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MITOCHONDRIA AS A DRUG SITE IN THE ISCHEMIC
REPERFUSION INJURY IN HEART
Krunal P. Pawar*1, Ashish V. Jaydeokar1, Dipak G. Gangurde1, Hitesh N. Thakar1,
Unnati S. Patil2, Pranav Samuel3, Chitrakala U. Dhuri4
M.E.S’s H.K. College of pharmacy, Prateeksha Nagar, Oshiwara, Jogeshwari (west),
Mumbai-400102
2
MGV’s S.P.H College of Pharmacy, Malegaon, Dist. Nashik.
3
Parshvanath Charitable Trust's V.M.H.P. Shah College of Pharmacy, Kasarvadavali,
Ghodbunder Road, Thane.
4
S.V.B. College of Pharmacy, Kalyan Shil Road, Dombivli (E), Dist:Thane - 421 203.
1
*Address for Correspondence
Krunal Pradeep Pawar
H.K. College of Pharmacy,
Oshiwara, Jogeshwari-west,
Mumbai- 400 102,
Maharashtra, India.
Phone: +91-9869925806
Email: [email protected]
ABSTRACT
Mitochondrial medicine is a unique disciple in the nascent stage and is growing with the
support of the technological advances and our knowledge of the role in the mitochondrial
role in the ischemic reperfusion injury as well. Cardioprotection operates with activation
of various signaling pathways. The MPTP is considered as the major mechanism of the
cardiac myocyte death. The pharmacological strategy inhibiting mitochondrial outer
membrane permeability and thus apoptosis, by manipulation of the Bcl-2 family proteins
and Mitochondrial Permeability Transition Pore to protect the myocardium against
ischemia-reperfusion is very recent. It has already provided interesting results, which
support the idea that a clinical benefit might be obtained in the near future. However,
with the current paucity of knowledge about the identity of the MPTP, preparing a
candidate for its inhibition needs more research and thus the major area of research in
future would be identifying the MPTP comprehensively.
Keywords: Mitochondria, free radicals, ischemia,apoptosis.
INTRODUCTION
Cardioprotection in ischemia has been the subject for research since last decade, but till
date the elucidated pathways still lack the understanding of the mechanism by which cells
reduce cell death. Considerable research led down the results that mitochondria is a
potential player in cell death. If mitochondrion is responsible for the apoptotic and
necrotic cell death then opposing the mitochondrial death pathways will help in
cardioprotection. The ischemic cell death is suggested due to the opening of a large pore
in the inner mitochondrial membrane known as mitochondrial permeability transition
pore. Inhibition of mitochondrial permeability transition pore and its components
enhances cardioprotection which thus leads to a potential drug target [1].
MITOCHONDRIA AND CELL
There are number of the mitochondria in the somatic cells. The mitochondria are called
as the ‘power house' of the cells [2]. Organs with more physiological energy need they
have more mitochondria such as muscles, brain, heart and liver.
Detailed structure of mitochondria
Mitochondria have dimensions of 0.5-1 µm in diameter and length of 7µm. The shape of
the mitochondria depends on the location of the tissue in which it is located and the
number of the mitochondria also depends on the energy requirement of the cell if the
organs has the requirement of more aerobic respiration then the number of mitochondria
will be more. The heart and kidneys which has more metabolic requirement has more
number of mitochondria.
Fig.1: structure of mitochondria: an overview
In fig.1, the mitochondrion contains outer membrane and inner membrane which are
made up of phospholipids bilayers and proteins. The two membranes have different
biochemical and physiological properties. There is an outer membrane, intermembrane
space and inner membrane, cristae space and matrix within the inner membrane space.
Outer membrane
The outer membrane is surrounds the entire organelle. The outer membrane is made up of
protein and lipids in the ratio 1:1. It contains numerous integrated proteins known porins.
