Cardiac proteomic responses to ischemiareperfusion ...

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Cardiac proteomic responses to ischemia–reperfusion injury and ischemic preconditioning Expert Rev. Proteomics 8(2), 241–261 (2011)

Hyoung Kyu Kim1, Vu Thi Thu1, Hye-Jin Heo1, Nari Kim1 and Jin Han†1 National Research Laboratory for Mitochondrial Signaling, Department of Physiology, College of Medicine, Cardiovascular and Metabolic Disease Center, Inje University 633-165 Gaegeum-Dong, Busanjin-Gu, Busan 613-735, Korea † Author for correspondence: Tel.: +82 518 906 727 Fax: +82 518 945 714 [email protected] 1

Cardiac ischemia and ischemia–reperfusion (I/R) injury are major contributors to morbidity and mortality worldwide. Pathological mechanisms of I/R and the physiological mechanisms of ischemic preconditioning (IPC), which is an effective cardiac protective response, have been widely investigated in the last decade to search for means to prevent or treat this disease. Proteomics is a powerful analytical tool that has provided important information to identify target proteins and understand the underlying mechanisms of I/R and IPC. Here, we review the application of proteomics to I/R injury and IPC to discover target proteins. We analyze the functional meaning of the accumulated data on hundreds of proteins using various bioinformatics applications. In addition, we review exercise-induced proteomic alterations in the heart to understand the potential cardioprotective role of exercise against I/R injury. Further developments in the proteomic field that target specialized proteins will yield new insights for optimizing therapeutic targets and developing a wide range of therapeutic agents against ischemic heart disease. Keywords : cardiac myocyte • exercise • ischemia–reperfusion injury • ischemic preconditioning • mitochondria

Ischemia–reperfusion injury

Myocardial ischemia is a heart condition caused by the cessation of blood flow supplying oxygen and nutrients to the heart. This results in a rapid drop of intracellular pH [1] , an increase in intracellular sodium (Na +) and calcium (Ca 2+) [2] , and an accumulation of lactic acid caused by increased glycolysis [1,3] . During ischemic periods, elevated reactive oxygen species (ROS) production [4] , loss of mitochondrial membrane potential (DYm) [5] , and cessation of oxidative phosphorylation (OXPHOS) [6] , with a concomitant fall in ATP [7,8] have also been reported (Figure 1) [9] . These events lead to cell death via apoptosis, autophagy and necrosis, and finally induce cardiac malfunction [4] . Restoration of blood flow to ischemic myocardium can re-establish metabolic and ionic homeostasis. However, reperfusion following prolonged ischemia can also lead to irreversible damage [10,11] . The functional recovery of the heart upon reperfusion is largely dependent on the duration of the ischemic period [9] ; the longer the period, the more likely the heart is to undergo irreversible damage [12] . Reperfusion alters ion f lux and rapidly renormalizes pH [13] , which in turn activates www.expert-reviews.com

10.1586/EPR.11.8

the Na+ –H+ exchanger and Na+ –HCO3- transporter, resulting in increased intracellular Na+ concentration. High Na+ accumulation leads to an increase in Ca 2+ influx into the cytosol via the Na + –Ca 2+ exchanger [14] and sarcolemmal L-type Ca 2+ channels [15] . The high intracellular Ca 2+ concentration not only enhances myocyte contractility but also promotes apoptosis. In reperfusion, especially during the first few minutes, a burst of ROS production can markedly alter intracellular proteins [4,16] , especially mitochondrial proteins [4,5,17] . Mitochondrial Ca 2+ overload and elevated mitochondrial ROS production can trigger the opening of the mitochondrial permeability transition pore (mPTP; Figure 2 ) [18] , which further impairs mitochondrial function [5,19,20] , subsequently leading to myocyte death via apoptosis, autophagy or necrosis (Figure 3) [9,21] . In addition, Murriel et al. showed that the translocation of PKCd to mitochondria during reperfusion plays a key role in regulating mitochondrial involvement in the signaling pathways leading to apoptosis and cell death [22] . Eventually, the heart undergoes cardiac arrhythmia [14] and irreversible postischemic contractile dysfunction [23] .

© 2011 Expert Reviews Ltd

ISSN 1478-9450

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Kim, Thu, Heo, Kim & Han

NHE inhibitors K Na

Ca

Na

Na

Ca

ATP

H Lactate

Ca VDAC

ATP

ANT

Ca

∆Ψ Ca

Glycolysis ATP F1ATPase Oligomycin

ADP

ATP

Cyp mPTP Low pH cyclosporin

Figure 1. Changes in ions and metabolites during ischemia. During ischemia, a decrease in ATP results in an increase in H +, stimulating Na + –H + and Na + –Ca2+ exchanges, which results in an increase in cytosolic Ca2+. NHE inhibitors attenuate the rise in Na + and subsequent rise in Ca2+, reducing ischemia–reperfusion injuries. Mitochondrial F1F0-ATPase, which is inhibited by oligomycin, uses ATP to generate mitochondrial membrane potential (DYm), which in turn can be used to transport Ca2+ into mitochondria. An elevation in mitochondrial Ca2+ can activate the mPTP pore, but the low pH associated with ischemia inhibits its opening. In addition to pH, cyclosporine, an mPTP inhibitor, also reduces the Ca2+ sensitivity of mPTP. ΔΨm: Mitochondrial membrane potential; ANT: Adenine nucleotide translocase; Cyp: Cyclophilin D; mPTP: Mitochondrial permeability transition pore; NHE: Na + –H + exchanger; VDAC: Voltage-dependent anion channel. Adapted from [9] with permission.

Mitochondrial dysfunction in ischemia–reperfusion injury

Owing to the abundance of mitochondria in the heart, the loss of mitochondrial function has a great impact on the heart, as ATP is needed to maintain contractile activity [12] . In addition to their central role in energy metabolism, mitochondria are involved in many cellular processes and are thought to be the main target of ischemia–reperfusion (I/R) injury [1,19] . Ischemia–reperfusion-treated cardiac mitochondria have altered structures and impaired functions [23] . In 2005, Solaini and Harris summarized the biochemical dysfunction of heart mitochondria that had undergone ischemia and reperfusion [24] . Recently, Cadenas et al. updated the pathways leading to mitochondrial reprogramming in ischemic heart disease [11] . As mentioned previously, during I/R, mitochondrial Ca 2+ overload from the mitochondrial Ca2+ uniporter and the mitochondrial Na+/Ca 2+ exchanger triggers the opening of the mPTP [25] , contributing to mitochondrial malfunction, including mitochondrial swelling and mitochondrial membrane disruption [26] . The impaired mitochondrial functions [5,19,20] include depressed respiratory chain complex activity [4,6,20,27] , decreased NADH dehydrogenase activity [28] , a lower membrane potential [5] , loss of ATP synthesis, increased ATP hydrolysis [1,9] , impaired ionic homeostasis 242

and formation of ROS [4–6,20,29] . These phenomena are all key factors that lead to irreversible damage [9] . Mitochondria are thought to be the main target of ROS generation and damage. Growing evidence indicates that most mitochondrial ROS are products of mitochondrial respiration chain complexes I [4] and III [30] . Significant mitochondrial ROS production [29] leads to extensive damage to the electron transport chain, which in turn results in further ROS generation [5] . Mitochondrial oxidative stress has been reported to suppress tricarboxylic acid (TCA) cycle enzymes, modify mitochondrial proteins [4,5] , trigger mPTP opening [13] , induce mitochondrial malfunction [20] , promote the release of proapoptotic proteins [31] and subsequently lead to cell death in the form of apoptosis or necrosis [4,32,33] . Together, these events help to explain why mitochondria are an important mediator and regulator of all forms of cell death in I/R [9] . Thus, mitochondria are a crucial target for treatments aimed at preventing cardiac damage following postischemic reperfusion. Ischemic preconditioning

Brief and repeated cycles of I/R preceding prolonged periods of ischemia, termed ischemic preconditioning (IPC), play a significant role in protecting the myocardium from subsequent sustained ischemic insult [34,35] . IPC protects the heart by preserving mitochondrial function and reducing the oxidative stress that occurs during ischemia and reperfusion. Ischemic preconditioning reduces postischemic myocardial hyperoxygenation by preserving NADH dehydrogenase and cytochome c oxidase [36] , preventing oxidative stress, decreasing infarction size and increasing tissue viability on reperfusion [17,37,38] . IPC treatment also limits the accumulation of cardiotoxic meta bolites, such as lactate, H+, NH3+ and inorganic phosphate, during ischemia [34] . Attenuated ATP depletion, reduced arrhythmia [19] and significantly improved heart contractile function have been documented in IPC samples [37] . Ischemic preconditioning also preserves tissue oxygen consumption [31,36] and prevents the specific alterations in mitochondrial structure and function that are associated with increases in Ca 2+ concentration and ROS production [31] . IPC causes a completely reversible increase in NADH during the preconditioning stimuli, and induces alteration of mitochondrial energy balance by decreasing the rate of NADH decline during prolonged ischemia and through reperfusion in comparison with the I/R-treated group [38] and prevents the release of cytochrome c into the cytosol [31] . Expert Rev. Proteomics 8(2), (2011)

