Myofiberbreak-up: A marker of ventricular fibrillation in sudden cardiac death

International Journal of Cardiology 100 (2005) 435 – 441 www.elsevier.com/locate/ijcard

Myofiberbreak-up: A marker of ventricular fibrillation in sudden cardiac death Giorgio Baroldia, Malcom D. Silverb, Marina Parolinia, Cristoforo Pomarac, Emanuela Turillazzic, Vittorio Fineschic,T a Institute of Clinical Physiology, National Research Council (CNR), Pisa and Milan, Italy Department of Laboratory Medicine and Pathobiology, University Health Network, University of Toronto, Toronto, Canada c Department of Forensic Pathology, University of Foggia, Ospedali Riuniti, Via L. Pinto, 1, 71100, Foggia, Italy

b

Received 28 March 2004; received in revised form 8 August 2004; accepted 4 October 2004 Available online 19 February 2005

Abstract Background: Electrophysiologically, ventricular fibrillation is defined as a bchaotic, random, asynchronous electrical activity of the ventricles due to repetitive re-entrant excitation and/or rapid focal dischargeQ. To this point its morphological equivalent has not been defined. Material and method: Several groups of different diseases and types of accidental death in normal subjects were studied. A complete autopsy was performed and the hearts were examined in 432 cases. A total of 16 myocardial samples per heart were processed for histological examination and sections were stained by haematoxylin and eosin or by specific stains. The frequency, location and extent of myocellular segmentation (stretching and/or rupture) of intercalated discs and associated changes of myocardial bundles and single myocells were investigated. A quantitative analysis was performed and the data were processed for statistical evaluation. Results: The frequency of MFB was maximal in coronary (88%) and Chagas (76%) groups followed by the intracranial brain haemorrhage group (52%). The extent of myofiberbreak-up was maximal in coronary/Chagas groups followed by intracranial haemorrhage and transplant groups. Conclusions: No correlation was seen between gender, age, heart weight, degree of coronary atherosclerosis, myocardial fibrosis, survival and MFB. If our postulate is correct, finding MFB in the myocardium might allow the diagnosis of a malignant arrhythmia followed by cardiac arrest due to ventricular fibrillation even in the absence of clinical information (sudden death out-of-hospital). D 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Ventricular fibrillation; Cardiac death; Myofiberbreak-up

1. Introduction Electrophysiologically, ventricular fibrillation is defined as a bchaotic, random, asynchronous electrical activity of the ventricles due to repetitive re-entrant excitation and/or rapid focal dischargeQ [1]. To this point its morphological equivalent has not been defined. In a previous study of selected cases of sudden and unexpected death [2], we learned [3] to distinguish the typical contraction band lesion [4] of catecholamine myo-

T Corresponding author. Tel.: +39 0881 733835; fax: +39 0881 732180. E-mail address: [email protected] (V. Fineschi). 0167-5273/$ - see front matter D 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2004.10.007

toxicity from a myocardial change marked histologically by a break-up of myocardial fibers (myofiberbreak-up or MFB) [5]. Fragmentatio miocardii in the old literature [6] meant a linear rupture along the myocardial cell course (fragmentation) or at the disc level (segmentation). A systematic autolysis investigation on the heart was performed by Nicolas [7]. The onset of fragmentation of heart muscle fibers occurred at 12–18 h post-mortem: this finding is easy to recognize in longitudinal cell sections [8]. If fragmentation is likely artifactual [9], however, a relationship between segmentation and contracted myocells was mentioned long ago [6] and related to ventricular fibrillation [10]. A possibility afterwards totally ignored.

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The aims of this report are to a) define the morphology and frequency of this structural break-up; b) exclude the possibility that it is an artefact of rigor mortis, autolysis or histological preparation; c) determine if it has any relationship to either therapy or other forms of myonecrosis; d) discuss its pathophysiology and e) comment on the significance of this change particularly in the forensic pathology setting.