The porins form channels that allow molecules 5000 Daltons or less in molecular weight
to diffuse freely from one side of membrane to the other side. Large protein move across
the membrane actively with the help of translocase enzyme [3].
Intermembrane space
The intermembrane space is between the inner and outer membrane. The intermembrane
space has ions and sugars same as that in cytosol but the protein constitution is different
as that of cytosol. The protein in the intermembrane space is cytochrome c [3].
Inner membrane
The inner membrane consists of 151 polypeptide and these peptides have high protein to
phospholipids ratio. The inner membrane consists of cardiopilin. In addition to this there
is membrane potential across the inner membrane formed by the action of the enzymes of
the of the electron transport chain [3].
Cristae
The inner mitochondria is compartmentalized in to structures called cristae which further
increases the surface area for the mitochondria enhancing the ability to produce ATP. The
inner membrane consists of F1 particles or oxysomes.
Matrix
As shown in fig.2, the matrix is enclosed in the inner membrane. It contains major part of
proteins. The matrix is necessary for the production of ATP with the help of ATP
synthase contained in the matrix. The matrix functions for the oxidation of pyruvate, fatty
acids and Citric Acid Cycle. The mitochondria has its own genetic material and
manufactures its own RNA and proteins
Fig.2: structure of mitochondria: internal
Energy production
The main functions of mitochondria are to produce ATP and to regulate cellular
metabolism. The core reaction involves Citric Acid Cycle or Krebs cycle. The production
of ATP is done by oxidizing glucose, pyruvate and NADH which is done in cytosol. This
process is called cellular respiration and aerobic respiration which requires oxygen.
Pyruvate and citric acid cycle
Pyruvate molecule which was produced by glycolysis is oxidized and combined with
coenzyme A to form CO2, Acetyl-CoA and NADH. The Acetyl-CoA then enters the
Citric Acid Cycle. Citric Acid Cycle oxidizes Acetyl-CoA to from CO2, three molecules
of NADH and one FADH2.
NADH and FADH2: The Electron Transport Chain
Fig.3: The electron transport chain
The redox energy from NADH and FADH2 is transferred to oxygen (O2) in several steps
via the electron transport chain. This electron transport chain is shown in fig.3. These
energy-rich molecules are produced within the matrix via the Citric Acid Cycle but are
also produced in the cytoplasm by glycolysis. Reducing equivalents from the cytoplasm
can be imported via the malate-aspartate shuttle system of antiporter proteins or feed into
the electron transport chain using a glycerol phosphate shuttle. Protein complexes in the
inner membrane (NADH dehydrogenase, cytochrome c reductase, and cytochrome c
oxidase) perform the transfer and the incremental release of energy is used to
pump protons (H+) into the intermembrane space. This process is efficient, but a small
percentage of electrons may prematurely reduce oxygen, forming reactive oxygen
species such as superoxide. As the proton concentration increases in the intermembrane
space, a strong electrochemical gradient is established across the inner membrane. The
protons can return to the matrix through the ATP synthase complex, and their potential
energy is used to synthesize ATP from ADP and inorganic phosphate (Pi) [4].
Role of mitochondria in ischemic heart injury
Mitochondria play a very important role in energy generation within the cell. Recent
literature states that mitochondria have roles in cardioprotection and apoptosis.
Fig.4: mitochondrial death pathway and apoptosis
Fig.4 simplifies the mitochondrial death pathway and apoptosis. The pathway is triggered
by various “death signals”, such as ROS, DNA damage, that promote binding of the
proapoptotic protein Bax with the outer mitochondrial membrane, most likely at the
contact sites between the two membranes, and its association with the PTP. This enables
the release of cytochrome c (•) and the apoptosis-inducing factor (AIF) (■) from the
intermembrane compartment to the cytosol. An increased intramitochondrial Ca2+ level
and ROS production facilitate this process by promoting PTP opening. Once in the
cytosol, cytochrome c and AIF, in cooperation with a cytosolic factor, Apaf-1 (not
indicated), activate caspase-9 and subsequently other members of the caspase family,
thus initiating self-digestion of the cell and nuclear DNA fragmentation, eventually
leading to apoptotic cell death. Association of Bax with mitochondria is prevented by the
antiapoptotic protein Bcl-2. ROS can be decomposed by Mn-containing (mitochondrial)
and Cu, Zn-containing (cytosolic) superoxide dismutases (SOD), catalase, and
glutathione peroxidase (GPx). Stimulation of ROS production is exemplified here by UV
and ionizing radiation and by two anticancer drugs, Adriamycin and BMD188.
