Use of 2-D DIGE analysis reveals altered phosphorylation in a tropomyosin mutant (Glu54Lys) linked...

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Use of 2-D DIGE analysis reveals altered

phosphorylation in a tropomyosin mutant (Glu54Lys)

linked to dilated cardiomyopathy

Chad M. Warren1, Grace M. Arteaga1, Sudarsan Rajan2, Rafeeq P. H. Ahmed2,David F. Wieczorek2 and R. John Solaro1

1 Department of Physiology and Biophysics, Center for Cardiovascular Research, College of Medicine,University of Illinois at Chicago, Chicago, IL, USA

2 Department of Molecular Genetics, Biochemistry, and Microbiology,University of Cincinnati Medical Center, Cincinnati, OH, USA

Current electrophoretic methods have not been optimized to fully separate post-translationallymodified mutant forms of tropomyosin (Tm) from wild-type cardiac samples. We describe here amethod employing a modified 2-D PAGE/2-D DIGE protocol, to fully separate native, mutant(E54K), and phosphorylated forms of Tm. Our data demonstrate the first evidence of a significant(,40%) decrease in Tm phosphorylation in transgenic compared to non-transgenic mousehearts, and indicate that altered phosphorylation may be a significant factor in the linkage of theE54K mutation to dilated cardiomyopathy.

Received: August 7, 2007Revised: November 1, 2007

Accepted: November 1, 2007

Keywords:

2-D DIGE / Familial cardiomyopathies / Kinases / Phosphatases / Sarcomere

100 Proteomics 2008, 8, 100–105

We report here an adapted approach for separatingcharged forms of tropomyosin (Tm) existing in hearts of non-transgenic (NTG) mice and transgenic (TG) mice expressinga Tm mutant (E54K), which is linked to dilated cardiomyo-pathy (DCM). Linkage of mutations in sarcomeric proteins tohypertrophic (HCM) and dilated cardiomyopathies (DCM) iswell established. However, the mechanisms by which amodification of contractile or regulatory proteins leads toremodeling, altered function and, in many cases sudden

death, remains poorly understood. Among the various the-ories, an attractive hypothesis is that a primary trigger forthese changes is an altered response of the sarcomeres toCa21. For example, there are data demonstrating that DCM islinked to sarcomeric mutations inducing a depressed sarco-meric response to Ca21, whereas HCM are linked to anenhanced sarcomeric response to Ca21 [1]. Our studies,which focused on Tm mutants linked to HCM, agree withthis hypothesis [2, 3]. More recently, we reported the firstevidence that detergent extracted fiber bundles from heartsexpressing the DCM-linked mutant aa-Tm(E54K) demon-strate significantly depressed cardiac function and sarco-meric Ca21-sensitivity [4]. These data support the conceptthat structural modifications in Tm induce an altered sarco-meric response to Ca21 that may be a significant primarycause of the altered function leading to remodeling of themyocardium.

In considering the mechanisms by which mutations insarcomeric proteins lead to an altered response to Ca21, it isnow apparent that one must include the potential for asso-ciated PTM that may either exacerbate or ameliorate the

Correspondence: Dr. R. John Solaro, Department of Physiologyand Biophysics (M/C901), College of Medicine, 835 S. WolcottAve, University of Illinois at Chicago, Chicago, IL 60612, USAE-mail: [email protected]: 11-312-996-1414

Abbreviations: cTnI, cardiac troponin I; DATD, N,N’-diallyltartar-diamide; DCM, dilated cardiomyopathy; HCM, hypertrophic car-diomyopathy; MYBP-C, myosin binding protein C; NTG, non-transgenic; TG, transgenic; Tm, tropomyosin; Tm-P, tropomyosinphosphorylation; TnT, troponin T

DOI 10.1002/pmic.200700772

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Proteomics 2008, 8, 100–105 Cell Biology 101

effects of the mutation. We have reported that a mutation incardiac troponin-C (TnC-G159D) linked to DCM blunts theCa21-desensitization induced by PKA-dependent phospho-rylation of cardiac troponin I (cTnI) [5]. We have also reportedthat PKC-phosphorylation of cTnI(R146G), an HCM-linkedmutation, had a significantly smaller effect on cTnC struc-ture, Ca21-binding and sarcomeric Ca21-sensitivity thanphosphorylation of cTnI [6]. These findings indicated thattropomyosin phosphorylation (Tm-P) in hearts of TG miceexpressing Tm(E54K) may be different from wild-type Tm.We therefore set out to adapt procedures for separating var-ious charged forms of Tm along with their respective phos-phorylated forms.

