tropomyosin dephosphorylation results in compensated cardiac

Tropomyosin Dephosphorylation Results in CompensatedCardiac Hypertrophy*

Received for publication, July 17, 2012, and in revised form, October 24, 2012 Published, JBC Papers in Press, November 12, 2012, DOI 10.1074/jbc.M112.402040

Emily M. Schulz‡, Richard N. Correll§, Hajer N. Sheikh‡, Marco S. Lofrano-Alves¶, Patti L. Engel�, Gilbert Newman**,Jo El J. Schultz**, Jeffery D. Molkentin§, Beata M. Wolska¶�, R. John Solaro�, and David F. Wieczorek‡1

From the ‡Department of Molecular Genetics, Biochemistry, and Microbiology and §Department of Pediatrics, Howard HughesMedical Institute and **Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati,Ohio 45267 and the ¶Department of Medicine and the �Department of Physiology and Biophysics, Center for CardiovascularResearch, University of Illinois at Chicago, Chicago, Illinois 60612

Background: Changes in phosphorylation status of sarcomeric proteins allows rapid alteration of cardiac function.Results: Tropomyosin dephosphorylation results in myocyte hypertrophy with increases in SERCA2a (sarcoplasmic reticulumCa2� ATPase 2a) expression and phospholamban phosphorylation but without functional changes.Conclusion: Tropomyosin phosphorylation can influence calcium regulatory proteins and cardiac remodeling in response tostress.Significance: This is the first report detailing that altering tropomyosin phosphorylation affects calcium handling proteins.

Phosphorylation of tropomyosin (Tm) has been shown to varyin mouse models of cardiac hypertrophy. Little is known aboutthe in vivo role of Tmphosphorylation. This study examines theconsequences of Tm dephosphorylation in the murine heart.Transgenic (TG) mice were generated with cardiac specificexpression of �-Tmwith serine 283, the phosphorylation site ofTm, mutated to alanine. Echocardiographic analysis and car-diomyocyte cross-sectional areameasurements show that�-TmS283ATGmice exhibit a hypertrophic phenotype at basal levels.Interestingly, there are no alterations in cardiac function, myo-filament calcium (Ca2�) sensitivity, cooperativity, or responseto �-adrenergic stimulus. Studies of Ca2� handling proteinsshow significant increases in sarcoplasmic reticulum ATPase(SERCA2a) protein expression and an increase in phospholam-ban phosphorylation at serine 16, similar to hearts under exer-cise training. Compared with controls, the decrease in phosphor-ylation of�-Tmresults in greater functional defects inTGanimalsstressed by transaortic constriction to induce pressure overload-hypertrophy. This is the first study to investigate the in vivo role ofTmdephosphorylation under both normal and cardiac stress con-ditions, documenting a role for Tm dephosphorylation in themaintenanceofacompensatedorphysiologicalphenotype.Collec-tively, these results suggest thatmodification of theTmphosphor-ylation status in the heart, depending upon the cardiac state/con-dition, maymodulate the development of cardiac hypertrophy.

Tropomyosin (Tm)2 is an � helical coiled coil proteininvolved in the Ca2�-dependent regulation of the thin filament

of the sarcomere. Upon binding of Ca2� to the troponin com-plex, a conformational change occurs that allows the Tm fila-ment to move away from the myosin-head binding site on thesarcomeric actin filament. Previous and recently publishedstudies show striated muscle �-Tm is phosphorylated at onesite, the penultimate amino acid, serine 283, by several potentialkinases including tropomyosin kinase, protein kinase A, andprotein kinaseC� (PKC�) (1–8). During fetal development, 70%of cardiac �-Tm in rat hearts is phosphorylated, whichdecreases to �30% post-natally (9). In vitro studies investigat-ing the functional role of Tm phosphorylation indicate that lowphosphorylation levels decrease the ability of �-Tm to polym-erize in a head-to-tail fashion; conversely, increasing phosphor-ylation enhances the interaction between the C- and N-termi-nal ends of adjoining Tm molecules. Additionally, changes in�-Tm phosphorylation status seem to alter sarcomeric func-tion, as shown by differential function of the actin-activatedmyosin S1-ATPases (4, 10). Taken together, these in vitro datasuggest that altering phosphorylation status affects the abilityof Tm to cooperatively activate the thin filament upon bindingof Ca2� to troponin (Tn).

In recent years in vivo studies performed on animal modelsindicate that changes in the phosphorylation status of sarcom-eric proteins such as troponin I (TnI),myosin binding proteinC(MyBPC), and the regulatory myosin light chain result in alter-ations in Ca2� sensitivity of the myofilament and changes incardiac function and may play a role in the development ofcardiac disease (11–15). Investigation of a dilated cardiomyop-athy transgenic (TG) mouse model bearing a human �-Tmmutation (E54K) shows that phosphorylation levels of Tmdecrease relative to non-transgenic (NTG) littermates (16, 17).Additionally, phosphorylation is increased in familial hyper-trophic cardiomyopathy �-Tm N175D mice generated by this

* This work was supported, in whole or in part, by National Institutes of HealthGrants RO1 HL-081680 (to D. F. W.), RO1 HL-064035 (to R. J. S. and B. M. W.),PO1 HL062427 (to R. J. S.),T32 HL-07382 (to E. M. S.), F32HL097551 (toR. N. C.), and T32 HL-07692 (to P. L. E.).

1 To whom correspondence should be addressed: Dept. of Molecular Genet-ics, Biochemistry and Microbiology, University of Cincinnati, College ofMedicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0524; Fax: 513-558-1885; E-mail: [email protected].