Activation is indicated as [+] and inhibition as [-] [5] [6].
HYPOXIA AND ISCHEMIA
Biology of hypoxia
Hypoxia occurs from conditions like ischemia and cardiovascular complications leading
to cell death. Hypoxia has shown to lead increase intracellular free calcium concentration,
[(Ca2+)i], 5-lipooxygenase, lipid peroxidation, cyclooxygenase constitutive nitric oxide
synthase (cNOS), leukotriene B4 (LTB4), prostaglandin E2 (PGE2), interleukins, tumor
necrosis factor-α (TNF-α), caspases, complement activation, kruppel-like factor 6
(KLF6), inducible nitric oxide synthase (iNOS), heat shock protein 70 kDa (HSP-70),
and hypoxia-inducible factor-1α [(HIF-1α). The sequence of their occurrence provides
understanding of the mechanism in hypoxia induced injury.
Calcium Effect
Intracellular calcium has considered to the most prominent secondary messenger. The
external signals are transmitted to the cell with the help of intracellular free Ca2+. The
resting [Ca2+]i is regulated at three different Levels:
1) At cell membrane;
2) At the intracellular Ca2+ pools in the cytoplasm; and
3) By Ca2+ -binding proteins in the cytoplasm and the nucleus.
First, there are Na+/Ca2+ exchanger, Ca2+/H+ antiporter, Ca2+-ATPase pumps, and Ca2+
channels present at the cell membrane. Normally, activation of the Na+/Ca2+ exchanger
stimulates the import of three molecules of Na+ and the export of two molecules of Ca2+.
However, this ions exchanger is concentration driven, so the Na+ and Ca2+ concentration
Gradients between the cytoplasm and the extracellular space can reverse the direction of
Na+/Ca2+ exchange. The Ca2+/H+ antiporter takes in two molecules of H+ and expels one
molecule of Ca2+. This antiporter is pH-sensitive. Ca2+ ATPase pumps are energy driven
and remove Ca2+ from the cell. Voltage-gated Ca2+ channels are activated by changes in
membrane potential. There are L-type, N-type, and T-type messenger- operated Ca2+
channels are activated by inositol 1,4,5-trisphosphate (IP3) or cAMP-stimulated protein
kinase (PKA); receptor-operated Ca2+ channels are activated by ligand-binding to
receptors on the membrane. Secondly there are intracellular Ca2+ pools in the cytoplasm.
The increase in [Ca2+]i induce Ca2+ mobilization from intracellular Ca2+ pools to further
elevate [Ca2+]i which could be simplified as this Ca2+ pools inducible. However, Ca2+
sequestration into the intracellular Ca2+ pools to lower [Ca2+]i also takes place. Third,
Ca2+-binding proteins are present in the cytoplasm and nucleus. The glutamic acid- and
phenylalanine-rich proteins, like calmodulin and S100 proteins are considered to exert
Ca2+-dependent actions in the nucleus or the cytoplasm. Hypoxia has been shown to
perturb calcium homeostasis. It is believed that a sudden increase in [Ca2+]i is associated
with cell death and the death is mediated by activation of Ca2+-dependent proteases [7].
Bcl-2 and p-53
Hypoxia alters expression of Bcl-2 and p-53. Expression of Bcl-2 is accompanied by
tumor progression protein p-53. p-53 inhibits cell growth and promotes programmed cell
death [7].
