Journal of Molecular and Cellular Cardiology 41 (2006) 406–409www.elsevier.com/locate/yjmcc

Editorial

TOM20 and the Heartbreakers: Evidence for the role of mitochondrialtransport proteins in cardioprotection

Ischemic heart disease (IHD) is the leading cause of deathin developed countries. In IHD, disruption of blood flow dueto blockage of coronary vessels leads to irreversiblemyocardial cell injury and death. In the past few decades,

significant research has been devoted to uncovering ways toprevent ischemic damage by increasing blood supply to themyocardial tissue at risk. A novel autoprotective phenomenoncalled ischemic preconditioning (IPC), first described byMurray et al. [1], has raised hopes that recruiting intrinsicmechanisms may reduce cellular damage during ischemia. InIPC, short ischemic periods protect against damage inducedby a longer subsequent ischemic event. The underlyingmolecular basis of IPC has been the subject of manyinvestigations and has centered on two major areas:determining the signaling pathway(s) that lead to protectionand characterizing the end-effector molecules in this process[2]. Multiple intracellular pathways that mediate IPC havebeen characterized, including the activation of protein kinases,production of nitric oxide, activation of cyclooxygenaseproteins, generation of reactive oxygen species, and changesin the intracellular calcium levels [2]. The downstream orend-effector molecules are still under investigation; mitochon-dria, however, are thought to be a key mediator of thisprocess.

Mitochondria are membrane-bound organelles in eukaryo-tic cells that are responsible for the production of energy inthe form of ATP. They are also critical in controlling thebalance between survival and death in response to a variety ofpathological and physiological conditions. Furthermore,mitochondria are shown to contribute to the pathogenesis ofboth apoptotic and necrotic cell death. It is believed that IPCinduces mitochondrial pathways that lead to cytoprotection,whereas prolonged ischemia shifts the balance towardmitochondrial death signaling.

Several mitochondrial mechanisms have been proposed tobe involved in IPC. These include preservation of respirationand ATP production, changes in the levels of reactive oxygenspecies, changes in the mitochondrial calcium levels, closureof the mitochondrial permeability transition pore (mPTP), andopening of the mitochondrial ATP-sensitive K+ channel(mitoKATP) [2,3]. mPTP is a mitochondrial inner membrane

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channel whose opening allows molecules smaller than 1500Daltons to enter the mitochondrial matrix [4]. The entry ofsmall molecules leads to increased mitochondrial permeabilityand ultimately to irreversible cell injury. mitoKATP, on theother hand, is a protective channel whose opening is believedto be crucial for IPC induction [2,3,5]. The precise molecularstructures of these two channels are not known but arethought to be multi-protein complexes [4,6].

The potential roles of other mitochondrial proteins andstructures in IPC and ischemic injury have been the subject ofmany investigations. In this issue of JMCC, Boengler et al.studied the role of members of the protein transportmachinery in both induction of cell death by ischemia andprotection by IPC. The protein levels of three members of themitochondrial protein transport system, translocase of theouter membrane (Tom)-20, Tom40, and translocase of theinner membrane (Tim)-23, were analyzed in minipigssubjected to low flow ischemia with and without precondi-tioning. The authors demonstrated that Tom20 levels decreaseafter 90 minutes of ischemia, and that IPC preserves thelevels of this protein. There was no significant difference seenin Tom40, Tim23 and adenine nucleotide translocator (ANT).The authors concluded that Tom20 may play a role inprotection against ischemic damage, providing a link betweenIPC and the mitochondrial protein transport machinery.

The mitochondria transport hundreds of nuclear-encodedproteins into their outer membrane, intermembrane space(IMS), inner membrane and matrix (7). Most mitochondrialprecursor proteins contain a mitochondrial targeting sequence(MTS) that resides in the N-terminus, although some containthis information more internally within the protein [8–12].MTSs are about 20-60 amino acids in length and haveabundant positive charges. They are predicted to formamphipathic α-helices in membranes and are recognized byprotein import machinery [7]. Fusion proteins containingthese peptides and non-mitochondrial proteins are importedinto the mitochondria [13–15]. The role of these sequences inmitochondrial protein trafficking has been studied usingmutational analysis.

