Table 2.

Summary of cardiovascular diseases investigated by proteomics and major findings

Disease	Methods	Tissue/cell type or Subcellular fraction	Model	Findings	Ref.
Acute coronary syndrome	2DE + MALDI-TOF/TOF + LC/MS/MS	Blood/monocytes	25 human patients with non-ST-elevation acute coronary syndrome were randomized to receive atorvastatin 80 mg/dL (n = 14) or conventional treatment (n = 11) for two months.	Expression of 20 proteins was modified by intensive treatment with atorvastatin including protein disulfide isomerase ER60 (PDI), annexin 1, prohibitin, and HSP-70.	[93]
	2DE, DIGE, MALDIM S/MS	Plasma/pool remaining after depletion of high-abundance proteins.	Plasma from forty patients with acute coronary syndrome were compared with twenty healthy volunteers and 10 stable CAD patients.	Besides proteins previously identified as upregulated in plasma from patients with acute coronary syndrome, four other potential biomarkers were identified: alpha-1-B-glycoprotein, Hakata antigen, tetranectin, and tropomyosin 4	[94]
	2DE + MALDI-TOF/TOF	circulating platelets	Platelets from 18 patients with non-ST segment elevation acute coronary syndrome and 10 matched stable coronary artery disease patients were compared	22 proteins differentially expressed including proteins involved in αllbβ3 and GPVI signaling. The number of differentially expressed proteins decreased at day 5 and further decreased 6 months after the acute event.	[77]
	iTRAQ + LC-MALDI-TOF/TOF	Cardiac/ventricular myocytes	Guinea pig ventricular myocytes were exposed to either 0 μM H2O2 for 5 min and then 10 units/ml catalase or 30 μM H2O2 for 5 min and then 10 units/ml catalase at 37 °C.	Altered expression of 35 proteins after transient exposure of myocytes to H2O2. Most protens altered were mitochondrial including malate dehydrogenase and cytochrome c oxidase subunit 2.	[95]
Adenaline and Reactive Oxygen species on cardiomyocytes	2DE + MALDI-TOF/TOF	Cardiac/myocytes and mitochondria from myocytes	Male rat cardiomyocytes under different conditions were compared: (i) control cells, with no exposure; (ii) cells incubated with adrenaline (ADR); (iii) cells exposed to ADR with XXO (xanthine with xanthine oxidase: a reactive oxygen species generating system); and (iv) cells exposed only to XXO.	Differential changes in myosin light chain-2, cytochrome c and voltage-dependent anion channel 1; redox regulation proteins (particular superoxide dismutase); energetic metabolism proteins (ATP synthase alpha chain and dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex); heat shock proteins.	[96]
Chronic model of type 1 diabetes	2DE + MALDI-TOF	Cardiac/none	Hearts of 4-to 5-mo-old control and 0VE26 mice were compared. 0VE26 mice are a chronic model of type 1 diabetes	Altered expression of 20 identified proteins, of which 12 were mitochondrial and included aconitase 2 and ATP synthase, Fl complex, a.	[97]
Congestive heart failure	2DE + DIGE/LC /MS/MS	Cardiac/Left atria myocytes	Dogs weighing 25-32 kg subjected to ventricular-tachypaced for 24hr (n=5) or 2 week periods (n=8). Sham-operated animals (instrumented but not paced) were used as controls (n=4 and 9 for 24hr and 2 week groups respectively).	Extensive changes (upregulation) in cardioprotective heat shock proteins, decreased antioxidant proteins (superoxide dismutase and peroxiredoxin), and desmin and filamin fragmentation in 2-week ventricular-tachypaced atrial cardiomyocytes.	[98]
Coronary atherosclerosis	2DE, LC/MS/MS	Coronary arteries/none	10 diseased and 7 normal human coronary arteries were compared.	Increased expression of ferritin light chain in diseased coronary arteries.	[99]
Dilated Cardiomyopathy (DCM)	Lable free LC/MS/MS	Cardiac/endomyocardial biopsies	Endomyocardial biopsies from 10 patients with inflammatory DCM as well as 7 controls with normal left ventricular function were compared.	174 proteins were differentially expressed. The major changes in protein expression were observed for mitochondrial and cytoskeletal proteins. Deregulation of proteins of carbohydrate metabolism, the actin cytoskeleton, and extracellular matrix remodeling was observed in DCM samples.	[81]
