Multiple Reaction Monitoring to Identify Site-Specific ... - Circulation

DOI: 10.1161/CIRCULATIONAHA.112.096388

Multiple Reaction Monitoring to Identify Site-Specific Troponin I Phosphorylated Residues in the Failing Human Heart

Running title: Zhang et al., Targeted quantitation of cardiac troponin I phosphorylation

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Pingbo Zhang, PhD1; Jonathan A. Kirk, PhD1;Weihua Ji, MS1; Cristobal G. dos Remedios, DSc2; David A. Kass, MD1; Jennifer E. Van Eyk, PhD1,4*; Anne M. Murphy, MD3*

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Dept of Medicine, Division of Cardiology, 4Depts of Biological Chemistry & Biomedical Engineering, John Jo hnss Ho hn H pk kin inss University, Baltimore, MD; 2Bo Johns Hopkins Bosch Bosc s h Institute, Univers sc University sit ityy of o Sydney, Sydney, 3 Australia; A usttraali l a; De Dept pt of Pediatrics, Johns Hopkins Hopk p ins University Uniiversity y Medical al Institutions, Inssti titu tuti tions,, Baltimore, MD **contributed *c onttrib on trib i ut u ed ed equ ual ally ly equally

Correspondence: Correspond den ence ce:: ce Jennifer Van Eyk, PhD The Hopkins Bayview Proteomics Center 5200 Eastern Ave, Mason F Lord Building Center Tower, Room 601 Baltimore, MD 21224 Tel: 410-550-8507 Fax: 410-550-8512 E-mail: [email protected]

Journal Subject Codes: [11] Other heart failure; [148] Heart failure - basic studies; [104] Structure; [105] Contractile function; [107] Biochemistry and metabolism; [108] Other myocardial biology; [155] Physiological and pathological control of gene expression 1


Abstract: Background - Human cardiac troponin I (cTnI) is known to be phosphorylated at multiple amino acid residues by several kinases. Advances in mass spectrometry (MS) allow sensitive detection of known and novel phosphorylation sites and measurement of the level of phosphorylation simultaneously at each site in myocardial samples. Methods and Results - Based on in silico prediction and LC/MS/MS data, 14 phosphorylation sites on cTnI, including 6 novel residues (S4, S5, Y25, T50, T180, S198), were assessed in explanted hearts from end-stage heart failure transplant patients with ischemic heart disease or idiopathic dilated cardiomyopathy and compared to samples obtained from non-failing donor Downloaded from http://circ.ahajournals.org/ by guest on April 24, 2018

hearts (n = 10 per group). Thirty MS-based multiple reaction monitoring quantitative tryptic assays and corresponding ppeptide p y were developed p for each pphosphorylatable p y p g non-phosphorylated p p y site. phosphorylation ite. The results show in heart failure there is i) a decrease in the extent of phosph phhorryl ylat atio at ionn of the io the known PKA sites (S22, S23) as well as other newly discovered phosphorylation sites located in the phosphorylation he N-terminal extension of cTnI (S4, S5, Y25); ii)) increase in phosphory ylation of the PKC sites (S41, residues (S76, S41 41,, S43, S 3,, T142); S4 T142) 2);; and an iii)) increase increeas a e in pphosphorylation hoospphoory ryla laatiion of of the t e IT-arm th IT-a -aarm ddomain omain re om esi s duess (S7 S76, T77) and C-terminal novel an nd CC terminal al ddomain om main aiin no nove vell phos ve pphosphorylation hos osppho phoryylatiion sites sitees off cTnI cTnI TnI (S165, (S1665, T180, T18 1800, S198). S19 198) 8).. In a ccanine an nin inee dyssynchronous model, sites dy yss ssyn y ch yn c rono nous no us heart heart eart failure faailure ree m odel od el,, enhanced el en nhanc hancced d pphosphorylation hossph sphor horyla rylaatiion at at threee of of tthe hee nnovel ovvell sit tess was was found towards foun undd to t decline dec ecline ne tow owar a ds ccontrol onntr trol ol ffollowing ollowi wing ng resynchronization res esyn ynchrooni niza zati tion on therapy. theera rapy py.. Conclusions functionally Conclusion ns - Selective, Sel elec ecti ec t ve ti ve, fu unc nction onnal ally ly ssignificant igni ig nifi fica fi cant ca nt phosphorylation pho hosp spho hory ho ry yla lati tion ti on alterations alt lter erat er atio at ions io ns occurred occur ccur u re r d on individual residues of cTnI in heart failure, likely reflecting an imbalance in kinase/phosphatase activity. Such changes can be reversed by cardiac resynchronization.

Key words: heart failure; mass spectrometry; cardiac troponin I; multiple reaction monitoring; phosphorylation

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Introduction Cardiac TnI (cTnI) plays a key role in the regulation of contraction and relaxation. As part of the thin filament, the Tn complex (cTnI, troponin T (TnT) and C (TnC)) along with tropomyosin (Tm) regulates the actin filament interaction with the thick filament (composed primarily of myosin) in a Ca2+-dependent manner. Human cTnI can be phosphorylated at multiple amino acid residues S22, S23, T30, S41, S43, S76, T77, T142, S149, and S165 (the numbering excludes the initiating methionine), and studies have shown that phosphorylation at different residues can alter function.1-2 For example, phosphorylation of S22/23 located in the cardiac-specific NDownloaded from http://circ.ahajournals.org/ by guest on April 24, 2018

terminus of cTnI (residues 1-32) by protein kinase A (PKA),3 C (PKC),4 D (PKD),5 or G (PKG)6 alters its interaction with TnC, reduces myofilament Ca2+-sensitivity, enhances sliding sliidiingg velocity, vel eloc ocit oc ity, y, and thus contributes to the acceleration of relaxation.7 Similarly, phosphorylation at known PKC itees significantly sign si gnif gn ific if fic icaantlly impacts im ddistinct i tinct from S22/ 2//23 2 pphosphorylation. hosp ho s horylation.2,7 Other sites function with effects dist S22/23 sites itees demonstrated demonstrrat ated ed inn vvitro ittro orr iinn an animal nim mal models moddells include: in nclludee: e: T30 T30 (mammalian (maammal mmal alia iann st sterile terril ilee 20-like 20-l 20 -lik -l ike kinase ik kinnas ki nase 1 10 (Mst1)); Mst s 1) 1)); );8 S4 S41/S43 S41/ 1/S4 S443 (PKC); ( KC (P KC); ) 9 S7 ); S76/T77 76/ 6/T7 T777 (kinases T7 (kkin nas asees es undetermined); und n eteerm ermine mine ned) d);10 d) T142 T 142 ((PKC-ȕ,, ; PKCPK C-ȕ, ȕ,, ȕ, , ;;11 S1 , S1499 (p21(p2 p 112,13 ,13 13 naase ((PAK)) PAK) PA K )12 K) aand an d S1 S S165 6 (PKA). 65 (PK PKA) A)..14 Ho A) However, Howe w veer, tthe we h rroles he oless ooff th ol thes these esee ph es phosphorylatable hos osph phor ph o ylatable or activated kina kinase

residues are not well understood. In end-stage failing hearts, a number of studies have found the phosphorylation state of S22 and/or S23 is significantly reduced compared to control myocardium, a change which may lead to an increase in Ca2+ sensitivity.3,15,16 The phosphorylation state of the other sites are less clear. Although there are several phospho-specific antibodies available (against S22/23, S23, S43, and T142), most have not been fully validated for specificity and with cross reactivity with neighboring phosphorylatable amino acid residues. Mass spectrometry (MS)-based methods are able to simultaneously measure the state of

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multiple phosphorylation sites.10,17 Our group has employed the newly developed multiplex quantitative proteomics approach, multiple reaction monitoring (MRM)18, which allows sitespecific quantification of each phosphorylatable residue. MRM assays have many desirable characteristics including: 1) quantification of both the phosphorylated and unphosphorylated peptides; 2) monitoring of the precursor (Q1) and several fragment (Q3) ions of the peptide provides a high degree of specificity; 3) sensitivity is greater than other MS-based techniques due to the two-stage filtering Q1 and Q3 which increases the signal-to-noise ratio; 4) the ability to provide absolute quantitation of each targeted phosphorylatable residue by incorporating Downloaded from http://circ.ahajournals.org/ by guest on April 24, 2018

stable isotope-labeled peptides as internal standards. Because both isotopes are chemically and 13 physically identical, the labeled peptides (N15) will co-elute with the endogenous us pep peptides e tide tide dess (N13 ),

and integration of the area beneath both peaks permits accurate quantification of the phosphorylation phhos osph phor ph oryl ylat yl atiionn le at lev level. vel. Using MRM, MRM, for forr the the first fir irst st time, tim imee, we we qquantified uantiffieed levels lev evel ev elss of of pphosphorylation ho pho hosp hory ylaati tioon on ooff 144 ssites i es oon it n cTnI S4, S5, S198 cT TnI n iincluding n lu nc ludi dinng di ng 6 nnovel oveel ov el ssites itees S it 4 S 4, 5, Y25, Y25 25,, T5 T50, 0, T180, T180, 180, and and S 1998 in in human hum uman n left lef eftt ventricles v nt ve ntri ricclees ri es ffrom rom ro m failing ischemic ischem mic (ISHD) (IS ISHD HD)) and HD an nd di ddilated late la tedd (I te ((IDCM) DCM) DC M) ccardiomyopathy a di ar d om omyo yo opa path thyy he th hear hearts a ts aand nd ddonor o or hhearts on eart ea rtss from rt apparently healthy individuals. This is the first determination of the relative phosphorylation of the cTnI PKC site T142, PKA site S165, and several novel sites in human heart in vivo. Overall, there is a switch in phosphorylation from the PKA to the PKC sites in ischemic and dilated cardiomyopathy, as well as modulation of several novel sites, a finding that was recapitulated in a canine pacing model of heart failure (HF). The present study provides important insight into the status of cTnI regulation in heart disease.

Methods Cardiac Myofibril and cTnI Protein Preparation 4


Human left ventricular free wall transmural tissue samples were obtained from explanted endstage failing hearts with ISHD and IDCM during heart transplant surgery, as well as from unutilized healthy donor hearts (n = 10 per group). Tissue was rapidly frozen and stored in liquid nitrogen (clinical information was shown in Supplemental Table 1). Ethics approval was provided by St. Vincent’s Hospital (#H03/118), Sydney Australia and by The University of Sydney (#09-2009-12146). Canine Model of HF and Reverse Remodeling after CRT Left ventricle tissue (LV) was collected from adult mongrel dogs; groups included: normal Downloaded from http://circ.ahajournals.org/ by guest on April 24, 2018

(Control), dyssynchronous pacing-induced heart failure (HF dys ), or cardiac resynchronization herapy (CRT) as previously published (n = 4 per group).19 All protocols were approved ap pprov pro ed bby y th thee therapy Animal Care and Use Committee of the Johns Hopkins Medical Institutions. At the conclusion of o the he pacing paci pa cing ci ng protocol, prooto oco col, l, hearts were extracted underr iice-cold cee-cold cardiopl cardioplegia plleg e iaa aand nd frozen in liquid nd nitr ni nitrogen rogen (see Su Supp Supplemental ppleement pp ntal nt al M Methods etho et hoods ds for for det details). taiils). MRM MR M Development D ve De velo lopm lo pm men nt aand ndd Op Opti Optimization t mi ti miza zati za tion ti on n Possible phosphorylation phoosppho h ry ryla l ti la tion o sites on sit i es e on on human huumaan cTnI cTnI were wer eree selected s leect se cted ed uusing sin ingg in ssilico ilic il icoo ph ic phosphorylation hos osph phor ph oryl or y ation prediction algorithm (NetPhos 2.0) and MS data obtained from the LTQ Orbitrap LC/MS/MS analysis of the isolated cTnI. Three to five MRM transitions per peptide were designed based on either MS/MS identification acquired data or prediction by MRMPilot™ Software 1.0 (AB SCIEX). Each MRM assay was optimized manually on a 4000 QTRAP MS instrument (Supplemental Methods). Synthesized Internal Standards (SIS) SIS peptides (unphosphorylated peptides had an N15 stable isotope label, while phosphorylated peptides were unlabeled and contained N13) were produced by solid-phase peptide synthesis

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(New England Peptide). For each phosphorylation site, a series dilution of light/heavy peptides were made to produce 6-point calibration curves at 0.125, 0.25, 0.5, 1, 5, and 10 fmol/μl with and without matrix comprising a pool digest of all tissue samples used (Supplemental Methods). A control peptide (NITEIADLTQK), which does not have any known post-translational modifications (confirmed by mass spectrometry and prediction software), was used to determine the total quantity of cTnI (fmol) in the sample. Q-Trap Nano-LC/MS/MS Analysis The mixture of peptides from the in gel digestion of cTnI proteins were reconstituted with 20 ȝl Downloaded from http://circ.ahajournals.org/ by guest on April 24, 2018

HPLC water containing 0.1% formic acid. MRM analyses were performed on a 4000 QTRAP Analyst 1.4.2 hybrid triple quadrupole/linear IT mass spectrometer (AB SCIEX) operating with th h An A alys al ystt 1. ys 1.4 4. 4.2 software oftware scheduled experiments in positive ion mode (Supplemental Methods). Statistical Analysis St tattis isti tica ti call An Anal lys ysiis is Peak area was P eaak ak detectionn and and quantification qua uant ntif nt ifiicat if icat atiion ion of ppeak eakk ar ea reaa wa as determined detter termin rmin i ed ed with witth Multiquant Mulltiq qua uant nt software sooft ftwa waree vversion wa errsiion 2.0 SCIEX) manually ensure correct 2. .0 (AB (AB SC CIE IEX) X)) aand nd iinspected nsspe p ctted m anua an uallly to ens nsur ns urre co cor rrec ectt pe peak a iidentification ak deent n ifficcat atio io on an aand d quan qquantification. uan ntiifi fica caatiion o . Measurements were performed then reduce technical variation. Measuremen ntss w eree pe er perf r orrme rf med in ttriplicate ripl ri plic icat ic atee an at andd th hen aaveraged veera rage gedd to red ge educ ed ucee te uc tech c ni ch nica call va ca vari r ation. Thee ri quantity (fmol) of each peptide was calculated based on the linear standard curve, and then the phosphorylated peptide was normalized to the total quantity of cTnI. For human samples, data was analyzed using one-way analysis of variance (ANOVA) on ranks, followed by Dunnett’s multiple comparisons post-hoc test. For canine samples, data was analyzed using a one-way ANOVA, followed by Bonferroni multiple comparisons post-hoc test. All calculations were done using SigmaPlot v11 (Systat), with a P-value less than 0.05 denoting significance. Human data are presented as median, 1st quartile, and 3rd quartile while canine data are presented as mean ± SEM.

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Validation of MRM Quantification by Immunoblotting Analysis The phosphorylation status of cTnI for subset of sites was verified by Western blotting (Supplemental Methods).

