Cardiomyocyte Apoptosis

Introduction
Cardiovascular disease is the leading cause of death in developing countries (WHO report, 2002), and the magnitude of an acute myocardial infarction (MI) or more specifically the number of dead cardiomyocytes, is a vital factor of subsequent heart function. Rational design of therapeutic interventions that protect the myocardium from cell death during ischemia has been a research priority for over thirty years. We need to understand the mechanisms underlying cardiomyocyte (CM) cell death, its timing, as well as research tools for its accurate and rapid quantification in order to do this effectively (Takemura et al., 2004).
Cardiomyocytes seem to be more sensitive to hypoxia and ischemia than other cell types (Takemura et al., 2004). Also, CM contain the highest mitochondrial volume of all cell types (Elsasser et al., 2000. Also CM secrete TNF-alpha, both of which have serious implications for CM cells in ischemia and MI (Kapadia et al., 1995; Giroir et al., 1994).
Myocardial ischemia is a condition in which oxygen deprivation to the heart muscle is accompanied by inadequate removal of metabolites because of reduced blood flow or perfusion. In contrast, mere oxygen deprivation without reduction in the clearance of metabolites is termed hypoxia or anoxia. Ischemia and hypoxia are relevant to pathologic cardiomyocyte (CM) cell death because this decrease in oxygen leads to a rapid decline in energy and ATP levels which are needed for the high energy requirements of these cells. Glycolysis is increased in these states leading to the formation of harmful byproducts such as lactate. Although prolonged myocardial ischemia has deleterious effects on mitochondrial function, evidence suggests that the generation of ROS during reperfusion (re-establishment of oxygen and blood flow after MI) is even more harmful through reaction of oxygen with reduced mitochondrial ubisemiquinone to produce high amounts of superoxides. It was concluded that oxygen deprivation enhances the susceptibility of the heart to ROS by increasing the amount of catalytic ferrous ion in the low molecular weight pool (McFalls et al., 2003).

Cardiomyocyte Cell Death
Until quite recently, cardiomyocyte death from ischemia was attributed to necrosis, or “accidental cell death” (Jugdutt et al., 2005). However recently, collective evidence from experimental and clinical studies has demonstrated that cardiomyocyte cell death following ischemia and/or reperfusion is now known to involve apoptosis, oncosis, autophagy, (and necrosis) in ischemic cardiac conditions. These conditions are thought to lead at later stages to necrosis (Takemura et al., 2004; Yan et al., 2005). This shift in thinking demonstrates that CM cell death prevention should target both CM apoptosis, oncosis, autophagy, and necrosis.

