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Two main patterns of cell death, necrosis and
apoptosis exist. Necrosis is a pathological process following cellular injury and affects
cell groups or a solid mass of tissue. Necrosis is due to various causative agents, the
most common being ischaemia.
Apoptosis on the contrary involves only scattered cells, like "dead leaves
falling from a tree in autumn"1 and is a part of a
"programmed" cellular process. It is intrinsic to normal cells in embryogenic
development, in normal tissue turnover and clones selection in lymphoid cells.
In the apoptotic process the cell undergoes volume reduction and shape distortion,
looses contacts with its viable neighbours and becomes isolated. Cytoplasm organelles
integrity is maintained, except some dilatation of smooth endoplasmic reticulum, but
striking nuclear changes occur. Nucleus becomes pycnotic, chromatin condenses under the
nuclear membrane usually in half-moon masses, and frequently breaks up into fragments,
which together with a few cytoplasmic organelles constitute a membrane-bounded apoptotic
body. The apoptotic bodies are immediately phagocytosed by neighbouring cells and
inflammatory response does not occur.
Apoptosis is an active energy requiring process controlled by several factors.
Apoptosis inhibitors include growth factors, cell matrix and sex steroids, while growth
factor withdrawal, loss of matrix attachment, glucocorticoids, free radicals, ionising
radiation and some viruses act as apoptosis inducers. From the biochemical point of view a
central role in determining the apoptotic process is played by an endogenous Mg++ and Ca++
dependent endonuclease which cut DNA into fragments which are multiple of nucleosomes. DNA
fragmentation at the internucleosomal level results in the ladder pattern which is visible
when agarose-gel-electrophoresis is performed2 and
which is a tool for the diagnosis of apoptosis. Another method to detect the process is an
"in situ" nick translation, known as TUNEL, which allows to detect single cell
apoptosis in formalin fixed and paraffin embedded tissues3.
Apoptosis is called also "programmed cell death" because genetic programmes
are involved in its regulation. A nematode, ceanorrhabditis elegans, provided the material
for extensively studying distinct stages of apoptosis and their controlling genes4. Among this set of genes, there is one, the ced-9 gene,
which determines whether or not the cell will die. The ced-9 gene is highly homologous to
the human bcl-2 gene, which is the major apoptosis suppressing gene in humans. Bcl-2
belongs to a family of genes including bax and bcl-x. The bcl-2 protoncogene encodes a
protein associated with intracellular membranes5. So
downregulation of bcl-2 may cause apoptosis, because leads to increase of Ca++ through the
ER membranes, releasing the DNAase6. In addition
increased cytosolic Ca++ activate transglutaminase which is involved in the alteration of
the cytoadhesive properties of apoptotic cells7,8.
Another enzyme involved in apoptosis in nematodes is a protein similar to
interleukin-1b-converting enzyme (ICE)9
which is functionally homologous to granzyme B, a protease responsible for apoptosis
caused by cytotoxic T cells by perforin/granzyme system10.
Cytotoxic T cells induce apoptosis also with another mechanism, the FAS/FASL system.
FAS is a type I membrane protein and its activation by binding the FAS ligand (FASL)
induces apoptosis in FAS-bearing cells. FAS, also called APO-1 or CD95, is a member of the
TNF/nerve growth factor receptor family and is expressed in a variety of cells, including
those of the thymus, liver, heart and kidney11; the FAS
ligand is a protein that belongs to the tumour necrosis factor family and is expressed in
activated T cells.
P53 and c-myc are primary apoptosis -promoting genes. Normally myc is required for
entrance into the cell cycle in response to a growth factor signal12.
When myc is expressed, withdrawal of growth factors results in apoptosis. The p53 protein
is involved in the cellular response to DNA damage. When DNA damage occurs, expression of
the p53 protein induces arrest of the cells in the G1 phase of the cell cycle13; if DNA repair mechanisms fail, apoptosis is triggered
in order to avoid that cells with aberrant genomic material survive.
Transforming growth factor beta (TGFb) belongs to a family
of multifunctional polypeptides which regulate normal cell growth, development and tissue
remodelling. It is a potent growth inhibitor and most probably it plays a role in limiting
the inflammatory response. It is also a mediator of the pathologic extracellular matrix
accumulation that features progression of tissue injury to end-stage organ failure14. It is well known that TGFb1
plays a key role in the fibrogenesis of liver and renal diseases, including adriamycin
nephropathy, and most probably it is involved also in myocardial fibrosis.
