Protecting Against Mitochondrial Membrane Permeability

Preventing the opening of the mitochondrial membrane permeability transition pore (mPTP) during the reperfusion period can potentially minimize tissue necrosis and apoptosis.

Membrane structures

Mitochondria are intracellular organelles found in most eukaryotic cells and range in size from 0.5 to 10 µm. The number of mitochondria varies widely both among species and by tissue type within a species; they can vary in number from one to thousands per cell. The mitochondrion has its own independent genome that is maternally transmitted. The mitochondrial DNA structure is similar to bacterial DNA supporting the theory that mitochondrial may be the symbiotic remnants of an ancestral free-living bacterium.   Mitochondria are responsible for the primary generation of adenosine triphosphate (ATP), the predominant chemical source of cellular energy for the body. As such, mitochondria are often considered the “power plants” of the cell. In addition to their role in generating energy, the organelles are active in the control of the cell cycle, cell growth, cellular differentiation, cellular signaling and cell death.   Mitochondria contain two membranes dividing the organelle into four distinct structures: the outer membrane, the intermembrane space, the inner membrane (which is folded into a series of “cristae”) and the internal matrix.   The outer mitochondrial membrane has a typical phospholipid structure seen in many eukaryotic plasma membranes. Many of its integrated proteins are porins that form channels allowing molecules <5 kDa to bidirectionally diffuse across the membrane. A large multi-subunit protein, translocase, allows for transport of proteins with specific N-terminus signaling sequences. The mitochondrial outer membrane, which can associate with the endoplasmic reticulum (ER) membrane, is important both in ER-mitochondria calcium signaling and in the transfer of lipids between the ER and mitochondria.   The inner mitochondrial membrane has a very high protein-to-phospholipid ratio (more than 3:1 by weight) and incorporates more than 150 polypeptides. It is extensively folded, giving rise to the cristae that substantially increase the surface area of the membrane. The amount of surface area is proportional to the amount of ATP that the organelle can produce and varies from tissue to tissue depending on its energy demands. Cells, such as muscle, have more folding compared to low energy requiring tissues. In general, the inner membrane is about five times the surface area of the outer membrane. The membrane proteins serve many functions but can be grouped in the following functions:

  • The five protein complexes of the electron transport chain that drive chemiosmosis, the transport of protons into the intermembrane space creating a pH and an electrical potential across the membrane
  • ATP synthase, the multi-subunit enzyme embedded in the membrane, utilizes the electromagnetic gradient across the membrane to synthesize ATP from adenosine diphosphate (ADP) and inorganic phosphate
  • Specific transport proteins that regulate metabolite passage into and out of the matrix, since the inner membrane lacks porins and is essentially impermeable to almost all ions and molecules
  • Protein import machinery that selectively transports large molecules via specific protein complexes
  • Mitochondria fusion and fission proteins that balance the opposing processes of mitochondrial fusion and fission that allow necessary mitochondrial interactions that determine function, development and apoptosis. The matrix is the space enclosed by the inner membrane. It contains about two-thirds of the total protein of the mitochondrion and, with the inner membrane, creates and stores ATP. Additionally, it contains hundreds of enzymes (including those involved with fatty acid and pyruvate oxidation and those of the citric acid cycle), mitochondrial ribosomes, tRNA, and several copies of the mitochondrial DNA genome.

Potential damage from increased mitochondrial membrane permeability

Under pathological conditions, the mitochondria can undergo an increase in the permeability of the mitochondrial membranes to relative small (<1.5 kDa) molecules, an event termed the “mitochondrial permeability transition.” This event is believed to result from the opening of a non-selective channel composed of several macromolecular components at sites where the inner and outer membranes of the mitochondrion meet. This channel is termed the mitochondrial membrane permeability transition pore (mPTP). There are numerous consequences to the opening of the mPTP including:

  • Loss of the electrochemical potential across the inner mitochondrial membrane
  • Increase in the intracellular calcium concentration
  • Loss of ATP content in the matrix
  • Leakage of mitochondrial proteins (including cytochrome c) leak into the cytosol
  • Mitochondria swelling

Although these processes are essential in many normal development and growth processes, there also appear to be the basis of the cellular injury and death in many pathological conditions. During intracellular acidosis induced by ischemia, the mPTP forms but remains closed. With reperfusion and correction of the acidosis, the mPTP opens, mitochondrial reactive oxygen species (ROS) are generated with the re-establishment of oxygen levels and the cascade leading to both cell necrosis and apoptosis (programmed cell death) are activated. It is believed that opening of the mPTP is a major contributor to the post-ischemic damage involved with reperfusion injury.

