Cardiovascular Research

Cardiovascular Research Advance Access first published online on August 21, 2007
This version [Corrected Proof] published online on September 27, 2007
Cardiovascular Research, doi:10.1093/cvr/cvm005

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Heart mitochondria: gates of life and death

Åsa B. Gustafsson^* and Roberta A. Gottlieb

BioScience Center San Diego State University 5500 Campanile Drive San Diego, CA 92182-4650, USA

^* Corresponding author. Tel: +1 619 594 8977; fax: +1 619 594 8984. E-mail address: agustafs{at}sciences.sdsu.edu

Time for primary review: 29 days

	Abstract

Top Abstract 1. Introduction 2. Mitochondria and reactive... 3. Reactive oxygen species... 4. Mitochondria and calcium 5. Mitochondrial permeability... 6. Mitochondrial mediators of... 7. Mitochondria and Bcl-2... 8. Consequences of mitochondrial... 9. Mitochondrial fission and... 10. Clinical perspective 11. Conclusions Funding References

 
Mitochondria are important generators of energy, providing ATP through oxidative phosphorylation. However, mitochondria also monitor complex information from the environment and intracellular milieu, including the presence or absence of growth factors, oxygen, reactive oxygen species, and DNA damage. Mitochondria have been implicated in the loss of cells in various cardiac pathologies, including ischaemia/reperfusion injury, cardiomyopathy, and congestive heart failure. The release of factors such as cytochrome c, Smac, Omi/Htr2A, and AIF from mitochondria serves to activate a highly complex and regulated cell death program. Furthermore, mitochondrial calcium overload might trigger opening of the mitochondrial permeability transition pore, causing uncoupling of oxidative phosphorylation, swelling of the mitochondria due to influx of water, and rupture of the mitochondrial outer membrane. In this review, we discuss the role of mitochondria in the control of cell death in cardiac myocytes.

KEYWORDS Apoptosis; Mitochondria; Myocytes; Necrosis; Reperfusion

Received April 29, 2007; revised July 10, 2007; accepted July 29, 2007

	1. Introduction

Top Abstract 1. Introduction 2. Mitochondria and reactive... 3. Reactive oxygen species... 4. Mitochondria and calcium 5. Mitochondrial permeability... 6. Mitochondrial mediators of... 7. Mitochondria and Bcl-2... 8. Consequences of mitochondrial... 9. Mitochondrial fission and... 10. Clinical perspective 11. Conclusions Funding References

 
Mitochondria play critical roles in both the life and death of cardiac myocytes. In healthy cells, their primary function is to meet the high energy demand of the beating heart by providing ATP through oxidative phosphorylation. Mitochondria occupy a large portion of each myocyte and are located between the myofibrils and just below the sarcolemma. The strategic positioning and abundance of mitochondria ensure a highly efficient localized ATP delivery system to support contraction, metabolism, and ion homeostasis.¹ However, mitochondria are also important regulators of cell death, responding to a wide variety of stress signals, including loss of growth factors, hypoxia, oxidative stress, and DNA damage. The switch to a cell death program can be mediated by opening of the mitochondrial permeability transition pore (mPTP) in the inner mitochondrial membrane, causing collapse of the membrane potential and swelling of the mitochondria,² often culminating in necrotic cell death, or permeabilization of the mitochondrial outer membrane (MOM) with release of proapoptotic proteins such as cytochrome c, Smac/Diablo, and apoptosis-inducing factor (AIF) to activate an energy-dependent apoptosis.³ It is important to remember that both forms of cell death are highly regulated and activated by mitochondria.

During apoptosis, the cell activates a signalling cascade which leads to cell death without triggering an inflammatory response. In contrast, necrosis is characterized by swelling of the cell and disruption of the plasma membrane. The resulting release of the cell's content into the extracellular space triggers an inflammatory response which can cause further damage to surrounding cells.⁴ Both processes have been implicated in loss of myocardial cells in pathologies such as ischaemia/reperfusion (I/R), cardiomyopathy, and congestive heart failure. This review discusses the mechanism(s) of mitochondrial dysfunction and how malfunctioning mitochondria might contribute to loss of cardiac myocytes.

	2. Mitochondria and reactive oxygen species

Top Abstract 1. Introduction 2. Mitochondria and reactive... 3. Reactive oxygen species... 4. Mitochondria and calcium 5. Mitochondrial permeability... 6. Mitochondrial mediators of... 7. Mitochondria and Bcl-2... 8. Consequences of mitochondrial... 9. Mitochondrial fission and... 10. Clinical perspective 11. Conclusions Funding References

