Mouse Model of a Familial Hypertrophic Cardiomyopathy Mutation in a-Tropomyosin Manifests Cardiac...

Greg P. Boivin, Beata Wolska, Christian Evans, R. John Solaro and David F. WieczorekMariappan Muthuchamy, Kathy Pieples, Prabhakar Rethinasamy, Brian Hoit, Ingrid L. Grupp,

Manifests Cardiac Dysfunction-TropomyosinαMouse Model of a Familial Hypertrophic Cardiomyopathy Mutation in

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Mouse Model of a Familial Hypertrophic CardiomyopathyMutation in a-Tropomyosin Manifests Cardiac Dysfunction

Mariappan Muthuchamy, Kathy Pieples, Prabhakar Rethinasamy, Brian Hoit, Ingrid L. Grupp,Greg P. Boivin, Beata Wolska, Christian Evans, R. John Solaro, David F. Wieczorek

Abstract—To investigate the functional consequences of a tropomyosin (TM) mutation associated with familialhypertrophic cardiomyopathy (FHC), we generated transgenic mice that express mutanta-TM in the adult heart. Themissense mutation, which results in the substitution of asparagine for aspartic acid at amino acid position 175, occursin a troponin T binding region of TM. S1 nuclease mapping and Western blot analyses demonstrate that increasedexpression of thea-TM 175 transgene in different lines causes a concomitant decrease in levels of endogenousa-TMmRNA and protein expression. In vivo physiological analyses show a severe impairment of both contractility andrelaxation in hearts of the FHC mice, with a significant change in left ventricular fractional shortening. Myofilamentsthat containa-TM 175 demonstrate an increased activation of the thin filament through enhanced Ca21 sensitivity ofsteady-state force. Histological analyses show patchy areas of mild ventricular myocyte disorganization andhypertrophy, with occasional thrombi formation in the left atria. Thus, the FHCa-TM transgenic mouse can serve asa model system for the examination of pathological and physiological alterations imparted through aberrantTM isoforms.(Circ Res. 1999;85:47-56.)

Key Words: tropomyosinn cardiomyopathyn troponinn hypertrophy

Familial hypertrophic cardiomyopathy (FHC) is an auto-somal dominant disease distinguished by myocyte disar-

ray and asymmetric ventricular hypertrophy that often resultsin death as a result of heart failure.1 Several genetic analyseshave demonstrated that FHC is a sarcomeric disease that isassociated with mutations in cardiac myosin heavy and lightchains (MHC, MLC),a-tropomyosin (TM), cardiac troponinT (cTnT), troponin I (TnI), and myosin-binding protein C.2–4

Both in vivo and in vitro studies on FHC-associated MHCmutations result in altered myofibrillar formation and func-tion.5,6 Also, a cTnT mutation causes decreased contractilityin adult cardiac myocytes.7 Although genetic studies haveprovided strong evidence that TM mutations are also respon-sible for FHC, the precise physiological consequences ofthese mutations in the disease process remain unknown.

Regulation of contractile activity in cardiac muscle isdependent on a cooperative interaction between thick and thinfilament sarcomeric proteins. TM, an essential thin filamentprotein, interacts with actin and the troponin complex (Tn) toregulate muscle contraction in a Ca21-dependent manner. Thestriated musclea-TM isoform (a-TMstr) is the predominantTM isoform in the adult mouse, rat, and human heart.8–10

Work in our laboratory shows that overexpression of wild-

typea-TM in the heart does not lead to pathological changesnor functional alterations in cardiac or myofiber performance(J.E. Oehlenschlager and D.F.W., unpublished data, 1997).Also, we have recently demonstrated that by use of a singletransgenic manipulation, the striated muscleb-TM isoform(b-TMstr) can be exchanged with the endogenousa-TMstr inthe mouse heart without alteration of the stoichiometric levelof total TM protein in the sarcomere.11 This ectopic expres-sion of b-TM in transgenic (TG) mouse hearts alters myo-cardial relaxation. In addition, the myofilaments from theseb-TM TG hearts exhibit an increase in the activation of thethin filament by strongly bound cross bridges and an increasein Ca21 sensitivity of steady-state force.12 Mice that have 75%b-TM in their cardiac myofibrils demonstrate severe cardiacpathological abnormalities, including thrombus formation inthe lumen of both atria and the subendocardium of the leftventricle.13 This TG approach allows us to study the in vivoeffect of alterations in the native TM population on myofil-ament activation, without altering relative levels of othersarcomeric proteins.

In this study, we adopted a TG strategy to address theeffect of the FHC Asp175Asna-TM mutation in cardiacdevelopment and function. The mutation causes a change in

Received July 24, 1998; accepted April 19, 1999.From the Department of Molecular Genetics, Biochemistry and Microbiology (M.M., K.P., P.R., D.F.W.), Department of Internal Medicine,

Division of Cardiology (B.H.), Department of Pharmacology and Cell Biophysics (I.L.G.), Department of Pathology and Laboratory Medicine(G.P.B.), University of Cincinnati College of Medicine, Cincinnati, Ohio; and the Department of Physiology and Biophysics (B.W., C.E., R.J.S.),University of Illinois, College of Medicine, Chicago. The current affiliation for M.M. is the Department of Medical Physiology, Texas A&MUniversity Health Science Center, College Station, Tex.