Apoptosis has an important role in morphogenesis15.
The normal morphogenesis of the human heart occurs not only in foetus but also after
birth. Myocyte apoptosis has been demonstrated in the early stages of postnatal
development in the rat heart and it was correlated with the expression of the protoncogene
bcl-216. Angiotensin II synthesis is increased in
immediate postnatal period in rat liver and it is known that this peptide can induce
apoptosis in vitro in ventricular rat myocytes: in fact ligand binding of angiotensin II
receptors on myocytes leads to the activation of an endogenous endonuclease17. Also in humans there is postnatal involution of the
right ventricular myocardium following to lungs inflation, diminution of the right
ventricular pressure and ductus arteriosus closure. It is highly probable that this
myocardial involution is due to myocyte apoptosis. Other cardiac structures like the
conduction system, sinus node, AV node, and His bundle, undergo morphological changes
after birth and James suggested many years ago, before the term apoptosis was created,
that changes were due to myocyte death18.
Apoptosis is also the mechanism for continuing control of organ dimension, maintaining
normal size in the face of cell turnover, or a reduction during atrophy. According to
their potential for renewal, cells can be classified as labile, which have a good capacity
to regenerate, stable, which usually divide at a very slow rate but retain the capacity to
divide when necessary, and permanent, which have not capacity to divide. Cardiac myocytes,
as well as nerve cells, are usually regarded as permanent, so when myocytes apoptosis
occurs there is reduction of myocytes number.
It is not clear if there is an age-related loss of myocytes in elderly. Recent studies
have shown that apoptosis can be triggered not only by the "internal clock", but
also by various external agents such as hyperthermia19-21,
hypoxia22-26, hepatic toxins24-26,
anti-cancer drugs27,28, and viruses29-32.
Apoptosis has also been demonstrated in end-stage heart failure33,
in experimentally-induced cardiomyopathy34,
atherosclerosis35, restenosis36,
acute myocardial infarction37 and acute and chronic
heart transplant rejection38,39. Apoptosis related with
changes in bcl-2 and BAX expression has been detected in failing human hearts40. A review of apoptosis in cardiovascular diseases has
been recently published by Davies41.
Apoptosis has been described in arrhythmic cardiac diseases. James demonstrated
apoptotic bodies in the sinus nodes surgically excised from patients with the long QT
syndrome42 and in an infant with right ventricular
failure and complete heart block43. According to this
author apoptotic bouts in the right ventricular myocardium may cause arrhythmias in two
ways: first because the dying cells have increased excitability and automaticity and
second, because the loss of function of died cells cause an alteration of the normal route
of right ventricular activation, providing an anatomic substrate for microreentrant or
macroreentrant circuits43.
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a primary heart muscle
disorder of unknown cause that is characterised clinically by left bundle branch block
ventricular arrhythmias and pathologically by progressive loss of myocardium with
fibro-fatty replacement44-47. It is often familial
(nearly 30%) with an autosomal dominant inheritance48.
Gene defects have been mapped both to chromosome 14 and 149-51.
Occurrence of apoptosis in this disease have been recently demonstrated by Mallat et
al52 in specimens obtained at autopsy and by our group
in endomyocardial right ventricular biopsies53,54. In
our study, apoptosis was seen in 7 of 20 ARVC cases and was related with acute symptoms.
Genetic predisposition to apoptosis is a very suggestive theory for pathogenesis of ARVC,
but it is also possible that in these cases apoptosis may be induced not by the programmed
internal clock but by external factors. The acute symptoms might be due to the presence of
a trigger such as a virus, and cellular apoptosis might be caused by cytotoxic T
lymphocytes or by antibody-dependent cytotoxic cells. Moreover in our cases of ARVC we
found evidence of apoptosis not only in myocytes but also in a few endothelial cells,
suggesting that both cell types were affected by the trigger of apoptosis at the same
time. Thus endothelial loss might cause some ischaemia which contributes to myo-cardial
loss. Whatever will be the etiopathogenetic mechanism it is clear that recurring or
continuated bouts of apoptosis lead to replacement of destroyed myocardium by fibro-fatty
tissue, which is the anatomical substrate of arrhythmias.
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