Postconditioning leads to an increase in protein S-nitrosylation

Abstract

Previous studies have shown a role for nitric oxide and S-nitrosylation (SNO) in postconditioning (PostC), but specific SNO proteins and sites have not been identified in the myocardium after PostC. In this study, we examined SNO signaling in PostC using a Langendorff-perfused mouse heart model. After 20 min of equilibrium perfusion and 25 min of global ischemia, PostC was applied at the beginning of reperfusion with six cycles of 10 s of reperfusion and 10 s of ischemia. The total period of reperfusion was 90 min. Compared with the ischemia-reperfusion (I/R) control, PostC significantly reduced postischemic contractile dysfunction and infarct size. PostC-induced protection was blocked by treatment with N^G-nitro-l-arginine methyl ester (l-NAME) (10 μmol/l; a constitutive NO synthase inhibitor), but not by either ODQ (10 μmol/l, a highly selective soluble guanylyl cyclase inhibitor) or KT5823 (1 μmol/l, a specific protein kinase G inhibitor). Two biotin switch based methods, two dimensional CyDye-maleimide difference gel electrophoresis (2D CyDye-maleimide DIGE) and SNO-resin-assisted capture (SNO-RAC), were utilized to identify SNO-modified proteins and sites. Using 2D CyDye-maleimide DIGE analysis, PostC was found to cause a 25% or greater increase in SNO of a number of proteins, which was blocked by treatment with l-NAME in parallel with the loss of protection. Using SNO-RAC, we identified 77 unique proteins with SNO sites after PostC. These results suggest that NO-mediated SNO signaling is involved in PostC-induced cardioprotection and these data provide the first set of candidate SNO proteins in PostC hearts.

ischemic postconditioning (PostC), the intermittent interruption of blood flow during the first minute of reperfusion, has been reported to effectively reduce myocardial ischemia-reperfusion (I/R) injury (30, 33, 34). Nitric oxide (NO) has been shown to be an important signaling molecule involved in PostC-induced cardioprotection (2, 20, 33). Early studies suggested that NO mediates PostC-induced protection through the classical soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP) signaling pathway (1, 32). A specific sGC inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), was shown to block the PostC-mediated reduction in infarct size in a Langendorff perfused heart model of I/R injury (8, 32). However, Cohen et al. (3) recently reported that pharmacological postconditioning with a NO donor, S-nitroso-N-acetyl- d,l-penicillamine (SNAP), was not blocked by ODQ, suggesting that a sGC/cGMP-independent NO signaling is important in mediating PostC (3).

Besides activating the sGC/cGMP signaling pathways, NO can directly modify protein sulfhydryl residues through protein S-nitrosylation (SNO), which has emerged as an important protein posttranslational modification in cardiovascular signaling (14, 23) and cardioprotection (24, 27, 28). SNO exerts cardioprotection not only through modulating protein structure and function, but also by shielding the S-nitrosylated thiol(s) from irreversible oxidative modification upon reperfusion (13, 28).

The goal of this study was to determine if PostC leads to an increase in protein SNO and to identify specific proteins and sites that undergo SNO following PostC. Using a modified biotin switch method with CyDye-maleimide monoreactive fluorescence dyes and a two-dimensional difference gel electrophoresis (2D DIGE), we found that PostC significantly increased protein SNO, and this increase was prevented with l-NAME treatment. SNO-resin-assisted capture (SNO-RAC) was utilized to identify those SNO-modified proteins and sites (i.e., 8 unique SNO-modified proteins in I/R hearts, while almost 10-fold more proteins in PostC hearts). These data are consistent with the hypothesis that PostC exerts its cardioprotection via NO-mediated protein SNO.

METHODS AND MATERIALS

Animals and compounds.

Male C57BL/6J mice obtained from Jackson Laboratories (Bar Harbor, ME) were used for all experiments. Mice were between 12 and 16 wk of age at the time of experimentation. All animals received humane treatment in accordance with National Institutes of Health guidelines and the “Guiding Principles for Research Involving Animals and Human Beings.” This study was reviewed and approved by the Institutional Animal Care and Use Committee of the National Heart, Lung, and Blood Institute. All compounds were obtained from Sigma (St. Louis, MO). The dose of each compound was based on previous studies (25, 26).

Langendorff perfused mouse hearts and I/R-PostC protocol.

After anesthesia with pentobarbital and anticoagulation with heparin, a thoracotomy was performed and the heart was quickly excised and placed in ice-cold Krebs-Henseleit buffer (in mmol/l: 120 NaCl, 11 d-glucose, 25 NaHCO₃, 1.75 CaCl₂, 4.7 KCl, 1.2 MgSO₄, and 1.2 KH₂PO₄). The aorta was cannulated and the heart was perfused with Krebs-Henseleit buffer (oxygenated with 95% O₂/5% CO₂ and maintained at pH 7.4) in retrograde fashion at a constant pressure of 100 cm of water at 37°C. The perfusion was performed in the dark to prevent light-induced SNO decomposition. After equilibrium perfusion for 20 min, mouse hearts were subjected to 25 min of no-flow global ischemia followed by 90 min of reperfusion. PostC was performed at the beginning of reperfusion with six cycles of 10 s of reperfusion and 10 s of ischemia. Each drug was infused during the first 7 min of reperfusion including the PostC period. Hearts that displayed a persistent irregular beating pattern after 20 min of reperfusion were excluded. The timing of drug administration is illustrated in the I/R-PostC protocol as shown in Fig. 1.