Correspondence to David F. Wieczorek, Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College ofMedicine, Cincinnati, Ohio 45267-0524. E-mail [email protected]

© 1999 American Heart Association, Inc.

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the charge of the encoded amino acid and occurs in a putativeTnT binding region. We established 4 distinct TG lines thatuse a cardiac-specific promoter to express thea-TM 175mutation in the heart. Results show that in all TG lines, anincrease ina-TM 175 mRNA and protein leads to reciprocaldecreases in endogenousa-TM levels. By use of a work-performing model, we determined that 2 lines, which havereplacement of.50% of endogenousa-TM with mutanta-TM175 protein, exhibit slower contraction and relaxationproperties. Echocardiographic analysis of these mice showsthat they exhibit a normal ventricular function but respondless to exercise andb-adrenergic stimulation. In addition, thecardiac myofilaments from these mice are more sensitive toCa21. Histopathological analyses of these hearts show vari-able occurrence of myocyte disarray, hypertrophy, and fibro-sis. This model provides an opportunity to enhance theunderstanding of the functional role of TM in the heart duringboth the normal and FHC condition.

Materials and MethodsGeneration of a-TM 175 TG ConstructMousea-TMstr cDNA was synthesized by use of reverse-transcrip-tase–polymerase chain reaction (RT-PCR), with total RNA isolatedfrom the FVB/N mice hearts. The resultant PCR product was clonedinto the pUC18 vector atPstI/SacI sites (which have been bluntended) and sequenced; the sequence identity was confirmed with thepublished murine skeletal musclea-TM cDNA sequences.14 Thisa-TM cDNA was used as starting material for the site-directedmutagenesis ofa-TM 175. The mutation at codon 175, GAC, wasconverted to AAC by PCR mutagenesis and ligated into ana-TMstrcDNA. The mutation was verified by sequencing. Thea-TM 175cDNA was linked at the 59 end to thea-MHC cardiac-specificpromoter, and the SV40 poly(A) signal was ligated at the 39 end ofthe construct. The complete TG construct was digested free of vectorsequence withSpeI/KpnI, purified, and used to generate TG mice aspreviously described.11 Founder mice were identified by use of PCRand confirmed by genomic Southern blotting by use of tail clip DNA.Stable TG lines were established by breeding the founder transgenicmice with non-TG cohorts.

S1 Nuclease MappingTotal RNA was isolated from hearts with RNAzol (Cinna Biotecx).RNA-DNA hybridization, followed by S1 nuclease analyses, wasperformed under the conditions used previously.11 To distinguish thea-TM 175 transcripts from endogenousa-TM mRNAs, we used aPCR probe that incorporated the third exon ofa-MHC (15 bp) and262 nucleotides of thea-TM coding region (Figure 1B). In S1nuclease mapping analyses, this probe protects 262 nucleotides ofendogenousa-TM mRNA, whereas a 277-bp fragment is protectedfor mutanta-TM 175 transcripts. A control GAPDH probe was usedfor quantitative purposes. Gels were autoradiographed on KodakX-Omat AR film and exposed for phosphorimaging analyses (Phos-phorImager, Molecular Dynamics). Quantification was performed bythe volume integration method, subtracting appropriate positions ofthe tRNA control sample lane as background. Three different S1 gelswere used to quantify the relative levels of transcripts.

Western Blot AnalysisMyofibrillar proteins were prepared as described,15 except that allsolutions contained the protease inhibitors leupeptin (0.5 mg/L),pepstatin A (0.5 mg/L), and PMSF (0.4 mmol/L). Equal amounts ofprotein (5mg) were run on 2 10% SDS-polyacrylamide gels. One gelwas stained with Coomassie blue to confirm equal protein loadingfor each sample, and the other gel was transferred to nitrocellulosewith a Bio-Rad transblot apparatus. The filters were incubated witha striated muscle TM-specific CH1 monoclonal antibody16 at 1:1000

dilution for 1 hour at room temperature. After being washed in PBS,filters were incubated with secondary antibody. For quantitativeWestern blot analyses, the reacting secondary antibody,35S-labeledanti-mouse IgG (Amersham Corp), was used at a specific activity of1 mCi per 10 mL of blocking buffer (5% nonfat dry milk powder inPBS) for 1 hour. Filters were dried, exposed to Kodak X-Omat ARfilm, and quantified by phosphorimaging analysis. For statisticalpurposes, Western blot analyses of myofibrillar proteins were per-formed 4 times with each sample, and mean6SEM values werecalculated.

