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Alcoholic Dilated Cardiomyophathy, Congestive Heart Failure and Apoptosis
Héctor L. Balbarrey1, Juan C. Picena2 and Edgardo E. Guibert3
2Cátedra de Anatomía y Fisiología Patológicas, Facultad de Ciencias Médicas, UNR
3Biología Molecular, Facultad de Ciencias Bioquímicas y Farmacéuticas, UNR
Material and Methods
Background: Severe congestive heart failure (C.H.F.) is a multisystemic syndrome of multiple etiology, with sombre prognosis. The cellular mechanisms responsible for the progressive deterioration of myocardial function in heart failure remain unclear and might be due to apoptosis (programmed cell death).
Objectives: to carry out a retrospective revision of patients deceased 30 years ago at the Hospital del Centenario, Rosario City, with C.H.F. due to alcoholic dilated cardiomyopathy (ADCM) and the search for apoptosis.
Methods: Six out of 14 male patients (p.) with ADCM and C.H.F., functional classification NYHA x= 3.28 died. Necropsies of 5 p. were performed (age 39 ± 2 years). The follow up from the last hospitalization due to C.H.F. and death was x = 9.7 ± 2.1 months. The necropsies were studied under three aspects: a) revision and analysis of clinical data, b) revision of histopathological findings of sections obtained from paraffin-embedded tissues preserved in Anatomía y Fisiología Patológicas, U.N.R., y c) in situ detection of apoptosis by labelling of DNA strand breaks associated with apoptosis using fluorescein-dUTP and Transferase Terminal enzyme, and specific DNA stain with D.A.P.I., a fluorescent compound. Samples from three other patients without myocardial disease, were used as controls.
Results: the revision of clinical records confirms the original diagnosis. Detailed histopathologic observation revealed the presence of apoptotic bodies, confirmed by specific reactions that detect fractured DNA.
Conclusions: the new methodology available, useful in spite of the elapsed time (30 years), allows us to deduct that in ADCM with C.H.F., apoptosis is present and could contribute to progressive deterioration and death of these patients.
Introduction: Almost thirty years ago twenty-four patients with alcoholic dilated cardiomyopathy were studied in the Hospital Nacional del Centenario (Rosario, Santa Fe)(1). The alcoholic cardiomyopathy is defined as "a lesion determined by the direct action of alcohol on the myocardial fiber" (2-4). Diagnosis was achieved taking into account the history of alcohol ingestion, namely, three to five liters of wine and 250 ml of distilled spirits (45 to 55 degrees) daily, during at least five years. Other known cardiomyopathies, such as Chagas disease, arterial hypertension, rheumatic heart disease, congenital heart diseases, coronary atheromatosis and valvular disorders were excluded (3). In ten patients, alcohol abstinence was followed by remission of signs and symptoms in a few weeks. In the rest of the patients (n=14) with severe cardiac failure signs and symptoms, the evolution was toward a progressive deterioration and irreversible damage, which led six patients to death in less than one year, after the last hospitalization. Five of them were autopsied and are analyzed in the present paper.
Since Kerr, J.F., Wyllie A.H., and Currie, AR describe the biological bases of apoptosis and their implications in human physiology, several reports have been presented in order to confirm its existence in different physiopathological processes (5). There are two main types of cell death: apoptosis and necrosis. Apoptosis, or its more current expression of programmed cell death, is an active process playing an essential role in the embryonic organogenesis and homeostasis in adult stage tissues (5,6). It is a genetically controlled process, and can be activated by an internal clock or by extracelular agents, such as hormones, citokines, reactive species of oxygen and toxic agents as alcohol (7). Necrosis is a passive accidental process in response to a severe hypoxia, ischemia, hyperthermia, viral infection or exposure to a great deal of chemical or physical agents (8). According to Narula et al. (9), who postulate that apoptosis is one of the cell mechanisms leading to end-stage heart failure, we thought in 1997, that applying techniques of molecular biology (10-12) to paraffin embedded material from autopsies of the above mentioned five patients, we could be able to confirm the presence of nuclear fragmentation. The latter were probable apoptotic myocardial bodies when examined by conventional light microscopy techniques. This could explain the rapid evolution and progressive deterioration, followed by death, in our patients with severe congestive heart failure (CHF), which is a multisystemic syndrome of varied etiology and somber prognosis. The cell mechanisms that could be responsible of the aggravated myocardial function are not clear indeed and could be due partly to the presence of apoptosis.
Objectives: To carry out a clinical and histopathological retrospective study of patients dead approximately thirty years ago in the Hospital del Centenario with CHF, functional class III-IV NYHA, produced by Alcoholic Dilated Cardiomyopathy (ADM) and demonstrate the presence of apoptosis.Top
Material and Methods: Myocardium samples, fixed in formaldehyde and embedded in paraffin, from the original 5 patients who died almost thirty years ago in the Hospital del Centenario, with CHF, functional class III-IV NYHA, produced by ADM. Cardiac tissue samples from patients dead also thirty years ago, but not from heart diseases, were utilized as controls.