2 The abbreviations used are: Tm, tropomyosin; Tn, troponin; TG, transgenic;NTG, non-transgenic; SERCA2a, sarcoplasmic reticulum Ca2� ATPase 2a;

PLN, phospholamban; TAC, transaortic constriction; CnA, calcineurin; LV,left ventricular; LVID, LV internal dimension; LVIDd, LVID diastole dimen-sion; LVIDs, LVID systole dimension; LVOT, LV outflow tract; LVAW, LV ante-rior wall dimension; LVPW, LV posterior wall dimension.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 53, pp. 44478 –44489, December 28, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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laboratory indicating a link between striated muscle Tm phos-phorylation, sarcomeric function, and cardiac disease (18).3 Toinvestigate the in vivo effect of decreased or ablated Tm phos-phorylation, we substituted serine 283 with an alanine (S283A),removing the phosphorylation site and effectively inhibiting theability of �-Tm to be phosphorylated. Several TG mouse linesexpressing this�-TmS283Amutationwere generated and ana-lyzed. These TG hearts show no changes in functional param-eters when investigated by echocardiography, myofilamentCa2�-tension relations, or in studies of work-performing heartduring �-adrenergic stimulus. However, these animals do havesex-specific differences in heart morphology likely due to thecardioprotective effects of estrogen that have been describedpreviously (19, 20). Male TGmice show a hypertrophic pheno-type as measured by echocardiography and supported by car-diomyocyte cross-sectional area measurements, whereasfemale mice do not. Male TG mice also show significant mod-ifications in proteins controlling Ca2� fluxes such as increasesin the expression of the sarcoplasmic Ca2� ATPase (SERCA2a)and phosphorylation of phospholamban (PLN). Thus, phos-phorylation of �-Tm may be part of a signaling cascade thatresults in changes in Ca2� handling protein levels and mayexplain the tight regulation of �-Tm phosphorylation levels.Additionally, when male TG animals are subject to pressureoverload via transaortic constriction (TAC), they exhibit a sig-nificant increase in hypertrophy as well as functional defectsincluding a striking decrease in fractional shortening comparedwith NTG litter mates. This is the first investigation to showthat alterations in the phosphorylation status of a thin filamentprotein, namely �-Tm, can cause a moderate hypertrophicresponseand increaseSERCA2aexpressionandPLNphosphor-ylation. Taken with our previous findings in cardiomyopathymodels, these results firmly establish that �-Tm phosphoryla-tion is necessary for an appropriate response during cardiacdisease.

EXPERIMENTAL PROCEDURES

Generation of S283A�-TmTGMice—Mouse striatedmuscle�-Tm cDNA was subjected to QuikChange II site-directedmutagenesis (Agilent Technologies) utilizing the primer5�-CACGCTCTCAACGATATGACTGCCATATAAGTTTCT TTGCTT CAC-3� mutating the penultimate serine to analanine. The mutation was verified through sequencing of theconstruct by Genewiz. The �-Tm S283A construct was thencloned into a vector containing the �-myosin heavy chain(�-MHC) promoter and a human growth hormone 3�-UTR andpoly-A tail sequence (21). Transgenic mice were generatedusing the FVB/N strain as previously described (22). Foundermice were identified using PCR. Copy number was determinedusing genomic Southern blot analysis. Nucleotide sequencingof TG mouse DNA verified the sequence of the �-Tm S283Atransgene.Genotyping—DNA samples were obtained from 14-day-old

mice, and PCRwas utilized to determine which animals carriedthe transgene. Primers specific for the transgene are �-MHCforward 5�-GCC CAC ACC AGA AAT GAC AGA-3� and

�-Tm reverse 5�-TCC AGT TCA TCT TCA GTG CCC-3�.GAPDH is used as an internal control, and primers are as fol-lows: GAPDH forward 5�-AGC GAG CTC AGG ACA TTCTGG-3� and GAPDH reverse 5� - CTC CTA ACC ACG CTCCTA GCA-3�.Transgenic Protein Quantification and Western Blot Analyses—

Myofibrillar proteins were extracted from NTG and TG malemouse ventricles as previously described (22). 30�g of themyo-fibrillar protein preparations were separated on a 10% SDS-PAGE gel. The Tm band was excised from the gel, reduced,alkylated, and subject to a tryptic digest. Recovered peptideswere desalted with a �-C18 ZipTip and spotted onto a matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) target plate. All spectra were acquired in reflector posi-tive ion mode on an ABSciex 4800 MALDI-TOF/TOFinstrument. The percentage TG protein was calculated afternormalization and subtraction of background contributions.Western blot analyses on myofibrillar protein preparations

(4�g) from 3-monthmaleNTG andTGhearts were conductedusing the Tm-specific antibody CH1 (Sigma), Tm Ser-283phosphorylation-specific antibody generated for this labora-tory (YenZyme), and sarcomeric �-actin antibody 5c5 (Sigma)as a loading control. To confirm the mutant Tm was properlyassembled into the sarcomere, cytoplasmic protein fractions (4�g) isolated from 3-month male NTG and TGmale mice, wereexamined by Western blot analyses.Whole ventricular homogenates from 3-month-old male