2. Material and method 2.1. Study population To explore the meaning of MFB several conditions were matched, each with some distinctive points that could aid interpretation. Thus, sudden and unexpected coronary and Chagas’s death and that from intracranial haemorrhage all showed a high frequency of focal myocardial necrosis typical of catecholamine myotoxicity. The AIDS group included young individuals who died of opportunistic infectious diseases with a long terminal period and with many types of therapeutic interventions. The end-stage congestive heart failure group represented three main categories of disease, i.e. ischemic heart disease, dilated cardiomyopathy of unknown origin and chronic valvulopathy; its common denominator was cardiac arrest by surgical excision without any further manoeuvres. Transplanted heart group is an example of denervated heart with consequent increased sensitivity of the myocardium to catecholamines [11,12]. The cocaine group was included due to a recognised increased adrenergic activity [13]. The other three groups were selected as healthy people who died of various types of accidents. Thus, hearts from the following groups of people were examined: 1. Coronary group. Twenty-five bhealthyQ subjects, who had no history of ischemic heart disease, or other cardiac or non-cardiac diseases, died suddenly and unexpectedly out-of-hospital while enjoying their usual lifestyle. Thirteen died within a few minutes

according to witnesses and were found dead. A rescue team made no resuscitation attempts. The other 12 received resuscitation and were monitored by electrocardiogram. In 21 cases pathologic findings other than coronary atherosclerosis, contraction band necrosis (CBN) old foci of myocardial fibrosis and cardiac hypertrophy were not found at autopsy. In four cases CBN was associated with a silent acute infarct with a histological age ranging from 8 h to 7 days. Tests for intoxication and poisoning were negative. 2. Chagas group. Thirty-four apparently healthy subjects who died suddenly and unexpectedly were serum positive for Chagas’s disease at autopsy [14]. None had a clinical history of cardiac disease, medical care or therapy, resuscitation attempts, significant postmortem changes in organs other than heart or a positive test for poisoning or intoxication. According to witnesses the terminal episode lasted a few minutes. 3. Intracranial haemorrhage group. These 27 patients died of a ruptured cerebral berry aneurysm, all without a history of cardiac disease. Thirteen survived less, and 14 more than one a day [15]. 4. Transplant group. Forty-six patients with irreversible congestive heart failure of various causes who had a heart transplant. The patients subsequently had a survival ranging from less than 1 week to more than 3 years. 5. AIDS group. Thirty-eight cases who had acquired immunodeficiency syndrome and died of non-cardiac opportunistic infectious disease[16]. 6. Congestive heart failure (CHF) group. This group included surgically excised hearts at transplantation from 63 patients with chronic ischemic heart disease, 63 with dilated cardiomyopathy of unknown cause and 18 with chronic valvulopathy, all patients being in end-stage congestive heart failure. At surgery none had electrocardiographic arrhythmias [17]. 7. Cocaine group. Twenty-six subjects dead from cocaine intoxication, 18 of whom died out-of-hospital without any emergency therapy and eight in hospital [18].

Table 1 Main characteristics of groups Groups Coronary Chagas Intracranial haemorrhage Transplant AIDS CHF Cocaine Head trauma Electrocution CO

Cases 25 34 27 46 38 144 26 45 21 26

Gender

Age (yrs)

M

W

21 26 6 39 33 130 20 37 21 20

4 8 21 7 5 14 6 8 – 6

T Hearts excised at transplantation with adjusted heart weight.

61F3 49F2 59F2 49F1.5 31F2 47F1 35F1 42F3 36F4 48F3

Heart weight (g) 562F25 464F28 427F19 419F19 368F11 473F10 351F10 364F10 347F9 375F14

Mode of death Sudden unexpected Sudden unexpected In hospital In hospital In hospital Hearts excised at surgeryT 18 in and 8 out of hospital Accidental Accidental Accidental

Resuscitation No

Yes

13 34 0 0 0 0 8 45 21 26

12 0 27 46 38 0 18 0 0 0

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8. Head trauma group. Forty-five individuals who died following head trauma; 26 dying instantaneously and 19 within a few hours [15]. 9. Electrocution group. Twenty-one subjects who died instantaneously of electrocution. 10. CO group. Twenty-six normal subjects found dead at home because of carbon monoxide intoxication [19]. The last three groups include normal subjects, who did not present any other pathology at autopsy, including critically obstructive coronary atherosclerosis. In all but CHF group, a complete autopsy was performed on a refrigerated body within 24 h of death. The main characteristics of the different groups are reported in Table 1. 2.2. Definitions Myofiberbreak-up includes the following histologic patterns: a) bundles of myocardial cells in distension alternated with hypercontracted ones. In the latter, widening or rupture (segmentation) of the intercalated discs occurs. Myocardial nuclei in the hypercontracted cells assume a bsquareQ aspect rather than the ovoid morphology seen in distended myocells (Fig. 1) in line hypercontracted myocells alternated with hyperdistended ones, divided by an often widened disc (Fig. 2A and C) non-eosinophilic band of hypercontracted sarcomeres alternated with stretched, often apparently separated sarcomeres (Fig. 2B). 2.3. Heart examination The method of heart examination has been published previously [3]. In brief, in the first six groups the heart was washed, weighed and, after 24 h of fixation in buffered 10% formaldehyde solution, coronary arteries and main branches were cross sectioned at 3 mm intervals along their whole course. Subsequently the entire, unopened heart was sliced