Results Human cTnI contains 209 amino acids, including 12 Ser, 8 Thr, and 3 Tyr residues. Using NetPhos 2.0, a phosphorylation prediction algorithm, we predicted 16 potential phosphorylatable residues. Fourteen of these predicted phosphorylatable residues were experimentally verified Downloaded from http://circ.ahajournals.org/ by guest on April 24, 2018

using LTQ Orbitrap MS analysis. The 8 known phosphorylation sites (S22, S23, S41, S43, S76, S198) T77, T142, S165) and additional 6 novel phosphorylation sites (S4, S5, Y25, T50, T5 50, T180, T18 180, 0, S 198) 19 8 were observed in the 30 different human heart tissues. To verify and quantify all phosphorylated amino tryptic amin inoo acids, acid ac ids, id s, MRM MRM assays were developed for the he ccorresponding orresponding trypt ptic ic ppeptides eptides that contained oone ne or o moree of tthese hesee rresidues. he esid es idue id uees.. MRM Optimization Phosphorylation Mapping MR M Workflow Workkfl flow ow w Development Deveelo Deve opm p ent ent aand ndd O ptim pt imiz izzattio ion n aand ndd P hosp ho spho sp horryl rylation nM apping app ping Thee wo workflow study Supplemental Fig. Th work rkfl rk flow fl ow uused sedd in tthis se hiss st hi stud udyy is ooutlined ud utli ut line li nedd in S ne uppl up plem pl emen em enta en tall Fi ta Fig g. 1 aand nd sschematically chem ch emat em atic at ical ic ally al ly iillustrates llus ll ustr us trat tr ates at es the process for developing and using MRM assays to quantify site-specific cTnI phosphorylation. The steps necessary to develop the pipeline were: i) development of MRM assays for each unphosphorylated and phosphorylated tryptic peptide containing a potential modified residue(s) and for a control peptide (NITEIADLTQK) not predicted to be modified that could be used to estimate the total quantity of cTnI in the sample, ii) optimization of sample preparation in order to isolate cTnI from LV tissue, and iii) optimization of tryptic digestion conditions in order to consistently generate the endogenous tryptic cTnI peptides for measurement by the MRM assays.

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For each MRM assay the MS parameters for each peptide were optimized and transitions selected to achieve the greatest sensitivity (individual peptide data shown in Supplemental Figs. 2 to 15). Figure 1 shows examples of in vivo MRM assays for representative cTnI peptides. Supplemental Table 2 and 3 lists all peptides and transitions used for the MRM assays to monitor and calibrate each phosphorylation site. To test the matrix effect in vivo, we used composite matrix composed of a tryptic digest of a pooled sample (10 μg each of the donor, ISHD, and IDCM), and the coefficient of variation (CV) was below 1% for the calibration curves with or without matrix. Based on this we determined the lower limits of detection and quantification Downloaded from http://circ.ahajournals.org/ by guest on April 24, 2018

(LLOD and LLOQ). Figure 2 shows the data for one of the novel sites, S198 (Supplemental Figs. 2-15 provide the MS/MS spectrum for all peptides and their MRM optimization). optimization) n).. Next, we determined the optimal sample processing to ensure consistency and preservation shown pr resservat atio tio ionn ooff pphosphorylation hosp ho s horylation status. As show wn inn Supplemental Supplementaal Fig.16, Fig. g 16 g. 16, there is essentially no difference between samples processed gel-based (SDS-PAGE di fer diff e ence bet tweeen n sa samp mp ple less pr proc oces esssed es sed uusing singg a gel l-bbaseed (S SDS-PA PAGE GE E ffollowed ollo owe wedd by iin-gel n-gel gel digestion) Samples prepared SDSdige di gest ge stio st ion) io n)) or or direct dire di reectt pprotein roteein ddigestion iges ig esti es tion ti on of of the the iisolated sol olat ol ated ed myofibrils. myo yofi fibbril fi br ls. s S am mpl p es were werre pr prep epaared ep d bby y SDS S DSSupplemental PAGE for the he ssubsequent ubse ub sequ se quen qu en nt analysis anal an a ys al ysis is of of the th he 30 human hum uman an ttissues issu is suues (See (Se Seee de ddetails tail ta ills in S u pl up plem emen em enta en t l Fig. 17 and Supplemental Table 4). The final step required for optimization of the pipeline was to ensure efficient and complete typtic digestion of cTnI. Importantly, despite efforts at optimization, by either extending the reaction time or adding a second batch of trypsin after 12 hours, several peptides were consistently miscleaved. For example, the peptide containing T142 is surrounded by basic residues and this resulted in the consistent generation of a miscleaved 5 amino acid peptide. Therefore, to ensure accurate quantitation at this PKC site, MRM assays were developed for both the non-tryptic and single-tryptic miscleaved phosphorylated and unphosphorylated peptides.

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Quantitation of Levels of Phosphorylation In total, we quantified levels of phosphorylation of 14 cTnI sites in left ventricle obtained from ISHD, IDCM and donor hearts (n = 10 per group), clinical data presented in Supplemental Table 1). Each assay was carried out in triplicate. The MRM quantitative data showed that phosphorylation status is decreased or increased in a site-specific manner (Fig. 3, Supplemental Table 5, Supplemental Fig. 18). Figure 3 shows the phosphorylation occupancy of each amino acid residue (fmol phosphorylation/fmol cTnI). Supplemental Fig. 18 summarizes the fold change for each site. Compared with the donor samples, the amount of phosphorylation of the NDownloaded from http://circ.ahajournals.org/ by guest on April 24, 2018

terminus cTnI sites was significantly reduced in ISHD and IDCM. The monophosphorylated S4 or S5 form were 25% and 21% lower in ISHD, and 14% (not significant) and 10% 100% lo llower w r in we IDCM, DCM, respectively, compared to cTnI isolated from the control donor myocardium (Supplemental exist primarily Sup uppl plem pl emen em enta en tal Fig. ta Fiig. g. 18). In the donor hearts, these se aadjacent djacent sites ex xist pr prim imarily as im diphosphorylated not heart di hos diph o phorylat ated ed species, spe peccies es, bu butt this thiss species speci peciees was wass no ot detected detec ecte ec tedd in n tthe he ffailing aili ai liing ng hea eart ea rt ggroups rooupps (F ((Fig. ig. 3, ig 3 Supplemental Supp Su pple pp leme le ment ntal nt al Table Tabl able le 55). ). PKA-mediated mono S22/S23 The PK KA-m med edia iate ia t d mo m no aand n ddi-phosphorylated nd i ph iphos osph os phor ph oryl or ylat yl a ed at d forms for orms ms of of S2 S22/ 2 S2 2/ S233 were were also als lsoo reduced in ISHD and IDCM compared to donor hearts, monophophosphorylated were 34% and 61% lower with ISHD, and 22% (not significant) and 76% lower for IDCM. While the diphosphorylated species was quantified in the donor hearts, levels were too low to quantify in the failing heart groups. Notably, the monophosphorylation of S23 was 857% greater than S22 in the control hearts, suggesting a potential priority of S23 phosphorylation over S22. There is also greater S23 phosphorylation than S22 in the failing hearts, but the ratio is reduced to 472% in ISHD and 195% in IDCM. Quantitation of the phosphorylation was verified by immunoblot for the PKA sites using the anti-S22 and/or 23 phospho-sensitive antibody (Supplemental Fig. 19) versus an

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antibody against total cTnI, 8I-7. The immunoblot results support the MRM data, showing a decrease in phosphorylation of the PKA sites in HF. The N-terminal novel site, Y25, is located near S22/S23 and is the only phosphorylatable Tyr residue in cTnI. Residue Y25 was more phosphorylated (Fig. 3) than S22 or S23 and dephosphorylated in ISHD (42%) and IDCM (43%) compared with non-failing donor samples (Fig. 3). Immunoblotting using an anti-phospho-Tyr antibody verified the reduction in phosphorylation of cTnI in HF (Supplemental Fig. 19). When compared to donor samples, the phosphorylation states of sites just downstream Downloaded from http://circ.ahajournals.org/ by guest on April 24, 2018

from the N-terminus were increased significantly in ISHD and IDCM. Monophosphorylation of PKC site S41 was increased in ISHD (22%) and IDCM (34%), as was S43 in IDCM ID DCM (56%) (56 56%) %) ((see see se Supplemental Table 5). T50 was phosphorylated, but there was not significant change between groups. grou gr ouups ps.. The The oother th her ssites ites located within the same ffunctional unnctional domain in n off cTnI cTnnI also had increased cT phosphorylation diphosphorylated (18%); IDCM: ph hosphorylatio os on (for (fforr ISHD: ISH HD: D: S76 S76 76 (44%), (44 44% %), T77 7 ((20%), 20% %), dip iphhos ip hospho h ry ho ylaate tedd (1 18%) %);; fo %) forr ID DCM CM:: S7 S76 (38%), (29%), (31%)). 38%) %),, T7 T777 (2 (29% 9% %),, ddiphosphorylated iphhosp ip hospho hory ho yla late tedd (3 te (31 1%) %))). IInterestingly, n ereesti nt es ing ngly ly,, similar ly siimi m la larr to t the thee other oth ther err pairs pair airs rs of of adjacent adjaace adja cent n phosphorylatable phosphorylat atab able ab l residues le res esid id due u s in cTnI, cTn TnI, I there I, the h re was was a ppreference r fe re fere r ncce fo re forr on onee of tthe he rresidue esid es idue ue ppair airr (S ai ((S41>S43 41>S43 and S76>T77) (Fig. 3). In the important inhibitory domain of cTnI, the sole phosphorylated residue (T142) was increased in ISHD (58%) and IDCM (82%) compared with the donor heart. Remarkably, this site had the greatest amount of phosphorylation of all sites (Fig. 3). The PAK target site S149 was not phosphorylated, as only the non-phosphorylated peptide was observed in the samples. It is, however, possible that the phosphorylated peptide is present but below the detectable limits of this assay (Supplemental Table 5). Similarly, the phosphorylated form of T30 was not observed. Finally, the three novel phosphorylation sites in the C-terminal region of cTnI were all

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increased in HF compared to donor hearts: S165, which was only significantly greater in IDCM (174%), T180 (44% and 36% greater for ISHD and IDCM), and S198 (136% and 127% greater for ISHD and IDCM). Phosphorylation of Novel Sites in Canine Model of HF and CRT The extent of phosphorylation of the three novel residues, S165, T180, and S198 were quantified in a well characterized canine model of HF dys and after treatment with CRT. These three sites were chosen because the tryptic peptides encompassing these phosphorylatable residues are conserved between dog and human. As shown in Fig. 4, HF dys led to greater phosphorylation of Downloaded from http://circ.ahajournals.org/ by guest on April 24, 2018

these three amino acid residues (S165: 659% increase, T180: 249% increase, S198: 479% increase). ncrease). This was reversed in dogs treated with CRT, with phosphorylation levels leve veelss ffalling alli alli ling ng relative elative to HF dys conditions by 83%, 30%, and 82% for S165, T180, and S198, respectively.

Discussion D isccus u sion This Th hiss study stu udyy provides pro rovi vide dess novel de noovel insights in nsi sigh g ts gh t into int n o site-specific si e-speci site pec ficc phosphorylation phhosphhorryl y atiion off cTnI cTn nI in i hhuman uman um an myocardial myoc my ocar oc ardi ar dial di al ssamples. ampl am ples pl es. It eestablishes es stab st abli ab lish li shes sh es tthat hatt hu ha huma human mann cT ma cTnI nI iiss more more extensively ext xten ensi en sive si vely ve ly phosphorylated pho hosp spho sp hory ho ryla ry late la tedd than te than previously reported, with 14 modifiable amino acid residues located throughout its entire length. We also provide evidence of HF-related phosphorylation changes for the established PKA- and PKC-modifiable amino acid residues and several newly identified phosphorylated sites, specifically: S4, S5, Y25, T50, T180, and S198. Finally, data was provided indicating the increased phosphorylation levels of three novel sites located in the C-terminus of cTnI are observed in an animal model of HF with dyssynchrony, but intriguingly reversed by CRT. Structure and Function of cTnI domains and impact on contractile function To provide context for the impact of altered phosphorylation in HF, a brief review of cTnI domains and function is necessary (Table 1).20-37 Cardiac TnI, the inhibitory protein of troponin, 11


forms a complex with TnT and TnC and is responsible for inhibiting activation of muscle via its interaction with actin-tropomyosin (Tm) at diastolic levels of calcium. When free calcium levels increase during systole, calcium rapidly binds to site II of cTnC and the affinity of cTnI for TnC is increased due to opening of the hydrophobic patch on the N terminal-lobe of TnC. The cTnIactin-Tm interaction occurs at two key domains of cTnI, the inhibitory region (128 to 147 in human cTnI) and a more C-terminal region (164 to 209) with a helical “switch peptide” (147163) bridging these two regions. Unique to cardiac muscle, the opening of the hydrophobic patch on TnC with calcium binding is facilitated by binding of the “switch” peptide of cTnI to the NDownloaded from http://circ.ahajournals.org/ by guest on April 24, 2018

terminal lobe of TnC.20 This is discussed in detail in numerous excellent reviews.2,21 Other key domains of cTnI include an N-terminal extension, unique to the hee cardiac car a di diac ac isoform. soform. Phosphorylation at the S22/S23 sites alters the shape of the cTnI, resulting in a bend at a hinge hi ing ngee do doma domain main ma in inn this this region, which permits an intramolecular in ntr t amolecular inter interaction erracti tion on bbetween etween the acidic region egiion o at the mo most s N-terminal N-ter -ttermi mina mi nall re na resi residues siidu dues es aand nd tthe hee inh inhibitory hib bitoory ory domain. doomain main n.22 P Physiological h siol hy olog ogic og ical all stu studies tudi tu d ess sspeak di peak pea too an an im impo important p rt po rtaant ant role rolle le ooff ph pho phosphorylation osph phor ph oryl or ylat yl atio at ionn of ccTnI Tn nI att tthe he S S22/S23 22/S 22 /S23 23 sites sit ites es in in augmenting au ugm men nti ting ng relaxation, rellaxat axatio io on,, acceleratingg fforce orce or ce ffrequency r qu re quen ency cy rresponse, esspo pons nse, ns e, aand nd ppotentially oten ot enti en t al ti ally ly increasing inc ncre reas re a in as ingg contractile co ont ntra ract ra ctil ct ille po powe power werr an we aand d lengthdependent contraction (Frank-Starling effects).27 The PKC sites lie within the IT-arm of cTnI (S41 and S43), a region that structural studies suggest is relatively rigid, and is involved in stabilization of cTnI to the thin filament, including interaction with TnT and the C-lobe of TnC. In contrast, T142 lies within the key inhibitory region that switches from actin-Tm to TnC binding as part of the activation process. Accumulating information from structural, biochemical in vitro and in vivo studies suggest that PKC phosphorylation at S41, S43 and T142 opposes effects produced by phosphorylation at the N-terminal extension residues S22 and S23 on relaxation kinetics.7,21 In addition to the PKC sites

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S41 and 43, sites T50, S76 and T77 lie within the IT-arm domain important for stabilizing cTnI on the actin filament. The most C-terminal region of cTnI contains a second actin-binding site which, though not directly involved in inhibiting acto-myosin interaction, may facilitate the transition to the activated state.29-30 Most of the C-terminal domain was not mapped in the crystal structure of troponin, suggesting it is highly mobile. Recent studies indicate this domain of cTnI influences the azimuthal position of troponin on Tm and that change in this region (193-209) as a result of mutation or proteolysis during ischemia/reperfusion or stretch injury, impairs muscle activation or relaxation respectively.31,35-37 Downloaded from http://circ.ahajournals.org/ by guest on April 24, 2018

