Journal of Molecular and Cellular Cardiology 87 (2015) 102–112

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Journal of Molecular and Cellular Cardiology

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Comprehensive assessment of chamber-specific and transmuralheterogeneity in myofilament protein phosphorylation by top-downmass spectrometry

Zachery R. Gregorich a,b, Ying Peng b, Nicole M. Lane b,c, Jeremy J. Wolff d, Sijian Wang e,f, Wei Guo b,1,Huseyin Guner b,g, Justin Doop b, Timothy A. Hacker h, Ying Ge a,b,c,g,⁎a Molecular Pharmacology Training Program, University of Wisconsin-Madison, Madison, WI 53706, USAb Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, WI 53706, USAc Cellular and Molecular Pathology Training Program, University of Wisconsin-Madison, Madison, WI 53706, USAd Bruker Daltonics, Billerica, MA 01821, USAe Department of Biostatistics & Medical Informatics, University of Wisconsin-Madison, Madison, WI 53706, USAf Department of Statistics, University of Wisconsin-Madison, Madison, WI 53706, USAg Human Proteomics Program, University of Wisconsin-Madison, Madison, WI 53706, USAh Department of Medicine, University of Wisconsin-Madison, Madison, WI 53706, USA

Abbreviations: PTMs, post-translational modificatiospectrometry; cTnI, cardiac troponin I; cTnT, cardiac trMLC2, myosin light chain 2; RV, right ventricle; LA, leftmid-myocardium; ENDO, sub-endocardium; EPI, sub-epMLC1, myosin light chain 1; LC, liquid chromatography; Fclotron resonance; pcTnI, mono-phosphorylated cTnI; pp

pcTnT, mono-phosphorylated cTnT; αTm, Tpm1.1;Tpm1.1; βTm, Tpm2.2; MLC1v, MLC1 ventricular isoformform; MLC1a, MLC1 atrial isoform; MLC2a, MLC2 aphosphorylated MLC2; LTQ, linear ion trap.⁎ Corresponding author at: 1300 University Ave., SMI 1

E-mail address: ge2@wisc.edu (Y. Ge).1 Present address: Department of Animal Sciences, Wy

82071, USA.

http://dx.doi.org/10.1016/j.yjmcc.2015.08.0070022-2828/© 2015 Elsevier Ltd. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 May 2015Received in revised form 4 August 2015Accepted 8 August 2015Available online 9 August 2015

Keywords:Transmural heterogeneityChamber heterogeneityPhosphorylationMass spectrometryMyofilament

The heart is characterized by a remarkable degree of heterogeneity, the basis of which is a subject of active inves-tigation. Myofilament protein post-translationalmodifications (PTMs) represent a critical mechanism regulatingcardiac contractility, and emerging evidence shows that pathological cardiac conditions induce contractile het-erogeneity that correlates with transmural variations in themodification status of myofilament proteins. Never-theless,whether there exists basal heterogeneity inmyofilament protein PTMs in theheart remains unclear. Herewe have systematically assessed chamber-specific and transmural variations in myofilament protein PTMs, spe-cifically, the phosphorylation of cardiac troponin I (cTnI), cardiac troponin T (cTnT), tropomyosin (Tpm), andmy-osin light chain 2 (MLC2). We show that the phosphorylation of cTnI and αTm vary in the different chambers ofthe heart, whereas the phosphorylation of MLC2 and cTnT does not. In contrast, no significant transmural differ-enceswere observed in the phosphorylation of any of themyofilament proteins analyzed. These results highlightthe importance of appropriate tissue sampling—particularly for studies aimed at elucidating diseasemechanismsand biomarker discovery—in order to minimize potential variations arising from basal heterogeneity inmyofila-ment PTMs in the heart.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

The heart is characterized by a remarkable degree of heterogeneity,which underlies normal pump function and is also altered in heart

ns; LV, left ventricle; MS, massoponin T; Tpm, tropomyosin;atrium; RA, right atrium; MYO,icardium; MS/MS, tandem MS;T-ICR, Fourier transform ion cy-cTnI, bis-phosphorylated cTnI;pαTm, mono-phosphorylated; MLC2v, MLC2 ventricular iso-trial isoform; pMLC2, mono-