Fig. 1.

Ischemia-reperfusion (I/R) and postconditioning (PostC) protocol. Mouse hearts were Langendorff perfused with Krebs-Henseleit buffer (oxygenated with 95% O₂/5% CO₂ and maintained at pH 7.4) at a constant pressure of 100 cm of water at 37°C in the dark. After equilibrium perfusion for 20 min, mouse hearts were subjected to 25 min of no-flow ischemia. PostC (6 cycles of 10 s of ischemia and 10 s of reperfusion) was applied immediately upon reperfusion, followed by an additional 90 min of reperfusion. Drug administration is illustrated for each PostC or I/R protocol. SNAP, S-nitroso-N-acetyl-d,l-penicillamine. l-NAME, N^G-nitro-l-arginine methyl ester.

Cardiac contractile function and infarct size measurements.

A latex balloon connected to a pressure transducer was inserted into the left ventricle of Langendorff perfused mouse hearts to monitor left ventricular developed pressure (LVDP). LVDP was recorded and digitized using a PowerLab system (ADInstruments, Colorado Springs, CO). The rate-pressure product (RPP = LVDP × heart rate) was used as an index of cardiac contractile function. The postischemic functional recovery was expressed as percentage of the preischemic RPP during the equilibrium period. For measurement of myocardial infarct size, at the end of the 90 min of reperfusion, hearts were perfused with 1% (wt/vol) of 2,3,5-triphenyltetrazolium chloride (TTC) and incubated in TTC at 37°C for 15 min, followed by fixation in 10% (wt/vol) formaldehyde. Infarct size was expressed as the percentage of the total cross-sectional area of the ventricles.

Total heart homogenate preparation.

Total heart homogenate was prepared for 2D DIGE and SNO-RAC proteomic analyses, and sample preparations were carried out in the dark to prevent SNO decomposition. Each snap-frozen mouse heart was powdered on liquid nitrogen followed by homogenization with a tight-fitting glass Dounce homogenizer on ice in 1.5 ml homogenate buffer containing (in mmol/l) 300 sucrose, 250 HEPES-NaOH (pH 7.8), 1 EDTA, and 0.1 neocuproine (a copper chelating agent). An EDTA-free protease inhibitor tablet (Roche Diagnostics, Indianapolis, IN) was added to the homogenate buffer just before use. Protein concentration of total heart homogenate was determined using the Bradford protein assay. Total heart homogenates were aliquoted in amber tubes, snap-frozen on dry ice, and stored at −80°C.

Identification of SNO proteins by 2D CyDye-maleimide DIGE.

We used a modified biotin switch method (10) with CyDye maleimide monoreactive sulfhydryl-reactive fluorescent dyes (GE Healthcare Life Sciences, Piscataway, NJ) to identify SNO proteins. After the CyDye-maleimide switch and 2D DIGE, each gel was scanned at the unique excitation/emission wavelength of each dye using a Typhoon 9400 imager (GE Healthcare Life Sciences) at a resolution of 100 μm. Images from each gel were aligned using the two internal anchor spots and analyzed with Progenesis Discovery software (Nonlinear Dynamics, Newcastle upon Tyne, UK). The gel was poststained with SYPRO Ruby (Sigma), and the protein spots that corresponded to the CyDye-maleimide fluorescent intensity with 25% or higher difference were picked. The Ettan Spot Handling Workstation (GE Healthcare Life Sciences) was used for automated extraction of the selected protein spots followed by in-gel trypsin digestion. After sample extraction from the spot handling workstation, each sample was manually desalted using Millipore (Billerica, MA) C18 Ziptips following the manufacturer's recommendation in preparation for liquid chromatography-tandem MS (LC-MS/MS) analysis.

SNO protein and site identification with SNO-resin assisted capture (SNO-RAC).

A modified version of the SNO-RAC protocol was developed to identify SNO-modified proteins and sites (5, 11, 12). All buffers were degassed before use with the SNO-RAC protocol to prevent oxidation of the thiopropyl Sepharose resin (GE Healthcare, Piscataway, NJ). In brief, after blocking free sulfhydryl group with 20 mmol/l N-ethylmaleimide (NEM), each total heart homogenate (0.25 mg) was loaded onto preequilibrated resin in the presence of 20 mmol/l Na-ascorbate, and incubated for 4 h at room temperature. After washing, resin-bound proteins were then subjected to trypsin digestion (sequencing grade modified; Promega) overnight at 37°C with rotation in the digestion buffer containing (in mmol/l) 50 NH₄HCO₃ and 1 EDTA. After washing, peptides were eluted for 30 min at room temperature in elution buffer containing (in mmol/l) 20 DTT, 10 NH₄HCO₃, and 50% methanol (vol/vol), followed by two volumes of H₂O. All fractions were combined and concentrated via Speedvac. Samples were then resuspended in 0.1% formic acid and cleaned with Millipore C18 Ziptips for LC-MS/MS analysis.