Caspases
Caspases are the front line executioner of apoptosis. Caspases are divided into functional
subgroups that are activated during the apoptosis and those which are implicate the
processing of the proinflammatory immune response. Caspases are in the inactive forms
which are known as procaspase which then turns into caspase after proteolytic cleavage
of the large and small units of the active enzyme [7].
fig.5: molecular abnormalities in a hypoxic event
Fig.5 describes the molecular abnormalities in a hypoxic event. Hypoxia increases KLF6
and NF-κB and decrease KLF-4 which results in an increased iNOS. This increases the
NO generation which then reacts with O2 to form ONOO- which in turns swells up the
mitochondria and release the cytochrome C and the caspase 9 to form apoptosome. This
apoptosome in turn activates caspase-3 leading to the apoptosis. KLF4: Kruppel-like
factor 4; KLF6: Kruppel-like factor 6; NF-κB: nuclear factor-kappa B; iNOS: inducible
nitric oxide; cyto-c: cytochrome c; casp-9: caspase-9; ↑: increase; ↓: decrease [7].
Adenosine Triphosphate (ATP)
ATP is considered as the energy cash of the cell. According to fig.6, the hypoxia
increases inflammatory mediators like LTB4 and decreases the enzyme activity and the
protein expression of pyruvate dehydrogenase (PDH) an enzyme that is responsible for
the oxidative decarboxylation of pyruvate molecule and formation of the Acetyl-CoA
which then moves in the Citric Acid Cycle. Thus the decrease enzyme activity of
pyruvate dehydrogenase leads to less generation of Acetyl-CoA molecule and low levels
of the ATP. Apoptosis requires energy if the levels of cellular ATP remains at levels high
than 15% of baseline, then apoptosis ensues. If the cellular ATP levels fall below 15%
baseline, then necrosis ensues [7].
Fig.6 effect of hypoxia on mitochondria
Free Radicals
Free radicals are molecules with unpaired electrons. These free radicals are highly
reactive which reacts with the biomolecules and causing oxidative damage to these
molecules. The hypoxia induced ATP depletion promotes the formation of free radicals.
These free radicals then react with the cellular targets which then change the major
configuration of the critical molecules. The free radicals which consist of reactive oxygen
and nitrogen species cause mitochondrial disruption so that the cytochrome c, AIF or
EndoG translocates to the cytoplasm. The cytochrome c then binds to Apaf-1 and caspase
-9 to form apoptosome that activates other caspase to initiate apoptosis. Understanding
the biology of hypoxia will certainly help to understand the mechanism of ischemic
injury and targeting the site for cardioprotection [7].
MYOCARDIAL ISCHEMIA AND INJURY
Introduction to ischemia and reperfusion
Myocardial ischemia is a clinical condition that is observed when there is reduced
coronary blood flow which then results in hypoxia and accumulation of the toxic cellular
metabolites. The accumulation of the lactic acid leads to the decrease in the intracellular
pH and increase in intracellular Na+ ([Na+]i) and Ca2+ ([Ca2+]i). The ionic and metabolic
disturbance leads to reduction of myocardial function. If the disturbance is resolved
quickly then the reestablishment and recovery occurs but if reperfusion is followed itself
by irreversible damage.
Depressed function and energy production during ischemia
The onset of ischemia there is a decline in developed pressure and increase in the enddiastolic pressure that is linked with regional and global akinesia. If the coronary flow is
not restored than there is increase in the pressure, full contracture develops and tissue
necrosis occurs. Ischemia leads to decrease in the energy producing mechanism since the
availability of oxygen is limited which in turns decreases the oxidative phosphorylation.
