Download FMM_Colin_Mitchell - Bioenergetic Failure

13 January 2011
No. 2
Bioenergetic Failure:
Mitochondrial Catastrophe or Survival
Coup
Colin Mitchell
Commentator:S. Kassie
Moderator:N. Kalafatis
Department of Anaesthetics
CONTENTS
INTRODUCTION ................................................................................................... 3
SEPSIS RELATED ORGAN DYSFUNCTION ....................................................... 3
THE MITOCHONDRION ....................................................................................... 5
CELLULAR RESPIRATION – ATP PRODUCTION .............................................. 7
Glycolysis ......................................................................................................... 8
Citric Acid Cycle (Krebs Cycle) ....................................................................... 8
Oxidative Phosphorylation .............................................................................. 9
MITOCHONDRIAL INJURY IN SEPSIS ............................................................. 12
Genetic Predisposition .................................................................................. 13
Endocrine Alterations .................................................................................... 13
DO2:VO2 Balance ............................................................................................. 14
Oxidative Stress ............................................................................................. 14
Reactive Oxygen Species ....................................................................................... 14
Reactive Nitrogen Species ..................................................................................... 15
Endogenous Antioxidant Protection ..................................................................... 16
Oxidative Stress Induced Mitochondrial Damage ................................................ 16
MITOCHONDRIAL CATASTROPHE HYPOTHESIS .......................................... 18
BIOENERGETIC FAILURE................................................................................. 19
TREATMENT STRATEGIES .............................................................................. 20
The prevention and reversal of early mitochondrial dysfunction ............... 20
The prevention of energetic failure once mitochondrial dysfunction is
established...................................................................................................... 22
The resolution of the mitochondrial dysfunction ......................................... 23
CONCLUSION .................................................................................................... 24
APPENDIX A – NOVEL ANTIOXIDANT THERAPIES ........................................ 25
REFERENCES.................................................................................................... 28
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INTRODUCTION
Sepsis is the systemic inflammatory response to a confirmed or suspected
infection.50 In the critically ill patient, it is the most common cause of both
morbidity and mortality. In uncomplicated sepsis, mortality may reach 25% and in
patients who develop organ failure this may increase up to 80%.1 Despite ongoing
research and an increase in the understanding of sepsis-related pathophysiology
as well as the development of novel approaches and treatments to deal with
sepsis, the deterioration in the function of various organs – the Multiple Organ
Dysfunction Syndrome (MODS) remains the most common cause of Intensive
Care Unit (ICU) mortality.1
For some time, the accepted aetiology of sepsis-induced organ dysfunction
centred on the role of the inflammatory response and the associated impairment
in vascular control, specifically in the micro circulation. It was felt that this would
result in impaired delivery of oxygen and other substrates to the tissues resulting
in progressive organ dysfunction, failure and ultimately death.25
The organs most commonly affected are the lungs, kidneys and liver and whilst
the clinical manifestations including arterial hypoxaemia, azotaemia and
cholestatic jaundice may be quite profound, on a histopathological level, fatal
sepsis is often much less dramatic. Histological specimens of the affected tissue
most commonly only demonstrate areas of focal necrosis or apoptosis and only
very rarely is there the massive loss of parenchymal tissue that might be expected
as a result of hypoperfusion or tissue anoxia.2
SEPSIS RELATED ORGAN DYSFUNCTION
The pathophysiology behind MODS is most certainly multifactorial with systemic
inflammation and its associated impairment in vascular and microcirculatory
control playing a central role especially in the earlier stages of the disease. They
are, however, unable to fully explain the full pathophysiology of this disease
process on their own.
Ninety years ago Barcroft proposed the mechanisms behind the three classic
causes of cellular hypoxia: low arterial oxygen tension (hypoxaemic hypoxia), low
circulating haemoglobin concentrations (anaemic hypoxia) and microvasular
hypoperfusion (stagnant hypoxia).3 In sepsis each of these mechanisms is
potentially occurring either in isolation or more commonly in combination.
The factors to ensure adequate oxygen delivery to the tissues are well described
by the Oxygen Flux Equation and, through manipulation of PaO2, haemoglobin
concentration and cardiac output; it attempts to address the contributors to cellular
hypoxia.
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DO2 = CO x ([Hb] x 1.34g/dl x SaO2 ) + (PaO2 x 0.003)
DO2 – Tissue Oxygen Delivery, CO – Cardiac Output, Hb – Haemoglobin (g/dl), SaO2 – Arterial Haemoglobin
Saturation, PaO2 – Arterial Partial Pressure of Oxygen (mmHg)
However, the well-described observation that MODS is often associated with
accelerated anaerobic metabolism despite a supranormal systemic oxygen
delivery adds weight to the concept of an additional intrinsic derangement in
cellular energy metabolism.
This phenomenon has been termed cytopathic hypoxia. And while it has been well
described with certain drug toxicities and poisonings, classically cyanide
poisoning, there has now been a resurgence in the plausibility of the concept that
sepsis-related organ dysfunction may be at least in part be due to a ‘bioenergetic
failure’ as a result of mitochondrial dysfunction.4
Other observations in support of this theory include the progressive decrease in
total body oxygen consumption as the severity of the sepsis increases,5 tissue
oxygen tension measurements are elevated in sepsis but will normalise during
recovery6 and the ability of organs with limited reservation ability (such as the
kidney) to fully recover to such an extent that organ support may not be required.
