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(Circulation Research. 1999;85:47-56.)
© 1999 American Heart Association, Inc.

Original Contribution

Mouse Model of a Familial Hypertrophic Cardiomyopathy Mutation in -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

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&M University Health Science Center, College Station, Tex.

Correspondence to David F. Wieczorek, Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524. E-mail david.wieczorek{at}uc.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—To investigate the functional consequences of a tropomyosin (TM) mutation associated with familial hypertrophic cardiomyopathy (FHC), we generated transgenic mice that express mutant -TM in the adult heart. The missense mutation, which results in the substitution of asparagine for aspartic acid at amino acid position 175, occurs in a troponin T binding region of TM. S1 nuclease mapping and Western blot analyses demonstrate that increased expression of the -TM 175 transgene in different lines causes a concomitant decrease in levels of endogenous -TM mRNA and protein expression. In vivo physiological analyses show a severe impairment of both contractility and relaxation in hearts of the FHC mice, with a significant change in left ventricular fractional shortening. Myofilaments that contain -TM 175 demonstrate an increased activation of the thin filament through enhanced Ca²⁺ sensitivity of steady-state force. Histological analyses show patchy areas of mild ventricular myocyte disorganization and hypertrophy, with occasional thrombi formation in the left atria. Thus, the FHC -TM transgenic mouse can serve as a model system for the examination of pathological and physiological alterations imparted through aberrant TM isoforms.

Key Words: tropomyosin • cardiomyopathy • troponin • hypertrophy

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease distinguished by myocyte disarray and asymmetric ventricular hypertrophy that often results in death as a result of heart failure.¹ Several genetic analyses have demonstrated that FHC is a sarcomeric disease that is associated with mutations in cardiac myosin heavy and light chains (MHC, MLC), -tropomyosin (TM), cardiac troponin T (cTnT), troponin I (TnI), and myosin-binding protein C.^{2 3 4} Both in vivo and in vitro studies on FHC-associated MHC mutations result in altered myofibrillar formation and function.^{5 6} Also, a cTnT mutation causes decreased contractility in adult cardiac myocytes.⁷ Although genetic studies have provided strong evidence that TM mutations are also responsible for FHC, the precise physiological consequences of these mutations in the disease process remain unknown.

Regulation of contractile activity in cardiac muscle is dependent on a cooperative interaction between thick and thin filament sarcomeric proteins. TM, an essential thin filament protein, interacts with actin and the troponin complex (Tn) to regulate muscle contraction in a Ca²⁺-dependent manner. The striated muscle -TM isoform (-TMstr) is the predominant TM isoform in the adult mouse, rat, and human heart.^{8 9 10} Work in our laboratory shows that overexpression of wild-type -TM in the heart does not lead to pathological changes nor 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 single transgenic manipulation, the striated muscle ß-TM isoform (ß-TMstr) can be exchanged with the endogenous -TMstr in the mouse heart without alteration of the stoichiometric level of total TM protein in the sarcomere.¹¹ This ectopic expression of ß-TM in transgenic (TG) mouse hearts alters myocardial relaxation. In addition, the myofilaments from these ß-TM TG hearts exhibit an increase in the activation of the thin filament by strongly bound cross bridges and an increase in Ca²⁺ sensitivity of steady-state force.¹² Mice that have 75% ß-TM in their cardiac myofibrils demonstrate severe cardiac pathological abnormalities, including thrombus formation in the lumen of both atria and the subendocardium of the left ventricle.¹³ This TG approach allows us to study the in vivo effect of alterations in the native TM population on myofilament activation, without altering relative levels of other sarcomeric proteins.