Functional AnalysesThe perfusion apparatus for the work-performing heart prepara-tions has been described previously.17 These preparations wereconducted on 7 control non-TG mice and 6a-TM 175 mice fromline 108. Preparations on 7a-TM 175 TG mice from line 69 werealso conducted. All physiological parameters were simulta-neously recorded on a 6-channel P7 Grass polygraph and ana-lyzed on an IBM-compatible computer. The program computedthe following parameters instantaneously: heart rate, mean aorticpressure, left intraventricular pressure (systolic, diastolic, and enddiastolic), time to one half relaxation (RT1/2), time to peakpressure (TPP), and rates of contraction (1dP/dt) and relaxation(2dP/dt). The data acquisition and analysis software enhancedboth the accuracy and precision of the data. When used inconjunction with polygraph output, the measurements provided acomplete and reliable record of the physiological performance ofeach heart. Individual points of the record were summarized asmean6SD, and the statistical difference was estimated by at test.Isoproterenol infusions were performed as described previously.11

In Vivo Echocardiographic Measurements ofCardiac FunctionEchocardiographic studies were performed in mice before and after8 weeks of exercise. The animals (5 control, 5 exercised control, 6a-TM 175 TG, and 8a-TM 175 TG exercised mice) were lightlyanesthetized with 2.5% avertin (0.01 mL/g) and were allowed tobreath spontaneously. Two-dimensionally targeted M-mode studieswere performed with a 9 MHz imaging transducer (Interspec-ATLApogee X-200) as previously described.18 Studies were performed atbaseline before and after the administration of 2.0mg/g IP isopro-terenol. M-mode measurements of end diastolic (EDD), end systolicdimensions (ESD), end diastolic thickness of the septum (IVSed), andposterior wall (PWed) were made from original tracings as previouslydescribed. Left ventricular (LV) mass was calculated by use of avalidated M-mode method19: LV Mass5(IVSDed1PWed1EDD)32(EDD)3. LV fractional shortening (FS) was calculated asFS51003(EDD2ESD)/EDD.

The percentage of change from baseline of fractional shorteningwith isoproterenol administration was computed as 1003(Isoproter-enol Value2Baseline Value)/Baseline Value.

Fiber Bundle Preparation andForce MeasurementsWe measured force developed by bundles of detergent-extractedfibers obtained from papillary muscle as previously described.20 Themice were anesthetized with pentobarbital (50 mg/kg body weight),and their hearts were quickly removed and immediately placed incold high relaxing (HR) buffer (10 mmol/L EGTA, 2 mmol/LMgCl2, 79.2 mmol/L KCl, 5.4 mmol/L Na2ATP, 12 mmol/L creatinephosphate, 20 mmol/L MOPS, pH 7.0 [ionic strength 150 mmol/L])plus protease inhibitors (2.5mg/mL pepstatin A, 1mg/mL leupeptin,and 50mmol/L PMSF). Papillary muscles were quickly dissectedfrom the heart, and bundles of fibers'150mm in diameter and 4 to5 mm long were prepared. These fiber bundles were glued betweena force transducer and a fixed post attached to a micromanipulator.The fiber bundles were extracted in the HR buffer that contained 10IU/mL creatine kinase and 1% Triton X-100 for 30 minutes. Thefibers were placed in HR buffer that contained 10 IU/mL creatinekinase, and the sarcomere lengths were set at 2.0mm, which was

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determined from the laser diffraction pattern. Isometric pCa-forcerelations were determined by bathing the skinned fiber bundlessequentially in low relaxing buffer, in which the EGTA concentra-tion was reduced to 0.1 mmol/L (versus the 10 mmol/L in HR), andthen in solutions of various pCa values as computed with a computerprogram. All solutions contained the cocktail of protease inhibitorsdescribed above. The results are presented as mean6SE. Theforce-pCa relation was fitted to the Hill equation with nonlinearregression analysis to derive the pCa50 and Hill coefficient. Shifts in

the pCa50 value were analyzed with an unpaired Studentt test withsignificance set atP,0.05.

Swimming ProtocolTwelve 8-week-olda-TM 175 TG mice and their age-matchednon-TG controls were subject to a swimming exercise program.21 Inbrief, animals were initially exercised for 20 minutes twice daily,with an increase of 10 minutes increments daily. The final durationof exercise was 90 minutes, twice daily, for 8 weeks. Groups of

Figure 1. a-TM 175 transgene construct and expression. A, Theconstruct used for the microinjection is shown. Site-directedmutagenesis at codon 175 in a-TM was made with the PCR asdescribed in Materials and Methods. The KpnI and SpeI enzymeswere used to release the transgene fragment. B, Double-strandedPCR probes for transgene construct and GAPDH were preparedwith the labeled 39 primer and strand-separated as described.11