30, Madison, WI 53706, USA.

oming University, Laramie, WY

failure [1–6]. Contractile heterogeneity, in particular, has taken on in-creased significance with the demonstration that transmural changesin myocardial contractility not only occur in the failing heart [1–3], butmay also be predictive of adverse cardiac events [7,8]. Myocardialcontractility is governed by a number of factors, including the size andduration of Ca2+ transients, as well as the intrinsic properties of thecontractile apparatus, which depend primarily on the expression ofmyofilament protein isoforms and post-translational modifications(PTMs) [9–12].

Myofilament protein PTMs have emerged as a keymechanism regu-lating cardiac contractility in health and disease [9–12]. Previous studiesin humans, as well as large and small animals, have shown that agingand pathological cardiac conditions induce contractile heterogeneityacross the free wall of the left ventricle (LV) that correlates with differ-ences in the modification status of myofilament proteins [1,2,5]. Yet,there is conflicting evidence in the literature regarding the existenceof basal heterogeneity in myofilament PTMs. Although several groups

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have observed transmural variations inmyofilament PTMs across the LVfree wall in small animals [4,13–15], such heterogeneity does notappear to be present in humans and large animals under basal condi-tions [1,2,16]. Comparatively, very little is known regarding the exis-tence of chamber-specific variations in myofilament protein PTMs inthe heart [17]. Knowledge of basal chamber-specific or transmural var-iations in myofilament protein PTMs will undoubtedly be essential forunderstanding cardiac physiology, elucidating PTM-associated diseasemechanisms, and biomarker discovery.

Top-down mass spectrometry (MS) is a powerful tool for the com-prehensive assessment of protein PTMs [18–20]. Intact proteins are an-alyzed in top-down MS, which allows for the detection of the entirecomplement of protein PTMs simultaneously without a priori knowl-edge [18–20]. Furthermore, the addition of PTMs to intact proteins hasrelatively little influence on their physiochemical properties, thus,allowing for the reliable quantification of modified and un-modifiedprotein forms present within the same spectrum [18–20]. Herein, wehave utilized quantitative top-down MS to systematically assesschamber-specific and transmural variations in myofilament proteinPTMs in the hearts of healthy pigs, which currently represent the goldstandard model system for human cardiovascular diseases [21]; with aspecial focus on the phosphorylation of cardiac troponin I (cTnI), cardiactroponin T (cTnT), tropomyosin (Tpm), and myosin light chain 2(MLC2).

2. Methods

A detailed Materials and methods section is provided in theSupplemental Material.

2.1. Tissue procurement

Pig heart tissuewas obtained fromYorkshire domestic pigs (approx-imately 3 months of age) as approved by the University of WisconsinAnimal Care and Use Committee. Excised hearts were quickly sectionedinto the LV, right ventricle (RV), left atrium (LA), and right atrium (RA),flash frozen in liquid nitrogen, and stored at−80 °C for later use. For ex-periments examining chamber-specific heterogeneity in myofilamentprotein phosphorylation, the LV samples consisted predominantly ofmid-myocardium (MYO) with little or no sub-endocardium (ENDO)or sub-epicardium (EPI). For experiments examining transmural het-erogeneity in myofilament protein phosphorylation, the free wall ofthe LV was further sectioned into thirds with the inner most third, themiddle third, and the outer most third representing the ENDO, MYO,and EPI, respectively, prior to flash freezing.

2.2. Immunoaffinity purification of cardiac troponin complex

Cardiac troponin complex was isolated from pig myocardial tissueby immunoaffinity purification as previously described [22].

2.3. Preparation of myofilament extracts

Myofilament proteins were extracted from pig myocardial tissueusing a two-step extraction procedure as previously described [17,23].