The study undertaking the combination of three aspects was as follows:
Results: Revision and analysis of registered data confirmed the alcohol etiology. Six out of 14 alcoholic patients with severe CHF, average NYHA functional class 3.38 and mean age 41.9 died, and autopsy was performed on 5 of them (Table 1). Evolution in these patients after the last hospitalization, until their death by CHF was 9.7 +- 2.2 months. The patients did not present cachexia. The necropsies showed increased heart weight (between 400 and 650g) with hypertrophy and global dilation, with neither valve lesions nor obstructive atherosclerosis of large coronary vessels (Table 2). The histopathological examination revealed hypertrofic areas and yuxtanuclear vacuolization of myocardial cells. The sarcoplasm exhibited granular degeneration with microfibrillar fragmentation (autolytic cell death) and lipofuscin deposits. Some myocardial cells showed increased cytoplasm acidophilia with nuclear pycnosis and without polymorphonuclear neutrophiles infiltration (Fig 1A). Deformed nuclei with burgeoning edges and thickened membrane were occasionally observed (Fig 1B). Absence of atherosclerosis was constant. Edema was present in the interstitium, more prominent in the subendocardium. None of these alterations were present in control samples. Fig 2A presents the TUNEL technique image in a patient who also died 30 years ago, but without myocardial pathology was used as control. A myocardial fiber with nuclear fragmentation and positive TUNEL reaction is shown in Fig 2B. The nuclear blue fluorescence with condensed and fragmented chromatin among the cardiac muscle cells stained with DAPI is displayed in Fig 2C.
Sections of ventricular myocardium in alcoholic patients.
|Figure 1 A- Oblique section. The arrow points a thinned myocardial fiber, with marked increase of cytoplasmatic acidophilia and nuclear pyknosis (apoptosis?). (H/E, 200x)|
Figure 1 B- Cross section of myocardial fibers with disperse cytoplasmatic vacuolation, and interstitium edema. A pyknosis nucleus is pointed, with a half moon shape (apoptosis?). (H/E, 200x)
Sections of ventricular myocardium preserved for thirty years
Figure 2 A- Control sample treated with TUNEL, as is described in Methods. Yellow granules of lipofuscin (L) are observed. The nuclei, tainted in orange, do not present fluorescence. (200x)
Figura 2 B- Sample from a dead patient, with ADCM and CHF, treated with TUNEL, as is described in Methods. The fluorescent nucleus of a cell in apoptosis is pointed. (200x)
Figure 2 C- Sample from a dead patient with ADCM and CHF stained with DAPI. The nucleus of a cell with apoptosis is pointed with blue fluorescence. (200x)
Discussion: Fibrillar lesions seen in the alcoholic dilated cardiomyopathy are common in other metabolic disorders. The histological complex image and the peculiar lesions of small vessels, conjointly with a proper clinical observation and the positive history of severe alcoholism contribute to diagnosis (1, 3). In the last five years, after Kerr et al. (5) works, who defined "a little known mechanism of controlled removal of cells, fulfilling a complementary role, opposed to mitosis, in the regulation of animal populations", the description of apoptosis in different physiopathological processes has remarkably increased (13, 14), particularly regarding papers dealing with heart failure (15, 17). Consequently, we attempt to cast a fresh look at an old problem. In the histological revision we point out the existence of cells with increased cytoplasmic acidophilia, without inflammatory infiltrate (Fig 1A), as well as voluminous nuclei with burgeoning borders, alternating with pycnosis. These qualitative findings could be interpreted as apoptosis (5). The TUNEL and DAPI reactions (Fig 2B and Fig 2C) evidence the DNA fragmentation, confirming the presence of apoptosis. We must admit that these techniques could fail to discriminate among apoptosis, necrosis and autolytic cell death (18). However, we think that the combination of both methods to detect in situ fragmented DNA, plus a thorough histological examination of the cardiac tissue, enable us to suggest that apoptosis is present in these patients. We did not find histological images of necrosis neither in the original description nor in the present revision. The images of apoptosis were absent in control samples. Our results are in agreement with Narula et al. (9) and Olivetti et al (15), who claimed that apoptosis is present in irreversible human CHF. Narula et al. (9) found only 0.2% of labelled cells by TUNEL method (10-12), which is pointed out in an editorial by Colluci (16). This scanty proportion of affected myocytes could be considered irrelevant regarding the cardiac function in patients with dilated myocardiopathy and terminal CHF. Nevertheless, Collucci (16) remarks that since those cells are present only for minutes or hours, it could be more proper to calculate the total number of lost cells through the time course. Assuming that a cell developing apoptosis is detectable during 24 hours, it could be estimated that a loss of 0.2 % of cell per day, persisting during a year, could result in more then 50 % of the total set of cardiac myocytes. In conclusion, the new available methodology, applied to our samples stored for approximately 30 years, enable us to infer that in dilated alcoholic myocardiopathy with severe congestive cardiac failure, apoptosis is present and might have contributed to deterioration and death in these patients.
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