Apoptosis of Cardiomyocytes in Ischemia
The morphological characteristics of apoptosis are that it is seen in single cells scattered in tissue. Microscopically, cells are visualized to have nuclear chromatin condensation, with the formation of small dense bodies. No cell membrane disruption occurs. The cell membrane undergoes budding as apoptotic bodies, with cell shrinkage occurring. Lysosomes are also left intact inside the cell. No inflammation or fibrosis results, as cells are “phagocytosed” by neighboring cells or sometimes by macrophages.
Morphological methods of apoptotic detection include the light microscopy and electron microscopy (EM) (Jugdutt et al., 2005; Edinger et al., 2004).
Apoptosis is usually induced by physiological stimuli, but can also be induced pathologically. It requires energy in the form of ATP, and gene transcription and protein synthesis are involved. Ions and fluids do not influx across membranes as the ion transporters and membrane dynamics are stable. As mentioned, apoptosis does not disrupt membranes and thus there is no leakage of intracellular enzymes or proteins (Jugdutt et al., 2005; Edinger et al., 2004).
Apoptosis is also characterized by non-random DNA fragmentation, which has used to biochemically detect cells undergoing apoptosis (). DNA ladering simply uses extracted DNA which is resolved on agarose gel electrophoresis, and is later stained to examine the DNA fragmentation pattern. Other methods of apoptosis detection include TUNEL or Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling, which is the most widely used histochemical technique for in situ labeling and localization of DNA breaks in individual nuclei on tissue sections. DNA is exposed by proteolysis and TdT (terminal deoxynucleotide transferase) incorporates biotinylated dUTP into sites of DNA breaks. The biotin signal is then amplified using avidin-peroxidase or fluorescent dye, and visualized (Jugdutt et al., 2005; Edinger et al., 2004; Dispersyn et al., 2001).
Although apoptosis was first used as a term in 1972 (Kerr et al., 1972), apoptosis in cardiomyocytes was not reported until 1994 (Gottlieb et al., 1994). Ischemia / reperfusion was induced in rabbit hearts, and apoptosis was identified using TUNEL, DNA fragmentation, and electron microscopy. Ischemia/reperfusion alone was shown to have significant CM apoptosis, whereas ischemia alone was not. Only ischemia coupled with reperfusion was able to induce apoptosis in the experiments.
Later studies demonstrated that ischemia alone, or even hypoxia can induce CM apoptosis. An in vitro experiment demonstrated that hypoxia induced apoptosis. Neonatal rat cardiomyocytes and nonmyocytes were cultured in 95% N2-5% CO2 atmosphere to produce hypoxic conditions. DNA fragmentation into integer multiples of the internucleosomal DNA length was observed in cardiomyocytes as early as 12 hours, whereas interestingly, nonmyocytes did not show fragmentation of DNA even up to 72 hours. (Tanaka et al., 1994).
Persistent ischemia alone even after 2.5 hours, without reperfusion was later shown to induce significant apoptosis in areas of acute infarction in rat hearts.
It was concluded that CM apoptosis is detected in both permanently ischemic and reperfused myocardium, however the onset of apotosis is accelerated by reperfusion
Thus, reperfusion helps prevent further apoptosis, accelerates the rate of apoptosis in those cells irreversibly destined to die. (Gattinger and Fliss, 1996).
In contrast with the experimental results indicated, Fujiwara et al. used immunogold TUNEL method combined with EM reported that in rabbit hearts, apoptosis was absent after coronary artery occlusion (Takemura et al., 1997). This was later confirmed by another experimental study suggesting that apoptosis did not play a role in myocardial infarction (Ohno et al., 1998). The experimental results thus give different conclusions, which may be due to the differences in models, species, and methods used for detecting PCD.

Data from Human Infarcts
DNA fragmentation was detected in humans from hearts autopsied following fatal MI. DNA was extracted from the H&E stained areas and agarose gel electrophoresis was conducted. It was demonstrated that in acute human infarction with coagulation, oncotic myocytes with phenotypic differences all showed positive staining for DNA fragmentation by DNA nick end labeling (Itoh et al., 1995)
It was shown that CM apoptosis and CM necrosis are independently contributing variables of infarct size in rats (Kajstura et al., 1996). Apoptotic cell death accounted for 2.8 million cells whereas necrosis accounting for only 90,000 cells after 2 hours. Myocyte necrosis peaked at 1 day with 1.1 million myocytes.
Olivetti et al. (1996) first demonstrated that in human infarcts, 12% of all myocytes were apoptotic in the border zone and 1% were apoptotic in remote (peripheral) areas. Apoptosis was measured quantitatively by the TUNEL assay, and confirmed biochemically by DNA extraction and agarose gel electrophoresis.
Using TUNEL and DNA laddering methods, another group showed PCD rates of 1% in border regions and about 0.5% in infarcts. Apoptotic values in remote areas were 0.01%, similar to controls (Saraste et al., 1997).
Still another group determined the rate of apoptosis to be 1.8-3.3% in infracted areas of the human heart, whereas the rate of apoptosis was 0.1% in control areas. This group also demonstrated increased PCNA labeling index compared to controls (Ottaviani et al 1999).
Apoptosis and necrosis were found to be present in 77 human hearts after autopsy with fatal infarction, and that the size of human infarcts may be underestimated because apoptosis is not represented in enzyme measurements used clinically, as apoptotic cells maintain their cellular integrity (James, 1998).
Thus the majority of studies demonstrated that apoptosis in the center of the infracted area is often negligible. Apoptosis in the peripheral border zones and in remote myocardium may play vital roles in ventricular remodeling and the final progression to heart failure (Elsasser et al., 2000). Furthermore, it is these peripheral CM cells that may be candidates for therapeutic interventions to prevent their death.