 
Mitochondria are a major source of reactive oxygen species (ROS), which are a byproduct of mitochondrial electron transfer activity. As much as 0.2–2% of the molecular oxygen consumed by mitochondria during respiration is converted to superoxide primarily by Complexes I and III.^5–7 Molecular oxygen (O₂) is highly electrophilic and superoxide production occurs when O₂ captures an electron from Complex I⁸ or from the ubisemiquinone located in Complex III.⁹ At higher oxygen concentrations, such as during reperfusion, diminished availability of reduced co-factors of the respiratory chain will increase mitochondrial ROS formation. In addition, a reduction in Complex I activity was shown to result in elevated levels of superoxide.¹⁰ The density of mitochondria in cardiac myocytes and the high rate of oxidative phosphorylation can result in generation of significant amounts of superoxide. Normally, superoxide is detoxified by the combined activity of the mitochondrial antioxidant enzymes manganese superoxide dismutase (MnSOD), catalase, and glutathione peroxidase (GPx).^11,12 Superoxide anions are quickly dismuted to hydrogen peroxide by MnSOD, which is converted to water by catalase and GPx. Oxidative stress occurs when excess ROS are generated that cannot be adequately countered by the antioxidant systems. Mice deficient in MnSOD develop normally but die within the first 10 days of life with cardiomyopathy.¹³ Moreover, an increase in superoxide caused by partial inhibition of SOD resulted in hypertrophy and apoptosis in isolated cardiac myocytes.¹⁴ These studies demonstrate the importance of SOD in the regulation of superoxide formed as a byproduct of oxidative phosphorylation.

Production of ROS at levels that exceed the detoxification capacity results in oxidative modification of mitochondrial proteins, lipids, and mtDNA, culminating in mitochondrial dysfunction and cell death. Oxidative stress has been associated with loss of cells in heart failure, I/R injury, and doxorubicin-induced cardiomyopathy, whereas reducing ROS production has been shown to be cardioprotective. For instance, increased generation of ROS was associated with mitochondrial dysfunction in failing hearts after myocardial infarction where mitochondria exhibited increased lipid peroxidation, decreased mtDNA copy number, and a reduced oxidative capacity due to lower activity of electron transfer enzymes.¹⁵ In addition, development of heart failure was shown to be associated with decreased antioxidant capacity and increased oxidative stress. For instance, SOD activity was decreased in an animal model of congestive heart failure,¹⁶ and catalase and GPx activities were reduced by 50% after myocardial infarction, whereas lipid peroxidation in the heart was increased by 50%.¹⁷ Moreover, transgenic mice overexpressing either MnSOD or the cytosolic Cu/ZnSOD had reduced infarct size compared to wild type,^18,19 and treatment with a ROS scavenger protected against hypoxia/reoxygenation and hydrogen peroxide treatment in isolated cardiac myocytes.²⁰ Doxorubicin, an anthracycline, is an effective anti-cancer drug known to cause severe cardiac toxicity. The toxicity is thought to be mediated through the generation of the ROS and impairment of mitochondrial function in cardiac myocytes. In support of this, antioxidants can protect isolated cardiac myocytes and intact hearts from anthracyline toxicity,^21–23 and transgenic mice overexpressing MnSOD had less mitochondrial damage at the ultrastructural level and reduced serum levels of creatine kinase and lactate dehydrogenase than wild type littermates after doxorubicin treatment.²⁴ Similarly, Xiong et al.²⁵ found that doxorubicin treatment led to uncoupling of electron transfer and oxidative phosphorylation in mitochondria and that mice with cardiac overexpression of Gpx1 were more resistant than wild type mice to acute cardiac toxicity of doxorubicin. These studies suggest that production of ROS and damage to mitochondria play an important role in doxorubicin-mediated cardiac toxicity.

	3. Reactive oxygen species-induced reactive oxygen species release (RIRR)

Top Abstract 1. Introduction 2. Mitochondria and reactive... 3. Reactive oxygen species... 4. Mitochondria and calcium 5. Mitochondrial permeability... 6. Mitochondrial mediators of... 7. Mitochondria and Bcl-2... 8. Consequences of mitochondrial... 9. Mitochondrial fission and... 10. Clinical perspective 11. Conclusions Funding References

 
ROS produced initially in mitochondria have been shown to act in a positive feedback, where mitochondria can respond to elevated ROS by increasing their own ROS production in a process known as ROS-induced ROS release (RIRR). Zorov et al.²⁶ initially discovered that ROS production in adult cardiac myocytes proceeded in two distinct phases; an initial slow increase in ROS production induced by photoactivation of mitochondrial dyes was followed by a second burst of ROS originating from the electron transport chain (ETC) occuring simultanously with dissipation of mitochondrial membrane potential (_m). Two different pathways of RIRR have been described. The first pathway of RIRR is dependent on the mPTP, where the initial enhanced ROS leads to opening of the mPTP and depolarization of mitochondria, yielding a short-lived burst of ROS originating from the mitochondrial ETC. Bongkrekic acid, an inhibitor of the mPTP, was shown to inhibit both _m collapse and the second mitochondrial ROS burst.²⁶ More recently, a second pathway of RIRR was described by O'Rourke and colleagues. This type of RIRR occurs through a mechanism involving opening of the inner membrane anion channel (IMAC) which is regulated by the mitochondrial benzodiazepine receptor (mBzR). They found that increased ROS in the mitochondria triggered opening of the IMAC, resulting in a brief increase in ETC-derived ROS. The RIRR was inhibited by ligands of the mBzR, but not by mPTP inhibitors or Ca²⁺ overload, suggesting that the RIRR occurred through a separate pathway that was independent of the mPTP.²⁷

Intracellular generation of mitochondrial ROS by both types of RIRR causes collapse of the _m and destabilization of the action potential, and is emerging as an important mediator of I/R injury. Several independent studies have demonstrated that RIRR is linked to hypoxia/reoxygenation-activated pathways of cell death via the mPTP. Cyclosporine A (CsA), a classic inhibitor of the mPTP, and sanglifehrin A, a recently described specific inhibitor of the mPTP, reduced infarct size following I/R in rat hearts²⁸ and reduced the responsiveness of the mPTP to photo-triggering via confocal imaging of the cardiac myocyte.^29,30 In addition, 4’-chlorodiazepam, an antagonist of the mBzR, shortened action potential duration and prevented arrhythmias during reperfusion, whereas the high affinity agonist, FGIN-1-27, amplified myocardial dysfunction.³¹ This suggests that the second pathway of RIRR participates in I/R-induced myocardial arrhythmias.