Fig. 2. (A) Stretching of myocells and widening of sarcomers in line with hypercontracted myofibers (arrows) (H & E250); (B) non-eosinophilic band of hypercontracted sarcomeres alternated with stretched, often apparently separated sarcomeres (H & E100).

by a machine in 1 cm thick sections from apex to base. Systematic samples of the coronary arteries were taken at their origins and where there was a lumen reduction greater than 50% to the naked eye. Samples of the myocardium were removed from the following eight sites, i.e. anterior, lateral and posterior whole wall of both left and right ventricles, anterior and posterior interventricular septum. They were also taken from a superior and an inferior heart slice. A total of 16 myocardial samples per heart were processed for histologic examination and sections were stained by haematoxylin and eosin or by specific stains (Mallory, Weigert elastic, Movat pentachrome, Afog) when needed. According to the surgical procedure, the hearts excised at transplantation were free of atria. For comparative purposes, with the end-stage congestive heart failure group, the heart weight recorded includes the theoretical atrial weight expressed as one-fourth of the total muscle mass (actual weight4/3) [20]. In the last four groups hearts were grossly examined in the same way but sampling of the myocardium was limited to one to four blocks of the whole anterior wall of the left ventricle with sections stained as above. 2.4. Quantitative analysis

Fig. 1. Square nuclei (arrows) in hypercontracted myocells (H & E400).

The area of each myocardial histologic slide was measured in square millimetres by a colour video camera connected to a

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Table 2 Frequency, location and extent of myofiberbreak-up Groups

Cases

Frequency

Location LV

Coronary Chagas’ Intracranial haemorrhage Transplant AIDS CHF Cocaine Head trauma Electrocution CO

Extent (sites)

RV

IVS

LV+RV

LV+IVS

RV+IVS

LV+RV+IVS

2

3

4

5

6

7

8

25 34 27

22 26 14

– 1 3

– – –

1 – 1

– 3 –

12 5 2

1 – 1

8 17 7

1 1 – 2

1 1 –

2 1 2

4 3 –

6 3 4

2 4 –

2 – –

4 14 6

46 38 144 26 45 21 26

20 9 – 11 29 19 5

1 2 – 11 29 19 5

– – – – – – –

– – – – – – –

2 1 – – – – –

9 5 – – – – –

1 – – – – – –

7 1 – – – – –

– 1 – 114 294 194 54

2 – –

1 2 –

5 1 –

6 4 –

– – –

– – –

6 1 –

LV, left ventricle; RV, right ventricle; IVS, interventricular septum. T In each heart eight samples sites were examined. Only the free wall of the anterior left ventricle was examined in cocaine and accidental death groups.

light microscope and converted to a digitalized image by an analysis system (Vidas, Zeiss, Germany). The number of foci and necrotic myocells were counted and normalised to 100 mm2. Myocardial fibrosis was calculated as a percentage of the total area examined. The extent of MFB was estimated in the first six groups. In all hearts eight sites with two samples for each site were histologically examined. Its grade was related to how many of the eight sites showed MFB. 2.5. Statistical evaluation Data are expressed as mean valueFone standard error. To assess statistical significance Student’s t-test for paired or unpaired data or one way analysis of variance and post hoc Scheffe’s test for continuous variables and chi-square test for discrete variables were used where appropriate. Linear regression analysis was used to assess the presence of

correlation between continuous variables. A probability p measured value b0.05 was considered significant.

