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(Circulation Research. 2001;88:383.)
© 2001 American Heart Association, Inc.

Integrative Physiology

Comparison of Two Murine Models of Familial Hypertrophic Cardiomyopathy

Bradley K. McConnell¹, Diane Fatkin¹, Christopher Semsarian¹, Karen A. Jones, Dimitrios Georgakopoulos, Colin T. Maguire, Michael J. Healey, James O. Mudd, Ivan P. G. Moskowitz, David A. Conner, Michael Giewat, Hiroko Wakimoto, Charles I. Berul, Frederick J. Schoen, David A. Kass, Christine E. Seidman, J. G. Seidman

From the Cardiovascular Division and Howard Hughes Medical Institute (D.F., C.E.S.), Brigham and Women’s Hospital, Boston, Mass; Department of Genetics (B.K.M., C.S., K.A.J., M.J.H., J.O.M., D.A.C., M.G., J.G.S.), Howard Hughes Medical Institute and Harvard Medical School, Boston Mass; Division of Cardiology (D.G., D.A.K.), Department of Medicine, The Johns Hopkins Medical Institutions, Baltimore, Md; Department of Cardiology (C.T.M., H.W., C.I.B.), Children’s Hospital and Department of Pediatrics, Harvard Medical School Boston, Mass; Department of Pathology (I.P.G.M., F.J.S.), Brigham and Women’s Hospital and Harvard Medical School, Boston, Mass.

Correspondence to Jonathan Seidman, PhD, Department of Genetics, Harvard Medical School, Alpert Bldg, 200 Longwood Ave, Boston, MA 02115. E-mail seidman{at}rascal.med.harvard.edu

	Abstract

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Abstract—Although sarcomere protein gene mutations cause familial hypertrophic cardiomyopathy (FHC), individuals bearing a mutant cardiac myosin binding protein C (MyBP-C) gene usually have a better prognosis than individuals bearing ß-cardiac myosin heavy chain (MHC) gene mutations. Heterozygous mice bearing a cardiac MHC missense mutation (MHC^403/+ or a cardiac MyBP-C mutation (MyBP-C^t/+) were constructed as murine FHC models using homologous recombination in embryonic stem cells. We have compared cardiac structure and function of these mouse strains by several methods to further define mechanisms that determine the severity of FHC. Both strains demonstrated progressive left ventricular (LV) hypertrophy; however, by age 30 weeks, MHC^403/+ mice demonstrated considerably more LV hypertrophy than MyBP-C^t/+ mice. In older heterozygous mice, hypertrophy continued to be more severe in the MHC^403/+ mice than in the MyBP-C^t/+ mice. Consistent with this finding, hearts from 50-week-old MHC^403/+ mice demonstrated increased expression of molecular markers of cardiac hypertrophy, but MyBP-C^t/+ hearts did not demonstrate expression of these molecular markers until the mice were >125 weeks old. Electrophysiological evaluation indicated that MyBP-C^t/+ mice are not as likely to have inducible ventricular tachycardia as MHC^403/+ mice. In addition, cardiac function of MHC^403/+ mice is significantly impaired before the development of LV hypertrophy, whereas cardiac function of MyBP-C^t/+ mice is not impaired even after the development of cardiac hypertrophy. Because these murine FHC models mimic their human counterparts, we propose that similar murine models will be useful for predicting the clinical consequences of other FHC-causing mutations. These data suggest that both electrophysiological and cardiac function studies may enable more definitive risk stratification in FHC patients.

Key Words: cardiomyopathy • hypertrophy • genetics • myosin • cardiac myosin binding protein C

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Familial hypertrophic cardiomyopathy (FHC) is inherited as an autosomal dominant trait that is characterized by myocardial hypertrophy in the absence of identifiable precipitating hemodynamic factors.^{1 2} The diagnostic hallmark of FHC is asymmetric hypertrophy of the interventricular septum; however, the distribution and severity of hypertrophy are variable.^{3 4} Affected individuals may present with symptoms ranging from dizziness and palpitations to sudden death. Molecular genetic studies have demonstrated that FHC is genetically heterogeneous; 10 disease genes have been identified.^{2 5} All 10 of these genes encode sarcomere proteins expressed in cardiac muscle. We and others have demonstrated that individuals bearing some sarcomere protein gene mutations have better prognoses than individuals bearing other mutations,^{6 7 8} but the basis for this correlation has not been elucidated.