LC-MS/MS analysis and database search.

LC-MS/MS was performed using an Eksigent nanoLC-Ultra 1D plus system (Dublin, CA) coupled to an LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, San Jose, CA) using CID fragmentation. Peptides were first loaded onto an Zorbax 300SB-C18 trap column (Agilent, Palo Alto, CA) at a flow rate of 6 μl/min for 6 min, and then separated on a reversed-phase PicoFrit analytical column (New Objective, Woburn, MA) using a short 15-min linear gradient of 5–40% acetonitrile for 2D gel spots and 40-min gradient for SNO-RAC in 0.1% formic acid at a flow rate of 250 nl/min. LTQ-Orbitrap Elite settings were as follows: spray voltage 1.5 kV; full MS mass range m/z 300 to 2,000. The LTQ-Orbitrap Elite was operated in a data-dependent mode; i.e., one MS1 high resolution (60,000) scan for precursor ions followed by six data-dependent MS2 scans for precursor ions above a threshold ion count of 500 with collision energy of 35%. The raw file generated from the LTQ Orbitrap Elite was analyzed using Proteome Discoverer v1.3 software (Thermo Fisher Scientific, LLC) using our six-processor Mascot cluster at NIH (v.2.4) search engine. The search criteria was set to: database, Swiss Institute of Bioinformatics (Sprot_103112, 16573 sequences); taxonomy, Mus musculus (mouse); enzyme, trypsin; miscleavages, 2; variable modifications, Oxidation (M), Deamidation (NQ), Acetyl (protein N-term), NEM (C); MS peptide tolerance 20 ppm; MS/MS tolerance as 0.8 Da. Protein identifications were accepted based on two or more unique peptides with a false discovery rate (FDR) of 99% or higher. Raw mass spectrometry data for SNO sites identified via SNO-RAC in PostC hearts (Table 3) can be accessed at Peptide Atlas (http://www.peptideatlas.org/PASS/PASS00392).

Table 3.

