OUP user menu

Mitochondrial depolarization and the role of uncoupling proteins in ischemia tolerance

Michael N. Sack
DOI: http://dx.doi.org/10.1016/j.cardiores.2006.07.010 210-219 First published online: 1 November 2006


Modest depolarization of the mitochondrial inner membrane potential is known to attenuate mitochondrial reactive oxygen species generation. Transient pharmacologic uncoupling of mitochondrial oxidative phosphorylation results in modest depolarization of the mitochondrial membrane potential and confers protection against subsequent cardiac ischemia–reperfusion injury. Whether cardiac mitochondria have an innate capacity to temporally self-modulate their membrane potential as a possible adaptive mechanism in the context of cardiac ischemia and early reperfusion is supported by emerging data and is an intriguing concept that warrants further investigation. The objective of this review is to explore the various mechanisms whereby mitochondrial depolarization can be evoked in the context of both cardiac ischemia and reperfusion and in response to the cardioprotective program of ischemic preconditioning. The potential regulatory pathways orchestrating this biological perturbation of mitochondrial function are explored from the level of signal transduction to potential transcription-mediated modulations of nuclear-encoded mitochondrial inner membrane proteins, emphasizing the potential function of the mitochondrial uncoupling proteins.

  • Cardiac ischemia
  • Mitochondrial respiration
  • Uncoupling proteins
  • Reactive oxygen species
  • Mitochondrial depolarization

1. Introduction

“It is a good thing for the entire enterprise that mitochondria and chloroplasts have remained small, conservative, and stable, since these two organelles are, in a fundamental sense, the most important living things on earth. Between them they produce oxygen and arrange for its use. In effect, they run the place.” This description by the physician scientist and commentator – Lewis Thomas [1] succinctly illustrates the centrality of mitochondrial function to sustaining life. The importance of this organelle is exemplified in the human heart where ATP in excess of ten times the cardiac mass, is produced and consumed daily [2], in large part produced by mitochondrial oxidative phosphorylation.

In the last few decades, characterization of the role of mitochondria in cellular biology has extended above and beyond oxygen utilization and ATP production. Mitochondria are now recognized as finely tuned rheostats that orchestrate and exquisitely modulate: energy production; reactive oxygen and nitrogen species homeostasis; calcium regulation; heme metabolism; programmed cell death (apoptosis) and most recently retrograde signaling to modulate signal transduction pathways [3] and nuclear regulation [4]. Historically, mitochondria were considered passive ‘victims’ of cardiac ischemia that contributed to subsequent cell death via ATP depletion and reactive oxygen species production. In light of the expanding role of mitochondria, a question posited is whether mitochondria actively modify the biological response to cardiac ischemia and reperfusion? The hypothesis explored in this review contends that cardiac ischemia mediated bioenergetic perturbations of mitochondrial oxidative phosphorylation directly modulate the biological responses to ischemia and reperfusion. The objective of this manuscript is to review the current knowledge pertaining to the dynamic biochemical, cellular and molecular responses of mitochondria to ischemia and reperfusion injury with respect to ischemia–reperfusion mediated changes in: oxidative phosphorylation; the energetic capacity of the mitochondria; the degree of mitochondrial coupled respiration and the regulation of reactive oxygen species. How these changes modify the final biological response to ischemia will be discussed. The mitochondrial role in mediating ischemia–reperfusion cell death via activation of the permeability transition pore, via apoptosis and necrosis have been recently reviewed [5,6] and are not discussed.

2. Coupling of electron transfer and ATP generation

The final common pathway for oxidative metabolism, which generates the bulk of cardiac ATP, is the sequential passage of electrons from high (NADH or FADH2) to low (molecular oxygen) redox potentials down the electron transfer chain (ETC – complexes I through IV). This step-wise electron transfer results in the active pumping of hydrogen ions out of the mitochondrial matrix into the inter-membranous space. The ensuing electrochemical gradient generated across in the inner mitochondrial membrane facilitates the translocation of protons from the inter-membranous space through the Fo/F1 ATPase (ATP-synthase) back into the mitochondrial matrix. This proton translocation is coupled to the phosphorylation of ADP to generate ATP. Collectively these reactions constitute oxidative phosphorylation and the direct synthesis of ATP from electron transfer encapsulates coupled respiration [7].