Necrosis of Cardiomyocytes in Ischemia
Necrosis is a general term used to describe another mode of cell death. Using the term necrosis is problematic due to the fact that dead cells are so severely degraded at the final stages that they cannot be morphologically determined whether they died via apoptosis or necrosis. Also, necrosis refers only to an irreversible stage of cell death, even though dying cells generally progress from a reversible to an irreversible stage.
Manjno and Joris (1995) used an old term “oncosis”, which refers to cell death accompanied by cell swelling. They proposed to substitute oncosis for necrosis in cells dying via a process involving cellular swelling, as contrasted to apoptosis which involves cellular shrinking. They then proposed then that the final stages of either apoptosis or oncosis be termed necrosis.

The Switch: Apoptosis or Oncosis (Necrosis)?
After prolonged myocardial ischemia, a critical depletion of energy levels leads to the loss of ionic homeostatsis, with cell swelling, rupture and necrotic death.
As programmed cell death (PCD) or apoptosis is energy-dependent, the switch in the decision between oncosis and apoptosis depends on ATP concentrations (24). Severe ischemia results in significant reduction and final depletion of adenine nucleotides accompanied by cellular injury. ATP loss of 70% as usually present in myocardial infarction is incompatible with apoptosis and causes oncosis (necrosis) 24.
In several models of myocardial ischemia and reperfusion, necrosis and apoptosis can coexist, though it is still unclear exactly what factors determine the ultimate fate of a cardiomyocyte. It has been proposed that myocardial segments exposed to the most severe areas of ischemia and reperfusion undergo necrosis, whereas cells on the periphery undergo apoptosis (Gottlieb et al., 1994; Fliss and Gattinger, 1996).
This is consistent with the notion that cells exposed to severe stimuli undergo necrosis, whereas cells exposed to less hypoxia become apoptotic and sustain partial recovery.

Apoptosis: Mitochondria and Cardiomyocytes
More recently, mitochondria have been shown to be involved with apoptosis and Mitochondria have been implicated in the generation of reactive oxygen species (ROS) during ischemia and apoptosis. Due to the fact that cardiomyocytes contain the highest volume density of all mammalian cells, (25% in human cells and 37% in mice) these quantitative differences significantly influence the role that mitochondria play in CM cell death (Elsasser et al., 2000). Moreover, ATP production is needed to fuel PCD and apoptosis, and the beating heart has energy requirements than other organs. Thus the sensitivity of apoptosis in CM may be higher in CM than other cells due to these cells having the highest mitochondrial volume (Jugdutt et al., 2005). As mentioned, cardiomyocytes showed DNA fragmentation as early as 12 hours, whereas non-myocyte fibroblasts did not show fragmentation of DNA even up to 72 hours with Fas stimulation. (Tanaka et al., 1994). Also, as demonstrated in an in vivo study, cardiac cells have a significantly lower sensitivity to apoptotic stimulation than liver cells of adult rat hearts, although the rate of clearance of cardiomyocytes was similar to that of apoptotic hepatocytes. (Hayakawa et al., 2003).

Signals and Mediators of Apoptosis in Ischemia
There is little information about the stimuli and mediators of apoptosis in the situation of cardiomyocyte ischemia, and even less information pertaining to the signaling mediators of oncosis in CM ischemia. We will describe the best characterized genes and the more significant pathways with respect to CM cell death here.