S-nitrosylation sites identified via SNO-RAC proteomic analysis in PostC hearts

Protein Name	ID	Peptide Sequence	SNO-Cys
Extracellular matrix and cell membrane
Galectin-1	P16045	ACGLVASNLNLKPGECLK	3, 17
Long-chain fatty acid transport protein 1	Q60714	VGSCGFNSR	406
PDZ and LIM domain protein 5	Q8CI51	ACTGSLNMTLQR	73
Protein-glutamine gamma-glutamyltransferase 2	P21981	YSGCLTESNLIK	553
Cytoplasm and cytoskeleton
6-Phosphofructokinase, muscle type	P47857	LPLMECVQVTK	351
		IFANTPDSGCVLGMR	709
Alpha-enolase*	P17182	VNQIGSVTESLQACK	357
Annexin A6	P14824	GTVCAANDFNPDADAK	358
Cytoplasmic dynein 1 heavy chain 1	Q9JHU4	VQYPQSQACK	631
		LQGATCSNNK	4568
Destrin	Q9R0P5	ASGVQVADEVCR	12
Dihydropyrimidinase-related protein 2	O08553	GLYDGPVCEVSVTPK	504
E3 ubiquitin-protein ligase UBR4	A2AN08	AVQCLNTSSK	2552
Elongation factor 2	P58252	ETVSEESNVLCLSK	591
Filamin-B	Q80X90	VAVTEGCQPSR	1326
Fructose-bisphosphate aldolase A*	P05064	ALANSLACQGK	339
Glutaredoxin-1	Q9QUH0	AQEFVNCK	8
Glyceraldehyde-3-phosphate dehydrogenase*	P16858	IVSNASCTTNCLAPLAK	150, 154
		VPTPNVSVVDLTCR	245
Heat shock protein HSP 90-β	P11499	FENLCK	564
l-Lactate dehydrogenase A chain	P06151	VIGSGCNLDSAR	163
Myosin-binding protein C, cardiac-type	O70468	ATNLQGEAQCECR	1260, 1262
Myosin light chain kinase, smooth muscle	Q6PDN3	VAGTQPITCK	1291
Obscurin	A2AAJ9	QADTGTVCATSPK/	3060
Rab GDP dissociation inhibitor β	Q61598	TDDYLDQPcCETINR	203
Selenium-binding protein 1	P17563	GGSVQVLEDQELTCQPEPLVVK	371
Serine/threonine-protein phosphatase 2A 55 kDa regulatory subunit B α isoform	Q6P1F6	AGAGGGNDIQWCFSQVK	13
Titin*	A2ASS6	VSECYVAR	25303
		VLDSPGPCGK	30933
Trifunctional purine biosynthetic protein adenosine-3	Q64737	QVLVAPGNAGTACAGK	41
Triosephosphate isomerase	P17751	IAVAAQNCYK	117
		IIYGGSVTGATCK	268
Tripartite motif-containing protein 72	Q1XH17	MQLQEACMR	144
		FCLVTSR	242
Tubulin β-4A chain	Q9D6F9	NMMAACDPR	303
		TAVCDIPPR	354
Ubiquitin-like modifier-activating enzyme 1	Q02053	DNPGVVTCLDEAR	234
Endo/sarcoplasmic reticulum, nucleus, ribosome
Bifunctional glutamate/proline-tRNA ligase	Q8CGC7	VACQGEVVR	910
Cellular nucleic acid-binding protein	P53996	TSEVNCYR	159
eIF4E-binding protein 1	Q60876	SAGSSCSQTPSR	7
Nascent polypeptide-associated complex subunit α	P70670	GTVVCLADSSLDTSVSASK	755
Poly(rC)-binding protein 1	P60335	INISEGNCPER	54
40S ribosomal protein S3	P62908	GCEVVVSGK	134
40S ribosomal protein S28	P62858	TGSQGQCTQVR	27
60S ribosomal protein L10	Q6ZWV3	MLSCAGADR	105
60S ribosomal protein L27a	P14115	NQSFCPTVNLDK	70
Sarco/endoplasmic reticulum calcium ATPase 2	O55143	SLPSVETLGCTSVICSDK	344, 349
		TGTLTTNQMSVCR	364
Mitochondria
2-Oxoglutarate dehydrogenase	Q60597	ICEEAFTR	566
3-Ketoacyl-CoA thiolase	Q8BWT1	YAVGSACIGGGQGIALIIQNTA	382
Acetyl-CoA acetyltransferase	Q8QZT1	QATLGAGLPISTPCTTVNK	116
ATP synthase subunit ϵ	P56382	FSQICAK	19
Aconitate hydratase	Q99KI0	VGLIGSCTNSSYEDMGR	385
Aspartate aminotransferase	P05202	VGAFTVVCK	295
Carnitine O-acetyltransferase	P47934	IYGQACATYESASLR	449
Carnitine O-palmitoyltransferase 1	Q924X2	SCTNESAAFVQAMMK	608
Citrate synthase	Q9CZU6	LPCVAAK	211
Creatine kinase S-type	Q6P8J7	GLSLPPACSR	180
Cytochrome b-c1 complex subunit 1*	Q9CZ13	LCTSATESEVTR	380
Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex	Q8BMF4	DVPLGAPLCIIVEK	290
Dynamin-1-like protein	Q8K1M6	FATEYCNTIEGTAK	351
Electron transfer flavoprotein-ubiquinone oxidoreductase	Q921G7	ASCDAQTYGIGLK	265
Enoyl-CoA hydratase	Q8BH95	TFQDCYSSK	111
		LVEEAIQCAEK	225
Glutathione S-transferase κ 1	Q9DCM2	LIENTDAACK	176
Iron-sulfur cluster assembly 2 homolog	Q9DCB8	LTDSCVQR	56
Isocitrate dehydrogenase [NADP]*	P54071	SSGGFVWACK	308
		VCVQTVESGAMTK	402
Isocitrate dehydrogenase [NAD] subunit α	Q9D6R2	IEAACFATIK	331
		cSDFTEEICR	359
Lactation elevated protein 1	Q3V384	VVQCLQK	100
Leucine-rich PPR motif-containing protein	Q6PB66	LIAAYCNVGDIEGASK	207
Malate dehydrogenase*	P08249	GYLGPEQLPDCLK	89
		EGVVECSFVQSK	275
Methylmalonate-semialdehyde dehydrogenase	Q9EQ20	VCNLIDSGTK	368
Mitochondrial tRNA-specific 2-thiouridylase 1	Q9DAT5	TPNPDINCNK	101
Mitofusin-1	Q811U4	LCQQVDVTQK	681
NADH-ubiquinone oxidoreductase 75 kDa subunit	Q91VD9	LSVAGNCR	75
		AVTEGAQAVEEPSIC	727
NADH-ubiquinone oxidoreductase chain 3	P03899	ANPYECGFDPTSSAR	39
Propionyl-CoA carboxylase α chain	Q91ZA3	MADEAVCVGPAPTSK	107
Short/branched chain specific acyl-CoA dehydrogenase	Q9DBL1	ASSTCQLTFENVK	261
Succinate dehydrogenase cytochrome b560 subunit	Q9CZB0	SLCLGPTLIYSAK	107
Succinate dehydrogenase [ubiquinone] flavoprotein subunit	Q8K2B3	TLNEADCATVPPAIR	654
Succinate-semialdehyde dehydrogenase	Q8BWF0	NAGQTCVCSNR	328, 330
Succinyl-CoA ligase subunit α	Q9WUM5	LIGPNCPGVINPGECK	172, 181
Succinyl-CoA ligase subunit β	Q9Z2I9	IcNQVLVCER	158
		ILACDDLDEAAK	430
Voltage-dependent anion-selective channel-1	Q60932	YQVDPDACFSAK	245
Voltage-dependent anion-selective channel-2*	Q60930	SCSGVEFSTSGSSNTDTGK	48
Voltage-dependent anion-selective channel-3	Q60931	cNTPTYCDLGK	8

