Received for publication, March 4, 2013, and in revised form, July 16, 2013 Published, JBC Papers in Press, August 19, 2013, DOI 10.1074/jbc.M113.466466
Emily M. Schulz‡, Tanganyika Wilder§, Shamim A. K. Chowdhury§, Hajer N. Sheikh‡, Beata M. Wolska§¶,R. John Solaro§, and David F. Wieczorek‡1
From the ‡Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine,Cincinnati, Ohio 45267, §Department of Physiology and Biophysics and ¶Section of Cardiology, Department of Medicine, Center forCardiovascular Research, University of Illinois, Chicago, Illinois 60612
Background: Increased calcium uptake can rescue congenital cardiomyopathies.Results: Decreasing tropomyosin phosphorylation increases phospholamban phosphorylation and improves cardiac functionand morphology in a cardiomyopathic mouse.Conclusion: Changing tropomyosin phosphorylation can influence calcium handling to adverse cardiac remodeling.Significance: This is the first report that altering tropomyosin phosphorylation can rescue a cardiomyopathic phenotype.
Studies indicate that tropomyosin (Tm) phosphorylation sta-tus varies in different mouse models of cardiac disease. Investi-gation of basal and acute cardiac function utilizing a mousemodel expressing an �-Tm protein that cannot be phosphory-lated (S283A) shows a compensated hypertrophic phenotypewith significant increases in SERCA2a expression and phosphor-ylation of phospholamban Ser-16 (Schulz, E. M., Correll, R. N.,Sheikh, H. N., Lofrano-Alves, M. S., Engel, P. L., Newman, G.,Schultz Jel, J., Molkentin, J. D., Wolska, B. M., Solaro, R. J., andWieczorek, D. F. (2012) J. Biol. Chem. 287, 44478–44489).Withthese results, we hypothesized that decreasing �-Tm phosphor-ylation may be beneficial in the context of a chronic, intrinsicstressor. To test this hypothesis, we utilized the familial hyper-trophic cardiomyopathy (FHC)�-TmE180Gmodel (Prabhakar,R., Boivin, G. P., Grupp, I. L., Hoit, B., Arteaga, G., Solaro, R. J.,and Wieczorek, D. F. (2001) J. Mol. Cell. Cardiol. 33, 1815–1828). These FHC hearts are characterized by increased heart:body weight ratios, fibrosis, increased myofilament Ca2� sensi-tivity, and contractile defects. The FHCmice die by 6–8monthsof age. We generated mice expressing both the E180G andS283A mutations and found that the hypertrophic phenotypewas rescued in the �-Tm E180G/S283A double mutant trans-genic animals; these mice exhibited no signs of cardiac hyper-trophy and displayed improved cardiac function. These doublemutant transgenic hearts showed increased phosphorylation ofphospholamban Ser-16 and Thr-17 compared with the �-TmE180Gmice. This is the first study to demonstrate that decreas-ing phosphorylation of tropomyosin can rescue a hypertrophiccardiomyopathic phenotype.
Tropomyosin (Tm)2 is an �-helical coiled coil proteininvolved in the Ca2�-dependent regulation of the thin filamentof the sarcomere. Once Ca2� binds to troponin C, a conforma-tional change occurs allowing the Tm to move away from themyosin head binding site on the sarcomeric actin filament,resulting in muscle contraction. �-Tm is the predominant Tmisoform found in cardiovascular muscle, making up �95% oftotal myofibrillar Tm (1). All striated muscle isoforms, includ-ing �-Tm, are phosphorylated at a single site, serine 283, byseveral potential kinases (2–7). Recent studies indicate thatmice expressing transgenic (TG) �-Tm with Ser-283 mutatedto alanine (S283A) show no major alterations in cardiac func-tion at basal levels with striking increases in the expression ofsarcoplasmic reticulum Ca2�-ATPase 2a (SERCA2a) and anincrease in phospholamban (PLN) Ser-16 phosphorylation (8).Additionally, thesemicemaintain a compensated hypertrophicphenotype throughout their lifetime.Previous studies demonstrate that TG animals expressing
Tmmutations that lead to cardiac disease show changes in Tmphosphorylation (9–11). It is also established that increasingSERCA2a expression and/or activity by decreasing PLNexpres-sion or increasing PLN phosphorylation at Ser-16 and/orThr-17 can rescue hypertrophic cardiomyopathy or heart fail-ure in mice (12–15). In addition, SERCA2a gene therapy hasbeen shown to be efficacious in human patients with end stageheart failure (10, 16–18).Familial hypertrophic cardiomyopathy (FHC) is considered a
disease of contractile proteins of the cardiac sarcomere, z-discproteins, and Ca2�-handling proteins (19). �-Tm has beenfound to encode 11 mutations that lead to FHC (20). Previous
* 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 HL-062426 (to R. J. S.), T32 HL-07382 (to E. M. S.), and T32 HL-07692 (toT. W.).
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: 513-558-0077; E-mail: [email protected].