Mitochondrial Pathway of Cardiomyocyte Cell Death in Ischemia
In the acute hypoxia (ischemia), over 100 genes are alternatively regulated at the transcriptional level, activated or inactivated in neonatal CM cells using a DNA microarray (Graham et al., 2004).
Only recently have papers shown a glimpse of the mitochondrial cardiomyocyte specific cell death pathways. It was previously shown that caspases, a family of proteases, and their activation was vital for the execution of apoptosis, especially caspase-3 (Nicholson et al., 1995). Immunohistochemical analysis of ischemic/reperfused left ventricle showed caspase-3 levels were substantially elevated and localized in the ischemic/reperfused region, and that caspase-3 co-localized to TUNEL positive myocytes in an in vivo rat model (Black et al., 1998).
Caspase-9 was later demonstrated to reside in the mitochondria of cardiomyocytes and neuronal cells, however only neuronal cell ischemia in an animal model was examined. Caspase-9 was shown to be released from neuronal mitochondria during ischemia (Krajewski et al., 1999)
ZVAD-fmk, a general caspase inhibitor, was shown to be effective in reducing myocardial reperfusion injury, which could at least be partially attributed to the attenuation of cardiomyocyte apoptosis (Yaoita et al., 1998). On the other hand, Okamura et al. examined the effect of caspase inhibitors on myocardial infarct size and CM DNA fragmentation in ischemia-reperfused rat hearts. Caspase inhibitors were able to inhibit myocyte DNA fragmentation and caspase activation, however there was no significant reduction of the infarct size in ischemia-reperfused rat hearts (Okamura et al., 2000).
When subjected to myocardial ischemia/reperfusion injury, caspase-3 over-expressing transgenic mice showed increased infarct size and a pronounced susceptibility to die. Caspase-3 expression levels were thus concluded to impact myocardial infarct size after ischemia/reperfusion injury, since cardiomyocyte-specific over-expression increased infarct size (Condorelli et al., 2001). However, Calpain and caspase-3 inhibitors reduce infarct size and post-ischemic apoptosis in rat heart without modifying contractile recovery (Perrin et al., 2003). There are several papers which are contradictory in this area, and thus the role of caspases in CM cell death and therapeutics remains controversial.
The Stat family of transcription factors has been shown to activate caspases. Ischemia/reperfusion was shown to induce STAT-1 activation and caspase-1 processing in ventricular myocytes in the intact heart ex vivo. STAT-1-transfected cells were more susceptible to ischemia-induced cell death than control transfected cells. A possible mechanism was shown where STAT-1 may activate the promoter of the pro-apoptotic caspase-1 gene in cardiomyocytes during ischemia. Antisense STAT-1 vectors reduced both ischemia- and overexpressed STAT-1-induced cell death in cardiac cells.
These results suggest that STAT-1 plays a critical role in the regulation of ischemia/reperfusion-induced apoptosis in cardiac cells, acting at least in part via a caspase-1 activation-dependent pathway (Stephanou et al., 2000)
Fluorometric assays of caspase activity demonstrated rapid activation of caspase-2 in chick cardiomyocytes within the first hour of reperfusion, whereas ischemia alone did demonstrate any increase in caspase activity. A caspase-2 specific inhibitor protected against I/R-induced cell death, whereas other caspase inhibitors failed to do so. The data determined that active caspase-2 may initiate cytochrome c release after reperfusion and that it is critical for the I/R-induced apoptosis in this model (Qin et al., 2004)
In post-ischemic mouse hearts, over-expression of procaspase-1 was associated with increased cardiac myocyte apoptosis in the peri- and non-infarct regions. Also, myocardial infarctions were 50% larger in area. Tissue culture studies revealed that procaspase-1 processing/activation is stimulated by hypoxia, and that caspase-1 acts in synergy with hypoxia to stimulate caspase-3 mediated apoptosis without activating upstream caspases (Syed et al., 2005).

BNIP3 is a novel subclass of death-inducing mitochondrial proteins with homology to the Bcl-2 gene family. Various methods were used including in vivo hypoxia and ex vivo primary CM cell hypoxia experiments. The data provide the first evidence for the involvement of BNIP3 as an inducible factor that provokes mitochondrial defects and cell death of ventricular myocytes during hypoxia (Regula et al., 2002).
An important factor in the initiation of necrosis or apoptosis is the opening of a nonspecific pore within the inner membrane of the mitochondria, the MPTP. Current data support the fact that stress stimuli induce the pore to open, releasing pro-apoptotic factors into the cytosol (McFalls et al., 2003). Neutral hypoxic conditions and cell fractionation experiments indicated that the BNIP3 protein is loosely bound to mitochondria, however it translocates into the membrane under acidotic conditions. Translocation of BNIP3 coincided with opening of the mitochondrial permeability pore (MPTP). Paradoxically, mitochondrial pore opening did not promote caspase activation, and broad-range caspase inhibitors did not block this cell death pathway. However, the pathway was blocked by antisense BNIP3 oligonucleotides and MPTP inhibitors (Graham et al., 2004).