	2DE + MALDITOF/TOF	Cardiac/none	Hearts from cows with bovine hereditary dilated cardiomyopathy were compared with nondiseased bovine hearts.	24 proteins (including myoglobin) are of decreased abundance in diseased tissue, whilst 11 proteins are of increased abundance in the diseased state.	[100]
	2DE + MALDITOF/TOF	Cardiac/affinit y purification of ubiquitinated proteins	12 DCM, 9 ischemic (IHD) and 12 unused human donor hearts were compared.	All DCM hearts showed significantly higher expression of certain key enzymes of the ubiquitinproteasome pathway.	[101]
	2DE + DIGE/LC/MS/MS	Cardiac/ventricular homogenates and myofibrils	Comparison of hearts from transgenic mice expressing a mutant tropomyosin (E54K) that is associated with dilated cardiomyopathy.	A significant (~ 40%) decrease in Tm phosphorylation in transgenic DCM hearts compared to nontransgenic mouse hearts. This suggests that altered phosphorylation may be a significant factor in the linkage of the E54K mutation to DCM.	[44]
	2DE + DIGE/LC/MS/MS	Coronary arteries/none	Comparison of nondiseased coronary arteries from human heart transplant donors and patients with DCM with no evidence of coronary artery disease, to coronary arteries from patients with ischemic heart disease (IHD).	Hsp27 showed decreased abundance in ischemic vessels. The expression of cytoskeletal proteins, such as vimentin was significantly reduced, while transgelin and Tm showed significantly increased abundance in vessels with IHD. Together with western blotting data, the results suggest that phospho-Hsp27 protects against vascular disease possibly by stabilizing the actin cytoskeleton within endothelial and/or smooth muscle cells.	[102]
Experimental alcoholic cardiomyopathy	iTRAQ/M ALDI TOF-TOF	Cardiac/ventricular nuclear, mitochondrial, sarcoplasmic and myofibrillar fractions	Male and female rats were maintained on either an alcohol-containing or alcohol-free diet for 18 wk and then compared.	Troponins were oppositely regulated by alcohol exposure in males (downregulated) vs. females (upregulated). Males consuming alcohol showed increased expression of proteins involved in oxidative phosphorylation (complexes I, III, IV, V) whereas females showed no change or decreased content.	[103]
Experimental Model of Type 1 Diabetes	iTRAQ + LC/MS/MS	Cardiac/mitochondria	Male rats treated with streptozotocin (60mg/kg) or saline and investigated after 120 days.	65 proteins differed significantly between the groups: up-regulation of several enzymes involved in the oxidation of long-chain fatty acid, in combination with down-regulation of short-chain fatty acid catabolism.	[104]
	in vitro stable isotope labeling, 2DE + MALDI-TOF	Cardiac/mitochondria	Diabetic rats and age-matched control rats were investigated at 1 or 4 weeks after STZ injection.	Up-regulation of the fatty acid β-oxidation. Down-regulation of protein levels for creatine kinase, voltage-dependent anion channel 1 (VDAC-1), HSP60, and Grp75.	[105]
	2DE + iTRAQ + MALDI-TOF/TOF	Cardiac/subsarcolemm al mitochondria and interf ibrillar mitochondria	Male mice injected with streptozotocin (50mg/kg) or saline daily for five consecutive days after 6 h of fasting. Five weeks after hyperglycemia onset, animals were euthanized for further experimentation.	Interf ibrillar mitochondria were impacted by type 1 diabetes mellitus to a greater extent than subsarcolemmal mitochondria, including a decrease in abundance of fatty acid oxidation and electron transport chain proteins. Mitochondrial phosphate carrier and adenine nucleotide translocator were decreased in the diabetic interf ibrillar mitochondria	[106]
Experimentaly induced cardiomyopathy	2DE + MALDI-TOF	Cardiac/none	Comparison of WT, 4 wk (asymptomatic) and 9 wk old (severe cardiomyopathy) frataxin knockout mice.	Pronounced changes in protein expression profile in 9 wk-old KO mice with few changes in 4 wk-old KO mice. Frataxin KO mice showed decreased expression of components of the iron-dependent complex-l and -II of the mitochondrial electron transport chain, enzymes involved in ATP homeostasis (creatine kinase, adenylate kinase) and a variety of chaperones. The KO hearts exhibited increased expression of enzymes involved in the citric acid cycle, catabolism of branched-chain amino acids, ketone body utilization and pyruvate decarboxylation.	[107]