Sites within the N-terminal extension of cTnI The data from the MRM assays confirm previous studies that have reported a sig significant igni nifi f caant fi reduction eduction in basal cTnI phosphorylation at S22/S23 in human HF,3,15-17 most likely reflecting the doown wnre regu re gula gu lattio la tion of of ȕ-adrenergic ȕ-adrenergic receptors and des esen es nsitization of si ignal allin ingg in the PKA pathway downregulation desensitization signaling with with h HF. Interestingly, Inteere rest s inglly, our our data dat ataa show shoow sh ow that thhat pho pphosphorylation hosphhoryllat atio ionn of of S23 S233 predominates pre redo domiina do nate tess over over e S S22 22 or or S22/S23 S222/ 2 S2 S233 inn donors don onoorss and and patient pat en pati nt samples. samp sa mple mp less. In aagreement grree eem ment w men with ithh th it this is ffinding, i di in ding ng,, several ng seveera rall st stud studies udiiess in ud n vvitro ittro using synthetic syntheetiic peptides pept pe ptid pt ides id es38 re reported repo p rtted ddifferent po i fe if fere rent re nt pphosphorylation hosp ho spho sp hory ho ryla lati la tion ti on kkinetics inet in e iccs be betw between twee tw e n S2 ee S222 an and S23 residues by PKA, with phosphorylation of S23 occurring 10-fold faster than S22. Zhang and colleagues also found the murine equivalent of S23 was phosphorylated several-fold more rapidly than S22 by PKA.39 In contrast, a top-down MS analysis of human cTnI10,17 isolated from human hearts revealed that S22 rather than S23 was the only monophosphorylated form and S23 was found only in diphosphorylated cTnI. Taken together, studies suggest potential differences in the biological status of individuals and may reflect alterations in turnover rate of the phosphorylation/dephosphorylation for S22 and S23 in vivo. There are three novel phosphorylatable residues in the N-terminus of cTnI: Y25, S4 and

13


S5. Y25 is of particular interest because it is the only Tyr residue modified indicating that a novel kinase signaling pathway may regulate TnI function. Y25 is one of the most highly phosphorylated residues in control hearts. Its close proximity to the well-studied S22/S23 phosphorylatable residues raises the intriguing possibility of synergy between these sites and that the phosphorylation state of one site could influence the degree of modification for the other sites. The other two novel phosphorylatable (S4/S5) residues are located at the extreme Nterminus of cTnI, a region unique to cardiac isoform and not present in either slow or fast skeletal TnI. A series of sequential N-terminal deletion mutants suggest that residues 1-15 play a Downloaded from http://circ.ahajournals.org/ by guest on April 24, 2018

role, albeit minor, in transmitting the phosphorylation signal to other myofilament proteins but are not directly involved in binding to cTnC.40 Potentially, S4 and S5 phosphorylation phosphoryl yllat atio i n could io coul co uldd ul enhance the interaction of the N-terminus and the internal inhibitory region of cTnI that specifically peccif ific ical ally lly ooccurs ccur urss wi ur w with th phosphorylation30 of S2 S22/S23. 22/S 2/S23. Interestingly, Interestinggly l , the th he S4/S5 S /S5 diphosphorylated S4 fform orm m is exclusively exclussiv ivel e y ffound ound ou nd iinn donor dono do noor myocardium. myoc my ocarddiuum. PKC PK KC si site sites tess of ccTnI Tn nI Our data dem demonstrate, mon onst sttra rate t , fo te for th thee fi first irs rstt ti time, ime me,, increased in ncr crea ease ea s d ph se phos phosphorylation osph phor ph o yl or y at atio ionn of tthe io he P PKC KC ssites ites it es S S41, 41, S43 (inn IDCM only) and T142 of cTnI in human HF, and a preference for the phosphorylation of S41 over S43. As debated by Solaro and van der Velden,2 the functional role of these sites in human heart and HF is not clear. However, in vitro and mouse mutant data suggest (Table 1) that increased phosphorylation at these sites would be expected to slow the kinetics of contraction, decrease Ca2+-sensitivity, and potentially depress both relaxation and contractility in vivo. Although T142 is a substrate of PKC in vitro, phosphorylation of this site has not been previously documented in the human heart. Strikingly, the extent of phosphorylation of T142 and the large change observed in ISHD and IDCM compared to donor hearts suggests that

14


phosphorylation at this site could have the largest functional impact amongst the PKC sites and those altered in HF. A transgenic mouse model with a pseudophosphorylated mutant of all three PKC sites (S43E, S45E, T144E equivalent to S41, S43, and T142 in human) resulted in depressed contractility and relaxation and decreased force generation in cardiac muscle even though the mutant is expressed at very low levels.28 These authors used computational analysis to propose that the effect of mimicking enhanced phosphorylation at these PKC sites in this model is related to delayed entry into the crossbridge cycle and persistence in the activated state. Thus, markedly enhanced phosphorylation at this site is likely to have significant impact in the Downloaded from http://circ.ahajournals.org/ by guest on April 24, 2018

human heart. Phosphorylation sites in the near amino terminal domain Phosphorylated sites in this region (T50, S76, and T77) could impact the interaction with the C21,30 1,3 ,30 30 obee of of cT cTnC nC and ndd C -terminal regions of cTnT.21 S41 S 41 and S43, when when phosphorylated, ph reduce lobe C-terminal 9,30 30 aacto-S1-ATPase cto o-S1-ATPase activity. acctiv i it i y.9, Whereas W here r as pprior re rior ttop opp dow down own an ana analysis alyysis is was ass una unable ablee to o ddifferentiate iffer eren ntiiat a e S7 S76 10,17 1 ,17 ,1 versus vers rsus us T T77 777 pphosphorylation, hosp sphhory rylaati t on,10 tthe he cu current urre rent nt stu study tudy dy w was ass aable blee to bl o ddefine efin inee th that hatt bot both othh S7 S766 and andd T77 T77 are are

phosphorylat ted ed.. The The ex eextent t nt ooff mo te mono noo and nd ddiphosphorylation ip pho hosp spho sp hory ho rylaati ry tion on of of S7 S76 and and T7 T777 is increased inc ncre reeas ased in both phosphorylated. forms of HF studied here. Phosphorylation of sites at the C terminus of cTnI There are three phosphorylation sites (S165, T180, and S198) located within the C-terminal region of cTnI. The domain includes residues 152-199 and is essential for full inhibitory activity and Ca2+-sensitivity of myofibrillar ATPase activity.32 The importance of this region is suggested by the fact that mutations in cTnI S165 and S198 cause hypertrophic cardiomyopathy.33 Based on MRM data, T180 is more highly phosphorylated than the other two residues in this region; however, phosphorylation of all three sites is increased in disease. S165 in human cTnI is

15


phosphorylated by PKA in vitro14 but our report is the first to show its phosphorylation in vivo. S165 is in the C-terminal region of cTnI that contains the Ca2+-sensitive TnC and actin-binding sites, and influences the affinity of the cTnI inhibitory region and TnC.30 In vitro phosphorylation of S165 reduces the affinity of cTnI for TnC.14 S165 is close to A163, which is a His in the skeletal isoforms. This residue has been implicated in the regulation of the acidosisresponsiveness conferred by cTnI.32,34 Mutation of the Ala to His at this position (163 in human or 164 in mice) protects against the deleterious effects of acidosis in a murine model of myocardial infarction.34 Downloaded from http://circ.ahajournals.org/ by guest on April 24, 2018

Residue S198 is localized within the “second” actin-binding site30 implicated in the tructural reorganization associated with Ca2+-activation, termed the “fly-casting”” m mechanism”. eccha hani nism sm””.41 structural We and others have shown the truncation of the C-terminus occurs in experimental global sch hem emia ia, ischemia-reperfusion, isch is chem mia ia-reperfusion, volume overload, d, aand nd ischemic hu umann m yocardium.31,35-37 In ischemia, human myocardium. vvivo ivo o and in vitro st studies tudi dies es showed sho howe w d th we that hat truncation trrunncattiio on re resulted esuulted ed in decreased deecreeased ma maxima maximal mall force foorcee ac activation, ctiivati tion on, a divergent dive verg rgen ent increase inccreasse in maximum in maxi ma ximu mu um Ca2-ac -activated accti tiva vate va tedd ac te aactin-Tm-myosin tiin-Tm m-m -myyosi sinn S11 ATPase si ATPas TPas a e activity, acti ac t viity ti ty,, and an nd a 35,37 ,37 ,3 37 tructural change cha hang ngee in tthe ng h llocation he occat atio i n of o aactin-Tm c in ct in--Tm ffilament. illam men ent. t 35 Phosphorylation Phos Ph osph phor o yl or ylat atio ionn of o S198 S19 1988 could co protectt structural

against proteolysis as previously suggested,31 which would reduce these potential negative functional effects of C-terminal proteolysis. Thus, we expect functional and structural alterations resulting from the phosphorylation on one or more of the C-terminal region residues of cTnI.

Conclusions We developed 30 peptide MRM assays in order to monitor the multiple phosphorylatable residues of cTnI and provided for the first site-specific quantitation of the level of phosphorylation of each site in human diseased and control samples. There are numerous functional studies that indicate the heart failure associated changes in phosphorylation including 16


reduced phosphorylation at the S22/S23 sites and enhanced phosphorylation at S41, S43, and T142 are likely to negatively impact function. These effects include impaired contractile power, impaired relaxation, and impaired responses to elevated heart rate. In addition, there may be functional implications of altered phosphorylation status of several residues not previously known to be phosphorylated. Overall, with HF there is a shift in phosphorylation involving decreases in novel residues in the N-terminal extension (S4, S5, S4/S5, Y25), increases at others in the near N-terminal region (S76, T77, S76/T77), and within the C-terminal domains (S165, T180, S198). Prior experiments in vitro and in vivo suggest that any disturbance in the balance of Downloaded from http://circ.ahajournals.org/ by guest on April 24, 2018

phosphorylation of cardiac proteins may lead to substantial functional consequences in heart disease, but it will be residue dependent. This implies that selectivity of various kinases/phosphatases for each residue will impact function. It is of interest that three of the novel sites dephosphorylated CRT, no ove vell si site tess located te loccate lo teed in the C-terminus were depho osp s horylated upon onn CRT RT T, reversing r versing the elevation re inn phosphorylation phosphoryla lati tion ion induced ind nduc ucced in in HF F dy This his suggests suggesstss th sug their heir ppotential hei oten ot enttiall cclinical liini n cal im impo importance. port rtan rt an nce ce.. Fi Fina Finally, naall l y, dyss . T this his study stu tudy dyy may may ay spur spu purr the th he development d ve de velo l pm lo pmen entt of en of nnovel ovel ov el bbiomarkers. io oma markkerrs. As ccTnI TnII is a vvaluable Tn aluuabl al uabl b e di dia diagnostic agnost agn nost s ic marker for m myocardial yoca yo card ca rdia rd i l in ia iinfarction, faarc rcti t onn, me meas measurement sur urem emen em entt of modified en mod odif ifie if iedd forms ie foorm rms of ccirculating ircu ir cula cu lati la ting ng ccTnI TnII may Tn provide a sensitive assay for the functional status of the myocardium.

Funding Sources: This work was supported by the National Heart Lung and Blood Institute: Proteomic Initiative contracts NHLBI-HV-10-05(2) (JEVE) and HHSN268201000032C (JEVE, AMA, DAK), P01HL081427 (JEVE), P01HL77189-01 (DAK, JEVE), and R01 HL63038 (AMM); Johns Hopkins Clinical Translational Science Award (CTSA); and American Heart Association Postdoctoral Fellowships 10POST4000001 (PZ) and 11POST7210031 (JAK). Conflict of Interest Disclosures: Patent: Dr. Zhang, Murphy and Van Eyk have a patent application pending on “Novel phosphorylation of cardiac troponin I as a monitor for cardiac injury.”

17


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4. Kobayashi T, Yang X, Walker LA, Van Breemen RB, Solaro RJ. A non-equilibrium isoelectric focusing method to determine states of phosphorylation of cardiac troponin I: identification of Ser-23 and Ser-24 as significant sites of phosphorylation by protein kinase C. J Mol Cell Cardiol. 2005;38:213-218. M.. Pr Protein 5. Haworth RS, Cuello F, Herron TJ, Franzen G, Kentish JC, Gautel M, Avkiran M Prot otei einn myofilament kinase D is a novel mediator of cardiac troponin I phosphorylation and regulatess m y fi yo fila lame la ment me n nt function. Circ Res. 2004;95:1091-1099. Layland 6. Lay yland J,, Li J-M, Shah AM. Role of cyclic GMP-dependent protein kinase in the contractile response myocytes. Physiol. 2002;540:457-467. esppon onse se ttoo exogenous exoggen ex enou o s nitric oxide in rat cardiacc m yocytes. J Phys ysiol.. 20 ys 2002 0 ;540:457-467. Ramirez-Correa GA, Cortassa S,, St Stanley Calcium 77.. Ramirez-Co R a orr rrea eaa G A, Co Cort rtas rt assa as sa S Stan an nley B, B, Gao Gaao WD, WD D, Murphy Murphhy AM. Murp AM. Calc C alcciu ium m sensitivity, sens se nsit ns itiv i it ity, y, force force or frequency relationship PKA PKC fr req eque u ncy re ela latioons onship p aand nd ccardiac arrdi d ac a ttroponin ropo ro ponin I: critical po critticcal ca ro role le ooff PK KA andd P KC phosp pphosphorylation h pho phory orylaationn sites. ite tes. s J Mo s. Moll Cell Cell Ce ll Cardiol. Car arddio diol. 22010;48:943-953. 0110; 0;48 48:9 48 :943 :9 43-9 -9953 53.. Zhang Z,, Ya Yan Lii J, G Gee Q, Sun Phosphorylation 8. You B, Yan Yaan G, G Z hang ha ng Z Y n L, L L Q JJin in JJP, P S P, un JJ.. Ph Phos o ph phor oryl or ylat yl atio at ionn of cardiac io car a di d ac troponinn I by m mammalian Biochem 2009;418:93-101. amma am mali lian an sterile ster st eril ilee 20-like 20-lik likee kinase kina ki nase se 1. 1 Bi Bioc oche hem m J. J 20 2009 09;4 ;418 18:9 :933-10 1011 9. Burkart EM, Sumandea MP, Kobayashi T, Nili M, Martin AF, E. Homsher, Solaro RJ. Phosphorylation or glutamic acid substitution at protein kinase C sites on cardiac troponin I differentially depress myofilament tension and shortening velocity. J Biol Chem. 2003;278:11265–11272. 10. Zabrouskov V, Ge Y, Schwartz J, Walker JW. Unraveling molecular complexity of phosphorylated human cardiac troponin I by top down electron capture dissociation/electron transfer dissociation mass spectrometry. Mol Cell Proteomics. 2008;7:1838-1849. 11. Wang H, Grant JE, Doede CM, Sadayappan S, Robbins J, Walker JW. PKC-ȕ,,VHQVLWL]HV cardiac myofilaments to Ca2+ by phosphorylating troponin I on threonine-144. J Mol Cell Cardiol. 2006;41:823-833. 12. Buscemi N, Foster DB, Neverova I, Van Eyk JE. p21-activated kinase increases the calcium sensitivity of rat triton-skinned cardiac muscle fiber bundles via a mechanism potentially 18


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21


Table 1. Summary the known and novel phosphorylation sites in this study and putative effect of altered phosphorylation in HF Residue

Kinase(s)


S4 & S5

Unknown

S22& S23

PKA,3 3.&ȕ DQGį 4 PKD,5 PKG 6

Y25

Unknown

Domain within cTnI20-26 Acidic region of N-terminal extension, and interacts with inhibitory region (IR) when S22 and S23 are phosphorylated.