In this study, we adopted a TG strategy to address the effect of the FHC Asp175Asn -TM mutation in cardiac development and function. The mutation causes a change in the charge of the encoded amino acid and occurs in a putative TnT binding region. We established 4 distinct TG lines that use a cardiac-specific promoter to express the -TM 175 mutation in the heart. Results show that in all TG lines, an increase in -TM 175 mRNA and protein leads to reciprocal decreases in endogenous -TM levels. By use of a work-performing model, we determined that 2 lines, which have replacement of >50% of endogenous -TM with mutant -TM175 protein, exhibit slower contraction and relaxation properties. Echocardiographic analysis of these mice shows that they exhibit a normal ventricular function but respond less to exercise and ß-adrenergic stimulation. In addition, the cardiac myofilaments from these mice are more sensitive to Ca²⁺. Histopathological analyses of these hearts show variable occurrence of myocyte disarray, hypertrophy, and fibrosis. This model provides an opportunity to enhance the understanding of the functional role of TM in the heart during both the normal and FHC condition.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of -TM 175 TG Construct
Mouse -TMstr cDNA was synthesized by use of reverse-transcriptase–polymerase chain reaction (RT-PCR), with total RNA isolated from the FVB/N mice hearts. The resultant PCR product was cloned into the pUC18 vector at PstI/SacI sites (which have been blunt ended) and sequenced; the sequence identity was confirmed with the published murine skeletal muscle -TM cDNA sequences.¹⁴ This -TM cDNA was used as starting material for the site-directed mutagenesis of -TM 175. The mutation at codon 175, GAC, was converted to AAC by PCR mutagenesis and ligated into an -TMstr cDNA. The mutation was verified by sequencing. The -TM 175 cDNA was linked at the 5' end to the -MHC cardiac-specific promoter, and the SV40 poly(A) signal was ligated at the 3' end of the construct. The complete TG construct was digested free of vector sequence with SpeI/KpnI, purified, and used to generate TG mice as previously described.¹¹ Founder mice were identified by use of PCR and confirmed by genomic Southern blotting by use of tail clip DNA. Stable TG lines were established by breeding the founder transgenic mice with non-TG cohorts.

S1 Nuclease Mapping
Total RNA was isolated from hearts with RNAzol (Cinna Biotecx). RNA-DNA hybridization, followed by S1 nuclease analyses, was performed under the conditions used previously.¹¹ To distinguish the -TM 175 transcripts from endogenous -TM mRNAs, we used a PCR probe that incorporated the third exon of -MHC (15 bp) and 262 nucleotides of the -TM coding region (Figure 1B). In S1 nuclease mapping analyses, this probe protects 262 nucleotides of endogenous -TM mRNA, whereas a 277-bp fragment is protected for mutant -TM 175 transcripts. A control GAPDH probe was used for quantitative purposes. Gels were autoradiographed on Kodak X-Omat AR film and exposed for phosphorimaging analyses (PhosphorImager, Molecular Dynamics). Quantification was performed by the volume integration method, subtracting appropriate positions of the tRNA control sample lane as background. Three different S1 gels were used to quantify the relative levels of transcripts.

View larger version (36K): [in this window] [in a new window]   Figure 1. -TM 175 transgene construct and expression. A, The construct used for the microinjection is shown. Site-directed mutagenesis at codon 175 in -TM was made with the PCR as described in Materials and Methods. The KpnI and SpeI enzymes were used to release the transgene fragment. B, Double-stranded PCR probes for transgene construct and GAPDH were prepared with the labeled 3' primer and strand-separated as described.11 Both probes were hybridized in the same reaction to RNAs from non-TG (NTG) and TG mouse hearts, and tRNA was used as a negative control. The band that corresponded to -TM 175 transcript is observed in all TG samples, and the endogenous -TM message is observed in both TG and NTG samples. A GAPDH message is detected in all samples, except in the negative control (the weak GAPDH signal is due to very low amounts of GAPDH probe [3000 cpm] used in this assay). The positions of -TM and -TM 175 are marked on the left; marker bands are shown on the right. A schematic representation of the transgene probe is given below. C, Autoradiogram of a Western blot of TG and NTG heart samples. Myofibrillar proteins from TG and NTG hearts were electrophoresed on SDS-polyacrylamide gels and the TM was detected by staining with CH1 antibody followed by 35S-labeled antimouse IgG. All TG hearts have an abundance of mutant protein -TM 175; however, the total TM content is similar in all the hearts. D, Quantification of TM content in the hearts of TG mice is shown. The radioactivity associated with -TM and -TM 175 proteins were quantified with a PhosphorImager system. Even in the relatively low copy number transgenic lines (69 and 113), 30% to 40% replacement of -TM with FHC mutant -TM 175 has occurred.