Intracellular creatine phosphate is rapidly decreased and intracellular phosphate is
increased. The glycolysis and lactic acid production are stimulated; the increase lactic
acid production leads to decrease of the intracellular pH decreases further inhibition of
glycolysis and reduction of ATP. The main reason for the ischemic injury is increase in
the intracellular phosphate and decreased intracellular pH [8].
Consequences of reperfusion
Consequences of the reperfusion injury include arrhythmias and loss of the intracellular
proteins. Reperfusion following prolong ischemia leads to the death of the cardiac
myocytes by necrosis or apoptosis. The mechanism of the necrosis and apoptosis in the
cardiac myocytes will be discussed in the proceding sections.
Disruption of ionic homeostasis during ischemic injury
Ischemia leads to decrease in the intracellular pH and decrease in ATP levels as well. The
increase in the acidic environment in the cell leads to activation of Na+/H+ exchanger as
the cell tries to restore the intracellular pH. The decrease in ATP levels and increase
intracellular pH leads inhibition of the Na2+/K+ ATPase so that the Na+ that enters the cell
cannot be pumped out through Na+/H+ exchanger and there is increases in the levels of
the [Na+]i. Prolong ischemia leads to the gradual increases in the intracellular Ca2+ due to
the inhibition of the Na+/Ca2+ exchanger, which normally pumps the Ca2+ out is stopped
due to increased [Na+]i. Elevated intracellular Ca2+ leads to the activation of the
degradation enzymes such as phospholipases, proteases and nucleases which ultimately
damages the tissue irreversibly. The irreversible tissue damage proceeds by blebbing of
plasma membrane and also by loss of ionic disturbances, swelling, de-energizing
mitochondria and release of intracellular enzymes which in turn exhibits loss of the
membrane integrity [8].
Effect of free radical on cellular components
a. Cell death
Two types of the cell death are necrosis and apoptosis [9]. Necrosis is associated with
inflammatory cell infiltration and subsequent collagen deposition and scar formation
whereas the apoptosis is accompanied by ultrastructural changes and biochemical
features, cytoplasmic and nuclear condensation.
b. Apoptosis
Apoptosis accounts for a great proportion of cell death associated with myocardial
ischemia/reperfusion injury. Apoptosis contributes to the impairment of cardiac
performance, and also plays an important role in the myocardial and vascular remodeling
processes. Radical oxygen species induced cardiac apoptosis which is mediated through
signaling systems, like intracellular Ca2 +, cytokines, lipid oxidation, and proto-oncogene
activation. The perturbation of intracellular Ca2 + homeostasis by the cellular redox state
can aggravate free radical reactions and can activate endonucleases via activation of
caspases-key enzymes in the apoptotic pathway. Additionally, intrinsic degree of oxidant
production regulates cellular susceptibility to apoptosis through both p53-dependent and
p53-independent pathways.
Effect of free radicals on lipids and lipid metabolism
Free lipids and membrane bound lipids are targets for ROS. ROS attacks on lipids leads
to lipid peroxidation thus leading to decreased membrane fluidity, increased membrane
permeability, inducing immune response and destabilized membrane receptors.
Effect of free radicals on long chain fatty free acids
Elevated plasma levels of free fatty acids (FFA) are culprit in myocardial ischemia.
Under normal physiological conditions of the heart, FFA is the first line source of energy
generated via β-oxidation within the mitochondrial matrix. FFA requires 60-70% of the
oxygen for energy production. However, β-oxidation cannot proceed under oxygen-
deprived conditions, such as in ischemia, where FFA and their metabolites become toxic.
When FFA metabolism is inhibited, toxic metabolic intermediates are accumulated and
incorporated into cell membranes, sarcolemma and mitochondrial membrane. High levels
of FFA and their metabolites impair Ca2 + homeostasis and ion gradients, which may lead
to cardiac arrhythmias during ischemia reperfusion [10].