Cardiac proteomic responses to ischemia–reperfusion injury & ischemic preconditioning

The protective mechanisms of IPC during the ischemic period and reperfusion involve a number of signaling pathways, including adenosine-mediated Akt phosphorylation and activation [39] , PKC activation [35,40,41] and the opening of ATP-dependent potassium channels (as discussed in the next section). Nitric oxide (NO) may also contribute to the protective role of IPC. The cardioprotective effects of NO as a trigger and mediator of IPC were reviewed by Cadenas et al. [11] . In addition, overexpression of antiapoptosis Bcl-2 in IPC could also contribute to cardioprotection by modulating energy metabolism and preventing acidification during ischemia [42] . Mitochondria: a potential target for IPC protection

A

At reperfusion: mPTP opens

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Cell death

Water and solutes Apoptosis Cytosol Outer mitochondrial membrane

VDAC

VDAC

ANT

ANT

Inter-membranous space Inner mitochondrial membrane

Mitochondrial matrix

ROS

Ruptured membrane Cytochrome c

Ca2+ Cyclophilin D

B

Mitochondria are the central target of myoIschemic preconditioning Cell survival At reperfusion: mPTP closed cardial protection measures [12] . Preserving mitochondrial function is essential to limit VDAC VDAC myocardial damage in ischemic heart disKATP channel Cytochrome c ease. The most effective mechanism of IPC protection is limiting mitochondrial ANT ANT malfunction. Halestrap et al. reviewed the important role of organelles in IPC [43] . Ischemic preconditioning releases the Cyclophilin D mPTP remains closed at 1. Reduction in matrix Ca2+ loading inactivation of redox-sensitive Krebs cycle reperfusion due to reduced 2. Improved energy production enzymes, reduces loss of mitochondrial [Ca2+] load, maintained ATP 3. Decreased release of ROS at respiration function, and prevents the levels and less oxidative stress time of reperfusion release of cytochrome c and alterations in mitochondrial structure [31] . One protective role of IPC is to reduce Figure 2. The opening of the mPTP at the time of reperfusion mediates cell death and the proposed scheme depicts the beneficial effects of ischemic the probability of mPTP opening [25] , preconditioning on mitochondria. (A) The mPTP opening allows water to enter, as the opening of these pores during the leading to rupture of the outer mitochondrial membrane and the release of cytochrome reperfusion period is thought to play a c and other proapoptotic proteins into the cytosol. mPTP is thought to be composed of major role in I/R injury. Halestrap et al. the VDAC, ANT and mitochondrial cyclophilin D. (B) Preconditioning confers beneficial summarized the signaling pathways that effects on mitochondrial function, which acts in concert to reduce the probability of mPTP opening during reperfusion, thereby mediating cellular protection. link IPC to mPTP inhibition, including ANT: Adenine nucleotide translocase; mPTP: Mitochondrial permeability transition pore; PKC, NO, cyclic GMP-dependent proROS: Reactive oxygen species; VDAC: Voltage-dependent anion channel. tein kinase, prosurvival kinase and AMPAdapted from [25] with permission. activated protein kinase [43] . In addition, connexin 43, a transmembrane protein, has been reported to contribute to the protective effects of IPC via mitochondrial Ca 2+ overload [2] , limiting ROS generation and its influence on the opening of these pores [44] . regulating mitochondrial volume. In addition to mPTP, ATP-dependent mitochondrial potassium (mitoKATP) channels are widely believed to play an Proteomic research important role in cardioprotection mechanisms. Activation Ischemia–reperfusion and IPC studies have revealed various of mitoKATP channels helps to reduce I/R injury [2,45–47] by functional and physiological responses of heart and cardiac myopreventing matrix condensation and enhancing ATP produc- cytes to I/R and IPC [9] . In addition, ‘omics’ analyses, including tion, thus limiting ischemia damage [9,43] . Furthermore, a par- genomics [48–52] and proteomics, have provided insights into the tially depolarized mitochondrial membrane potential (mild underlying molecular mechanisms of the responses. uncoupling) induced by opened mitoKATP channels is reported In cardiac I/R and IPC research, in 2001, Arrell et al. first to be involved in inhibiting the opening of mPTPs by suppressing reported the novel phosphorylation of myosin light chain 1 in www.expert-reviews.com

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electrophoresis, 1D-liquid chromatography [LC]-MS/MS and 1D-isobaric tag for rela↓ pH tive and absolute quantitation-LC MS/MS; mPTP Supplementary Table 1). In order gain a better ↑ Mito Ca ↑ Ca understanding of the integrative-systemic ↓ ATP responses of the heart to IPC/I/R, we created an IPC/I/R-heart proteome pool with ↑ ∆Ψm ↑ Cyto c the selected proteome sets, and then ana↑ Mito Ca lyzed systemic alterations within the pool. The pool consisted of 363 proteins that ↑ pH changed in abundance or underwent any ↑ mPTP Reperfusion kind of PTM (Supplementary Table 2) (tables available at: www.expert-reviews.com/doi/ ↑ Calpain ↑ Na ↑ Ca ↑ Caspase suppl/10.1586/epr.11.8). After we removed ↓ ATP overlapping protein entries, 200 non ↑ O2 ↑ ROS redundant proteins remained (Supplementary Table 3) (tables available at: www.expertreviews.com/doi/suppl/10.1586/epr.11.8). Membrane damage Different protein IDs were converted to Death and rupture HUGO Gene Nomenclature Committee official human protein symbols for systemic analysis. The systemic properties of the proteome set were analyzed using ‘Search Figure 3. Pathways leading to cell death in ischemia–reperfusion. During ischemia, as much as 50% of ATP is used via reversal of the F1F0-ATPase to maintain Tool for the Retrieval of Interacting Genes/ ΔΨm, which uptake of Ca2+. However, the low pH during ischemia inhibits mPTP Proteins’ (STRING 8.3) [72] , ‘Protein Ana opening. On reperfusion, the rapid restoration of pH causes cytosolic Na + and Ca2+ lysis Through Evolutionary Relationships’ 2+ accumulation, subsequently leading to mitochondrial Ca overload and opening of (PANTHER 7.0) classification system [73] , mPTP. The returned oxygen results in generation of ROS, a trigger of mPTP opening. The the ‘NCBI Clusters of Orthologous opening of mPTP causes cyto C release, initiating the cell death program. Furthermore, an increase in Ca2+ -activated calpain also contributes to cell death. Groups’ (COG) database [74] , ‘Cytoscape’ ΔΨm: Mitochondrial membrane potential; Cyto c: Cytochrome c; Mito: Mitochondrial; and ‘ClueGO’ [75] , ‘Kyoto Encyclopedia of mPTP: Mitochondrial permeability transition pore; ROS: Reactive oxygen species. Genes and Genomes’ (KEGG) database [76] Adapted from [9] with permission. and ‘WoLF PSORT’ [77] . rabbit cardiomyocytes that were pharmacologically preconFirst, we constructed the Ischemic Heart Interaction Network ditioned with adenosine, using 2DE and MALDI-TOF mass (IHIN), which consisted of 196 protein nodes and 985 protein– spectrometer analysis [53] . This study demonstrated an impor- protein interaction edges, using STRING 8.3 software. Each protant advantage of 2DE proteomics in enabling analysis of post- tein in the IHIN was categorized with a COG functional cluster. translational modifications (PTMs) of multiple proteins. Also The reported abundance of the protein in the total proteome pool in 2001, Vondriska et al. suggested an innovative strategy to use was assigned a node size of 1–9 (Figure 4) . In the COG category, functional proteomics to investigate PKCe signaling pathway over half (57%) of the total modified proteins in IHIN were proteins [54] . Because PKCe signaling is a key mechanism of involved in three major functional categories: energy production IPC, this review and the associated proteomic results offered and conversion (29% of total); PTM, protein turnover, chapernew insights [55] . ones (16% of total); and cytoskeletal (12%) (Figure 5A) . Subcellular During the last decade, various proteomic approaches have localization analysis using WoLF PSORT revealed that, together revealed potential alterations of some cardiac proteins in different with cytosolic proteins (37.7%), a large number of proteins in the I/R or IPC states [17,56–71] . We selected 18 different proteomic data- mitochondrial proteome (34.3%) responded to various ischemic sets from previously published studies to elucidate the proteomic stimulations (Figure 5B) . responses of the heart to IPC/I/R (Supplementary Table 1) (tables available at: www.expert-reviews.com/doi/suppl/10.1586/epr.11.8). Energy production & conversion These studies have been performed using various species (e.g., Ischemic stimulation (IS), comprising ischemia, I/R and any kind H9C2 cell line, mouse, rat, rabbit, dog and swine), conditions of ischemic precondition, most markedly modified expression level (ischemia preconditioning, adenosine and diazoxide pretreat- and PTM of proteins, which was implicated in ‘energy production ment, long and short ischemic periods, resveratrol, P38 MAPK and conversion’ (COG code C). Modified proteins in the C catinhibition, Src inhibitor and glycogen synthase kinase [GSK] egory comprised 29% of total IHIN proteins (Figure 5A) . Because inhibitor VIII) and protein identification tools (2DE-MALDI- heart tissue expends the most energy of any tissue in the human TOF, electrospray ionization [ESI]-MS/MS, 2D-difference gel body, the remarkable changes in energy production and conversion Ischemia