Uncoupled oxidative phosphorylation results from electron transfer and proton influx into the mitochondrial matrix independent of the phosphorylation of ADP and results from either damage to ATP-synthase or via active or passive increased permeability of the inner-mitochondrial membrane [8]. Inner mitochondrial membrane proteins such as the uncoupling proteins (UCPs) [9], the adenine nucleotide translocases (ANTs) [10,11] and cytochrome oxidase itself [12] can facilitate proton leak/slip into the mitochondrial matrix to promote partial uncoupled respiration. Additionally, ionic cycling and the phospholipid fatty acids composition of the inner mitochondrial membrane contribute to a modest component of uncoupled respiration and proton conductance respectively [13,14]. Together these various mechanisms result in various degrees of depolarization of the inner mitochondrial membrane and possible concurrent de-energization which can represent regulated physiologic respiratory control [15] or, if in excess, facilitate mitochondrial and cellular damage [16].

The physiologic role of uncoupled respiration is best exemplified by the adaptive non-shivering thermogenesis induced by uncoupling protein 1 (UCP1) to generate heat in brown adipocytes [14]. Partial mitochondrial uncoupling has also been implicated as a potential mechanism promoting enhanced longevity in mice [11] and functions to diminish reactive oxygen species (ROS) production [7]. Furthermore, it is suggested that proton leak accounts for up to 20% of mitochondrial oxygen consumption as an integral component of the standard metabolic rate [15,17]. Of note, physiologic uncoupling and modest mitochondrial depolarization are proposed to concurrently maintain the energetic capacity of mitochondria and modulate mitochondrial inner membrane dependent ROS production (reviewed [18]).

3. Effects of ischemia reperfusion on mitochondrial oxidative phosphorylation

Mitochondrial ultrastructural and functional injury during cardiac ischemia are both time-dependent and progressive [8,19]. Fig. 1 schematizes the perturbations of inner mitochondrial membrane protein function in a temporal response to ischemia. The earliest perturbations in ETC complex function are evident within 10 min of ischemia where ATP-synthase [20] and ANT activity [21] are diminished. This is followed by the reduction of activity in complex I [8,20] within 20 to 30 min of ischemia. In contracting cardiomyocytes the electrochemical gradient is shown to be modestly diminished in the first 30 min of simulated ischemia [16]. These perturbations are reversible if reperfusion occurs within this timeframe [22]. The time-span of reversibility of mitochondrial injury is variable and dependent on the heart rate and on the species being studied [23]. More prolonged ischemia begins to result in irreversible myocyte damage and is associated with defects in complexes III and IV [8,24]. Cardiomyocytes exposed to prolonged ischemia demonstrate a robust reduction in inner mitochondrial membrane potential [16]. During ischemia, ATP-synthase reverses direction to facilitate the maintenance of the proton-motive force by ATP hydrolysis. A higher rate of ATP hydrolysis reduces the time to irreversible ischemic injury and mitochondrial dysfunction. Interestingly, in slow heart rate animals, an endogenous antagonist of ATP-synthase activity, ATPase inhibitor factor [25] is shown to protect mitochondria by delaying ischemic ATP hydrolysis [26]. Prolonged ischemia and depletion of ATP function in concert to disrupt mitochondrial oxidative phosphorylation.

Fig. 1

Temporal effects of ischemia and reperfusion on mitochondrial ETC function and respiratory coupling.

The restoration of blood flow restores oxygen and substrate supply and serves to remove residual metabolites that are detrimental to mitochondrial and myocytes recovery [27]. Consistent with the temporal recovery of mitochondrial function, brief ischemic periods result in the rapid restoration of oxidative phosphorylation at reperfusion [22]. Conversely once ischemic damage to complexes III and IV is evident energetic recovery is limited. Reperfusion, at this point may exacerbate injury including further disruption of oxidative phosphorylation [28].