Bax, another mitochondrial apoptosis pathway protein, was shown to be involved in myocardial cell death. Electron microscopy revealed reduced damage to mitochondria and the nuclear chromatin structure in Bax-deficient mice. Bax heart knockouts had superior tolerance to I/R injury and thus this gene may be a potential target for therapeutic intervention in patients with myocardial ischemia (Hochhauser et al., 2003).
Capano et al. (2005) visualized GFP-tagged Bax translocation from the cytosol to mitochondria, commencing within 20 min of simulated ischaemia and progressing for several hours in ischemic cardiomyocytes. Using inhibitors, it was shown AMP-activated protein kinase and p38 MAPK are important for Bax translocation.
Hou et al. (2005) also demonstrated similar results.
Mitochondrial Bcl-2, an anti-apoptotic protein, is known to protect cells from apoptosis. Bcl-2 over-expression in transgenic mouse hearts was examined. The infarct sizes, expressed as percentages of the area at risk, were significantly smaller in the transgenic mice than in the non-transgenic mice Over-expression of Bcl-2 using transgenic mice rendered the CM cells more resistant to apoptosis and I/R injury in cardiomyocytes (Chen et al., 2001)
Comparative microscopy of mitochondrial flavoproteins, membrane potential, and calcium-sensitive probes demonstrated homogeneity and clear co-localization in both isolated cardiomyocytes and permeabilized myocardial fibers. After ischemia/reperfusion however, significant heterogeneity of all these parameters was detected. (Kuznetsov et al., 2004).

Death Domain Pathway of Cardiomyocyte Cell Death in Ischemia
TNF alpha is known to be produced by cardiac cells themselves (Kapadia et al., 1995; Giroir et al., 1994).
Due to the fact that TNF-alpha can trigger apoptosis in many cell types, it was thought that endogenous TNF alpha may contribute to apoptosis in cardiac cells.
TNF-alpha induced apoptosis in rat cardiomyocytes in vitro was quantified by single cell microgel electrophoresis of nuclei ("cardiac comets" – or comet assay) as well as by morphological and biochemical criteria. It was also shown that TNF-alpha stimulated production of the endogenous second messenger, sphingosine, suggesting sphingolipid involvement in TNF-alpha -mediated cardiomyocyte apoptosis. TNF-alpha levels have been shown to be increased in ischemic myocardial disorders, and thus may contribute to TNFalpha-induced cardiac cell death (Krown et al., 1996)
It was later shown that in vivo transfer of soluble TNF-alpha receptor 1 gene improved cardiac function and reduced infarct size after myocardial infarction in rats
(Sugano et al., 2004).

Oncosis of Cardiomyocytes in Ischemia
Little is known about CM cell apoptosis in ischemia, and even less is known about oncosis or programmed necrosis in ischemia. It was thought that caspases are vital for apoptosis or PCD, however caspase-independent cell death was also observed in many systems such as caspase inhibition. In some cases, caspase-independent necrotic death could be prevented with antioxidant treatment or inhibition of the protein kinase, RIP. These results led to the hypothesis of “programmed” necrosis – where necrosis is a signaling pathway which can be blocked by inhibiting certain factors.
Interestingly, apoptosis seems to inhibit the oncotic pathways by cleaving key factors required for programmed necrosis such as RIP and PARP (Edinger et al., 2004). However little was understood about these processes until very recently, and most of this work has been outside of the cardiac field. More recently, using PARP inhibitors several groups demonstrated quite significant inhibition of CM cell death (Alexy et al., 2004).
A novel PARP inhibitor, L-2286 was administered during ischemia-reperfusion in in isoproterenol-induced myocardial infarction. Thereafter, the cardiac energy metabolism, oxidative damage, and the phosphorylation state of Akt and MAPK cascades were monitored. L-2286 exerted significant protective effect against ischemia-reperfusion-induced myocardial injury in the ischemic model (Palfi et al., 2005)