2 The abbreviations used are: Tm, tropomyosin; MHC, myosin heavy chain; Tn,troponin; SERCA2a, sarcoplasmic reticulum Ca2�-ATPase 2a; PLN, phos-pholamban; FHC, familial hypertrophic cardiomyopathy; TG, transgenic;DMTG, double mutant transgenic; NTG, non-transgenic; LV, left ventricle;E/A ratio, maximal velocity of blood flow in the early diastole (E)/maximalvelocity of blood flow in the late diastole (A); Em, myocardial velocity in theearly diastole; BNP, brain natriuretic peptide; ANP, atrial natriuretic pep-tide; cTn, cardiac troponin.
work in our laboratories established a transgenic mouse modelexpressing mutant FHC �-Tm E180G protein. This mousemodel develops severe concentric cardiac hypertrophy withsignificant ventricular fibrosis and atrial enlargement. Signifi-cant physiological alterations in cardiac function, includingdiastolic dysfunction, occur in addition to myofilaments thatdemonstrate an increased activation of the thin filamentthrough enhanced calcium sensitivity of steady-state force (21,22). Recent studies show that Tm phosphorylation levelschange from 1.5 to 15 months of age; however, by 4–5months,the levels of Tm phosphorylation are higher in �-Tm E180Gtransgenic hearts than in their controls (8). Furthermore, theseresults of increased Tm phosphorylation in hypertrophic car-diomyopathy mice were confirmed with another FHC mousemodel, namely the �-Tm D175N mice (23). Interestingly, adilated cardiomyopathy mouse model (�-Tm E54K) shows a40% decrease in Tm phosphorylation in the heart; myofila-ments from thesemice exhibit a decrease inCa2� sensitivity (9).Unknown issues concerning the FHC Tm mice are whether
the increased Tm phosphorylation levels are a contributingcause or consequence of the disease phenotype. To address thisquestion, we hypothesized that decreasing Tm phosphoryla-tion through a Tm S283A mutation may attenuate cardiachypertrophy and improve cardiac function in mice expressingthe �-Tm E180G mutation alone. To that end, we generateddouble mutant transgenic (DMTG) mice expressing Tmencoding both the �-Tm E180G and S283A mutations on thesame molecule; several TG mouse lines were generated andanalyzed. DMTG animals had no change in heart:body weightratios and no deposition of fibrotic material characteristic ofthe �-Tm E180G hypertrophic cardiomyopathy phenotype.Also, echocardiographic studies indicated rescued cardiacfunction with improved performance in the DMTG animalscompared with non-transgenic (NTG) mice. Also, the DMTGmyofilaments exhibited intermediate levels of Ca2� sensitivitybetween those found in NTG and �-Tm E180G hearts. Inter-estingly, the DMTG hearts did not show the dramatic increasein SERCA2a expression seen in the �-Tm S283A hearts but didexhibit increased phosphorylation of PLN at both Ser-16 andThr-17 when compared with the �-Tm E180G levels. This isthe first study demonstrating that decreasing phosphorylationof �-Tm can rescue a hypertrophic cardiac disease phenotype.
EXPERIMENTAL PROCEDURES
Generation of TGMice—Mouse striatedmuscle�-TmcDNAwas subjected to QuikChange II site-directed mutagenesis(Agilent Technologies) utilizing the primer 5�-CAC GCT CTCAAC GAT ATG ACT GCC ATA TAA GTT TCT TTG CTTCAC-3� mutating the penultimate serine to an alanine andthe primer 5�-ACG TGC AGA GGG GCG GGC TGA-3�mutating glutamic acid 180 to a glycine. Themutation was ver-ified through sequencing of the construct by Genewiz. The�-Tm E180G/S283A DMTG construct was then cloned into avector containing the cardiac-specific �-myosin heavy chain(�-MHC) promoter and a human growth hormone poly(A) tailsequence (see Fig. 1A) (24). DMTGmice were generated usingthe FVB/N strain, and founder mice were identified using PCR
(25). Nucleotide sequencing of DMTGmouse tail DNA verifiedthe sequence of the �-Tm E180G/S283A transgene.The �-Tm E180G TG mice and the �-Tm S283A mice were
generated previously and extensively characterized withrespect to their cardiac phenotype and function (8, 21, 22). Weconfined our study to male mice because previous resultsshowed sex-specific differences in the development of a com-pensated hypertrophic phenotype in the �-Tm S283Amice (8).Genotyping—DNA samples were obtained from 5-day-old
mice, and PCRwas utilized to determine which animals carriedthe transgene. The following primers specific for the transgenewere used: �-MHC Forward, 5�-GCC CAC ACC AGA AATGAC AGA-3�; �-Tm Reverse, 5�-TCC AGT TCA TCT TCAGTGCCC-3�. GAPDHwas used as an internal control, and thefollowing primer set was used: GAPDHForward, 5�-AGCGAGCTCAGGACATTCTGG-3�; GAPDH Reverse, 5�-CTC CTAACC ACG CTC CTA GCA-3�.Transgenic Protein Quantification and Western Blot Ana-
lyses—Myofibrillar proteins were extracted from NTG, �-TmE180G, �-Tm S283A, and DMTG mouse ventricles as de-scribed previously (25). 25 �g of the myofibrillar protein prep-arations were separated by 12% SDS-PAGE and stained withCoomassie Blue. The presence of the �-Tm E180G mutationresults in differential mobility in SDS-PAGE (23, 25), allowingquantification of endogenous and DMTG proteins in the sam-ple. Measurements were performed using ImageQuant 5.1. Toconfirm that the double mutant Tm was properly assembledinto the sarcomere, cytoplasmic protein fractions (25 �g) iso-lated from 3-month-old NTG and DMTGmale mice were sep-arated by 12% SDS-PAGE and visualized with Coomassie Bluestain.Western blot analyses on myofibrillar protein preparations