The exact mechanisms underlying ETC complex dysfunction and the degree and rate of recovery during ischemia and reperfusion are not well established. However, ETC complex protein modifications via: redox or phosphorylation signaling; perturbations in electrolyte concentrations; dysregulation of substrate supply and following depletion of cardiolipin content of the inner mitochondrial membrane all contribute to ischemia–reperfusion mediated disruption of the functional integrity of electron transfer [8,29–31]. Interestingly, following an ischemic insult of sufficient duration to result in persistent modest contractile dysfunction, coupling of mitochondrial oxidative phosphorylation to oxygen consumption is restored to baseline ratios 20 min into reperfusion, in the isolated perfused heart [32].

Additional dysregulation in energetics is also operational during cardiac ischemia and reperfusion, including modulations in substrate or metabolic pathway utilization [27] and in extra-mitochondrial high-energy phosphocreatine transduction [33–35]. Moreover, perturbations in high-energy phosphocreatine transduction and ATP utilization are also integral to persistent contractile dysfunction following the acute perturbations associated with early reperfusion [32].

4. Ischemia–reperfusion and the generation of reactive oxygen species

In the heart, the electron transfer chain is the main source of reactive oxygen species (ROS) production. It is generally believed that the superoxide radical anion (O2) is produced by the transfer of an unpaired electron from complex I and from ubisemiquinone of complex III to molecular oxygen [4,18]. Minimal to no ROS is generated under anoxic conditions [36], but myocardial ischemia is associated with a progressive reduction in tissue oxygen levels and then a rapid restoration and/or overshoot of oxygen levels at reperfusion [37]. During ischemia free radicals are generated in the myocardium [16,38]. At cardiac reperfusion, following a short ischemic stress, up to a 6-fold induction of ROS generation can occur [38,39]. As prolonged myocardial ischemia irreversibly disrupts electron transfer chain activity additional extra-mitochondrial sources of ROS probably become the major source of reperfusion ROS generation. One such source results from the infiltration of neutrophils into the infarcting tissue [40]. Additional sources include oxyradical generation from xanthine oxidase, activation of the arachidonate cascade, autoxidation of catecholamines, activation of various NAD(P)H oxidases and from reactive nitrogen species [41].

Additionally mitochondrial matrix Ca2+ overload is postulated to enhance ETC ROS generation during cardiac ischemia and reperfusion [42]. This hypothesis is less well established, with some discordant data as discussed below. As background, the primary effect of mitochondrial Ca2+ is the stimulation of oxidative-phosphorylation via allosteric activation of tricarboxylic acid cycle and ETC enzymes [43]. Under normoxic circumstances Ca2+ influx into the mitochondrial matrix is augmented in response to energetic demand with Ca2+ uptake primarily driven by the electrochemical gradient established by the mitochondrial membrane potential and by the relatively low intramitochondrial Ca2+ concentration [44]. In response to cardiac ischemia and reperfusion, Ca2+ influx into the mitochondrial matrix is facilitated, in part by ROS mediated calcium release from the sarcoplasmic reticulum [45]. Evidence to support a role for Ca2+ influx in the generation of additional ROS includes: 1) a direct correlation between Ca2+ levels and ROS generation when complex I is pharmacologically inhibited [46,47]; 2) similarly, in isolated heart mitochondria ROS generation is calcium concentration dependent following complex III inhibition [48] and 3) mitochondria extracted from hypoxic hearts demonstrate a direct correlation between ROS generation and the Ca2+ concentration [49]. Contrary data, which fails to demonstrate Ca2+-dependent mitochondrial ROS production is evident under different experimental conditions where complex I was inhibited by rotenone [48,49].

5. Strategic control points of mitochondrial respiration to modulate cardiac ischemia tolerance

In general mitochondrial ROS generation increases with a more negative inner mitochondrial membrane potential [18,50]. When the mitochondrial membrane potential is high, superoxide production increases as a result on non-specific single-electron reduction of molecular oxygen at complexes I and III of the ETC [18]. In contrast, modest reduction in mitochondrial membrane potential (mitochondrial depolarization), more tightly associates electron transfer to the ETC complexes, thereby limiting random electron disassociation and superoxide production [9]. A second proposed mechanism of modest depolarization-mediated reduction in ROS generation is via decreased membrane potential dependent Ca2+ influx into the mitochondria [51]. This reduction in mitochondrial Ca2+ may attenuate ischemia-mediated ROS production.