	4. Mitochondria and calcium

Top Abstract 1. Introduction 2. Mitochondria and reactive... 3. Reactive oxygen species... 4. Mitochondria and calcium 5. Mitochondrial permeability... 6. Mitochondrial mediators of... 7. Mitochondria and Bcl-2... 8. Consequences of mitochondrial... 9. Mitochondrial fission and... 10. Clinical perspective 11. Conclusions Funding References

 
Cardiac mitochondria also play an important role in regulating calcium homeostasis and can act as a sponge to buffer Ca²⁺. Since elevated [Ca²⁺] may be deleterious, it is necessary to maintain a physiologic Ca²⁺ concentration in the cytoplasm; much of the cellular Ca²⁺ is stored in the sarcoplasmic/endoplasmic reticulum (SR/ER). Mitochondria have been shown to be positioned near Ca²⁺-release sites on the SR/ER and can capture a substantial amount of the released Ca²⁺.³² The ability of mitochondria to accumulate Ca²⁺ helps to prevent the level of calcium in the cytosol from becoming too high and to prevent SR/ER depletion by recycling Ca²⁺ to the SR/ER.³³ Mitochondrial calcium can be stored as calcium phosphate precipitates, permitting sequestration of surprisingly large amounts of Ca²⁺.³⁴ Mitochondrial Ca²⁺ homeostasis also regulates energy metabolism to synchronize ATP generation with cell function. For instance, uptake of Ca²⁺ results in the activation of three dehydrogenases in the mitochondrial matrix.³⁵ This causes an increase in mitochondrial NADH/NAD⁺ ratio which may result in increased energy available for mitochondrial functions. In addition, matrix Ca²⁺ has been suggested to activate ATP synthesis through a direct effect on F₀F₁-ATPase.³⁶ Mitochondrial matrix [Ca²⁺] is controlled by a Ca²⁺ uniporter channel for influx and by a Na⁺/Ca²⁺ exchanger (NCX) for efflux.³⁷ The activity of NCX saturates as mitochondrial matrix [Ca²⁺] increases, whereas the uniporter acts as a channel and is not saturated with increasing [Ca²⁺]. As a result, the [Ca²⁺] increases in the matrix and beyond a certain threshold, the mitochondria can no longer regulate matrix [Ca²⁺], resulting in mitochondrial overload.³⁷ If the mitochondrial Ca²⁺ load is substantially increased, Ca²⁺ can promote opening of the mPTP thereby triggering cell death.² However, the role of Ca²⁺ as a mediator of mPTP opening in cardiac myocytes has recently come into question. Most experiments demonstrating mPTP opening in response to Ca²⁺ has been done in vitro using isolated mitochondria, but Juhaszova et al.²⁹ found that increased uptake of Ca²⁺ into mitochondria did not result in mPTP opening in isolated cardiac myocytes. Similarly, another study using a model of simulated I/R found that mPTP opening during reperfusion was not dependent on mitochondrial uptake of Ca²⁺ during ischaemia.³⁸ Clearly, the role of Ca²⁺ in mediating mPTP opening needs to be further investigated in vivo.

It is well established that myocardial I/R injury is accompanied by mitochondrial Ca²⁺ overload which contributes to mitochondrial dysfunction and cell death. During ischaemia, there is a drop in ATP/ADP and a build-up of lactic acid, with a consequent drop in intracellular pH. In an attempt to restore the pH, the cell utilizes the Na⁺/H⁺ exchanger, but this results in loading of the cell with Na⁺. This Na⁺ cannot be pumped out via the Na⁺/K⁺-ATPase due to compromised ATP levels. Consequently, the plasma membrane NCX which normally extrudes Ca²⁺ operates in reverse and the cell becomes loaded with Ca²⁺.³⁹ Upon reperfusion, Ca²⁺ re-enters the mitochondria and consequently produces mitochondrial Ca²⁺ overload, which may lead to cell death. Strategies that limit mitochondrial Ca²⁺ accumulation have significant beneficial effects on cardiac function following I/R. For instance, treatment of hearts with ruthenium red (RR) or Ru360, inhibitors of the Ca²⁺ uniporter, improved functional recovery and reduced infarct size in hearts subjected to I/R.^40,41 In addition, isolated heart mitochondria were more resistant to Ca²⁺-induced swelling in the presence of Ru360 or RR, suggesting that pore opening during Ca²⁺ overload is modulated by activity of the Ca²⁺ uniporter.⁴⁰ Other studies have demonstrated the beneficial effects of plasma membrane NCX inhibitors on myocardial I/R injury. For instance, KB-R7943 or SEA0400, two different inhibitors of the NCX, reduced infarct size and improved post-ischaemic recovery of function,^42,43 whereas enhancing expression of NCX in the heart increased susceptibility to I/R injury.⁴⁴