Decreasing Damage of Ischemia Reperfusion Injury

Protecting against mitochondrial membrane permeability transition may decrease the amount of infarcted tissue following reperfusion in heart attacks and strokes.

Effects of ischemia

Ischemia is defined as the inadequate supply of oxygen and nutrients to maintain normal cellular aerobic metabolism. It arises primarily from inadequate blood flow to tissue and is seen in a variety of clinical conditions including myocardial infarction, strokes, acute and chronic kidney injury, systemic shock, liver shock, mesenteric ischemia and organ transplantation. In tissues with high metabolic activity, such as the brain and heart, the high-energy ATP stores are depleted within the first few minutes of ischemia. This forces the cell to switch from the oxygen dependent, highly efficient ATP generation in the mitochondrial, to relatively inefficient cytoplasmic anaerobic glycolysis in which glucose produces two ATP molecules and lactic acid.

The buildup of lactic acid and carbonic acid (H2CO3) that results from accumulation of carbon dioxide leads to a substantial fall of cellular and extracellular pH. Within two minutes of ischemia, extracellular pH can drop from 7.3 to 6.7.   The electrolyte gradients across the cell membrane are maintained by ATP-dependent membrane pumps that keep intracellular sodium levels low and potassium levels high compared to the extracellular compartment. Loss of ATP interferes with these pumps’ operation leading to the rapid influx of sodium and chloride ions, efflux of potassium ions and passive influx of water into the cells. Similarly, calcium is present in the extracellular milieu at a concentration 10,000 times greater than within the cell.

This differential is maintained by an active membrane calcium pump but is also maintained by ATP-driven sequestration of calcium in the endoplasmic reticulum, the transmembrane differential in sodium ion concentrations and by an oxidation-dependent calcium sequestration inside the mitochondria. During ischemia all of these mechanisms are hampered leading to dramatic increases in intracellular calcium levels. Elevated intracellular calcium ion levels activate membrane phospholipases and protein kinases and, in turn, the phospholipase activation produces free fatty acids including the potent prostaglandin inducer, arachidonic acid. The degradation of the membrane by phospholipases compromises membrane integrity and further interferes with calcium regulation.

Reperfusion as a primary focus of treatment

Re-establishment of adequate oxygen and nutrients can limit the size of the ultimate area of infarction. Thus, rapid reperfusion and maintenance of the re-established blood flow has become a primary focus of treatment in myocardial infarction, stroke and transplantation. Paradoxically, however, restoration of normal blood flow to an area of ischemia results in a complex cascade of inflammation and oxidative stress. Increasing evidence points to the fact that reperfusion injury exists and can lead to incremental cell necrosis and apoptosis.

At reperfusion, the vascular endothelium upregulates the production of adhesion proteins and releases leukocyte attractants. Attraction and accumulation of leukocytes (primarily neutrophils and monocytes) to these areas trigger multiple mediator cascades leading to cytokine and chemokine release, the generation of reactive oxygen species (ROS), the release of proteolytic enzymes from white blood cells and increased vascular permeability. Leukocyte accumulation can create physical plugs in the microvasculature that already have slow blood flow due to tissue edema. The net effect is exacerbation of already compromised tissue perfusion. During ischemia, the hydrolysis of ATP via AMP leads to an accumulation of hypoxanthine. Increased intracellular calcium enhances the conversion of xanthine dehydrogenase to xanthine oxidase which, upon reperfusion and reintroduction of oxygen, may produce superoxide and xanthine from the accumulated hypoxanthine and restored oxygen. This results in further oxidative stress that can compromise cellular function and survival.

Increasing data suggest that a critical pathway in reperfusion injury is damage to the mitochondria consisting of an influx of small (<1 kDa) molecules into the organelle leading to swelling of the mitochondrial matrix, rupture of the outer mitochondrial membrane and leakage of mitochondrial associated proteins such as cytochrome c into the cell cytoplasm. This initiates the apoptotic death pathway as well as cell necrosis due to disruption of mitochondrial oxidative phosphorylation and depolarizing of the mitochondrial membrane potential. These changes are thought to result from opening of a mitochondrial permeability transition pore (mPTP) that spans the inner and outer mitochondrial membrane. The pore itself appears to consist of an interaction of the voltage-dependent anion channel of the outer mitochondrial membrane, the adenine nucleotide translocase of the inner mitochondrial membrane and cyclophilin D in the mitochondrial matrix. The mPTP remains closed during ischemia, presumably because of the low intra-mitochondrial pH. In the first few minutes following reperfusion there is a further influx of calcium into the mitochondria, a burst of oxidant stress and rapid correction of the acidosis which permit the mPTP to open.

