Apoptosis is an organized, energy dependent process, which leads to cell death. 
Its definition is based on distinct morphological features [10] and 
demonstration of internucleosomal DNA degradation [27], executed by selectively 
activated DNAses [4, 22]. The morphologic hallmarks of apoptosis include 
chromatic margination, nuclear condensation and fragmentation, and condensation 
of the cell with preservation of organelles. The process is followed by 
fragmentation of the cell into membrane-bound apoptotic bodies, which undergo 
phagocytosis by nearby cells without associated inflammation [10, 11]. Apoptosis 
characteristically occurs in insolated single cells. The duration of apoptosis 
is estimated to be from 12 to 24 hours, but in cell culture visible morphologic 
changes are accomplished in less than two hours [10, 16]. Non-apoptotic cell 
death, a prototype of which is cell death due to ischemia (oncosis), is 
characterized by depletion of intracellular ATP stores, swelling of the cell 
with disruption of organelles and rupture of the plasma membrane [15]. Groups of 
necrotic cells and inflammation are found in tissues [10, 15]. The significance 
of apoptosis has mostly been studied using the TUNEL assay that detects DNA 
strand breaks in tissue sections and allows quantification of apoptotic cells by 
light microscopy [6]. Common experience seems to be that the TUNEL assay is 
prone to false positive or negative findings. This has been explained by the 
dependence of the staining kinetics on the reagent concentration [17], fixation 
of the tissue [2] and the extent of proteolysis [17]. Active RNA synthesis [12] 
and DNA damage in necrotic cells [17, 19] may cause non-specific staining. To 
obtain reliable and reproducible results, TUNEL assay should be carefully 
standardized by using tissue sections treated with DNAse (positive control of 
apoptosis). Quantification of apoptosis should include enough microscopic fields 
and identification of the cell type undergoing apoptosis. The specificity of the 
results can be substantiated by combining other methods with TUNEL, such as 
assessment of the pattern of DNA fragmentation or evaluation of the 
morphological features. Even though there is high variation in the results 
obtained in consecutive studies under the same circumstances, increasing 
evidence shows that TUNEL-positive cardiomyocytes and internucleosomal DNA 
fragmentation are associated with various cardiac diseases, including acute 
myocardial infarction and heart failure [reviewed in 5, 9]. Some morphological 
features of apoptosis have been observed in TUNEL-positive cardiomyocytes using 
light microscopy (Figure 1) or confocal microscopy [20]. Electron microscopic 
evidence of apoptosis has been found in the degenerating conduction system [7], 
in experimental heart failure [23], and in human hibernating myocardium [3]. In 
acutely ischemic myocardium the interpretation of the findings remains 
controversial, since only non-apoptotic cell morphology has been found in 
electron microscopy [8, 19]. One explanation might be abortion of the apoptotic 
program due to the lack of ATP before the morphologic features are fully evident 
[14]. Another explanation is the possibility that non-apoptotic cell death and 
apoptosis share common mechanisms in the early phases of the processes [14, 19]. 
The exact mechanisms of ischemic cell death remain to be clarified and the 
classification between apoptosis and non-apoptosis cell death to be specified. 
Recently, caspase activation has emerged as the central molecular event leading 
to apoptosis, preceding DNA degradation and the development of apoptotic 
morphology [22, 25]. New methods have been developed to demonstrate caspase 
activation [1, 13]. Inhibition of caspase may be an efficient way to prevent 
apoptotic cardiomyocyte death as well as to define and specifically probe the 
significance of apoptotic cell death in cardiac diseases.