S-nitrosylation (SNO) cysteine residues (SNO-Cys) are labeled in upper case and underlined (C); cysteine residues blocked by N-ethylmaleimide (NEM) are labeled in lower case (c). Protein identifications were accepted based on two or more unique peptides with a FDR of 99% or higher. *SNO-modified proteins and peptides were also identified from 2 of 3 SNO-RAC/LC-MS/MS proteomic analyses. SNO-Cys indicates the amino acid in the protein that is SNO.

Data analysis.

Results are expressed as means ± SE. Statistical significance was determined by one-way ANOVA followed by a post hoc Bonferroni test.

RESULTS

Ischemic PostC exerted a protective effect.

We first confirmed that a protocol of PostC with six cycles of 10 s of reperfusion and 10 s of ischemia applied at the beginning of reperfusion after 25 min of global no-flow ischemia (Fig. 1) was cardioprotective. As shown in Table 1 and Fig. 2, PostC significantly increased the postischemic functional recovery, as the rate-pressure product (RPP) recovery was 46.1 ± 2.9% (n = 14) in PostC hearts compared with 20.9 ± 3.7% (n = 9) in I/R-control hearts. The postischemic myocardial infarction was 33.8 ± 2.4% (n = 14), significantly smaller than I/R-control hearts (56.7 ± 4.2%, n = 9).

Table 1.

Evaluation of cardiac contractile function in Langendorff-perfused mouse hearts

			Preischemic Equilibration					End of Reperfusion
Heart Samples	n	BW, g	FR, ml/min	HR, beats/min	LVDP, cmH2O	+dP/dt, cmH2O/ms	−dP/dt, cmH2O/ms	FR, ml/min	HR, beats/min	LVDP, cmH2O	+dP/dt, cmH2O/ms	−dP/dt, cmH2O/ms
I/R-Control	9	27.2 ± 0.6	2.6 ± 0.2	346 ± 18	144 ± 8	8.1 ± 0.5	−5.8 ± 0.2	1.7 ± 0.2	338 ± 10	33 ± 6	4.2 ± 0.4	−3.4 ± 0.3
PostC	14	26.6 ± 0.5	2.5 ± 0.2	351 ± 12	135 ± 4	8.5 ± 0.3	−5.9 ± 0.2	1.7 ± 0.1	320 ± 11	67 ± 5†	5.6 ± 0.3	−4.3 ± 0.2
I/R-SNAP	8	27.9 ± 0.3	2.7 ± 0.2	389 ± 12	125 ± 9	8.0 ± 0.3	−5.2 ± 0.3	1.8 ± 0.1	345 ± 11	54 ± 3*	4.7 ± 0.2	−3.9 ± 0.3
I/R-l-NAME	6	27.2 ± 0.5	2.6 ± 0.1	380 ± 20	147 ± 8	9.1 ± 0.4	−6.2 ± 0.2	2.1 ± 0.2	301 ± 19	44 ± 8	4.2 ± 0.5	−3.7 ± 0.4
l-NAME + PostC	8	26.5 ± 0.6	2.9 ± 0.2	374 ± 11	143 ± 4	9.0 ± 0.4	−6.2 ± 0.4	1.8 ± 0.3	309 ± 13	47 ± 6	4.2 ± 0.5	−3.6 ± 0.4
I/R-ODQ	5	27.3 ± 0.3	2.7 ± 0.3	386 ± 26	129 ± 7	8.1 ± 0.6	−5.5 ± 0.4	1.7 ± 0.2	344 ± 13	30 ± 6	4.0 ± 0.5	−3.3 ± 0.2
ODQ + PostC	7	28.3 ± 0.5	2.5 ± 0.2	379 ± 20	130 ± 7	9.0 ± 0.6	−5.7 ± 0.4	2.0 ± 0.2	334 ± 10	57 ± 4‡	4.7 ± 0.3	−3.9 ± 0.3
I/R-KT5823	4	27.6 ± 0.3	2.5 ± 0.1	416 ± 24	124 ± 6	8.4 ± 0.6	−5.6 ± 0.3	1.9 ± 0.1	314 ± 11	39 ± 5	3.9 ± 0.3	−3.1 ± 0.4
KT5823 + PostC	5	27.6 ± 0.6	2.4 ± 0.1	408 ± 21	127 ± 5	8.5 ± 0.4	−6.3 ± 0.4	1.9 ± 0.1	344 ± 19	65 ± 3§	5.5 ± 0.3	−4.0 ± 0.2

Values are means ± SE; n = no. of hearts. BW, body weight; FR, flow rate; HR, heart rate; LVDP, left ventricular developed pressure; ±dP/dt, rates of pressure rise and fall, respectively; I/R, ischemia-reperfusion; SNAP, S-nitroso-N-acetyl-d,l-penicillamine. l-NAME, N^G-nitro-l-arginine methyl ester; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; PostC, postconditioning;.