Physiologic effect of phosphorylation Unknown

N-terminal extension. Phosphorylation results in bending of hinge region and cTnI more compact; Promotes acidic region at extreme N terminus interaction with inhibitory region N-terminal extension

Desensitizes to Ca2+;May accelerate crossbridge kinetics; Decrease Ca2+ affinity of cTnC; Increases contractile power and relaxation and length dependent activation. unknown

S41 & S43

3.&ȕDQGį

IT Arm

T50

Unkn Un Unknown k ow wn

IT Arm

S76* S7 76* & T 77** T77* T142 T T1 42 2

Unknown Un nknow ownn PKC

IT-arm IT T-ar arm m re region egi gion o on Inte In tera te raactts wi ith t ccTnT Tn nT Interacts with Within With hin n iinhibitory nhib bit itor ory region reggion re

S165†

1 PKA PK KA14

T180 & S198

Unknown

“Switch “S Swi witc tch pe tc pept peptide” p id pt de” bbetween etwe et ween we en 2 acti tin bi ndi ding re gions; i actin binding regions; Adjacent to key residue for acidosis impact on contractile function.30-34 C-terminal region involved in position of troponin along tropomyosin; Also location of HCM mutants

2+

Putative Effect of altered phosphorylation in HF May inhibit interaction of acidic region of N- terminus with IR;22,25-26 Speculated to have similar impact as decrease in S22 and S23 phosphorylation.3 Inhibits lusitropic response to ȕ$5VWLPXODWLRQLPSDLUV relaxation and force frequency response; May inhibit length dependent activation (Frank-Starling Law).3-4,7,24,27 Putatively similar to S22,S23

Decreases Ca sensitivity; Slow kinetics in motility assays and stabilizes inhibition of activation of thin filament;9pseudophosphorylat ion decreases contractility and relaxation in vivo.7,28 Unkn Un Unknown k own

Unknown

Unknown U nkknow wn

Unknown Unnkn know ownn ow

Conflicting Connflicttin ingg ev evide evidence encee onn effec effect ct onn Ca Ca2++ se sensitivity; ens nsit itiiviity; Decreases Deccreasess cooperativity co oopperaativ vit ityy ooff aactivation; c iv ct ivat atio ion; io n9 n; Pseudophosphorylation Pseudo Ps doph phhos osph phor oryl ylat a io on in vivo vi ivo impa iimpairs mpaiirs irs re rel laxati laxa tion ti on.28 relaxation. Unknown Unkn Un know kn o n ow

,28 ,2 Im Impairs mpaairs rela relaxation; axaati t onn;99,28 Impairs Im mpaair i s con contractile ntra raactiile force. forcee.

Unknown

Impairs relaxation and force 7,9,217,9 ,21-22,2 22, 8 frequency response. resspo p nsse. e 7,9,21-22,28

Potential Pote Po tent ntia nt iall impact ia im mpa pact c on response ct tto o aacidosis cid idosis i andd switching swit itchi hing off binding from actin to TnC with activation.29-30 Reduced proteolysis31 Could impact on position of troponin along tropopmyosin, effects on activation.31,3537 Reduced proteolysis31

*These sites had been noted by researchers,10 however it was unclear which of the two sites were phosphorylated. † Had only been noted in vitro. Arabic numbers are referred to the citations in References.

Figure Legends:

Figure 1. Development of MRM assays for the representative cTnI peptide and its corresponding phosphorylated peptide. A typical Total Ion Chromatogram is shown for the 22


multiplex consisting of the peptides, S (p) SNYR, RPTLR, RP (p) TLR, ISASR, and I(p) SASR (Panel A). The extracted ion chromatogram for the unphosphorylated peptide (RPTLR with m/z 322.2/389.4) and its corresponding phosphorylated peptide (RP (p) TLR with m/z 362.2/ 468.5) eluting at 7.44 and 8.53 min (Panel B). Three transitions for the unphosphorylated and phosphorylated peptide corresponding to RPTLR at m/z 362.2/288.4, 362.2/468.5, and 362.2/565.8 (Supplemental Table 2 lists the other transitions used) (Panel C). The plot of peak area ratio vs. observed concentration (panel D) or calculated vs. observed concentration (panel E) with linear calibration curve slopes for the three transitions of the phosphorylated peptide Downloaded from http://circ.ahajournals.org/ by guest on April 24, 2018

RP (p) TLR. The ratio is the peak area of phosphorylated peptide RP (p) TLR (unlabeled peptide) six-ppoi oint n different nt dif iffe fere fe rent re n nt divided by the corresponding unphosphorylated (heavy labeled peptide) at six-point dilution ratio of concentrations 0.125, 0.25, 0.5, 1, 5, and 10 fmol/μl. The concentration of unnla labe belled be led ph phos sph phor orylation site T142 peptide ranges raang n es from subfemtomole subfem mto t mo ole to 10s of femtomole unlabeled phosphorylation levels, levvels, ve whereas wherea eass the thhe concentration conc co ncen nc entr en trat tr atio at ionn of of hheavy eavy la labeled abeled ed int internal terrna nall st sstandard an ndaard iiss kept kept cconstant onst on stan st an nt aatt 1 ffmol/μl. mol/μ mo Three Thre Th reee re re repl replicate plic pl icat ic atee measurements meas me asur urem e en em nts are are represented rep epre rese re sennted se nt d at at each eaach h cconcentration once on cent ntra nt raatiion ppoint. oiint nt.. Ci Circ Circle r lee = tra rc transition raansit itio io on 1 (m/z 362.2/288.4, 362.2/28 2888. 8 4, y2 = 0.3 0.3789x 378 789xx - 00.0931, . 9331,, R .0 R²² = 0.9975); 0..99 9 75 7 );; square squ quar aree = tr ar transition ran ansi siti si tion ti on 2 ((m/z m/zz 3362.2 m/ 6 .2 /468.5, 62 y3 = 0.2868x - 0.0745, R² = 0.9940); triangle = transition 3 (m/z 362.2/565.8, y4 = 0.1005x 0.0267, R² = 0.9915). The plots demonstrates good linearity, with slopes falling close to the diagonal, black line (theoretical slope = 1), and good agreement between the three transitions at each concentration point. Inset plots show the lower end of the concentration range. The lower limits of detection and quantification (LOD and LOQ) calibration curve generated for this peptide in buffer was 0.1 and 0.3 fmol/μl, respectively.

Figure 2. Alignment of cTnI sequence and the representative peptide containing a novel phosphorylation site S198 in peptide NIDAL (p) SGMEGR. A schematic of the human cTnI 23


sequence (1-209 amino acid) illustrating function domains and linear positions of the known and novel phosphorylation sites20-26(Panel A). Residues 1-15 interact with the inhibitory region (IR). The domain H1-H4 stands for 4 helices of cTnI protein. The helix (H) 1 binds the Cterminal of cTnC and cTnT; H2 binds TnT; H3 binds the N-domain of TnC in response to Ca2+ and is referred to as the switch region; and H4 binds actin-Tm. The inhibitory region (IR) binds both TnC and actin-tropomyosin in a Ca2+-dependent manner. The representative MS spectrum of the phosphorylated peptide NIDAL (p) SGMEGR (Panel B) containing the site S198 and the corresponding unphosphorylated peptide NIDALSGMEGR (Panel C) of cTnI in left Downloaded from http://circ.ahajournals.org/ by guest on April 24, 2018

ventricular tissues. Representative MRM spectrum of the phosphorylated peptide NIDAL (p) SGMEGR in matrix (Panel D) or without matrix (Panel E). A six-poi six-point int ddilution i ut il utio ionn ratio io rati ra tio of light/heavy peptide was run for each MRM assay, illustrating the co-elution time and relative intensity inte in tens te nsit ns ityy for it fo thee nati nnative at ve light peptide phosphorylated phosphory yla lateed S198 peptides, peptide dees, respectively. res esppectively. pe MRM ttransitions ran ansitions off t1-t5 an t1-t55 were wer eree arranged arra ar raange gedd for fo th the he na native atiive peptide peptide de as as inn an an order orde derr (Q1>Q2), (Q Q1> >Q2 Q2), ), tt1:622.8 1:62 1: 622..8+2> 62 549.7 5449. 9.77+1 ((y5); y5); y5 ); t2: t2: 622.8 622 22..8++22> 57 573.7 73. 3.77+2 (y ((y11-98); y111-9 -98) 8);; t3 8) t3: 3: 6622.8 222.88+2> 716. 7716.8 16. 6 8++11 ((y6); y6); y6 );; t4 t4: 4: 6622.8 22.8 22 . +2> 91 .8 918.8 18..8+1 (y (y9(y9 92.88+22> 10 1016 1016.9 166.9 . +11 ((y9); y ); for y9 forr tthe hee hheavy eavy ea vy llabeled ab bel e ed e ppeptide epti ep t de aass in ti n aan n or orde order derr of tt1: de 1 1: 98); t5: 622 622.8 627.8+2>242.4+1(y2); t2: 627.8+2> 559.6+1 (y5); t3: 627.8+2> 726.8+1 (y6); t4: 627.8+2> 839.7+2 (y7); t5: 627.8+2> 928.8+1 (y8), respectively.

Figure 3. Quantitation of phosphorylation sites in myocardium obtained from donor and failing hearts. The stoichiometric quantity (fmol phosphorylation/fmol protein) phosphorylation of cTnI by MRM assay for Donor (light gray), ischemic heart failure (ISHD, white) and dilated cardiomyopathy (IDCM, dark gray) (n = 10 per group) for all of the sites (panel A) and the eleven least abundant sites (panel B). All raw data was calibrated by the synthesized internal standard peptides, converted for fmol based on standard curves and normalized to the total 24


quantify of troponin I in the sample (fmol). Values are median, 1st quartile and 3rd quartile. *, P < 0.05 for ISHD or IDCM versus Donor. #, values were below the lower limit of quantification (LLOQ). Phosphorylated T30 and S149 were not detected in any of the three groups, and were omitted from the figure. See Supplemental Table 5 and Supplemental Figure 18 for complementary data.

Figure 4. Quantitation of phosphorylated sites in canine model of HF. The stoichiometric quantity (fmol phosphorylation/fmol protein) of phosphorylation sites on cTnI by MRM assay Downloaded from http://circ.ahajournals.org/ by guest on April 24, 2018

in Con (Control, black), HF dys (dyssynchronous heart failure, white), and CRT (cardiac resynchronization therapy, gray) (n = 4 per group). All raw data was calibrated d by the the synthesized internal standard peptides, then normalized to total troponin I. Error bars indicate thee st stan standard anda an dard da rdd err error ror ooff mean (SEM). *, P < 0.0011 HF HF dys or CRT versuss C Con. o . #, P < 0.001 CRT on vversus errsus rs HF dy by y oneoone-way nee-wa waay AN ANOV ANOVA OVA OV A fo foll followed llow ll owed ow ed bby y Bo Bonf Bonferroni ferro erro roni ni multiple mul ulti tipl plee comparisons pl compar com aris ar ison is onss post-hoc on post po st-ho st hocc te ho test test. s. dyss b d

25

D

Peak area a ratio (light/heavy)

RP(p)TLR, 362.2/ 468.5 I(p)SASR, 307.2/333.2 ISASR, 267.2/439.2

6 Time (min)

0 2 4 6 8 10 12 14

Intensity (cps)

E3 1.0E3

0 2 4 6 8 10 112 2 14 Time (min) (min)

0 Fig.1

TLR TL R 362.2/288.4 362 2/2 /288 88 4 RP(p)TLR,

8.53

5.0E4

RP(p)TLR, 362.2/468.5

RP(p)TLR, 362.2/565.8

8.53 4

8

12

2.4

0.24 0.08 0.0 0.2 0.4 0.6 0.8 1.0 O.C (fmol/ȝl)

1.6 0.8

0.0

0

6 2 4 8 Observed concentration (O.C) (fmo (fmol/ȝl)

E

8 53 8.53

5.5E4

12

RP(p)TLR 362.2/ 468.5 468..5 8.53 min

5.5E4

me (min) (miin)) Time C

10

3.2

16

20

Time (min)

24

28

32

C.C (fmol/ȝl) C C (fmo C. m l/ l/ȝ ȝl)

RPTLR 322.2/ 389.4 7.444 m in min

2.0E5

8

Calculated concentration(C.C) (fmol/ȝl) C) (fmo fm l/ l/ȝ ȝl)

4

Intensity Intens n ityy (cps) ( ps) (c

2

0

Intensity (cps)

B

S(p)SNYR, 353.6/ 619.3

0.40

4.0

RPTLR, 322.2/ 389.3

Intensity (cps)


2.0E5

Peak area ratio

A

12 10 8

10

1.00 0.66 08 0. 0.8 0.4 00.2 .22 00 0. 0.0 0 0 0.2 0 2 0.4 0 4 00.6 6 0.8 0 8 1.0 10 0.0 O.C (fmol/ȝl)

6 4 2 0

0

2

4 6 8 10 Observed concentration (O.C) (fmol/ȝl)

12

+Actin+TnC+ Tm T50

Y25

Ac Ac S22/23 T30

Known

T180 H1

S5 H2

IR

H3

H4

H1

S5 H2

IR

H3

H4

S41/43

S76 T77

T142 S149 D

B

Phospho-S198, NIDAL(p)SGMEGR

100%

100%

N

D E

I R

G

L

A M

S+8 80 80 S+80 S+80

G

M

G L

A

E D

Relative Rela Re lati tivee intensity

Relative intensity

parent+2H-98 R

G I

N

parent+2H-NH pa p rent nt+2 nt +2 2H-NH3 y9 y3

b2 b 2 y2 y 2

b3

0%

y5 5 b5 5

b4

2500

0

y6 y7 y8 b8 8

b6 6 b7 7

50 500

750 750

m/z

b9 b9 b10 0

Relative intensity

I G

D E

A

L M

S G

G S

y6

M L

A

E D

R I

y5 parent+2H-H2O b5 b6 b2 y1 y2

0% 0 Fig.2

100%

Unphospho-S198, NIDALSGMEGR R

250

y3 b4 b3

y4

500

y7 y8 b8

b7

m/z

750

b9

b10

1000

t4

t5

11.11 11.11

11.11 11 11.11

11.11 11 11.11

11 11. 1 11 11.11

11.11

11.11 11 11.11

11.11 11 11.11

11.11 11 11.11

11.10 11 11.10

11.11

110 0 20 2 30

100 20 300

10 20 30

10 20 30

100%

E

N

With matrix t3

0%

1000 1000

y9 G

t2

S165

0%

C 100%

t1

S198

N

t1

t2

100 20 300 Tiime (min) Time

With Wi thou outt matrix m trix ma Without t4 t3

t5

11.00

11.01

11.01

11.01

11.01

11.01

11.01

11.01

11.01

11.01

10 20 30

10 20 30

10 20 30

10 20 30

0% 100%

0% 10 20 30 Time (min)