Western Blot Analysis
Myofibrillar proteins were prepared as described,¹⁵ except that all solutions contained the protease inhibitors leupeptin (0.5 mg/L), pepstatin A (0.5 mg/L), and PMSF (0.4 mmol/L). Equal amounts of protein (5 µg) were run on 2 10% SDS-polyacrylamide gels. One gel was stained with Coomassie blue to confirm equal protein loading for each sample, and the other gel was transferred to nitrocellulose with a Bio-Rad transblot apparatus. The filters were incubated with a 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 quantitative Western blot analyses, the reacting secondary antibody, ³⁵S-labeled anti-mouse IgG (Amersham Corp), was used at a specific activity of 1 µCi per 10 mL of blocking buffer (5% nonfat dry milk powder in PBS) for 1 hour. Filters were dried, exposed to Kodak X-Omat AR film, and quantified by phosphorimaging analysis. For statistical purposes, Western blot analyses of myofibrillar proteins were performed 4 times with each sample, and mean±SEM values were calculated.

Functional Analyses
The perfusion apparatus for the work-performing heart preparations has been described previously.¹⁷ These preparations were conducted on 7 control non-TG mice and 6 -TM 175 mice from line 108. Preparations on 7 -TM 175 TG mice from line 69 were also conducted. All physiological parameters were simultaneously recorded on a 6-channel P7 Grass polygraph and analyzed on an IBM-compatible computer. The program computed the following parameters instantaneously: heart rate, mean aortic pressure, left intraventricular pressure (systolic, diastolic, and end diastolic), time to one half relaxation (RT_1/2), time to peak pressure (TPP), and rates of contraction (+dP/dt) and relaxation (-dP/dt). The data acquisition and analysis software enhanced both the accuracy and precision of the data. When used in conjunction with polygraph output, the measurements provided a complete and reliable record of the physiological performance of each heart. Individual points of the record were summarized as mean±SD, and the statistical difference was estimated by a t test. Isoproterenol infusions were performed as described previously.¹¹

In Vivo Echocardiographic Measurements of Cardiac Function
Echocardiographic studies were performed in mice before and after 8 weeks of exercise. The animals (5 control, 5 exercised control, 6 -TM 175 TG, and 8 -TM 175 TG exercised mice) were lightly anesthetized with 2.5% avertin (0.01 mL/g) and were allowed to breath spontaneously. Two-dimensionally targeted M-mode studies were performed with a 9 MHz imaging transducer (Interspec-ATL Apogee X-200) as previously described.¹⁸ Studies were performed at baseline before and after the administration of 2.0 µg/g IP isoproterenol. M-mode measurements of end diastolic (EDD), end systolic dimensions (ESD), end diastolic thickness of the septum (IVS_ed), and posterior wall (PW_ed) were made from original tracings as previously described. Left ventricular (LV) mass was calculated by use of a validated M-mode method¹⁹ : LV Mass=(IVSD_ed+PW_ed+EDD)³- (EDD)³. LV fractional shortening (FS) was calculated as FS=100x(EDD-ESD)/EDD.

The percentage of change from baseline of fractional shortening with isoproterenol administration was computed as 100x(Isoproterenol Value-Baseline Value)/Baseline Value.

Fiber Bundle Preparation and Force Measurements
We measured force developed by bundles of detergent-extracted fibers obtained from papillary muscle as previously described.²⁰ The mice were anesthetized with pentobarbital (50 mg/kg body weight), and their hearts were quickly removed and immediately placed in cold high relaxing (HR) buffer (10 mmol/L EGTA, 2 mmol/L MgCl₂, 79.2 mmol/L KCl, 5.4 mmol/L Na₂ATP, 12 mmol/L creatine phosphate, 20 mmol/L MOPS, pH 7.0 [ionic strength 150 mmol/L]) plus protease inhibitors (2.5 µg/mL pepstatin A, 1 µg/mL leupeptin, and 50 µmol/L PMSF). Papillary muscles were quickly dissected from the heart, and bundles of fibers 150 µm in diameter and 4 to 5 mm long were prepared. These fiber bundles were glued between a force transducer and a fixed post attached to a micromanipulator. The fiber bundles were extracted in the HR buffer that contained 10 IU/mL creatine kinase and 1% Triton X-100 for 30 minutes. The fibers were placed in HR buffer that contained 10 IU/mL creatine kinase, and the sarcomere lengths were set at 2.0 µm, which was determined from the laser diffraction pattern. Isometric pCa-force relations were determined by bathing the skinned fiber bundles sequentially in low relaxing buffer, in which the EGTA concentration was reduced to 0.1 mmol/L (versus the 10 mmol/L in HR), and then in solutions of various pCa values as computed with a computer program. All solutions contained the cocktail of protease inhibitors described above. The results are presented as mean±SE. The force-pCa relation was fitted to the Hill equation with nonlinear regression analysis to derive the pCa₅₀ and Hill coefficient. Shifts in the pCa₅₀ value were analyzed with an unpaired Student t test with significance set at P<0.05.