^*P < 0.05,

^†P < 0.01 vs. I/R-control;

^‡P < 0.05 vs. I/R-ODQ;

^§P < 0.05 vs. I/R- KT5823.

Fig. 2.

PostC-induced cardioprotection was dependent upon nitric oxide (NO)/S-nitrosylation (SNO) signaling. A: postischemic left ventricular rate-pressure product (RPP) functional recovery. B: infarct size, measured at the end of reperfusion by 1% TTC staining. Results are expressed as means ± SE. Statistical significance was determined by one-way ANOVA followed by a post hoc Bonferroni test. *P < 0.05; **P < 0.01 vs. I/R-control; #P < 0.05 vs. I/R + 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ); ##P < 0.01 vs. I/R + KT5823. The number of animals in each group is indicated in the column.

Reperfusion with SNAP protected the heart against I/R injury.

Cohen et al. (3) recently demonstrated that infusion of an NO/SNO donor SNAP at reperfusion has a protective effect in an intact rabbit coronary artery occlusion and reperfusion model. To test whether an NO donor can mimic PostC in our Langendorff perfused mouse heart I/R model, we infused the NO donor, SNAP (10 μmol/l), into the perfusate during the first 7 min of reperfusion. As shown in Fig. 2, treatment with SNAP upon reperfusion significantly improved cardiac postischemic functional recovery (40.0 ± 3.4%, n = 8) and decreased postischemic myocardial infarct size (36.8 ± 2.7%, n = 8).

PostC increased myocardial SNO.

To test whether PostC increased SNO, PostC hearts were collected and snap frozen in liquid N₂ after 2 min of PostC plus 5 min of additional reperfusion and I/R-control hearts after 7 min of reperfusion. The total heart homogenate was prepared in the dark as described in methods and materials. A modified biotin switch method using CyDye-maleimide monoreactive fluorescence dyes and 2D DIGE proteomic analysis was carried out for SNO detection (25, 26). As shown in Fig. 3, SNO proteins in I/R-control hearts were labeled by Cy3-maleimide (pseudocolored in green) and PostC hearts were labeled with Cy5-maleimide (pseudocolored in red). SNO protein spots showing a change of at least 25% or higher in PostC hearts compared with I/R-control were picked for identification via mass spectrometry. As shown in Table 2, PostC-treated hearts showed an increase in SNO for a number of proteins, and most of these SNO proteins were previously found in IPC hearts (25, 26), including aconitase, ATP synthase subunit α, creatine kinase S/M type, α-cardiac muscle actin, cytoplasmic malate dehydrogenase, electron transfer flavoproteins α/β, myosin light chain 1, and myoglobin.

Fig. 3.

PostC increased myocardial protein SNO. Top: representative two-dimensional (2D) CyDye-maleimide difference gel electrophoresis (DIGE) gel from three independent experiments was scanned at each of the distinct wavelengths of the fluors, showing a pattern of protein SNO for that particular treatment group. Bottom: overlaid image of Cy3-maleimide (I/R control, green) vs. Cy5-maleimide (PostC, red). Protein spots (1–13) showing a change of at least 25% or higher in PostC hearts compared with I/R-control were picked for MS/MS analysis and are listed in Table 2.

Table 2.

Proteins identified by 2D CyDye-maleimide DIGE with increased SNO level in PostC hearts

					SNO Level (Arbitrary Ratio)
Spots	Protein Name	Accession No.	MW, kDa	Protein pI	PostC vs. I/R	PostC vs. PostC + l-NAME
1	2-Oxoglutarate dehydrogenase	Q60597	116.4	6.83	1.48 ± 0.15	1.40 ± 0.16
2	Aconitate hydratase, mitochondrial	Q99KI0	85.4	8.08	1.50 ± 0.19	1.38 ± 0.14
3	Mitochondrial F1-ATPase subunit α	Q03265	59.7	9.22	1.47 ± 0.18	1.50 ± 0.13
4	Creatine kinase S-type	Q6P8J7	47.8	8.64	1.45 ± 0.18	1.53 ± 0.14
5	Creatine kinase M-type	P07310	43.0	6.58	1.51 ± 0.28	1.41 ± 0.11
6	α-Cardiac muscle actin	P68033	42.3	5.23	1.45 ± 0.17	1.44 ± 0.16
7	Malate dehydrogenase, cytoplasmic	P14152	36.5	6.16	1.46 ± 0.15	1.49 ± 0.19
8	Glyceraldehyde-3-phosphate dehydrogenase	P16858	35.8	8.25	1.28 ± 0.04	1.35 ± 0.09
9	Malate dehydrogenase, mitochondrial	P08249	35.6	8.68	1.57 ± 0.14	1.45 ± 0.08
10	Electron transfer flavoprotein α	Q99LC5	35.0	8.62	1.54 ± 0.12	1.55 ± 0.14
11	Electron transfer flavoprotein β	Q9DCW4	27.8	8.24	1.46 ± 0.12	1.49 ± 0.13
12	Myosin light chain 1	P09542	22.5	5.03	1.43 ± 0.13	1.50 ± 0.13
13	Myoglobin	P04247	17.1	7.07	1.30 ± 0.05	1.31 ± 0.07