Minimizing damage

Thus, prevention of the opening the mPTP during the reperfusion period by a small cell-permeable compound such as Bendavia™ has the potential to minimize tissue necrosis and apoptosis. This could decrease the amount of infarcted tissue following reperfusion in heart attacks and strokes as well as improve organ function following transplant surgery.

Acute Myocardial Infarction: Minimizing Infarct Size

According to the American Heart Association, in 2009 the estimated annual incidence of heart attack (myocardial infarction, MI) was 610,000 new events and 325,000 recurrent events. The average age of a person having a first heart attack was 64.5 years old for men and 70.3 years old for women. The lifetime risk of developing coronary heart disease (CHD) after age 40 is 49 percent for men and 32 percent for women. CHD caused approximately one of every five deaths in the United States in 2005 and remains the largest single killer of American males and females. For more statistics on this deadly disease, see “The Top 5 Killers of Men,” Men’s Health, June 2011.

Infarct size is a major determinant of mortality in myocardial infarction. Limitation of infarct size has therefore been an important objective of strategies to improve clinical outcomes. Currently, the most effective way to limit infarct size is to reperfuse the jeopardized myocardium as soon as possible with the use of coronary angioplasty or thrombolysis and to prevent re-occlusion of the coronary artery with the use of anti-platelet therapy.

When blood flow to cardiac myocytes is disrupted by occlusion of a coronary artery, the absence of oxygen forces the heart to provide its adenosine triphosphate (ATP) through glycolysis causing further inhibition of ATP synthesis. The decrease in pH stimulates Na+-H+ exchange activity which represents a major mechanism for H+ extrusion and pH regulation during ischemia reperfusion. The ATP depletion results in Ca+2 overload in the cytoplasm and increased Ca+2 in mitochondria.

Reperfusion of the heart

ATP depletion and high inorganic phosphate levels induce mitochondrial permeability transition pore (mPTP) opening by sensitization to Ca+2. However, during ischemia, the low pH inhibits the pore from opening. With reperfusion, the intracellular acidosis is corrected and the a opens leading to mitochondrial swelling, loss of the potential across the mitochondrial membrane, cytochrome c release, depletion of ATP store and, ultimately, cell necrosis or apoptosis

Reperfusion of the heart is accompanied by re-energization of mitochondria that can then take up the accumulated calcium. Associated with this is a burst of reactive oxygen species (ROS) generation in the cytoplasm and mitochondria. These free radicals, which include superoxide anion (•O2), hydrogen peroxide, hypochlorous acid, nitric oxide-derived peroxynitrite, and hydroxyl radical (•OH) are produced within minutes of reperfusion and continue to be generated for hours after the restoration of blood flow to ischemic tissue.    The most concerning consequence of reperfusion injury is myocyte death. Animal data suggest that up to 50 percent of an infarct size may be attributable to reperfusion injury. This observation highlights the potential value of therapies that reduce or eliminate reperfusion injury.

New approaches

Previous attempts to individually target known mediators of myocardial reperfusion injury in patients have used antioxidant therapy, calcium-channel blockers, sodium–hydrogen exchange inhibitors, and anti-inflammatory compounds with largely disappointing results. This has led to the concept that new, multi-targeted mechanistic approaches to ischemia reperfusion injury are required to successfully translate experimental interventions into clinical therapy. Many of these are specifically targeting the opening of the mPTP since inhibition of this pore is related to the preservation of normal mitochondrial functioning and cardioprotection and is thought to be the basis for the beneficial infarct-limiting effects of both ischemic pre- and post-conditioning.

Cardiac remodeling is an early and progressive response of the heart to insults such as ischemia and stimulation by cytokines and enzymes. It can continue for months or years after an acute myocardial infarction. Initially, cardiac remodeling can have beneficial compensatory effects that help to maintain forward blood flow and subsequent perfusion of critical organs. However, in the longer term, it is deleterious to the heart resulting ultimately in pulmonary venous hypertension and alveolar congestion, low cardiac output and organ dysfunction, and the clinical syndrome of symptomatic congestive heart failure.

Preventing left ventricular remodeling is of key importance after acute myocardial infarction because it may be related to a reduction in adverse cardiac events including new onset or worsening of congestive heart failure and cardiac mortality rates.

 

 

Stealth Peptides’ lead compound, the small cell-permeable peptide drug Bendavia™, targets the mitochondria with the goal of preventing such ischemia reperfusion damage.

 

Publications

The following articles may be of interest with regard to Bendavia™ and EMBRACE-STEMI™. If these publications are unavailable to you, please Contact Us to obtain copies for your personal use.

 

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