Membrane lipids
Free radicals also react with membrane bound lipids leading to lipid peroxidation. These
reactive species react with polyunsaturated lipid membrane generating products which
lead to inhibition of protein synthesis and alter enzyme activities. Oxidation of
polyunsaturated fatty acids can cause membrane disintegration, mitochondrial
dysfunction and Ca2+ overload. Free radicals may act as signal transduction molecules.
Cellular H2O2 was transiently increased upon activation of platelet derived growth factor
[11]
. Numerous signaling pathways are affected by the platelet-derived growth factorinduced increase in H2O2 concentration. H2O2 activates apoptotic and hypertrophic
signaling pathways in cardiac myocytes [12]
Ca2+ dysregulation and mitochondrial Ca2+ transport
In normal cardiac function, mitochondrial Ca2+ transport pathways are thought to play a
significant role in the coordination of ATP supply and demand [17]. The relationship of the
[Ca2+]i and energy demands is illustrated in fig.6 [6]
Increased energy demand and
physical and metabolic activity of
cardiac myocytes
Increased
mitochondrial Ca2+
Increased
cytosolic Ca2+
Increased NADH
Increased energy supply
fig.6: The relationship of the [Ca2+]i and energy demand
Mitochondrial Ca2+changes in ischemia
The mitochondria have a capacity to take large quantities of Ca2+. However excessive
accumulation of Ca2+ in mitochondria leads to damage of the mitochondria by competing
for ATP production and inducing the Mitochondrial Permeability Transition Pore [6] [8].
MITOCHONDRIAL TRANSITION PERMEABILITY PORE
Introduction to Mitochondrial Transition Permeability Pore
Mitochondrial dysfunction is the underlying cause of ischemia-reperfusion injury.
Dramatic events occur in the mitochondria which lead to a chain of events which leads
apoptotic and necrotic cardiomyocyte death. The mPTP is a large conductance pore
formed apparently through a conformational change of several constituent proteins of
mitochondrial membrane. The pore opens most clearly under specific and usually
pathological conditions, and spans the inner and outer mitochondrial membranes [18]. This
section will elaborate on the on the role of the MPT pore in ischemia reperfusion injury,
the mechanisms involved, and regarding the pore's molecular composition.
Mitochondrial death pathways
There is molecular machinery in the mitochondria which leads to the cardiac myocytes
death. The mitochondrial death mechanism is the so-called “intrinsic” pathway that
mediates apoptosis. Toxic stimuli such as oxidative stress induce translocation and
integration of the pro-death members of the Bcl-2 family. These proteins, by a
mechanism permeabilize the outer membrane to an extent that allows the release of proapoptotic proteins from the inter-membrane space, cytochrome c, Smac/DIABLO,
htrA2/Omi protease, and endonuclease-G (endoG). Cytochrome c binds to the cytosolic
protein apaf1 and the resultant “apoptosome” activates the caspase-9 and -3 protease
system. Smac/DIABLO and htrA2/Omi activate caspases by either sequestering or
degrading caspase-inhibitory proteins. EndoG translocates to the nucleus and mediates
DNA fragmentation. The coordinated action of these proteins subsequently results in the
death of the cardiomyocyte by apoptosis. Mitochondrial rupture can lead to the release of
pro-apoptotic inter-membrane proteins described above, which would initiate the
apoptosis. However, if the stress is severe or prolonged, ATP which is required for
apoptosis to occur will be depleted and the cell will instead die by necrosis. Indeed, the
relative contribution of MPT to apoptosis versus necrosis is still the subject of debate [19].