The high copy DCM TG aa-Tm(E54K) and NTG linesused for this study were previously characterized [4]. TheInstitutional Animal Care and Use Committee approved thehandling and maintenance of animals. The high copy DCMTG aa-Tm(E54K) mutation is lethal shortly after 1 month ofage. The hearts were extracted from animals of about1 month of age due to the lethality of the mutation and theseparated ventricles were immediately placed in liquidnitrogen. The liquid nitrogen frozen mouse ventricles werethen used to prepare either myofibrils or ventricular homo-genate samples.

Ventricular homogenates were prepared by cutting asmall piece of ventricle tissue (,50 mg) and placing it in a2-mL dounce homogenizer with universal sample buffer(8 M urea, 2 M thiourea, 4% CHAPS, and 10 mM EDTA pH8.0) at a 1:20 w:v ratio. The tissues were homogenized firstwith pestle A (loose clearance) once and then pestle B (tigh-ter clearance) twice with at least 15 strokes per homogeniza-tion cycle. The samples were clarified by microfuge cen-trifugation 18 0006g for 10 min. Myofibrils were purifiedfrom the TG and NTG samples as previously described [7].Myofibrils were also solubilized in universal sample buffer ata 1:20 w:v ratio using the dounce homogenizers similarly toventricular homogenates. After clarification by microfugecentrifugation (18 0006g for 10 min), both myofibril andventricular homogenate sample concentrations were deter-mined using the RC-DC assay kit (Bio-Rad, Hercules, CA).Samples were diluted in universal sample buffer to 5 mg/mLin a total volume of 50 mL. To insure a pH above pH 8.0, weadded 1 mL of 1.5 M Tris-HCl pH 8.8 to the samples.

Samples used for 2-D DIGE were labeled by adding100 pmoles of CyDye to 50 mg of total protein. The proteinwas labeled at room temperature for 2 h protected from light.After labeling, the reaction was quenched by adding 0.2 mL of10 mM lysine. The samples were stored in the 2807C freezeruntil running the gels. The samples were randomly labeledwith Cy3 and Cy5 in order to correct for any differences. NTGand TG samples were mixed with 340 mL of IEF buffer [8 Murea, 2 M thiourea, 4% w/v CHAPS, 10 mM EDTA pH 8.0,250 mM DTT, 0.25% v/v 3–11 NL, 0.25% v/v 4.5–5.5 IPGbuffer (GE Healthcare) and 2 mM tributylphosphine (TBP)]to give 10 mg total protein from each sample. Mixed samplewas added to 18-cm IPG strips pH 4.5–5.5 (GE Healthcare)

and rehydrated in the Bio-Rad Protean IEF cell, after which,the focusing method was initiated as follows: step 1, 250 Vrapid ramp for 15 min; step 2, 10 000 V slow ramp for 2 h;step 3, 10 000 V rapid ramp for 45 000 Vh; and step 4, a holdat 500 V.

After focusing, both ends of the strip were cut to fit in the2-D gel. Strips were equilibrated in 6 M urea, 2% SDS,50 mM Tris-HCl, pH 8.8, 20% v/v glycerol, and 2% DTT for15 min and then for additional 15 min placed in the samebuffer except DTT was replaced with 2.5% iodoacetamide.Strips were placed onto the stacking gel without an agaroseplug. The resolving gel was 12% total acrylamide, 0.5% bis-acrylamide, 10% v/v glycerol [8, 9], pH 8.8; the stacking gelwas 2.95% acrylamide, 15% N,N’-diallyltartardiamide(DATD) [8, 10], 10% v/v glycerol [8, 9], pH 6.8 and 0.01%bromophenol blue. The stacking gel composition used arelatively high concentration of DATD (15%) [10] for thespecial purpose of allowing for a lower total acrylamide con-centration (2.95%) and retaining mechanical strength. TheDATD crosslinker allows for a large pore size thus facilitat-ing the movement of the protein from the IPG strip into thesecond dimension gel, while still allowing efficient stackingof the proteins. The inclusion of 10% glycerol into both thestacking and resolving gels helps with resolution of the spotsdue to the increased viscosity of the gel matrix and sub-sequent lower diffusion rate during the run [8, 9].