(4 �g) from 3-month-old male NTG, �-Tm E180G, �-TmS283A, and DMTG hearts were conducted using the Tm-spe-cific antibody CH1 (Sigma-Aldrich), Tm Ser-283 phosphoryla-tion-specific antibody generated for our laboratory (YenZyme),and sarcomeric �-actin antibody 5C5 (Sigma-Aldrich) as aloading control.Whole ventricular homogenates from 3-month-old NTG,
�-Tm E180G, �-Tm S283A, and DMTG mice were utilized tovisualize Ca2�-handling protein expression levels. Westernblots were used to visualize SERCA2a (Abcam), troponin I(TnI) (Cell Signaling Technology), pTnI23/24 (Cell SignalingTechnology), PLN (Thermo Scientific), phosphorylated PLNSer-16 (Badrilla), and phosphorylated PLN Thr-17 (Badrilla).Sarcomeric actin (Sigma) was used as a loading control. Phos-phorylated PLN levels are given as a ratio of the phosphorylatedform over total PLN expression. Total PLN expression was cal-culated by adding the monomeric and pentameric species ofPLN for each sample and normalizing to actin.Two-dimensional Isoelectric Focusing PAGE—Two-dimen-
sional isoelectric focusing PAGE was performed on mousehearts as described previously (9). 3 �g of myofibrillar prepara-tions were resolved on a 24-cm 4.0–5.0 immobilized pH gradi-ent isoelectric focusing strip. The sampleswere then resolved inthe second dimension by 10% SDS-PAGE, transferred to nitro-cellulose, and subjected to immunoblotting. Tm muscle-spe-cific antibody CH1 was used to visualize both the unphosphor-
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ylated and phosphorylated NTG and �-Tm E180G TG proteinspecies.Histopathological Analyses and Cardiomyocyte Cross-sec-
tional Area Analyses—Mouse hearts from 3 to 13 months wereisolated, weighed, and examined for histopathological changes.Heart weight to body weight ratios were calculated to deter-mine evidence of cardiac hypertrophy. For histological analy-ses, the hearts were stained with hematoxylin/eosin (H&E) orMasson’s trichrome and evaluated for the presence of fibrosis,myocyte disarray, and calcification. Images were taken on aNikon SM2–2T dissecting microscope and an Olympus BX4Ccompound microscope.To quantify changes in cardiomyocyte cross-sectional area,
tissue sections were stained with wheat germ agglutinin conju-gated with Texas Red (Sigma-Aldrich) to visualize cardiomyo-cyte membranes. DAPI was used to stain the nuclei of car-diomyocytes. Randomized images of the left ventricular freewall were taken using a fluorescence camera mounted on aZeiss Axioskop, and cardiomyocyte cross-sectional area wasmeasured using NIH ImageJ.Quantitative Real Time PCR Analyses—RNA was isolated
from 3-month-old NTG, �-Tm E180G, �-Tm S283A, andDMTGmouse ventricular tissue using TRIzol reagent (Invitro-gen). cDNA was generated using the Superscript III kit (Invit-rogen). Real time RT-PCR was performed using an Opticon 2real time RT-PCR machine (MJ Research). Each sample wasmeasured in triplicate, and each experiment was repeatedtwice. Target mRNA was normalized to GAPDH expression asdescribed by Pfaffl (26). The following specific primers wereused: atrial natriuretic peptide (ANP) Forward, 5�-GCTTCCA-GGCCATATTGGAG-3�; ANP Reverse, 5�-GGGGGCATGA-CCTCATCTT-3; �-MHC Forward, 5�-TCATCCGAATCCA-TTTTGGG-3�;�-MHCReverse, 5�-CATAATCGTAGGGGT-TGTTG-3�; brain natriuretic peptide (BNP) Forward, 5�-GAG-GTCACTCCTATCCTCTGG-3�; BNP Reverse, 5�-GCCATT-TCCTCCGACTTTTCTC-3�; GAPDH Forward, 5�-TGACC-ACAGTCCATGCCATC-3�; GAPDH Reverse, 5�-GACG-GACACATTGGGGGTAG-3�.Echocardiographic Measurements—Echocardiographic mea-
surements were performed utilizing a high resolution trans-ducer (Vevo 770High Resolution Imaging Systemwith a centerfrequency of 30 MHz) after anesthetization of 3-month-oldmice as described previously (27). M-mode images of the leftventricle (LV), LV outflow tract, and left atrium were takenfrom the left parasternal long axis view. The parasternal shortaxis view at the level of the papillary muscles was used to mea-sure the LV internal dimension, anterior wall thickness, andposterior wall thickness. NTG and TG 12–16-week-old micewere examined. Pulsed Doppler was performed with the apicalfour-chamber view. The mitral inflow was recorded with theDoppler sample volume at the tip of themitral valve leaflets. Tomeasure time intervals, the Doppler sample volumewasmovedtoward the LV outflow tract, and both themitral inflow and LVoutflowwere obtained in the same recording. Three parametersof the LV diastolic function were evaluated: 1) E/A ratio, whichis the maximal velocity of blood flow in the early diastole (E)/maximal velocity of blood flow in the late diastole (A); 2) Ewavedeceleration time, which is the time from E to the end of the
early diastole; and 3) LV isovolumic relaxation time, which isthe time measured from the aortic valve closure to the mitralvalve opening. Additional information about the diastolic func-tion was obtained with tissue Doppler imaging. Peak myocar-dial velocities in the early (Em) diastole were obtained with thesample volume at the septal side of the mitral annulus in thefour-chamber view. All measurements and calculations wereaveraged from three consecutive cycles and performed accord-ing to the American Society of Echocardiography guidelines(28, 29). Data analysis was performed with Vevo 770 analyticsoftware.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 (27) and mounted between a forcetransducer and a micromanipulator. The sarcomeric lengthwas adjusted to 2.3 �m using laser diffraction patterns, andisometric tension was measured. Fiber bundles were then sub-jected to sequential Ca2� solutions (pCa), and isometric ten-sion was again measured. All experiments were carried out at22 °C.Statistics—All statistics are presented as mean � S.E. Where