2.4. Offline and online top-down high-resolution MS and tandem MS(MS/MS)

For MS analysis of cTnI and cTnT, desalting and offline MS analysiswere carried out as previously described [22]. Top-down MS and MS/MS analyses ofMLC2 andmyosin light chain 1 (MLC1)were also carriedout as previously described [23], with minor modifications. For onlineMS analysis of Tpm, myofilament extracts were diluted 1:1 (v/v) withmobile phase A (mobile phase A: 0.1% formic acid in water; mobilephase B: 0.1% formic acid in methanol) prior to liquid chromatography

(LC)-MS. Myofilament extracts were separated using a Dionex U3000LC system (Thermo Scientific, Boston, MA, USA) equipped with ahome-packed PLRP column (PLRP-S, 200 mm × 500 μm, 10 μm,1000 Å; Varian, Lake Forest, CA, USA) and a gradient going from 20% Bto 90% B over 55 min, at a flow rate of 12.5 μL/min. The Dionex U3000LC system was coupled online with a 12 T Fourier transform ion cyclo-tron resonance (FT-ICR)mass spectrometer (Bruker Daltonics, Billerica,MA, USA) using the Bruker electrospray ionization source. Sampleswere introduced into the mass spectrometer using a capillary voltageand an endcap offset of −4.5 kV and −5 kV, respectively. A resolvingpower of 250,000 (at m/z 400) and a fixed ion accumulation time of0.02 s were used for spectral acquisition. Mass spectra obtained usingthe methods described above were highly reproducible (Supp. Fig. 1).

2.5. Protein identification

Identification of the atrial isoforms of MLC2 and MLC1 from pig wascarried out as previously described [23].

2.6. Western blot

Myofilament extracts were separated by SDS-PAGE and transferredto PVDF membranes (Millipore, Billerica, MA, USA). Membranes wereblocked with Protein-Free Blocking Buffer (Thermo Scientific) and blot-tedwith antibodies against cTnI (ThermoScientific) and cTnI phosphor-ylated at Ser22/23 (Cell Signaling Technology, Beverly, MA, USA).Western blots were analyzed using ImageJ.

2.7. Quantitative analysis

Offline and online mass and tandem mass spectra were analyzedusing in-house developed MASH Suite software [24] and Bruker DataAnalysis software, respectively, and manually validated to ensure dataaccuracy. Themost abundant relativemolecularmasses andmonoisoto-pic masses are reported for intact proteins and fragment ions, respec-tively. The relative abundances of each protein form, as well as thetotal protein phosphorylation, were calculated based on the signal in-tensities from the mass spectra as previously described [17,23,25,26],taking into account oxidized protein forms and those associated withnon-covalent adducts.

2.8. Statistical analysis

Data are from at least three biological replicates (n = 3), each withtwo technical replicates. For the chamber analysis, the relative abun-dance of each protein form and the total protein phosphorylation valuesfrom the EPI, MYO, and ENDOwere included as additional replicates forthe LV (n = 12 for LV in the chamber comparison). The relative abun-dance data were analyzed in R 3.2.1 using linear mixed effects modelswith random intercepts incorporating 2 main effects (either chamberor transmural layer and phosphorylation type) and their interaction.One-wayANOVAwas used to evaluate the statistical significance of var-iance for the total protein phosphorylation in either the different cham-bers of the heart, or in the different layers of the LV freewall. TheMann–WhitneyU test was used for group comparison of theWestern blot data.All values are reported as mean ± SEM. Differences between meanswere considered significant at p b 0.05.

3. Results

3.1. cTnI phosphorylation varies in a chamber-specific but not transmuralmanner in the heart

To determine whether cTnI phosphorylation varies in the differentchambers of the heart, cTnI was immunoaffinity purified from the LV,RV, LA, and RA of healthy pig hearts, and analyzed by top-down MS.

