It has many pores based on protein, which allow passage of ions and molecules. It drives APO.

This is the action site of the already mentioned pro-death members of the Bcl-2 family. Under the response to noxious stimuli, Bax which normally resides in the cytosol, is translocated to MOM and the endoplasmic reticulum (ER).

Thus it causes MOM permeabilization and the release of pro-apoptotic proteins from the inter-membrane space into the cytosol, such as cytochromes, Smac/DIABLO and endonuclease-G (endo G). Cytochrome c binds to the cytosolic protein apaf 1 and thus causes the formation of the “apoptosome” which activates the caspase-9 and −3 system [7].

This has more restricted permeability, much like the cell plasma membrane. It is involved in electron transport and ATP synthesis, and is spanned by the Mitochondrial Permeability Transition Pore (MPTP) which drives N.

This pore is inhibited by low pH <7:0 thus it is closed during ischemia, which corresponds clinically to persistent artery occlusion. When reperfusion occurs, there is restitution of pH and acceleration of cytosolic Ca²⁺ increase (which is slow during ischemia). The cell deals with this explosive Ca²⁺ increase by taking it up into the mitochondria via the MITO Ca²⁺ uniporter a protein that uses the negative ΔΨm to drive Ca²⁺ into the matrix [12].

There exist many MPTP activators, such as Ca²⁺, ROS, Pi inorganic phosphate which uses after adenine nucleotides are depleted after the onset of ischemia and a reduction of the ATP/ADP ratio. From the pharmacological agents cyclosporine-A is a typical inhibitor; its beneficial effects on I/R injury [8] will be discussed later.

The MPTP is very well described by Baines [7], Crompton [8], Lemasters et al. [10], and Weiss et al. [11]: They state that the MITO use electron transport to develop an electrochemical gradient across the MIM (membrane space and matrix). This is formed by the MIM membrane potential (ΔΨm or ΔCm according to Weiss et al. [11] ≈ −200 mV), and a proton gradient (ΔpH). The electrochemical gradient is employed by ATP synthase to phosphorylate ADP to ATP. When MPTP opens, ΔΨm depolarizes, and the synthase actually starts consuming ATP in a (futile) attempt the restore the proton gradient; solutes up to 15 kDa pass the MIM, and cause MITO swelling. As MITO swell, the MIM stretches, but actually it is the MOM which ruptures: The surface of MIM is enhanced by the cristae, parallel invaginating membrane structures. The MOM rupture effects the release of pro-apoptotic proteins from the inter-membrane space, notably cytochrome c, Smac/DIABLO and endonuclease – G (endoG). Cytochrome c binds to the cytosolic protein apaf1, creating the “apoptosome” which activates the Caspase-9 and 3 protease system, while EndoG translates to the nucleus, mediating DNA fragmentation, as described by Baines [7], who also points out that the Bcl-2 proteins permeabilize the MOM while ROS and Ca²⁺ the MIM. The role of ROS production is complex. MITO ROS can trigger MPTP opening, with further ROS release creating a cascading process known as ROS-induced ROS release mechanism.

Some further molecular characteristics of the MPTP should be given:

This protein resides in the MITO matrix. Its activation opens the MPTP, while its inhibition (by cyclosporine-A or sangliferin A) is cardioprotective [12]. Interestingly according to Lemasters et al. [10] CsA prevents both N and APO.

The MITO phosphate carrier (PiC) is a CypD-interacting protein which regulates ATP synthesis. Its overexpression induces APO.

Apart from the MPTP, another death channel has been described, the mitochondrial apoptosis channel, which is regulated by the Bcl-2 protein family [12]: The Mitochondrial calcium uniporter (MCU), through which. normally, Ca²⁺ enter the MITO. As Pan et al. [13] describe, under normal conditions entry of Ca²⁺ in the MITO augments their ATP production to match its demand up to 10–20 fold. However, larger increases can induce CD. Normally, most unstimulated cells maintain Ca²⁺ in the range of 100–200 nM in the cytosol and the MITO through the function of pumps in the plasma membrane and the ER.