NTG and TG mice were utilized to visualize Ca2� handlingprotein expression levels. Western blots were used to visualizesarcoplasmic ATPase 2a (SERCA2a) (AbCam), TnI (Cell Sig-naling), pTnI23/24 (Cell Signaling), PLN (Thermo Scientific),phosphorylated serine 16 PLN (PLN Ser-16) (Badrilla), phos-phorylated PLN threonine 17 (PLN Thr-17) (Badrilla), and cal-cineurin (CnA) (Sigma). Sarcomeric actin (Sigma) was used as aloading control.Two-dimensional Isoelectric Focusing-PAGE—Two-dimen-

sional isoelectric focusing-PAGE was performed on mousehearts as previously described with modifications (17). 3 �g ofmyofibrillar preparations were resolved on a 24-cm 4.0–5.0immobilized pH gradient isoelectric focusing strip. After iso-electric focusing, the samples were resolved in the seconddimension on a 10% SDS-PAGE gel and transferred to nitrocel-lulose membrane for Western blotting. Tm muscle-specificantibody CH1was used to visualize both the unphosphorylatedTm and phosphorylated Tm species. Percentage of Tm phos-phorylationwascalculatedas:(phosphorylatedTm)/(phosphor-ylated Tm � nonphosphorylated Tm) � 100, where the valuesfor the two protein species have been determined using ImageQuant v5.1.Histopathologial Analyses and Cardiomyocyte Cross-sec-

tional Area Analyses—Malemouse hearts at 3, 6, and 9monthswere analyzed. Heart weight to body weight ratios were calcu-lated to evaluate for the presence of cardiac hypertrophy. Forhistological analyses, the heartswere stainedwith hematoxylin/eosin orMasson’s Trichrome and evaluated for the presence ofnecrosis, fibrosis, myocyte disarray, and calcification. Imageswere taken on a Nikon SM2–2T dissecting microscope and anOlympus BX4C compound microscope.3 H. N. Sheikh and D. F. Wieczorek, unpublished information.

Tm Dephosphorylation Results in Compensated Cardiac Hypertrophy

DECEMBER 28, 2012 • VOLUME 287 • NUMBER 53 JOURNAL OF BIOLOGICAL CHEMISTRY 44479


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To quantify changes in cardiomyocyte cross-sectional area,tissue sections were stained with wheat germ agglutinin fromTriticum vulgaris conjugated with Texas Red (Sigma) to visu-alize cardiomyocyte membranes. DAPI was used to stain thenuclei of cardiomyocytes. Randomized images of the left ven-tricular free wall were taken using a fluorescent cameramounted on a Zeiss Axioskop, and the cardiomyocyte cross-sectional area was measured using ImageJ (NIH).Quantitative Real-time PCR Analyses—RNA isolated from

3-month-old NTG and TG male mouse ventricular tissue wasisolated using TRIzol reagent (Invitrogen). Real time RT-PCRwas performed using an Opticon 2 real time RT-PCR machine(MJ Research). Each sample was measured in triplicate, andeach experiment was repeated twice. Target mRNA was nor-malized to GAPDH expression as described by Pfaffl (23).Echocardiographic and Pressure Overload Measurements—

Echocardiographic measurements were performed utilizing a30-MHz high resolution transducer (Vevo 770 high resolutionimaging system) after anesthetization of 3-month-old mice aspreviously described (24). Echocardiographic dimensions andthicknesses were taken from two-dimensional guidedM-Modefrom the parasternal long axis view in triplicate on NTG andTG 12–16-week-old mice. Fractional shortening (in %) wasobtained by the formula 100 � (LVIDd � LVIDs)/LVIDd,where LVIDd and LVIDs are left ventricular (LV) internal dimen-sions in diastole and systole, respectively. The relative wall thick-ness indices were calculated by the formula (LVAW� LVPW)/LVIDd, where LVAW and LVPW indicate anterior andposterior wall thicknesses, respectively, and LVIDd is the LVdiastolic internal dimension. The LV outflow tract (LVOT)diameter (D) was measured to calculate the LVOT cross-sec-tional area (LVOT CSA � �(D/2)2). The velocity-time integral(VTI, in cm) was calculated by integrating the Doppler veloci-ties in the LVOT. The product LVOT CSA � VTI is the LVstroke volume, which multiplied by heart rate gives us the car-diac output (ml/min). 12–16-Week-old male mice of both gen-otypes were subject to TAC or sham operation as previouslydescribed (25). Echocardiographic measurements were takenin M-Mode. Because only male TG mice exhibit changes inechocardiographic measurements, only male animals are usedin this study. Pressure gradients across the constriction weremeasured using Doppler echocardiography as previouslydescribed (25). Two weeks post-surgery, mice were again sub-ject to echocardiography and sacrificed.Calcineurin/Protein Phosphatase Activity Assay—Calcineu-

rin activity (CnA), also known as protein phosphatase 2B, wasmeasured using a calcineurin/protein phosphatase 2B activitykit (Calbiochem). Cardiac homogenates from 3-month-oldmale NTG and TG hearts were used. CnA activity is measuredas the rate of dephosphorylation of a synthetic peptide in thepresence and absence of EGTA, okadaic acid, and EGTA withokadaic acid. Phosphate release was measured by the colori-metric Green Reagent (Calbiochem).Measurements of Ca2�-dependentActivation of Tension—Fi-

ber bundles from papillary muscles of 5-month-old male NTGand TG hearts were detergent-extracted in high relaxing bufferas described previously (26) and mounted between a forcetransducer and a micromanipulator. The sarcomeric length

was adjusted to 2.0 and 2.2 �m using laser diffraction patterns,and isometric tension was measured. Fiber bundles were thensubjected to sequential Ca2� solutions (pCa), and isometrictension was again measured. All experiments were carried outat 22 °C.Isolated Work-performing Heart Model—Three-month-old

male NTG and TG were anesthetized and treated with heparinto prevent microthrombi as previously described (27). Theaorta was cannulated, preserving the aortic valve and the coro-nary artery. To measure intraventricular systolic and diastolicpressures, an intraventricular catheter was inserted into the leftventricle. A cannula was also inserted into the left pulmonaryvein, allowing the direction of the perfusate to be switched fromretrograde (Langendorff) to anteriograde (working). COBEpressure transducers were utilized to measure aortic pressure,atrial pressure, and left ventricular pressure and were recordedusing a Grass polygraph and digital acquisition system.Statistics—All statistics are presented as the mean � S.E.