Both probes were hybridized in the same reaction to RNAs fromnon-TG (NTG) and TG mouse hearts, and tRNA was used as anegative control. The band that corresponded to a-TM 175 tran-script is observed in all TG samples, and the endogenous a-TMmessage is observed in both TG and NTG samples. A GAPDHmessage is detected in all samples, except in the negative control(the weak GAPDH signal is due to very low amounts of GAPDHprobe [3000 cpm] used in this assay). The positions of a-TM anda-TM 175 are marked on the left; marker bands are shown on theright. A schematic representation of the transgene probe is givenbelow. C, Autoradiogram of a Western blot of TG and NTG heartsamples. Myofibrillar proteins from TG and NTG hearts were elec-trophoresed on SDS-polyacrylamide gels and the TM wasdetected by staining with CH1 antibody followed by 35S-labeledantimouse IgG. All TG hearts have an abundance of mutant pro-tein a-TM 175; however, the total TM content is similar in all thehearts. D, Quantification of TM content in the hearts of TG mice isshown. The radioactivity associated with a-TM and a-TM 175proteins were quantified with a PhosphorImager system. Even inthe relatively low copy number transgenic lines (69 and 113), 30%to 40% replacement of a-TM with FHC mutant a-TM 175 hasoccurred.

Muthuchamy et al FHC Mutation in a-Tropomyosin 49




control and conditioned animals were subject to physiological andmorphological analyses. There was no increased mortality ofa-TM175 TG mice as a consequence of increased exercise.

Histological AnalysisThe heart tissue was fixed in Bouin’s solution (75% saturatedaqueous picric acid, 25% formaldehyde [40%], and 5% glacial aceticacid) for 24 hours and stored in 70% ethanol until processed.Sections (5mm) were prepared and stained with hematoxylin andeosin or trichrome stain. To assess myocyte hypertrophy, 1 section atthe apex of the right ventricle was examined, blinded to genotype, at3400 magnification. This area was identified. Images were trans-ferred to a PowerMac 9600/350 by use of a Sony DXC930 colorvideo camera. Images were captured with Scion Image 1.6 softwareand a Scion C-G7 RGB PCI frame grabber. Scion Image software isa variant of the public domain NIH Image program (developed at theUS National Institutes of Health and available on the Internet athttp://rsb.info.nih.gov/nih-image). Adobe Photoshop 4.0 was used tomanipulate images, and Excel 4.0 was used for data collection andanalysis. Approximately 50 to 70 myocytes were counted in thesection, and the average area of the myocytes was determined.

ResultsConstruction of FHC Mutant a-TM 175Transgenic Micea-TM cDNA was used in site-directed mutagenesis to gen-erate a GAC to AAC transition at codon 175, which resultedin an Asp to Asn amino acid change. Thea-TM 175 cDNAwas then inserted downstream of thea-MHC promoter,which confers cardiac-specific expression11 (Figure 1A). TheSV40 polyadenylation/termination signal sequences were li-gated 39of the a-TM 175 to insure that the transcript wasprocessed correctly. This construct was isolated from vectorsequences, purified, and injected into male pronuclei toproduce TG mice. Several founder mice were generated, andgerm-line transmission of the transgene was confirmed byPCR analysis of the DNA. Southern blot analysis wasperformed to determine the copy numbers of the transgenewithin the various lines11; 4 lines with copy numbers of 4, 30,26, and 3 (TG lines 69, 108, 109, and 113, respectively) wereselected for additional studies. Neither these founder mice northeir progeny demonstrate any gross phenotypic alterations orreduced viability.

Replacement of Nativea-TM With a-TM 175Mutant Protein in TG HeartsIn previous studies, we have shown that overexpression ofb-TM in TG mouse hearts causes a concomitant decrease inendogenousa-TM. To ascertain whether a similar phenom-enon occurs with overexpression ofa-TM 175, we analyzedmRNA and protein expression of both endogenousa-TM andTG a-TM 175 in the TG hearts. S1 nuclease protection assayswere conducted to quantify the levels of the TM mRNAs withrespect to the control GAPDH transcripts. Values weremeasured in a single reaction mixture in which both probeswere hybridized to an equal amount of RNA (20mg) from TGor non-TG mouse hearts. Hybridization products were thendigested with S1 nuclease and analyzed as described.8 Asobserved in Figure 1B, the single-stranded 320-nucleotide(nt) TM probe can distinguish the TGa-TM 175 mRNA (277nt) from the endogenousa-TM transcript (262 nt). The bandthat corresponded toa-TM 175 message is detectable in all

TG heart RNA samples. In addition, there is a reciprocaldecrease in the TG heart samples of the endogenousa-TMtranscript levels when compared with the non-TG samples.Thus, a compensatory change occurs to regulate TM levels atthe molecular level in thea-TM 175 mice. Additionally,results show that steady-state levels ofa-TM 175 transcriptare dependent on the copy number of the transgene. Aquantitative phosphorimaging analysis shows a 5-fold in-crease ofa-TM 175 message over endogenousa-TM tran-script in TG lines 108 and 109, which corresponds to 85% oftotal TM message. In TG lines 69 and 113, there is a low levelof a-TM 175 transcript expression when compared withnativea-TM mRNA.