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COG A B C D E F G H I J M N O P Q R S T U V

Node Size

W

Node size

abundance

Abundance

1

1

9

9

Z

Figure 4. National Center for Biotechnology Information Clusters of Orthologous Groups-based functional categories of Ischemic Heart Interaction Network. The Ischemic Heart Interaction Network consists of 196 protein nodes and 985 protein interaction edges. Each node color represent the Clusters of Orthologous Group of the protein, and the node size represents detected abundance of the protein in the total proteome pool. Protein lists were acquired from 18 different proteomic datasets. (A) RNA processing and modification. (B) Chromatin structure and dynamics. (C) Energy production and conversion. (D) Cell cycle control, cell division and chromosome partitioning. (E) Amino acid transport and metabolism. (F) Nucleotide transport and metabolism. (G) Carbohydrate transport and metabolism. (H) Coenzyme transport and metabolism. (I) Lipid transport and metabolism. (J) Translation, ribosomal structure and biogenesis. (M) Cell wall/membrane/envelope biogenesis. (N) Cell motility. (O) Post-translational modification, protein turnover, chaperones. (P) Inorganic ion transport and metabolism. (Q) Secondary metabolites biosynthesis, transport and catabolism. (R) General function prediction only. (S) Function unknown. (T) Signal transduction mechanisms. (U) Intracellular trafficking, secretion and vesicular transport. (V) Defense mechanisms. (W) Extracellular structures. (Z) Cytoskeleton. COG: Clusters of Orthologous Groups.

function are serious risk factors of cardiac cell death and heart failure after IS. Normally, energy production and use is tightly regulated and balanced in the heart. However, IS leads to a decrease in energy production and an imbalance in the utilization of www.expert-reviews.com

energy sources in the heart between fatty acids (FAs) and glucose. Biologically, oxygen availability is reduced during ischemia, resulting in an increase of anaerobic glycolysis, which leads to an accumulation of intracellular lactate and protons (H+). In addition, free 245

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1.1% 1.4% 1.7% 2.2% 2.8%

0.6% 0.8% 0.3%

28.8%

3.3% 3.6%

Mitochondrial OXPHOS proteins

4.7%

5.5%

5.8% 15.8% 7.2% 11.9% Energy production and conversion Cytoskeleton General function prediction only Lipid transport and metabolism Carbohydrate transport and metabolism Amino acid transport and metabolism Secondary metabolites biosynthesis, transport and catabolism RNA processing and modification Extracellular structures Chromatin structure and dynamics Cell motility

Post-translational modification, protein turnover, chaperones Signal transduction mechanisms Inorganic ion transport and metabolism Translation, ribosomal structure and biogenesis Cell cycle control, cell division, chromosome partitioning Intracellular trafficking, secretion and vesicular transport Function unknown Cell wall/membrane/envelope biogenesis Defense mechanisms Coenzyme transport and metabolism Nucleotide transport and metabolism

1.5% 4.9% 6.4%

0.5% 37.7%

13.2% Cytosol

Mitochondria

Nucleus

Extracellular

Cytosol nucleus

Plasma membrane

Cytoskeletal

Endoplasmic reticulum

34.3%

Figure 5. Functional overview of the Ischemic Heart Interaction Network. (A) Clusters of Orthologous Groups category-based functional properties of Ischemic Heart Interaction Network (IHIN) proteins. Over half (57%) of the total modified proteins in IHIN involved in three major functional categories: energy production and conversion (29% of total); post-translational modification, protein turnover and chaperones (16%); and cytoskeletal (12%). (B) Subcellular localization of IHIN proteins. Subcellular localization analysis using WoLF PSORT revealed that together with cytosolic proteins (37.7%), a large proportion of mitochondrial proteome (34.3%) responded to various ischemic stimulations.

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FAs increase rapidly in the plasma during ischemia, and are then promptly oxidized in the reperfusion period, with accompanying oxidative damage to cardiac myocytes [78] . AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism in the normal and ischemic heart [79] . The central biological process of cardiac energy metabolism is carried out in the OXPHOS system in the mitochondrial inner membrane. Thus, the alteration of major OXPHOS proteins is responsible for modifying all cardiac energy metabolism. KEGG pathway mapping and a gene ontology (GO) enrichment assay of IHIN demonstrated that changes in OXPHOS are a remarkable feature of the IS heart. Overall, 21 OXPHOS-related proteins (15.67% of total associated genes; GO term p-value corrected with Bonferroni = 3.42 × 10-10) in OXPHOS complexes I, III, IV and V were greatly modified in various IS regimes (Table 1 & Figure 6) . Complex I

Mitochondria OXPHOS complex I, also referred to as NADH dehydrogenase, is the largest and most complicated enzyme complex in OXPHOS. It is the entry site to OXPHOS and the NADH binding site. Mutation, deficiency or disorder of any complex I subunit can cause various diseases, including neurodegenerative disease, metabolic syndrome and cardiovascular disease [80] . The IHIN revealed changes in the expression of eight key subunits of complex I (the number of times detected in IHIN in parent heses): NADH dehyd rogenase (ubiquinone) 1 a subcomplex 10 (NDUFA10; three), NADH dehydrogenase (ubiquinone) 1 a/b subcomplex 1 (NDUFAB1; once), NADH dehydrogenase (ubiquinone) Fe-S protein (NDUFS)1 (five times), NDUFS2 (two times), NDUFS3 (six times), NDUFS8 (two times), NADH dehydrogenase (ubiquinone) flavoprotein (NDUFV)1 (once), and NDUFV2 (five times ; Supplementa ry Ta ble 3) . The most frequently detected protein was NDUFS3 (six times). In one study, it was detected five times in different locations and expression patterns [62] . Expert Rev. Proteomics 8(2), (2011)


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Table 1. Energy production and conversion-related Kyoto Encyclopedia of Genes and Genomes pathways and associated proteins in the Ischemic Heart Interaction Network. GOID

GO term

Genes (n) Associated genes (%)

Term p-value

Term p-value Associated genes found in IHIN corrected with Bonferroni

KEGG:00190 Oxidative phosphorylation

21

15.671641

6.58 × 10 -12 3.42 × 10 -10

ATP5A1, ATP5B, ATP5D, ATP5H, ATP5J, COX4I1, COX5A, COX5B, COX6B1, NDUFA10, NDUFA2, NDUFAB1, NDUFS1, NDUFS2, NDUFS3, NDUFS8, NDUFV1, NDUFV2, UQCRC1, UQCRFS1, UQCRH

KEGG:00010 Glycolysis/ gluconeogenesis

14

23.4375

2.11 × 10 -11

1.10 × 10 -9

ACSS1, ALDH2, ALDH9A1, DLAT, DLD, ENO1, ENO3, GAPDH, LDHA, LDHB, PDHA1, PDHB, PGAM1, TPI1

KEGG:00071 Fatty acid metabolism

8

19.0476

6.42 × 10 -5

3.33 × 10 -3

ACAA2, ACADL, ACADVL,ACAT1, ALDH2, ALDH9A1, ECHS1, HADHA

KEGG:00020 Citrate cycle (tricarboxylic acid cycle)

12

38.70968

3.09 × 10 -12 1.61 × 10 -10

ACO2, DLAT, DLD, DLST, IDH2, IDH3A, MDH1, MDH2, OGDH, PDHA1, PDHB, SUCLA2

GO: Gene ontology; GOID: Gene ontology ID; IHIN: Ischemic Heart Interaction Network; KEGG: Kyoto Encyclopedia of Genes and Genomes.

Another study demonstrated that both adenosine and diazoxide conditioning may be effective at preserving NDUFS3 expression in rabbit cardiac myocytes [63] . IS-induced protein alteration may be attenuated by ischemic preconditioning [17,59,71] , GSK inhibitor VIII [71] , adenosine or diazoxide [63] , or Src inhibitor [69] .