Where appropriate, paired and unpaired t tests, analysis of vari-ance with Bonferroni correction, and analysis of variance withrepeated measures were used to detect significance. Signifi-cance was set at p � 0.05.

RESULTS

Generation of �-Tm S283A TG Mice—To determine thefunctional significance of Tm phosphorylation, we generatedTG mice in which the Tm phosphorylation site (serine residue283) was replaced with a non-phosphorylatable alanine residue(S283A). The transgene construct used to generate �-TmS283A TG mice is shown in Fig. 1A. Multiple TG lines weregenerated and studied. Line 2 has the highest TG mRNAexpression and the second highest copy number of all trans-genic animals generated (17 copies) determined by genomicSouthern blot analysis. Forward and reverse sequencing of theconstruct indicates no mutations or deletions in the transgene.Cardiac �-Tm S283A Protein Expression and Phosphoryla-

tion inTransgenicMice—Often,mutations inTm isoforms leadto differential migration on SDS-PAGE gels (16, 18, 22, 28).However, because serine is only 16 daltons larger than alanineand has nearly an identical isoelectric point, expression levels ofTG and endogenous protein cannot be separated using tradi-tional methods. Instead, myofibrillar protein preparations ofage-matched NTG and TG mouse hearts as well as recombi-nantly expressed NTG or TG protein are resolved by a combi-nation of SDS-PAGE and MALDI-TOF analyses (Fig. 1B). Theratio of serine containing peptides (endogenous Tm) and ala-nine containing peptides (TG Tm) is calculated after normal-ization and background subtraction. As an additional control,the peptides corresponding to both the serine and alanine pro-files are further fractionated to ensure that the proper trypticpeptide is being analyzed. Line 2 has �93.7% TG proteinexpression, Line 25 has �86% TG protein expression, and Line97 has �88% TG protein expression (Fig. 1C), with a concom-itant decrease in NTG protein, maintaining total Tm levels at100%. Investigation of the cytosolic fraction shows no signifi-cant accumulation of either endogenous or TG Tm, indicatingthat the TG protein is properly incorporated into the myofibril(data not shown). Additionally, myofibrillar protein prepara-




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tions run on an SDS-PAGE gel show that all myofibrillar pro-teins are present in the proper ratio in all three TG lines, indi-cating that the myofibrils are being properly assembled andthere is no change in total Tm levels (data not shown).TmPhosphorylation inNTGand�-TmS283AMouseHearts—

To study Tm phosphorylation in TG mice, it was necessary toestablish the basal level of Tm phosphorylation in NTG heartsusing two-dimensional isoelectric focusing-PAGE. Resultsshow that an unphosphorylated and a single-phosphorylatedspecies of Tm appears in NTG heart samples (Fig. 2A). Uponcalf intestinal phosphatase treatment, the phosphorylated Tmprotein species is lost. These results are in agreement with pre-viously published studies that identify Ser-283 as the phosphor-ylation site in striated muscle Tm (1, 8, 9, 17). Further analysisshows a trend of decreasing Tm phosphorylation from 6 weeksto 5 months of age with an average of �30%. At 15 months ofage, animals show a significant increase in Tm phosphoryla-tion, indicating a possible return to fetal gene programs due tosenescence (Fig. 2B) (29–31).To determine the phosphorylation status of Tm in TGmyo-

fibrillar preparations, we generated a Tm Ser-283 phosphory-lation-specific antibody. As seen in Fig. 2, C and D, there is a

clear decrease in the phosphorylation status of Tm in the TGmyofibrillar preparations compared with the NTG prepara-tions. As TG Line 2 had the greatest decrease in phosphoryla-tion and exhibited the same phenotype in comparison to theother TG lines, we chose to focus on Line 2 TG mice. Whenconsidering the phosphorylation status of these S283A TGmice, it is important to remember that endogenous Tm inNTGmice is phosphorylated at 30%. Line 2 has �5-fold less (or 80%less) phosphorylation than NTG littermates, corresponding to6% endogenous Tm available for phosphorylation in this line.We believe Line 25 has more endogenous Tm phosphorylationbecause of its lower level of transgene expression. These resultssuggest that most, if not all, of the endogenous Tm in the TGmice is being phosphorylated.Gravimetrics and Cardiac Morphology of �-Tm S283A TG

Hearts—Morphological analyses of the left ventricular wallshows a very mild increase in cardiomyocyte disarray and dis-organization as indicated by centrally located nuclei and partialloss of the typical cobblestone shape of the cardiomyocyte (Fig.3A). Staining the membrane with wheat germ agglutinin andmeasurement of cross-sectional area shows a significantincrease in TG cardiomyocyte area (445.5 � 17.4 �m2 versus

FIGURE 1. A, �-TM S283A construct is shown. The �-MHC promoter drives cardiac-specific expression of the striated muscle �-Tm cDNA with an encodedsubstitution at amino acid 283 (S283A). B, shown are MALDI-TOF profiles of NTG and TG recombinant �-Tm and NTG and TG Line 2, Line 25, and Line 97 �-TmS283A from myofibrillar preparations of 3-month-old mouse hearts. C, shown is quantification of data presented in panel B.




