Mitochondrial Death Channels

In heart attacks, cells die if they aren’t perfused with fresh oxygen—and kill themselves if they are. Understanding cell suicide may greatly improve outcomes

Keith A. Webster

Gates of Death

The mitochondrial death channels include the mitochondrial permeability transition pore (mPTP) and the mitochondrial apoptosis channel (mAC). The mPTP has been under intense study for almost half a century, but its composition and mechanism of action are still controversial. Most experts agree that the mPTP is a voltage-dependent channel that is regulated by calcium and oxidative stress.Most but not all agree that three distinct mitochondrial proteins can influence the function of the mPTP. Two of these proteins are normally engaged in transporting metabolites into and out of the mitochondria. The first and outermost component of the mPTP is the voltage-dependent anion channel (VDAC), present in the mitochondrial outer membrane and required for the transport of ATP and ADP between mitochondria and the cytoplasm. The second component is the adenine nucleotide translocator, a transporter protein that exchanges ATP for ADP across the inner mitochondrial membrane, thus ferrying ATP in the matrix to the VDAC for export to the rest of the cell. The third component, cyclophilin D (CypD) ensures the proper folding of newly synthesized mitochondrial proteins and has attracted special interest because of its response to certain drugs. CypD is localized on the matrix side of the inner membrane. Together the three proteins of the mPTP span both mitochondrial membranes and provide a pathway between the matrix and the cell cytoplasm.

Under physiological conditions, the mPTP is closed to molecules that are not substrates of VDAC or the adenine nucleotide translocator. Conditions prevailing during a severe heart attack cause the mPTP to open, probably during early reperfusion. Opening of the mPTP allows the unregulated transport of materials out of the mitochondrion, and initiates both necrotic and apoptotic death. Death regulated by the mPTP channel causes 50 percent or more of the lethal injury caused by a heart attack.

The second death channel, known as the mAC, was discovered only recently (in 2004). It is responsible for classical apoptosis. Multiple life-threatening stimuli cause the mAC to open, allowing the release of the suicide inducers. The activity of the mAC is under tight control by a family of proteins collectively known as the Bcl-2 family, of which an important member is called Bax. Bcl-2 derives its name from B-cell lymphoma 2, the second member of a family of proteins whose over-expression is linked with lymphoma. The combined actions of the mPTP and the mAC are responsible for most and possibly all of the deaths caused by reperfusion during a heart attack.

The individual roles of the two death channels in promoting infarction have been dissected using specific inhibitors and genetic experiments in which individual genes associated with the channels are deleted, and the effects of the deletion on channel function and programmed death are determined. These studies have been done mainly using mice. The matrix component of the mPTP, CypD, is selectively bound and inhibited by an immunosuppressant drug called cyclosporine A. For many years we have known that treatment of animals with cyclosporine A reduces the damage of acute myocardial infarction. Most researchers in the field thought that this was because cyclosporine A suppressed apoptosis. However, recent studies on mice with homozygous CypD gene deletions (CypD –/–) indicate that this may not be true. CypD –/– mice were found to be resistant to lethal injury caused by a heart attack, but the effect was due to decreased necrosis, not decreased apoptosis.

The studies confirmed previous findings that calcium and oxidative stress caused the mPTP to open, and significantly more calcium and oxygen free radicals were required to open the mPTP when CypD was absent. However, CypD deficiency had no effect on apoptotic cell death when cells were treated with classical inducers of apoptosis. This means that necrosis but not apoptosis requires an intact mPTP and a functional CypD to be activated during a heart attack. It also suggests that this form of necrosis is voluntary (programmed) because it only occurs when CypD is present and can be prevented by genetic deletion of CypD or inhibition of CypD with cyclosporine A. Together these studies support a primary role for the mPTP, regulated by calcium and oxidative stress, in causing necrotic death during a heart attack. They indicate that the mAC can function independently of the mPTP to promote apoptosis, but they do not eliminate the possibility that both channels have roles in the regulation of apoptosis during a heart attack.

Necrosis and apoptosis both contribute to myocardial infarction. A major contribution of apoptosis is indicated by direct measurement of apoptotic markers in tissue of heart attack victims, as well as by the effects of specific inhibitors of apoptosis. The former indicate that between 20 and 50 percent of cardiac myocytes in the area of the left ventricle exposed to ischemia are actively undergoing apoptosis shortly after reperfusion. Consistent with this, selective inhibitors of apoptosis added during a heart attack can reduce infarction by 20 to 50 percent. Numerous studies indicate that opening of the mAC initiates this death pathway.

The mAC launches apoptosis by forming a channel in the outer mitochondrial membrane that allows for the release of cytochrome c, a mobile electron carrier in the electron-transport chain that is associated with the inner membrane and intermembrane space. Cytochrome c interacts with other proteins in the cell cytoplasm to form a death-dealing complex called an apoptosome. The apoptosome in turn mediates the activation of a network of proteases called caspases and DNAses that digest and destroy cellular proteins and DNA. Like the mPTP, the mAC is opened by calcium and oxidative stress during reperfusion, but it appears to be a more sensitive channel that opens at lower levels of stress. Reperfusion has the ingredients to open both death channels, and it seems likely that both death pathways are activated simultaneously. Necrosis caused by the opening of mPTP may be responsible for a major part of the early infarction caused by the heart attack, whereas mAC-mediated apoptosis may contribute more to the extended infarction that develops over time after reperfusion. The pathways leading to activation of the death channels are linked; it has been shown that Bax, the activator of the mAC complex, can interact directly with the sarcoplasmic reticulum, causing the release of calcium that is taken up by mitochondria and that may contribute to opening of the mPTP. There is also compelling evidence that both pathways are regulated by other Bcl-2 proteins.

An underlying feature of the death channels is that they are not inevitable. Observations that CypD- and Bax-deficient mice are significantly protected against the damage of heart attacks suggest that the damage can be largely prevented by blocking the death channels. Indeed, preclinical studies have confirmed that a number of pharmacological agents mitigate the opening of the mitochondrial death channels and can reduce tissue damage from heart attack or angioplasty, with potentially dramatic decreases in infarction and mortality. Along with cyclosporine A, currently approved as an immunosuppressant and widely used during organ transplant procedures, one of the most extensively tested and potent agents is low-dose sildenafil citrate, an agent already approved for erectile dysfunction and marketed as Viagra. It may not be long before an older individual, feeling unwell before bed, perhaps having barely worrisome chest pains, will recall a consultation in the doctor’s office and reach for cyclosporine A, against the chance that angina is about to become something more urgent. The threat can be minimized if medical care is near at hand, and if the mitochondrial death channels can be coaxed into remaining closed.

Bibliography

Anversa, P., and J. Kajstura. 2001. Myocyte cell death in the diseased heart. Circulation Research 82:1231–1233.
Daniel, P. T., K. Schulze-Osthoff, C. Belka, and D. Guner. 2003. Guardians of cell death: the Bcl-2 family proteins. Essays in Biochemistry 39:73–88.
Giordano, F. J. 2005. Oxygen, oxidative stress, hypoxia, and heart failure. Journal of Clinical Investigation 115:500–508.
Green, D. R., and J. C. Reed. 1989. Mitochondria and apoptosis. Science 281:309–312.
Webster, K. A., and N. H. Bishopric. 2003. Apoptosis inhibitors for heart disease. Circulation 108:2954–2956.
Webster, K. A. 2007. Programmed death as a therapeutic target to reduce myocardial infarction. Trends in Pharmacological Science 9:492–499.