Fig. 1.Basal cTnI phosphorylation varies in a chamber-specific but not transmuralmanner in theheart. (A) Representativemass spectra for cTnI from the four chambers of the heart. Circlesrepresent the theoretical isotopic abundance distribution of the isotopomer peaks corresponding to the assigned mass. Squares and stars represent oxidized cTnI and potassium adducts,respectively. (B) Graph showing the relative abundances of cTnI, pcTnI, and ppcTnI in the four chambers of theheart. (C)Graph showing total cTnI phosphorylation in the LV, RV, LA, and RA.(D) Representativemass spectra for cTnI from the three layers of the LV freewall. Stars representpotassiumadducts. (E) Graph showing the relative abundances of cTnI, pcTnI, and ppcTnI inthe EPI, MYO, and ENDO of the LV free wall. (F) Graph showing total cTnI phosphorylation in the three layers of the LV free wall. cTnI corresponds to NCBI RefSeq accession numberABF84065. *p b 0.05, **p b 0.001.

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Three major cTnI protein forms, with relative molecular masses thatmatched closely with those previously reported for un-phosphorylated,mono-phosphorylated (pcTnI), and bis-phosphorylated (ppcTnI) cTnIfrom pig [22,23], were detected in all four chambers of the heart(Fig. 1A). Several additional protein forms, including cTnI containing theV116A polymorphism reported previously [22], as well as oxidized cTnIand cTnI associated with non-covalent adducts, were also observed(Fig. 1A).MS-basedquantification of the cTnI protein forms in each cham-ber revealed significant differences in the relative abundances of un-phosphorylated cTnI, pcTnI, and ppcTnI (Fig. 1B, Supplemental Results).Calculation of the total phosphorylation revealed that cTnI phosphoryla-tion was significantly greater in the ventricles than in the atria (Fig. 1C,Supplemental Results).

Next, cTnI from the EPI, MYO, and ENDO of the LV free wall was an-alyzed using top-downMS to determine whether cTnI phosphorylationalso varies transmurally in the heart. Un-phosphorylated cTnI, pcTnI,and ppcTnI were the predominant cTnI protein forms detected by top-downMS in the EPI, MYO and ENDO (Fig. 1D). Additionally, minor pro-tein forms corresponding to cTnI associated with non-covalent adductswere also observed (Fig. 1D). Quantitative top-down MS analysis re-vealed no significant differences in either the relative abundances ofcTnI protein forms, or total cTnI phosphorylation, across the LV freewall (Fig. 1E and F, Supplemental Results).

Western blot analysis of cTnI-Ser22/23 phosphorylation, which wehave previously shown are the only sites basally phosphorylated inpig myocardium [22], was used to confirm the results of the top-downMS analysis. cTnI-Ser22/23 phosphorylation was significantly greaterin the LV than in the LA and RA (Fig. 2A), which corresponds wellwith the significantly increased relative abundance of ppcTnI in the LV(compared to the LA and RA) observed by MS analysis (Fig. 1B). Like-wise, cTnI-Ser22/23 phosphorylation in the RV was increased relativeto the LA and RA byWestern blot (Fig. 2A); however, the difference be-tween the RV and RA was not statistically significant as it was based onthe MS analysis (Fig. 1B). Similarly, cTnI-Ser22/23 phosphorylation, asassessed by Western blot, was not significantly different across thelayers of the LV free wall (Fig. 2B), which is also in good agreementwith the results of our top-down MS analysis (Fig. 1E). Collectively,

Fig. 2. cTnI-Ser22/23 phosphorylation is significantly increased in the LV in comparison to thecTnI phosphorylated at Ser22/23 and total cTnI, aswell as the associated quantification results fo(B) RepresentativeWestern blots for cTnI phosphorylated at Ser22/23 and total cTnI, aswell as thlower panels, respectively (n = 3). *p b 0.05.

these results clearly demonstrate that basal cTnI phosphorylation variesin a chamber-specific but not transmural manner in the heart.