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686.9 � 66.9 �m2 p � 0.05, NTG and TG, respectively) (Fig.3B). Gravimetric analysis was performed on TG animals from 1month to 9 months of age. Interestingly, results show nochanges in heart weight to body weight ratios, likely due to themoderate nature of this hypertrophy (Fig. 3C). There are nodifferences in the survival of NTG and TG mice.Cardiac Function of �-Tm S283A TGHearts—To determine

whether the relationship between Ca2� concentration andforce-tension development is altered in myofilaments at thesarcomeric level in TG hearts with significantly decreasedphosphorylation of Tm, we analyzed skinned fiber bundlesfrom the papillarymuscle of 5-month-old hearts. No significantchanges in absolute tension or normalized tension in NTG ver-susTGmice were found (Table 1, Fig. 4,A and B). Additionally,there are no significant differences in pCa50 or the Hill coeffi-cient (nH), a measure of the cooperative activation of the thinfilament of the sarcomere.The work-performing heart model was utilized to determine

ex vivo functional effects of the decrease inTmphosphorylationstatus. These measurements were performed in mice at 3months of age. At basal levels, there are no changes in contrac-tion and relaxation parameters in TG hearts. Additionally,when isoproterenol is administered to determine whether the�-adrenergic response is impaired, there are no significant dif-ferences in contraction and relaxation (Fig. 4, C and D).To assess whether decreasing the phosphorylation level of

�-Tm has an effect on in vivo cardiac function, we performedechocardiographic analysis on 3-month-old NTG and TGmice. There are no physiological changes in heart functionbetween the NTG and TG mice as shown by fractional short-ening, cardiac output, or ejection fraction (Table 2). However,there are sex-specific differences in cardiac morphology. Male

TG animals show significant increases in LVmass, LV anteriorwall thickness, LV posterior wall thickness, and LV relative wallthickness index, indicating that TG mice have a hypertrophicphenotype without attendant functional defects. Female TGmice show no changes when compared with female or maleNTG hearts. Differences in the development of cardiac hyper-trophy between sexes have been previously noted (20, 32).Thus, the increase in cardiomyocyte area and left ventricularhypertrophy with no change in heart weight to body weightratio or female cardiac enlargement demonstrates the moder-ate nature of this hypertrophic phenotype.Gene Expression and Protein Changes in �-Tm S283A TG

Hearts—Given that histological and echocardiographic analy-ses indicate that TGmice exhibit amoderate hypertrophic phe-notype at 3 months of age, altered gene expression was deter-mined in�-TmS283ATGhearts. Real timeRT-PCR analysis ofthe RNA isolated from ventricular tissue indicate a trendtoward an increase in �-MHC, brain natriuretic peptide (BNP)and atrial natriuretic peptide (ANP) without significant statis-tical increases (Fig. 5A).Genes involved in cardiomyocyte Ca2� handling were also

examined. Interestingly, there are no changes in gene expres-sion of SERCA2a, the L-type Ca2� channel, NCX, PLN or RyR2(Fig. 5B). However, there is a significant increase (p � 0.05) inthe gene expression of MCIP1, a protein involved in modulat-ing CnA activity in vivo (33).To determine whether real time RT-PCR levels of Ca2� han-

dling genes correlate with corresponding protein expression inthe TGmyocardium, protein expression fromwhole hearts wasdetermined by Western blot analysis (Fig. 5, C and D). Resultsindicate that the phosphorylation site at PLN Ser-16 andSERCA2a protein expression are increased by30% over NTGlevels. There are no changes in total PLN, phosphorylation atPLNThr-17, TnI, or phosphorylation at TnI 23/24 or CnA. Thelack of increased SERCA2a gene expression by real time RT-PCR analysis compared with the significant increase in proteinexpression suggests that increased protein stability or transla-tion may be operative. MICP1 gene expression is utilized as amarker of alterations in CnA protein expression or activity.Although MICP1 gene expression is increased, there is noincrease in CnA expression. However, it is possible that CnAactivity may be altered without altering protein expression.Calcineurin Activity Assay—As MIC1P can be both a facili-

tator and an inhibitor of CnA activity, it is necessary to deter-mine whether changes in mic1p gene expression alters CnAactivity, as CnA is an important regulator in the Ca2� handlingprocess. A CnA/ protein phosphatase 2B activity assay was per-formed (Calbiochem) on whole heart preparations from3-month-old mouse hearts. There are no significant changes inCnA activity (Fig. 5E).Cardiac Function in �-TM S283A Pressure Overload Mouse

Hearts—To determine the effect of decreased �-TM phospho-rylation during cardiac stress and disease, 12–16-week-old TGmouse hearts were subject to TAC along with NTG littermatesand sham-operated animals from both genotypes.Animals were subject to echocardiography before surgery as

well as 2 weeks after TAC andwere then sacrificed for histologyand gravimetric studies. NTG and TG TAC-operated animals

FIGURE 2. A, two-dimensional isoelectric focusing-PAGE gels show that Tmhas one phosphorylation site indicated by the arrow (upper panel) that can beremoved after calf intestinal phosphatase treatment (lower panel). Cardiacmyofibrillar protein preparations were probed with the CH1 striated muscleTm antibody. B, percent of total Tm phosphorylated in NTG mice was mea-sured using two-dimensional isoelectric focusing on myofibrillar prepara-tions taken at 1.5, 3, 5, and 15 months of age. C, shown is Western blot analysisof in �-Tm phosphorylation (pTm) from hearts at 3 months of age. n � 3. D,shown is quantification of phosphorylation levels of �-Tm found in panel C.