Swimming Protocol
Twelve 8-week-old -TM 175 TG mice and their age-matched non-TG controls were subject to a swimming exercise program.²¹ In brief, animals were initially exercised for 20 minutes twice daily, with an increase of 10 minutes increments daily. The final duration of exercise was 90 minutes, twice daily, for 8 weeks. Groups of control and conditioned animals were subject to physiological and morphological analyses. There was no increased mortality of -TM 175 TG mice as a consequence of increased exercise.

Histological Analysis
The heart tissue was fixed in Bouin's solution (75% saturated aqueous picric acid, 25% formaldehyde [40%], and 5% glacial acetic acid) for 24 hours and stored in 70% ethanol until processed. Sections (5 µm) were prepared and stained with hematoxylin and eosin or trichrome stain. To assess myocyte hypertrophy, 1 section at the apex of the right ventricle was examined, blinded to genotype, at x400 magnification. This area was identified. Images were transferred to a PowerMac 9600/350 by use of a Sony DXC930 color video camera. Images were captured with Scion Image 1.6 software and a Scion C-G7 RGB PCI frame grabber. Scion Image software is a variant of the public domain NIH Image program (developed at the US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image). Adobe Photoshop 4.0 was used to manipulate images, and Excel 4.0 was used for data collection and analysis. Approximately 50 to 70 myocytes were counted in the section, and the average area of the myocytes was determined.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Construction of FHC Mutant -TM 175 Transgenic Mice
-TM cDNA was used in site-directed mutagenesis to generate a GAC to AAC transition at codon 175, which resulted in an Asp to Asn amino acid change. The -TM 175 cDNA was then inserted downstream of the -MHC promoter, which confers cardiac-specific expression¹¹ (Figure 1A). The SV40 polyadenylation/termination signal sequences were ligated 3' of the -TM 175 to insure that the transcript was processed correctly. This construct was isolated from vector sequences, purified, and injected into male pronuclei to produce TG mice. Several founder mice were generated, and germ-line transmission of the transgene was confirmed by PCR analysis of the DNA. Southern blot analysis was performed to determine the copy numbers of the transgene within the various lines¹¹ ; 4 lines with copy numbers of 4, 30, 26, and 3 (TG lines 69, 108, 109, and 113, respectively) were selected for additional studies. Neither these founder mice nor their progeny demonstrate any gross phenotypic alterations or reduced viability.

Replacement of Native -TM With -TM 175 Mutant Protein in TG Hearts
In previous studies, we have shown that overexpression of ß-TM in TG mouse hearts causes a concomitant decrease in endogenous -TM. To ascertain whether a similar phenomenon occurs with overexpression of -TM 175, we analyzed mRNA and protein expression of both endogenous -TM and TG -TM 175 in the TG hearts. S1 nuclease protection assays were conducted to quantify the levels of the TM mRNAs with respect to the control GAPDH transcripts. Values were measured in a single reaction mixture in which both probes were hybridized to an equal amount of RNA (20 µg) from TG or non-TG mouse hearts. Hybridization products were then digested with S1 nuclease and analyzed as described.⁸ As observed in Figure 1B, the single-stranded 320-nucleotide (nt) TM probe can distinguish the TG -TM 175 mRNA (277 nt) from the endogenous -TM transcript (262 nt). The band that corresponded to -TM 175 message is detectable in all TG heart RNA samples. In addition, there is a reciprocal decrease in the TG heart samples of the endogenous -TM transcript levels when compared with the non-TG samples. Thus, a compensatory change occurs to regulate TM levels at the molecular level in the -TM 175 mice. Additionally, results show that steady-state levels of -TM 175 transcript are dependent on the copy number of the transgene. A quantitative phosphorimaging analysis shows a 5-fold increase of -TM 175 message over endogenous -TM transcript in TG lines 108 and 109, which corresponds to 85% of total TM message. In TG lines 69 and 113, there is a low level of -TM 175 transcript expression when compared with native -TM mRNA.