Experimental acute myocardial ischemia	isobaric tag labelling, iTRAQ, OFFGEL fractiona tion, LC/MS/MS	Cardiac/sarcomeric, nuclear, and cytoplasmic enriched fractions	Comparison of rat ventricular tissue from ischemic and non-ischemic regions of rat hearts induced by acute myocardial ischemia by ligatingthe left-anterior descending coronary artery in vivo for lhr without reperfusion.	22 unique proteins in the sarcomeric enriched fraction had changed at least 20% including a decrease in ryanodine receptor 2 in ischemic regions.	[108]
Experimental ischemia-reperfusion injury	2DE, LC/MS/MS	Cardiac/none	Isolated male rat hearts were perfused under aerobic conditions or subjected to ischemia-reperfusion in the presence or absence of the cardioprotective Rho kinase inhibitor, Y-27632.	Y-27632 treatment affected four proteins: lactate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase were significantly increased in the Y-27632 treated group, while creatine kinase and two different molecular fragments of ATP synthase were normalized to control levels by Y-27632. The cardioprotective effect of Y-27632 likely involves increased energy production.	[109]
Experimental hyperdynamic mouse hearts	2DE, MALDI-TOF, LC-MS/MS	Cardiac/Ventricle	Ventricular proteins from phospholamban-KO and WT mice were compared.	Loss of phospholamban is associated with MLC-1 isoform switching and increased MLC-2v, HSP27 and aB-crystallin phosphorylation.	[110]
Left ventricular remodeling (LVR)	SELDI-TOF	Plasma/plasma albumin depleted	Human plasma samples from 93 patients (obtained on day 5 of hospitalization) were divided into three groups (no, low, or high remodeling) and compared.	Post-translational variants of the al-chain of haptoglobin were more elevated in remodeling patients.	[111]
	2DE + MALDI-TOF	Cardiac and plasma/left ventricle	Comparison of plasma from 10-week-old male rats which had myocardial infarction induced by left coronary ligation and plasma from 16 sham-operated rats.	2D phosphoproteomics showed that troponin T phosphorylation was decreased in the left ventricle from rats with LVR. Western blotting with anti-phosphoserine residue 208 of troponin T showed that phosphorylation of this site was decreased in plasma and left ventricles from rats and humans.	[46]
Hypertension	iTRAQ + LC/MS/MS	Cardiac/Mitochondria	20-month-old spontaneously hypertensive rat (SHR) and Wistar-Kyoto controls were compared	79 proteins were differentially expressed between groups. Changes in proteins involved in several metabolic pathways, chaperone and antioxidant systems. Multiple subunits of the oxidative phosphorylation complexes were increased (complexes 1, III and IV) or decreased (complexes II and V) in SHR heart mitochondria.	[112]
Myocardial Infarction	SELDI-TOFMS	Plasma/lmmu no-purification of cardiac troponin 1 forms from plasma	Cardiac Troponin 1 forms present in the human plasma from 64 patients with acute myocardial infarction	Several forms of cardiac troponin 1 were detected with intact and bis-phosphorylated troponin 1 mostly present by itself.	[113]
Reperfusion arrhythmias	isobaric tag labeling, iTRAQ, MALDI TOF-TOF	Cardiac/Left ventricular membrane enriched fraction	Rat myocardial ischemia reperfusion (IR) model was induced by 30 min coronary occlusion and 120 min reperfusion in the presence and absence of grape seed proanthocyanidin (GSPE) extract	92 differentially expressed proteins. Na+/K+ ATPase al subunit was decreased in IR group while it was significantly increased in GSPE group compared to sham group.	[24]
Type 2 diabetes mellitus.	iTRAQ + MALDI TOF-TOF	Cardiac/subsarcolemm al mitochondria and interf ibrillar mitochondria	Pooled subsarcolemmal and interf ibrillar mitochondria subpopulations from 18wt old db/db and WT mouse hearts were compared	Inner mitochondrial membrane proteins and mitochondrial protein import machinery were predominantly decreased in diabetic mitochondria. Subsarcolemmal mitochondria from db/db showed greater differences than interfibrillar mitochondria when compared to their respective WT controls.	[114]