Mitochondrial proteins are believed to be released fromribosomes into the cytoplasm as completed chains. A number

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of cytosolic proteins (such as the chaperone protein Hsp70)interact with these precursor peptides and guide them to themitochondria [7]. The lipophilicity of mitochondrial mem-branes prevents the entrance of these precursors directly intothe mitochondria; thus their transport requires aqueous cation-sensitive channels. Many different Tom and Tim proteins areinvolved in protein entry into the mitochondria (Fig. 1) [10–12]. Proteins comprising the TOM complex in the outermitochondrial membrane consist of at least eight differentproteins, which work together to recognize cytosolic pre-cursors and in conjunction with cytosolic chaperone proteinsto facilitate their entry into the mitochondria. Tom20, Tom22,Tom37, and Tom70 recognize precursor peptides and move

Fig. 1. Mitochondrial protein import pathway (adapted from reference 20).Nuclear encoded precursor proteins are synthesized in the cytosol and eventuallyunfold with the help of cytosolic chaperones, such as Hsp70. There are two maintypes of precursor peptides, those with an MTS (cleavable N-terminuspresequence) and noncleavable hydrophobic peptides with an internal targetingsequence (see text for details). Peptides with MTS are recognized by the TOMcomplex and subsequently interact with Tom20/Tom22 (Tom70/Tom37 for non-MTS peptides) which transfer these proteins to the protein-conducting channelof the TOM complex, Tom40. Once these precursors are translocated to the IMS,they associate with the Tim17/Tim23 complexes. Mitochondrial heat shockprotein 70 (mtHSP70) and Tim44 facilitate the translocation of these precursorsfrom the inner membrane into the matrix. The transport across the innermembrane is dependent on mitochondrial membrane potential and ATPhydrolysis. Inside the matrix the precursor peptides are further processed bythe mitochondrial-processing peptidase, which cleave the MTS and allow theprotein to fold into its natural configuration with the help of chaperone proteins.The numbers in the illustration correspond with the molecular mass of theproteins in kDa. IM=inner membrane, OM=outer membrane.

them from the cytosol into the IMS [16]. One component ofthe TOM complex, consisting of Tom20 and Tom22,selectively binds precursor proteins with a cleavable N-terminal MTS. Peptides containing non-cleavable internaltargeting sequences are delivered to the IMS by a Tom70-Tom37 complex. The other TOM proteins (Tom40, Tom5,Tom6 and Tom7) form a pore on the mitochondrial surfacecalled the general import pore (GIP), with Tom40 functioningas the cardinal component. Tom5 is the smallest Tom proteinand functions as an intermediary between Tom40 and theother members of the TOM complex [10,16]. Tom6 andTom7 appear to function as regulators of GIP formation:Tom6 promotes the association between Tom22 and Tom40,while Tom7 has the opposite activity [10].

The translocases of the inner mitochondrial membraneconsist of a multiple subunit complex, the TIM complex,which routes precursor proteins from the IMS into the matrix.The central portion of the TIM complex includes the subunitsTim17, Tim23, and Tim44 [17]. TOM and TIM complexes tendto localize to the contact areas between the outer and innermembranes, thus allowing a more direct transfer of peptidesfrom one compartment to the next. Tim17 and Tim23 span themitochondrial inner membrane and form a pore through whichproteins may cross into the matrix. The transport of proteinsfrom IMS to the matrix is dependent on both ATP hydrolysisand an intact mitochondrial membrane potential [16,18].Addition of the uncoupler carbonyl cyanide m-chlorophenylhy-drazone (CCCP) to isolated mitochondria results in a decreasein protein transport [19]. Furthermore, only the membranepotential and not the proton motive force is required for thetransport of proteins, suggesting that protein translocationacross the inner membrane is not driven by the movement ofprotons. The final step in the translocation of the precursors intothe matrix involves the binding to the mitochondrial heat shockprotein 70 (mtHsp70) and mtHsp70-dependent hydrolysis ofATP. mtHsp70 intracts with Tim44, and this complex facilitatesthe translocation of precursors into the matrix [20]. It is nottotally clear how mtHsp70 carries out this process [20,21].

Upon transport into the mitochondrial matrix, precursorpeptides are transformed into their mature protein configuration.This maturation process enables mitochondrial proteins to carryout their intended mitochondrial function. The first step is theproteolytic cleavage of the MTS by a matrix processingpeptidase [22]. This cleavage results in a mature protein thatmust then bind to Hsp60 and its co-chaperonin, cpn10, twoproteins that help refold the cleaved protein into its functionalconformation [16]. In summary, protein transport into themitochondria is a complicated yet orchestrated process thatrequires interaction and cross-talk among multiple cytoplasmicand mitochondrial proteins. Furthermore, intact mitochondrialmembrane potential and readily available ATP are needed forthis process to be completed.

