Dysfunction of the Bioenergetic Pathway

M. Singer

Introduction

Sepsis represents a whole-body inflammatory response to infection that often progresses to multiple organ failure (MOF). In this condition, organ function is altered in an acutely ill patient such that homeostasis cannot be maintained without interventions, including pharmacological and mechanical support, in the hope that recovery will eventually ensue once the severe inflammatory insult has subsided.

Despite three decades of intensive research and billions of pharmaceutical company dollars searching for immunotherapeutic magic bullets, mortality rates for sepsis-induced MOF have not changed dramatically. The incidence of sepsis is rising [1], and predicted to increase still further as elderly and immunosuppressed populations grow.

The syndrome of sepsis presents numerous paradoxes and many questions that remain unanswered. Why are some people more susceptible than others to a septic insult? How does excessive inflammation actually cause the organs to fail?

Why do these failed organs look remarkably normal with minimal evidence of cell death [2]? Unlike many other conditions that cause single organ failure, why is there, in general, (near-) total recovery of organ function should the patient survive? Why is this recovery seen even in organs with poor regenerative capacity [3]?

This chapter will attempt to address at least some of these points, adopting the stance that mitochondrial dysfunction lies at the core of organ dysfunction (Fig. 1.). Recovery is thus contingent on the restoration of an adequately function- ing bioenergetic pathway. I will suggest that this assumed ‘failure’ may actually represent an adaptive, last-ditch, protective response to enable eventual survival of the patient should they be ‘fit’ enough to endure the prolonged inflammatory insult.

How Does Inflammation Lead to Organ Failure?

Genetic, immune, and exogenous factors (e. g., more pathogenic bacteria) are responsible for triggering an excessive degree of inflammation, yet precise mecha- nisms and interactions remain poorly understood. There is an increasing apprecia-

Fig. 1. A postulated mechanism of organ dysfunction and recovery

tion of the importance of other systems, such as the hormonal, immune, metabolic, and bioenergetic pathways in inducing organ dysfunction. The degree of pertur- bation of each of these systems has been independently associated with increased mortality. Furthermore, many of the conventional paradigms that have attempted to explain the underlying pathophysiology of organ failure have been successfully challenged in recent years. For example, the traditional belief held that organ fail- ure was directly related to inflammatory mediator-induced release of vasoactive agents, activation and aggregation of neutrophils and platelets, and a disseminated intravascular coagulation (DIC) that would result in an abnormal microvasculature with consequent tissue hypoxia, cell death, and organ dysfunction. However, this dogma has been undermined by the findings of normal histology in the majority of affected organs [2], and a paradoxical rise in oxygen tension in tissues as varied as gut epithelium, bladder, and skeletal muscle [4–6]. As the tissue oxygen tension represents the local balance between supply and demand, this infers that oxygen is actually freely available but is not being utilized. Thus, the major problem in sepsis may lie at a cellular level. As cytochrome oxidase is by far the predominant

consumer of molecular oxygen (> 90% of total body utilization), a mitochon- drial pathology is, therefore, implicated in the pathophysiology of sepsis-induced MOF.

Mitochondria in Health

The mitochondrion is the powerhouse of the body, being responsible for > 95%

of ATP in most cell types. A number of other roles are increasingly appreciated, such as initiation of cell death pathways (apoptosis and necrosis), intracellular calcium regulation, oxygen sensing, steroidogenesis, and signaling. The latter is probably mediated by production of reactive oxygen species (ROS). Production of superoxide at complexes I and III of the electron transport chain is considered to be responsible for approximately 2% of mitochondrial oxygen consumption in healthy cells. As will be discussed later, production of ROS increases significantly during sepsis and may be responsible for mediating many of the pathological effects seen in this syndrome.

Mitochondrial ATP production is a highly sophisticated and regulated pro- cess (Fig. 1). It was first described as the chemiosmotic proposal (the coupling of biological electron transfer to ATP synthesis) by Peter Mitchell in the 1960s.