appropriate, paired and unpaired t tests, analysis of variancewith 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 E180G/S283A DMTG Mice—To deter-mine the role of �-Tm phosphorylation in the context of a pro-gressive, genetic cardiac disease, we generated a DMTGmousemodel that expressed a non-phosphorylatable alanine at aminoacid 283, the location of the sole known phosphorylation site instriatedmuscle�-Tm (2, 4, 5). On the same construct, we intro-duced the �-Tm E180G FHC mutation (22). The transgeneconstruct used to generate these DMTG mice is shown in Fig.1A. Four DMTG lines were generated and studied.Cardiac �-Tm E180G/S283A DMTG Protein Expression—
DMTGprotein expressionwas determined as the percentage oftotal Tmprotein expression in all fourDMTG lines. These linesexhibited a range of 50–64% DMTG protein expression whencomparedwith total Tmprotein. Line 325 and Line 335 (64 and50% DMTG protein expression, respectively) did not differ intheirmorphological or physiological results; for that reason, wefocused on Line 325. Line 325mice also exhibited a similar levelof Tmphosphorylation as the�-TmS283Amice.As seen in Fig.1B, the �-TmE180Gmutation conferred differential migrationto the DMTG protein when visualized by SDS-PAGE, allowingquantification of endogenous and TG protein levels (Fig. 1C).Of particular interest is a feedback mechanism present in car-diac muscle when striated muscle Tms are expressed exoge-nously, whereas the endogenous Tm protein level decreasesconcomitantly with increasing DMTG Tm protein expression;there are no changes in total Tm protein (25, 30). Examinationof the cytoplasmic preparations from these DMTG miceshowed no significant accumulation of DMTG protein in thecytoplasm, indicating that the DMTG protein is being properlyincorporated into the sarcomere (data not shown).
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Phosphorylation Status of �-Tm E180G and �-Tm E180G/S283A DMTG Mice—Utilizing a Tm phosphorylation-specificantibody (8), we investigated the phosphorylation status of boththe �-Tm E180G and DMTG cardiac myofibers. Interestingly,at 3 months of age, �-Tm E180G hearts showed a significantincrease in Tm phosphorylation when compared with NTGlevels, whereas Line 325 DMTG hearts exhibited a significantdecrease in phosphorylation (Fig. 2). Both theNTGprotein andthe TG �-Tm E180G protein were phosphorylated when sam-pleswere separated using two-dimensional isoelectric focusing,indicating that there is no strong bias toward phosphorylatingendogenous or TG protein incorporated into the sarcomere(Fig. 2E). Additionally, the phosphorylation status of theDMTGhearts was very similar to the phosphorylation status ofthe �-Tm S283A mouse hearts.Histopathological, Gravimetric, and Cardiomyocyte Cross-
sectional Area Analyses of �-Tm E180G and �-Tm E180G/S283A DMTG Mice—To ascertain whether there were histo-logical abnormalities in the hearts of the DMTG animals, weconducted a detailed morphological analysis of the left ventric-ular free wall of 3-month-old mice. Results show that theDMTGhearts exhibited a phenotype that is very similar to age-matched NTG mice with no cardiomyocyte disarray, enlarge-ment, and excessive fibrosis, which are pathological changescharacteristic of the �-TmE180G hearts (Fig. 3A).Wheat germagglutinin staining of the cell membrane of the cardiomyocytesshowed no significant differences between cross-sectionalareas from NTG and DMTG left ventricular cardiomyocytes(Fig. 3B). As expected, given the degree of hypertrophy thatoccurs in the �-Tm E180G model, there was a significantincrease in cross-sectional area compared with both NTG andDMTG. There were also no differences in heart weight:bodyweight ratios between NTG and DMTG animals. In contrast
and as we reported previously, the �-TmE180G animals exhib-ited significant increases in heart weight:body weight ratioscompared with NTG littermates (21, 22, 31, 32). We hypothe-size that the presence of the S283A mutation is responsible fornormalizing the cardiac pathological phenotype in the DMTG
FIGURE 1. A, �-Tm E180G/S283A DMTG construct. The �-MHC promoter drives cardiac-specific expression of the striated muscle �-Tm with encoded substi-tutions at amino acids (aa) 180 (E180G) and 283 (S283A) on the same molecule. B, myofibrillar preparations resolved by SDS-PAGE and stained with CoomassieBlue. Note the small shift in migration between the �-Tm E180G and DMTG Tm samples. Arrows indicate DMTG endogenous and TG protein. C, quantificationof endogenous Tm protein remaining in NTG, �-Tm E180G, �-Tm S283A, and DMTG samples. Error bars represent S.E.
FIGURE 2. A, immunoblot of phosphorylation status of �-Tm E180G hearts at3 months of age. B, immunoblot of phosphorylation status of �-Tm S283Ahearts at 3 months of age. C, immunoblot of phosphorylation status of DMTGhearts at 3 months of age. D, quantification of �-Tm phosphorylation in heartsfrom 3-month-old NTG, �-Tm E180G, �-Tm S283A, and DMTG mice. *, versusNTG, p � 0.05. E, two-dimensional SDS-PAGE of Tm from �-Tm E180G TGmyofibers. Error bars represent S.E. pTm, phosphorylated Tm; �-Tm-p, phos-phorylated �-Tm.