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show significantly increased pressure gradients at 2weeks, indi-cating the efficacy of the pressure overload model (Fig. 6A).LVIDd, LVIDs (LVID systole), and percent fractional shorten-ing are the only parameters that show significant alterations infunction between NTG TAC- and TG TAC-operated animals(Fig. 6, B and D). Interestingly, although NTG TAC operatedhearts have a greater pressure gradient, TG TAC-operatedhearts are the only group that experiences a significant decreasein percent fractional shortening (Fig. 6D). NTG sham, TGsham, and NTG TAC all have fractional shortening at 33%,whereas the TGTAC-operated group has fractional shorteningat 24%, indicating impairment in cardiac function in that group.These data indicate that significantly decreasing the phospho-rylation status of �-Tm impairs the ability of the myocardiumto properly respond to acute stress.Gravimetrics and Cardiac Morphology in �-TM S283A TAC

and Sham-operated Animals—NTG TAC operated animalshave a significant increase in heart weight to body weight whencompared with NTG sham-operated animals (p� 0.0001) (Fig.6E). Additionally, TG TAC-operated animals show an increasein heart weight to body weight ratios compared with TG sham-operated animals (p � 0.05).

Hematoxylin/eosin staining of TG sham-operated heartsshow a mild increase in disorganization compared with NTGsham-operated hearts. Masson’s Trichrome staining of both

NTG and TG sham-operated hearts show no significantincreases in the deposition of fibrotic tissue in the left ventric-ular free wall (Fig. 6G, i and ii). NTG TAC- and TG TAC-operated heart sections stained with hematoxylin/eosin showcardiomyocyte disorganization and centrally located nuclei.BothNTGandTGTAC-operated hearts stainedwithMasson’sTrichrome show increases in fibrosis. Cardiomyocyte cross-sectional analyses demonstrate significant increases in TGshamandNTGTAC- andTGTAC-operated hearts (p� 0.001)compared with NTG sham mice (Fig. 6Giii). TG TAC car-diomyocytes show greater increases in size comparedwith bothTG shamandNTGTACcardiomyocytes (p� 0.0001, p� 0.01)(Fig. 6F).

DISCUSSION

Post translational modifications, such as alterations in thephosphorylation status of sarcomeric and Z-disc proteins canresult in altered cardiac contractility with progression to dis-ease and death (11–14). This is the first in vivo study investigat-ing the functional role of cardiac �-Tm phosphorylation. Toaddress this, the single Tm phosphorylation site, serine 283,was changed to an alanine, and TG animals were generated forstudy. In vivo assessment of basal cardiac function of �-TmS283A TGmice shows that the hearts exhibit a moderate com-pensated hypertrophic phenotype with an increase in myocytesize due to a stimulus initiated by decreased Tm phosphoryla-tion. It is possible that the increase in cardiomyocyte size occursin response to mechanical defects induced by autophagy orapoptosis, two cell death processes involved in the transitionfrom compensated to decompensated hypertrophy (34, 35).However, the data suggest this compensatory response is an

FIGURE 3. A, shown is immunohistochemistry of 3-month �-Tm S283A TG hearts stained with hematoxylin and eosin (i) and wheat germ agglutinin (ii). B,cardiomyocyte cross-sectional area measurements are shown. * indicates p � 0.05; TG, n � 4; NTG, n � 4. C, shown is heart weight (HW) to body weight (BW)ratios of 3-month-old mice. TG, n � 6; NTG, n � 6.

TABLE 1Parameters involved in Ca2�-tension relations in skinned fiber bundles

Group pCa50 nH n

NTG 5.280 � 0.19 1.64 � 0.09 4TG 5.251 � 0.01 1.81 � 0.07 4




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attempt to normalize LV wall stress and preserve pump func-tion, which may point toward the role that autophagy places inmaintaining cell and tissue homeostasis (36). Increases in LVmass, LVAW, LVPW, relative wall thickness, and increases incardiomyocyte cross-sectional area coupledwith a lack of func-tional defects in contractility indicate the TG hearts are in acompensated or adaptive state of hypertrophy in response todecreased Tm phosphorylation. Further characterization ofcontractile function assayed by skinned fiber preparations indi-cate that TGmyofibers develop force-tension relations that aresimilar to NTG controls. Also, TGmyofibers lacking Tm phos-phorylation do not exhibit changes in Ca2� sensitivity or coop-erative activation of the thin filament.Tm phosphorylation has long been speculated to play a role

in themodulation of the structural and functional properties ofthe thin filament given that the site of phosphorylation is serine283 and is located in the 7–11-amino acid head-to-tail overlapregion between neighboring Tm molecules. Although cooper-ativity is not entirely understood, studies suggest that Tmhead-to-tail interactions between contiguous Tm dimers play a role.A study by Gaffin et al. (37) indicates that substitution of neg-