IHIN detected expressional modifications of COX 1 (one), 4i1 (one), 5A (three), 5B (one) and 6B1 (four). The modifications were effectively attenuated by treatment with preconditioning and GSK inhibitor VIII [71] . Complex V

Complex III

Also known as cytochrome c oxidoreductase or cytochrome bc1 complex, this complex is a dimer, with each subunit complex containing 11 proteins. It catalyzes electron transfer from ubiquinol to cytochrome c and pumps protons across the mitochondrial inner membrane [81] . I/R injury decreases complex III activity, with loss of cardiolipin content [82] . IHIN detected changes in three out of 11 complex III subunits: ubiquinol-cytochrome c reductase core subunit 1 (UQCRC1; five), ubiquinolcytochrome c reductase iron-sulfur subunit 1 (UQCRFS1; two), and ubiquinol-cytochrome c reductase subunit 6 (UQCRH; two). The most frequently detected protein was UQCRC1, which has been observed five times in four different studies. IS-induced expression changes are effectively attenuated by treatment with PC and GSK inhibitor VIII [71] , adenosine or diazoxide [63] . Complex IV

The final protein complex in the electron transport chain is cytochrome c oxidase (COX), also known as complex IV. Mammalian complex IV has a complicated structure with 13 subunits [83] . This enzyme mediates the final reaction in the electron transport chain and transfers electrons to oxygen, while pumping protons across the membrane [84] . The degradation of the COX I, IVi and Vb subunits following I/R injury in isolated rabbit heart, which involves PKA-mediated phosphorylation, has been investigated [85] . I/R-induced loss of COX I and Va sub unit and PC-induced prevention were reported by Yu et al. [86] . www.expert-reviews.com

The expressions of ATP synthase, H + -transporting mitochondrial F1 complex and a-subunit (ATP5A1) were most frequently modified by IS (nine times in 18 studies). ATP5A1 is part of the F1 enzymatic complex that binds ADP, phosphate and ATP, and it plays a major role in the mitochondrial energyproducing mechanism [87] . ATP5A1 is also the target of other intrac ellular transducers, effectors and modulators [88] . The expression of ATP5A1 is significantly altered by various I/R treatments, with modification prevented or attenuated by IPC [59] , complement inhibition [64] and p38 MAP kinase inhibition [67] . IHIN also revealed altered levels of four other subunits of the F1–F0 complex of ATP synthase: ATP5B (three), ATP5D (three), ATP5H (five) and ATP5J (two). IS-induced changes in the expression of complex V subunit proteins were effectively attenuated by treatment with preconditioning and GSK inhibitor VIII [71] , adenosine or diazoxide [63] , and MAPK inhibition [67] . The mitochondrial OXPHOS subunit proteins are closely localized with each other and are functionally implicated. Thus, given the proteomic responses of these important OXPHOS subunits to IS, we presume that impaired electron and proton transport lead to the loss of inner-membrane potential to produce ATP and to the rapid generation of ROS in IS heart mitochondria. Glucose, FA metabolism & TCA cycle proteins

We found changes in the expression of 25 proteins implicated in three other major energy production and conversion pathways: glucose, FA metabolism and the TCA cycle, which yielded 247

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times in four different studies [17,61,62,67] . PDHB, an E1 multisubunit of the PDH NDUFS3 complex, is a mitochondrial multienzyme NDUFS1 MT-CO1 complex that catalyzes the conversion of ATP5H COX5B pyruvate to acetyl CoA and CO2 and plays UQCRC1 ATP5D NDUFS2 NDUFV1 a central role in linking glycolysis and the COX5A UQCRFS1 ATP5J TCA cycle [92] . PDH activity was regulated COX4I1 UQCRH ATP5A1 COX6B1 by the relative ratios of ATP/ADP, NADH/ NDUFA10 ATP5B NDUFS8 NAD + and acetyl-CoA/CoA via pyruvate NDUFV2 dehydrogenase kinase and pyruvate dehydrogenase phosphatase [93] . Decreased activity of the PDH complex in ischemic Complex I Complex III Complex IV Complex V heart has been reported in rats [28,94] and rabbits [95] . An increased NADH/NAD + ratio during the development of ischemia Ischemic period Ischemic precondition Src inhibition MAPK inhibition has been suggested to cause the loss of Complement C1 inhibition GSK inhibition Adenosine/diazoxide PDH activity  [94] . The expression of PDHB is mostly upregulated by IS and attenuated by PC [17] and MAPK inhibiFigure 6. Proteomic response of electron transport chain component proteins to tors [67] . Although the exact reasons for, ischemic stimulations. Modified proteins in each complex were visualized with various and physiological consequences of, PDHB effectors and interactions. Proteins were mapped on the pathway by using a Kyoto Encyclopedia of Genes and Genomes pathway-mapping algorithm. Protein node size upregulation during IS are not clear, we indicates the detected frequency in Ischemic Heart Interaction Network. Lines between assumed that the energy source shift from proteins indicate protein–protein interactions. Small colored circles around proteins FA to glucose in the ischemic heart may indicate various treatment conditions. give the signal to increase PDHB gene ATP5J, 5A1, 5B, 5D and 5H: F-type H + -transporting ATPase subunit 6, subunit a, b, d and transcription in the nucleus. In addition, subunit d; COX4I1, 5A, 5B and 6B1: Cytochrome c oxidase subunit IV, Va, Vb and VIb ; MT-CO1: Cytochrome c oxidase subunit I; NDUFA10: NADH dehydrogenase (ubiquinone) the reported proteomic alteration of two 1a subcomplex 10; NDUFAB1: NADH dehydrogenase (ubiquinone) 1a/b subcomplex 1; other PDH complex subunits, PDHA and NDUFS1, 2, 3 and 8; NADH dehydrogenase (ubiquinone) Fe-S protein 1, 2, 3 and 8; DLAT, suggested that the PDH complex is NDUFV1 and 2: NADH dehydrogenase (ubiquinone) flavoprotein 1 and 2; vulnerable to I/R injury. Thus, protection UQCRC1: Ubiquinol-cytochrome c reductase core subunit 1; UQCRFS1: Ubiquinolof PDH activity during the I/R period is an cytochrome c reductase iron-sulfur subunit; UQCRH: Ubiquinol-cytochrome c reductase subunit 6. important clinical target to diminish I/R heart injury [28] . 14, 8 and 11 altered proteins, respectively. Five proteins were The IHIN detected the second most common protein, ALDH2, included in more than two pathways: pyruvate dehydrogenase three times. ALDH2 is a therapeutic target that protects the heart (PDH) E1 component subunit-a (PDHA), dihydrolipoamide against ischemic injury [96,97] . Chen et al. demonstrated that S-acetyltransferase (DLAT), PDH E1 component subunit-b ALDH2 activation via the small-molecular activator Alda-1 suc(PDHB), dihydrolipoamide dehydrogenase (DLD) and aldehyde cessfully protects the heart against ischemic injury. A subsequent dehydrogenase, mitochondrial precursor (ALDH2). study suggested that the protective effect of ALDH2 may be derived from the detoxification of aldehyde and differential reguGlucose metabolism lation of autophagy via AMPK– and Akt–mTOR signaling during Glucose metabolism appears to be an effective target in lessen- ischemia and reperfusion [97] . I/R decreased the expression level of ing the severity of ischemic injury [89–91] . Although many experi ALDH2 [68] whereas GSK inhibition increased it [71] . Diazoxide mental studies have addressed the changes in cellular metabolism treatment decreased the ALDH2 level [63] . that occur during and subsequent to ischemia [90,91] , little is known about the glucose metabolism-related proteomic response FA metabolism to ischemia. When oxygen supplementation is limited during ischemia, FA The IHIN detected changes in the expression of 14 glycoly- metabolism, more specifically FA b-oxidation, decreases, and sis- and gluconeogenesis-related proteins (23.4% of total asso- oxidative phosphorylation for ATP production is impaired. As ciated genes; p = 1.10 × 10 -9) (Table 1) , l-lactate dehydrogenase a result, cellular free FAs accumulate and are then rapidly oxiA and B, ALDH2, 4-trimethylaminobutyraldehyde dehydro dized by the restoration of oxygen supply during the reperfusion genase (ALDH9A1), glyceraldehyde 3-phosphate dehydro period. A rapid increase of FA b-oxidation produces an excess genase, PDHA, PDHB, DLD, DLAT, enolase 1 and 3, triose- of acetyl CoA residues, saturating the TCA cycle and leading phosphate isomerase, phosphoglycerate mutase and acetyl-CoA to a rise in the mitochondrial acetyl CoA/CoASH ratio. This synthetase (Figure 7A) . PDHB was detected most frequently: five increase of acetyl CoA activates the PDH complex kinase, which NDUFAB1