Values are means ± SE. Protein identifications were accepted based on two or more unique peptides with a false discovery rate (FDR) of 99% or higher and a correct molecular mass identification. SNO protein spots showed a change of at least 25% or higher in PostC hearts compared with I/R-control or PostC + l-NAME hearts (P < 0.05, n = 3 in each group).

l-NAME abolished the PostC-induced increase in protein SNO.

To confirm that the changes in SNO observed in Fig. 3 and Table 2 are involved in the protection associated with PostC, we performed additional experiments using l-NAME to determine whether the inhibition of NOS with l-NAME, which has been previously shown to block PostC-induced protection, would also block the PostC-mediated increase in protein SNO. Langendorff-perfused mouse hearts were treated with 10 μmol/l l-NAME during the first 7 min of reperfusion. Compared with I/R-control, perfusion of non-PostC hearts with l-NAME had no significant effect on postischemic functional recovery (Table 1, Fig. 2A) or infarct size (Fig. 2B). However, treatment with l-NAME blocked PostC-induced cardioprotection (Fig. 2). After confirming that PostC-induced protection was blocked by l-NAME, we compared the SNO level of PostC hearts with and without l-NAME treatment to determine whether the PostC-induced increase in SNO could be prevented by l-NAME. As shown in Table 2, PostC + l-NAME hearts have significantly lower SNO levels compared with PostC hearts, suggesting that inhibition of NOS with l-NAME prevented the PostC-induced increase in SNO.

S-nitrosylation site identification in PostC hearts via SNO-RAC analysis.

Because of dynamic range issues and the limited amount of protein that can be loaded onto the 2D gel, the SNO proteins identified in 2D DIGE are biased toward detection of high-abundance proteins (Table 2). Furthermore, although 2D fluor-maleimide DIGE is useful in screening and quantifying SNO proteins, unfortunately we have not been able to identify SNO sites with this method (12). Therefore, we utilized a SNO-RAC protocol to identify SNO proteins and sites in total heart homogenates. As shown in Fig. 4, SNO-RAC detected only 10 SNO peptides from 8 unique proteins in I/R hearts, while 91 SNO peptides from 77 proteins were identified in PostC hearts. Almost half (37 of 77) of the identified SNO proteins are mitochondrial, suggesting mitochondria are a major organelle targeted by PostC-mediated SNO. Most of these proteins contain only one SNO modified cysteine, while ∼20% of them contain two or more SNO sites (Table 3).

Fig. 4.

PostC increased total number of SNO-modified proteins and sites. Total number of SNO-modified proteins (A) and sites (B) from SNO-RAC were identified via LC-MS/MS for I/R and PostC hearts (n = 3 in each group). Protein identifications were accepted based on two or more unique peptides with a false discovery rate (FDR) of 99% or higher. Each protein/peptide was identified from at least 2 of 3 SNO-RAC/LC-MS/MS proteomic analyses.

ODQ or KT5823 treatment did not block PostC-mediated protection.

In non-PostC hearts, perfusion with either ODQ (a sGC inhibitor) or KT5823 (a specific protein kinase G inhibitor) during the first 7 min of reperfusion did not significantly affect postischemic functional recovery or infarct size (Fig. 2). In contrast to l-NAME treatment, which abolished the protection of PostC, 10 μmol/l ODQ or 1 μmol/l KT5823 treatment did not block PostC-induced cardioprotection, i.e., postischemic RPP recovery was 40.2 ± 2.2% (n = 7) for PostC + ODQ and 43.5 ± 3.0% (n = 5) for PostC + KT5823, infarct size was 35.1 ± 3.7% (n = 7) for PostC + ODQ and 32.5 ± 2.3% (n = 5) for PostC + KT5823, which were comparable to the protective effect induced by PostC.

DISCUSSION

NO signaling has been suggested to play an important role in PostC-induced protection. Inhibition of NOS by l-NAME has been shown to block protection in a variety of postconditioning models (18, 29, 33). Furthermore PostC was blocked by reducing agents such as N-acetyl-l-cysteine or 2-mercaptopropionylglycine (19), suggesting that a redox-sensitive mechanism is also involved in the protection afforded by PostC. In addition, a recent study has suggested that PostC prolongs early acidosis, and this would favor the formation of protein SNO (22). Therefore, all of these studies suggest a possible role for protein S-nitrosylation in PostC-induced cardioprotection.

