.
7. 7. Grimm W, Rudolph S, Christ M, Pankuweit S, Maisch B. Prognostic significance of morphometric endomyocardial biopsy analysis in patients with idiopathic dilated cardiomyopathy. Am Heart J, 2003; 146: 372–376. doi: 10.1016/S0002-8703(03)00148-0.
8. 8. Bowles NE, Bowles KR Towbin JA. The “final common pathway” hypothesis and inherited cardiovascular disease. The role of cytoskeletal proteins in dilated cardiomyopathy. Herz, 2000; 25:168–175. doi: 10.1007/s000590050003.
9. 9. Fruchier L Alcoholic cardiomyopathy and ventricular arrhythmias. Chest, 2003; 123: 1320–1325.
10. 10. Rimm EB, Williams P, Fosher K, Criqui M, Stampfer MJ. Moderate alcohol intake and lower risk of coronary heart disease: meta-analysis of effects on lipids and haemostatic factors. BMJ, 1999; 319: 1523–1528.
11. 11. Wezeman FH, Gong Z. Adipogenic effect of alcohol on human bone marrow-derived mesenchymal stem cells. Alcohol Clin Exp Res, 2004; 28: 1091–1110. doi: 10.1097/01.ALC.0000130808.49262.F5.
12. 12. Fumeron F, Betoulle D, Luc G et al. Alcohol intake modulates the effect of a polymorphism of the cholesterol ester transfer protein gene on plasma high density lipoprotein and the risk of myocardial infarction. J. Clin. Invest, 1995; 96: 1664–1671.
13. 13. Wojnicz R, Nowalany-Kozielska E, Wojciechowska C et al. Randomized, placebo-controlled study for immunosuppressive treatment of inflammatory dilated cardiomyopathy: two-year follow-up results. Circulation, 2001; 104: 39–45.
14. 14. Mason JW. Myocarditis and dilated cardiomyopathy. Cardiovasc Res, 2003; 60: 5–10.
15. 15. Kajander OA, Kupari M, Laippala P et al. Dose dependent but non-linear effects of alcohol on the left and right ventricle. Heart, 2001; 86: 417–423.
16. 16. Piano MR. Alcoholic cardiomyopathy: incidence, clinical characteristics, and pathophysiology. Chest, 2002; 121: 1638–1650.
17. 17. Nicolas JM, Fernandez-Sola J, Estruch R et al. The effect of controlled drinking in alcoholic cardiomyopathy. Ann Intern Med, 2002; 136: 192–200.
18. 18. Ga V Azzi A, De Maria R, Parolini M, Porcu M. Alcohol abuse and dilated cardiomyopathy in men. Am J Cardiol, 2000; 85: 1114–1118. doi: 10.1016/S0002-9149(00)00706-2.