The concept that the mitochondrial membrane potential is regulated as a component of the cellular biological program modulating cellular tolerance to ischemic injury is actively being explored. Direct evidence supporting post-translational modulation of ETC complex proteins, with subsequent changes in their respiratory activity during cardiac ischemia and reperfusion is not well established. However, accruing evidence demonstrates that numerous signaling pathways govern ETC flux and may play a role in modulating mitochondrial energetics and membrane potential. Complex IV activity appears to be the most susceptible to post-translational modification of various complex protein moieties. Tyrosine phosphorylation of the subunit I of cytochrome oxidase results in inactivation of the enzyme and with a reduction in overall cellular ATP levels [52]. Protein kinase A also inhibits complex IV via phosphorylation of multiple subunits within the cytochrome oxidase complex [53]. Conversely, protein kinase Cε activates cytochrome oxidase via phosphorylation of subunit IV [54]. Whether any of these modifications are operational during ischemia and reperfusion require investigation.

The most extensive characterization of signal transduction mediated modulation in electron transfer chain flux is underscored by the regulation of cytochrome oxidase activity by nitric oxide (NO) [55]. NO competitively inhibits cytochrome oxidase potently and reversibly and reduces the affinity of this enzyme for O2. This inhibitory effect of NO is inversely dependent on O2 levels, with greater inhibition of cytochrome oxidase activity at lower oxygen concentrations [56,57]. Furthermore, the temporal inhibitory effect of NO has diverse phenotypic outcomes. Short-term exposure to NO in mitochondria results in transient and reversible, mitochondrial depolarization [58]. In contrast, persistent inhibition of respiration [59], or high concentrations of NO [60] result in the collapse of the mitochondrial membrane potential, ATP depletion, and cell death. The potential biological relevance of NO mediated control of mitochondrial respiration is evident where NO donors confer protection against ischemia–reperfusion injury in cardiomyocytes in parallel with modest mitochondrial depolarization and reduced mitochondrial Ca2+ uptake [61]. This control of ETC flux by NO supports the concept of modest mitochondrial depolarization as an adaptive mechanism against ischemia–reperfusion injury. Interestingly, modest mitochondrial respiratory inhibition using pharmacologic inhibitors similarly demonstrates enhanced tolerance to cardiac and skeletal muscle ischemic injury [62–65].

Together, these data identify potential signaling pathways that may contribute to the modulation of ETC activity during ischemia and reperfusion. Direct inhibition of ETC flux using pharmacologic agents that are known to diminish the inner mitochondrial membrane potential confers tolerance against ischemic injury [61,66,67]. This concept is further supported where modest depolarization by the administration of low concentrations of uncoupling agents such as carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) or 2,4 dinitrophenol evokes protection against ischemic damage in the intact heart [68], in cardiomyocytes [69] and in the brain [70] in parallel with a diminution in mitochondrial ROS production. Interestingly, the potassium channel opening compounds that promote cardioprotection also partially uncouple mitochondrial respiration [13].

The paradigm being established is that transient modest mitochondrial depolarization, whether via modulation in ETC flux or via pharmacologic uncoupling, confers protection against ischemia–reperfusion injury. In an analogous fashion to innate regulatory control of ETC flux, the question arises as to whether endogenous modulation of mitochondrial uncoupling may be a component of the regulatory machinery governing cellular ischemia tolerance. This question is explored below where the role of mitochondrial uncoupling proteins function as a mediator of proton leak in the heart is discussed.

6. A proposed role for cardiac enriched mitochondrial uncoupling proteins

Mitochondrial uncoupling proteins are integral membrane proteins localized to the inner mitochondrial membranes. Uncoupling protein 1 (UCP1), is the most extensively characterized member of this family and plays a central role in adaptive thermogenesis in brown adipocytes [71]. UCP1 allows the influx of protons back into the mitochondrial matrix thereby uncoupling mitochondrial respiration from ATP-synthesis. This in turn, leads to more rapid respiration and heat production. This prototype of the uncoupling proteins is directly activated by fatty acids and intrinsically inhibited by purine nucleotides (reviewed [9]).