3. Results The frequency of MFB was maximal in coronary (88%) and Chagas (76%) groups followed by the intracranial brain haemorrhage group (52%). No relation was found with resuscitation therapy. In all groups the anterior left ventricular wall was more frequently involved ( pb0.06). The extent of myobreakup was maximal in coronary/ Chagas groups followed by the intracranial haemorrhage and transplant groups (Table 2). No statistical significance was found when the frequency and extent of MFB were compared with the number of foci and myocells with CBN (Table 3). However, when only the anterior left ventricle was considered in order

Table 3 Frequency and extent of myofiberbreak-up versus contraction band necrosis (CBN) foci (F) and myocells (M)100 mm2 Groups

Coronary +Silent acute infarct No infarct ResuscitationTT No resuscitationTT Chagas Intracranial haemorrhage Transplant AIDS CHF Cocaine Head trauma Electrocution CO

Myofiberbreakup Absent

Present

b4 sites

N4 sites

CBN

CBN

CBN

CBN

F

M

F

M

F

M

F

M

– – – – – 2F1.8 38F12 27F8 2F0.7 2F0.3 1F0.3 1F0.4 – 2

– – – – – 14F12 82F26 172F41 10F5 11F2 2F0.3 4F0.4 – 9

28F8 29F10 27F10 39F16 9F2 4F2 15F5 29F7 9F8 – 8F2 13F7 8 1F0.6

508F200 1717F698 185F48 241F65 102F59 40F21 52F34 273F72 21F14 – 25F6 28F13 46 4F2

18F4 25F4 15F5 24F5 6F2 19 13F11 35F13 14F14 – –T –T –T –T

224F91 551F199 115F56 177F106 52F27 78 18F12 287F120 26F24 – – – – –

35F14 33F23 36F16 47F24 13F4 2F1.3 15F6 25F10 2F1.5 – – – – –

715F332 2883F404 233F68 273F86 152F118 37F22 63F45 266F95 14F13 – – – – –

T Only left anterior ventricle examined. TT Four silent acute infarct cases excluded.

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Table 4 Myofiberbreak-up in anterior left ventricle vs contraction band necrosis foci (F) and myocells (M)100 mm2 Groups

Cases

Myofiberbreakup in ALV Present

Absent CBN

Coronary +Silent acute infarct No infarct Resuscitation44 No resuscitation44 Chagas’ Intracranial haemorrhage Transplant AIDS CHF Cocaine Head trauma Electrocution CO

25 4 21 10 11 34 27 46 38 144 26 45 21 26

17 3 14 7 7 26 14 20 9 – 11 29 19 5

CBN

F

M

51F21 31F16 57F27 81F48 28F8 5F3 13F7 35F14 12F10 – 8F2 13F7 8 1F0.6

1160F643 3564F2225 358F152 468F267 221F94 65F51 56F38 453F166 22F16 – 25F7 28F13 46 4F2

8 1 7 3 4 8 13 26 29 144 17 16 2 21

F

M

15F7 – 15F7 3.8 19F9 3F1.3 41F14 57F19 5F2.3 3F0.7 1F0.2 1F0.4 – 2

47F18 – 47F18 19 56F22 8F4 135F71 229F69 21F13 19F4 2F0.4 4F0.4 – 9

TT Four silent acute infarct cases excluded.

to compare all groups, a higher extent of CBN was seen in the presence of MFB in most groups (Table 4). This finding was significant in the cocaine group ( pb0.01). No correlation was seen between gender, age, heart weight, degree of coronary atherosclerosis, myocardial fibrosis, survival and MFB. In all hearts excised at transplantation this change was absent. In normal controls, it was present in a few CO intoxication cases (19%), in most cases of electrocution (90%) and in 64% of the subjects with head trauma (Table 2). Fifteen hearts, one sudden death in a bsilentQ AIDS case, seven from patients with intracranial haemorrhage and seven sudden and unexpected coronary death cases—the only ones with some vital signs—all monitored by electrocardiogram at the time of death, showed malignant arrhythmia/ventricular fibrillation. All had a histologic pattern of extensive (more than four cardiac sites) MFB.