To examine the effects ofa-TM 175 transgene expressionon the protein profile of the myofilament, we analyzedmyofibrillar protein fractions of TG and non-TG hearts.Western blot analysis (Figure 1C) shows that the non-TGsample only contains thea-TM protein, whereas doubletbands are clearly visible in all TG samples. The slowmigrating band in the TG samples represents the nativea-TMstriated protein. The point mutation in codon 175 changes anegatively charged aspartic acid to a neutral asparagine,

Figure 2. Physiological analyses of LV function. A, Comparisonof contractile parameters in a-TM 175 TG and NTG mousehearts. Baseline values are set at 5 mL/min cardiac output and50 mm Hg mean aortic pressure (Table 1). Rates of contraction(1dP/dt) and relaxation (2dP/dt) are significantly reduced inFHC mutant hearts, and TPP and time to half relaxation (RT 1/2)are prolonged in these hearts (P,0.001). B, Isoproterenolresponse of TG and NTG mouse hearts in the work-performingheart model. Dose responses in cardiac performance weredetermined with isoproterenol concentrations that ranged from1310210 to 131027 mol/L. TG hearts respond to isoproterenol ina similar fashion as NTG control hearts; however, cardiac per-formance is still markedly different from controls.





Figure 2. Continued.





which results in thea-TM 175 protein that contains 1negative charge less than nativea-TM protein. In vitrobiochemical studies indicate that this specific amino acidcharge change affects the local unfolding of the region andalters the conformation of this mutanta-TM 175 molecule.22

Other investigators have also found that changes in a singleamino acid of TM results in aberrant migration of the proteinwith gel electrophoresis (M. Fizman, PhD, oral communica-tion, April 5, 1997). Recent studies ona-TM 175 proteinanalysis in skeletal muscle confirm a differential migration inSDS-PAGE similar to our results.23

To negate the possibility that a deletion ofa-TM 175 DNAsequences occurred during transgenesis, we conducted RT-PCR analyses on RNAs from TG lines 108 and 109 withSV40 as a 39primer. The PCR fragments that resulted werecloned and sequenced. Results show that there is no deletionin the a-TM 175 nucleotide sequence in the transgeneconstruct.

To characterize TM expression in thea-TM 175 TG mice,we quantified endogenous and mutant TM protein incorpo-ration in the myofibrils (Figure 1D). In TG lines 69 and 113,40.365.2% and 32.263.1% of the total TM isa-TM 175protein, respectively. This level of mutant TM protein syn-thesis is increased disproportionately over its associatedmRNA level; this situation may be due to an increasedstability or translation of the mutanta-TM transcripts. Inlines 108 and 109,a-TM 175 accounts for 63.268.3% oftotal a-TM. As mentioned previously, the expression ofa-TM 175 is associated with a decrease in nativea-TMlevels. Summation ofa-TM 175 protein levels with nativea-TM protein levels show no net change in total TMmyofibrillar protein content. Also, examination of cytosolicTM levels show no alterations from endogenous cytosolicprotein levels (data not shown). The variance in expressionamong TG lines is copy number dependent, which is similarto previous studies that used thea-MHC promoter.11 Addi-tional experiments demonstrate that there are no quantitativechanges in MHC, actin, or troponin levels nor is thereexpression ofb-TM in the a-TM 175 TG mice (data notshown).

The a-TM 175 Hearts Are HypodynamicTo assess whether any functional changes in cardiac perfor-mance occur in the FHC mutant mice, we conducted severalphysiological studies by use of the work-performing heartmodel,17 echocardiography, and skinned fiber preparations.Results demonstrate that with 60%a-TM 175 protein in theheart (ie, lines 108 and 109), there are significant functionaldifferences in many physiological parameters. As seen inFigure 2A, contractile function was altered in the FHC micewhen compared with non-TG littermate controls: the maxi-mum rate of contraction (1dP/dt) was reduced and the TPPwas prolonged. In addition, relaxation properties were al-tered: the maximum rate of relaxation (2dP/dt) was de-creased and the RT1/2 was increased. The determined mea-surements for these functions are shown in Table 1. Inaddition to these functional differences, intraventricular pres-sures were also significantly different between the TG andnon-TG mouse hearts. These results demonstrate that thecardiac performance of the TG mice is altered drastically bythis change in TM expression, which results in thea-TM 175FHC mice exhibiting diminished rates of contraction andrelaxation. Surprisingly, the mice that express,40% of thea-TM 175 protein do not show any differences in physiolog-ical performance, as determined by the work-performingheart (data not shown) or in skinned fiber analyses (seebelow). The explanation as to why.40%a-TM 175 proteinis needed before functional changes are observed is unknown;however, we speculate that a decreased protein stability fora-TM 175 protein or a functional compensation by wild-typea-TM would account for the observations.