Two UCP1 homologues UCP2 and UCP3, which are present in the heart [72], share high amino acid sequence identity (73%) and are similar to UCP1 [9]. As these inner mitochondrial membrane carrier proteins are most closely aligned to UCP1, it is suspected that they may have similar biochemical functions.

The abundance of UCP2 gene expression in the heart appears to be species specific with relatively higher levels in mice compared to humans [73]. Interestingly the presence of UCP2 protein in the murine heart has been questioned [74], however, it is present in human and rat myocardium [72,75]. UCP3, has a more restricted tissue distribution, and is found predominantly in skeletal muscle, with lower levels in the heart [76].

The functional roles of UCP2 and UCP3 are less well characterized than that of UCP1. In general it is accepted that these proteins are not constitutive uncouplers of oxidative phosphorylation and that they are not crucial for non-shivering thermogenesis, except in response to specific pharmacologic activation [77]. The functional characterization of the UCP homologues has been complicated by experimental artifact which has arisen in overexpression studies. Overexpression of UCP2 and UCP3 in the inner mitochondrial membrane can compromise the inner membrane integrity [78] with a resultant excess uncoupling of oxidative phosphorylation [79–81]. Furthermore, tissue specific functions are operational in that UCP2 overexpression demonstrates divergent cell-type specific activities [9,82].

In rat neonatal cardiomyocytes, UCP2 overexpression does not have intrinsic ETC uncoupling activity, but does confer tolerance to oxidative stress via diminished mitochondrial Ca2+ overload and reduced ROS generation [83]. This lack of constitutive effect on mitochondrial coupling is consistent with the revelation that UCP2 and UCP3 are functionally quiescent until activated by either ROS signaling [72,84], by fatty acid peroxidation products [85] or via reactive aldehydes [86]. Interestingly, ectopic overexpression of UCP1 in cardiac derived H9c2 cells [87] and in the murine heart [88] mirrors the cytoprotective effect of UCP2 overexpression against ischemia and reperfusion injury. Genetic depletion of the uncoupling proteins should give greater insight into their role in protection against ischemic injury. No studies to date have explored this in the intact heart. However, RNA interference studies where UCP2 and or UCP3 was partially depleted in cardiac derived H9c2 cells demonstrate diminished tolerance to anoxic stress in association with excess ROS production [72]. In this study the hierarchical effects of these homologues were established where UCP2 plays a functionally greater role than UCP3 in promoting tolerance to anoxia and reoxygenation injury [72].

Further evidence to support tissue specific cytoprotective effects of UCP2 is evident in the brain where UCP2 is shown to play a similar role in modulating neuronal tolerance to ischemic stress (reviewed [89]). The ROS modulating effects of UCP2 are also evident in UCP2 knockout mice which exhibit: (1) increased ROS production in response to infectious agents; (2) excess ROS in response to organ injury and (3) demonstrate increased susceptibility to ROS-mediated atherosclerosis (reviewed [90]).

An additional proposed role for UCP3 is as a conduit to export fatty acid anions from the mitochondria to prevent accumulation of fatty acid metabolic by-products in the mitochondria. This hypothesis, although attractive, is currently supported predominantly by associational studies which cannot distinguish between primary fatty acid export and ROS-mediated effects (see recent reviews [9,90]). Studies designed to directly test this is are an active area of investigation. Interestingly, the ischemic heart is exposed to elevated levels of free fatty acids [27] and an innate mechanism to attenuate lipotoxic effects via cardiac UCP3 activation is an attractive hypothesis that deserves exploration.