A critical aspect was pouring the gels into 1868-cm glassplates with custom-made 8 mm61 mm68 cm spacers. Thespacers can be made easily by using Bio-Rad Protean II XLspacers and then cutting them to length for the 8-cm heightof the plates. The importance of the gel size was to allow theuse of a narrow pH range and an 18-cm strip with only cut-ting the ends of the strip allowing it to still fit on top of thesecond dimension gel. The separation of the NTG Tm fromthe Tm-P TG was not possible without the combination ofthe narrow range and length of the strip. We felt it wasimportant not to cut the strip in the middle due to possiblemigration differences between the pieces allowing for addi-tional inconsistencies between gels. To separate TnT and Tmspecies we had to utilize 16 cm of the strip, which will just fitthe width of the gel. Due to the full utilization of the gel therewas no space available to run an external size standard in aseparate lane. However, identified spots within the gel canserve as an internal size standard. The gel was run at 16 mAwith constant cooling (87C) in a Hoefer SE600 unit until thedye front reached the bottom of the gel.

The gels were washed in ddH2O twice for 30 min andthen imaged using a Typhoon 9410 Imager (GE Healthcare)with Cy3 (532-nm laser) and Cy5 (633-nm laser) at a resolu-tion of 100 mm. Gels were stained with Pro-Q Diamond stain[7] (Molecular Probes, Eugene, OR) and imaged with theTyphoon 9410. After staining with Pro-Q diamond stain, thegels were silver-stained as previously described [11].

SDS-PAGE gels and transfer onto PVDF membraneswere performed as described [7, 8]. The primary antibodiesand dilutions used were tropomyosin-CH1 1:500 (Iowa


102 C. M. Warren et al. Proteomics 2008, 8, 100–105

Hybridoma Bank), TnI-pan M46 1:2500 (Fitzgerald), MYBP-C-pan 1:80 000 (a generous gift of Dr. Richard Moss), phos-pho-cTnI 1:500 (Cell Signaling), phospho-serine PKC-sub-strate 1:500 (Cell Signaling), phospho-PKA-substrate 1:500(Cell Signaling), and a rabbit phospho-serine-283-specific Tm(custom produced by 21st Century Biochemicals) 1:100 000.The secondary antibodies and dilutions used were anti-mouse-IgG (FAB) specific 1:80 000 Sigma and anti-rabbit-IgG 1:30 000 (GE Healthcare). The immunoblots were pro-cessed and developed as previously described [7].

All data are represented as means 6 SEM with a level ofsignificance set at p ,0.05. The 2-D DIGE gel spots were ana-lyzed using PDQuest (Bio-Rad), which determined the quan-tity of the spots and Excel (Microsoft) was used to ratio thequantities and perform t-tests. The ratio of Tm-P to total Tmwas used to get the percent of Tm that was phosphorylated.The percent reduction from NTG was calculated by (%NTG -%TG)/%NTG. The immunoblots were analyzed with Image-quant v5.2 (GE Healthcare) to obtain the band intensities andExcel (Microsoft) was used to ratio and perform t-tests.