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animals. Additionally, DMTG animals maintained the sameheart weight:body weight ratios as NTG littermates for at least18 months of age. Importantly, although �-Tm E180G animalsdo not typically survive past 6–8 months of age (22, 31), theaddition of the S283A mutation considerably extended the lifeexpectancy of these mice to that of NTG animals (data notshown).Cardiac Function in �-Tm E180G and �-Tm E180G/S283A
DMTG Animals—To assess whether decreasing phosphoryla-tion of �-Tm in a genetic model of hypertrophic cardiomyopa-thy improves cardiac function, we performed echocardio-graphic analysis on 3-month-old NTG, �-Tm E180G, andDMTG mice (Table 1). DMTG mice showed only a slightincrease in the size of the left atria; however, the increase in sizewas not as large as the increase in atrial size typically found in�-Tm E180G animals (21, 22, 32). Most striking is the signifi-cant increase in ejection fraction, fractional shortening, andvelocity of circumferential fiber shortening in the DMTG ani-mals comparedwith bothNTGand�-TmE180Ganimals, indi-cating that the DMTG hearts have improved systolic function
and are hypercontractile compared with NTG littermates.Additionally, diastolic function (E/Em and E/A ratios) was sig-nificantly improved in DMTG animals and showed rescue ofthe extreme diastolic dysfunction seen in the �-Tm E180Gmice (Table 1). In short, the DMTG animals demonstrate theprevention of the dysfunctional cardiac phenotype seen in the�-Tm E180G FHC model and actually show improved cardiacperformance. Recent studies have shown that there are nophysiological differences in cardiac function between �-TmS283A mice and controls (8).To determine whether the myofilament Ca2� sensitivity can
contribute to the observed improved phenotype in DMTGmice, we measured force-Ca2� relations in skinned, detergent-extracted fiber bundles from the papillary muscle of 3-month-old NTG, �-Tm E180G, and DMTG hearts. Although myofila-ments from DMTGmice showed an increased Ca2� sensitivitycompared with NTG controls, the Ca2� sensitivity of theDMTG was significantly lesser than that of �-Tm E180Gmyo-filaments (Fig. 4). Myofilaments exhibited a pCa50 of 5.94 �0.01 forDMTG, 5.81� 0.02 forNTG, and 6.01� 0.03 for�-Tm
FIGURE 3. A, histological studies of NTG, �-Tm E180G, and DMTG hearts at 3 months of age stained with H&E, Masson’s trichrome, and wheat germ agglutinin(WGA). Images were taken at 40�, and scale bars indicate 50 �m. B, cardiomyocyte cross-sectional area measurements (n � 6). *, versus NTG, p � 0.05; #, versus�-Tm E180G, p � 0.05. C, heart weight to body weight (HW:BW) ratios of 3-month-old mice (n � 6). Error bars represent S.E.
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E180G (Table 2). The maximum tension and Hill coefficient(nH) were not significantly different among the groups.Gene Expression Changes in �-Tm E180G and �-Tm E180G/
S283ADMTGHearts—With the improvement in cardiac func-tion in the DMTG hearts, we determined whether the rescuedhearts also show improvements at themolecular level by assess-ing gene expression of cardiomyopathy markers. Real time RT-PCR analysis of RNA isolated from ventricular tissue showedthat �-Tm E180G mice exhibited significant increases in�-MHC, BNP, and ANP as expected given the level of hyper-trophy present in the hearts of those animals (Fig. 5, A–C).Interestingly, DMTG hearts showed a significant decrease in�-MHC compared with both NTG and �-Tm E180G hearts.Moreover, DMTG hearts exhibited similar levels of ANP andBNP in comparison with NTG hearts and significant decreaseswhen comparedwith�-TmE180G hearts, whereas levels in the�-Tm S283A hearts did not significantly differ from normalvalues.Changes in Proteins Involved in Ca2� Handling in �-Tm
E180G and �-Tm E180G/S283A DMTG Hearts—To deter-mine whether there are changes in Ca2�-handling proteins inthe DMTG hearts, we conducted Western blot analyses.Results show that there were significant decreases in phosphor-ylated PLN in the �-Tm E180G and DMTG animals comparedwith NTG controls (Fig. 6). However, the DMTG samplesshowed significant increases in phosphorylation at both Ser-16andThr-17 comparedwith�-TmE180G samples (Fig. 6B). The�-Tm S283A animals exhibited a significant increase in PLNphosphorylation at Ser-16 as reported previously (8). Surpris-ingly, there was no increase in SERCA2a expression in theDMTG animals unlike that seen in the �-Tm S283A hearts.Therewas an increase in total PLNexpression in both the�-TmE180G and DMTG hearts when compared with �-Tm S283A(Fig. 6C). With the increase in PLN phosphorylation, it is pos-sible that there was a relief of the inhibition by PLN onSERCA2a, thus increasing Ca2� resequestration into the sarco-plasmic reticulum and helping rescue the FHC phenotype inthe DMTG hearts.We also examined phosphorylation of TnI, as this protein
can be considered a master regulator of sarcomeric function
and Ca2� sensitivity. Although there was an upward trend inTnI amino acid 23/24 phosphorylation in the DMTG mice,there were no significant differences in TnI expression or TnIphosphorylation at amino acids 23 and 24 (Fig. 6,D and E). Theabsence of alterations indicates that the shift of DMTGmyofiber Ca2� sensitivity towardNTG levels was notmediatedby TnI phosphorylation but occurred through some othermechanism.