7. Mitochondrial uncoupling, uncoupling proteins and ischemic preconditioning

It has been shown that the heart can be protected from ischemic injury through activation of innate pro-survival programs. Tolerance against otherwise lethal ischemia is most powerfully induced following repetitive transient non-lethal periods of myocardial ischemia and reperfusion termed ischemic preconditioning (reviewed [91]). This preconditioning effect is evident in two temporally distinct phases which correlate with their cognate regulatory control mechanisms. Classic preconditioning is an acute adaptive phase evident within minutes to 2–4 h following ischemic preconditioning and is regulated at the post-translational level. Delayed-preconditioning is an ischemia-tolerant phase evoked 12–72 h following the preconditioning ischemia and is primarily regulated at the transcriptional level [92]. The veracity of mitochondrial coupling as an integral component in modulating cardiac tolerance to ischemia would be reinforced if mitochondria from preconditioning mirror the mitochondrial cytoprotective phenotype described above.

The initial description of the classical preconditioning phenomenon conforms with the central paradigm presented in this review in that immediately prior to an index ischemic event, preconditioned hearts are modestly depleted in ATP levels (1/3 reduction) compared to non-preconditioned control hearts [19]. Further studies in cell culture show that acute preconditioned mitochondria in situ are uncoupled as evident by a lower inner membrane potential, reduced ATP levels and greater oxygen consumption versus non-preconditioned control cells [93]. The association of classic preconditioning with proton leak has been directly confirmed by measuring concurrent oxygen consumption and mitochondrial membrane potential in mitochondria extracted from preconditioned rat hearts prior to the onset of a prolonged ischemia–reperfusion insult [94]. The putative modifiers that abolish this phenotype implicate activation of the uncoupling proteins and of ANT as this proton leak is blocked by GDP and carboxyattractyloside respectively [94].

The mitochondrial bioenergetic milieu of hearts following delayed preconditioning has been less well characterized, however it is known that the basal metabolic function of delayed preconditioned mitochondria does not exhibit increased proton leak [92]. However, in parallel with the acutely preconditioned mitochondria [95], these mitochondria are more tolerant of an anoxia–reperfusion insult compared to non-preconditioned mitochondria [92]. Interestingly, the transcriptional activator peroxisome proliferator activated receptor co-activator 1 alpha (PGC1α) is upregulated as a component of the delayed preconditioning program [92]. Transient activation of PGC1α in skeletal muscle cells upregulates UCP2 and UCP3, in concert with increased mitochondrial proton leak [96]. Although, the direct link between PGC1α activation and proton leak in delayed preconditioning has not been established, upregulation of UCP2 and UCP3 is evident in the delayed preconditioned rat heart [72]. This regulation is associated with increased GDP-sensitive proton leak in the presence of ROS and these mitochondria generate less ROS at reperfusion following a prolonged anoxic insult [72]. This prerequisite of ROS intermediates to induce mitochondrial uncoupling is consistent with the requirement of signaling intermediates to activate UCP2 and UCP3.

The role of uncoupling proteins in augmenting tolerance to delayed ischemic preconditioning is also evident in the brain. Here sub-lethal ischemia upregulates UCP2 expression and confers protection to the brain against a delayed ischemia injury [97]. Moreover, overexpression of UCP2 confers protection against neuronal ischemia injury and is proposed to mediate these salutary effects through control of ROS generation and possibly via the mediation of redox signaling [97,98].

The direct demonstration that ROS signaling activates UCPs and results in subsequent attenuation in subsequent ROS levels has begun to be explored during delayed preconditioning [72]. The role of ROS activation of UCPs during acute preconditioning has not to my knowledge been investigated. However, the real time demonstration that superoxide levels are necessary in ischemic preconditioning signal transduction and that subsequent superoxide levels are markedly attenuated during ischemia and reperfusion following preconditioning compared to non-preconditioned ischemia–reperfusion controls [99] is compatible with the paradigm of ROS-mediated UCP activation resulting in diminution of subsequent ROS generation. In this study by Stowe and colleagues [99] the preponderance of the attenuation of ROS generation following preconditioning occurs in the latter half of the ischemic period and even more robustly during reperfusion. Whether this aligns with the regulatory control of UCP activation has not been established.