Light chain

S4/5

+Actin+Tm

+TnC

Heavy chain

Novel

IR

+TnT

+TnC+TnT

Light chain

+IR

Heavy chain

Function

Relative intensity


A



Multiple Reaction Monitoring to Identify Site-Specific Troponin I Phosphorylated Residues in the Failing Human Heart Pingbo Zhang, Jonathan A. Kirk, Weihua Ji, Cristobal G. dos Remedios, David A. Kass, Jennifer E. Van Eyk and Anne M. Murphy


Circulation. published online September 12, 2012; Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2012 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7322. Online ISSN: 1524-4539

The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circ.ahajournals.org/content/early/2012/09/11/CIRCULATIONAHA.112.096388

Data Supplement (unedited) at: http://circ.ahajournals.org/content/suppl/2012/09/12/CIRCULATIONAHA.112.096388.DC1

Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation is online at: http://circ.ahajournals.org//subscriptions/

1

SUPPLEMENTAL MATERIAL

SUPPLEMENTAL METHODS

Cardiac Myofibril and cTnI Protein Preparation Human Left ventricular transmural tissue samples were obtained from explanted endstage failing hearts hearts with ISHD and IDCM during heart transplantation surgery as well as from unutilized healthy donor hearts (n = 10 per group), rapidly frozen and stored in liquid nitrogen. Ethics approval was provided by St. Vincent’s Hospital (#H03/118), Sydney Australia and by The University of Sydney (#09-2009-12146). To preserve the endogenous phosphorylation status, frozen tissue samples each 0.1 g weight were used to purify cardiac myofibrils. Briefly, 0.1 g each of frozen LV heart tissue was collected in fresh relaxing buffer (containing 75 mM KCl, 2 mM MgCl2, 2 mM EGTA, 1 mM NaN3, 10 mM imidazole, pH 7.2, 4 mM Phosphocreatine, 1 mM ATP, 50 mM BDM, 1 mM DTT, 1 mM benzaamidine-HCI, 0.1 mM PMSF, 1 µg/ml leupeptine, 1 µg/ml pepstatin, and 1% Triton X-100) rapidly minced using a scissor and homogenized on ice for 10 s in the buffer containing 0.1 M EDTA plus the complete protease and phosphatase inhibitor cocktail tablets (Roche) using a Polytron homogenizer at 55% max speed. Resultant homogenates were centrifuged at 3,000 × g for 8 min at a temperature of 4 ºC and the supernatant fraction removed. The pellet was then suspended in a rigor buffer (containing 75 mM KCl, 2 mM MgCl2, 2 mM EGTA, 1 mM NaN3, 10 mM imidazole, pH 7.2) with 1% Triton X-100. Transferred the solution into a Duall-glass homogenizer for homogenization and centrifuged two times at 3,000 × g for 8 min, followed by washing two times with the rigor buffer without Triton X-100. Following washes the pellet was suspended in a K-60 buffer (containing 60 mM KCl, 20 mM MOPS, and 2 mM MgCl2) and centrifuged two

2

times at 2,000 × g for 8 min. Purified myofibril proteins were extracted in buffer (4 M urea, 0.3 M NaCI, 4% SDS, and 50 mM Tris-HCI pH 8.8) and centrifuged twice at 12, 000 × g for 15 min. The resultant soluble fraction was frozen at -80ºC until analysis. Canine Model of HF and Reverse Remodeling after CRT Left ventricle tissue (LV) was collected from adult mongrel dogs (n = 4 per group); groups included: normal (Control), dyssynchronous pacing-induced heart failure (HFdys), or cardiac resynchronization therapy (CRT) as previously published.1 All protocols were approved by the Animal Care and Use Committee of the Johns Hopkins Medical Institutions. Briefly, a left bundle-branch ablation (LBBB) by radiofrequency and subsequent right atrial pacing for 6 weeks at 200 bpm was used to induce HFdys. LBBB was confirmed by intra-cardiac electrograms, with surface QRS widening from 50±7 to 104±7 ms (P < 0.001). For CRT, the HFdys protocol was stopped after 3 weeks, at which time biventricular pacing was carried out 5 for 3 weeks at the same pacing rate of 200 bpm. At terminal study the hearts were extracted under cold cardioplegia, dissected into endocardial and mid/epicardial segments from the septum (i.e. LV and RV septum) and LV lateral wall, and frozen in liquid nitrogen. Tissue samples obtained from the upper third of the LV lateral wall were used in the present study. Protein Quantification and Digestion and Peptide Extraction Accurate determination of protein concentration was assessed by a bicinchoninic acid (BCA) assay kit form from Pierce (Rockford) according to the company’s instruction. Extracted myofilament proteins were separated on 1D SDS-PAGE using 4-12% NuPAGE Bis-Tris gels (1mm, Invitrogen). Electrophoresis was performed according to manufacturer’s protocols and individual gels were stained using the coomassie blue R-250. Protein bands relevant to cTnI

3

were excised from fresh gels and digested as described previously. Samples were monitored to ensure consistent and complete digestion after optimization of the method. Briefly, protein bands were excised, cut into 1 mm3 pieces, and washed 3 times with 50% acetonitrile/25 mM ammonium bicarbonate for 15 min with shaking. Gel pieces were incubated with 25 mM ammonium bicarbonate +10 mM dithiothreitol for 60 min at 55 ºC, washed with acetonitrile (ACN), then incubated with 25 mM ammonium bicarbonate + 55 mM iodoacetamide (freshly made) for 30 minutes in the dark. After these incubations, the gel pieces were washed with acetonitrile, dried, and rehydrated with 10 ng/µl trypsin (Promega, sequencing grade) in 25mM ammonium bicarbonate then placed on ice for 30 min. Excess trypsin was removed then 20 µL of 25 mM ammonium bicarbonate was added. Samples were digested at 37 ºC for 18 hr. The liquid was transferred to a clean tube. The peptides were extracted twice using 50% ACN+0.1 TFA% for 20 min at 25 ºC with a shaking, and combined two times of extraction with the liquid from the previous step. The final peptide mixture was performed off-line cleanup or desalting with a C18 reversed-phase chromatography in C18 ZipTips (Millipore) using 0.1% TFA. The peptides were then eluted three times with 10 µl of 70% ACN, 0.1% TFA. The combined solution was dried using a vacuum centrifuge SpeedVac (Thermo Electron) and frozen at -80 ºC until analysis. Quantitative Comparison of Sample Processing. Comparison between direct digestion with trypsin or first separated by SDS PAGE with cTnI band excised and undergo in-gel digestion was carried out in five replicates to ensure extent of phosphorylation was preserved during processing. Comparison of the phosphorylated and unphosphorylated peptides containing T142 was between a gel-based and direct homogenizationbased protein digestion by MRM. In the gel-based method, 30 μg of purified myofilament

4

proteins from heart failure LV tissues was applied to 4-12% Bis-Tris NuPAGE gels as above, followed by Coomassie staining and tryptic in-gel digestion. Alternatively, whole proteins were extracted directly from the purified myofilament, and then 30 µg of the protein mixture was treated as above without the separation by NuPAGE gels. All peptide mixtures were desalted prior to analysis by MRM assays of peptides (RPTLR or RP(p)TLR) and compared to the peptide NITEIADLTQK which thus normalizes to the total protein concentration. MALDI-TOF MS and Orbitrap Mass Spectrometry To ensure the correct band with cTnI was excised from 1 D gels, we performed protein identifications by peptide mass fingerprint (PMF) technique for all major 12 protein bands from a heart failure sample using a mass spectrometry 4800 MALDI-Tof/Tof mass spectrometry (ABI) MALDI-TOF/TOF for protein identification by peptide mass fingerprint (PMF) technique. MS spectra were acquired on the 4800 MALDI TOF/TOF Analyzer (ABI) using 1000 shots/spectra and a laser power between 4300–4700 units. PMF was conducted with the database search tool Mascot. Peptides were searched in against the SwissProt human database using the following criteria: fixed modification carbamidomethyl; variable modifications_oxidation (methionine); maximum missed cleavages_2; peptide tolerance_ ± 0.5Da; MS/MS tolerance_ ± 0.8 Da. The band 9 was successfully identified as cTnI about 70% sequence coverage with Mascot score 160 (Supplemental Fig.17). LTQ Orbitrap (Thermo Scientific) mass spectrometer was used for initial verification of the phosphorylated sites. For the LTQ Orbitrap, desalted peptides were reconstituted in 10 µl 0.1% v/v aqueous formic acid (FA). Each 5 µl sample was injected and analyzed on an Agilent 1200 nano-LC system (Agilent) connected to an LTQ-Orbitrap mass spectrometer equipped with a nanoelectrospray ion source (Thermo Scientific). Peptides were separated on a BioBasic (New

5

Objective, Woburn, MA) C18 RP-HPLC column (75 μm x 10 cm) using a linear gradient from 5% B to 65% B in 60 minutes at a flow rate of 300 nl/min, where mobile phase A was composed of 0.1% v/v aqueous FA and mobile phase B was 90% acetonitrile, 0.1 % FA in water. MS/MS data from the LTQ LC/MS/MS (Thermo Scientific) were analyzed using a Sorcerer 2™SEQUEST®, with post-search analysis performed using Scaffold (Proteome Software). All raw data peak extraction was performed using Sorcerer 2™-SEQUEST® default settings. Data was searched against the Swissprot human database, using a full trypsin digestion, with the following criteria: fixed modification_carbamidomethyl; variable modifications_phosphorylation (Ser, Thr, and Tyr) and oxidation (Met); Peptide mass tolerance was set to either 100 ppm or 0.1 amu, fragment mass tolerance set to 1 amu, fragment mass type set to monoisotopic, maximum number of modifications set to 4 per peptide. All MS/MS spectra were manually examined using Scaffold (Proteome Software). The analyses for biological and technical replicates were conducted in triple. Synthesized Internal Standards (SIS) A series of SIS peptides were designed on detection of the appropriate peptide for the known and novel to be phosphorylated including mono or di-phosphorylated and chemically produced using solid-phase peptide synthesis (>95% pure) (New England Peptide; NEP). The corresponding non-phosphorylated sequences were isotopically labeled by incorporating 15N/13C in either Lys (K) or Arg (R) amino acid residue at the C-terminus (mass difference of 8 and 10 daltons, respectively for K or R). The phosphorylated peptides were unlabeled. The synthetic reference peptides were useful for confirming the identity of the target peptide through perfect co-elution and by exhibiting identical fragmentation patterns between the internal standards and endogenous analytes. Amino acid analysis was carried out both on labeled and unlabeled

6

peptides, and all were dried for storage and delivered to us by company. The peptide was suspended at 10 pmol/µl with 0.1% formic acid in HPLC water and aliquot each 10-50 µl for a storage at -80 ºC. The optimized parameters included declustering potential (DP), collision energy (CE), and Collision Cell Exit Potential (CXP) for the synthetic peptides. Optimization was obtained by ramping the parameters DP (0-400 volts), CE (5-130 volts) and CXP (0-66 volts) from low to high with a step of 1 for all parameters and a fixed setting of 10 volts for entrance potential (EP) and are listed in table. A total 30 assays and 145 transitions were designed based on detection of the appropriate peptide and matching transition (Q1/Q3) for each mono or di-phosphorylated and the corresponding unphosphorylated sequence. Standard curves Using these optimized MRM parameters, we made calibration curves for each mono or di-phosphorylated and the corresponding non-phosphorylated tryptic peptide by nano-LCMS/MS in triplicate. For each phosphorylation site, a series dilution of light/heavy peptides were made to produce 6-point calibration curves at 0.125, 0.25, 0.5, 1, 5, and 10 fmol/µl. All analytes were spiked in with or without a mix matrix containing 10 µg digest of the donor, ISHD, and IDCM LV at a six-point dilution ratio of different concentration 0.125, 0.25, 0.5, 1, 5, and 10 fmol/µl in triplicates. A mixture containing 10 heavy labeled peptides of cTnI at known concentrations (fixed at 1 fmol/µl) to mimic a digest matrix of human cTnI, then followed analysis by a 4000 QTRAP hybrid triple quadrupole/linear IT mass spectrometer (AB SCIEX) operating with Analyst 1.4.2 software, scheduled experiments in the optimized parameters and positive ion mode. Each standard curve for the phosphorylated peptides was then generated from

7

precise quantities of each of these internal standard peptides. The lower limit of detection and quantification (LLOD and LLOQ) was determined for each peptide. Nano-LC/MS/MS Analysis Digested protein samples were analyzed using a 4000 QTRAP hybrid triple quadrupole/linear IT mass spectrometer (AB SCIEX) operating with Analyst 1.4.2 software scheduled experiments in positive ion mode. The cleanup mixture of peptides from the in gel digestion of cTnI proteins were reconstituted with 20 µl water containing 0.1% Formic acid. Peptides were separated by an Eksigent Tempo nano-LC system (Eksigent Technology) onto a BioBasic C18 reverse-phase PicoFrit column (300 Å, 5 µm, 75 µm ×10 cm, 15 µm tip, New Objective). Peptides were eluted 36-min linear gradient from 5 to 40% B (mobile phase A: 2% v/v ACN containing 0.1% v/v formic acid; mobile phase B: 98% v/v ACN containing 0.1% v/v formic acid) at 500 nl/min flow rate. The column eluent solution was directed into the NanoSpray source spray head via coupling to a distal coated PicoTip fused silica spray tip (360 µm od, 75 µm id, 15 µm diameter emitter orifice; New Objective). Samples were analyzed using the following settings curtain gas (CUR):15; collision gas (CAD): high; ion spray voltage (IS): 2.5 kV; ion source gas1 (GS1): 25; ion source gas 2 (GS2): 0; resolution Q1 and Q3: unit; heater interface temperature: 150 ºC. Each sample was run in triplicates. Data Processing and Statistical Analysis Peak detection and quantification of peak area was determined with Multiquant software version 2.0 (AB SCIEX) and inspected manually to ensure correct peak identification and quantification. Measurements were performed in triplicate and then averaged to reduce technical variation. In cases where the peptide was detected, but not quantifiable (above LLOD but below

8

LLOQ), the quantity was tagged as LLOQ and was replaced with 100 as a lower cutoff point. If the peptide was not observed it was recorded as not detected (ND).The quantity of each peptide (in fmoles) was determined based on the corresponding standard curve using data points above the LLOQ. Values were then normalized against the total quantity of cTnI (phosphopeptide (fmol)/ cTnI (fmol)), and then the fold change between control and the heart failures groups was calculated. For human samples, data was analyzed using one-way analysis of variance (ANOVA) on ranks, followed by Dunnett’s multiple comparisons post-hoc test. The canine samples were analyzed using a one-way ANOVA, followed by Bonferroni multiple comparisons post-hoc test. All calculations were done using SigmaPlot v11 (Systat), with a P-value less than 0.05 denoting significance. Validation of MRM Quantification by Immunoblotting Analysis Immunoblotting analysis was optimized for each antibody, by altering blocking conditions and concentrations of antibodies (primary and secondary). The phosphorylation status of cTnI was monitored by western blotting a specific antibody against phosphorylated PKA-sites anti-pS22 and/or 23 and Phospho-Tyrosine Mouse mAb (P-Tyr-100) (used at 1:1000) (Cell Signaling). For normalization, after visualizing the S22/23, membranes were striped and reprobed using a mouse antibody clone 8I-7 (used at 1:10000) from International Point of Care Inc, Toronto, Canada. Phosphorylated cTnI (S22/23) was calculated as phosphor signal/total protein signal in triple experiments. Secondary antibodies were alkaline phosphatase conjugated donkey anti-rabbit and alkaline phosphatase conjugated goat anti-mouse, both from Jackson Immuno Research (1:10,000 dilution). Briefly, SDS-PAGE gels were incubated in transfer buffer (25 mM bis-Tris

9

and 1mmol/L EDTA, pH 7.2) for 10 min prior to being transferred to PVDF (Millipore, 45 μm) via the Mini-Transblot Cell (Bio-Rad) for 1h at 100V. Membranes were blocked with 5% v/v western blocking reagent (Roche) in TBST (20 mM Tris, 150 mM NaCl and 0.1% v/v Tween, pH 7.5) at 4C for 1h. Blots were then incubated with primary antibody (1:5,000) for 1h at room temperature, washed for 30 min with TBST, incubated with secondary antibody for 1h and then washed for 1h with TBST (200 mM Tris, 1.5M NaCl, 0.1% v/v Tween-20, pH 7.5). Chemiluminescence was used in its linear range. Densitometrical analysis was done with a Progenesis image software (Nonlinear Dynamics), and the ratio of phosphor cTnI to total cTnI was calculated by Microsoft Excel software.