To further investigate factors determining the clinical response to a sarcomere protein gene mutation, we have created two strains of mice, using homologous recombination in embryonic stem cells that bear precise analogues of FHC-causing mutations. Individuals bearing the ß-cardiac myosin heavy chain (MHC) gene Arg403Gln missense mutation have an earlier disease onset and a shorter life expectancy than individuals bearing mutations in the cardiac myosin binding protein C (MyBP-C) gene.^{7 8} Transgenic mice expressing cDNAs encoding mutant forms of both MyBP-C and MHC have been produced previously.^{9 10} These mice are of limited value for directly comparing the consequences of FHC-causing mutations because the amount and temporal pattern of mutant cDNA expression, even when controlled by a cardiac specific promoter, varies even between different strains of mice bearing the same mutant transgene. We have previously created a mouse bearing the Arg403Gln mutation in one allele of the -cardiac MHC gene, the murine analogue of the human ß-cardiac MHC gene.¹¹ Heterozygous Arg403Gln -cardiac MHC (MHC^403/+) mutant mice have been shown to develop physiological and histological features typical of human FHC.^{11 12 13} Recently, we created a strain of mice bearing a neomycin resistance gene inserted in the cardiac MyBP-C gene.¹⁴ This mutant allele encodes a truncated MyBP-C protein that closely resembles the truncated MyBP-C protein that causes FHC in some individuals (Family NN in Figure 1A and Reference14¹⁴ ); heterozygous mice bearing the mutant allele were designated MyBP-C^t/+ mice. Although we have previously demonstrated that both homozygous MHC^403/403 and MyBP-C^t/t mice produce mutant polypeptides and develop dilated cardiomyopathy, the cardiac phenotype of older heterozygous mice bearing these mutations has not been compared.

View larger version (38K): [in this window] [in a new window]   Figure 1. Structures of three MyBP-C alleles. A, Schematic of exons 29 to 32 of the wild-type, MyBP-C(Neo), and family NN alleles, the RNAs produced by each allele, and the structure of the carboxyl ends of MyBP-C protein encoded by the wild-type (WT), MyBP-C(Neo), and NN alleles. The MyBP-C(Neo) allele was created by inserting the neomycin resistance gene (PGK-Neo-polyA gene) into exon 3014 whereas the defective MyBP-C gene found in family NN contains mutation in the RNA splice donor sequence of exon 30. The black lines above each gene segment reflect the structure of the encoded RNA indicated in the 5'' orientation; thick lines are exons incorporated into the RNA, thin lines indicate skipped segments of the gene not found in RNA. Amino acid residues 1064 to 1111 of the wild-type protein are encoded by exon 30. Novel amino acid residues at the carboxyl end of the mutant protein (bold) are encoded by altered reading of exon 31. The carboxyl end of the analogous human mutation found in family NN28 is shown for comparison. Differences between the family NN protein and the MyBP-C(Neo) protein reflect sequence differences between the mouse and human MyBP-C gene exon 31. B, Northern blot analyses of MyBP-C and GAPDH RNAs in LV tissue of wild-type (+/+) and MyBP-Ct/+ (t/+) mice. C, Western blot analyses of total protein extracts from left ventricles of wild-type (+/+) and MyBP-Ct/+ (t/+) mice. Western blot analyses identified the 150-kDa MyBP-C protein in the left ventricles of mutant and wild-type mice.

We have characterized cardiac structure and function of MHC^403/+ and MyBP-C^t/+ mice at several ages to define the development of hypertrophic cardiomyopathy in these mice. We demonstrate in the present study that both mutations cause cardiac hypertrophy. However, MHC^403/+ mice develop hypertrophy much earlier than MyBP-C^t/+ mice consistent with the pathologies observed in humans bearing these mutations. In addition, we demonstrate that hearts bearing truncated MyBP-C polypeptide have normal cardiac function and do not demonstrate inducible ventricular tachycardia whereas age-matched hearts bearing -cardiac MHC Arg403Gln missense polypeptide have significant deficits in cardiac and electrophysiological function.^{12 15} We speculate that deficits in cardiac function, rather than increased cardiac hypertrophy, are responsible for the more severe symptoms observed in individuals with the Arg403Gln missense mutation.

	Materials and Methods

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Animals
Mice bearing the MyBP-C(Neo) allele were generated using homologous recombination techniques and a targeting construct containing exons 28 to 32 of the murine cardiac MyBP-C gene with a 2-kb neomycin gene insertion in exon 30 as described.¹⁴ Heterozygous mice bearing the MyBP-C(Neo) allele are designated MyBP-C^t/+. The structure of mutant-derived MyBP-C RNA was defined by reverse transcription–polymerase chain reaction (RT-PCR) amplification and DNA sequencing of RNA derived from homozygous mice bearing the MyBP-C(Neo) allele.¹⁴ Mouse genotypes were determined by PCR amplification or restriction enzyme digestion of tail DNA from each animal.^{11 14} MHC^403/+ mice were generated as described.¹¹ Both strains of mice were bred and maintained on the 129SvEv genetic background. Electrophysiological analyses were performed on mice bearing these mutations on a 129/BS genetic background. All mice were maintained according to protocols approved by the Institutional Animal Care and Use Committee of Harvard Medical School.

Cardiac Physiology and Pathology
Cardiac tissues from male mice were subjected to histological examination using methods described previously.^{11 16} A single experienced pathologist who was unaware of the mouse genotype reviewed all histological specimens.

Echocardiographic studies were performed on male mice using a 12-MHz linear array probe with a Sonos 5500 ultrasonograph (Hewlett-Packard) as described.¹⁴ Left ventricular (LV) end-diastolic (LVDD) and end-systolic (LVSD) chamber dimensions and wall thickness were obtained from M-mode tracings using measurements averaged from 3 separate cardiac cycles. LV fractional shortening (%) was derived as follows: (LVDD-LVSD)/LVDDx100. A single observer, who did not know the mouse’s genotype, made all echocardiographic measurements. Heart rates were determined from electrocardiographic recordings performed during echocardiography.