Substrate is provided by oxidation of glucose (glycolysis), fats, and amino acids (notably alanine) from which acetyl co-enzyme A (acetyl CoA) is produced within the mitochondrion. This is incorporated within the tricarboxylic acid (Krebs’) cycle, generating reducing equivalents in the form of NADH and FADH₂. These molecules provide electrons to complexes I and II, respectively, of the electron transport chain. Passage of electrons from NADH to complex I is considered the primary route. As electrons are passed down the chain to complexes III and IV, pro- tons move across the inner mitochondrial membrane to generate a proton gradient that can drive ATP synthase to generate ATP from ADP. Complex IV (cytochrome oxidase) is the only point in the whole process where oxygen is consumed. For every glucose molecule metabolized, 2 molecules of ATP are generated during glycolysis, 2 in the Krebs’ cycle, and approximately 25 in the electron transport chain.

Rich [7] assumed that if 90% of human power is provided by the protons trans- ferred through ATP synthase, then the trans-membrane proton flux would have to represent roughly 3× 10²¹protons per second and ATP would be reformed at a rate of around 9× 10²⁰molecules per second. This is equivalent to an ATP turnover rate of 65 kg per day, a figure that would rise considerably during periods of activity.

He further calculated that to support an average adult oxygen consumption rate of 380 liters O₂ per day, 2× 10¹⁹ molecules of cytochrome oxidase are needed.

The amount of inner mitochondrial membrane necessary to hold this amount of respiratory enzyme equates to a surface area of approximately 14,000 M².

Mitochondria in Sepsis

Mitochondrial abnormalities – both biochemical and ultrastructural – have been recognized in in vivo and in vitro models of sepsis for more than 30 years. Of note, a systematic review of these models [8] showed variable findings in short- term models of varying severity lasting several hours, with increased, decreased, or unaltered mitochondrial function being reported. However, when the study duration exceeded 16 hours, dysfunction and/or injury were consistent features.

Corresponding functional changes were noted that supported the concept of mi- tochondrial dysfunction. For example, Rosser et al. found that maximal oxygen consumption was markedly increased in a hepatocyte cell line exposed to endo- toxin after six hours, but was significantly depressed by 24 hours [9]. In a patient study, Kreymann et al. noted that increasing sepsis severity was associated with progressive falls in oxygen consumption [10].

Human data are still relatively scanty. Initially, small case series reported de- creases in ATP or decreased activity of various respiratory chain complexes [11–16].

A larger study published in 2002 consisted of a group of 28 patients in septic shock, and a control group of patients undergoing elective hip surgery [17]. A significant association was seen between sepsis severity and complex I inhibition in muscle biopsies taken within 24 hours of admission to intensive care. Interestingly, there was a clear delineation between subsequent survivors and non-survivors, with ATP levels being preserved in the former (compared to the orthopedic controls) and significantly reduced in the latter. This was found notwithstanding the lack of any clinical differentiation between the two septic groups at the time of biopsy. This human study prompted the development of a long-term (3-day) rat model of fecal peritonitis that remained adequately fluid resuscitated throughout to ensure an adequate circulating blood volume. This model mimics many of the physiological, biochemical, histological, and outcome characteristics of the human patient and enables comparison of muscle data with other ‘vital’, deeper organs, such as liver and kidney [18]. Mitochondrial results were comparable to the human muscle data in both liver and kidney with the more severely septic animals also demonstrat- ing greater degrees of complex I inhibition and a fall in ATP levels. Importantly, recovery in mitochondrial function paralleled clinical and biochemical recovery.

A crucial question that will be addressed later in this review is whether such mito- chondrial recovery is fundamental to the restoration of organ function and, if so, how this could arise?

Nitric Oxide: A Mechanism for Mitochondrial Inhibition

An important step in unraveling the mechanism of mitochondrial inhibition in sepsis arose from the discovery of nitric oxide (NO). This reactive species is pro- duced in greater quantities in sepsis than in any other clinical condition and is largely responsible for the characteristic hypotension and vascular hyporeactivity (i. e., decreased responsiveness to vasoconstrictor catecholamines) of septic shock.