Limitations of the Study The potential limitations of this study include the fact that human samples may have lost of phosphorylation (or have artificially-induced phosphorylation) during harvesting or while in 80oC storage. However, there is no reason to suspect a priori differential changes in donor and heart failure samples and the MRM quantitative data shows that different amino acid residues either increase or decrease in failing and donor hearts. Thus, this is difficult to rationalize as a storage effect.

10

SUPPLEMENTAL TABLES Supplemental Table 1. Characteristics of samples in human heart failure Code #

Type

Gender

Age

LVEF (%)

NYHA

1

ISHD

M

31

23

III

2

ISHD

M

45

-

IV

3

ISHD

M

46

25

III

4

ISHD

M

47

20

III/IV

5

ISHD

M

50

-

-

6

ISHD

M

52

30-35

III/IV

7

ISHD

M

54

-

-

8

ISHD

M

54

35

III/IV

9

ISHD

F

43

35

IIIB

10

ISHD

F

49

35

III

11

IDCM

M

43

-

III

12

IDCM

M

46

15-20

III

13

IDCM

M

56

15

IV

14

IDCM

M

57

10

III

15

IDCM

M

58

20

III/IV

16

IDCM

M

60

15

IV

17

IDCM

F

23

15

III

18

IDCM

F

31

20

III

19

IDCM

F

53

20

III

20

IDCM

F

54

22

III

21

Donor

M

19

-

-

22

Donor

M

23

-

-

11 23

Donor

M

23

-

-

24

Donor

M

26

-

-

25

Donor

M

39

-

-

26

Donor

M

44

-

-

27

Donor

M

52

-

-

28

Donor

F

27

-

-

29

Donor

F

41

-

-

30

Donor

F

45

-

-

The average age of the ISHD, IDCM, and donor group is 47, 48, and 34, respectively. M: male; F: female; LVEF: left ventricular ejection fraction, normal range 55-79%; NYHA: New York Heart Association; A functional classification of cardiac failure according to severity of disease and the need for therapeutic intervention. I Asymptomatic heart disease; II Comfortable at rest; symptomatic with normal activity; III Comfortable at rest; symptomatic with < normal activity; IV Symptomatic at rest—Criteria Committee, NYHA, Inc: Diseases of Heart & Blood Vessels, 6th ed, Little Brown, Boston 1964.

12

Supplemental Table 2. List of all peptides and transitions used for MRM assays to monitor the known phosphorylation of cTnI site Residue S22

Peptide (p)SSNYR

S23

S(p)SNYR

S22/23

(p)S(p)SNYR

S22/23

SSNYR

S22/23

SSNYR*

S22/23

RRSSNYR

S22

RR(p)SSNYR

S41

I(p)SASR

S43

ISA(p)SR

Q11 353.9+2 353.9+2 353.9+2 353.9+2 353.9+2 353.6+2 353.6+2 353.6+2 353.6+2 353.6+2 394.0+2 394.0+2 394.0+2 394.0+2 394.0+2 313.2+2 313.2+2 313.2+2 318.2+2 318.2+2 318.2+2 318.2+2 318.2+2 469.8+2 469.8+2 469.8+2 509.8+2 509.8+2 509.8+2 306.9+2 306.9+2 306.9+2 306.9+2 306.9+2 307.0+2 307.0+2 307.0+2 307.0+2 307.0+2

Q32 175.3+1 304.9+2 338.4+1 452.4+1 539.5+1 338.4+1 452.5+1 521.5+1 549.5+1 619.5+1 296.1+2 338.3+1 345.0+2 452.3+1 521.8+1 338.4+1 452.5+1 539.5+1 185.1+1 175.1+1 348.4+1 462.5+1 549.5+1 338.2+1 539.2+1 269.6+1 338.2+1 619.2+1 310.1+1 258.4+2 262.3+1 333.4+1 402.4+1 500.5+1 333.4+1 342.2+1 402.3+1 420.3+1 500.4+1

RT3 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0

ID y1 y5-98 y2 y3 y4 y2 y3 y4-98 y4-80 y4 y5-2×98 y2 y5-98 y3 y4-98 y2 y3 y4 y1 b2 y2 y3 y4 y2 y4 y4 y2 y4 y4 y5-98 y2 y3 y4-98 y4 y3-80 y2 y4-98 y4-80 y4

CE4 32.2 13.6 22.7 22.1 22.2 22.6 22.0 23.0 21.5 23.0 18.9 27.2 14.0 26.0 27.4 21.6 17.0 18.4 28.1 16.2 21.6 17.0 18.4 28.5 28.5 28.5 30.5 30.5 30.5 15.5 25.7 19.9 21.8 16.4 19.2 27.7 20.3 25.1 14.8

13

S41/43

I(p)SA(p)SR

S41/43

ISASR

S41/43

ISASR*

S41/43

SKISASRK

S41/43

ISASRKLQLK

S41

SKI(p)SASRK

S41

I(p)SASRKLQLK

S76

AL(p)STR

T77

ALS(p)TR

S76/T77

AL(p)S(p)TR

S76/T77

ALSTR

S76/T77

ALSTR*

347.5+2 347.5+2 347.5+2 347.5+2 347.5+2 267.0+2 267.0+2 267.0+2 272.0+2 272.0+2 272.0+2 272.0+2 272.0+2 438.2+2 438.2+2 438.2+2 572.9+2 572.9+2 572.9+2 478.2+2 478.2+2 478.2+2 612.9+2 612.9+2 612.9+2 313.9+2 313.9+2 313.9+2 313.9+2 313.9+2 313.9+2 313.9+2 313.9+2 313.9+2 313.9+2 354.6+2 354.6+2 354.6+2 354.6+2 354.6+2 274.2+2 274.2+2 274.2+2 278.9+2 278.9+2

298.3+1 384.7+1 412.4+1 482.5+1 580.2+1 262.2+1 333.6+1 420.4+1 185.3+1 272.2+1 254.2+1 343.6+1 430.4+1 381.3+1 225.7+1 563.4+1 515.3+1 643.4+1 872.6+1 461.3+1 265.7+1 643.4+1 595.3+1 723.4+1 872.6+1 276.3+1 345.3+1 443.2+1 458.5+1 556.5+1 256.4+1 345.4+1 443.4+1 458.5+1 556.6+1 256.7+1 327.5+1 425.4+1 523.4+1 538.7+1 276.2+1 363.2+1 476.3+1 286.3+1 373.3+1

22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0

y3-98-17 y4-2×98 y3 y4-98 y4 y2 y3 y4 y1 y2 y2-18 y3 y4 y4 y5 y6 b5 b6 y7 y4 y5 y6 b5 b6 y7 y2 y3-98 y3 y4-98 y4 y2-98 y3-98 y3 y4-98 y4 y2-98 y3-2×98 y3-98 y3 y4 y2 y3 y4 y2 y3

25.0 24.8 21.4 20.0 16.1 12.0 17.0 17.0 27.1 12.0 26.0 17.0 17.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0 27.3 22.1 15.5 21.1 16.6 16.3 19.2 14.6 21.6 15.8 18.4 24.1 18.3 14.9 18.9 17.7 16.1 17.5 17.7 16.1

14

T142

P(p)TLR

T142

PTLR

T142

RP(p)TLR

T142

RPTLR

T142

RPTLR*

S149

I(p)SADAMMQALLGAR

S149

ISADAMMQALLGAR

S165

AKE(p)SLDLR

S165

AKESLDLR

S165

AKESLDLR*

Total

NITEIADLTQK*

278.9+2 283.7+2 283.7+2 283.7+2 243.7+2 243.7+2 243.7+2 362.2+2 362.2+2 362.2+2 362.2+2 362.2+2 322.2+2 322.2+2 322.2+2 327.2+2 327.2+2 327.2+2 327.2+2 327.2+2 764.4+2 764.4+2 764.4+2 764.4+2 724.4+2 724.4+2 724.4+2 724.4+2 506.8+2 506.8+2 506.8+2 506.8+2 506.8+2 466.7+2 466.7+2 466.7+2 471.7+2 471.7+2 471.7+2 471.7+2 471.7+2 627.4+2 627.4+2 627.4+2 627.4+2

486.5+1 279.1+1 392.2+1 469.3+1 199.1+1 312.2+1 389.3+1 288.4+1 313.3+2 370.3+1 468.5+1 565.8+1 288.2+1 389.4+1 486.6+1 185.2+1 298.2+1 327.2+2 399.4+1 496.6+1 1061.6+1 1176.6+1 1247.6+1 1414.7+1 1061.6+1 1176.6+1 1247.6+1 1334.7+1 288.4+1 458.2+2 507.2+2 585.6+1 715.8+1 288.2+1 603.8+1 732.9+1 298.5+1 436.3+2 526.6+1 613.8+1 742.9+1 612.9+1 683.8+1 797.0+1 926.0+1

22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0

y4 b2 b3 y3 b2 b3 y3 y2 y5-98 y3-98 y3 y4 y2 y3 y4 y1 y2 y5 y3 y4 y10 y11 y12 y13 y10 y11 y12 y13 y2 y8-98 y8 y5-98 y6-98 y2 y5 y6 y2 y7 y4 y5 y6 y5 y6 y7 y8

17.5 19.2 19.2 19.2 17.2 17.2 17.2 22.9 21.2 22.1 26.2 22.8 29.3 28.7 24.0 29.3 29.3 12.9 28.7 24.0 43.2 43.2 43.2 43.2 41.2 41.2 41.2 41.2 23.2 25.9 14.7 33.0 31.0 32.2 31.0 24.9 32.3 26.4 25.1 26.7 24.9 38.3 28.1 30.0 29.1

15

627.4+2

1027.1+1

22.0

y9

28.0

Note: For MRM transition, characteristic parameters were used in assay including: 1. Q1, parental ion and its charge is in superscript; 2. Q3, fragment ion and it charge in superscript; 3. Retention time (RT), unit in msec; 4. Collision energy (CE). Each transition was optimized, as described in Supplemental Methods. Star (*) indicates the isotopically labeled by incorporating 15 13 N/ C in either Lys (K) or Arg (R) amino acid residue at the C-terminus, in mass difference of 8 and 10 daltons respectively for K or R. Total means unmodified tryptic peptide used to quantify the total amount of cTnI present in samples.

16

Supplemental Table 3. List of all peptides and transitions used for MRM assays to monitor the novel phosphorylation sites Residue S4

Peptide Ac-ADG(p)SSDAAR

S5

Ac-ADGS(p)SDAAR

S4S5

Ac-ADGS(p)S(p)DAAR

S4S5

Ac-ADGSSDAAR

S4S5

Ac-ADGSSDAAR*

T50

(p)TLLLQIAK

T50

TLLLQIAK

T50

TLLLQIAK*

T180

ED(p)TEK

Q11 486.7+2 486.7+2 486.7+2 486.7+2 486.7+2 486.7+2 486.7+2 486.7+2 486.7+2 486.7+2 526.7+2 526.7+2 526.7+2 526.7+2 526.7+2 446.7+2 446.7+2 446.7+2 451.7+2 451.7+2 451.7+2 451.7+2 451.7+2 490.6+2 490.6+2 490.6+2 490.6+2 490.6+2 450.2+2 450.2+2 450.2+2 454.2+2 454.2+2 454.2+2 454.2+2 454.2+2 351.6+2 351.6+2 351.6+2 351.6+2 351.6+2

Q32 430.3+2 437.8+2 686.5+1 645.6+1 743.6+1 317.5+1 599.5+1 686.8+1 743.7+1 430.4+2 363.4+2 412.2+2 421.2+2 627.9+1 570.6+1 606.6+1 663.7+1 778.8+1 442.8+1 529.6+1 616.6+1 673.7+1 788.8+1 441.8+2 459.6+1 572.7+1 685.9+1 799.0+1 572.7+1 685.9+1 799.0+1 339.5+1 467.6+1 580.7+1 693.8+1 807.0+1 293.6+2 227.4+2 284.6+2 359.6+1 474.5+1

RT3 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0

ID y8 y9-98 y6 y7-98 y7 y3 y5 y6 y7 y8 y7-98 y7 y8-98 y7-2×98 y6-98 y6 y7 y8 y4 y5 y6 y7 y8 y8-98 y4 y5 y6 y7 y5 y6 y7 y3 y4 y5 y6 y7 y5-98 y3 y4 y3-98 y4-98

CE4 17.0 17.0 18.0 21.0 16.0 36.1 17.7 17.5 20.0 18.7 30.3 26.5 24.5 31.8 29.9 21.3 18.9 18.3 13.9 18.7 21.3 18.9 18.3 17.6 26.2 26.0 23.9 23.8 22.6 21.3 22.4 30.0 26.0 22.6 21.3 22.4 17.2 24.8 19.2 21.9 23.6

17

T180

EDTEK

T180

EDTEK*

S198

NIDAL(p)SGMEGR

S198

NIDALSGMEGR

S198

NIDAL(p)SGMEGR*

Total

NITEIADLTQK*

310.9+2 310.9+2 310.9+2 314.9+2 314.9+2 314.9+2 622.8+2 622.8+2 622.8+2 622.8+2 622.8+2 582.8+2 582.8+2 582.8+2 627.8+2 627.8+2 627.8+2 627.8+2 627.8+2 627.4+2 627.4+2 627.4+2 627.4+2 627.4+2

276.4+1 377.4+1 500.5+1 284.4+1 385.4+1 500.5+1 414.6+1 549.7+1 716.8+1 918.8+1 1016.9+1 636.8+1 820.8+1 936.9+1 726.8+1 559.6+1 839.7+1 928.8+1 1026.9+1 612.9+1 683.8+1 797.0+1 926.0+1 1027.1+1

22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0 22.0

y2 y3 y4 y2 y3 y4 y7 y5 y6 y9-98 y9 y6 y8 y9 y6 y5 y7 y9-98 y9 y5 y6 y7 y8 y9

16.9 18.7 19.9 16.9 18.7 19.9 27.8 25.9 33.0 12.1 34.8 27.8 34.8 26.1 28.4 27.8 35.3 12.5 26.2 38.3 28.1 30.0 29.1 28.0

Note: For MRM transition, these characteristic parameters were used in assay including: 1. Q1, parental ion and it charge in superscript; 2. Q3, fragment ion and it charge in superscript; 3. Retention time (RT), unit in msec; 4. Collision energy (CE). Each transition was optimized, as described in Supplemental Methods. Star (*) indicates the isotopically labeled by incorporating 15 13 N/ C in either Lys (K) or Arg (R) amino acid residue at the C-terminus, in mass difference of 8 and 10 daltons respectively for K or R. Total means unmodified tryptic peptide used to quantify the total amount of cTnI present in samples.