To examine the effects of -TM 175 transgene expression on the protein profile of the myofilament, we analyzed myofibrillar protein fractions of TG and non-TG hearts. Western blot analysis (Figure 1C) shows that the non-TG sample only contains the -TM protein, whereas doublet bands are clearly visible in all TG samples. The slow migrating band in the TG samples represents the native -TM striated protein. The point mutation in codon 175 changes a negatively charged aspartic acid to a neutral asparagine, which results in the -TM 175 protein that contains 1 negative charge less than native -TM protein. In vitro biochemical studies indicate that this specific amino acid charge change affects the local unfolding of the region and alters the conformation of this mutant -TM 175 molecule.²² Other investigators have also found that changes in a single amino acid of TM results in aberrant migration of the protein with gel electrophoresis (M. Fizman, PhD, oral communication, April 5, 1997). Recent studies on -TM 175 protein analysis in skeletal muscle confirm a differential migration in SDS-PAGE similar to our results.²³

To negate the possibility that a deletion of -TM 175 DNA sequences occurred during transgenesis, we conducted RT-PCR analyses on RNAs from TG lines 108 and 109 with SV40 as a 3'primer. The PCR fragments that resulted were cloned and sequenced. Results show that there is no deletion in the -TM 175 nucleotide sequence in the transgene construct.

To characterize TM expression in the -TM 175 TG mice, we quantified endogenous and mutant TM protein incorporation in the myofibrils (Figure 1D). In TG lines 69 and 113, 40.3±5.2% and 32.2±3.1% of the total TM is -TM 175 protein, respectively. This level of mutant TM protein synthesis is increased disproportionately over its associated mRNA level; this situation may be due to an increased stability or translation of the mutant -TM transcripts. In lines 108 and 109, -TM 175 accounts for 63.2±8.3% of total -TM. As mentioned previously, the expression of -TM 175 is associated with a decrease in native -TM levels. Summation of -TM 175 protein levels with native -TM protein levels show no net change in total TM myofibrillar protein content. Also, examination of cytosolic TM levels show no alterations from endogenous cytosolic protein levels (data not shown). The variance in expression among TG lines is copy number dependent, which is similar to previous studies that used the -MHC promoter.¹¹ Additional experiments demonstrate that there are no quantitative changes in MHC, actin, or troponin levels nor is there expression of ß-TM in the -TM 175 TG mice (data not shown).

The -TM 175 Hearts Are Hypodynamic
To assess whether any functional changes in cardiac performance occur in the FHC mutant mice, we conducted several physiological studies by use of the work-performing heart model,¹⁷ echocardiography, and skinned fiber preparations. Results demonstrate that with 60% -TM 175 protein in the heart (ie, lines 108 and 109), there are significant functional differences in many physiological parameters. As seen in Figure 2A, contractile function was altered in the FHC mice when compared with non-TG littermate controls: the maximum rate of contraction (+dP/dt) was reduced and the TPP was prolonged. In addition, relaxation properties were altered: the maximum rate of relaxation (-dP/dt) was decreased and the RT_1/2 was increased. The determined measurements for these functions are shown in Table 1. In addition to these functional differences, intraventricular pressures were also significantly different between the TG and non-TG mouse hearts. These results demonstrate that the cardiac performance of the TG mice is altered drastically by this change in TM expression, which results in the -TM 175 FHC mice exhibiting diminished rates of contraction and relaxation. Surprisingly, the mice that express <40% of the -TM 175 protein do not show any differences in physiological performance, as determined by the work-performing heart (data not shown) or in skinned fiber analyses (see below). The explanation as to why >40% -TM 175 protein is needed before functional changes are observed is unknown; however, we speculate that a decreased protein stability for -TM 175 protein or a functional compensation by wild-type -TM would account for the observations.

View larger version (51K): [in this window] [in a new window]   Figure 2. Physiological analyses of LV function. A, Comparison of contractile parameters in -TM 175 TG and NTG mouse hearts. Baseline values are set at 5 mL/min cardiac output and 50 mm Hg mean aortic pressure (Table 1). Rates of contraction (+dP/dt) and relaxation (-dP/dt) are significantly reduced in FHC mutant hearts, and TPP and time to half relaxation (RT 1/2) are prolonged in these hearts (P<0.001). B, Isoproterenol response of TG and NTG mouse hearts in the work-performing heart model. Dose responses in cardiac performance were determined with isoproterenol concentrations that ranged from 1x10-10 to 1x10-7 mol/L. TG hearts respond to isoproterenol in a similar fashion as NTG control hearts; however, cardiac performance is still markedly different from controls.