248



Glucose metabolism

Review

Fatty acid metabolism

TPI1

GAPDH ACADVL PGAM1 ECHS1 ENO1 DLAT

PDHA1

ENO3

PDHB

ACAT1

ACADL LDHA

LDHB ACAA2

HADHA

ACSS1 DLD

ALDH2

ALDH9A1

ALDH2

ALDH9A1

TCA cycle

PDHB PDHA1 DLAT

ACO2 IDH3A

MDH2

IDH2 MDH1

DLST SUCLA2

OGDH DLD

Ischemic period

Ischemic precondition

Src inhibition

GSK inhibition

Adenosine/diazoxide

MAPK inhibition

Figure 7. Proteomic response of glucose, fatty acid and tricarboxylic acid cycle metabolism component proteins to ischemic stimulations. Modified proteins in (A) glucose metabolism, (B) fatty acid metabolism and (C) the TCA cycle were visualized with various effectors and interactions. Proteins were mapped on the pathway by using a Kyoto Encyclopedia of Genes and Genomes pathway mapping algorithm. Protein node size indicates the detected frequency in the Ischemic Heart Interaction Network. Lines between proteins indicate protein–protein interactions. Small colored circles around proteins indicate various treatment conditions. ACAA2: 3-Ketoacyl-CoA thiolase, mitochondrial; ACADL: Long-chain-acyl-CoA dehydrogenase; ACADVL: Very long chain acyl-CoA dehydrogenase; ACAT1: Acetyl CoA acetyltransferase, mitochondrial precursor; ACO2: Aconitate hydratase, mitochondrial precursor; ACSS1: Acetyl CoA synthetase 1; ALDH2: Aldehyde dehydrogenase, mitochondrial precursor; ALDH9A1: 4-Trimethylaminobutyraldehyde dehydrogenase; DLAT: Pyruvate dehydrogenase E2 component (dihydrolipoamide acetyltransferase); DLD: Dihydrolipoamide dehydrogenase; DLST: 2-oxoglutarate dehydrogenase E2 component (dihydrolipoamide succinyltransferase); ECHS1: Enoyl-CoA hydratase 1; ENO: Enolase; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; GSK: Glycogen synthase kinase; HADHA: Hydroxyacyl-CoA dehydrogenase/3ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), a subunit; IDH2: Isocitrate dehydrogenase [NADP], mitochondrial precursor; IDH3A: Isocitrate dehydrogenase [NAD] subunit, mitochondrial precursor; LDHA: l-lactate dehydrogenase; MDH: Malate dehydrogenase; OGDH: 2-oxoglutarate dehydrogenase E1 component; PDH: Pyruvate dehydrogenase; PGAM1: Phosphoglycerate mutase; SUCLA2: Succinyl-CoA synthetase b subunit; TCA: Tricarboxylic acid; TPI: Triosephosphate isomerase.

www.expert-reviews.com

249

Review


Table 2. Cytoskeleton-related Kyoto Encyclopedia of Genes and Genomes pathways and associated proteins in the Ischemic Heart Interaction Network. GOID

GO term

Genes (n)

Associated genes (%)

Term p-value

Term p-value Associated genes found corrected with in IHIN Bonferroni

KEGG:04260

Cardiac muscle contraction

17

21.25

4.89 × 10 -12

2.54 × 10 -10

ACTC1, ATP1B3, COX4I1, COX5A, COX5B, COX6B1, MYH6, MYH7, MYL2, MYL3, RYR2, TNNI3, TNNT2, TPM1, UQCRC1, UQCRFS1, UQCRH

KEGG:05410

Hypertrophic cardiomyopathy

12

14.117647

9.21 × 10 -7

4.79 × 10 -5

ACTC1, DMD, LMNA, MYBPC3, MYH6, MYH7, MYL2, MYL3, RYR2, TNNI3, TNNT2, TPM1

KEGG:05414

Dilated cardiomyopathy

12

13.043478

2.19 × 10 -6

1.14 × 10 -4

ACTC1, DMD, LMNA, MYBPC3, MYH6, MYH7, MYL2, MYL3, RYR2, TNNI3, TNNT2, TPM1

KEGG:05412

Arrhythmogenic right ventricular cardiomyopathy

8

10.526316

5.27 × 10 -4

0.027411037

ACTN2, ACTN3, CTNNA1, CTNNB1, DMD, JUP, LMNA, RYR2

KEGG:04810

Regulation of 9 actin cytoskeleton

4.1666665

0.113277497

1

ACTN2, ACTN3, BCAR1, EZR, MAP2K1, MYL2, MYL9, PDGFRB, PTK2

GO: Gene ontology; GOID: Gene ontology ID; IHIN: Ischemic Heart Interaction Network; KEGG: Kyoto Encyclopedia of Genes and Genomes.

phosphorylates and inactivates PDH, disabling the uptake of pyruvate into the mitochondrion and preventing effective oxidation of glycolysis products. At this point, the lack of the energy and lactate accumulation results in cessation of contraction, which is called cardiac ‘stunning’ [98] . The IHIN detected changes in the expression of eight FA metabolism-related proteins (19% of total associated genes, p = 0.0033) (Table 1) : acetyl CoA acyltransferase 2, long-chainacyl CoA dehydrogenase (ACADL), very-long-chain-acyl CoA dehydrogenase, acetyl CoA acetylt ransferase, mitochondrial (ACAT1), ALDH2, ALDH9A1, enoyl CoA hydratase, and hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoylCoA hydratase (trifunctional protein), a subunit (HADHA) (Figure 7B) . Matched with the biological response of the rapid increase in FA b-oxidation, the increased expression of ACADL after I/R and IPC considerably attenuated protein alteration [17,71] . Interestingly, IPC treatment downregulated the levels of ACADL, very-long-chain-acyl CoA dehydrogenase and HADHA, compared with I/R, whereas GSK inhibition upregulated these and two additional proteins, acetyl CoA acyltransferase 2 and enoyl CoA hydratase [71] . GSK inhibition has been suggested to increase the protein stability of the mitochondrial proteome, similar to its modulation of protein stability in the Wnt pathway and its downstream target, b-catenin [71,99,100] . Logically, IPC-induced reduction of FA b-oxidation-implicated proteins delays rapid oxidation during the reperfusion period, probably giving the ischemic heart time to gradually recover functionality with minimum oxidative stress. TCA cycle

Owing to the imbalanced energy substrate and metabolic shift, decreased glycolysis and increased FA b-oxidation are typical 250

outcomes of a cardiac I/R period. During ischemia, decreased acetyl CoA supplements are coupled with depressed TCA cycle activity, which gradually recovers during the reperfusion period [101] . Increased hydrogen peroxide (H 2O2 )-induced oxidative stress impairs aconitase activity, resulting in suppressed TCA cycle activity during the I/R period [102,103] . The IHIN revealed changes in the expression of 12 TCA cycle metabolism-related proteins (38.7% of total associated genes; p = 3.09 × 10 -12 ) (Table 1) : aconitate hydratase, DLAT, DLD, 2-oxoglutarate dehydrogenase E2 component, isocitrate dehydrogenase 2 and 3A (IDH2 and IDH3A), malate dehydrogenase 1 and 2 (MDH1 and MDH2), 2-oxoglutarate dehydrogenase, PDHA1, PDHB and ADP-specific succinyl-CoA ligase b-chain (Figure 7C) . Among these, changes in IDH3A and MDH2 were most frequently reported (four times). IS and I/R treatment decreased the levels of IDH3A [68] and MDH2 [17] , which are enzymes directly involved in NADH production in the TCA cycle. In addition, changes in the protein levels of all three components of the oxoglutarate dehydrogenase complex, NADH production enzyme complex, 2-oxoglutarate dehydrogenase [17] , DLD [62] and 2-oxoglutarate dehydrogenase E2 component [71] were detected in separate studies. Considering that NADH is both a major product and regulator of the TCA cycle, as well as a key substrate for OXPHOS ATP production, these proteomic responses result in impaired NADH production, leading to impaired energy metabolism during a prolonged post-I/R period [103] . Various pretreatments, including PC [17,59] , GSK inhibition [71] and adenosine/ diazoxide [63] , effectively compensated for the changes in protein level (Figure 7C) . Aconitate hydratase is the enzyme most vulnerable to oxidative stress during I/R [102,103] , and GSK inhibition increased its level, compared with the I/R level [71] . Expert Rev. Proteomics 8(2), (2011)


Review

Table 3. Cardiac proteomic alteration in response to various exercise training. Protein identification

Symbol

IPI

Exe/Con COG

Exercise condition

Ref.