18

Supplemental Table 4. Identification results of major LV myofibril proteins by MALDI-TOF/TOF mass spectrometry analysis Band #

Protein name

Peptide Sequence

Observed m/z

Calculated m/z

Error ppm

Score

Expect

Queries /matched

1

Titin

2

Myosin

3

α-Actinin

LIPGQEYIPR EILGYWVEYR WEQFYVMPLPR VTGLVEGLEYQFR VENLTEGAIYYFR IPGPPETLQIFDVSR IDQLQEGCSYYFR TLEATISGLTAGEEYVFR LPYTTPGPPSTPWVTNVTR NLTEGEEYTFQVMAVNSAGR ILYGDFR MFNWMVTR DSLLVIQWNIR NNLLQAELEELR IEELEEELEAER VIQYFAVIAAIGDR ILNPAAIPEGQFIDSR DLEEATLQHEATAAALR SEAPPHIFSISDNAYQYMLTDR TIPWLENR EGLLLWCQR LASELLEWIR GITQEQMNEFR VGWELLLTTIAR GYEEWLLNEIR ILASDKPYILAEELR

1234.70 1326.70 1464.76 1509.82 1573.82 1667.91 1677.80 1956.01 2083.11 2215.06 882.46 1083.50 1355.75 1440.76 1487.71 1534.85 1739.93 1837.93 2554.21 1027.57 1173.62 1228.71 1351.65 1370.82 1420.73 1730.01

1234.67 1326.66 1464.76 1509.78 1573.78 1667.88 1677.74 1956.01 2083.07 2215.02 882.45 1083.49 1355.75 1440.75 1487.69 1534.85 1739.92 1837.93 2554.18 1027.54 1173.59 1228.68 1351.61 1370.79 1420.69 1729.96

28.5 34.5 28.5 26 31.7 18.2 33.0 17.5 18 21.7 3.0 1.0 2.4 4.6 10.8 3.5 5.8 10.3 14.0 29.6 26.2 27.2 26.5 25.9 26.4 30.5

37 36 51 37 56 31 51 55 57 36 46 44 57 106 94 55 63 130 106 61 60 63 70 78 71 96

6.6e-03 8.3e-03 2.0e-04 6.0e-03 7.2e-05 1.8e-02 2.1e-04 7.1e-05 2.0e-03 5.2e-03 8.6e-04 1.5e-03 7.0e-05 8.6e-10 1.2e-08 8.5e-05 1.5e-05 2.8e-12 3.9e-10 3.0e-05 4.4e-05 1.9e-05 3.8e-06 3.5e-07 2.4e-06 4.7e-09

2/10 6/10 8/10 10/10 14/10 16/10 18/10 21/10 27/10 29/10 1/9 3/9 5/9 7/9 9/9 11/9 20/9 22/9 29/9 2/10 4/10 6/10 9/10 11/10 15/10 20/10

19

4

5

6

7

8

9

ETADTDTAEQVIASFR ISSSNPYSTVTMDELR ACLISMGYDLGEAEFAR Desmin VELQELNDR VAELYEEELR FASEASGYQDNIAR RIESLNEEIAFLK EINLPIQTYSALNFR FLEQQNAALAAEVNR TFGGAPGFPLGSPLSSPVFPR LQEEIQLKEEAENNLAAFR Actin GYSFVTTAER AVFPSIVGRPR QEYDEAGPSIVHR SYELPDGQVITIGNER VAPEEHPTLLTEAPLNPK DLYANNVLSGGTTMYPGIADR TTGIVLDSGDGVTHNVPIYEGY ALPHAIMR Troponin T YEINVLR VLAIDHLNEDQLR DLNELQALIEAHFENR Tropomyosin LVIIESDLER IQLVEEELDR SIDDLEDELYAQK IQLVEEELDRAQER AISEELDHALNDMTSI Translocase GAWSNVLR EQGFLSFWR YFPTQALNFAFK Troponin I AYATEPHAK AKESLDLR

1752.86 1798.89 1901.92 1114.53 1249.61 1527.68 1560.82 1648.89 1672.85 2087.09 2245.15 1129.50 1197.66 1499.65 1789.85 1954.99 2227.02

1752.81 1798.84 1901.86 1114.56 1249.61 1527.69 1560.85 1648.89 1672.85 2087.08 2244.13 1129.54 1197.69 1499.70 1789.88 1955.03 2227.05

28.9 29.7 30.7 25.2 5.4 9.1 14.5 0.7 0.2 3.9 2.2 30.3 23.7 29.4 14.3 20.5 14.4

118 95 91 31 71 94 30 92 132 96 142 69 49 99 97 94 122

4.2e-11 7.4e-09 1.9e-08 3.3e-02 3.2e-06 9.8e-09 2.7e-02 1.5e-08 1.9e-12 5.1e-09 1.2e-13 4.6e-06 5.0e-04 3.2e-09 5.1e-09 8.3e-09 1.3e-11

22/10 24/10 28/10 8/8 10/8 16/8 18/8 21/8 23/8 27/8 29/8 4/7 6/7 10/7 13/7 17/7 22/7

3195.57

3195.60

7.3

98

1.8e-09

29/7

905.49 1534.79 1910.94 1185.64 1242.63 1537.67 1726.86 1757.78 901.46 1168.55 1445.70 986.46 930.59

905.49 1534.81 1910.94 1185.66 1242.64 1537.71 1726.88 1757.81 901.47 1168.56 1445.73 986.48 930.56

4.2 7.3 4.1 15.1 7.1 23.8 12.6 18.6 13.6 7.7 17.3 20.2 32.2

47 90 107 78 85 94 58 62 45 65 53 50 57

9.0e-04 3.3e-08 5.1e-10 7.1e-07 1.3e-07 9.2e-09 4.7e-05 1.4e-05 2.1e-03 1.2e-05 1.0e-04 3.0e-04 1.3e-05

1/3 12/3 25/3 3/5 5/5 16/5 19/5 21/5 3/3 16/3 23/3 1/7 2/7

20

10

MLC-2

11

MLC-1

12

Troponin C

TLLLQIAK NITEIADLTQK NIDALSGMEGR ISADAMMQALLGAR CQPLELAGLGFAELQDLCR EAFMLFDR ITYGQCGDVLR ALGQNPTQAEVLR DTGTYEDFVEGLR IEFTPEQIEEFK AAPAPAPPPEPERPK NKDTGTYEDFVEGLR DTFAALGR DGFIDKNDLR EAFTIMDQNR GADPEETILNAFK SEEELSDLFR

898.71 1244.69 1161.43 1446.78 2189.05 1027.45 1280.65 1395.80 1500.72 1508.77 1523.87 1742.85 849.36 1191.60 1223.58 1403.70 1223.57

898.78 1244.66 1161.44 1446.73 2189.06 1027.47 1280.61 1395.74 1500.67 1508.73 1523.80 1742.81 849.43 1191.58 1223.56 1403.69 1223.56

70.7 40.1 8.6 29.5 4.5 22.2 26.5 39.3 35.4 22.1 40.1 27.1 85.3 15.0 18.4 10.7 5.7

43 64 57 77 118 58 76 96 74 42 52 79 76 50 38 65 84

5.3e-03 3.0e-05 1.1e-06 1.6e-06 6.6e-11 4.1e-05 7.6e-07 6.4e-09 1.3e-06 2.0e-03 1.0e-04 3.3e-07 7.3e-02 3.0e-03 4.7e-03 9.8e-06 1.1e-07

3/7 3/7 4/7 10/7 25/7 1/7 7/7 10/7 14/7 16/7 18/7 23/7 2/4 13/4 15/4 19/4 17/1

Note: Human left ventricular proteins of heart failure (30µg/lane) were resolved by NuPAGE 4-12% Bis-Tris gel and stained with Coomassie R-250. The protein identification was based on Mascot search results by peptide mass fingerprint (PMF) technique. MS spectra were acquired on the 4800 MALDI TOF/TOF Analyzer (ABI) using 1000 shots/spectra and a laser power between 4300–4700 units. MLC: Myosin light chain

21

Supplemental Table 5. Summary for the quantitation of site-specific cTnI phosphorylation by MRM assay in ISHD and IDCM failing and normal human heart (n = 10 for each group)

Site S4 S5 S4 & S5 S22 S23 S22 & S23 Y25 T30 S41 S43 S41 & S43 T50 S76 T77 S76 & T77 T142 S149 S165 T180 S198

Donor Q1, Q 3 Median 0.0052 0.0049, 0.0058 0.0046 0.0045, 0.0049 0.0078 0.0075, 0.0082 0.0022 0.0021, 0.0024 0.0212 0.0208, 0.0222 0.0070 0.0066, 0.0074 0.0770 0.0714, 0.0830 N.D. 0.0094 0.0090, 0.0096 0.0039 0.0026, 0.0050 0.0027 0.0026, 0.0029 0.0223 0.0214, 0.0231 0.0774 0.0763, 0.0789 0.0301 0.0300, 0.0309 0.0034 0.0031, 0.0036 0.0974 0.0756, 0.1188 N.D. 0.0023 0.0021, 0.0027 0.0337 0.0330, 0.0362 0.0017 0.0017, 0.0019

ISHD Median 0.0039 * 0.0036 * N.D. 0.0015 * 0.0083 * LLOQ 0.0444 * N.D. 0.0115 * 0.0040 0.0027 0.0230 0.1115 * 0.0360 * 0.0040 * 0.1534 * N.D. 0.0032 0.0487 * 0.0040 *

IDCM Q1, Q 3

0.0036, 0.0045 0.0035, 0.0037 0.0014, 0.0016 0.0081, 0.0092 0.0425, 0.0468 0.0111, 0.0128 0.0037, 0.0043 0.0026, 0.0028 0.0204, 0.0241 0.1069, 0.1210 0.0355, 0.0368 0.0037, 0.0041 0.1329, 0.1651 0.0022, 0.0038 0.0465, 0.0490 0.0034, 0.0048

Median 0.0045 0.0041 * N.D. 0.0017 0.0051 * LLOQ 0.0436 * N.D. 0.0126 * 0.0060 * 0.0028 0.0244 0.1070 * 0.0389 * 0.0045 * 0.1776 * N.D. 0.0064 * 0.0460 * 0.0039 *

Q1, Q 3 0.0043, 0.0049 0.0038, 0.0044 0.0016, 0.0019 0.0045, 0.0053 0.0307, 0.0458 0.0108, 0.0141 0.0056, 0.067 0.0026, 0.0032 0.0204, 0.0281 0.1051, 0.1080 0.0362, 0.0456 0.0040, 0.0055 0.1589, 0.2036 0.0051, 0.0098 0.0447, 0.0498 0.0036, 0.0041

The ratio of each phosphorylated residue of cTnI (fmol phosphorylation/fmol total protein) determined by MRM assay for Donor, ischemic heart failure (ISHD) and dilated cardiomyopathy (IDCM) (n = 10 per group) for all of the sites. Phosphopeptides observed, but below lower limited of quantification (LLOQ); Phospho-peptides that were not detected by MS are denoted as not detected (N.D.). Q1, first quartile; Q3, third quartile; *, P < 0.05 ISHD or IDCM versus Donor by one-way ANOVA on ranks followed by a Dunnett’s multiple comparisons post-hoc test.

22

SUPPLEMENTAL FIGURES

Supplemental Figure 1. Schematic process in developing multiple reaction monitoring (MRM) assays for cardiac troponin I phosphorylation.

23

Supplemental Figure 2. Optimization of the MRM assay for phosphorylated peptides containing S4 or S5. Panel A: MS/MS spectrum of the novel monophosphorylated site S4 in vitro. MS/MS of the doubly charged 486.7 ion allows for selection of product ions for monitoring the native (light) S4 monophosphorylated peptide ((ac)ADG(p)SSDAAR). Panel B: Optimization of MRM on the S4 monophosphorylated peptide ((ac)ADG(p)SSDAAR). Panel C: MS/MS spectrum of the novel monophosphorylated site S5 in vitro. MS/MS of the doubly charged 486.7 ion allows for selection of product ions for monitoring the native (light) S6 monophosphorylated peptide ((ac)ADGS(p)SDAAR). Panel D: Optimization of MRM on the S5 monophosphorylated peptide ((ac)ADGS(p)SDAAR). Optimization was obtained by ramping the parameters declustering potential (DP)(0-400 volts), collision energy (CE)(5-130 volts), and Collision Cell Exit Potential (CXP)(0-66 volts) from low to high with a step of 1 for all parameters and a fixed setting of 10 volts for entrance potential (EP).

24

Supplemental Figure 3. Optimization of the MRM assay for phosphorylated and unphosphorylated peptides containing S4/5. Panel A: MS/MS spectrum of the novel diphosphorylated sites S4/5 in vitro. MS/MS of the doubly charged 526.7 ion allows for selection of product ions for monitoring the native (light) S4/5 diphosphorylated peptide (ac)ADG(p)S(p)SDAAR). Panel B: Optimization of MRM on the S4/5 diphosphorylated peptide (ac)ADG(p)S(p)SDAAR). Panel C: MS/MS of the doubly charged 415.7 ion allows for selection of product ions for monitoring the labeled (heavy) S4/5 unphosphorylated peptide ((ac)ADGSSDAAR). Panel D: Optimization of MRM on the S4/5 unphosphorylated peptide ((ac)ADGSSDAAR). Optimization was obtained by ramping the parameters declustering potential (DP)(0-400 volts), collision energy (CE)(5-130 volts), and Collision Cell Exit Potential (CXP)(066 volts) from low to high with a step of 1 for all parameters and a fixed setting of 10 volts for entrance potential (EP).