3.2. cTnT phosphorylation does not vary in the different chambers of theheart or across the LV free wall

We next sought to determine whether the phosphorylation of cTnTvaries in the different chambers of the heart. Two cTnT protein forms,with relative molecular masses similar to those previously publishedfor un-phosphorylated and mono-phosphorylated (pcTnT) cTnT frompig [23], were detected by top-down MS in all four chambers of theheart (Fig. 3A). Additional protein forms corresponding to cTnT associ-ated with non-covalent adducts were also observed in the spectra(Fig. 3A). Quantification of cTnT protein forms revealed that the relativeabundances of cTnT and pcTnT did not differ significantly between theLV, RV, LA, and RA (Fig. 3B, Supplemental Results). Likewise, total cTnTphosphorylation was similar across the chambers of the heart (Fig. 3C,Supplemental Results).

Similarly, top-downMS analysis of cTnT phosphorylation across theLV free wall showed no significant differences in either the relativeabundances of cTnT protein forms or total cTnT phosphorylation inthe EPI, MYO, or ENDO (Fig. 3D–F, Supplemental Results). These resultsclearly show that cTnT phosphorylation does not vary in a chamber-specific or transmural manner in the heart.

3.3. Tpm phosphorylation exhibits chamber-specific but not transmuralheterogeneity in the heart

We next examined Tpm phosphorylation in the atria and ventriclesto determine whether there is chamber-specific heterogeneity in Tpmphosphorylation in the heart. Three Tpm protein forms, with relativemolecular masses similar to those previously reported for un-phosphorylated Tpm1.1 (αTm), mono-phosphorylated Tpm1.1(pαTm), and un-phosphorylated Tpm2.2 (βTm) from pig [23,27], weredetected by top-down MS analysis in the LV, RV, LA, and RA (Fig. 4A).Minor protein forms corresponding to oxidizedαTm and pαTm, respec-tively, were also observed in the spectra (Fig. 4A). Quantification of the

LA and RA, but does not vary across the LV free wall. (A) Representative Western blots forr the chamber comparison, are shown in the upper and lower panels, respectively (n=3).e associated quantification results for the LV layer comparison, are shown in theupper and

Fig. 3. cTnT phosphorylation does not vary in the different chambers of the heart or across the LV free wall. (A) Representative mass spectra for cTnT from the LV, RV, LA, and RA. Circlesrepresent the theoretical isotopic abundance distribution of the isotopomer peaks corresponding to the assigned mass. Triangles and diamonds represent sodium and phosphoric acid(+H3PO4) adducts, respectively. (B) Graph showing the relative abundances of cTnT and pcTnT in the four chambers of the heart. (C) Graph showing total cTnT phosphorylation in theLV, RV, LA, and RA. (D) Representative mass spectra for cTnT from the EPI, MYO, and ENDO of the LV free wall. Triangles and diamonds represent sodium and phosphoric acid(+H3PO4) adducts, respectively. (E) Graph showing the relative abundances of cTnT and pcTnT across the layers of the LV free wall. (F) Graph showing total cTnT phosphorylation inthe EPI, MYO, and ENDO. cTnT corresponds to NCBI RefSeq accession number ADY80031. *p b 0.05, **p b 0.001.

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Fig. 4.αTmphosphorylation exhibits chamber-specific but not transmural heterogeneity in the heart. (A) Representativemass spectra for Tpm from the LV, RV, LA, and RA. Circles representthe theoretical isotopic abundance distribution of the isotopomer peaks corresponding to the assignedmass. Squares represent oxidized Tpm. (B) Graph showing the relative abundances ofαTm, pαTm, and βTm in the four chambers of the heart. (C) Graph showing total αTm phosphorylation in the LV, RV, LA, and RA. (D) Representative mass spectra for Tpm from the threelayers of the LV free wall. Squares represent oxidized Tpm. (E) Graph showing the relative abundances of αTm, pαTm, and βTm in the EPI, MYO, and ENDO. (F) Graph showing total αTmphosphorylation in the EPI, MYO, and ENDO. αTm and βTm correspond to NCBI RefSeq accession numbers NP_001090952 and XP_005660265, respectively. *p b 0.05, **p b 0.001.