The subsequent recognition that NO and, more particularly, peroxynitrite (formed from the reaction between NO and superoxide), were potent inhibitors of the elec- tron transport chain [19–21] suggested its likely relevance in sepsis. In both the septic patient and fecal peritonitis animal model studies described earlier [17, 18], raised NO production (measured as tissue nitrite/nitrate) correlated with the de- gree of sepsis severity and complex I dysfunction. Glutathione, an endogenous mitochondrial antioxidant that protects complex I from nitrosative damage, was correspondingly reduced and the inability to reverse this inhibition with exogenous glutathione suggested nitration of the enzyme leading to a longer-lasting, if not irreversible, inhibition. In a macrophage cell line incubated with endotoxin [22], a progressive decrease in oxygen consumption and complex I inhibition was found.

In conjunction with these findings, early nitrosylation was followed by a progres- sive increase in nitration which was accentuated in the presence of concurrent hypoxia. Similar findings have been reported in isolated rat aorta exposed to en- dotoxin and interferon (IFN)-

γ

If the above findings can be extrapolated to patients, coexisting tissue hypoxia (for example, due to delayed fluid resuscitation) would have a synergistic effect with systemic inflammation and would reduce the competition between NO and oxygen for the same binding site on cytochrome oxidase. Boulos et al. [24] incubated an endothelial cell line with plasma taken from septic patients and found a decrease in mitochondrial respiration and ATP levels compared to that seen following incubation with plasma from healthy controls. This depression could be reversed by inhibition of either NO synthase (NOS) or poly-ADP-ribose polymerase (PARP), a nuclear repair enzyme that depletes NAD stores yet also has anti-inflammatory actions. Excessive NO has also been implicated in the skeletal and cardiac muscle contractile failure seen in sepsis, for which a mitochondrial etiology has been suggested [25, 26].

Influence of Hormones on Mitochondrial Activity in Health and Sepsis

Numerous hormones impact on different aspects of mitochondrial function in health, for example, oxidative phosphorylation activity (insulin, thyroid, cate- cholamines), efficiency and uncoupling (thyroid, growth hormone, testosterone), free radical formation, and lipid peroxidation (insulin, dehydroepiandrosterone) and biogenesis (leptin).

During sepsis and other critical illness, there are well-recognized perturbations in endocrine, metabolic, and bioenergetic activity. For example, most patients de- velop the low T3 (‘sick euthyroid’) syndrome, the severity of which will distinguish eventual non-survivors from survivors, even on admission to intensive care [27].

As many of the actions of thyroid hormones on metabolism are mediated through their actions on mitochondrial activity, the low T3 syndrome in sepsis may also have direct implications on cellular respiration. Furthermore, thyroid status also

has effects on NO production. In hypothyroidism, hypothalamic expression of NOS is significantly reduced [28], whereas liver and skeletal muscle mitochondrial NOS activities are significantly increased, correlating inversely with both serum T3 lev- els and oxygen uptake [29]. Adrenal insufficiency is also well recognized in sepsis, and corticosteroid replacement is now broadly applied to critically ill populations.

However, while short-term dosing of corticosteroids increased rat skeletal muscle mitochondrial mass and respiratory complex activity [30], chronic administration had the opposite effect [31]. This may be pertinent in view of the critical illness myopathy that has been linked with steroid use. Yet another example of a hormonal perturbation in sepsis is that of leptin. Plasma leptin levels are increased in even- tual survivors of sepsis [32]. As prolonged starvation is associated with marked decreases in leptin levels, fasted mice will show increased lethality to endotoxin which can be partly reversed by exogenous leptin administration [33]. We have reproduced this finding in a long-term mouse model and found an associated improvement in mitochondrial function (unpublished data).

The Role of Mitochondria in Sepsis-induced Cell Death

Although most organs affected in sepsis demonstrate minimal, if any, evidence of cell death, a notable exception is the immune pathway. In post-mortem studies of patients dying from MOF, Hotchkiss et al. found evidence of increased apoptosis in spleen, lymphocytes, and gut epithelium [2]. In subsequent studies of isolated lymphocytes, they reported evidence of activation of mitochondrial cell death pathways [34]. Inhibition of caspase activation was related to improved outcomes in a mouse model of sepsis [35]. On the other hand, a number of investigations have shown that neutrophil apoptosis is profoundly depressed in sepsis [36] and burns [37]. Decreased activation of mitochondria-related death pathways was noted, with maintenance of mitochondrial transmembrane potential. Reasons for these contrasting responses in different immune cells need to be better understood, particularly in respect to potential application of therapies, e. g., caspase inhibitors that may affect both cell types. Increased lymphocyte apoptosis is associated with a shift in Th1:Th2 ratios and enhanced immunosuppression, whereas resolution of neutrophil (PMN)-mediated inflammation occurs through apoptosis.