4. Discussion Amongst the three types of cardiac arrest described in the literature, i.e. asystole secondary to neurally-mediated bradycardia [21], electromechanical dissociation with loss of pulse, heart sounds, blood pressure and consciousness in the presence of a normal electrocardiogram [22] and ventricular fibrillation, the latter is particularly frequent in ischemic heart disease as shown in defibrillated people [23,24] and patients dying suddenly while monitored by a Holter machine [25]. The MFB here described could be interpreted as a morphologic counterpart of a terminal dysfunction ending in ventricular fibrillation (VF), giving a structural background to the chaotic electrical asynchronous activity [1]. Two aspects should be considered: a) the first is the pathogenic mechanism which starts syncytial myocardial

dysfunction; b) MFB appears vital, even if a terminal phenomenon and it is totally different from those changes seen in post-mortem decomposition or autolysis by enzymatic softening and liquefaction of a putrefying myocardium [26]. a) The morphologic aspect of MFB indicates a structural chaotic state of contraction at different level (bundles or myocells). However, the extent of this morpho-functional disruption ranges from small foci in one area to a diffuse involvement of most or all cardiac regions. One may speculate that, independent from its extent, ventricular fibrillation may be triggered (re-entry or rapid focal discharge) by a small focus or a large one, respectively in expression of short or long-lasting terminal malignant arrhythmia. In turn, successful or unsuccessful defibrillation could be related to the extent of this damage. All our cases monitored electrically at the time of death showing malignant arrhythmia with unsuccessful resuscitation, had MFB extended at more than four sites. One notes that all morphologic findings as focal myocardial necrosis or fibrosis, reported in the literature and supposed to be the cause of re-entry or focal discharge are inconsistent, since they are too often unrelated to electrical instability. b) The MFB changes we described seem to be vital. Their absence in all hearts excised at transplantation questions the belief that they are artifactual or secondary to histological processing; the artifactual myofiber fragmentation due to the knife in cutting the histologic section being easily distinguished from MFB. On the other hand, similar changes were never described as part of rigor mortis of the myocardium [26]. Furthermore, if rigor mortis was the cause, MFB should be a regular finding in all cases autopsied after 1–24 h. One notes that ventricular fibrillation per se is not the cause of MFB even if it is prolonged 25 min in dog (unpublished data)

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or for hours [27]. In fact, when ventricular fibrillation starts, almost immediately there is a loss of pump function with consequent severe reduction or abolishment of coronary flow [28]. The maintenance of some contractility in the absence of nutrient flow is unlikely as shown in experimental coronary occlusion where regional sudden loss of blood flow determines loss of contractility within a few seconds [29]. The main cause of malignant arrhythmia and ventricular fibrillation is generally considered to be ischemia. Rather than the latter, we believe that an unbalanced neuroregulation within the myocardium is the cause. This opinion is based on the following points: a) selected sudden and unexpected death cases show a unique acute myocardial lesion pathognomonic of catecholamine myotoxicity, i.e. CBN as a signal of acute adrenergic stress; b) experimental prevention of both catecholamine myotoxicity and ventricular fibrillation by betablockers following acute coronary occlusion [30] c) a higher extent of histologic catecholamine damage in the presence of MFB. The absence of malignant arrhythmia in experimental catecholamine intravenous infusion despite myocardial damage proportional to an increasing dose [31,32] suggests that only a regional or zonal intramyocardial discharge from nerve endings may be responsible for syncytial disruption. In dogs, noradrenaline infusion in one coronary artery determined an immediate, severe electrical instability with an episode of ventricular fibrillation (experiment in progress). The morphologic demonstration of catecholamine myotoxicity, independently of its extent, is a hallmark of a local adrenergic overdrive unrelated to ischemia [33]. The adrenergic lesions are present in ischemic and non-ischemic zones following experimental acute coronary occlusion in dog, associated with MFB in most animals (presented for publication). In man, the adrenergic damage may depend from several factors such as nerve reflexes in response to regional asynergy, lymphoplasmacell inflammation around nerves adjacent of the coronary media associated with atherosclerotic plaque [3] or possible intramural nerve destruction as in Chagas’s disease [14] or in stunned myocardium [34]. A variety of conditions—coronary heart disease, congestive heart failure, global or regional denervation, etc—and pathogenetic mechanisms with a common denominator i.e. adrenergic stress linked with an overexpression of b-adrenergic receptors in turn related with free radicals lipid peroxidation and intramyocellular calcium influx [35–38]. If our postulate is correct, finding MFB in the myocardium might allow the diagnosis of a malignant arrhythmia followed by cardiac arrest due to ventricular fibrillation even in the absence of clinical information (sudden death out-of-hospital). The obvious limitation of the study is that this autopsy findings only rarely can be confirmed by a terminal electrocardiographic record. A systemic study to confirm

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