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Tpm protein forms in spectra from the different chambers of the heartrevealed significant differences in the relative abundances of un-phosphorylatedαTm, pαTm, and un-phosphorylated βTm (Fig. 4B, Sup-plemental Results). Overall, the total phosphorylation of αTm was in-creased in the atria relative to the ventricles (Fig. 4C, SupplementalResults), although the difference in total phosphorylation between theRA and LV did not reach statistical significance (p=0.050013) (Fig. 4C).

Top-down MS analysis of Tpm in the EPI, MYO, and ENDO revealedno significant differences in either the relative abundances of Tpm pro-tein forms, or total αTm phosphorylation, across the free wall of the LV(Fig. 4D-F, Supplemental Results). These findings confirm that αTmphosphorylation is increased in the atria in comparison to the ventricles,but does not vary across the LV free wall.

Fig. 5. Identification of the atrial isoforms of MLC1a and MLC2a from pig. Fragmentation mapsfragment ions that were matched to the sequences for MLC1a (53 c and 44 z∙ ions total) and Mmass discrepancies of 28.04 Da and 42.01 Da localized to the N-termini of MLC1a and MLC2a,for representative fragment ions are shown for each protein. The P value represents the probano less than the score for the assignedmatch. Thus, the low P values obtained forMLC1a andMLin the identification of these proteins. MLC1a and MLC2a correspond to NCBI RefSeq accession

3.4. The phosphorylation of MLC2 does not vary in a chamber-specific ortransmural manner in the heart

We next sought to assess differences in myosin light chain proteinforms in the different chambers of the heart and across the LV freewall. However, while the relative molecular masses of the ventricularisoforms of MLC1 and MLC2 (MLC1v and MLC2v, respectively) frompig have previously been determined [23], the relative molecularmasses of the atrial isoforms of MLC1 andMLC2 (MLC1a andMLC2a, re-spectively), calculated based on sequences from the NCBI database, didnot match with those experimentally determined for presumed peakscorresponding to MLC1a and MLC2a. Thus, to confirm the identity ofMLC1a and MLC2a, peaks presumably corresponding to these protein

and tandem mass spectra for (A) MLC1a and (B) MLC2a. Fragmentation maps show theLC2a (39 c and 49 z∙ ions total) from Sus scrofa in the NCBI database. There were minor

respectively. Bracket indicates removal of the N-terminal methionine. Zoomed-in spectrability that the match between the spectrum and a random protein has a similarity scoreC2a (4.3E-53 and 3.5E-54 forMLC1a andMLC2a, respectively) translate to high confidencenumbers XP_003131354 and XP_003134933, respectively.

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forms were isolated and fragmented by electron capture dissociation.Information from the tandem mass spectra was then searched againstthe pig database usingMS-Align+ [28], which allowed for the high con-fidence identification ofMLC1a (relativemolecularmass: 21,502.94 Da)and MLC2a (relative molecular mass: 19,384.70 Da) from pig (Fig. 5).Thus, we have confirmed the expression of different MLC1 and MLC2isoforms in the atria and ventricles of the pig heart (Figs. 6 and 7).

After confirming the identity of peaks corresponding to MLC1a andMLC2a, the phosphorylation of MLC2 was assessed in the differentchambers of the heart. A total of four major MLC2 protein forms weredetected by top-down MS analysis with forms corresponding to un-phosphorylated and mono-phosphorylated MLC2v detected in the LVand RV and forms corresponding to un-phosphorylated and mono-phosphorylated MLC2a detected in the LA and RA, respectively(Fig. 7A). Additional protein forms corresponding to oxidized MLC2and MLC2 associated with non-covalent adducts were also observed inthe spectra (Fig. 7A). Quantification of MLC2 protein forms revealedthat the relative abundances of un- and mono-phosphorylated(pMLC2) MLC2 did not differ significantly between the LV, RV, LA, andRA (Fig. 7B, Supplemental Results). Likewise, although there was a

Fig. 6.DifferentMLC1 isoforms are expressed in the atria and ventricles of pig heart. Representarepresent oxidation, potassium adducts, sodium adducts, and phosphoric acid adducts (+H3POdium and potassium. No phosphorylation was detected for MLC1. MLC1v corresponds to NCBI

minor increase in the total MLC2 phosphorylation in the RV (in compar-ison to the LV, LA, and RA), these differences were not significant(Fig. 7C, Supplemental Results).