The effect of reduced contractile and relaxation perfor-mance by thea-TM 175 TG mouse hearts is maintainedduring maximal stimulation with isoproterenol, ab-adrenergic agonist that stimulates muscle contraction andrelaxation (Figure 2B). In the work-performing heart model,the response to isoproterenol was similar in the TG andnon-TG hearts. However, because of the inherent differencesin contraction and relaxation between the 2 genotypic groups,the functional differences remained even after isoproterenoladministration at multiple concentrations.

TABLE 1. Cardiac Parameters of a-Tropomyosin Mutant Overexpression(TG) vs NTG

NTG (n57) TG (n56) P

Mean aortic pressure (afterload), mm Hg 49.560.17 50.060.13 Set

Intraventricular pressure

Systolic 99.760.76 96.660.97 0.029*

Diastolic 211.360.47 21.0660.85 0.000†

End diastolic 4.160.34 7.560.52 0.000†

Heart rate, bpm 344.465.53 326.967.33 0.080

Venous return, mL/min 5.160.01 5.160.02 Set

LV minute work, mm Hg z mL/min 252.560.66 255.360.83 Set

1dP/dt, mm Hg/s 4222.6630.21 3922.5642.56 0.000†

2dP/dt, mm Hg/s 23810.4662.48 22563.5691.99 0.000†

TPP, ms/mm Hg 0.404960.003 0.442860.005 0.000†

RT1⁄2, ms/mm Hg 0.462760.009 0.624460.023 0.000†

*P,0.05; †P,0.001. Set indicates fixed for experimental protocol.





In addition to the use of the work-performing heart model,we assessed in vivo cardiac function with echocardiography.These experiments were conducted in control and TG micebefore and after implementation of an 8-week swimmingexercise program. Table 2 shows the echocardiographicparameters of LV function determined in these mice bothbefore and after swimming exercise. At baseline, there is atendency for fractional shortening to be greater (13%) in TGmice than their wild-type littermates; however, this did notachieve statistical significance (P,0.10). Isoproterenol pro-duced a significant decrease in LV end-systolic dimensionand an increase in shortening fraction in both groups; how-ever, this increase was greater in controls than in TG animals(15.463.6 versus 8.562.7%, P,0.001). These results sup-port measurements found with the work-performing heart,specifically, the hypodynamic aspects of thea-TM 175hearts. Septal and posterior wall thickness, LV mass, andheart rates were similar in both groups.

After the swimming exercise, the shortening fraction in-creased significantly in control animals but was unchanged inTG mice. As a result, there was no difference in fractionalshortening between TG and control mice. Exercise had noapparent effect on LV mass in either group.

We also conducted pCa-force measurements to determinewhether the functional alterations found in vivo would bereflected in myofilaments of the FHC myocardium. Myofil-ament activation via Ca21 binding to troponin C (TnC) wasmeasured in skinned fiber preparations (line 108 mice).Results demonstrate that the force developed by FHC myo-filaments is significantly more sensitive to Ca21 than non-TGmyofilaments (Figure 3). The pCa50 (ie, pCa for half-maximal tension) is 5.8360.01 for FHC preparations and5.7360.01 for non-TG control preparations. This leftwardshift in the pCa-force relationship of FHC myofilamentscorroborates the decreased functional properties noted in theFHC work-performing heart preparations because this in-crease in Ca21 sensitivity may act to decrease the rate ofrelaxation (2dP/dt) and increase the RT1/2. There were nosignificant differences in the pCa-force relationship betweenmyofilaments from TG line 69 and non-TG control mice(data not shown).

In a separate series of experiments, we perfused heartsfrom mice (treated with ab-adrenergic blocking agent)with Tyrode’s solution for 20 minutes before preparationof the fiber bundles. Previous experiments24 have shownthat TnI phosphorylation is minimal under these condi-tions. The fiber bundles prepared from non-TG and TGhearts did not differ in Ca21 sensitivity from the previousresults. In an additional set of experiments, we comparedpCa-force relations of skinned fiber bundles from non-TGand TG hearts before and after phosphorylation by proteinkinase A (PKA). Both before and after PKA phosphoryla-tion of TnI, the myofilaments from TG mice were stillmore sensitive to Ca21 (Reference 25 and C.E., D.F.W.,R.J.S., B.W., unpublished data, 1999). Increased Ca21

sensitivity, particularly under the phosphorylating condi-tions that occur duringb-adrenergic stimulation, maypredispose the myocardium to impaired relaxation andcontribute to altered cardiac function in patients with FHCduring exercise or stress.

Figure 3. Normalized pCa-force relationof skinned fiber bundle preparationsfrom NTG and a-TM 175 TG (line 108)mouse hearts at sarcomere length2.0 mm. Force was normalized to thecorresponding maximum force at pCa4.50. Data are presented as mean6SE.In NTG fiber bundle preparations, n57from 4 different hearts. In TG prepara-tions, n57 from 4 different hearts. pCa50

was 5.7360.01 in NTG and 5.8360.01 inTG preparations. Other conditions aredescribed in Materials and Methods.