View this table: [in this window] [in a new window]   Table 1. Cardiac Parameters of -Tropomyosin Mutant Overexpression (TG) vs NTG

The effect of reduced contractile and relaxation performance by the -TM 175 TG mouse hearts is maintained during maximal stimulation with isoproterenol, a ß-adrenergic agonist that stimulates muscle contraction and relaxation (Figure 2B). In the work-performing heart model, the response to isoproterenol was similar in the TG and non-TG hearts. However, because of the inherent differences in contraction and relaxation between the 2 genotypic groups, the functional differences remained even after isoproterenol administration at multiple concentrations.

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 mice before and after implementation of an 8-week swimming exercise program. Table 2 shows the echocardiographic parameters of LV function determined in these mice both before and after swimming exercise. At baseline, there is a tendency for fractional shortening to be greater (13%) in TG mice than their wild-type littermates; however, this did not achieve statistical significance (P<0.10). Isoproterenol produced a significant decrease in LV end-systolic dimension and an increase in shortening fraction in both groups; however, this increase was greater in controls than in TG animals (15.4±3.6 versus 8.5±2.7%, P<0.001). These results support measurements found with the work-performing heart, specifically, the hypodynamic aspects of the -TM 175 hearts. Septal and posterior wall thickness, LV mass, and heart rates were similar in both groups.

View this table: [in this window] [in a new window]   Table 2. Echocardiographic Parameters of LV Function

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

We also conducted pCa-force measurements to determine whether the functional alterations found in vivo would be reflected in myofilaments of the FHC myocardium. Myofilament activation via Ca²⁺ binding to troponin C (TnC) was measured in skinned fiber preparations (line 108 mice). Results demonstrate that the force developed by FHC myofilaments is significantly more sensitive to Ca²⁺ than non-TG myofilaments (Figure 3). The pCa50 (ie, pCa for half-maximal tension) is 5.83±0.01 for FHC preparations and 5.73±0.01 for non-TG control preparations. This leftward shift in the pCa-force relationship of FHC myofilaments corroborates the decreased functional properties noted in the FHC work-performing heart preparations because this increase in Ca²⁺ sensitivity may act to decrease the rate of relaxation (-dP/dt) and increase the RT_1/2. There were no significant differences in the pCa-force relationship between myofilaments from TG line 69 and non-TG control mice (data not shown).

View larger version (12K): [in this window] [in a new window]   Figure 3. Normalized pCa-force relation of skinned fiber bundle preparations from NTG and -TM 175 TG (line 108) mouse hearts at sarcomere length 2.0 µm. Force was normalized to the corresponding maximum force at pCa 4.50. Data are presented as mean±SE. In NTG fiber bundle preparations, n=7 from 4 different hearts. In TG preparations, n=7 from 4 different hearts. pCa50 was 5.73±0.01 in NTG and 5.83±0.01 in TG preparations. Other conditions are described in Materials and Methods.

In a separate series of experiments, we perfused hearts from mice (treated with a ß-adrenergic blocking agent) with Tyrode's solution for 20 minutes before preparation of the fiber bundles. Previous experiments²⁴ have shown that TnI phosphorylation is minimal under these conditions. The fiber bundles prepared from non-TG and TG hearts did not differ in Ca²⁺ sensitivity from the previous results. In an additional set of experiments, we compared pCa-force relations of skinned fiber bundles from non-TG and TG hearts before and after phosphorylation by protein kinase A (PKA). Both before and after PKA phosphorylation of TnI, the myofilaments from TG mice were still more sensitive to Ca²⁺ (Reference 25²⁵ and C.E., D.F.W., R.J.S., B.W., unpublished data, 1999). Increased Ca²⁺ sensitivity, particularly under the phosphorylating conditions that occur during ß-adrenergic stimulation, may predispose the myocardium to impaired relaxation and contribute to altered cardiac function in patients with FHC during exercise or stress.