Subsequent analysis of MLC2v phosphorylation across the EPI, MYO,and ENDO of the LV free wall revealed no significant differences in therelative abundances of MLC2v protein forms, or total MLC2v phosphor-ylation (Fig. 7D–F, Supplemental Results). Thus, these results demon-strate that MLC2 phosphorylation does not vary significantly acrosseither the chambers of the heart or the LV free wall in pigs.

4. Discussion

Myofilament protein PTMs have emerged as a keymechanism regu-lating cardiac contractility, and increasing evidence has shown that thealteration of myofilament protein PTMs, particularly phosphorylation,can contribute to contractile dysfunction and heart failure [9–12,25,26,29,30]. Additionally, studies in humans, as well as large and small ani-mals, have shown that aging and pathological cardiac conditions inducetransmural changes in contractile function that correlate with differ-ences in the modification status of myofilament proteins [1,2,5].

tivemass spectra forMLC1 from the LV, RV, LA, and RA. Square, star, triangle, and diamond4), respectively. Star and triangle combination represents MLC1 associated with both so-RefSeq accession number NP_001265702.

Fig. 7. The phosphorylation ofMLC2 does not vary in a chamber-specific or transmural manner in the heart. (A) Representative mass spectra for MLC2 from the LV, RV, LA, and RA. Circlesrepresent the theoretical isotopic abundance distribution of the isotopomer peaks corresponding to the assigned mass. Squares, triangles, stars, and crosses represent oxidation, sodiumadducts, potassium adducts, and a co-eluting protein, respectively. (B) Graph showing the relative abundances of MLC2 and pMLC2 in the four chambers of the heart. (C) Graph showingtotal MLC2 phosphorylation in the LV, RV, LA, and RA. (D) Representativemass spectra for MLC2v from the EPI, MYO, and ENDO of the LV free wall. (E) Graph showing the relative abun-dances ofMLC2v and pMLC2v across the layers of the LV free wall. (F) Graph showing totalMLC2v phosphorylation the EPI, MYO, and ENDO.MLC2v corresponds to NCBI RefSeq accessionnumber AAM47004. *p b 0.05, **p b 0.001.

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Nevertheless, whether there is basal heterogeneity inmyofilament pro-tein PTMs in the heart remains an important question\\particularlygiven the potential usefulness of myofilament PTMs as biomarkers forheart disease [26,31].

In the present study, we have systematically assessed regional andtransmural variations in myofilament protein PTMs in the hearts ofhealthy pigs by top-down MS, with a focus on the phosphorylation ofcTnI, cTnT, αTm, and MLC2. Here, we have demonstrated that, whilethe phosphorylation of certain myofilament proteins (cTnI and αTm)varies in a chamber-specific manner within the heart, the phosphoryla-tion of others (cTnT and MLC2) does not. Additionally, we have shownthat the phosphorylation of cTnI, cTnT, αTm, and MLC2v do not differsignificantly across the LV free wall in healthy pigs. The observed differ-ences in cTnI and αTm phosphorylation in the atrial and ventricularmyocardium are likely due to chamber-specific variations in the expres-sion or activity of kinases or phosphatases in the heart [32].