Two major inferences can be drawn from the process of themitochondrial protein transport. First, given the importance ofthis process, any defect in protein transport into the mitochon-dria may lead to a pathological condition. This is supported byseveral studies that have shown that defects in mitochondrial

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protein transport result in clinical disorders. Aguirre et al. [23]found mutations in the gene of a member of the TIM complex(Tim8a) in a Spanish familial case of Mohr-Tranebjaerg (a rareX-linked condition characterized by dystonia and progressivepostlingual sensorineural hearing impairment). Additionally,the levels of Hsp60 were found to be significantly reduced in aninfant with multi-organ failure and multiple mitochondrialenzymatic defects [24]. Decreased synthesis and mitochondrialtransport of Hsp60 has also been found in a patient withmitochondrial encephalomyopathy [25]. Schapira et al. [26]have reported a case of mitochondrial myopathy with adeficiency in mitochondrial protein transport. These studiessuggest that defects in protein transport into the mitochondriamay lead to mitochondrial dysfunction and severe pathologicalconditions.

Second, since increased activity of certain mitochondrialproteins is needed in conditions that require high mitochondrialbiogenesis, protein transport may be enhanced under theseconditions to improve protein delivery to the mitochondria.There is indirect evidence supporting this possibility. In skeletalmuscle, contractile activity, which induces mitochondrialbiogenesis, is associated with increased expression of theprotein import machinery components, especially Tom20 andHsp70 [27]. Furthermore, Tom20 appears to play a particularlyimportant role in protein transport in these cells. Overexpressionof this protein in C2C12 skeletal muscle cells results in anincrease in the import of malate dehydrogenase, while areduction in the levels of Tom20 using antisense DNA leads to adecrease in protein import [28]. Treatment of rats with thyroidhormone T3 leads to an enhanced rate of protein transport intothe mitochondria, which is also associated with increased levelsof Tom20 protein [29].

These studies suggest that increased protein transport isneeded in conditions of high biogenesis. Since increasedmitochondrial biogenesis has also been suggested in IPC [30],Boengler et al. attempted to study the effects of IPC onmembers of the protein transport machinery. The demonstrationthat Tom20 decreases after 90 minutes of ischemia but ispreserved in IPC provides evidence for the role of mitochondrialtransport proteins in the adaptation to ischemia and IPC.

If the effects of ischemia and IPC on Tom20 are to increaseprotein transport to the mitochondria, why did these processesnot change the levels of Tom40 and Tim23 proteins? Threepossibilities could explain this observation. First, ischemia andIPC may not result in an increase in protein transport and thechanges in Tom20 levels under these conditions may not berelated to Tom20′s protein transport activity. Second, Tom20may catalyze the rate limiting step in protein transport into themitochondria in ischemia and IPC. Finally, the levels of othermembers of the TIM and TOM complexes, which were notstudied in the current manuscript, may be altered in ischemiaand IPC and may contribute to increased protein transport to themitochondria. Further studies are needed to determine whichone of these possibilities (or others) is correct.

Although the findings by Boengler et al. are interesting andhave addressed an important question, they have also raisedmany new questions. Does Tom20 overexpression protect

cardiomyocytes in vitro and in vivo? Accordingly, would areduction in its level in the heart lead to an increase in cell deathand results in heart failure in intact animals? Are the changes inthe levels of Tom20 in ischemia and IPC due to a change intranscription, translation, protein degradation or other mechan-isms? What is the signal transduction pathway betweenmitochondria and the nucleus that leads to this phenomenon?Are other mitochondrial transport proteins (besides those thatwere studied in this paper) involved in ischemic damage andIPC? The answers to these questions may better delineate therole of protein transport machinery in normal human physiologyand pathological conditions.

In summary, the current manuscript by Boengler et al.demonstrates that ischemia is associated with a decrease inTom20, while IPC preserves the levels of this protein. Thesefindings provide, for the first time, evidence for the role of themitochondrial transport proteins in the pathogenesis of ischemicdamage in the heart and the mechanism of protection by IPC.Although the underlying mechanism for this process is notclear, it is tempting to speculate that changes in Tom20 levelsleads to the transport of certain proteins that are needed to altermitochondrial biogenesis. If true, this hypothesis would suggestthat adjustments in the mitochondrial activity in ischemia andIPC are dependent on the protein transport machinery.

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Mark BowersHossein Ardehali⁎

Division of Cardiology,Department of Medicine,

Northwestern University Medical Center, Chicago, IL, USATarry 12-725, 303 E Chicago Ave,

Chicago, IL 60611, USAE-mail address: h-ardehali @northwes tern.e du.⁎Corresponding author. Tel.: +1 312 503 2296;

Fax: +1 312 503 0137.