	5. Mitochondrial permeability transition pore (mPTP)

Top Abstract 1. Introduction 2. Mitochondria and reactive... 3. Reactive oxygen species... 4. Mitochondria and calcium 5. Mitochondrial permeability... 6. Mitochondrial mediators of... 7. Mitochondria and Bcl-2... 8. Consequences of mitochondrial... 9. Mitochondrial fission and... 10. Clinical perspective 11. Conclusions Funding References

 
The mPTP is a voltage-dependent, high-conductance channel with a diameter of 3 nm allowing for rapid equilibration of ions and water, as well as passage of molecules up to 1.5 kDa.⁴⁵ The mPTP is regulated by cyclophilin D (CycD) in the inner mitochondrial membrane, and is important for mPTP function.⁴⁶ Various studies have implicated VDAC and ANT as elements of the mPTP, yet mitochondria from knockouts for multiple isoforms of VDAC or ANT are still able to demonstrate pore opening albeit with higher thresholds.^47,48 Other studies have revealed a role for Complex I in mPTP function.^49,50 Under normal conditions, the inner mitochondrial membrane is impermeable to all but a few ions and metabolites for which specific transport mechanisms are present. This tight regulation is necessary for mitochondria to maintain a pH gradient and membrane potential which drives oxidative phosphorylation. Opening of the pore causes collapse of the proton gradient and electrical potential across the inner mitochondrial membrane, leading to uncoupling of oxidative phosphorylation. The high colloid osmotic pressure in the matrix causes influx of water, and swelling of the matrix; whereas the inner mitochondrial membrane can expand through cristae remodelling, the outer mitochondrial membrane is unable to expand resulting in rupture and release of cytochrome c and other pro-apoptotic proteins into the cytosol.

Many studies have found that opening of the mPTP occurs during I/R. During ischaemia, depletion of adenine nucleotides and accumulation of inorganic phosphate (Pi) sensitizes the mPTP to opening. The mPTP is regulated by pH, and the low pH during ischaemia prevents opening of the pore. Upon reperfusion, the mitochondria resume respiration and generate a membrane potential, resulting in an increase in the production of ROS. Reperfusion also triggers rapid accumulation of Ca²⁺ within the mitochondria. Within a few minutes of reperfusion, the pH returns to normal and mPTP opening occurs. Halestrap et al.⁵¹ demonstrated that mPTP opening did not take place during ischaemia but occurred approximately 2 min into reperfusion. Opening of the mPTP contributes to mitochondrial dysfunction in cardiac myocytes and inhibition of pore opening is cardioprotective. For instance, inhibitors of the mPTP protected against hypoxia/re-oxygenation-mediated cell death in isolated cardiac myocytes,^52,53 and reduced infarct size in the ex vivo model of I/R.^28,30,54 Treatments that reduce mPTP opening, such as decreasing oxidative stress with free radical scavengers, and maintaining a lower pH during reperfusion are also cardioprotective.⁵¹ Moreover, CycD deficient mice, whose mPTP is more resistant to Ca²⁺- and oxidative stress-induced opening, had reduced injury after myocardial I/R compared to wild-type litter mates.^46,55 Interestingly, cells lacking CycD were still sensitive to apoptotic stimuli, suggesting that mPTP opening is not required for induction of apoptosis via the mitochondrial pathway.⁴⁶ In contrast, cardiac mitochondria from transgenic mice overexpressing CycD in the heart displayed a lower threshold for mPTP opening in vitro, and hearts displayed increased signs of apoptosis, hypertrophy, and decreased cardiac function.⁴⁶ Moreover, the mPTP has been shown to contribute to loss of myocytes during progression to heart failure. For instance, transgenic mice overexpressing the alpha subunit of Gq develop cardiac hypertrophy and subsequent lethal cardiomyopathy.⁵⁶ Adams et al.⁵⁷ reported that overexpression of a constitutively active form of Gq in isolated cardiac myocytes caused mitochondrial dysfunction and cell death, which was prevented in the presence of bongkrekic acid. These studies demonstrate that opening of the mPTP contributes to loss of cardiac myocytes in various cardiovascular diseases.

Reduced mPTP opening has also been suggested to play a role in pre- and post-conditioning of the heart brief, i.e. periods of ischaemia performed at the prior to ischaemia or at onset of reperfusion, respectively. Pre-conditioning was shown to be associated with reduced mPTP opening in Langendorff-perfused hearts.⁵⁸ Studies using isolated cardiac myocytes and mitochondria have implicated the inhibition of the mPTP in both calcium- and diazoxide-mediated pre-conditioning.^53,59,60 Similarly, mPTP inhibition has been suggested to be responsible for the protection provided by post-conditioning,⁶¹ which agrees with the study by Hausenloy et al.⁵⁹ who found that CsA treatment at the start of reperfusion reduced infarct size.