The results contained herein provide the first demonstration that PostC leads to an increase in protein SNO. We further show that this PostC mediated increase in protein SNO is blocked with l-NAME, which also blocks the protective effects of PostC. Comparing the SNO proteins measured by SNO-RAC in PostC hearts (Table 3) with the proteins that show SNO in IPC hearts [Table 1 from Kohr et al. (13)], we find that ∼50% of those proteins that were SNO with IPC also show SNO with PostC (25, 26), suggesting that there might be a common set of proteins targeted by NO/SNO signaling with both IPC and PostC. Therefore, the increase in SNO in IPC and PostC may play a similar role in cardioprotection against I/R injury. For example, we have shown that IPC led to an increase in SNO of the mitochondrial F1-ATPase subunit α. In this study, we also found that PostC induced an increase in SNO of the mitochondrial F1-ATPase (Table 2). In addition, the IPC-induced increase in SNO could shield critical cysteine residue(s) from further oxidative damage upon reperfusion (13). Interestingly, a similar finding has been reported in a recent study, in which Cys294 of the mitochondrial F1-ATPase was found to form a disulfide bond with another cysteine residue in dyssynchronous heart failure, while cardiac resynchronization therapy leads to SNO of Cys294 and prevents disulfide formation (31).

The sGC/cGMP/protein kinase G (PKG) signaling pathway has been suggested to mediate PostC-induced cardioprotection (4, 15), and the main supportive evidence is that inhibition of the cGMP-dependent signaling pathway with selective inhibitors such as ODQ or KT5823 blocks the protection induced by PostC (8, 18, 32). However, a recent study questioned the role of NO-mediated sGC/cGMP/PKG-dependent signaling in PostC by demonstrating that the addition of SNAP, an NO donor, at reperfusion produced protection that was not blocked by ODQ (3). In addition, a role for cGMP-independent NO-induced cardioprotection against I/R injury has also been demonstrated in studies using isolated cardiomyocytes (6, 9). In the present study, treatment of perfused mouse hearts with either ODQ or KT5823 did not alter postischemic functional recovery or infarct size in non-PostC hearts. However, mouse hearts treated with either ODQ or KT5823 were still protected by PostC, suggesting that blockade of the sGC/cGMP/PKG pathway does not abolish NO-dependent PostC-induced cardioprotection. In addition, Methner et al. (16) have shown that protection through postconditioning is unaffected by cardiomyocyte-selective ablation of protein kinase G.

Cardioprotection similar to that obtained with PostC could be also achieved with pharmacological agents given upon reperfusion, i.e., pharmacological postconditioning. By using an intact I/R heart model, Cohen et al. (3) showed that SNAP, an S-nitrosothiol agent, was protective when administered upon reperfusion. In this study, we found that SNAP also elicits pharmacological postconditioning effects in Langendorff perfused mouse hearts (Fig. 2 and Table 1). A mitochondria-targeted S-nitrosothiol agent, mito-SNO, has also been shown to be protective when given at reperfusion in a recent study with an open-chest mouse I/R model. Furthermore, the protection afforded by PostC or mito-SNO has been found to be unaffected by cardiomyocyte-selective ablation of PKG, suggesting an important role for SNO signaling in PostC (16). In addition, Penna et al. (21) showed that pharmacological PostC by diazoxide induced mitochondrial protein S-nitrosylation.

Limitations and perspectives.

The data in this paper demonstrate that PostC leads to an increase in SNO and myocardial protection. Further, the protection afforded by PostC was abolished by l-NAME, suggesting a key role for NO/SNO signaling in PostC-induced protection. However, treatment with either ODQ (a specific sGC inhibitor) or KT5823 (a specific PKG inhibitor) did not block PostC-induced protection, suggesting that NO-mediated protein SNO, rather than activation of the sGC/cGMP/PKG signaling pathway, plays an essential role in PostC. These results together with similar findings from IPC hearts (25) suggest that NO-mediated protein SNO plays a common protective role in the myocardium. These studies have identified a number of proteins that undergo SNO with PostC. However, future studies will be needed to demonstrate the functional impact of SNO on these protein targets and their specific role in PostC.

GRANTS

This work was supported by the National Institutes of Health Intramural Program (A. M. Aponte, J. Sun, and E. Murphy), the American Heart Association (12BGIA11780030, M. J. Kohr), and National Heart, Lung, and Blood Institute Grants 5R-01-HL-039752 (C. Steenbergen) and 1K99-HL-114721 (M. J. Kohr). G. Tong was supported by the China Scholarship Council No. 2011659007.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: G.T., A.M.A., M.J.K., C.S., E.M., and J.S. conception and design of research; G.T. and J.S. performed experiments; G.T. and J.S. analyzed data; G.T., A.M.A., M.J.K., C.S., E.M., and J.S. interpreted results of experiments; G.T. and J.S. prepared figures; G.T. and J.S. drafted manuscript; G.T., A.M.A., M.J.K., C.S., E.M., and J.S. edited and revised manuscript; G.T., A.M.A., M.J.K., C.S., E.M., and J.S. approved final version of manuscript.