Mitochondria in ischemia-reperfusion injury
The following description is focused on the main factors that contribute to the induction
of MPT and cardiomyocyte death.
a. Adenine nucleotides
The ischemia can be explained as the lack of oxygen supply to the affected area of the
myocardium. As the electrons generated by the electron transfer chain in the
mitochondria cannot be transferred to molecular oxygen there is a cessation of oxidative
phosphorylation and inhibition of mitochondrial ATP synthesis. Also the inhibition of
electron transfer prevents the pumping of H+ across the inner membrane, which is
required to generate the Δψm. In an effort to maintain the Δψm the mitochondrion runs
the F1F0 ATP synthase in reverse thereby hydrolyzing the remaining ATP. [14]
b. Mitochondrial Ca2+
After inhibition of the of oxidative phosphorylation in ischemic condition, the cardiac
myocytes has to rely on anaerobic glycolysis which leads to generation of the lactate
which leads to acidification of the cytosol, this leads ionic disturbances leading to slow
increase of cytosolic Ca2+ that is then accelerated upon reperfusion. Application of a
contractile stimulus to smooth muscle produces an elevation in the free cytoplasmic Ca2+
concentration ([Ca2+]i), which subsequently leads to activation of the contractile
machinery. Depending upon the nature of the contractile stimulus, the increase in [Ca2+]i
may reflect influx of Ca2+ from the extracellular medium, and/or release of Ca2+ from the
sarcoplasmic reticulum (SR) (17).
c. Mitochondrial ROS
It is paradoxical that the restoration of the oxygen supply to the ischemic region by
reperfusion is most likely the biggest cause of myocyte death [20]. The reperfusion is
associated with outburst of mitochondrial derived free oxygen radicals. These radicals
will damage the mitochondrial proteins which includes electron transfer chain and lipid
peroxidation. The free oxygen radicals are the inducer for the MPTP.
d. Apoptotic Bcl-2 proteins
Ischemia reperfusion induces cardiac myocyte death. The activation of Bcl-2 proteins
such as Bax, Bid, Puma and BNIP3, translocation of these proteins in the mitochondrial
membranes leads to death of cardiomyocyte. The activation of pro-death Bcl-2 proteins
such as Bax, Bid, Puma, and BNIP3, and the translocation and integration of these
proteins into mitochondrial membranes has been causing ischemic damaged to myocytes.
The MPT pore
The MPT phenomenon is mediated by the MPT pore which is a non-specific channel and
is thought to span the inner mitochondrial membrane. The pore itself is permeable to
solutes up to 1.5 kDa. This pore helps in the equilibration of H+. The mitochondrial pore
is sensitive to redox, Ca2+, voltage, adenine nucleotide, Pi, and pH sensitive [21][22][23]. The
pore consist the voltage-dependent anion channel (VDAC) [24] in the outer membrane,
the adenine nucleotide translocase (ANT) in the inner membrane, plus cyclophilin-D
(CypD) [25] in the matrix. The increased Ca2+ concentration and the presence of the free
radical species induce pore opening and the adenine nucleotides inhibit the pore opening.
PHARMACOLOGICAL STRATEGIES TO PREVENT ISCHEMIC
REPERFUSION INJURY
The importance of limiting myocardial ischemia–reperfusion injury in clinical practice
has been appreciated for a very long time. Unfortunately, till date no therapeutic
approach has been demonstrated clinically effective in protecting heart muscle at risk of
ischemic and reperfusion injury. Following strategies can be hypothesized in order to
prevent the ischemic and reperfusion [20].
a. Proteins of the mitochondrial outer membrane
 Targeting mitochondrial permeability transition pore
Triggering mitochondrial permeability transition pore induces the release of cell death
effectors and the loss of mitochondrial functions which are necessary for cell survival [26].
Thus, drugs should be design in a fashion which will be able to block or to limit
mitochondrial membrane permeabilization during ischemia-reperfusion and can be a
possible cytoprotective. Another approach could be determining the genetic makeup of
the mitochondrial permeability transition pore and targeting the drug [27].