Autophagic Cardiomyocyte Cell Death in Ischemia
It was thought that apoptosis was a synonym for programmed cell death, however an increasing number of studies demonstrate the existence of caspase-independent forms of PCD (Lockshin and Zakeri, 2002). Autophagy was thought to be a physiological process for eliminating unnecessary subcellular components, however recently autophagy as a form of cell death is attracting more attention. Cardiomyocytes are thought to be more sensitive to autophagy than most cell types (Sybers et al., 1976; Larsen and Sulzer, 2002). Vacuolar degeneration is autophagy’s most characteristic feature, with cells exhibiting large vacuoles. Although autophagy has been shown to be even more prevalent than apoptosis in some models of heart failure, there has been scarcity of studies on autophagy and ischemia (Takemura et al., 2004; Knaapen et al., 2001).
A few papers were found which shed some light on CM autophagy. In one study, fetal mouse hearts were maintained for up to four hours in glucose-free media in an atmosphere of 95% N/5% CO2 followed by resupply of O2 and glucose. Twenty-four hours later, many cells recovered without residual injury. Many others however revealed autophagic vacuoles ranging from those in which organelles were readily identified, to those characteristic of residual bodies. It was stated that autophagy has not been emphasized as an important mechanism in transient ischemia in adult myocytes, but it may play a role in repair of sublethal injury (Sybers et al., 1976).
Another study used rabbit hearts perfused under hypoxic conditions. The CM cells underwent progressive subcellular damage, which becomes irreversible by one hour. One hour of hypoxia yielded irreversibly injured myocytes. Upon reoxygenation, some of these cells displayed typical changes of necrosis, but others apparently underwent an abortive repair process involving the formation of large, probably nonfunctional lysosomes. The data suggested that lysosomal autophagy may be important in the cardiac cell repair initiated during and after hypoxia. (Decker et al., 1980)
More recently, a proteomic approach to chronic ischemia was taken. Autophagy-related proteins were found to be up-regulated in response to ischemia. The changes in protein expression were not evident after one episode, and only began to appear after two or three episodes. Expression was most marked after six episodes of ischemia, when EM demonstrated autophagic vacuoles in chronically ischemic myocytes. Conversely, apoptosis, which was most marked after three episodes, decreased strikingly after six episodes, when autophagy had increased. It was concluded that autophagy triggered by ischemia could be a homeostatic mechanism, by which apoptosis is inhibited and the deleterious effects of chronic ischemia are limited (Yan et al., 2005).

Discussion
The ultimate fate of a cardiac cell, whether it lives or dies, seems to be a delicate balance of the factors promoting cell death and those factors promoting cell survival. There has been a recent explosion of information regarding the analysis of these factors, and we were able to touch upon select pathways due to restrictions in space.
Although an explosion of data has emerged from the growing field of cardiomyocyte cell death in ischemia, many questions are left unanswered. Results regarding the role of apoptosis in acute coronary occlusion are divergent. Even the rates of apoptosis and cell death vary widely from study to study. Differences in experimental data could be the result of the use of different models (occlusion v constriction), ischemia and reperfusion time lengths, species, inhibitors, transgenic models, and the methods used in detection of cell death (some ideas taken from Elsasser et al., 2000).
Apoptosis is characterized by a long cascade of events that ultimately produces the final DNA fragmentation product that is used often for assaying apoptosis or cell death. Most of the papers cited have indirect evidence of apoptosis as they are based on detection of DNA fragmentation and/or so-called apoptosis-related factors. Few have examined evidence for apoptotic ultrastructure, which is the gold standard for diagnosing apoptosis (Takemura et al., 2004). TUNEL is problematic for several reasons including possessing high false positive and negative rate, and oversensitivity. TUNEL also may not distinguish between apoptosis and necrosis, and lacks standardization of apoptotic counts (Jugdutt et al., 2005).
Also DNA ladders may not be specific for cardiomyocyte apoptosis in vivo since non-myocyte cells which greatly exceed CM cells in number, may contaminate tissue samples (Takemura et al., 2004).
Attempts have been made to characterize CM cell ultrastructure in different modes of cell death however EM morphology may vary depending on the conditions and evaluation of membrane ultrastructure. Another compounding problem is the fact that all microscopy is limited by the fact that apoptotic cell appearance only lasts for a few minutes and apoptotic bodies may be seen for only a few hours before they are phagocytozed (Jugdutt et al., 2005).

Therefore in summary, easier methods of cell death identification in CM cells need to be developed. Furthermore, an easier and rapid method of quantification of the different cell modes needs to be developed. In addition, a high-throughput method would greatly enhance the rate of data coming from this field.