Other specific cell types also show increased apoptosis. In one septic rat model [38], increased apoptosis was detected in neurons within the hippocampus, choroid plexus, and cerebellar Purkinje cells. Both mitochondrial Bax (a member of the pro-apoptotic Bcl-2 family) and cytochrome c were upregulated in the early stages of sepsis (6–12 hrs), but decreased later on (48–60 hrs). Increased neu- ronal and glial apoptosis was also found within the autonomic centers of the brain in post-mortem studies of patients dying of septic shock, and this was strongly associated with endothelial iNOS expression [39].

Changes in Mitochondrial Phenotype

Other than direct inhibition of mitochondrial respiratory enzymes, recent data suggest that changes in phenotype during sepsis will also decrease the amount of mitochondrial protein expressed. In two related papers using a rat endotoxin model studied after 48 hours [40, 41], Callahan and Supinski described a down- regulation of genes encoding components of both the electron transport chain and glycolysis, with corresponding decreases in enzyme activities, mitochondrial oxygen consumption, and ATP formation. Calvano and colleagues [42] assessed changes in leukocyte gene expression patterns over a 24 hour period in volunteers given a single dose of endotoxin, thus allowing precise timing of the initiating inflammatory insult. Of 44,000 probe sets, the signal intensity of 5,093 probe sets (representing 3,714 unique genes) was significantly affected. A minority of probe sets was induced by 2 hours; over half showed reduced abundance at 2–9 hours but returned to baseline by 24 hours, while the remainder showed a delayed response, peaking at 4–9 hours but returning to baseline by 24 hours. Of note, these authors reported a suppression of genes involved in energy production (e. g., components of mitochondrial respiratory chain complexes I, III, and V) with a concurrent down- regulation of genes encoding for both protein synthesis and protein degradation.

Mitochondrial Recovery

In the afore-mentioned studies in septic shock patients [16] and in long-term rat fe- cal peritonitis [17], a decrease in complex I activity was noted, however complex IV activity showed a tendency to rise. This may be misleading, as rapid reversibility of competitive NO inhibition of this enzyme in the room air environment in which the in vitro assay was prepared and performed may belie any in vivo inhibition present in an environment where the oxygen tension is more than 20-fold lower.

On the other hand, it may possibly represent a true result and be due to an increase in activity of the enzyme per unit mass due, for instance, to a conformational change. More likely, however, is the possibility that total enzyme protein has in- creased. Although other recent studies have reported a decrease in complex IV protein [43] and mitochondrial content [44], these were performed in severe, high mortality rodent models. On the other hand, Suliman et al. found that bacterial lipopolysaccharide (LPS, endotoxin) injected into rats produced early DNA dam- age followed by stimulation of new mitochondrial protein (‘biogenesis’) [45]. NO has been recently shown to be a crucial component in the production of new mi- tochondria [46, 47]. This is consistent with the finding of Elfering et al. [48] that nitration induced a greatly accelerated turnover in new mitochondrial protein.

Thus, prolonged and excessive NO production may result in an initial inhibition of mitochondrial activity followed, if the organism survives, by a stimulation of recovery of function. This dual role is consistent with the notion we have proposed that MOF may actually represent a protective, adaptive response to a prolonged and severe inflammatory insult [49]. The acute phase of sepsis is marked by an

abrupt rise in secretion of stress hormones with an associated increase in mito- chondrial and metabolic activity. The combination of severe inflammation, with excess ROS formation, and secondary changes in the endocrine profile and an alteration in phenotype, will then diminish energy production, metabolic rate, and normal cellular processes, leading to multiple organ dysfunction. However, reduced cellular metabolism could increase the chances of survival of cells, and thus organs, in the face of this overwhelming insult. This is analogous to hiberna- tion, estivation, and other environmental stressors. Levy and colleagues [50] have recently demonstrated biochemical, functional, and bioenergetic changes in septic hearts that reflect the hibernatory response to a cold environment.