TABLE 2. Echocardiographic Parameters of LV Function

NTG TG

Baseline(n55)

Exercise(n57)

Baseline(n56)

Exercise(n58)

LVEDD, mm 3.460.2 3.960.3 3.460.3 3.460.2

LVESD, mm 2.160.1 1.860.2* 2.060.2 1.960.3

LVFS, % 37.664.9 46.565.1* 42.463.7 45.969.4

LV mass, mg 37.6611.6 35.169.3 38.268.6 40.7610.0

LVEDD indicates LV end-diastolic dimension; LVESD, LV end-systolic dimen-sion; and LVFS, LV fractional shortening.

*P,0.05 vs baseline.





Cardiac Histopathology of a-TM FHC HeartsA notable feature of FHC is the striking phenotypic variationobserved between and within families segregating the dis-ease.26,27 Also, within the heart, myocyte hypertrophy anddisarray can be patchy.28 In humans, thea-TM FHC muta-tions are associated with variable ventricular hypertrophy anda low incidence of sudden cardiac death.29,30Specifically, theAsp175Asna-TM mutation demonstrates large differences inmagnitude of the hypertrophic response, which range frommarked to mild hypertrophy dependent on genetic back-ground and environmental influences. Because the FHCphenotype in human patients is associated with a variablehypertrophic response, it was important to determine whethercardiac morphology is altered in thesea-TM mutant mice.Measurements show there are not significant differences inthe heart:body weight ratio in the TG to non-TG mice (0.65%versus 0.60%). Histological examination of the heart wasperformed on 20-week-old male TG and littermate controlmice. Morphological analysis on myocardial sections from 11of 17 (65%) TG mice exhibit various mild pathologicalalterations. Characteristic features include occasional mildmyocyte hypertrophy, disorganization of LV and apical rightventricular septal myocytes, and thrombi formation in the leftatrium (Figure 4). To confirm the qualitative assessment of

myocyte hypertrophy, we performed morphometric analysisof 4 non-TG and 9 TG mice. We examined the myocytes atthe apex of the right ventricular lumen, because this wasidentified as one of the areas of greatest change. The meanarea of the myocytes from the control hearts was778619.8mm2 and in the TG mice, it was 926642.9 mm2;these values are statistically significant at theP,0.05 level.No dramatic increase existed in cardiomyocyte necrosis.Nuclear gigantism, increased interstitial cellularity, and fibro-sis were additional features consistent with myocyte hyper-trophy (Figure 4). Because only 5% of the total myocardiumis affected by the hypertrophy, the net increase in heartweight in this region is masked by the remaining myocardiumof the TG mice. No abnormal lesions are found in non-TGhearts. This mild and patchy phenotype is in agreement withthe clinical features exhibited by patients who encode thea-TM 175 FHC mutation30 and other FHC mutations.31,32

Humans who carry FHC mutations are susceptible tolife-threatening episodes with strenuous exercise.33 To assessthe effect of exercise on the FHC mutant mice, we exercisedFHC TG and control mice by swimming. After 8 weeks ofswimming as exercise,a-TM 175 TG mice and controls werekilled and a morphological examination of their hearts wasconducted. Results show a slight increase in the incidence of

Figure 4. Histopathology of FHC mutant a-TM 175 hearts. A, Left ventricle of an a-TM 175 TG mutant heart. There is mild myocytehypertrophy, disorganization increased interstitial cellularity, and increased myocyte nuclear size (nuclear gigantism). B, Control regionof the left ventricle. C, Apex of the right ventricular lumen of a different a-TM 175 mutant heart that shows severe fibrosis (trichromestained). D, a-TM 175 TG mutant left atrium with thrombi and mineralization. Magnification was 350 for A and B; magnification was325 for C and D.





fibrosis and myocyte disorganization but no substantialchange in the degree of hypertrophy in the exercised FHCmice.

DiscussionMissense mutations (Ala63Val, Asp175Asn, and Glu180Gly)in the a-TM gene have been linked to FHC. The phenotypicseverity associated with the TM mutations in human patientsand the altered sarcomeric function associated with these TMamino acid changes are not well established. To address theseissues, we developed a TG mouse that overexpressed amutant a-TM molecule demonstrated to cause FHC in hu-mans. This missense mutation encodes a substitution of anaspartic acid for an asparagine at amino acid position 175.Results indicate that overexpression of thea-TM 175 muta-tion in the heart alters the amounts of nativea-TM at themRNA and protein levels. Hearts of adult TG mice areassociated with both structural and functional perturbations.The structural changes are focal mild cardiomyocyte disor-ganization and hypertrophy. Physiological alterations includean impairment in both cardiac contractile and relaxationfunctions. Thus, the development of this FHC animal modelwill enhance our understanding of the role of TM in thesarcomere during both normal and pathological conditions.