The debate between the relative contributions of microvascular dysfunction and
bioenergetic failure could possibly be explained by a single theory. Shunting in the
microvasculature will result in regional tissue hypoxia together with decreased
ATP production.
In addition further mitochondrial dysfunction (from oxidative stress, decreased
expression of mitochondrial proteins and hormonal alterations) may result in an
energy demand versus supply imbalance. Should this imbalance continue to a
point at which ATP concentrations will fall below a critical level, the cellular death
pathways are initiated.
This means a combination of shunting and decreased mitochondrial oxygen
utilisation may be responsible for the increased venous oxygen saturations seen
in resuscitated sepsis.
The high oxygen content in tissue beds may be explained by a reduction in
metabolic activity in an attempt to match the reduced energy supply (as a result of
decreased oxygen delivery or mitochondrial dysfunction).7
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THE MITOCHONDRION
The mitochondrion is an intracellular organelle that is found in almost all
eukaryotic cells. It ranges in size from 0.5 to 10 µm and is enclosed by a double
membrane. Unlike other cellular organelles, it possesses its own DNA and it has
been postulated that they may once have been free living organisms or developed
from endosymbiotic proteobacteria.8 More than 90% of the body’s oxygen
utilisation is by the mitochondria in the process of energy production.8
1-inner membrane
2-outer membrane
3-cristae
4-matrix
Fig 1: The Mitochondrion9
Mitochondrial Structure
The mitochondria consist of a number of different compartments each of which will
carry out a number of specific functions.
I. The Outer Membrane
This is a phospholipid bilayer which is similar in structure to the plasma
membrane. It contains transmembranous proteins or porins that form
channels to allow for the passive diffusion of small molecules (<5 kD)
across the membrane.
There is a potential difference across the membrane of approximately 150 to -180mV with respect to the outside. This is formed as a result of
proton movement associated with the electron transfer chain. The
importance of this electrical gradient is also important for some of the
new therapies in which drug delivery is targeted directly to the
mitochondrion as discussed later.
II.
The Intermembranous Space
This space is located between the outer and inner membranes. As a
result of the relative permeability of the outer membrane to small
molecules, it has the same ionic concentrations as the cytosol.
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Large proteins, on the other hand, require specific transport mechanisms
to enter or leave this compartment and as such the protein content will be
different from that of the cytosol.
Cytochrome C, an essential component of the electron transfer chain, is a
protein that under normal conditions is localised to the intermembranous
compartment. Disruption of the outer membrane for any reason, will allow
leakage of this protein into the cytosol, and is one of the triggers which
will result in apoptosis (programmed cell death).10
III.
The Inner Membrane
This is also a phospholipid bilayer, but with a protein to phospholipid ratio
of 3:1 by weight (cf. a ratio of 1:1 in the plasma membrane) i.e. it is highly
protein dense. It contains a specific phospholipid, cardiolipin, which is
characteristic of mitochondrial and bacterial plasma membranes.
It does not contain porins and this results in the inner membrane being
totally impermeable to all molecules. Any substance required to enter or
leave the mitochondrial matrix therefore requires a specific membrane
transporter.
The inner membrane contains proteins that will perform one of five
specific functions, namely:
i. The reduction-oxidation (REDOX) reactions of oxidative
phosphorylation
ii. ATP Synthase which generates ATP in the matrix
iii. Transport proteins that allow passage of molecules into and
out of the matrix
iv. Protein assembly units
v. Fusion and fission proteins
IV.
Cristae
The inner membrane is highly convoluted, which allows for a greater
surface area for the production of ATP. Mitochondria in cells with a
greater demand for ATP will have more cristae (e.g. hepatic mitochondria
may have an inner surface area in excess of 5 times that of the outer
membrane)
V.
Matrix
This is the space enclosed by the inner membrane and, together with the
membrane- bound ATP Synthase, is the site of ATP production. It also
contains the enzymes responsible for the citric acid cycle and lipid
oxidisation together with mitochondrial ribosomes, transport RNA and the
mitochondrial DNA.
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VI.
Mitochondrial DNA (mtDNA)
The mitochondrion has its own genetic material as well as the capability
to manufacture its own RNA and proteins. mtDNA has a remarkable
similarity to bacterial genomes.
Mitochondrial Function
The mitochondria are responsible for a wide variety of functions which include 33:
 Regulation of the cell membrane potential
 Apoptosis
 Calcium homeostasis
 The regulation of cellular proliferation
 Regulation of cellular metabolism
 Certain haem synthesis reactions
 Steroid synthesis
However, the mitochondrion is best known for and classically described as being
the ‘Powerhouse’ of the cell. This is due to one of its most prominent functionsthat being the production of Adenosine Triphosphate (ATP) which is the primary
source of cellular chemical energy.
In fact, gram for gram, a mitochondrion generates more energy than the sun.
CELLULAR RESPIRATION – ATP PRODUCTION
Cellular respiration is the metabolic process that take place within cells in order to
convert the biochemical energy found in nutrients (glucose, fatty acids and amino
acids) into an energy source that can be used by the cell, namely ATP.