8. Mitochondrial uncoupling/depolarization and the maintenance of oxidative phosphorylation capacity

The premise underlying the cardioprotective role of transient activation of uncoupling proteins predicates that mitochondrial depolarization can occur to a sufficient level to attenuate ETC ROS generation without further disrupting the capacity to generate cardiac ATP production during ischemia and reperfusion. The stoichiometric efficiency of oxidative phosphorylation is defined by the P/O ratio, or the amount of inorganic phosphate incorporated into ATP per amount of oxygen consumed. In classical equilibrium thermodynamics the maximal efficiency of energy conversion is achieved when the energy transduction systems are fully coupled [18]. In contrast, biological systems exhibit variable levels of efficiency that display non-ohmic characteristics [100]. This efficiency variation is orchestrated by numerous extrinsic and intrinsic factors that modulate coupling between the proton-motive force and the rate of respiration (reviewed by Kadenbach [18]). A concept posited by Kadenbach [18,101] which possibly relates to the mechanism of action of uncoupling proteins is that: i) a relatively minor alterations of the inner mitochondrial membrane potential at high physiologic inner mitochondrial membrane potential significantly alter ETC ROS generation and ii) the maximal rate of ATP-synthase energy generating capacity occurs at low physiologic mitochondrial membrane potential with no augmentation with further increasing membrane potential. Continuous temporal analysis of these mitochondrial perturbations during ischemic preconditioning has not been undertaken. However, acute ischemic preconditioning is associated with both increased proton leak immediately following the preconditioning ischemia and with increased efficiency of cardiac oxygen consumption to the degree of contractile recovery, as a measure of preserved energetics, compared to non-preconditioned ischemic control hearts [94]. Although UCP2 and UCP3 have been associated with these mitochondrial alterations as discussed in this review, there is no direct demonstration that UCP2 and or UCP3 can both increase proton leak and concurrently maintain oxidative phosphorylation. This potential mechanism of action of the uncoupling proteins under ischemic stress conditions requires investigation to definitively prove the hypothesis presented here.

9. Limitations in the data supporting the mitochondrial uncoupling hypothesis in augmenting resilience to ischemic stress

Insertion of exogenous proteins into the mitochondrial inner membrane may give rise to artifactual uncoupling, as shown following the insertion of multiple UCP homologues [79–81,102] and by the insertion of other related mitochondrial carrier proteins [103]. This insertion of proteins into the inner mitochondrial membrane may give rise to improper insertion or folding or to abnormal interaction with native proteins, thereby conferring unphysiologic action. This is supported where overexpression of UCP3 does not exhibit superoxide mediated proton conductance [78] and where robust overexpression of UCP2 promotes mitochondrial uncoupling without the need for redox activation [104]. Thus, although the uncoupling protein overexpression data presented is one component of the data supporting modest uncoupling as a mechanism to diminished ROS-mediated cellular sensitivity to ischemic injury, we should remain cognizant of the potential experimental artifact that can contribute to this phenotype.

The quantization of modest ‘physiologic’ depolarization versus robust ‘pathologic’ depolarization is not well characterized, nor reviewed in this manuscript. The physiologic mitochondrial membrane potential is in the order of 200 mV and is subject to some debate due to experimental limitations in its direct measurement [105]. Interestingly, the maximal attenuation of mitochondrial membrane potential with robust overexpression of UCP2 approaches 15 mV [104]. Thus, the degree of mitochondrial uncoupling, that is termed modest, is most likely to be a modulation of less than 10% and probably much less than this under physiologic conditions. However, the absolute modulation of the inner mitochondrial membrane potential has not been determined in the studies presented in this review.

Additional inner membrane carrier proteins and channels may play a role in modulating the inner mitochondrial membrane potential in response to ischemia injury. Examples of this include the ANT [106] and the ‘elusive’ mitochondrial KATP channel [107]. However, these and other potential membrane potential modulatory proteins have not been discussed in this review.

Finally, although numerous groups have demonstrated that ‘preconditioned’ mitochondria exhibit enhanced recovery from anoxia and re-oxygenation [92,95], extra-mitochondrial energy transduction also contributes to overall cellular protection [108] and hence modulation of mitochondrial biology itself, is only part of the overall metabolic program modulating ischemia tolerance.