DISCUSSION
Previouswork has indicated that in hearts of two FHCmousemodels that exhibit severe cardiomyopathic disease �-Tmphosphorylation levels are significantly higher than in NTGage-matched controls (11). To test whether the increased Tmphosphorylation levels may be contributing to the disease, wegenerated a cardiac-specific construct expressing a knownhuman FHC mutation (�-Tm E180G) and a mutation respon-sible for decreasing Tm phosphorylation at the sole knownphosphorylation site (�-Tm S283A) (8). This DMTG approachwas taken to ensure equivalent levels of both modifications inthe same Tm molecule, which may not occur in sarcomeres of�-TmE180G and�-TmS283ATGcrossed F1 generationmice.Four DMTG lines were generated expressing both the E180Gand S283Amutations on the same �-Tmmolecule. These linesshowed alterations in �-Tm phosphorylation without signifi-cant differences in cardiac phenotype. DMTG Line 325 waschosen for further study given the similarity in Tm phosphory-lation status with the �-Tm S283A animals. We reported pre-viously that �-Tm E180G TG animals have a short life span(6–8months) compared with NTG controls (21, 22, 31). Inter-estingly, DMTGanimals had a longer life span,measured out to18months of age, indicating a significant improvement in over-all cardiac health of the DMTG animals. Although the DMTGhearts did not exhibit the striking increases in SERCA2aexpression and PLN Ser-16 phosphorylation evident in the�-Tm S283A mice, the DMTG animals had significantincreases in PLN phosphorylation at Ser-16 and Thr-17 com-
TABLE 1Cardiac function of NTG, �-Tm E180G, and �-Tm E180G/S283A DMTGat 3 months of age as assessed by echocardiographyLVIDd, left ventricular internal dimension in diastole; EF, ejection fraction; FS,fractional shortening; Vcf, velocity of circumferential shortening; IVRT, isovolu-metric relaxation time; DT, deceleration time; E/A ratio, ratio of early-to-late ven-tricular filling velocities; E/Em ratio, ratio of early pulse Doppler filling velocity toearly tissue Doppler velocity; bpm, beats/min.
FIGURE 4. A, Ca2�-tension relations in skinned fiber bundles from NTG, �-TmE180G, and DMTG hearts. B, normalized force relations in skinned fiber bun-dles from NTG, �-Tm E180G, and DMTG hearts. Error bars represent S.E. mN,millinewtons.
TABLE 2Myofilament properties in NTG, �-Tm E180G, and �-Tm E180G/S283Amice
paredwith the�-TmE180Gmice, suggesting that DMTGmicemay have more active SERCA2a. The DMTG hearts expressedNTG levels of the cardiac hypertrophic markers �-MHC, BNP,and ANP. Increased re-expression of these genes in adults isassociatedwith the cardiac fetal gene programoften foundwithcardiac remodeling and hypertrophy.Our data show that�-TmE180G hearts expressed very high levels of these transcripts,which were reduced to NTG levels in the DMTG mice in thecase of all threemarkers studied. The decrease in�-MHC levelsmay partially account for the improvement in cardiac functionin the DMTG versus the �-Tm E180G hearts. Specifically, theimprovement in diastolic function and increased speed of ven-tricular shortening could result from a larger �- to �-MHCratio. These finding are in agreement with previous work show-ing that improved ventricular function is associated withincreased �- to �-MHC expression (33, 34). That decreasingTm phosphorylation can result in decreasing expression ofthese hypertrophy markers illustrates the comprehensive res-cue of the hypertrophic phenotype at the biochemical level.Investigation of cardiac performance in NTG, �-Tm E180G,
and DMTGmice showed that the �-Tm E180Gmice exhibitedsevere cardiac dysfunction, specifically in relaxation parame-ters such as E/Em and E/A ratios, possibly due to alterations inSERCA2a activity and PLN expression/phosphorylation thatcan lead to a reduction in the rate of Ca2� transit decay, whichis known to contribute to a slower relaxation rate (35). Interest-ingly, the introduction of the S283A dephosphorylation muta-tion returned E/Em and E/A ratios to NTG levels, rescuing therelaxation defect found in �-Tm E180G animals. These find-ings are similar to those observed in TG mice expressing bothchimeric �-/�-Tm and �-Tm E180G proteins (36). The moststriking result shown in the echocardiographic studies is the
FIGURE 5. A–C, quantitative RT-PCR analysis of expression of the cardiomyopathy marker genes in 3-month-old NTG, �-Tm E180G, �-Tm S283A, andDMTG hearts. For NTG, n � 12; for E180G, n � 8; for S283A, n � 8; and for DMTG, n � 10. *, versus NTG, p � 0.05; #, versus �-Tm E180G, p � 0.05; @, versus �-TmS283A, p � 0.05. Error bars represent S.E.
FIGURE 6. A, Western blots of sarcoplasmic Ca2� flux proteins in NTG, �-TmE180G, �-Tm S283A, and DMTG hearts. B, quantification of phosphorylationlevels of PLN Ser-16 and PLN Thr-17. C, quantification of expression of PLN andSERCA2a. D, Western blots of TnI and phosphorylated TnI (pTnI) from NTG,�-Tm E180G, �-Tm S283A, and DMTG hearts. E, quantification of data from D.*, versus NTG, p � 0.05; #, versus �-Tm E180G, p � 0.05; @, versus �-Tm S283A,p � 0.05; n � 8. Error bars represent S.E.