These metabolic reactions are catabolic in nature and involve reduction-oxidation
(REDOX), with molecular oxygen being the most common oxidising agent under
conditions of aerobic metabolism.
By accepting electrons, the oxygen itself is being reduced, but it is causing the
oxidisation of something else. Cellular respiration is algebraically summarised by
the equation:
C6H12O6 + 6O2 → 6CO2 + 6H2O + 38ATP
In reality, the actual energy yield is in the region of 30 to 32 molecules of ATP per
mole of glucose. Cellular respiration consists of three interdependent pathways:
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Glycolysis
Glycolysis is a metabolic process that occurs within the cytoplasm. It involves the
conversion of one molecule of glucose into two molecules of pyruvate. It can also
occur under anaerobic conditions with a net production of 2 molecules of ATP (4
ATP molecules produced with 2 consumed), as well as 2 molecules of the
reduced electron carrier, Nicotinamide Adenine Dinucleotide (NADH). NADH is
utilised later to generate ATP during the process of oxidative phosphorylation.
Glucose + 2NAD+ + 2Pi + 2ADP → 2Pyruvate + 2NADH + 2ATP + 2H+ + 2H2O
Fig 2: The Glycolytic Pathway9
In the presence of oxygen, the pyruvate is transported into the mitochondrion
where it is oxidised to the 2-carbon compound, Acetyl CoA. This results in the
generation of one molecule of CO2 and one molecule of NADH. In the absence of
oxygen, pyruvate fermentation occurs resulting in the formation of waste products
including lactate, in skeletal muscle.
Citric Acid Cycle (Krebs Cycle)
Acetyl CoA will then enter the Citric Acid Cycle within the mitochondrial matrix.
This is an 8 step process that utilises some 18 different enzymes. The net energy
yield (per molecule of glucose i.e. 2 molecules of Acetyl CoA) is 6 NADH, 2
reduced Flavin Adenine Dinucleotide molecules (FADH2) and 2 ATP with the
formation of two waste products, H2O and CO2.
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Fig 3: The Citric Acid Cycle9
Oxidative Phosphorylation
The final step of cellular respiration takes place on the mitochondrial cristae.
Energy in the form of ATP is produced from a process that commences with the
flow of electrons along 5 inner membrane-bound molecular complexes (a process
known as the electron transfer chain).
These electrons are generated by oxidising the NADH (and FADH2) produced by
the citric acid cycle to NAD+ (and FAD). The subsequent transfer of electrons from
complex to complex results in a reciprocal transfer of protons from the
mitochondrial matrix into the intermembranous space.
This generates the mitochondrial membrane potential that is then used to drive
the phosphorylation of ADP by the enzyme ATP synthase, which will result in the
formation of ATP.
Since this process is potentially the origin of mitochondrial dysfunction, we will
look at it in some detail.
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Fig 4: Schematic Representation of Oxidative Phosphorylation in theMitochondrion.8
Complex I (NADH Dehydrogenase)
This is a large enzyme that catalyses the 2 electron reduction of coenzyme Q10
(ubiquinone) to QH2 (ubiquinol) by NADH. The net result of this electron transfer is
4 protons being pumped from the mitochondrial matrix into the intermembranous
space. Complex I has been found to be a potent source of superoxide free
radicals.11
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Fig 5: Complex I (NADH Dehydrogenase)9
Complex II (Succinate Dehydrogenase)
This is the only enzyme found in both the electron transfer chain and the citric acid
cycle. It oxidises succinate to fumarate and in so doing, also reduces coenzyme
Q10 to QH2. This reaction releases less energy that the Complex I oxidisation of
NADH and as a result it does not result in proton transport across the membrane.
Complex III (Cytochrome C Reductase)
The reaction catalysed here is the oxidation of QH2 and the reduction of
cytochrome C. This is a complex, two- step process and is the second important
source of superoxide free radicals, which are formed via the production of the
highly reactive free radical intermediate, ubisemiquinone (Q-). The net effect is 4
protons entering the intermembranous space
Fig 5: Complex III (Cytochrome C Reductase)9
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Complex IV (Cytochrome c Oxidase)
The final protein complex of the electron transfer chain pumps protons across the
inner membrane whilst accepting electrons from the reduced cytochrome C onto
the terminal electron acceptor, oxygen, which is subsequently reduced to water.
.
Fig 6: Complex IV (Cytochrome C Oxidase)9
Complex V (ATP Synthase)
This protein, whilst not specifically involved in the electron transfer chain, is the
final enzyme in the oxidative phosphorlyation pathway. It uses the energy from the
proton electrochemical gradient across the inner membrane to drive the formation
of ATP from ADP
MITOCHONDRIAL INJURY IN SEPSIS
There are a number of animal studies that have been able to demonstrate the
presence of mitochondrial dysfunction in sepsis.12 Ultra-structural damage has
been seen in the hepatic mitochondria of patients who have died as a result of
sepsis.13 The depletion of endogenous antioxidants have been associated with
mitochondrial dysfunction as well as related to the severity of organ failure and
eventual outcome.14
As mitochondria utilise over 90% of the body’s oxygen consumption in the process
of ATP production, any abnormalities in oxygen consumption are therefore likely
to be as a result of mitochondrial dysfunction. In sepsis there are a number of
potential causes for mitochondrial dysfunction. These may include genetic
predisposition and down-regulation of mitochondrial protein synthesis, the
associated endocrine changes, imbalances between oxygen supply (DO2) and
oxygen uptake (VO2)as well as the ubiquitous role of Oxidative Stress.