The MCU blockade can prevent abnormal increase Ca²⁺ (and Fe²⁺ entry).

Thus it is suggested that MCU is required to cope with increased Ca²⁺ cycling.

During ischemia, the closed state of the MPTP, effected by a decrease in pH, activation of Pi, and a mild increase of Ca²⁺ and ROS results into limited damage. At reperfusion, the opening of the MPTP is characterized by an increase of the pH, the Pi, the Ca²⁺ and an explosive increase of ROS resulting into irreversible damages.

Lemasters et al. [10] recapitulate that the MPTP is involved in both APO and N.

Another question which has been advanced is whether APO can be induced without the all-important caspase-3 activation [3, 12].

Studies in mice and C. elegans provide evidence for caspase independent CD. The apoptosis-inducing factor (AIF) is such a candidate.

Susin et al. [14] have shown that AIF can cause a picture of mitochondrial- effected APO which cannot be prevented by a wide ranging caspase inhibitor. It does not need Apaf 1 or procaspase-9 for its action. However, AIF is required for cell death mediated by poly-ADP ribose polymerase (PARP), which is a main component of N, as will be further described. It also interacts with cyclophilin A, and triggers the release of cytochrome c. They suggest that it has a direct effort on isolated nuclei, effecting chromatin condensation and fragmentation which can be caspase-independent. They also postulate that BH3-domain only protein may act independently of Apaf-1 and caspases.

According to Whelan et al. [3] N can be signaled by two pathways:

This process is regulated by Cyclophilin D. MPTP can be opened by increases in oxidative stress, increased phosphate concentration, adenine nucleotide depletion, and importantly an increase of the matrix Ca²⁺.

Its opening causes the break-down of intracellular ATP.

Cyclophilin-D regulates MPTP pore opening and swelling of the mitochondria hallmark of N and cell death.

Cytoplasmic Ca²⁺ through MPTP opening can lead to N, in the worm and in the mammal through the proteases calpains, which are activated by Ca²⁺, and cathepsins which are released from the lysosomes during stress.

According to Zong and Thompson [19] calpain activation leads to N through cleavage of the Na⁺/Ca²⁺ exchanger in the plasma membrane and a sustained secondary intracellular Ca²⁺ overload.

However, according to Whelan et al. [3] calpain can also induce APO through Bax and Bid, and autophagy, through cleavage of Atg 5 (autophagy related 5 homolog) which will be described later. According to the same authors, the lysosomal cathepsins leak out explosively of the lysosomes, digest molecules and lead to N; under less abrupt conditions; partial lysosomal rupture leads to APO. They point out that N, APO and AUTO can be initiated by the lysosomes, whose integrity is preserved by HSP70.

Syntichaki et al. [22] point out that they are the result of perturbation of intercellular Ca²⁺.

Hotchkiss et al. [12] delineate the following mediators of N: ROS, Ca²⁺-activated non-lysosomal proteases calpains and cathepsins. These proteases are activated by oxidative stress and Ca²⁺ overload. Prolonged elevation of catecholamines Ang II and other vasoactives hormones may well be involved.

Whelan et al. [3] describe how ischemia-induced hypoxia increases intracellular acidosis, which extrudes H⁺ from the cell by Na⁺/H⁺ exchange leading to an increase of intracellular Na⁺, which through the Na⁺/Ca²⁺ exchanger leads to an intercellular Ca²⁺ increase, with the cell swelling effects which have already been described.

At reperfusion, Ca²⁺ induced Ca²⁺ release is effected through the ER(SR), or sarcoplasmic reticulum and MPTP opening is induced in some forms of N, such as DNA damage from alkylating agents mainly through ATP depletion. They also describe that HMGβ1, which as already stated is released as an inflammatory elicitor during N, can provide a switch from APO to N.

Opening of the MPTP pore is a major component of necrotic cell death. At R, APO cell death occurs at a lower incidence than N, but for a more extended time: APO occurs as early as 30 min and as late as 3 days after R initiation [23].