10. Conclusions

The preponderance of evidence supports modest mitochondrial depolarization as both an early and reversible event during cardiac ischemia. This probably occurs in part due to modest inhibition of electron transfer and possibly via the activation of endogenous inner membrane proteins including the uncoupling proteins. These modest changes in the rate of coupled oxidative phosphorylation would reduce electron transfer to molecular oxygen and thereby diminish ROS production. An additional function of modest mitochondrial depolarization includes a reduction in mitochondrial Ca2+ uptake. Interestingly, modulation of mitochondrial coupling is also evident in ischemic preconditioning, the biological phenomenon known to maximally augment cell survival programming in the heart. Hence, short-lived modest mitochondrial depolarization is associated with the delay and the attenuation of subsequent ischemia–reperfusion mediated cell death. The postulated mechanisms are schematized in Fig. 2.

Fig. 2

Mediators of modest mitochondrial depolarization and potential adaptive responses to this depolarization.

The regulatory mechanisms orchestrating the modulation in coupled respiration are complex, but appear to span the regulatory spectrum from transcriptional regulation (in delayed preconditioning), to post-translational signal transduction which directly modulates ETC complex protein activities and which may activate inner mitochondrial membrane transport proteins such as the uncoupling proteins. The complexity and role of the mitochondria as both a recipient and a mediator in signal transduction is further underscored where retrograde signaling from de-energized mitochondria signals the nucleus to modulate additional adaptive responses [4,109].

Overall these data demonstrate that the mitochondrion is truly a finely tuned and highly regulated organelle which functions as a multi-dimensional rheostat controlling biological responses including the cellular capacity to respond to pathophysiologic insults such as cardiac ischemia and reperfusion injury. Our understanding of the complex regulatory mechanisms governing mitochondrial function and their role in retrograde signaling to the cytosol and nucleus is in its infancy. The further dissection of regulatory control of mitochondrial function and the role of mitochondria in molecular regulation should identify novel targets to mediate cardiac tolerance to oxidative stress.


I would like to thank Toren Finkel, M.D., Ph.D. and Paul Hwang, M.D., Ph.D. for their critical review of this manuscript. Funding for my laboratory is generously provided by the National Heart Lung and Blood Institute, Division of Intramural Research of the National Institutes of Health, USA.


  • Time for primary review 26 days


  1. [1]
  2. [2]
  3. [3]
  4. [4]
  5. [5]
  6. [6]
  7. [7]
  8. [8]
  9. [9]
  10. [10]
  11. [11]
  12. [12]
  13. [13]
  14. [14]
  15. [15]
  16. [16]
  17. [17]
  18. [18]
  19. [19]
  20. [20]
  21. [21]
  22. [22]
  23. [23]
  24. [24]
  25. [25]
  26. [26]
  27. [27]
  28. [28]
  29. [29]
  30. [30]
  31. [31]
  32. [32]
  33. [33]
  34. [34]
  35. [35]
  36. [36]
  37. [37]
  38. [38]
  39. [39]
  40. [40]
  41. [41]
  42. [42]
  43. [43]
  44. [44]
  45. [45]
  46. [46]
  47. [47]
  48. [48]
  49. [49]
  50. [50]
  51. [51]
  52. [52]
  53. [53]
  54. [54]
  55. [55]
  56. [56]
  57. [57]
  58. [58]
  59. [59]
  60. [60]
  61. [61]
  62. [62]
  63. [63]
  64. [64]
  65. [65]
  66. [66]
  67. [67]
  68. [68]
  69. [69]
  70. [70]
  71. [71]
  72. [72]
  73. [73]
  74. [74]
  75. [75]
  76. [76]
  77. [77]
  78. [78]
  79. [79]
  80. [80]
  81. [81]
  82. [82]
  83. [83]
  84. [84]
  85. [85]
  86. [86]
  87. [87]
  88. [88]
  89. [89]
  90. [90]
  91. [91]
  92. [92]
  93. [93]
  94. [94]
  95. [95]
  96. [96]
  97. [97]
  98. [98]
  99. [99]
  100. [100]
  101. [101]
  102. [102]
  103. [103]
  104. [104]
  105. [105]
  106. [106]
  107. [107]
  108. [108]
  109. [109]
View Abstract