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Genetic Predisposition
Many studies have demonstrated various genetic polymorphisms that are linked to
outcomes in sepsis including increased activation of Nuclear Factor κβ (NF-κβ)
which is responsible for regulating many of the proinflammatory mediators. As a
result, genetic associations with enhanced NF-κβ activation are associated with
poor outcomes.15
There is also evidence that sepsis may result in a decreased synthesis of new
mitochondrial proteins, specifically decreased expression of the respiratory chain
complex and ATP synthetase genes.16
Endocrine Alterations
Following the initial stress response associated with an inflammatory insult, sepsis
is associated with adrenal insufficiency, the sick euthyroid syndrome, insulin
resistance and hypogonadism.8
Mitochondria have receptors for both
adrenocortical and thyroid hormones, with the thyroid being a key modulator of
mitochondrial function.27
Hyperthyroidism increases ATP production at the expense of reduced efficiency of
production with the opposite being true of hypothyroidism. The ‘sick euthyroid
syndrome’ seen in sepsis is associated with a decreased, although more efficient,
cellular respiration.27 The effects of corticosteroids on the mitochondria appear to
depend on duration of exposure to steroids. Acute stress increases complex IV
activity (in a rat model),17 However hypercortisolaemia associated with chronic
intermittent stress has resulted in a decrease in mitochondrial functioning.27
Insulin resistance and the associated hyperglycaemia than occurs in septic
patients may also contribute to mitochondrial dysfunction through the absence of
the permissive effects of insulin on mitochondrial protein synthesis and cellular
respiration. The associated hyperglycaemia may also result in the increased
production of reactive oxygen species (ROS). This may contribute towards the
improved outcomes in critical care patients who have been rendered
normoglycaemic with insulin therapy.13
Circulating Leptin, a hormone secreted by adipose tissue involved in the
regulation of food intake and energy balance, has been shown to be decreased in
non-survivors when compared to survivors.18 The administration of exogenous
leptin to diabetic rats has been shown to increase mitochondrial proliferation in
white adipose cells.19
A similar effect has been demonstrated by administering oestrogen following
trauma haemorrhage which resulted in an increase in mitochondrial protein
production, enzyme activity and ATP levels when compared to a control.20
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DO2:VO2 Balance
Much has been written about the importance of the re-establishment of tissue
perfusion and oxygen delivery specifically in the early stages of sepsis. The
optimisation of the contributors to tissue oxygen delivery, namely cardiac output,
haemoglobin levels and PaO2, remain central to the management of sepsis.
Conceptually sepsis may be considered in two distinct phases.
Early sepsis (shutdown phase) at which stage the mitochondria have not yet been
damaged and resuscitation, in an attempt to re-establish organ perfusion, has
been demonstrated to have improved outcomes.21
The stage of late sepsis is characterised by established mitochondrial damage
and attempts at aggressive resuscitation at this stage have been shown to have
adverse outcomes.27 This opens the door to looking at possible treatment options
that may decrease the body’s energy requirements, thereby decreasing VO2, in an
attempt to conserve cellular function and energy.
Oxidative Stress 33
Reactive Oxygen Species
ROS are chemically active substances that contain oxygen. They also include
molecules with an unpaired electron (the so-called free radicals) which includes
the superoxide anion, as well as strong oxidizing agents such as hydrogen
peroxide.
Whilst ROS can be formed by exogenous sources such as ionising radiation, they
are also naturally-occurring by-products of oxidative phosphorylation formed as a
result of the incomplete reduction of oxygen to water. As molecular oxygen is such
a strong oxidising agent (readily accepts electrons), its reduction does involve the
production of these potentially harmful intermediates.
As much as 1% of oxygen may be converted to ROS via a process known as
electron leakage in which electrons are transferred directly to oxygen to form
superoxide.
O2 + e- → O2- + e- → O22O2- = Superoxide
O22- = Peroxide
ROS have important physiological roles to play in normal cell signalling and as
such their production needs to be seen as important for cellular function and
survival. Under normal circumstances ROS production is very tightly controlled by
a number of endogenous antioxidants because, whilst short-lived, they are also
highly reactive and have the potential to interact indiscriminately with surrounding
molecules. Therefore, if not controlled, they can be extremely damaging to the
same mitochondria in which they are produced.
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This is a process known as Oxidative Stress and occurs whenever ROS
production exceeds the capacity of the body’s regulatory systems.
Oxidative stress has been known to contribute to a number of disease processes
including Sickle Cell Disease, Alzheimer’s and Parkinson’s as well as the
retinopathies and neuropathies cause by diabetes.
There is also a well-defined association of the role of oxidative stress in
cardiovascular disease. Specifically, oxidised Low Density Lipoprotein (LDL)
appears to trigger atherogenesis which ultimately leads to atherosclerosis and
ultimately cardiovascular disease.22
Reactive Nitrogen Species
Oxidative phosphorylation can also result in the production of Nitric Oxide (NO-)
which, by virtue of its unpaired electron, is also a free radical. The NO- in turn has
the ability to form by-products known as Reactive Nitrogen Species (RNS).