	6. Mitochondrial mediators of cardiac protection

Top Abstract 1. Introduction 2. Mitochondria and reactive... 3. Reactive oxygen species... 4. Mitochondria and calcium 5. Mitochondrial permeability... 6. Mitochondrial mediators of... 7. Mitochondria and Bcl-2... 8. Consequences of mitochondrial... 9. Mitochondrial fission and... 10. Clinical perspective 11. Conclusions Funding References

 
The mitochondrial ATP-sensitive K⁺ (mK_ATP) channel plays a central role in mediating protection in ischaemic pre-conditioning (IPC). Pharmacological activation of this channel protects against I/R injury, whereas inhibition of the mK_ATP prevents the protective effects of IPC.⁶² Opening of the mK_ATP channel has been reported to reduce mitochondrial Ca²⁺ uptake and to inhibit mPTP opening,^60,63 as well as to increase ROS production by the ETC.^64,65 Whereas excess levels of ROS triggers cell death, lower levels of ROS produced during brief periods of ischaemia and reperfusion have been shown to play a role in IPC.⁶⁶ The exact mechanism for opening of the mK_ATP during pre-conditioning is still under intense investigation. Recently, it was demonstrated that mK_ATP and PKC directly interact in the mitochondrial inner membrane and that PKC is required for the opening of the mK_ATP.⁶⁷ Previous studies have found that activation and translocation of PKC to the mitochondria are important for pre-conditioning.^68–70 Gray et al.⁷¹ demonstrated that treatment with a PKC selective inhibitor abolished the protective effects of pre-conditioning. In addition, transgenic mice overexpressing PKC in the heart had reduced infarct size after myocardial I/R,⁷² and PCK knockout mice did not retain the protective effects of pre-conditioning.⁷³ These studies suggest that PKC mediates protection of mitochondria in the myocardium by activation of the mK_ATP.

Activation of the PI 3-kinase/Akt signalling pathway has also been demonstrated to provide cardiac protection against various stressors by preserving mitochondrial integrity and function. PI 3-kinase signalling has been shown to provide protection against I/R injury during pre-conditioning⁷⁴ and Akt activation has been shown to decrease apoptosis, as well as to reduce infarct size and to improve cardiac function after I/R.⁷⁵ Furthermore, adenovirally infecting mouse⁷⁶ or rat hearts⁷⁷ with constitutively active Akt gene constructs confers protection against I/R injury. Akt has been shown to protect mitochondrial integrity and inhibit cytochrome c release following an apoptotic stimulus.^78–80 However, the exact mechanism for Akt-mediated cardioprotection is still unclear and it appears that Akt acts on multiple target to provide its protective effects. For instance, preservation of mitochondrial integrity and function by Akt has been shown to be dependent on the presence of glucose and hexokinase. Akt was shown to preserve mitochondrial membrane potential in response to growth factor deprivation by increasing glucose transporter expression and glytolytic activity resulting in greater substrate availability for mitochondrial electron transport.⁸¹ Akt also elevated mitochondrial hexokinase association and activity at the mitochondria.⁷⁸ In addition, Akt has been shown to provide protection by phosphorylation and inactivation of the BH3-only protein Bad⁸² and prevention of Bax translocation to the mitochondria,⁸³ as well as induction of anti-apoptotic Bcl-2 proteins.^84,85 Akt has also been shown to phoshorylate and inactivate glycogen synthase kinase-3ß (GSK-3ß),⁸⁶ and inhibition of GSK-3ß prevents opening of the mPTP.²⁹ GSK-3ß is thought to contribute to I/R-mediated cell death,^87,88 and has been shown to be inhibited by pre-conditioning.^88,89

	7. Mitochondria and Bcl-2 family proteins

Top Abstract 1. Introduction 2. Mitochondria and reactive... 3. Reactive oxygen species... 4. Mitochondria and calcium 5. Mitochondrial permeability... 6. Mitochondrial mediators of... 7. Mitochondria and Bcl-2... 8. Consequences of mitochondrial... 9. Mitochondrial fission and... 10. Clinical perspective 11. Conclusions Funding References

 
The Bcl-2 family proteins are important regulators of the mitochondrial apoptotic pathway. This family is composed of both pro- and anti-apoptotic proteins that share up to four conserved regions known as Bcl-2 homology (BH) domains. Anti-apoptotic members such as Bcl-2 and Bcl-X_L contain all four subtypes of BH domains, and promote cell survival by inhibiting the function of the pro-apoptotic Bcl-2 proteins. The pro-apoptotic members can be separated into two structurally distinct subfamilies. The ‘multidomain’ proteins (Bax and Bak) share three BH regions and lack the BH4 domain. They are structurally similar to the anti-apoptotic proteins.^90,91 In contrast, ‘BH3-only’ proteins which include Bid, Bnip3, Nix/Bnip3L, and Puma, share only the BH3 domain and are structurally diverse.⁹² The BH3-only proteins function as death signal sensors in the cell and play a major role in transducing signals from the cytosol to the mitochondria. All BH3-only proteins initiate cell death through the activation of Bax and Bak, and studies using cells derived from knock-out mice lacking both Bax and Bak have demonstrated that Bax and Bak are essential for initiation of cell death through the mitochondrial pathway. In response to an apoptotic stimulus, BH3-only proteins bind to and neutralize the anti-apoptotic Bcl-2 proteins, thereby releasing Bax and Bak. Certain BH3-only proteins, such as tBid, can directly interact with the pro-apoptotic Bax and Bak to trigger apoptosis. In healthy cells, Bax is localized in the cytosol and upon a cell death signal, Bax rapidly translocates to the mitochondria, whereas Bak is already present in the mitochondria as an integral membrane protein. Although the precise mechanisms of membrane permeabilization are unclear, it has been proposed that it can result from a conformational change of Bax or Bak, their full insertion into mitochondrial membranes as homo-oligomerized multimers, and formation of giant protein-permeable pores.⁹³