Other future directions include further defining the differences and similarities in cell death pathways between cardiomyocytes and other more investigated cell types.
Another direction to follow is the role of free radicals and what relative role they play in CM cells versus other cell types.

Other more fundamental questions remain unanswered:

At what point is cardiomyocyte apoptosis induced by ischemia reversible? (Elsasser et al., 2000). As this plays into the usefulness of therapeutic interventions, this would be an important question from a drug development therapy standpoint.

Another question is, is autophagy a major player in CM cell death? At this point this question cannot be answered. To our knowledge no study has shown the percentage of cell death based on morphology (with quantification) with respect to time after ischemia and/or reperfusion injury - i.e what percentage of cells are undergoing autophagic cell death, apoptosis, oncosis, necrosis at each period?
Furthermore is autophagy in CM cells distinct from other cell types, and therefore are the molecular pathways the same or distinct from other cell types?
Although mechanisms have been postulated, little is known as to why cells undergo apoptosis versus oncosis. As there seems to be programmed necrosis or oncosis, more experimental evidence is need to delineate the pathways involved and also differentiate this from programmed cell death or apoptosis/autophagy.
Also, as this form of cell death has been only observed in conditions where apoptosis is inhibited either chemically or genetically, more work needs to be done in this area (Edinger et al., 2004).

During Ischemia, how are PCD CM cells removed from the intercalated disc from neighboring myocytes and what is the time frame of this process? (Elsasser et al., 2000). One paper may give clues to this process as phagocytosis of apoptotic cells and apoptotic bodies occurred with engulfment by either neighboring myocytes or macrophages, but predominantly the latter (Jason TN, 1998).

Cardiomyocytes as mentioned secrete TNF-alpha, which may also be involved in their cell death. As TNF-alpha levels have been shown to be increased in ischemic myocardial disorders, this may increase the rate of apoptosis and/or necrosis in cells already experiencing forms of cellular stress such as ischemia (Krown et al., 1996).

Mitochondria play an important role in the apoptotic cascade, and in context of cardiomyocytes, which have the highest volume fraction of mitochondria of all cells (25% in human, and 39% in mice), mitochondria may play an even greater role in CM cell death (Elsasser et al., 2000). This was shown in several instances in the experimental evidence where CM cells under hypoxia die much sooner and experience a higher rate of cell death than other cell types.

Cardiomyocytes, by virtue of their nature, require a great deal of energy which is supplied by their enormous mitochondrial capacity, the highest of all cell types. As ATP is needed for the two forms of programmed cell death, apoptosis and oncosis, this high ATP requirement may cause cardiomyocytes to be more prone or have a heightened sensitivity for necrosis during periods of stress or ischemia/hypoxia. Experiments should be conducted to examine if CM cells have a greater need for ATP during ischemia than other cell types, and addition of ATP energy sources to CM cells blocks necrosis. To our knowledge this has not been examined.
Also, to our knowledge, no experiments have shown the critical ATP energy levels during CM ischemia which is the threshold for necrotic cell death. An examination of the minimum level of ATP that leads to necrosis would be important for examining the ATP hypothesis of necrosis during ischemia.

As mentioned, the generation of ROS during reperfusion (re-establishment of oxygen and blood flow after MI) is even more harmful than ischemia. This is due to the reaction of oxygen with mitochondrial ubisemiquinone which was reduced during ischemia to produce high amounts of superoxides (McFalls et al., 2003). Due to their increased mitochondrial presence, CM cells may experience higher superoxide, free radical, and ROS loads than other cell types under similar conditions as they may have higher levels of ubisemiquinones per cell. This has not been studied to our knowledge, and should be examined using various cell types by measuring free radical formation rates (or ubisemiquinone /Fe levels) under hypoxia/ischemia.

In conclusion, this increased mitochondrial presence may be in fact the cardiomyocytes’ ultimate downfall, the very reason why cardiomyocytes are so sensitive to hypoxia/ischemia induced cell death, as the balance of life versus death may be in fact already shifted in favor of the pro-apoptotic factors, or death. It remains to be seen whether therapeutics may in fact be able to reverse the cellular death pathways that may have already been activated, and thus attempt to shift the balance in favour of cardiac cell survival.