Peroxynitrite, a highly toxic RNS, is formed from the interaction between NO- and
the superoxide anion (O2-).
Under normal circumstances the superoxide anion would be rapidly converted into
Hydrogen Peroxide (H2O2) and then to water by the endogenous antioxidant
systems. However, in the presence of increased NO- , the reaction between the
superoxide anion and NO- to form peroxynitrite occurs even more rapidly. It is this
peroxynitrite that is thought to account for most of the cytotoxic effects of NO-.
Nitric oxide is formed via the action of the enzyme Nitric Oxide Synthase (NOS)
on L-Arginine.
L-Arginine + NADPH + H+ + 2O2
NOS
Citrulline + NO- + NADP+
There are a number of different isoforms of the NOS enzyme, including:
1. Neuronal NOS (nNOS or NOS-1), produced by neurons in the central and
peripheral nervous systems and involved in cellular communication.
2. Endothelial NOS (eNOS or NOS-3), a constitutive form that is released by
vascular endothelium and is involved in the regulation of vascular tone.
3. Inducible NOS (iNOS or NOS-2) is involved in the immune response.
Activation by inflammatory mediators including Nuclear Factor κβ (NF κβ) will
result in iNOS transcription and the production of large quantities of NO -. The
induction of the high output iNOS often occurs in an oxidative environment and
as such the high levels of NO- have the ability to interact with superoxide anion
producing the toxic RNS peroxynitrite. This has a role in the killing abilities of
the macrophage respiratory burst.
4. Whilst as yet unconfirmed, there is growing interest in the potential for there to
be a mitochondrial form of NOS (mtNOS).23 This may further support the
central role of mitochondrial dysfunction in sepsis.
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Endogenous Antioxidant Protection
ROS and RNS are naturally occurring by-products of oxidative phosphorylation
and play an important role in a number of physiological processes within the
mitochondria. These include calcium and iron homeostasis and certain cellsignalling pathways. They are also highly toxic and therefore have the potential to
cause damage to surrounding molecules and organelles.
It stands to reason that there must be some endogenous protective mechanism
from these entities. Indeed, under normal circumstances their activity is tightly
regulated by an interacting system of antioxidants. This includes the enzymatic
pathways Manganese Superoxide Dismutase (MnSOD), Thioredoxin (TRX),
Peroxiredoxins, Sulphiredoxins, Cytochrome C, Peroxidise and Catalase, as well
as a number of non-enzymatic pathways, such as Glutathione (GSH), Ascorbic
Acid (Vit. C), Tocopherol (Vit. E) and Uric Acid.
As described earlier, an example of the intricate interaction between the various
endogenous pathways is that of the superoxide anion. This ROS is formed within
the mitochondrial matrix and as it is unable to cross the impermeable inner
membrane, is rapidly converted to H2O2 (itself a ROS), by the action of the
enzyme MnSOD.
H2O2 is converted to water by mitochondrial GSH or TRX. It appears that GSH is
the most abundant antioxidant in mitochondria whilst the TRX system is more
efficient. At low levels of oxidative stress both systems appear to keep H2O2 levels
under control equally. When oxidative stress increases (e.g. during sepsis) the
TRX system appears to become dominant in mitochondrial protection.24
Oxidative Stress Induced Mitochondrial Damage
Oxidative stress has been defined as an imbalance with the over-production of
ROS and the underproduction of the protective antioxidant systems and has been
well described in sepsis. Potential sources for oxidative stress in the critically ill
patient include the electron transfer chain, xanthine oxidase activation, neutrophil
activation respiratory burst and the metabolism of arachadonic acid.23
The superoxide anion is the ROS initially produced in the mitochondrion. It is
formed as a result of electron leak from the oxidative phosphorylation reactions
associated mainly with complex I and to a lesser degree with complex III.
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From in-vivo work with isolated mitochondria, it has been determined that the rate
of O2- production is critically dependant on:
1. The proton motive force (∆p) i.e. the mitochondrial membrane potential
2. The NADH/NAD+ ratio
3. The CoQH2/CoQ ratio
4. The local O2 concentration
In any situation in which the mitochondria are not producing ATP, they will have
an increased ∆p as well as an increased NADH/NAD+ ratio and reduced
Coenzyme Q10 pool. All of these factors then promote the formation of O2production.
This oxidative stress may then result in damage to lipids, proteins and the nucleic
acids of both within the mitochondria as well as the cell by any one of a number of
mechanisms 33.
 Direct toxicity of the ROS to the mitochondrial membrane. The peroxication
of the mitochondrial membrane lipid cardiolipin, which is located on the inner
membrane, will result in the dissociation of cytochrome C away from the
intermembranous space,25 the net effect of which will include decreased
ATP synthesis and further increase in ROS production.
 Nitric oxide competes with oxygen binding to complex IV, thus decreasing
the activity of the enzyme. This blocks the electron transfer chain with a
resultant overproduction of superoxide.
 The ROS may also have a direct inhibitory effect on the antioxidant system
itself via the oxidisation or peroxidation of the component enzymes.