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significant increase in ejection fraction, velocity of circumfer-ential fiber shortening, and fractional shortening (%) in theDMTG animals compared with NTG littermates, indicatingthat the DMTG hearts exhibited improved contractile param-eters possibly due to an increase in SERCA2a activity. Theresults of our skinned fiber study indicate that some of the res-cue is attributable to the sarcomere because of the decrease inthe pCa-tensionmeasurements when comparedwith increasedCa2� sensitivity of �-Tm E180G myofilaments. Also, therewere no differences in nH from NTG values.Tm and the Tn complex function as a Ca2�-sensitive switch-
ing mechanism in the thin filament. Ca2� binding to TnCcauses Tm to move from a position blocking strong cross-bridge binding to actin to a position facilitating strong cross-bridge binding. Strongly bound cross-bridges enhance activa-tion by pushing Tm away from the myosin binding sites, andactivation is cooperatively transmitted through the thin fila-ment via Tm overlap regions. The fact that the �-Tm E180Gmutation involves a charge change suggests that it may disruptnormal Tm-actin binding, which is dependent on polar andhydrophobic interactions. Our results show that by changingthe Tm phosphorylation site (S283A) in the context of �-TmE180G the DMTGmyofilaments exhibit a decrease in the pCa-tension curve. This change occurs without affecting maximumtension and is in agreement with increased relaxation (as mea-sured by echocardiography) when compared with the �-TmE180G values. Furthermore, because no changes in cooperativ-ity were found among the NTG, �-Tm E180G, and DMTGmyofilaments, it appears that there are no differences in Tminteractions with actins under the control of Tm and that thereis no effect on end-to-end interactions linking near neighborfunctional units consisting of actin-Tm-Tn.The carboxyl end of Tm is involved in multiple protein-pro-
tein interactions, including the head-to-tail overlap of Tmmol-ecules, binding to actin, and binding to troponin T. Previousstudies show that the carboxyl end of Tm is involved in theregulation of Ca2� sensitivity (37, 38). Recent in vitro dataexamining the properties of recombinant, non-phosphorylat-able �-Tm (S283A), and a phosphomimetic (�-Tm S283D)indicate that although the S283D mutation slows deactivationof the thin filament the S283A mutation does not alter myofil-ament function (39); these data support our in vivo myofila-ment Ca2� sensitivity work regarding the effect of the S283Amutation. Additional studies show that when fiber bundlesfrom �-Tm S283D mice are analyzed there are no changes inmyofilament Ca2� sensitivity.3 Thus, myofilament Ca2� sensi-tivity may involve interactions between the Tm 180 and 283amino acid regions probably through TnT binding. We find itsurprising that both in vivo and in vitro studies indicate that�-Tm S283A myofilaments did not exhibit a change in Ca2�
sensitivity, yet the �-Tm E180G/S283A did exhibit a decreasein Ca2� sensitivity from the values obtained with Tm E180Galone; additional studies are in progress to address this issue.Tm is phosphorylated at amino acid Ser-283 located in the
overlap region of Tm; this region interacts with the T1 con-
served region of the amino terminus of cTnT (40). The �-TmE180G mutation would be expected to influence T2 binding incTnT. Recent work demonstrates that the interplay betweenTm and cTnT in the overlap region effectuates different statesof Tm on the actin filament (41, 42). These interactions facili-tate the binding of Tm to actin, promote Tm-Tm polymeriza-tion on the actin filament, and regulate cooperative activationof the thin filament. Based on our observations, reducing phos-phorylation of Tm together with the �-Tm E180G mutationoffsets the cardiac dysfunction/impairment of the FHC �-TmE180G mutation by itself possibly by altering interactions ofTm with cTnT in both the T1 and T2 regions. This hypothesisis supported by previous work showing that FHC �-Tm E180Gmice can also be rescued by modifications in the Tm carboxylterminus (36).Recent studies have indicated that the �-Tm E180G muta-
tion results in a strikingly more flexible striated muscle �-Tmcompared with WT �-Tm (43). Moreover, it has been sug-gested that this mutation leads to an increase in local flexibility,likely partially unwinding or relaxing the coiled coil aroundamino acid 180 (44, 45). The greater global flexibility of mutant�-Tm indicates that a lower concentration of Ca2� is necessaryto induce conformational changes needed to move Tm off themyosin head binding sites on actin, leading to increased Ca2�
sensitivity of the thin filament (46). Also, recent work byTardiffsuggests that mutations in Tm can elicit functional effects bypropagation of structural effects through the Tm helix (47). Assuch, it is possible that the S283A mutation at the carboxylterminus restores �-Tm to normal levels of flexibility, whichmay account for the near complete rescue at the level of thesarcomere, although this possibility demands further study.Previous studies have indicated that normalization of Ca2�
flux dynamics and altered Ca2� uptake by the sarcoplasmicreticulummay play a role in the rescue of the FHCphenotype inthe DMTGmice. Although there was no significant increase inSERCA2a expression in the DMTG animals, unlike the S283Aanimals, there were alterations in the phosphorylation status ofPLN, whichmay reduce the inhibition of SERCA2a by PLN andresult in increased activity. It is also possible that the decrease inSERCA2a in DMTG animals compared with the �-Tm S283Aanimals may be related to the presence of the disease-causingE180Gmutation as previous studies have shown that decreasesin SERCA2a expression occur in the �-Tm E180G mice at dif-ferent ages (21). Indeed, previous studies that altered proteinsinvolved in Ca2� fluxes in the context of the �-Tm E180Gmicehave shown thatCa2� buffering using parvalbumin or knockingout PLN in the �-Tm E180G TG animals can rescue the severecardiomyopathic phenotype (16, 48). Similar to theDMTGani-mals studied here, PLN knock-out/Tm E180G (PLNKO/�-TmE180G) mice show reversals in hypertrophic marker geneexpression compared with the significant increase seen in�-Tm E180G mice. The PLNKO/�-Tm E180G animals alsoshow rescue of relaxation parameters by echocardiography. Onthe other hand, the PLNKO/�-Tm E180G animals did not dis-play the improved functional performance seen in the DMTGanimals (10). Additionally, rescue of the �-Tm E180G pheno-type via adenoviral delivery of SERCA2a normalizes heart3 B. J. Biesiadecki, R. J. Solaro, and D. F. Wieczorek, unpublished data.