Mitochondrial Protection

If mitochondrial dysfunction and/or damage is central to the pathogenesis of MOF, strategies to protect the mitochondria may prevent the progression, or at least ameliorate, the development of organ failure.

An important advance in the clinical management of patients either with, or at high risk of developing sepsis, is the concept of intensive insulin therapy. The package of tight glycemic control (4.5–6.1 mmol

/

A variety of putative mechanisms of action for the beneficial effects of intensive insulin therapy have been suggested. In an important follow-up paper examining liver and muscle biopsies taken soon after death [52], minimal cell death was seen in both intensive insulin therapy and conventionally-treated groups. However, inten- sive insulin therapy resulted in almost complete protection against the significant ultrastructural damage to liver mitochondria and the corresponding decrease in respiratory enzyme activity frequently seen in the control group. This suggests that better glycemic control, leading to reduced glycation of mitochondrial pro- tein, and/or additional insulin and glucose, enhancing glycolysis and having direct effects on mitochondrial function, represent potential protective mechanisms.

There are a number of other possible protective approaches to maintain mi- tochondrial function. As much of the injury to the organelle is considered to be mediated by reactive species, levels of intra-mitochondrial antioxidants, such as glutathione [53] or manganese superoxide dismutase [54], could be supplemented at an early stage. Melatonin is also an efficient scavenger of reactive oxygen and nitrogen species and will increase the mitochondrial glutathione pool. After sep- sis, administration of melatonin reduced expression and activity of the inducible isoform of NOS (iNOS) and improved mitochondrial function [55]. There may be a role for specific inhibitors of iNOS which have been shown to ameliorate cardiac depression and improve mitochondrial activity and structure in an endotoxic rat model [56].

Induction of heat shock protein (HSP) may also prove beneficial. A short period of hyperthermia 24 hours before a septic insult was protective for both heart [26]

and liver [57] mitochondrial function. The precise mechanism(s) of action remains to be determined as the heat shock response offers broad cytoprotective properties with effects on apoptosis, NO production, and heme oxygenase induction. Among the various HSPs, HSP32 (heme oxygenase 1) has received considerable attention as its induction generates significant amounts of carbon monoxide and the potent antioxidant bilirubin which have been shown to have protective effects in a variety of shock models. Recently, hemin, a pharmacological inducer of heme oxygenase, was shown, in an endotoxic rat model, to increase tissue heme oxygenase levels, prevent alterations in mitochondrial function, and attenuate increases in plasma nitrite/nitrate levels and tissue markers of free radical generation [58].

PARP-1 is a DNA repair enzyme that is activated when nuclear damage oc- curs. It consumes NAD and may potentially deplete this vital electron carrier, and thus ATP production, within the mitochondria. PARP inhibitors were developed with the aim of preventing this energy depletion but, more recently, they have also been recognized to have potent anti-inflammatory properties. For example, in an oxidative stress model, PARP inhibition provided mitochondrial protection through inducing phosphorylation and activation of Akt [59]. In various sep- tic models, PARP inhibition prevented mitochondrial dysfunction and improved outcomes [24, 60].

In the situation of established sepsis, such protective strategies may prove less effective. Consideration should be given to approaches that provide substrate able to bypass the site of mitochondrial inhibition (e. g., succinate [61]) or stimulate recovery pathways such as accelerating mitochondrial biogenesis, e. g., through activation of the transcriptional coactivator, peroxisome proliferator-activated re- ceptor gamma coactivator-1

γ

γ

Conclusion

Prolonged sepsis will induce mitochondrial dysfunction and damage. As a con- sequence of decreased energy availability, metabolism must decrease or the cell will soon die. As cell death is not a major feature, it is thus feasible that the cells enter a hibernation-like state as a late protective response and this biochemi- cal/physiological shutdown is manifest as multiple organ dysfunction/failure. Re- covery would then be contigent upon restoration of mitochondrial function, either through repair of existing damaged mitochondria or production of new organelles.

Excess production of NO and other reactive species appears likely to be the main

‘culprit’ of the initial injury and altered bioenergetics, yet, paradoxically, may pro- vide the stimulus for eventual recovery of function. Therapeutic strategies could thus be geared towards mitochondrial protection or accelerating recovery.

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