 Finally ROS may result in direct damage to mtDNA. This is a very important
effect of oxidative stress for the following reasons:
1. The mtDNA is in very close proximity to the electron transport chain, the
source of ROS
2. mtDNA encodes for a number of tRNA species, rRNA species as well as
proteins that are crucial to the electron transfer chain and energy
generation
3. The entire mtDNA encodes for expressed genes (in comparison with the
Genomic DNA which has a high proportion of non-coding sequences).
Therefore damage to mtDNA is much more likely to result in a functional
mutation.
Any of these mechanisms will in turn, ultimately result in a self amplifying cycle of
more ROS production (aptly named ROS-induced ROS release), until finally the
production of ROS will completely overwhelm the antioxidant system with ensuing
cell death. This is a concept known as Toxic Oxidative Stress or the Mitochondrial
Catastrophe Hypothesis.26
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Fig 7: Overview of Mitochondrial ROS Production31
MITOCHONDRIAL CATASTROPHE HYPOTHESIS
Under normal circumstances, the inner mitochondrial membrane is impermeable.
However there are certain triggers which include oxidative stress, calcium
overload and apoptotic protein expression that can result in a concept known as
‘Permeability Transition’ 33.
These triggers result in an increase in inner membrane permeability, and the
subsequent release of cytochrome C and Apoptosis Inducing Factor. These in
turn will activate the caspase cascade. The Caspases (Cysteine-dependent
aspartate-directed proteases) are a family of proteases that play an essential role
in apoptosis giving them their alternative, somewhat dramatic name of
‘Executioner Proteins’.
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The critical point of apoptosis appears to be the initial increase in the membrane
permeability as once the caspase activators are released the process becomes
irreversible.
BIOENERGETIC FAILURE
To this point, the discussion has been around the progressive deterioration in
mitochondrial function which, if not prevented or reversed, will result in a reduction
in cellular metabolism to a point at which the organ is no longer able to perform its
basic function.
An interesting point first raised by Singer in 200427 was the possibility that this
bioenergetic failure should possibly not be seen as ‘failure’, at least not initially,
but rather as a metabolic shutdown aimed at shifting the cell from normal
functioning into a survival mode. Should one subscribe to the theory that
mitochondrial dysfunction is not actually mitochondrial failure, then it should be
considered to rather be a potential survival mechanism potentially aimed at
inducing cellular ‘hibernation’ by triggering a state of suspended animation.
This thus possibly protects the organism from the energetic failure until such time
as the inflammatory insult has resolved and a recovery can be made. Animals
that hibernate or aestivate have the ability to significantly reduce their metabolic
energy expenditure. Certain amphibians and reptiles have the ability to withstand
periods of hypoxia by suppressing ATP turnover. Whilst humans neither hibernate
or aestivate, organs such as the heart have the ability to exhibit abnormalities in
contractile function in the face of chronic ischaemia that is potentially reversible
once perfusion if re-established, so called myocardial hibernation or stunning,
which could represent an adaptive response to hypoxia.
Nonetheless, should the mitochondrial dysfunction be persistent, there will be a
tipping point at which the minimal threshold level for ATP production is reached
and the apoptotic pathways are initiated.27
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Fig 8: Postulated progression of mitochondrial dysfunction during sepsis and recovery 28
TREATMENT STRATEGIES
Within this model of sepsis-induced MODS, the result of cellular energetic failure,
potential treatment strategies may be divided into 3 broad categories:
The prevention and reversal of early mitochondrial dysfunction
 Optimising DO2 – In early sepsis, cellular hypoxia will result in decreased
aerobic ATP production and contribute to mitochondrial dysfunction. As a
result, the early optimisation of oxygen delivery may prevent bioenergetic
failure if instituted whilst the mitochondria are still able to produce ATP. This
approach, however, may no longer be beneficial once the mitochondria have
already been damaged and energy production has been further affected.
It is clear that this dysfunction may also occur even with adequate fluid
resuscitation and re-establishment of tissue oxygenation and, in this
scenario, failure of oxygen consumption to improve (despite the reestablishment of delivery) has been associated with worse outcomes in
septic patients.29
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 Glucose Control – As discussed previously, both the hyperglycaemia and
insulin resistance seen in sepsis pose a threat to mitochondrial functionality.
The maintenance of normoglycaemia using insulin was demonstrated by
Vanhorebeek to have beneficial effects of hepatic mitochondrial structure
and function.13
 Antioxidants – The association between sepsis-related organ dysfunction
and oxidative stress-mediated mitochondrial dysfunction has opened the
door to the possibility of there being a role for the therapeutic administration
of antioxidants in critically ill patients.
Whilst there is evidence that systemic antioxidant therapy, in particular
selenium, glutamine and the Ω-3 fatty acids,30 may improve outcomes in
sepsis, systemic antioxidant supplementation has yet to prove specifically
successful in critically ill patients.31 It has been postulated that a possible
reason for this may be because they did not target the specific intracellular
site most affected by oxidative damage, the mitochondria.32
This then has lead to the search for mechanisms in which delivery of the
antioxidant directly to the mitochondrion can be achieved consistently and
reliably. These approaches are currently undergoing tests in-vitro and in
animal models.33
There are a number of methods by which therapeutic delivery to the
mitochondrion may be achieved:
1. Delivery of the antioxidant directly to the mitochondria by attaching it to a
specific carrier molecule.