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weight to body weight ratios and improves cardiac function,similar to our observations in the DMTG animals (16).This report clearly demonstrates that alterations in the phos-
phorylation status of �-Tm can improve a genetically inducedcardiomyopathic phenotype. Other studies have also demon-strated that modifications in contractile protein phosphoryla-tion can improve cardiac and/or sarcomeric function. In vivocardiac function was improved in a restrictive cardiomyopathymousemodel (cTnI193His) when thesemice were crossed with aTG mouse expressing an amino-terminal truncated cTnI(cTnI-ND); two phosphorylation sites on cTnI (Ser-23 and Ser-24) are deleted in the cTnI-ND mice (49). In addition, deletionof the cTnI amino-terminal domain also improves cardiac con-tractility in aged mice (50). Furthermore, studies show thatmyosin light chain kinase-induced phosphorylation of skinnedmuscle fibers from an FHC regulatory light chain model(D166V) reverses the overly sensitive responsiveness to calcium(51). Thus, altering phosphorylation of sarcomeric proteinmayoffer a potential therapeutic approach to improve cardiac func-tion under specific cardiac stress conditions.Themechanism bywhich decreasing�-Tmphosphorylation
improves function in the context of the �-Tm E180Gmutationis unknown. Gaffin et al. (10) have shown that �-Tm E180Ganimals exhibit significant decreases in ERK1/2 phosphoryla-tion that are normalized when the animals are crossed withPLNKO mice. ERK1/2 is one of the MAPK pathways involvedin cardiac hypertrophy, and MEK1, an upstream effector ofERK1/2, has been shown to regulate increased levels of ERK1/2phosphorylation in a compensated hypertrophic phenotype,similar to what is seen in the �-Tm S283A animals (52). It ispossible that the alterations occurring at the level of the sar-comere, specifically changes in Tm nearest neighbor interac-tions, may result in alterations in the activation of proteinssuch as protein kinase C � (PKC�), which binds actin and isan upstream activator of the MEK1-ERK1/2 pathway viac-Raf (53–55). These questions will be addressed in futureinvestigations.Another signaling pathway that might possibly be activated
to prevent the disease phenotype in the DMTG mice involvesprotein phosphatase PP2a and casein kinase-2-interacting pro-tein (CKIP-1). Previous work shows that �-Tm E180G micehave increased levels of PP2a and casein kinase-2 (31, 56).Morerecent work by Ling et al. (57) demonstrates that CKIP-1 andPP2a directly interact, which facilitates the binding of PP2a toHDAC4 and promotes HDAC4 dephosphorylation. ThisHDAC4 dephosphorylation suppresses cardiac hypertrophyand the fetal cardiac gene program. If decreased �-Tm phos-phorylation leads to increased levels of CKIP-1 expression, thenthis signaling pathway may be activated to rescue the �-TmE180G/S283A mice and suppress the cardiac fetal geneprogram.The pathways bywhich the�-TmE180Gmutation promotes
a pathological phenotype have not been well elucidated, butmyofibers from FHCmodels exhibit increased Ca2� sensitivityand diastolic dysfunction possibly leading to stress-sensitivepathways of hypertrophic growth. It has been suggested thatdecreasing myofilament sensitivity would be a straightforwardapproach to treating FHC, although no pharmacological inter-
ventions are currently available. Targeting Tm phosphoryla-tion specifically, given the rescue of the FHC phenotype shownin this study, may provide a starting point for research intopossible therapeutics. However, significant preliminary studieswould first need to address the appropriateness of decreasingTm phosphorylation in treating other cardiomyopathies, thespecific phosphatase involved in Tm dephosphorylation, and ameans of targeting that phosphatase to Tm while restrictingpotential secondary targets. To conclude, our data show thatexpression of an�-Tmprotein expressing both the E180GFHCmutation and the phosphorylation-reducing S283A mutationrescues a cardiomyopathic hypertrophic phenotype. Changesin Ca2�-handling proteins may additionally be responsible forthe improved functional performance found in the DMTGhearts. Changes in local flexibility of the Tm molecule con-ferred by the replacement of Ser-283 with an Ala residue andthe significant loss of phosphorylation may be responsible forthe restoration of Tm to proper flexibility. Although alterationsin actin-Tn-Tm interactions could play a vital role, the precisemechanism whereby decreased Tm phosphorylation rescuescardiac hypertrophy remains to be elucidated.
Acknowledgments—We thank Hannah Yaejee Hong for technicalassistance and Maureen Bender for excellent animal husbandryskills.
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Decreasing Tropomyosin Phosphorylation and Cardiac Disease
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