25

Supplemental Figure 4. Optimization of the MRM assay for phosphorylated peptides containing S22 or S23. Panel A: MS/MS spectrum of the monophosphoylated site S22 in vitro. MS/MS of the doubly charged 353.9 ion allows for selection of product ions for monitoring the native (light) S22 monophosphorylated peptide ((p)SSNYR). Panel B: Optimization of MRM on S22 monophosphorylated peptide ((p)SSNYR). Panel C: MS/MS spectrum of the monophosphoylated site S23 in vitro. MS/MS spectrum of the doubly charged 353.6 ion allows for selection of product ions for monitoring the native (light) S23 monophosphorylated peptide (S(p)SNYR). Panel D: Optimization of MRM on S23 monophosphorylated peptide (S (p)SNYR). Optimization was obtained by ramping the parameters declustering potential (DP)(0-400 volts), collision energy (CE)(5-130 volts), and Collision Cell Exit Potential (CXP)(0-66 volts) from low to high with a step of 1 for all parameters and a fixed setting of 10 volts for entrance potential (EP).

26

Supplemental Figure 5. Optimization of the MRM assay for phosphorylated and unphosphorylated peptides containing S22/23. Panel A: MS/MS spectrum of the diphosphorylated sites S22/23 in vitro. MS/MS spectrum of the doubly charged 394.0 ion allows for selection of product ions for monitoring the native (light) S22/23 diphosphorylated peptide ((p)S(p)SNYR). Panel B: Optimization of MRM on the signature S22/23 diphosphorylated peptide ((p)S(p)SNYR). Panel C: MS/MS spectrum of the doubly charged 318.2 ion allows for selection of product ions for monitoring the labeled (heavy) S22/23 unphosphorylated peptide (SSNYR*). Panel D: Optimization of MRM on S22/23 unphosphorylated peptide (SSNYR*). Optimization was obtained by ramping the parameters declustering potential (DP)(0-400 volts), collision energy (CE) (5-130 volts), and Collision Cell Exit Potential (CXP)(0-66 volts) from low to high with a step of 1 for all parameters and a fixed setting of 10 volts for entrance potential (EP).

27

Supplemental Figure 6. Optimization of the MRM assay for phosphorylated peptide containing Y25 and an unmodified peptide for quantification of the total protein. Panel A: MS/MS spectrum of the novel monophosphorylated site Y25 in vitro. MS/MS of the doubly charged 353.9 ion allows for selection of product ions for monitoring the native (light) Y25 monophosphorylated peptide (SSN(p)YR). Panel B: Optimization of MRM on the Y25 monophosphorylated peptide (SSN(p)YR). Panel C: MS/MS spectrum of the doubly charged 627.4 ion allows for selection of product ions for monitoring the labeled (heavy) peptide (NITEIADLTQK*) for quantification of the total protein. Panel D: Optimization of MRM on the peptide (NITEIADLTQK*) for the total protein. Optimization was obtained by ramping the parameters declustering potential (DP)(0-400 volts), collision energy (CE)(5-130 volts), and Collision Cell Exit Potential (CXP)(0-66 volts) from low to high with a step of 1 for all parameters and a fixed setting of 10 volts for entrance potential (EP).

28

Supplemental Figure 7. Optimization of the MRM assay for phosphorylated peptides containing S41 or S43. Panel A: MS/MS spectrum of the monophosphoylated site S41 in vitro. MS/MS spectrum of the doubly charged 306.9 ion allows for selection of product ions for monitoring the native (light) S41 monophosphorylated peptide (I(p)SASR). Panel B: Optimization of MRM on S41 monophosphorylated peptide (I(p)SASR). Panel C: MS/MS spectrum of the monophosphoylated site S43 in vitro. MS/MS spectrum of the doubly charged 307.0 ion allows for selection of product ions for monitoring the native (light) S43 monophosphorylated peptide (ISA(p)SR). Panel D: Optimization of MRM on the S43 monophosphorylated peptide (ISA(p)SR). Optimization was obtained by ramping the parameters declustering potential (DP)(0-400 volts), collision energy (CE)(5-130 volts), and Collision Cell Exit Potential (CXP)(0-66 volts) from low to high with a step of 1 for all parameters and a fixed setting of 10 volts for entrance potential (EP).

29

Supplemental Figure 8. Optimization of the MRM assay for phosphorylated and unphosphorylated peptides containing S41/43. Panel A: MS/MS spectrum of diphosphorylated sites S41/43 in vitro. MS/MS spectrum of the doubly charged 346.8 ion allows for selection of product ions for monitoring the native (light) S41/43 diphosphorylated peptide (I(p)SA(p)SR). Panel B: Optimization of MRM on the S41/43 diphosphorylated peptide (I(p)SA(p)SR). Panel C: MS/MS spectrum of the doubly charged 272.0 ion allows for selection of product ions for monitoring the labeled (heavy) S41/43 unphosphorylated peptide (ISASR*). Panel D: Optimization of MRM on the S41/43 unphosphorylated peptide (ISASR*). Optimization was obtained by ramping the parameters declustering potential (DP)(0-400 volts), collision energy (CE)(5-130 volts), and Collision Cell Exit Potential (CXP)(0-66 volts) from low to high with a step of 1 for all parameters and a fixed setting of 10 volts for entrance potential (EP).

30

Supplemental Figure 9. Optimization of the MRM assay for phosphorylated and unphosphorylated peptides containing T50. Panel A: MS/MS spectrum of the novel monophosphorylated site T50 in vitro. MS/MS of the doubly charged 490.6 ion allows for selection of product ions for monitoring the native (light) T50 monophosphorylated peptide ((p)TLLLQIAK). Panel B: Optimization of MRM on the T50 monophosphorylated peptide ((p)TLLLQIAK). Panel C: MS/MS of the doubly charged 454.2 ion allows for selection of product ions for monitoring the labeled (heavy) T50 unphosphorylated peptide (TLLLQIAK*). Panel D: Optimization of MRM on the T50 unphosphorylated peptide (TLLLQIAK*). Optimization was obtained by ramping the parameters declustering potential (DP)(0-400 volts), collision energy (CE)(5-130 volts), and Collision Cell Exit Potential (CXP)(0-66 volts) from low to high with a step of 1 for all parameters and a fixed setting of 10 volts for entrance potential (EP).

31

Supplemental Figure 10. Optimization of the MRM assay for phosphorylated peptides containing S76 or T77. Panel A: MS/MS spectrum of the novel monophosphorylated site S76 in vitro. MS/MS of the doubly charged 313.9 ion allows for selection of product ions for monitoring the native (light) S76 monophosphorylated peptide (AL(p)STR). Panel B: Optimization of MRM on the S76 monophosphorylated peptide (AL(p)STR). Panel C: MS/MS spectrum of the novel monophosphorylated site T77 in vitro. MS/MS of the doubly charged 313.9 ion allows for selection of product ions for monitoring the native (light) T77 monophosphorylated peptide (ALS(p)TR). Panel D: Optimization of MRM on the T77 monophosphorylated peptide (ALS(p)TR). Optimization was obtained by ramping the parameters declustering potential (DP)(0-400 volts), collision energy (CE)(5-130 volts), and Collision Cell Exit Potential (CXP)(0-66 volts) from low to high with a step of 1 for all parameters and a fixed setting of 10 volts for entrance potential (EP).

32

Supplemental Figure 11. Optimization of the MRM assay for phosphorylated and unphosphorylated peptides containing S76/T77. Panel A: MS/MS spectrum of the novel diphosphorylated sites S76/T77 in vitro. MS/MS of the doubly charged 354.6 ion allows for selection of product ions for monitoring the native (light) S76/T77 diphosphorylated peptide (AL(p)S(p)TR). Panel B: Optimization of MRM on the S76/T77 diphosphorylated peptide (AL(p)S(p)TR). Panel C: MS/MS of the doubly charged 278.9 ion allows for selection of product ions for monitoring the labeled (heavy) S76/T77 unphosphorylated peptide (ALSTR*). Panel D: Optimization of MRM on the S76/T77 unphosphorylated peptide (ALSTR*). Optimization was obtained by ramping the parameters declustering potential (DP)(0-400 volts), collision energy (CE)(5-130 volts), and Collision Cell Exit Potential (CXP)(0-66 volts) from low to high with a step of 1 for all parameters and a fixed setting of 10 volts for entrance potential (EP).

33

Supplemental Figure 12. Optimization of the MRM assay for phosphorylated and unphosphorylated peptides containing T142. Panel A: MS/MS spectrum of the monophosphoylated site T142 in vitro. MS/MS spectrum of the doubly charged 362.2 ion allows for selection of product ions for monitoring the native (light) T142 monophosphorylated peptide (RP(p)TLR). Panel B: Optimization of MRM on T142 monophosphorylated peptide (RP (p)TLR); Panel C: MS/MS spectrum of the doubly charged 327.2 ion allows for selection of product ions for monitoring the labeled (heavy) T142 unphosphorylated peptide (RPTLR*). Panel D: Optimization of MRM on T142 unphosphorylated peptide (RPTLR*). Optimization was obtained by ramping the parameters declustering potential (DP)(0-400 volts), collision energy (CE)(5-130 volts), and Collision Cell Exit Potential (CXP)(0-66 volts) from low to high with a step of 1 for all parameters and a fixed setting of 10 volts for entrance potential (EP).

34

Supplemental Figure 13. Optimization of the MRM assay for phosphorylated and unphosphorylated peptides containing S165. Panel A: MS/MS spectrum of the novel monophosphorylated site S165 in vitro. MS/MS of the doubly charged 506.8 ion allows for selection of product ions for monitoring the native (light) S165 monophosphorylated peptide (AKE(p)SLDLR). Panel B: Optimization of MRM on the S165 monophosphorylated peptide (AKE(p)SLDLR). Panel C: MS/MS of the doubly charged 471.7 ion allows for selection of product ions for monitoring the labeled (heavy) S165 unphosphorylated peptide (AKE(p)SLDLR*). Panel D: Optimization of MRM on the S165 unphosphorylated peptide (AKE(p)SLDLR*). Optimization was obtained by ramping the parameters declustering potential (DP)(0-400 volts), collision energy (CE) (5-130 volts), and Collision Cell Exit Potential (CXP)(0-66 volts) from low to high with a step of 1 for all parameters and a fixed setting of 10 volts for entrance potential (EP).

35

Supplemental Figure 14. Optimization of the MRM assay for phosphorylated and unphosphorylated peptides containing T180. Panel A: MS/MS spectrum of the novel monophosphorylated site T180 in vitro. MS/MS of the doubly charged 351.6 ion allows for selection of product ions for monitoring the native (light) T180 monophosphorylated peptide (ED(p)TEK). Panel B: Optimization of MRM on the T180 monophosphorylated peptide (ED(p)TEK). Panel C: MS/MS of the doubly charged 314.9 ion allows for selection of product ions for monitoring the labeled (heavy) T180 unphosphorylated peptide (EDTEK*). Panel A: Optimization of MRM on the T180 unphosphorylated peptide (EDTEK*). Optimization was obtained by ramping the parameters declustering potential (DP)(0-400 volts), collision energy (CE)(5-130 volts), and Collision Cell Exit Potential (CXP)(0-66 volts) from low to high with a step of 1 for all parameters and a fixed setting of 10 volts for entrance potential (EP).

36

Supplemental Figure 15. Optimization of the MRM assay for phosphorylated peptides containing S198. Panel A: MS/MS spectrum of the monophosphoylated site S198 in vitro. MS/MS spectrum of the doubly charged 622.8 ion allows for selection of product ions for monitoring the native (light) S198 monophosphorylated peptide (NIDAL(p)SGMEGR). Panel B: Optimization of MRM on S199 monophosphorylated peptide (NIDAL(p)SGMEGR). Panel C: MS/MS spectrum of the doubly charged 627.8 ion allows for selection of product ions for monitoring the labeled (heavy) S198 monophosphorylated peptide (NIDAL(p)SGMEGR*). Panel D: Optimization of MRM on the heavy labeled S198 monophosphorylated peptide (NIDAL(p)SGMEGR*). Optimization was obtained by ramping the parameters declustering potential (DP)(0-400 volts), collision energy (CE)(5-130 volts), and Collision Cell Exit Potential (CXP)(0-66 volts) from low to high with a step of 1 for all parameters and a fixed setting of 10 volts for entrance potential (EP).

37

Supplemental Figure 16. Quantitative comparison of sample processing. Comparison of the phosphorylated and unphosphorylated peptides containing T142 was between a gel-based and direct homogenization-based protein digestion by MRM. In the gel-based method (green), 30 μg of purified myofilament proteins from heart failure LV tissues was applied to 4-12% Bis-Tris NuPAGE gels (Invitrogen), followed by Coomassie staining and tryptic in-gel digestion. Alternatively, whole proteins were extracted directly from the purified myofilament, and then 30 µg of the protein mixture was treated as above without the separation by NuPAGE gels (blue). All peptide mixtures were desalted prior to analysis by MRM assays of peptides (RPTLR or RP(p)TLR) to the peptide NITEIADLTQK for the total protein. All the data shown was an average value of five replicates with standard errors. The result of MRM assays in five replicates showed no significant difference between two sample preparation methods. As well, the gelbased method demonstrated greater intensity of signals and reduced percent coefficient of variation (CV% = 5-6%) compared to the direct protein digestion.

38

Supplemental Figure 17. Preparation and identification of cTnI. Lane L is the protein marker of molecular size; lanes 1-9 are proteins of human left ventricular tissues with heart failure. Proteins of 30 µg were loaded for each lane and resolved by NuPAGE 4-12% Bis-Tris gel and stained by Coomassie R-250. Protein identification was completed by peptide mass fingerprint (PMF) technique on MALDI-TOF/TOF instrument (see Supplemental Table 4).

39

Supplemental Figure 18. Quantitation of the fold change of phosphorylation sites in myocardium obtained from donor and failing hearts. The fold change of the phosphorylation sites of cTnI by MRM assay in Donor (light gray), ISHD (white) and IDCM (dark gray) heart

40

(Panel A); The fold change of the novel phosphorylation sites of cTnI (n = 10 per group) (Panel B). Values are median, 1st quartile and 3rd quartile. The quantity of each peptide was determined in the linear range of standard curve and then the fold change was calculated between the various groups. S149 phosphorylation was not detected. Note: S22 & S23 diphosphorylated peptide was detected but below LLOQ in the failing heart (indicated with a #).

41

Supplemental Figure 19. Quantitatively determine phosphorylation of cTnI at S22/23 by immunoblot. Panel A: Quantitation of the phosphorylation was verified by immunoblot for PKA-sites of S22 and/or 23 and the potential Y25. Image of immunoblot result of extracts from human left ventricular (LV) tissues using anti-phospho-troponin I (Cardiac) (Ser22 and/or 23) antibody and Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Cell signaling). The loading control is monoclonal anti-desmin antibody (Sigma). Panel B: Average ratio of phosphorylated PKA-sites of S22 and/or 23 decreased in ISHD and IDCM. Experiment results were acquired in triplicate on 1 µg each of proteins from LV myofibrils isolated from the end-stage failing hearts of ISHD, IDCM, and non-failing donor hearts (n = 10). Densitometrical analysis was done with a Progenesis image software (Nonlinear Dynamics). Phosphorylated cTnI (S22/23) was calculated as phospho signal/total protein signal in triple experiments.

42

SUPPLEMENTAL REFERENCES 1. Spragg DD, Leclercq C, Loghmani M, Faris OP, Tunin RS, DiSilvestre D, McVeigh ER, Tomaselli GF, Kass DA. Regional alterations in protein expression in the dyssynchronous failing heart. Circulation. 2003; 108: 929–932.

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