 The MAC/Bak channel
Mitochondrial membrane permeabilization is under the control of the Bcl-2 family of
proteins. Proapoptotic members (e.g. Bax, Bak or the BH3-only subfamily proteins Bid,
Bad, Bnip3) facilitate membrane permeabilization and promote the release of cytochrome
c and other intermembrane space components. Cytochrome c triggers formation of the
apoptosome complex and activation of caspase-9. In addition to this, there is another
pathway which can be activated by two other proteins, apoptosis inducing factor (AIF)
and endonuclease G, which are also released after outer membrane permeabilization, can
induce apoptosis in a caspase-independent manner. The anti-apoptotic members Bcl-2
and Bcl-xL are mainly localized to the mitochondrial outer membrane where they
antagonize the pro-apoptotic effect of Bax and Bak. Designing the anti-apoptotic
intervention targeting the Bcl protein may lead to successful lead.
 BH3-only Bcl-2 proteins
The activation of Bak and Bax is regulated by Bcl-2 proteins that consist of BH-3
domains [28]. These proteins regulate the signals of the apoptosis of the mitochondria and
hence can be the perfect intervention as the drug target which would prevent ischemicreperfusion injury. Another potential target from outer mitochondrial membrane
permeabilization might involve VDAC which is the major permeability pathway for
metabolites through the mitochondrial outer membrane. Different isoforms of VDAC
have been identified as mentioned in the above section. The role of VDAC in cell death
induced by mitochondria seems to be isoform specific. VDAC1 has been implicated in
the mitochondrial release of proapoptotic protein whereas VDAC2 displays antiapoptotic properties [29] [30], however VDAC is the hypothetical component of the MPTP
therefore intense research is to be done.
b. Proteins of the mitochondrial inner membrane
The inner membrane of the mitochondria consists of the proteins which regulate the
electron transport chain (ETC) and oxidative phosphorylation. The inner membrane has
high ψm, the lipid cardiopilin and is compartmentalized with the cristae. The ETC is the
important source of the mitochondrial free radical generation that occurs due to leakage
of the molecular oxygen. The free radicals include superoxide anion (O-2) and hydrogen
peroxide which are continuously generated, as the byproducts of the normal aerobic
metabolism. Normally the byproducts are detoxified by the help of manganese
superoxide peroxidase (MnSOD) and mitochondrial GSH-dependent peroxidase which
ensures that free radical levels are normal and do not reach the toxic levels. However, the
rise in the free radicals production beyond the cellular defenses then the condition is
termed as ‘oxidative stress’ which can cause cell damage and lead to cell death. The
respiratory complex Ι, known as the NADH dehydrogenase generates significant levels of
free radicals. Hence ETC can be a target in preventing the myocyte death. The complex
ΙΙΙ (bc1) regulates the MPTP and also controls the mitochondrial calcium influx. The
other targets could be the UCP (uncoupling protein) which regulates the protons gradient
and also the optic atrophy protein (OPA1) has been recently identified as the regulator of
the cytochrome c release and thus can be the potential target in the ischemic reperfusion
injury. In the recent work it has been found that the OPA1 is regulated by the MPTP.
Thus, the membrane dynamics of the inner mitochondria are involved in apoptotic
regulation and can be the potential targets for therapeutic intervention [31] [32].
Other possible Interventions to prevent the ischemic reperfusion injury:
 Conjugation of the antioxidants with lipophilic compounds and delivering it in the
cardiac myocytes to prevent the ischemic reperfusion injury and efficient methods
to regulate drug delivery to the cardiac myocytes.
 Extensive research on the PKCε [33] which focuses and modulates the
functionality of MPTP.
 Designing the agents in order to lower the pH in the cardiac myocyte which helps
in inhibiting the opening the MPTP.
 Inhibiting the xanthine oxidase with the help of designing a candidate with
specificity to the enzyme in the cardiac myocytes that leads to production of the
free radicals which in turn opens the MPTP further initiating the apoptotic events
in the myocyte.
 Use of proteomic approach to examine the MPTP structure and try to isolate the
‘event’ of the MPTP opening and isolating the proteins in the event.
 Designing the specific Ca2+ inhibitors in the myocytes which are responsible for
the opening of the MPTP.
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