Hsp20

HSPB6

IPI00205489.1

Up

O

[128]

Dihydrolipoamide S-succinyltransferase

DLST

IPI00551702.2

Up

C

Glyceraldehyde 3-phosphate dehydrogenase

GAPDH

IPI00554039.1

Up

G

ATP synthase D-chain

ATP5H

IPI00200053.3

Down

C

Triosephosphate isomerase 1

TPI1

IPI00231767.5

Up

G

Chronic exercise: 6 weeks, 5 days/week, 10– 60 min duration, 15–66 m/min speed increase

a-cardiac myosin heavy chain

MYH6

IPI00189809.2

Up

Z

[129]

a-cardiac actin

ACTC1

IIPI00194087.3

Up

Z

Nebulette

NEBL

IPI00364938.3

Down

Z

Heart fatty acid-binding protein

FABP3

IPI00231971.5

Up

I

Long-chain acyl-CoA dehydrogenase

ACADL

IPI00211225.1

Up

I

Endurance exercise: 6 weeks, 4 days/week, speed and incline adjust to 70–75% VO2 peak for 30 min

Short-chain acyl-CoA dehydrogenase

ACADS

IPI00231359.3

Up

I

Acyl-CoA thioesterase

ACOT2

IPI00421885.3

Up

C

Malate dehydrogenase, cytoplasmic

MDH1

IPI00198717.8

Up

C

Aspartate aminotransferase

GOT2

IPI00210920.1

Up

E

Electron transfer flavoprotein subunit-a

ETFA

IPI00205332.4

Down

C

NADH dehydrogenase (ubiquinone) flavoprotein 1

NDUFV1

IPI00191913.1

Up

T

NADH dehydrogenase (ubiquinone) a subcomplex

NDUFA1

IPI00769025.1

Up

C

Creatine kinase M-type

CKM

IPI00211053.6

Down

C

Creatine kinase basic-type

CKMT2

IPI00188313.1

Down

C

Aconitase

ACO2

IPI00421539.3

Down

C

Heat-shock 20-kDa-like protein p20

HSPB6

IPI00205489.1

Up

O

Four and a half Lim domains protein 2

FHL2

IPI00206193.1

Up

T

26S proteosome non-ATPase regulatory subunit 1

PSMD1

IPI00212512.1

Up

O

a-2-HS-glycoprotein precursor (fetuin-A)

AHSG

IPI00327469.1

Down

U

ATP synthase O subunit, mitochondrial precusor

ATP5O

IPI00195123.1

Down

C

[130]

Citrate synthase mitochondrial precusor

CS

IPI00206977.1

Up

C

Malate dehydrogenase, mitochondrial precusor

MDH2

IPI00197696.2

Up

C

Endurance exercise: 5 days, 60 min/day, 30 m/min, ~70% of VO2 peak

Long-chain specific acyl-CoA dehydrogenase, mitochondrial precursor

ACADL

IPI00211225.1

Down

I

d(3,5)-d(2,4)-dienoyl-CoA isomerase, mitochondrial precusor

ECH1

IPI00326561.3

Down

I

Trifunctional enzyme subunit-a, mitochondrial precursor

HADHA

IPI00212622.1

Up

I

Hydroxyacyl-CoA dehydrogenase, mitochondrial precusor

HADHSC IPI00205157.1

Down

I

Methylmalonate-semialdehyde dehydrogenase mitochondrial precusor

ALDH6A1 IPI00205018.2

Up

E

Aspartate aminotransferase, mitochondrial precusor

GOT2

IPI00210920.1

Up

E

Coiled-coil-helix-coiled-coil-helix domain-containing 3 CHCHD3

IPI00869949.2

Down

L

Amine oxidase (flavin-containing) A

IPI00202370.3

Down

Q

MAOA

COG: Clusters of Orthologous Groups; Exe/Con: Expressional changes of protein in exercise compared with control; Hsp: Heat-shock protein; IPI: International protein index; VO2: Volume of oxygen.


251

Review


Table 3. Cardiac proteomic alteration in response to various exercise training. Protein identification

Symbol

IPI

Exe/Con COG

Exercise condition

Enoyl-CoA hydratase

ECH1

IPI00326561.3

Down

I

Short-chain acyl-CoA dehydrogenase

ACADS

IPI00231359.3

Up

I

Triosephosphate isomerase

TPI1

IPI00231767.5

Down

G

Electron transfer flavoprotein subunit-b

ETFB

IPI00364321.3

Up

C

Malate dehydrogenase

MDH1

IPI00198717.8

Up

C

Short-chain specific acyl-CoA dehydrogenase

ACADS

IPI00231359.3

Up

I

Ndufa10 protein

NDUFA10 IPI00421549.3

Up

T

High-intensity swimming – 8 weeks, 6 days/week, twice/day, increment of duration 30 min/week until 120 min. Load increase 1% of bodyweight in week 5, 1.5% at week 6, and 2% at weeks 7 and 8

Triosephosphate isomerase

TPI1

IPI00231767.5

Up

G

ATP synthase subunit-a

ATP5A1

IPI00396910.1

Up

C

Isocitrate dehydrogenase (NAD) subunit-a

IDH3A

IPI00198720.1

Up

E

Myosin light polypeptide 3

MYL3

IPI00231788.5

Down

Z

A kinase (PRKA) anchor protein 3

AKAP3

IPI00201274.1

Down

O

Stress 70 protein

HSPA9

IPI00363265.3

Down

O

Glutathione peroxidase 1

GPX1

IPI00192301.2

Up

O

Hydroxysteroid dehydrogenase-like 2

HSDL2

IPI00367240.3

Down

I

Prohibitin

PHB

IPI00211756.1

Up

O

Aldehyde reductase

AKR1B1

IPI00231737.5

Down

R

Antioxidant protein 2 – peroxiredoxin-6

PRDX6

IPI00231260.5

Up

O

Ref. [131]

COG: Clusters of Orthologous Groups; Exe/Con: Expressional changes of protein in exercise compared with control; Hsp: Heat-shock protein; IPI: International protein index; VO2: Volume of oxygen.

Cytosolic & mitochondrial creatine kinase

The creatine kinase/phosphocreatine (CK/PCr) system plays a complex role in heart energy metabolism, including energy storage and supply, depending on the energetic condition of the heart. The CK/PCr system may play important roles in myocardial ischemia and reperfusion. Suppression of mitochondrial CK activity by ischemia has been reported, with a close correlation between mitochondrial CK loss and functional depression in isolated rabbit heart after global I/R [104] . Moreover, the functional uncoupling between the ADP/ATP carrier and mitochondrial CK was suggested as a possible cause of cardiac failure during the early ischemic period in isolated guinea pig heart [105] . Similarly, a recent study has demonstrated that inhibition of CK activity impairs myocardium contractility recovery after I/R in rabbit [106] . The IHIN showed changes in the protein levels of three CK isoforms: CKB, CKM and sarcomeric mitochondrial CK (CKMT2). In line with previous CK activity studies, I/R treatment decreased CKM [60,62,64,67] and CKMT2 [62] protein levels. Moreover, GSK inhibition [71] and complement C1 inhibition [64] effectively prevented protein loss. However, the CKB level increased after both short-term (15 min) and long-term (60 min) treatments of I/R [62] . Although the concentration of creatine kinase (CK)-MB enzyme released in serum is widely used as a biomarker of cardiac infraction, the physiological consequences of elevated CKB in cardiac tissue after I/R are unknown. Nevertheless, these proteomic alterations indicate 252

that CK/PCr-mediated energy storage and conversion is also impaired during and after IS. PTM, protein turnover & chaperones

Of the COG functional categories, PTM, protein turnover and chaperones (COG code ‘O’) was the second most markedly involved in IS (Figure 5) , with modified O-category proteins comprising 16% of the IHIN total. After proteins are translated from mRNA, they are manipulated by complex regulatory mechanisms to acquire specific functions. Regulation of protein folding and removal of abnormal proteins are essential for cell survival and cellular repair. PTM and protein folding are regulated by specialized functional proteins, termed chaperones, which include heat-shock proteins (HSPs) [107] . Defective or dysfunctional chaperones are involved in various human diseases and conditions, including diabetes, myocardial infarction, neurodegenerative disease and aging [108,109] . Defense mechanisms based on the expression, activation or redistribution of molecular chaperone proteins counteract a fundamental mechanism of ischemic injury, the unfolding, misfolding or pathological modification (e.g., peroxidation) of critical proteins in various intracellular compartments [110] . HSPs play an important role in the defense mechanism against I/R injury. The major inducible stress protein HSP70 protects directly against myocardial ischemic damage, improves metabolic recovery, enhances functional recovery and reduces infarct size in transgenic mouse hearts [111] . A previous study extensively investigated Expert Rev. Proteomics 8(2), (2011)


the protective functions of inducible HSP70, mitochondrial HSPs 60 and 10, and small HSPs 27 and aB-crystallin against ischemic reoxygenation-mediated injury, using transgenic animals and isolated cardiac myocyte-derived cells [112] . Likewise, reversible damage after simulated or myocardial ischemia can be diminished by the overexpression of the small HSPs aB-crystallin [113,114] and HSP27 [115] . In these responses, heat-shock transcription factor (HSF)1 was suggested as the primary mediator, in association with three other HSFs (HSFs 2–4) [116] .

