There are two types of cell death that can be identified in eukaryotic cells. Necrosis is an uncontrolled event where a cell dies rapidly, losing membrane integrity and allowing leakage of intracellular contents. This uncontrolled ‘bursting’ of cells can cause a potentially damaging immune response and infiltration of macrophages to clear up the cellular debris (Darzynkiewicz et a l, 1997). Necrosis can be detected by the loss of membrane integrity which allows release of cytoplasmic enzymes such as lactate dehydrogenase (LDH) or creatinine kinase (CK) (Lowe et a l, 1988). Loss of membrane integrity can also be detected using dyes such as Trypan Blue which can only enter cells where the membrane is not intact (Leite et a l, 1999; Mascotti et a l, 2000). Techniques for measuring necrotic cell death cannot exclude detection of apoptotic cells so are generally thought of as measuring total cell death.
Apoptosis, also known as programmed cell death is a controlled event triggered by a signalling cascade. Cell death from apoptosis is a slower process than necrosis and usually takes 12-24 hours, compared to just a few minutes for necrosis (Collins et a l,
1997).
Apoptosis results in rapid cell shrinkage and phagocytosis by neighbouring cells. In apoptosis there is no cytoplasmic leakage and therefore no immune response
(Darzynkiewicz et a l, 1997; MacLelian & Schneider, 1997). Apoptosis can be detected in cells in a number of ways. One of the first events in apoptosis is the ‘flipping’ of a phosphatidyl-serine residue from the inside of the plasma membrane to the outside. This event can be detected with Annexin V labelling which stains cells with the phosphatidy-serine residue on the outer surface of their plasma membranes (Willingham, 1999). Another characteristic of apoptotic cells is DNA cleavage. This can be detected by the appearance of a DNA ladder on an agarose gel or by specific labelling of 3’OH ends with techniques such as TUNEL labelling (using terminal transferease) (Willingham, 1999; Mesner-PW & Kaufmann, 1997). Despite the many quantitative methods for the detection of apoptosis, detection of cellular morphological changes remains the most well recognised indicator of apoptosis. Apoptotic morphological changes include; cell shrinkage, nuclear hypersegmentation and chromatin condensation, cell surface blebbing, and formation of apoptotic bodies (Collins e ta l, 1997).
The first genes to be connected to apoptosis were the ced (cell death abnormal) genes in
Caenorhabditis elegans (Yuan et a l, 1993), These genes are required for apoptosis in
the worm and share a high degree of homology to the human gene for Interleukin-1 converting enzyme (ICE), which is a protease that cuts out ILl from its precursor protein (Yuan et a l, 1993). More than ten human CED/ICE family members have since been identified and are known as caspases (Alnemri et a l, 1996). All caspases are cysteine proteases that have a cysteine residue in their active site and cleave their target proteins at specific aspartic acid residues. Caspases are synthesised as large precursors (procaspases) and are cleaved to their active form by other caspases in a proteolytic cascade. There are many targets for activated caspases including cleavage of structural and adhesion proteins, helping the cell to round up and be taken up by adjacent cells (Nicholson & Thomberry, 1997). The caspase cascade can be activated by many different mechanisms, a few of those known are briefly described below.
Apoptosis can be triggered from outside the cell, for example by a killer lymphocyte, which may recognsise a cell as being virally infected. Lymphocytes can secrete proteins such as perforin onto the target cell membrane causing channels to open up. These channels allow the entry of apoptotic agents such as granzyme B to activate the caspase cascade (Atkinson & Bleackley, 1995).
Receptor mediated apoptosis can be caused by binding of extracellular death signals to death receptors on the cell membrane (for review see (Ashkenazi & Dixit, 1998)), The best characterised death receptors are Fas (also called CD95), which binds to Fas ligand and TNFRl (also called p55) which binds to IN F (tumour necrosis factor). Death ligands and] receptors can be produced by lymphocytes or by cells subjected to stress. Activated Fas receptors aggregate and via an adapter molecule recruit procaspase 8 proteins, which when brought into close proximity with one another cleave and activate each other. Activation of TNFRl activates the [transcription factors AP-1 and NF-kB. Unlike Fas, it rarely causes apoptosis unless protein synthesis is blocked (Ashkenazi & Dixit, 1998). Increased levels of TNF have been detected in patients with heart failure (Levine et a l, 1990).
Damage to mitochondria causes the electron transport chain protein cytochrome c to be released into the cytoplasm. Once in the cytoplasm, cytochrome c binds to, and induces oligomerization of the adapter protein Apaf-1 which binds to procaspase 9. Apaf-1 bound procaspase 9 molecules then cleave each other resulting in activation. Active caspase 9 then cleaves and activates procaspase 3, active caspase 3 mediates the apoptotic response (Li et a l, 1997). Cytochrome c release occurs in most forms of apoptosis, in some cases it may be the trigger and in others a downstream effect (Green & Reed, 1998).
It is important for survival that cells can regulate their apoptotic machinery to ensure that cell death is not triggered accidentally. For this reason there are many endogenous regulators of the apoptotic cascade. The bcl2 family of proteins are important cellular regulators of apoptosis. Bcl2 and bclx inhibit apoptosis, while bax, bad and bid are pro- apoptotic (Adams & Cory, 1998; Vaux et a l, 1992). The tumour suppressor p53 is a DNA binding protein that has been implicated in cell cycle arrest in some forms of apoptosis (Dulic et a l, 1994). Additionally, p53 induces apoptosis in response to DNA damage, so preventing the propagation of faulty genes (Lane, 1993). The anti-apoptotic effects of bcl2 are partly due to inhibition of p53 (Miyashita et a l, 1994). This shows that control of apoptosis depends on a balance of pro and anti apoptotic genes.
Animal models of heart failure provide a useful tool in the study of the diseased heart. Ischemia reperfusion injury can be induced in vivo by coronary artery ligation for a defined ischemic period followed by reperfusion. Coronary artery ligation in the rat resulted in 2.8x10^ cells that were labelled as apoptotic and only 90,000 necrotic after two hours. Up regulation of the apoptotic marker genes bcl2 and Fas was also detected in the infarcted area (Kajstura et a l, 1996). Apoptosis has also been demonstrated in human heart failure. A 232 fold increase in apoptotic myocytes was shown in failing hearts compared with control hearts (Olivetti et a l, 1997). In this study, apoptosis was identified by DNA fragmentation and morphological characteristics such as chromatin condensation. In a separate study, Narula et al characterised apoptosis in human failing hearts by cytochrome c release, caspase 3 activation and ultrastructural examination (Narula et a l, 1999). This study also showed that many cells which had cytochrome c release and activated caspases did not have any morphological markers of apoptosis. This implies that either heart cells have some mechanism to block apoptosis following activation of caspase 3 or the cells are commited to apoptosis but have not begun the physical process (Reed & Patemostro, 1999). These studies show that apoptosis is likely to be an important factor in the myocyte loss seen in heart failure.
1.4.0 Cardiac Hypertrophy