TG and NTG samples were labeled separately andequally mixed and separated on a modified 2-D PAGE gelcropped to only show region of interest as shown in Fig. 1A.The samples were equally mixed in order to have all species

represented for subsequent Pro-Q Diamond staining andimmunoblots to show which spots are phosphorylated andidentify the Tm spots. The phosphorylated proteins wereidentified using Pro-Q Diamond stain, which specificallydetects phosphorylated proteins (Fig. 1B). The 2-Dimmunoblot of a TG sample (Fig. 1C) identified all fourspots as Tm. In Fig. 2A an NTG sample labeled with Cy3 hadtwo spots, and in (Fig. 2B), a TG sample labeled with Cy5 hadfour spots. TG samples had four spots (Fig. 2B) becausethere was approximately 50% replacement of the endoge-nous Tm with the high copy mutant E54K in the myofila-ments [4]. The E54K mutation induced a strong positivecharge, which causes the phosphorylated and non-phospho-rylated mutated protein to migrate at a different isoelectricposition on the gels. Due to only one additional P-Tm spot inthe TG and one in the NTG that was identified as phospho-rylated (Fig. 2B), there is likely only one phosphorylated siteat position 283 in both NTG and TG [12]. Moreover, thephospho-specific Tm antibody (epitope site is phospho-ser-ine-283) reacted with both spots, indicating that the TG andNTG are probably the same (Fig. 2D).

Using 2-D DIGE, we were able to clearly distinguishwhich spots were NTG and TG Tm when two channels weremerged as demonstrated in Fig. 2C. For quantification, the

Figure 1. The 2-DE and immunoblot. (A) Silver-stained 2-D gel of NTG and TG ventricularhomogenates labeled separately and equallymixed. (B) Pro-Q-stained 2-D gel from panel (A),which stains phosphorylated proteins. (C) The2-D immunoblot of a transgenic myofibrillarsample using a specific tropomyosin antibodyCH1 to identify tropomyosin spots. (D) The 2-Dimmunoblot of a transgenic myofibrillar sampleusing a specific Tm phospho-serine 283 anti-body to identify phosphorylated spots. TnT3 or 4,troponin T (isoform 3 or 4); p, phosphorylated;MLC-2, regulatory myosin light-chain.



Figure 2. The 2-D DIGE of ventricular homogenates. (A) Non-transgenic sample labeled with Cy3. (B) Transgenic samplelabeled with Cy5. (C) Merged images from panels (A) and (B). P,phosphorylated. Only the Tm area of the gel is shown. The NTGand TG samples were labeled separately and equally mixed.

channels were separated and analyzed in order to differ-entiate the NTG from the TG overlay (TG samples haveabout 50% NTG Tm). The benefit of using DIGE over othermethods is that the gel-to-gel variation is significantlyreduced due to the separation of both NTG and TG samplesin the same gel.

Immunoblot analysis using a phospho-specific cTnI andMYBP-C (myosin binding protein C) antibody in conjunc-tion with a pan TnI M46 and MYBP-C pan antibody did notindicate any significant differences between NTG and TG(Supporting Information Table 1). Based on our 2-D DIGEanalysis, there were no significant differences in expressionor phosphorylation of MLC-2 and TnT (troponin T) (Sup-porting Information Table 2). Data from the 2-D DIGE anal-ysis of Tm from ventricular homogenates (n = 8) (Table 1)indicated that compared to the NTG Tm, there was a signifi-cant (,40%) decrease in mutant Tm(E54K) phosphorylation.Interestingly, the myofibrillar preparations (n = 3) analyzedin the same way as the ventricular homogenates had similarreductions in Tm-P (Table 1). Thus, it is apparent that Tm-Pwas similar in sarcomeric myofilaments and the ventricular

Table 1. Quantification of Tm-P from 2-D DIGE

NTG TG % reduction from NTG

VH 39 6 3.0%* 24 6 4.0%* 38.5MF 42 6 1.2%** 27 6 1.9%** 35.7

Values are means 6 SEM. VH, ventricular homogenate; MF,myofibril preparation. *p = 0.014 between NTG vs. TG ventricularhomogenates; **p = 0.004 between NTG vs. TG myofibrils.

homogenates (containing 50% exogenous Tm). We thereforeconclude that the unincorporated cytoplasmic Tm-P wasaltered similarly to the sarcomeric Tm-P.