According to Whelan et al. [3], Poly-ADP-ribose-polymerase (PARP) is a main player in N occurring through DNA damage from alkylating agents.

It is often mentioned, but its actual role has not been adequately defined. Calise and Powell [34] provide a recent inclusive review.

This system, in parallel with the various mechanisms involved in CD has definite roles in cell-cycle control, protein turnover and quality control, cell signaling and APO.

According to the authors, the system is closely involved in myocardial ischemia. Its four components include the proteasome, ubiquitin (UB), the ubiquitination machinery and the deubiquitinases. It is an energy-requiring (ATP) process.

The proteasome can be involved in the removal and clearing of oxidized and damaged proteins. It can also form a hybrid, the “immunoproteasome”, since the ubiquitin system is also involved in immune response and antigen presentation.

Ubiquitin (Ub) is involved in protein degradation.

The UPS and specifically the proteasome, becomes dysfunctional in ischemia, both myocardial and cerebral. Its ATP-dependent activity is mainly affected, decreasing proteasome activity. It is also affected by oxidative damage. As in so many aspects of CD, the authors describe that whether a proteasome inhibitor has beneficial or detrimental effects is dose and time dependent. Thus, the inhibitor bortezomib, indicated for multiple myeloma treatment, may demonstrate cardiovascular toxicity.

The UPS becomes dysfunctional during ischemia, while its function is preserved by ischemic preconditioning.

The UPS exerts a parallel function to AUTO, which also prevents proteotoxic stress, but targets mostly macromolecular structures and organelles. Moreover, AUTO is upregulated while the UPS becomes dysfunctional at ischemia. Actually, it is suggested that there is an inverse relationship between UPS and autophagic flux.

It must be pointed out that the efforts to further clarify the various modalities of CD continue.

Thus the nomenclature Committee on Cell Death has issued Recommendations in 2005 and 2009 [2]. They state that it is important to discriminate between dying as a process and death as an end point (Table 15.1).

They also suggest that exact description of various characteristics should replace the mention of the modality itself i.e. TUNEL positive cells % instead of APO %. Similarly they recommend that “double membraned microvesicles” should be described instead of AUTO.

Galluzzi et al. [1] as many other authors have, describe that N is a negative definition i.e. cell death with no signs of APO or AUTO. Moreover they suggest limiting the use of the term oncosis. However, Kostin [5] extensively uses this term.

The Committee authors [2] also describe the term: Mitotic catastrophe which occurs after a dysregulated of failed mitosis and is morphologically accompanied by micro- and multinucleosis.

Also in their Table 15.2 they describe “cornification”.

They also comment on the validity of the term “programmed cell death”.

They point out that “genetic programming” characterizes APO during development and aging. They recommend that the terms developmental, etoposide, osmotic shock-induced cell death etc. should be used.

Moreover, they give a useful suggestion as to when a cell can be considered dead (Table 15.1).

In their Table 15.2 they give morphologic characteristics of APO, AUTO, and N.

Before finishing this description of the various forms of CD, the common pathways of the crosstalk among the three processes, which has already been referred to, and addressed by most expert groups [1–3, 12] must be further discussed.

Kung et al. [35] described experiments both in C. elegans and mammalian cells.

They stress that the process of N is as old as APO, and that Virchow first described the “unregulated” or ‘accidental” form of N. They describe that in APO the main mechanism is permeabilization of the MOM, while in N the opening of the MPTP, which spans the MIM. They also point out that the same death ligands that can activate APO (TNFα, Fas ligand TRAIL-TNF-related APO-inducing ligand) can also stimulate N, and that TNFα can induce survival, APO or N. They also remind how, when caspase-8 is deleted or inhibited, APO cannot be initiated and programmed N emerges through TNFR1 ligation. They also remind that apart from Ca²⁺ overload, Fe²⁺ cystolic increase can lead to TNFα-induced N. They also remind that APO and N are connected at the death receptor and MITO pathways, and that the MOM and MIM are separated by only microns, promoting these cross-talks.