Previous studies have observed that there is transmural heterogene-ity in the modification status of myofilament proteins across the ven-tricular wall in small animals [4,13–15]. In particular, MLC2vphosphorylation has been shown to vary across the free wall of the LVin rats and mice [4,13,15]; however, here we found no difference inMLC2v phosphorylation (or in the phosphorylation of cTnI, cTnT, andαTm) across the layers of the LV free wall in pigs, which is in goodagreement with previous studies in pigs and humans [1,2]. Whilethere is the possibility that increased workload may induce transmuraldifferences in myofilament protein phosphorylation in large animalsand humans, as it does in rodents [13], this, at least, does not appearto be the case in pigs [2]. Thus, the data presented here, as well asdata from other groups [1,2,16], suggests that transmural heterogeneityin myofilament protein phosphorylation does not exist in large animalsand humans under basal conditions. Although it is possible that thephosphorylation of other myofilament proteins, such as cardiac myosinbinding protein C, may vary transmurally in the heart, it seems likelythat different mechanisms regulate transmural differences in mechani-cal function across the LV freewall in large animals and humans. Indeed,Stelzer et al. have shown that a transmural gradient in the expression ofαmyosin heavy chain can account for the higher rates of stretch activa-tion and force redevelopment in the EPI in comparison to the ENDO inpig hearts [16]. It should be noted that this gradient is also present inhuman hearts [33]. Another similarity between pig and human heartsis thatαTmphosphorylation is increased in the atria relative to the ven-tricles, which we show here in pigs, and in our previous study usinghuman donor hearts [17]. Although the significance of increased αTmphosphorylation in the atria remains unclear, consistency at the molec-ular level, as well as in the mechanisms regulating mechanical activitywithin the heart, highlight the benefits of using large animals, whichmore faithfully recapitulate human cardiac physiology and pathophysi-ology [21,34], to model human cardiovascular diseases.

Furthermore, given that the phosphorylation of both cTnI and αTmhave been linked to altered Ca2+-handling by the contractile apparatus[11,35], chamber-specific differences in the phosphorylation of theseproteins could underlie differences in myofilament Ca2+ sensitivity be-tween the atria and ventricles. Analysis of skinned multicellular prepa-rations from the atria and ventricles of pig by Locher et al. revealedsignificant differences in the rate of force redevelopment, the rate ofATP hydrolysis, the tension cost, and the calcium sensitivity betweenthese preparations [36].While differences in the rate of force redevelop-ment, rate of ATP hydrolysis, and tension cost could be explained by dif-ferences in the expression of the α and β isoforms of myosin heavychain in the atria and ventricles, the differences in calcium sensitivitycould not be explained, as no differences were found in myofilamentprotein phosphorylation by ProQ Diamond analysis [36]. Here, wehave shown that cTnI-Ser22/23 phosphorylation, a well-known modu-lator of myofilament Ca2+ sensitivity [11], is reduced in the atria byboth top-down MS analysis and Western blot, which could partly ac-count for the differences in Ca2+ sensitivity between atrial and

ventricular preparations observed by Locher et al. The reason that wewere able to detect differences in cTnI phosphorylation in the atrialand ventricular myocardium, while they were not, may be due to thesensitivity of the ProQ Diamond stain to negatively charged aminoacids within proteins [37]. Differences in myofilament Ca2+ sensitivityin the atria and ventriclesmay be necessary for adaptingmechanical ac-tivity to differences in intracellular Ca2+-handling in atrial and ventric-ular myocytes [38].

In summary, these results demonstrate that myofilament proteinPTMs vary in a chamber- and protein-specific manner in the heart. Ad-ditionally, transmural variations in myofilament protein phosphoryla-tion do not appear to be present in pigs under basal conditions,although the possibility that there is transmural heterogeneity in thephosphorylation of myofilament proteins that were not analyzed inthis study remains a possibility. Collectively, these findings highlightthe importance of appropriate tissue sampling, particularly in studiesaimed at elucidating PTM-associated diseasemechanisms and biomark-er studies, in order to minimize potential variations arising from basalheterogeneity in myofilament PTMs in the heart.

Disclosures

None.

Acknowledgments

The authors would like to thank Matthew Lawrence of the HumanProteomics Program at UW-Madison for technical assistance. The au-thorswould also like to thankDeyang Yu for critical reading of thisman-uscript. Financial support was kindly provided by NIH R01 HL109810and R01 HL096971 (to Y.G.).

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.yjmcc.2015.08.007.

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