Cardiac Histopathology of -TM FHC Hearts
A notable feature of FHC is the striking phenotypic variation observed between and within families segregating the disease.^{26 27} Also, within the heart, myocyte hypertrophy and disarray can be patchy.²⁸ In humans, the -TM FHC mutations are associated with variable ventricular hypertrophy and a low incidence of sudden cardiac death.^{29 30} Specifically, the Asp175Asn -TM mutation demonstrates large differences in magnitude of the hypertrophic response, which range from marked to mild hypertrophy dependent on genetic background and environmental influences. Because the FHC phenotype in human patients is associated with a variable hypertrophic response, it was important to determine whether cardiac morphology is altered in these -TM mutant mice. Measurements show there are not significant differences in the heart:body weight ratio in the TG to non-TG mice (0.65% versus 0.60%). Histological examination of the heart was performed on 20-week-old male TG and littermate control mice. Morphological analysis on myocardial sections from 11 of 17 (65%) TG mice exhibit various mild pathological alterations. Characteristic features include occasional mild myocyte hypertrophy, disorganization of LV and apical right ventricular septal myocytes, and thrombi formation in the left atrium (Figure 4). To confirm the qualitative assessment of myocyte hypertrophy, we performed morphometric analysis of 4 non-TG and 9 TG mice. We examined the myocytes at the apex of the right ventricular lumen, because this was identified as one of the areas of greatest change. The mean area of the myocytes from the control hearts was 778±19.8 µm² and in the TG mice, it was 926±42.9 µm²; these values are statistically significant at the P<0.05 level. No dramatic increase existed in cardiomyocyte necrosis. Nuclear gigantism, increased interstitial cellularity, and fibrosis were additional features consistent with myocyte hypertrophy (Figure 4). Because only 5% of the total myocardium is affected by the hypertrophy, the net increase in heart weight in this region is masked by the remaining myocardium of the TG mice. No abnormal lesions are found in non-TG hearts. This mild and patchy phenotype is in agreement with the clinical features exhibited by patients who encode the -TM 175 FHC mutation³⁰ and other FHC mutations.^{31 32}

View larger version (124K): [in this window] [in a new window]   Figure 4. Histopathology of FHC mutant -TM 175 hearts. A, Left ventricle of an -TM 175 TG mutant heart. There is mild myocyte hypertrophy, disorganization increased interstitial cellularity, and increased myocyte nuclear size (nuclear gigantism). B, Control region of the left ventricle. C, Apex of the right ventricular lumen of a different -TM 175 mutant heart that shows severe fibrosis (trichrome stained). D, -TM 175 TG mutant left atrium with thrombi and mineralization. Magnification was x50 for A and B; magnification was x25 for C and D.

Humans who carry FHC mutations are susceptible to life-threatening episodes with strenuous exercise.³³ To assess the effect of exercise on the FHC mutant mice, we exercised FHC TG and control mice by swimming. After 8 weeks of swimming as exercise, -TM 175 TG mice and controls were killed and a morphological examination of their hearts was conducted. Results show a slight increase in the incidence of fibrosis and myocyte disorganization but no substantial change in the degree of hypertrophy in the exercised FHC mice.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Missense mutations (Ala63Val, Asp175Asn, and Glu180Gly) in the -TM gene have been linked to FHC. The phenotypic severity associated with the TM mutations in human patients and the altered sarcomeric function associated with these TM amino acid changes are not well established. To address these issues, we developed a TG mouse that overexpressed a mutant -TM molecule demonstrated to cause FHC in humans. This missense mutation encodes a substitution of an aspartic acid for an asparagine at amino acid position 175. Results indicate that overexpression of the -TM 175 mutation in the heart alters the amounts of native -TM at the mRNA and protein levels. Hearts of adult TG mice are associated with both structural and functional perturbations. The structural changes are focal mild cardiomyocyte disorganization and hypertrophy. Physiological alterations include an impairment in both cardiac contractile and relaxation functions. Thus, the development of this FHC animal model will enhance our understanding of the role of TM in the sarcomere during both normal and pathological conditions.

This is the first demonstration that exogenous expression in the heart of TM encoding FHC mutations result in pathological and physiological defects. C. Seidman and colleagues^{30 34} showed that human patients with the -TM Asp175Asn mutation also exhibit variable myocyte hypertrophy, myofiber disarray, and replacement fibrosis. The severity and distribution of the pathological phenotype vary substantially in affected members and range from marked to mild hypertrophy, which is similar to FHC family members who share other FHC mutations.³² Patients with -TM mutations have a normal life expectancy that we also observe in these TG mice. Interestingly, in results similar to ours, TG mice that express FHC cTnT mutations also demonstrate variable hypertrophy.³⁵ We speculate that different genetic backgrounds, environmental influences, and physical activities play an important role in the development and severity of the FHC phenotype.