Our data are the first to demonstrate that altered phos-phorylation of Tm may be an important aspect of the mech-anisms linking the Tm(E54K) mutation to DCM. In our pre-vious study, we reported that, when compared to NTG con-trols, detergent-extracted (skinned) fibers regulated byTm(E54K) demonstrated a depression in maximum tensionand Ca21-sensitivity [4]. The present data indicate that analtered PTM of Tm must be taken into account in the deter-mination of the mechanism of the effect of Tm-E54K todepress the response of the myofilaments to Ca21.

Evidence of modifications in Tm-P and other chargemodifications associated with altered cardiac function offerssome insights into the significance of our data. Tm-P sig-nificantly increases in myocytes with development to theadult [13]. Compared to adults, tension development of neo-natal heart myofilaments is more sensitive to Ca21 [13].There is also evidence that specific charge modificationsassociated with switching from alpha to beta Tm, also altersmyofilament sensitivity to Ca21 [14, 15]. The charge differ-ences in beta-Tm compared to alpha-Tm are a Ser for Glu atTm-229 and a His for Asn at Tm-276. These amino acid sub-stitutions make beta-Tm more negative than alpha-Tm.Thus, in both cases (phosphorylation and isoform switch-ing), the more negatively charged Tm induces an increase inmyofilament sensitivity to Ca21. The E54K mutation resultsin more positively charged Tm and an associated depressionin myofilaments response to Ca21 [4]. Our hypothesis is thatthe depression in Tm-P documented in the present studycontributes to this depression in myofilament response toCa21 and to the overall pathology leading to DCM. We thinkit is significant that we found a similar depression in myofi-lament response to Ca21 and a correlated depression in Tm-Poccurring in skinned fibers prepared from hearts of TG miceexpressing active MKK6be, the upstream activator of p38MAPK [16]. Activation of p38 MAPK also leads to DCM.

Although there is a lack of detailed knowledge regardingthe molecular mechanisms by which phosphorylation affectsthe structure-function relations of Tm, there are some in vitrodata relevant to our findings. The interpretation of thesefindings is couched in current knowledge regarding molec-ular mechanisms by which Ca21 activates tension in cardiacmyofilaments, which are reviewed in Kobayashi and Solaro


104 C. M. Warren et al. Proteomics 2008, 8, 100–105

[17]. Ca21 binding to troponin C (TnC) releases a functionalunit ( actin:Tn:Tm in a 7:1:1 ratio) of the thin filament from aprevailing inhibition by promoting altered interactions ofTnI (the inhibitory unit) with actin and TnT (the Tm-bindingunit). These protein-protein interactions trigger a release ofTm from a position on the thin filament that hinders theactin-cross-bridge reaction. End-to-end interactions of Tmalong the thin filament are also important in the spread ofthe activation of a functional unit to a near neighbor. N-ter-minal structural changes, which may alter the end-to-endinteractions have been determined in Tm(E54K) [18]. More-over, S283 the penultimate amino terminal residue is locatedin this region, which is not only important in end-to-endinteractions of Tm, but also interacts with actin and TnT insustaining activation. There is in vitro evidence from studiesusing reconstituted thin filament preparations that phos-phorylation of Tm affects its affinity for TnT, and affects end-to-end interactions as determined by studies of Tm polymer-ization [19, 20]. Moreover, Sano et al. [21], who employed anS283E mutation to mimic Tm-P, reported that a-TmS283Ehad a lower affinity for actin, while retaining an enhancedpolymerization reported earlier [22]. Enhanced end-to-endinteractions by phosphorylation of Tm as well the othereffects noted above would be expected to ease activation ofthe myofilaments by Ca21. These effects of Tm-P fit with ourhypothesis that Tm-P increases myofilament activity and thatthe fall in Tm-P associated with expression of the E54Kmutant contributes to the depression in cardiac function thatprogresses to DCM.

Special thanks to Chao Yuan for many discussions pertainingto 2-D DIGE. This work was supported by National Institutes ofHealth Grants R37 HL22231 and P01-HL62426 (RJS), HL-71952 (DFW), and K01 HL-67709 (GMA).

The authors have declared no conflict of interest.

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