Review

The IHIN detected changes in the expression of 24 PTM regulation, protein turnover and chaperone-related proteins (Supplementary Table 3 & Figure 8) . Among these, the most frequently detected were aB-crystallin (CRYAB) and HSP27, at seven times each. The expression level of CRYAB increased in a relatively short period (15 min) of ischemia and in the reperfused heart [62] but decreased in I/R models of more than 30 min–1 h [57,62,64,65,67] . Complement inhibitors [57,64] and resveratrol [65] effectively attenuated the loss of CRYAB protein in I/R, whereas MAPK inhibition had no effect in

PSMB3

PRDX1 PRDX6

CRYAB PRDX2 PSMB4

PDIA3 TCP1

PHB HSPA9

PSMB5

HSPA4 HSPB1

HSPD1 HSPA8 DNAJC7

PRDX3 GSTM5 HSPB6 HSPA2

GSTM1 UBLCP1

UCHL3

TRIM72

Ischemic period

Ischemic precondition

Src inhibition

Complement C1 inhibition

GSK inhibition

Adenosine/diazoxide

Resveratrol

MAPK inhibition

Complement inhibition (FUT-175)

Figure 8. Proteomic response of post-translational modification, protein turnover and chaperones-implicated proteins to ischemic stimulations. Proteins were mapped on the pathway by using a Kyoto Encyclopedia of Genes and Genomes pathway mapping algorithm. Protein node size indicates the detected frequency in the Ischemic Heart Interaction Network. Lines between proteins indicate protein–protein interactions. Small colored circles around the proteins indicate various treatment conditions. CRYAB: aB-crystallin; DNAJC7: DNAJ homolog subfamily C member 7; GSTM: Glutathione S-transferase µ; HSP: Heat-shock protein; PDIA3: Protein disulfide isomerase A3 precursor (ERP60); PHB: Prohibitin 1; PRDX: Peroxiredoxin; PSMB: Proteasome subunit b; TCP1: T-complex protein 1; TRIM72: Tripartite motif-containing 72 LOC365377; UBLCP1: Ubiquitin-like domain-containing CTD phosphatase-1; UCHL3P: Ubiquitin carboxyl-terminal hydrolase isozyme L3.


253

Review


TUBA8

TUBB4 KIF5C

KIF5A MYLC2B PFN1 TPM1

OBSCN

TNNT2 MYL2

MYH6

MYL9

ACTA2

MYL3

CAPZB

(proteasome subunit b type 3, 4 and 5, ubiquitin-like domain-containing CTD phosphatase-1, ubiquitin carboxyl-terminal hydrolase isozyme L3). Supplementary Table 2 lists the detailed conditions and expression patterns of the proteins. The dynamic changes in the expression of these PTM, turnover and chaperone proteins reflect the active response of cardiomyocytes in reducing oxidative stress and repairing damage to prevent cell death. Various preconditioning measures could be helpful in triggering the molecular defense mechanism earlier and boosting recovery mechanisms after I/R injury.

ACTC1

MYBPC3

Cytoskeleton ACTN3

In addition to energy metabolism, and PTM, protein turnover, and chaperones, expresNRAP sion levels of cardiac cytoskeleton proteins (COG code ‘Z’) were widely modified in IS MYH7 CTTN (Figure 9) , comprising 12% of modified proteins in IHIN. The intracellular structure of cardiomyocytes is essential in maintaining BCAR1 its intrinsic function of supplying blood to the whole body. Electron microscope studIschemic period Ischemic precondition Src inhibition ies of ultrastructural change in the ischemic heart have revealed various structural modiComplement C1 inhibition Adenosine/diazoxide MAPK inhibition H2O2 fications of T tubules, nuclei, mitochondria and myofilaments [117,118] . Disruption of plasma membrane integrity, with loss of Figure 9. Proteomic response of cytoskeletal component proteins to ischemic vinculin, during myocardial ischemia has stimulations. Proteins were mapped on the pathway by using a Kyoto Encyclopedia of Genes and Genomes pathway mapping algorithm. Protein node size indicates the been reported [119] . Various contractile and detected frequency in Ischemic Heart Interaction Network. Lines between proteins cytoskeletal proteins, myosin, actin, tropoindicate protein–protein interactions. Small colored circles around proteins indicate myosin, troponin T, myomesin, desmin, various treatment conditions. tubulin and vinculin have been shown to ACTA2: Actin, aortic smooth muscle; ACTC1: a-actin, cardiac (mouse); ACTN: a-actinin; be altered during myocardial ischemia, BCAR1: p130CAS/breast cancer anti-estrogen resistance protein-1; CAPZB: F-actin capping protein, b1-subunit; CTTN: Cortactin-Src substrate/CTTN; KIF: Kinesin heavywith damage to contractile proteins occurchain isoform; MYBPC3: Myosin-binding protein C, cardiac; MYH: Myosin heavy chain; ring sooner than that to the cytoskeleton MYL1: Myosin light chain; MYL2: Myosin light chain 2; MYL3: Ventricular myosin light and subcellular organelles [120] . The loss of chain 1; MYL9: Myosin regulatory light polypeptide 9; MYLC2B: Myosin regulatory light contractile and cytoskeletal proteins was chain RLC-A; NRAP: Nebulin-related anchoring protein isoform C; OBSCN: Similar to suggested to be due to a Ca 2+ -activated proobscurin, cytoskeletal calmodulin and titin-interacting RhoGEF; PFN1: Profilin-1; TNNT2: Troponin T cardiac muscle; TPM1: Tropomyosin 1 a-chain (a-tropomyosin); tein degradation process during myocardial TUBA8: Similar to tubulin s-8 chain; TUBB5: Tubulin-b5. ischemia [121] . The Ischemic Heart Interaction Network preventing its loss [67] . HSP27 expression levels increased in both showed changes in the expression of 24 cytoskeletal proteins (see short- and long-period I/R [61,62] but were decreased by H2O2- Supplementary Table 3 & Figure 9 ). The modification of cardiac muscle induced oxidative stress. SRC inhibition attenuated the decrease [69] . troponin T (TNNT2) was most remarkable, being reported nine In addition, IHIN detected changes in the expression of seven HSP times. Clinically, TNNT2 is widely used as a biomarker to detect proteins (DNAJC7, HSPA2, HSPA4, HSPA8, HSPA9, HSPB6 and cardiac infarction, along with several other markers, including HSPD1), two other chaperones (tripartite motif-containing 72 myoglobin and the MB fraction of creatine kinase [121] . As a caland t-complex protein 1), eight antioxidative proteins (glutathione cium-dependent, tropomyosin-binding subunit protein, TNNT2 S-transferase µ family proteins [GSTM1 and 5], peroxiredoxin regulates cardiac muscle contraction. Thus, genetic mutations members [PRDXZ1, 2, 3 and 6], prohibitin, protein disulfide iso- of, or defects in, this protein can cause severe cardiac contractilmerase A3 precursor), and five protein turnover-regulated proteins ity dysfunction, such as idiopathic dilated cardiomyopathy [121] . ACTN2

254

MYL1



Interestingly, smooth muscle a-actin 2 (ACTA2) was also frequently modified by ischemic stimulation, suggesting that IS causes proteomic modification of coronary artery smooth muscle cells as well as cardiomyocytes. Because ACTA2 mutations cause various cardiovascular diseases that lead to cardiac infarction and heart failure, IS-induced alteration of ACTA2 could be another serious risk factor for ischemic cardiac damage [80] . In the KEGG pathway database, changes in the expression of various actin–myosin filament component proteins and their regulatory proteins have also been implicated in various cardiac disease pathways, including hypertrophic cardiomyopathy, dilated cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy (Table 2) . Thus, IS-induced proteomic alterations in the cardiac cytoskeleton may further lead to secondary cardiomyopathies in the post-I/R period. Cardiac proteomic response to exercise: strengthening the innate cardioprotective mechanism

Review

evaluated in several recent studies [128–131] , from which we extracted 54 proteins whose expression changed. From these, we created the Exercised Heart Interaction Network (EHIN), using STRING 8.0 and Cytoscape (version 2.7.0). EHIN consisted of 44 nonredundant protein nodes and 91 interaction edges. It included 25 upregulated, 17 downregulated and two expression-ambiguous proteins (Table 3 & Figure 11A) . As in IHIN, the COG category of ‘energy production and conversion’-related proteins were most frequently modified (15 proteins, 28% of total) by exercise treatments. Interestingly, the second most frequently detected proteins were those involved in lipid transport and metabolism (11 proteins, 20% of total) (F igure 11B) . Under physiological conditions, the heart prefers to use FA b-oxidation rather than glycolysis to produce ATP. Thus, tight regulation of lipid metabolism in the heart is essential. Genetic defects of any b-oxidation enzymes in mitochondria may lead to heart dysfunction and failure. Although exercise training significantly reduced plasma-free FAs, triglyceride concentration, total cholesterol and high-density lipoproteins, data on exercise-induced lipid metabolism alteration in the heart are scarce. The EHIN showed that ACADL was upregulated [129] or downregulated [130] whereas short-chain acyl-CoA dehydrogenases were upregulated in different exercise studies [129,131] . Enoyl-CoA hydratases were downregulated after endurance exercise [130] and high-intensity swimming [131] . EHIN also showed upregulation of heart FA-binding protein [129] , HADHA [130] , and downregulation of hydroxyacyl-CoA dehydrogenase, mitochondrial precursor [130] and hydroxysteroid dehydrogenase-like 2 [131] . However, whether these proteomic alterations can lead to enhanced lipid metabolism in the exercise-trained heart or if altered lipid metabolism has a protective effect against ischemic damage remain to be determined.