The Bcl-2 family proteins play a central role in regulating apoptosis in the heart. The anti-apoptotic Bcl-2 proteins provide protection of mitochondria by acting on multiple targets. For instance, Bcl-2 has been shown to prevent permeabilization of the outer mitochondrial membrane by inhibiting activation of Bax/Bak,⁹⁴ and to increase the calcium threshold for mPTP opening in heart mitochondria by blocking opening of the pore.⁹⁵ Moreover, transgenic mice overexpressing Bcl-2 in the heart had fewer apoptotic cells, reduced infarct size and improved recovery of cardiac function after I/R.^96–98 Mice null for desmin develop cardiomyopathy characterized by ultrastructural abnormalities in mitochondria and extensive cell death, but overexpression of Bcl-2 in these hearts corrected the mitochondrial defects and improved cardiac function.⁹⁹ During ischaemia, electron transport and mitochondrial generation of ATP are inhibited, and the F₁F₀-ATPase runs in reverse, consuming glycolytically generated ATP.^100,101 Interestingly, transgenic mice overexpressing Bcl-2 in the heart had a decreased rate in decline of ATP during ischaemia as well as reduced acidification, suggesting that Bcl-2 provides protection by inhibiting consumption of glycolytically generated ATP by the F₁F₀-ATPase.⁹⁶ Moreover, elevated expression of Bcl-X_L by adenoviral gene transfer inhibited Bax translocation from the cytosol to the mitochondria, reduced cytochrome c release from mitochondria and decreased apoptosis after I/R.¹⁰² Similarly, the BH4 domain of Bcl-X_L has been shown to be necessary and sufficient for protection against mitochondrial dysfunction and apoptosis and recently was shown to interact with truncated Bid.¹⁰³ Perfusion of hearts with this BH4 peptide linked to a protein transduction domain (TAT-BH4) reduced infarct size and creatine kinase release after I/R.¹⁰⁴ These studies demonstrate a cardioprotective role for the anti-apoptotic Bcl-2 proteins in preventing mitochondrial dysfunction and cell death in cardiac myocytes in response to various stressors.

Many of the pro-apoptotic Bcl-2 proteins have also been implicated in the pathogenesis of various cardiac diseases including myocardial hypertrophy, infarction, and heart failure. For instance, chronic hypoxia, stretch, and chronic pressure overload in rat hearts correlated with increased Bax and decreased Bcl-2 levels accompanied by mitochondrial dysfunction and cell death.^105,106 Moreover, Bax has been reported to be activated in cardiac cells in response to oxidative stress¹⁰⁷ and during ischaemia.¹⁰⁸ Hearts from Bax deficient mice had reduced mitochondrial damage and decreased infarct size after I/R compared to wild type, implicating Bax as a major player of mitochondrial dysfunction in I/R.¹⁰⁹ Among the BH3-only proteins, Bid, Puma, Bnip3, and Nix/Bnip3L have been implicated in cardiac myocyte death. For instance, Bid has been reported to be subjected to proteolytic cleavage during myocardial I/R. The activated truncated Bid (tBid) then translocates to the mitochondria causing activation of Bax/Bak and release of cytochrome c into the cytosol.^104,110,111 Puma was upregulated in cardiac myocytes in response to hypoxia/reoxygenation, whereas Puma knockout mice had reduced infarct size and improved cardiac function after I/R.¹¹² Moreover, Bnip3 and Nix/Bnip3L have been associated with mitochondrial dysfunction and cell death in the heart. Bnip3 has been shown to contribute to I/R injury via activation of Bax^113,114 and was found to be upregulated in failing hearts,¹¹⁵ whereas Nix/Bnip3L has been implicated in cardiac hypertrophy and development of cardiomyopathy.¹¹⁶

	8. Consequences of mitochondrial permeabilization

Top Abstract 1. Introduction 2. Mitochondria and reactive... 3. Reactive oxygen species... 4. Mitochondria and calcium 5. Mitochondrial permeability... 6. Mitochondrial mediators of... 7. Mitochondria and Bcl-2... 8. Consequences of mitochondrial... 9. Mitochondrial fission and... 10. Clinical perspective 11. Conclusions Funding References

 
Permeabilization of the MOM and loss of _m are universal features of cell death and are generally considered as the ‘point of no return’ in the events leading to cell death.¹¹⁷ Once permeabilization has occurred, it leads to rapid cell death through a variety of independent and redundant mechanisms. These include caspase activation, irreversible metabolic changes, disruption of oxidative phosphorylation, and the release of caspase independent effectors such as apoptosis inducing factor (AIF).