2. Administration of an antioxidant that naturally accumulates in or acts on the
mitochondrion.
3. Augmentation of the mitochondrial endogenous antioxidant defences via
either pharmacological means or by genetic manipulation 33.
Currently, in the majority of studies on mitochondrial-targeted antioxidants,
treatment has been given prior to the septic insult. This is clearly not
congruent with the clinical situation, which would require delaying treatment
until sepsis has been established.
However, ongoing refinement to these early in-vivo and animal models will
allow development of studies that have more relevant applications in
humans.26 It should be borne in mind that there are, however, a number of
situations in which the timing of the oxidative stress trigger is known in
advance, and as such the encouraging trends demonstrated by a number of
these studies should not be discounted as entirely clinically irrelevant.
Page 21 of 31
Interesting questions that will need to be addressed at some point in the
development of this novel treatment modality include the potential ability of
the antioxidant to decrease, stop or even reverse established ‘bioenergetic
failure’; what the optimal dose and timing of each intervention will be; does
the possibility that we may need to target specific modalities to specific
organs exist, and what is the place of this modality in other forms of
oxidative stress including ischaemia-reperfusion injury, haemorrhagic shock
and non-infective SIRS?
A more detailed look at the novel treatments currently under investigation
can be noted in appendix A.
The prevention of energetic failure once mitochondrial dysfunction is
established
 Electron Donors – Succinate dimethyl ester has been shown to reduce
hepatic ATP contentin septic models.34 This is achieved by bypassing
complex I (which is more significantly affected in sepsis) and allowing the
relatively preserved action of complex II to increase the flow of electrons
through the electron transfer chain by the oxidation of succinate to
coenzyme Q. This will increase ATP generation if there is no rate-limiting
inhibition distal to complex II.8
 Suspended Animation – Once mitochondrial dysfunction is established, a
novel treatment strategy would be to decrease cellular energy expenditure
by inducing a state of cellular hibernation.Mice exposed to hydrogen
sulphide (H2S) have demonstrated significant decreases in metabolic rate.
H2S is a specific, potent, reversible inhibitor of Complex IV (Cytochrome C
Oxidase) and exposure in a concentration of 80ppm resulted in body
temperature approaching that of the environment and oxygen consumption
and carbon dioxide production both decreasing as much as 90% from
baseline.35 On reversal of the hydrogen sulphide, this state of suspended
animation is reversed with no permanent behavioural or functional sequelae.
This radical approach would carry with it a number of complexities; the
cooling associated with suspended animation would negate the
cytoprotective effects of heat shock proteins which require the hyperthermic
response to infection in order to be expressed. A further complication may
then be timing of the reversal of ‘hibernation’, with premature stimulation
before the mitochondria are ready to resume energy production resulting in
cellular compromise.8
 β Blockers – Sepsis has also been described as the ‘rude unhinging of
metabolism’36 which results in activation of the sympathetic nervous system,
hyperglycaemia and insulin resistance, breakdown of proteins and systemic
vasodilatation amongst other things. All of these effects would be potentially
attenuated with the use of Beta Blockers.37
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It may seem reasonable to consider a treatment strategy that deviates from
maximising oxygen delivery to one which attempts to minimise oxygen
requirements. Whilst controversial, the use of Beta Blockers would
potentially fulfil this role in decreasing cardiac workload and VO2 assuming
DO2 has been optimised. There are no current human studies that address
the issue of Beta Blockade in sepsis, with most of the evidence either being
extrapolated from non septic populations (including medical, burns and
trauma) or from animal based septic models.38
The resolution of the mitochondrial dysfunction
Whilst the trigger for this step has yet to be discovered, the repair and
replacement of the damaged and dysfunctional mitochondria are likely to be
controlled at the transcriptional level.
 Nitric Oxide – whilst high quantities of NO- produced during the inflammatory
response are cytotoxic and result in mitochondrial dysfunction, it has
recently been established that small quantities of cNOS-derived endothelial
NO- may be a major trigger in mitochondrial biogenesis.8
 Hormone supplementation – Thyroid hormone stimulates mitochondrial
activity and has been shown to up-regulate mitochondrial related
transcription factors in mice.39 The low circulating T3 seen in chronic illness
may be the result of neuroendocrine dysfunction and the correct timing of
thyroid hormone supplementation may be a potent stimulant of
mitochondrial activity and metabolic rate and may promote early organ
recovery. As mentioned preciously, premature stimulation of ‘hibernating’
mitochondria may have detrimental effects on the cell as well.
The potential benefits of other hormone supplementation, namely Leptin and
Oestrogen,have been mentioned previously.
Page 23 of 31
Fig 9: Potential therapeutic Interventions in mitochondrial dysfunction8
CONCLUSION
The principles of intensive care are supportive and still almost all interventions
undertaken in the critically ill patients including mechanical ventilation, inotropic
support, endocrine supplementation and immunonutrition have all been
associated with adverse outcomes.