The cardiac performance of the FHC mutant mice that have a predominance of -TM 175 protein is abnormal but compatible with life. At the whole heart level, there are decreases in the rates of contraction and relaxation, in addition to prolonged TPP and RT_1/2. Also, LV fractional shortening was greater in the FHC mice, which is compatible with the human condition. In skinned fiber preparations, the force developed by FHC myofilaments is significantly more sensitive to Ca²⁺ than non-TG myofilaments. The slower myocardial dynamics of these FHC mice resembles the physiological performance found in human FHC muscle.^{4 23} Interestingly, even at the maximal isoproterenol stimulation, a difference in the cardiac performance exists between these TG and normal mice. Although the percentage of response to isoproterenol is similar between FHC TG mice and control mice, the maximum level of stimulation is less in the TG hearts. With regard to myofilament-related mechanisms, during ß-adrenergic stimulation, the accelerated rate of relaxation may be attributed to an increase in the "off rate" for Ca²⁺ exchange with TnC when TnI is phosphorylated by PKA.^{36 37 38} This similar percentage response to ß-adrenergic stimulation fits with data that indicate that the binding affinity of Tn to TM is the same in wild-type controls and in preparations that contain the Asp175Asn mutation. The relative loss of response to a maximal level of ß-adrenergic stimulation may be related to other effects of switching from wild-type TM to -TM 175. For example, the mutation may induce a change in TM flexibility and response to myosin S1 binding.

Two -TM mutations associated with FHC (Asp175Asn and Glu180Gly) occur in a putative TnT binding region. Our results with myofilaments that contain mutant -TM demonstrate an increase in Ca²⁺ sensitivity, with a hypodynamic in vivo cardiac function. The C-terminal half of -TM (specifically the region around amino acid Cys190) is quite flexible in comparison to the N-terminal region of the molecule.^{39 40} In addition, there is a notch in the -helical structure near amino acid residues 169 to 172 in which 2 bulky hydrophobic residues are adjacent.⁴¹ Interestingly, Asp175Asn and Glu180Gly are located in this region and cause an amino acid charge change in the -TM protein. It is likely that these mutations disrupt the already weak TnT-TM interactions in this region and alter the Ca²⁺ sensitivity of the myofilaments. Biochemical analyses of bacterially expressed FHC TM proteins indicate that the mutant TMs have a different structural conformation when interacting with actin in the "on state."⁴² Also, the FHC TM mutant proteins increase the actin filament velocity compared with native -TM in the in vitro motility assay.⁴³ Thus, the increase in the Ca²⁺ sensitivity of the FHC myofilaments could be due to the changes in the cooperative activation of thin filaments by altering the interaction of TM and actin, by affecting the TnT binding to TM, or through both mechanisms.

Numerous studies have demonstrated the importance of posttranscriptional regulation in the control of contractile protein synthesis. Whereas alternative splicing mechanisms are known to regulated tissue- and developmental-specific isoform expression, the importance of translational regulation is being realized more fully. This situation is particularly evident with respect to TM expression. A heterozygous knockout condition results in normal protein levels despite the presence of only a single functional allele.^{20 44} With overexpression of ß-TM in TG mice, translational compensation also occurs by decreasing the expression of -TM to maintain normal protein levels.¹¹ In both this current study, which examines TM expression in cardiac muscle in TG mice, and in an analysis of TM in skeletal muscle from FHC patients by Bottinelli et al,²³ proper stoichiometric protein synthesis is maintained after overexpression of exogenous TM or synthesis of aberrant -TM. Also, TG mice that overexpress wild-type -TM maintain normal levels of TM protein without alterations in structure or function (J.E. Oehlenschlager and D.F.W., unpublished data, 1997). Thus, a feedback mechanism is operative in striated muscle that carefully regulates and maintains proper stoichiometric levels of TM, presumably to ensure regulated sarcomere assembly and function.

	Acknowledgments

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

Received July 24, 1998; accepted April 19, 1999.

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