This is the first demonstration that exogenous expression inthe heart of TM encoding FHC mutations result in patholog-ical and physiological defects. C. Seidman and colleagues30,34

showed that human patients with thea-TM Asp175Asnmutation also exhibit variable myocyte hypertrophy, myofi-ber disarray, and replacement fibrosis. The severity anddistribution of the pathological phenotype vary substantiallyin affected members and range from marked to mild hyper-trophy, which is similar to FHC family members who shareother FHC mutations.32 Patients witha-TM mutations have anormal life expectancy that we also observe in these TG mice.Interestingly, in results similar to ours, TG mice that expressFHC cTnT mutations also demonstrate variable hypertro-phy.35 We speculate that different genetic backgrounds,environmental influences, and physical activities play animportant role in the development and severity of the FHCphenotype.

The cardiac performance of the FHC mutant mice that havea predominance ofa-TM 175 protein is abnormal butcompatible with life. At the whole heart level, there aredecreases in the rates of contraction and relaxation, inaddition to prolonged TPP and RT1/2. Also, LV fractionalshortening was greater in the FHC mice, which is compatiblewith the human condition. In skinned fiber preparations, theforce developed by FHC myofilaments is significantly moresensitive to Ca21 than non-TG myofilaments. The slowermyocardial dynamics of these FHC mice resembles thephysiological performance found in human FHC muscle.4,23

Interestingly, even at the maximal isoproterenol stimulation,a difference in the cardiac performance exists between theseTG and normal mice. Although the percentage of response toisoproterenol is similar between FHC TG mice and controlmice, the maximum level of stimulation is less in the TGhearts. With regard to myofilament-related mechanisms, dur-ing b-adrenergic stimulation, the accelerated rate of relax-

ation may be attributed to an increase in the “off rate” forCa21 exchange with TnC when TnI is phosphorylated byPKA.36–38 This similar percentage response tob-adrenergicstimulation fits with data that indicate that the binding affinityof Tn to TM is the same in wild-type controls and inpreparations that contain the Asp175Asn mutation. Therelative loss of response to a maximal level ofb-adrenergicstimulation may be related to other effects of switching fromwild-type TM to a-TM 175. For example, the mutation mayinduce a change in TM flexibility and response to myosin S1binding.

Two a-TM mutations associated with FHC (Asp175Asnand Glu180Gly) occur in a putative TnT binding region. Ourresults with myofilaments that contain mutanta-TM demon-strate an increase in Ca21 sensitivity, with a hypodynamic invivo cardiac function. The C-terminal half ofa-TM (specif-ically the region around amino acid Cys190) is quite flexiblein comparison to the N-terminal region of the molecule.39,40

In addition, there is a notch in thea-helical structure nearamino acid residues 169 to 172 in which 2 bulky hydrophobicresidues are adjacent.41 Interestingly, Asp175Asn andGlu180Gly are located in this region and cause an amino acidcharge change in thea-TM protein. It is likely that thesemutations disrupt the already weak TnT-TM interactions inthis region and alter the Ca21 sensitivity of the myofilaments.Biochemical analyses of bacterially expressed FHC TMproteins indicate that the mutant TMs have a differentstructural conformation when interacting with actin in the “onstate.”42 Also, the FHC TM mutant proteins increase the actinfilament velocity compared with nativea-TM in the in vitromotility assay.43 Thus, the increase in the Ca21 sensitivity ofthe FHC myofilaments could be due to the changes in thecooperative activation of thin filaments by altering the inter-action of TM and actin, by affecting the TnT binding to TM,or through both mechanisms.

Numerous studies have demonstrated the importance ofposttranscriptional regulation in the control of contractileprotein synthesis. Whereas alternative splicing mechanismsare known to regulated tissue- and developmental-specificisoform expression, the importance of translational regulationis being realized more fully. This situation is particularlyevident with respect to TM expression. A heterozygousknockout condition results in normal protein levels despitethe presence of only a single functional allele.20,44 Withoverexpression ofb-TM in TG mice, translational compen-sation also occurs by decreasing the expression ofa-TM tomaintain normal protein levels.11 In both this current study,which examines TM expression in cardiac muscle in TGmice, and in an analysis of TM in skeletal muscle from FHCpatients by Bottinelli et al,23 proper stoichiometric proteinsynthesis is maintained after overexpression of exogenousTM or synthesis of aberranta-TM. Also, TG mice thatoverexpress wild-typea-TM maintain normal levels of TMprotein without alterations in structure or function (J.E.Oehlenschlager and D.F.W., unpublished data, 1997). Thus, afeedback mechanism is operative in striated muscle thatcarefully regulates and maintains proper stoichiometric levelsof TM, presumably to ensure regulated sarcomere assemblyand function.





AcknowledgmentsThis work was supported by National Institutes of Health grantsHL-54912 (D.F.W.) and HL-22231 (R.J.S.). B.W. is supported bythe American Heart Association of Metropolitan Chicago Grant-in-Aid. We are grateful to Dr J. Robbins for providing the mousecardiac a-MHC promoter. The authors thank J. Neumann fortechnical assistance.

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