The most well known protein released from mitochondria during apoptosis is cytochrome c. The function of cytochrome c in the mitochondria is to shuttle electrons from complexes III to IV in the respiratory chain. However, during apoptosis mitochondria release cytochrome c into the cytosol, where it associates with Apaf-1 along with dATP and caspase-9 to form the macromolecular complex known as the apoptosome.¹¹⁸ The formation of this complex triggers activation of caspase-9 which in turn cleaves and activates the effector, caspase-3, culminating in cell death. Caspases are responsible for the proteolytic inactivation of crucial cellular targets, including mitochondria. For instance, caspases have been shown to enter the permeabilized outer membrane of mitochondria and cleave essential subunits in Complex I, disrupting mitochondrial respiration.¹¹⁹ Interestingly, it has been shown that inhibition of caspases has no effect on loss of _m and delays—but does not prevent—cell death induced by stimuli that activate the mitochondrial pathway.¹²⁰

Other apoptotic factors released from the mitochondria are Smac/Diablo, Omi/Htr2A, AIF, and endonuclease G (EndoG). Inhibitors of apoptosis proteins (IAPs) are endogenous inhibitors of initiator caspase-9 and downstream effector caspases-3 and -7. Once released, Smac/Diablo and Omi/HtrA2 antagonize IAPs, thereby permitting activation of caspases.^121–123 Another mitochondrial protein that promotes cell death upon release into the cytosol is AIF. In the mitochondria, AIF is critical for optimal detoxification of ROS and the assembly and stabilization of Complex I.^124,125 Cardiac myocytes isolated from harlequin mutant mice that have downregulated expression of AIF were found to be more sensitive to hydrogen peroxide-induced cell death, and hearts displayed increased injury after I/R.¹²⁴ In addition, targeted disruption of AIF in the heart leads to severe defects in Complex I activity and development of dilated cardiomyopathy.¹²⁶ These studies demonstrate that AIF is essential for normal mitochondrial function. However, upon induction of apoptosis, release of AIF into the cytosol is followed by rapid translocation to the nucleus where it facilitates chromatin condensation and large-scale DNA fragmentation. For instance, Siu et al.¹²⁷ found AIF and Omi/Htr2A in the cytosol in an animal model of salt-induced heart failure. Another study reported that AIF was detected in the cytosolic and nuclear fractions of hearts subjected to I/R, and that IPC attenuated AIF release, implicating AIF as a contributor to myocardial cell death in I/R.¹²⁸ EndoG is a mitochondrion specific nuclease that also translocates to the nucleus during apoptosis, where it cleaves chromatin DNA into nucleosomal fragments.¹²⁹ Bahi et al.¹³⁰ found that ischaemia induced the release of EndoG from mitochondria and translocation to the nucleus in isolated neonatal myocytes, and that knock-down of EndoG by siRNA reduced DNA laddering suggesting that EndoG is an important executor of DNA degradation during ischaemia in cardiac myocytes. However, the importance of EndoG in cell death has recently been questioned. Two independent studies reported no obvious effect on cell death and DNA degradation in EndoG deficient mice. The mice are viable and there is no effect on mitochondrial DNA copy number or mutation rate.¹³¹ Moreover, cells isolated from these mice show no difference in susceptibility in response to various apoptotic stimuli.¹³² However, these studies did not assess whether the hearts were less susceptible to injury in response to stress such as I/R or pressure overload. Clearly, more studies are required to elucidate the role of EndoG in apoptosis. In our laboratory, we found that some antibodies to EndoG cross-reacted strongly with creatine kinase (unpublished data), which may add to the confusion.

	9. Mitochondrial fission and ‘mitophagy’

Top Abstract 1. Introduction 2. Mitochondria and reactive... 3. Reactive oxygen species... 4. Mitochondria and calcium 5. Mitochondrial permeability... 6. Mitochondrial mediators of... 7. Mitochondria and Bcl-2... 8. Consequences of mitochondrial... 9. Mitochondrial fission and... 10. Clinical perspective 11. Conclusions Funding References

 
Mitochondria are dynamic organelles that are constantly undergoing fission and fusion to adapt to changing conditions of the cell. Recently, several studies have reported that mitochondrial morphology changes during apoptosis, resulting in small round mitochondrial fragments.^133–135 For instance, Karbowski et al.¹³⁵ reported that Bax co-localized with Dynamin-Related Protein-1 (Drp-1), a protein involved in mitochondrial fission, at defined foci on the mitochondrial membrane at the onset of apoptosis. More importantly, a dominant negative of Drp-1 inhibited fragmentation and apoptosis, but not Bax translocation to the foci in response to staurosporine treatment. Drp-1 has also been reported to be required for optimal release of cytochrome c induced by the BH3-only protein Bik, presumably through remodelling of the cristae.¹³⁶ Moreover, the mitochondrial network in HL-1 cardiac myocytes was shown to undergo extensive fragmentation in response to simulated I/R or overexpression of Bnip3.^113,137 The Bnip3-mediated mitochondrial fragmentation correlated with accumulation of Bax and Drp-1 at distinct clusters on the mitochondrial fragments.¹¹⁴ These studies suggest that the Bcl-2 proteins can mediate apoptosis through the mitochondrial fission pathway.