atively charged amino acids at the C terminus causes a signifi-cant change in the distance between Tmmonomer strands andpossibly alterations in contiguous Tmmolecule interactions. Invitro studies investigating the striated muscle Tm phosphor-ylation site indicate that changing the phosphorylation status of�-Tm alters the head-to-tail interaction between neighboringTmmolecules (4). Contrary to expectation, removing the phos-phorylation of �-Tm at Ser-283 in an in vivo system does notresult in any alterations in cooperative activation of the thinfilament. Thus, phosphorylationmay not be amajormodulatorof cooperative spread of activation in the myofilament latticeand may be more significantly related to the actin filamentindependent of Tmhead-to-tail interactions (38). NMR studiesof the interaction between the N- and C-terminal dimers indi-cate that the last 2–5 amino acids at the C terminus are flexible(39). The fact that the very last C-terminal residues are mildlydisordered may offer some explanation as to why loss of addi-tional negative charges in the formof phosphorylated serine hasno effect on cooperativity or Tm head-to-tail interactions. Thephosphorylated Ser-283 may be in an area too flexible to allowfor strong interaction with residues in the N terminus of thesubsequent Tm molecule.The lack of change in cardiac function, myofilament cooper-

ativity, and Ca2� sensitivity coupled with the development ofcompensated hypertrophy in the TG animals warranted aninvestigation into the possible mechanisms involved in thehypertrophic response. The gene expression profile of the�-Tm S283A TG found that mcip1 significantly increases.Mcip1 gene expression is utilized as a marker for CnA activityand has been alternatively shown as both a facilitator and inhib-itor of CnA activity in vivo (33, 40, 41). Increases inCnA activityhave been shown to induce cardiac hypertrophy inmousemod-els, and conversely, the development of a hypertrophic pheno-type can be prevented via CnA inhibition (42, 43). Interestingly,

FIGURE 4. A, normalized force in skinned fiber bundles from NTG and TG hearts is shown. B, Ca2�-tension relations in NTG- and TG-skinned fiber bundles. C andD, isoproterenol dose-response curves in TG and NTG mouse hearts are shown. Hearts were subjected to isolated heart analysis with increasing concentrationsof isoproterenol (10�11–10�6 mol/liter).

TABLE 2Cardiac function of NTG and �-TM S283A TG mice at 3 months of ageas assessed by M-Mode echocardiographyLVISd, LV internal systolic dimension; RTW, relative wall dimension; CO, cardiacoutput; HR, heart rate; EF, ejection fraction.

Parameters NTG (n � 4) TG (n � 4)

LVIDd, cm 0.40 � 0.11 0.42 � 0.13LVISd, cm 0.28 � 0.14 0.29 � 0.24LV mass, mg 82.71 � 3.34 112.5 � 9.99aLVAW, cm 0.07 � 0.04 0.08 � 0.01aLVPW, cm 0.07 � 0.014 0.09 � 0.06aRTW, cm 0.03 � 0.02 0.04 � 0.02aCO, ml/min 25.13 � 2.48 25.33 � 2.29HR, bpm 469.5 � 24.82 453.3 � 24.88EF 58.39 � 1.99 61.49 � 6.75FS % 30.54 � 1.29 33.50 � 4.65

a p � 0.01 to 0.05.




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in the �-TM S283A TG animals, the increase ofmcip1mRNAdid not result in changes of CnA expression or activity, indicat-ing another effector downstream of Tm phosphorylation lossmay be responsible for the hypertrophic phenotype.As numerous studies have demonstrated the importance of

Ca2� in the modulation of cardiac hypertrophy, we examinedwhether alterations in expression of Ca2� proteins occurred inTGmice. Although there are no changes in total TnI expressionor TnI phosphorylation at amino acids 23 or 24, there are alter-ations in sarcoplasmic reticulumproteins. Increasing SERCA2aprotein expression and/or activity can rescue multiple diseasephenotypes and improvemyofibrillar efficiency and contractileparameters both in human cardiomyocytes and rodent hearts(44–47). Conversely, in animal models of cardiac disease aswell as human patients with heart failure, SERCA2a proteinlevels and activity often decrease (48, 49). Surprisingly,SERCA2a protein levels are increased in the �-Tm S283A TGhearts by�30%overNTG levels. Additionally, the 30% increase

in PLNphosphorylation at Ser-16 indicates that further restric-tion on SERCA2a activity has been released, as PLN in anunphosphorylated state results in inhibition of the pump (50–52). Similar to increasing SERCA2a expression and activity,phosphorylation of PLN Ser-16 results in a hypercontractileheart. However, there are no changes in cardiac contractility inthe �-Tm S283A mice. Rather, normal cardiac function asmeasured by multiple methods is preserved in the TG heartsrather than enhanced. This indicates that the increase inSERCA2aprotein levels aswell as the increase inPLNSer-16phos-phorylationmaybenecessary tomaintainnormal cardiac functionin a heart in which Tm has largely been dephosphorylated. It ispossible, therefore, that if SERCA2a activity was inhibited, agreater degree of hypertrophy and/or a progression to decompen-sated cardiomyopathy and heart failure would result.Increased SERCA2a protein and activity levels are associated

with physiological hypertrophy. In most exercise trained mod-els, PLNprotein levels are unchanged, although changes in PLN

FIGURE 5. A, shown is quantitative real-time-PCR analysis of cardiomyopathy marker genes in 3-month-old mouse hearts. There is no significant difference ingene expression for any of the genes included in this profile. NTG n � 12, TG n � 10. B, shown is quantitative real-time-PCR analyses of cardiac gene expressionnormalized to GAPDH. SERCA2a, Mcip1: NTG n � 12, TG n � 10. NCX, PLN, L-type, RyR2: NTG n � 6, TG n � 5. C, Western blot analysis of Ca2� handling proteinsin NTG and TG hearts is shown. D, shown is quantification of Ca2� handling protein levels. PLN Ser-16 and PLN Thr-17 phosphorylation levels were normalizedto total PLN. All other protein expression was normalized to actin. n � 8. E, shown is phosphatase activity in mouse heart extract. NTG, n � 3; TG, n � 3. *,indicates p � 0.05; **, indicates p � 0.01.