The increase in ischemic heart disease as a major cause of death worldwide has resulted in a great deal of research on reducing ischemic and I/R injury of the heart. Most studies have sought to understand and mimic the mechanism of ischemic pre conditioning, the powerful endogenous cardioprotective mechanism. For example, PI3K, AKT, p70S6K, mTOR, GSK, endothelial nitric oxide synthase/NO, PKC-e, ERK, p38MAPK and JNK are target molecules of IPC [9] . Other studies have focused on the relationship between heart mitochondrial function and chronic exercise [122] . Several studies have suggested that endurance exercise training protects cardiomyocytes against I/R-induced oxidative stress [123,124] via increased endothelial nitric oxide synthase myocardial content [125] , improves myocardial contractile function and effectively prevents impaired coronary perfusion after an I/R episode in senescent rats [126] . In addition, exercise-induced HSP overexpression also participates in myocardial protection against unfavorable Regular exercise training consequences that result from other modes of cardiac damage and dysf unction [122] . Importantly, endurance exercise training Coronary ER stress Cardiac Myocardial also protects mitochondria against I/Rcirculation proteins HSPs antioxidants induced damage [122,127] . In a recent review, Powers et al. extensively discussed various potentially exercise-induced mechanisms Mito KATP Sarco KATP COX-2 activity channels channels that protect the heart against I/R injury, including enhanced coronary circulation, elevated myocardial HSPs, increased myocardial cyclooxygenase-2 activity, elevated endoplasmic reticulum stress proteins, enhanced function of sarcolemmal ATPsensitive potassium channels, elevated levels Cardioprotection against I/R injury of mitoKATP channels and increased myocardial antioxidant capacity (Figure 10) [127] . Figure 10. Previously suggested numerous exercise-induced In addition to physiological and molecucardioprotective mechanisms. lar-based analysis of the beneficial effect of COX: Cyclooxygenase; ER: Endoplasmic reticulum; HSP: Heat-shock protein; I/R: Ischemia–reperfusion; Mito: Mitochondrial; Sarco: Sarcolemmal. exercise on cardiac protection, cardiac proAdapted from [127] with permission. teomic responses to various exercises were www.expert-reviews.com

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chaperones were the third most frequently detected changes in EHIN (eight proteins, 15% of total), including increased levels of HSP20, prohibitin, peroxiredoxin 6 and glutathione peroxidase 1. The fourth most detected exercisealtered expression was of heart cytoskeletal proteins (five proteins, 9% of total). Except for lipid metabolism, the major altered functional categories and their orders of dominance were the same in IHIN and EHIN. This suggests that the exerciseinduced proteomic response may attenuate I/R injury to the heart by strengthening innate cardioprotective mechanisms.

PHB AKR181 HSPA9 PRDX6 ATP5H GPX1

ATP5O

MAOA HSPB6

TPI1

ATP5A1

NDUFV1

ACTC1

MDH2 GAPDH

MDH1 DLST ACO2

MYH6

CS

GOT2

CKMT2

MYL3

CKM

FHL2 IDH3A NDUFA10

FABP3

ACADL ECH1 HADHA

Up

Expert commentary & five-year view

HADH ETFA

Cardiac ischemia and I/R injury contribute to morbidity and mortality. In order to ACADS ETFB understand the pathophysiological mechaAmbiguous nisms underlying I/R injury and to prevent ALDH6A1 critical heart damage, various studies have been underway in recent decades. Proteomic analysis is a powerful tool analyzing celluACOT2 PSMD1 AKAP3 HSDL2 AHSG CHCHD3 NEBL lar processes based on analysis of multiple alterations of proteins rather than evaluating a single protein at a time. Moreover, proteins rarely act alone but rather interact Energy production and conversion 2% 2% with other proteins and comprise specific 2%2% Lipid transport and metabolism 6% pathways responsible for various biologi28% Post-translational modification, cal processes [132] . Therefore, proteomic protein turnover, chaperones 7% analysis is capable of providing a broadCytoskeleton based portrait of the dynamic changes in Amino acid transport and metabolism the cardiac proteome during ischemia and 7% Carbohydrate transport and metabolism reperfusion, and offers an effective means of Signal transduction mechanisms identifying protein targets that are relevant Replication, recombination and repair to disease [133] . Various proteomic analyses 9% Secondary metabolites biosynthesis, have been applied to this disease to iden20% transport and catabolism tify target proteins that result in cardiac 15% General function prediction only damage during and after an I/R period. Intracellular trafficking, secretion Furthermore, proteomic approaches in a and vesicular transport large number of pharmacologically and physiologically preconditioned hearts have Figure 11. Exercise-induced cardiac proteome alteration. (A) The Exercised Heart revealed target proteins that have protecInteraction Network (EHIN) consists of 44 protein nodes, which were extracted from four tive effects against I/R injury (Supplementary representative sets of proteomic studies. A total of 25 upregulated, 17 downregulated Table 2) . However, focusing on the long list of and two expression-ambiguous proteins were detected in EHIN. (B) Clusters of differentially expressed proteins is challengOrthologous Groups category-based functional properties of EHIN proteins. Over half (63%) of total modified proteins in EHIN are involved in three major functional ing to elucidate the molecular mechanisms categories: energy production and conversion (28% of total); lipid transport and of cellular processes [134] . Since the relationmetabolism (20%); and post-translational modification, protein turnover and chaperones ship between genotype and phenotype is (15%). Impressively, proteins implicated in lipid transport and metabolism were highly too complex to be ascribed to a change of an ranked in EHIN. individual protein, a comprehensive dataOne of the most predominant effects of exercise training was integrative approach is needed to interpret the interrelationships an increase in antioxidant and chaperone proteins [122] . Likewise, between proteins. Data-integrative approaches have been successchanges in proteins involved in PTM, protein turnover and fully applied to analyze a set of differentially expressed proteins by 256

Down



mapping them onto the protein functional network [135,136] . Thus, we did not only collect large amounts of the proteome dataset but also analyzed its data-integrative properties to reveal alterations in the functional network of IHIN (Supplementary Table 3 & Figure 5) . Although present and previous proteomic studies have shown alterations in hundreds of IS implicated cardiac proteins and their functional network, a number of important questions still remain to be answered. First, most proteomic applications were performed at one point in time. Because protein alteration is very dynamic, especially in the heart, which is the most dynamic organ in the human body, proteomic results from a single point in time cannot explain dynamic cardiac responses during or after IS. Thus, proteomic alterations must be evaluated continuously and include both shortand long-term periods. The short-term period might include early (within 10 min of reperfusion), middle (10–120 min) and late (>120 min) periods. Longer term periods might include 1 day, 1 week and 1 month after I/R. Second, although various important pathways involving IS have been revealed in recent decades, the regulatory mechanisms of a large number of proteins are still unclear, owing to the complexity of up- and downregulation mechanisms. Changes in protein levels are modulated by complex factors, including target gene promotion rate, gene translation, PTM, protein turnover and degradation rate. Thus, identifying the mechanisms that regulate altered proteins should be the next step after substantial proteomic data acquisition. Third, whether proteomic alteration is a result of cellular injury or a compensatory mechanism of cells or tissues is not clear. A careful evaluation of this question is critical for supporting cardioprotective strategies based on proteomic results. Fourth, various essential signaling responses to IS are modulated by membrane-located proteins, including ion channels and

Review

receptors. However, proteomic approaches rarely detect changes in these important membrane proteins because of the relatively small protein yield and hydrophobicity of most membrane proteins. Moreover, the activities of these channels and receptors are more often modulated by interacting molecules or physiological signaling than by quantitative changes in protein level. Fortunately, recently developed high-sensitivity proteomic tools are capable of analyzing PTM proteins in extremely small amounts. Thus, specially targeted proteomic approaches are necessary to assess changes in these proteins. Signalosomes, which are newly proposed cardiac protective signaling delivery complexes, could be an excellent target for a specialized proteomic approach [137] . Signalosomes are multimolecular signaling complexes that deliver cardioprotective signals from cells to mitochondria; however, the details of their component proteins are still unclear [137,138] . In the near future, developments in proteomic applications and instruments are expected to improve multidimensional proteomic assessments, providing detailed data on the molecular basis of cardiac response to I/R injury and IPC. The accumulated proteomic information and associated functional meanings will be highly useful for optimizing therapeutic targets and developing a wide range of therapeutic agents for ischemic heart disease. Financial & competing interests disclosure

This work was supported by the Priority Research Centers Program (20100020224) and the Basic Science Research Program (R0A-2007-000-20085) through the National Research Foundation of Korea, funded by the Ministry of Education, Science and Technology. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Key issues • The cardiac proteomic response to ischemia reperfusion and ischemic preconditioning is related to four major functional categories: energy production and conversion; post-translational modification, protein turnover and chaperones; cytoskeleton; and signal transduction mechanisms. • Cardiac mitochondrial proteomic alteration makes up the majority of total cardiac proteome responses. • Exercise-induced cardiac proteomic responses may strengthen cardioprotective mechanisms against ischemia–reperfusion injury. • Proteomic approaches that are time dependent, mechanistic and target specific are the most helpful for unveiling the potential key proteins in the onset and progression of ischemic heart diseases.

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