Mitochondrial dysfunction has also been reported to lead to upregulation of autophagy. Autophagy is a process important in cellular homeostasis and in removing excess or damaged organelles.¹³⁸ For instance, mitochondrial fragmentation correlated with upregulation of autophagy and sequestration of mitochondrial fragments in autophagosomes (mitophagy) in HL-1 myocytes overexpressing Bnip3.¹¹³ Autophagy plays an important role in the cellular response to stress and has been shown to be upregulated in the myocardium in response to I/R.^137,139 Upregulation of autophagy in HL-1 myocytes was found to protect against simulated I/R cell death in HL-1 myocytes,¹³⁷ while enhanced autophagy during chronic myocardial ischaemia correlated with decreased apoptosis, suggesting that induction of autophagy is a protective response.¹⁴⁰ In contrast, Beclin-1 is a protein essential in the autophagic pathway and heterozygous knockout mice had reduced autophagy, smaller infarcts, and apoptosis after I/R compared to wild type,¹³⁹ suggesting that upregulation of autophagy in response to I/R may be detrimental. Clearly, further studies are required to elucidate the roles of mitochondrial fragmentation and autophagy in myocardial cell death.

	10. Clinical perspective

Top Abstract 1. Introduction 2. Mitochondria and reactive... 3. Reactive oxygen species... 4. Mitochondria and calcium 5. Mitochondrial permeability... 6. Mitochondrial mediators of... 7. Mitochondria and Bcl-2... 8. Consequences of mitochondrial... 9. Mitochondrial fission and... 10. Clinical perspective 11. Conclusions Funding References

 
Many pharmacological agents that directly or indirectly protect mitochondrial integrity in response to stress have been used in clinical trials for the treatment of various cardiovascular diseases. For instance, beta-blockers such as carvedilol have been shown to be very effective for the treatment of ischaemic heart disease and congestive heart failure. Carvedilol has been demonstrated to have additional effects independent of blocking the beta-adrenergic receptor and has been shown to directly protect against mitochondrial permeability transition by reducing oxidative stress.^141,142 Although most antioxidants protect against myocardial cell death in animal models, clinical trials have produced mixed results. For instance, large clinical trials using antioxidant vitamins have failed to show any clinical benefits on the heart.^143–145 In contrast, clinical studies have demonstrated that infusion of N-acetyl cysteine (NAC) duing thrombolysis correlated with a reduced infarct size and increased preservation of left ventricular function.^146,147 In addition, administration of NAC in combination with streptokinase reduced oxidative stress and improved left ventricular function in patients with acute myocardial infarction.¹⁴⁸

Pharmacological openers of the mK_ATP channel were developed for the treatment of angina pectoris and hypertension even before this channel was identified as an important mediator of protection during IPC. Clinical studies have demonstrated that infusion of nicorandil, a hybrid K_ATP channel opener and nitrate, improved left ventricular function in patients with acute myocardial infarction.¹⁴⁹ Although pre-conditioning provides great protection against myocardial injury in animal models of I/R, its clinical application is limited since the coronary artery is already occluded in patients with acute myocardial infarction by the time they are admitted to the hospital. In contrast, post-conditioning offers greater clinical potential and can be applied to a number of clinical procedures such as cardiac transplantation and coronary angioplasty. The study by Staat et al.¹⁵⁰ demonstrated that patients subjected to four cycles of 1 min ischaemia and 1 min reperfusion immediately upon reperfusion during coronary angioplasty for acute myocardial infarction had reduced infarct size compared to control patients.

	11. Conclusions

Top Abstract 1. Introduction 2. Mitochondria and reactive... 3. Reactive oxygen species... 4. Mitochondria and calcium 5. Mitochondrial permeability... 6. Mitochondrial mediators of... 7. Mitochondria and Bcl-2... 8. Consequences of mitochondrial... 9. Mitochondrial fission and... 10. Clinical perspective 11. Conclusions Funding References

 
Mitochondria are critical to cardiac cell survival through their essential roles in energy production and calcium homeostasis, but they also function as central regulators of apoptotic and necrotic cell death (Figure 1). Bax/Bak-mediated permeabilization of the MOM leads to apoptosis, while opening of the mPTP leads to a caspase-independent (necrotic) form of cell death. In either case, failure of mitochondrial ATP production also contributes to cell death in these energy-intensive cells. To protect cardiac myocytes from death, it is important to preserve mitochondrial integrity. Loss of mitochondrial membrane integrity is generally considered a point of no return, as inhibiting the post-mitochondrial phase of apoptosis does not rescue the cardiomyocyte. Preservation of mitochondrial integrity is of utmost importance in the design of cardioprotective therapies.

View larger version (34K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 1 Mitochondria as a central integrator of multiple stress signals.

 

	Funding

Top Abstract 1. Introduction 2. Mitochondria and reactive... 3. Reactive oxygen species... 4. Mitochondria and calcium 5. Mitochondrial permeability... 6. Mitochondrial mediators of... 7. Mitochondria and Bcl-2... 8. Consequences of mitochondrial... 9. Mitochondrial fission and... 10. Clinical perspective 11. Conclusions Funding References

 
This manuscript was supported by funds from the California Tobacco-Related Disease Research Program of the University of California, New Investigator Award #14KT-0109 (to ÅBG) and NIH grants HL060590, and HL085577 (to RAG).

Conflict of interest: none declared.

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Top Abstract 1. Introduction 2. Mitochondria and reactive... 3. Reactive oxygen species... 4. Mitochondria and calcium 5. Mitochondrial permeability... 6. Mitochondrial mediators of... 7. Mitochondria and Bcl-2... 8. Consequences of mitochondrial... 9. Mitochondrial fission and... 10. Clinical perspective 11. Conclusions Funding References

 

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