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phosphorylation are seen (53, 54). This is similar to the resultsfoundwith the�-Tm S283Amice. Additionally, in animals thathave compensated or physiological hypertrophy due to exercisetraining, there are no changes in gene expression of commoncardiomyopathy markers, identical to what is seen in the TGmice investigated here (55). Exercise training improves car-diomyocyte contractility and calcium handling and oftenimproves disease in both animal and human models of cardiacdisease (56–58). Although the mechanisms responsible for thedevelopment of physiological hypertrophy during exercisetraining are not well elucidated, we speculate that ablating thephosphorylation site of �-Tm results in a similar signaling cas-cade that occurs in response to exercise training. In a physio-logically exercised heart, the signaling pathways that are acti-vated result in improved cardiac function, whereas in responseto Tm dephosphorylation, the TG hearts are able to functionnormally with a mild hypertrophic phenotype.

Increases in the level of SERCA2a appear to be responsiblefor cardiac dysfunction in the TG TAC-operated animals com-pared with NTG TAC-operated animals. Previous work indi-cates that SERCA2a up-regulation by �20% did not result inincreased energy consumption by the heart at basal levels (59).However, when those animals were subjected to pressure over-load hypertrophy, SERCA2a overexpressing TGmice show sig-nificant decreases in contractile force and free energy, leadingto increased morbidity. The adaptive response involvingSERCA2a up-regulation and increased PLN Ser-16 phosphor-ylation ensures that Tm dephosphorylated hearts remain com-pensated in a physiological state and do not progress to car-diomyopathy and heart failure under normal conditions.However, this increased SERCA2a expression and PLNphosphorylationmost likely leads to cardiac dysfunction andpathology after TAC operation. Animals were sacrificed 2weeks after TAC, but we speculate, based on the increase in

FIGURE 6. Echocardiographic analyses of NTG sham, TG sham, NTG TAC, and TG TAC hearts from 12–16-week-old mice. A, pressure gradients in NTGsham, TG sham, NTG TAC, and TG TAC hearts are shown. B and C, diastolic and systolic left ventricular internal dimensions (LVIDd, LVIDs), respectively. D,fractional shortening (% FS) is shown. E, heart weight (HW) to body weight (BW) ratio of NTG and TG sham-operated hearts and NTG and TG TAC-operatedhearts is shown. F, cardiomyocyte cross-sectional area measurements are shown. n � 6 for all groups. * indicates p � 0.05, ** indicates p � 0.01, *** indicatesp � 0.001. G, shown are tissue sections from NTG sham, TG sham, and NTG TAC- and TG TAC-operated hearts stained with hematoxylin and eosin (i), Masson’sTrichrome (ii), and wheat germ agglutinin (iii). All images were taken at 40�. The scale bar indicates 50 �m.




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hypertrophy in the TG TAC operated animals, that thisgroup would exhibit increases in hypertrophic markers aswell as increased lethality.This is the first study indicating that dephosphorylating a

sarcomeric protein can result inmaintenance of a compensatedor physiological hypertrophic phenotype. Additionally, to ourknowledge, this is the first study in which a TG animal withalterations in a sarcomeric protein results in increases inSERCA2a protein expression and PLNSer-16 phosphorylation.Tmphosphorylation appears to be involved in the developmentof compensated or physiological hypertrophy, possibly throughproteins involved in signaling at the z-disc. Novel PKC� andPKC� are two molecules shown to promote physiologicalhypertrophy. Both molecules translocate to the z-disc uponcardiomyocyte stimulation (60, 61). PKC�, specifically, hasbeen shown to associate with the myofilament and bindstrongly to actin, resulting in constitutively active PKC� (62).Previous studies indicate that dephosphorylated Tm bindsactin differentially from phosphorylated Tm, and it is possiblethat the replacement of Ser-283 with an Ala residue affectsnearest neighbor interactions in the sarcomere, allowing PKC�greater access to the binding site on actin (4, 10). Mice express-ing a PKC�-specific activator exhibit normal cardiac functionand a compensated hypertrophic phenotype indicating thatPKC� can be a positive modulator of compensatory cardiachypertrophy (63). Additionally, activated PKC� can activateMEK1 through Raf1, which has been shown to also result incompensated hypertrophy, indicating that the PKC�-Raf1-MEK1-ERK1/2 pathwaymay be playing a role in the S283A TGmouse phenotype (64–66). Studies examining the potentialsignaling pathways activated byTmdephosphorylation are cur-rently in progress. In summary, the results presented herefirmly establish that the status of Tm phosphorylation caninfluence expression of Ca2� regulatory proteins and theresponse of the heart to acute cardiac stress.

Acknowledgments—We thank Dr. Vikram Prasad and Dr. TracyPritchard for technical assistance and close reading of themanuscriptand Maureen Bender for excellent animal husbandry skills.

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John Solaro and David F. WieczorekEngel, Gilbert Newman, Jo El J. Schultz, Jeffery D. Molkentin, Beata M. Wolska, R.

Emily M. Schulz, Richard N. Correll, Hajer N. Sheikh, Marco S. Lofrano-Alves, Patti L.Tropomyosin Dephosphorylation Results in Compensated Cardiac Hypertrophy

doi: 10.1074/jbc.M112.402040 originally published online November 12, 20122012, 287:44478-44489.J. Biol. Chem.

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