The potential causes of ‘bioenergetic’ failure in sepsis are multifactorial and range
from genetic predisposition to imbalances between oxygen delivery and oxygen
use to mitochondrial damage as a result of oxidative stress.
Mitochondrial dysfunction may be as a result of direct or indirect damage from
both ROS and NOS (oxidative stress), decreased expression of mitochondrial
proteins or as a result of the metabolic and hormonal disruptions that occur in
sepsis.
As in many of the body’s responses to an insult, sepsis should possibly be viewed
not as an uncontrolled, damaging process but possibly as an intricate,
multisystemic condition that includes both protective and damaging pathways.
Page 24 of 31
Although MODS may be the end result of ‘bioenergetic failure’, it may also
represent an adaptive process to the reduced energy supply in an attempt to allow
the organ to recover enough function to allow long-term recovery, should the initial
insult be controlled.
Should this be the case, we need to understand that any treatment or intervention
we institute may be deleteriously affecting this adaptive attempt and in so doing,
may result in detrimental consequences.7 We should also be aware that as a
result of sepsis being a dynamic process, the timing of interventions may be
crucial to the outcome.
The renewed interest in attempting to address the imbalance between ROS
overproduction and antioxidant deficits in sepsis, especially by targeting delivery
of antioxidants directly to the source of the ROS production, the mitochondria,
may have potential as novel future treatments in MODS- associated sepsis.
APPENDIX A – NOVEL ANTIOXIDANT THERAPIES
Lipophilic cations
Liphophilic cations will accumulate in the mitochondria as a result of its negative
membrane potential (-150 to -180mV). By covalently binding a specific antioxidant
molecule to the cation, delivery to the mitochondrion can be ensured. A number of
different formulations are being utilised e.g. MitoQ attaches the antioxidant
coenzyme Q10 (ubiquinone) to the cation triphenylphosphonium (TPP).
Accumulation of MitoQ in the mitochondrion is 500 times the levels of those found
in the cytoplasm. Once inside, the MitoQ will be absorbed into the inner
membrane and recycled into ubiquinol which is then active in the respiratory
chain.40 MitoQ is the most well-studied of the mitochondrial targeted antioxidants.
Whilst some reservations have been expressed as to the potential efficacy of
these cations in septic patients on the basis that the dysfunctional mitochondria
may have a reduced ability to accumulate them, MitoQ has been shown to provide
better protection to oxidative stress-mediated injury in treated when compared
with non treated tissue both in-vitro, and in animal models.41, 30
In addition to its antioxidant effects, MitoQ has anti-inflammatory effects. In
conditions of sepsis induced in human epithelial cells, it has been shown to be
able to decrease ROS production and offer protection to the mitochondrial
membrane. It has also been shown to decrease Interleukin-6 (IL-6) and IL-8
release during in vitro lipopolysaccharide stimulation.42 Sepsis-induced cardiac
dysfunction has also been attenuated in rat models following administration of
MitoQ.43
Other compounds that have been conjugated to TPP include:
 Tocopherol (MitoVitE)
 The peroxidase compound Ebselen (MitoPeroxidase)
 Plastoquinone (SkQ)
Page 25 of 31
Fig 9: The Lipophillic Cations 26
Antioxidant Peptides
SS Peptides, so called because they were designed by Szeto and Schiller42 are
small, synthetic, positively-charged peptides containing less than 10 amino acids.
They are able to freely enter cells by passive diffusion and as a result of their
positive charge; they will accumulate within the mitochondria. The peptide
sequence is a proprietary formulation and based on ischaemia-reperfusion models
of oxidative stress, they appear to have the ability to scavenge a number of
different ROS.44
Hemigramacidin–TEMPOL Conjugates
TEMPOL is a compound that has the ability to scavenge ROS. Conjugation of this
molecule to hemigramacidin (which is part of the antibiotic Gramacidin-S that has
a high affinity for the mitochondrial membrane) will result in the compound being
targeted directly to the mitochondria. Studies in rat haemorrhagic shock models
have shown benefit in TEMPOL treated tissues when compared with nontreated.45
Page 26 of 31
Increasing Endogenous Mitochondrial Antioxidants
Glutathione is the most common of the endogenous antioxidant mechanisms. It is
synthesised in the cytoplasm from its 3 component amino acids: cysteine, glycine
and glutamate.
After synthesis it is transported into the mitochondrion. Compounds such as
glutathione N-acetyl-L-cysteine choline esters have been shown to be able to
increase the availability of endogenous glutathione by acting as a source for the
component amino acids. In vitro studies are promising but this has yet to be
studied in models of sepsis.46
Genetic Manipulation
Novel genetic approaches to increasing endogenous antioxidant production
include adenoviral transfection of human MnSOD into rats. This has resulted in
increased hepatic MnSOD activity with a reduction in oxidative damage induced
by both alcohol and by ischaemia-reperfusion. 47,48
Melatonin
Melatonin is synthesised from the amino acid tryptophan. The highest
concentrations of melatonin within the cells are found in the mitochondria. Both
melatonin and a number of its metabolites have been shown to have profound
antioxidant activity. Melatonin has been shown to prevent mitochondrial
dysfunction, energy failure and apoptosis as well as attenuating the inflammatory
cytokine release in animal models of oxidative injury.49
Page 27 of 31
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