LV hemodynamic studies were performed in male mice as described previously.^{12 17} In brief, anesthetized mice were intubated, artificially ventilated, and real-time LV pressure-volume relationships were measured using a newly developed miniaturized impedance/micromanometer catheter (Millar Instruments). Aortic flow was measured by an ultrasound perivascular probe (Transonics, 1RB) placed around the thoracic aorta. Pressure-volume signals were recorded at steady state and during transient reduction of cardiac preload achieved by inferior vena caval occlusion. Data were digitized at 2 kHz for subsequent analysis.

Surface resting electrocardiograms and electrophysiological studies were performed in anesthetized male wild-type and MHC^403/+ mice as described.^{15 18} Standard procedures for pacing and extra-stimulus testing were used to assess baseline conduction parameters and arrhythmia induction.^{15 18} The PR, QRS, RR, and QT intervals were measured in 6 surface limb ECG leads by two independent observers who were blinded to mouse genotype. Inducible ventricular tachycardia was assessed as described previously.^{18 19}

RNA and Protein Analyses
Northern blot analyses were performed as described previously.^{14 20} Total RNA was isolated from the left ventricle using Trizol (Gibco BRL) and analyzed by standard Northern blot procedures.¹⁴ MyBP-C RNA was detected using ³²P-labeled insert from a 2.4-kb mouse MyBP-C cDNA plasmid clone designated pcMyBPC. The MyBP-C cDNA probe consisted of a 1584-bp segment encoding amino acid residues 582 to 1110.¹⁴ Other RNAs were detected using 5'-³²P-labeled oligonucleotide probes and hybridized to nylon membranes using standard hybridization conditions.¹⁴ The oligonucleotides used as transcript specific probes were as follows:

atrial natriuretic factor: 5'-AATGTGACCAAGCTGCGTGACACACCACAAGGGCTTAGGATCTTTTGC-3'

brain natriuretic factor: 5'-CAGCTTGAGATATGTGTCACCTTGGAATTTTGAGGTCTC-TGCTGGACC-3'

-skeletal actin: 5'-TGGCTTTAATGCTTCAAGTTTTCCATTTCCTTTCCACAG-GG-3'

GAPDH: 5'-GGAACATGTAGACCATGTAGTTGAGGTCAA-TAAG-3'

The hybridization signal for each oligonucleotide probe was quantified using ImageQuant software (Molecular Dynamics) and normalized to the signal intensity observed with an oligonucleotide specific for GAPDH RNA. MyBP-C polypeptides were identified by Western blot analyses using antibody raised against chicken cardiac MyBP-C.¹⁴

Statistical Analysis
The statistical significance of differences between groups of wild-type, MyBP-C^t/+, and MHC^403/+ mice in continuous variables was determined by one-factor ANOVA and the unpaired Student’s t test. Differences in categorical variables were assessed with the ² test. Data are expressed as mean±SD. A value of P<0.05 was considered significant.

	Results

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Expression and Structure of RNAs and Proteins Expressed in MyBP-C^t/+ and MHC^403/+ Hearts
MyBP-C mRNA expressed from the MyBP-C(Neo) allele was characterized by Northern blot analyses of heterozygous MyBP-C^t/+ cardiac RNA (Figures 1A and 1B). The 4.5-kb MyBP-C RNA transcript was found in the left ventricle of both wild-type and MyBP-C^t/+ mice; however, MyBP-C mRNA expression in MyBP-C^t/+ mice (n=6) was 49.2±16.6% the level of expression in wild-type mice (n=6). MyBP-C (150 kDa) protein was identified in myofibrillar extracts from cardiac left ventricles of wild-type and MyBP-C^t/+ mice. MyBP-C (mutant and wild type) protein expression in MyBP-C^t/+ left ventricles (n=4) was 89.9±8.5% the level found in wild-type left ventricles. Because the mutant MyBP-C(Neo) allele produces a reduced amount (10% of wild type) of truncated MyBP-C peptide,¹⁴ we hypothesize that the heterozygous MyBP-C^t/+ hearts contain 90% wild-type MyBP-C and 10% truncated peptide.

The structure of the carboxyl end of the MyBP-C polypeptide encoded by the MyBP-C(Neo) allele was characterized by nucleotide sequence analyses of RT-PCR–amplified products derived from RNA found in the left ventricle of homozygous MyBP-C^t/t mice (Figure 1A and Reference14¹⁴ ). MHC^403/+ left ventricles contain equivalent amounts of mutant and wild-type -cardiac MHC mRNA and protein (our unpublished results).

Myocardial Histopathology
Previous studies¹¹ demonstrated that hearts from 15-week-old MHC^403/+ mouse hearts have mild myocyte hypertrophy, interstitial fibrosis, and myofibrillar disarray that become much more severe by 30 to 50 weeks of age. No histological abnormalities were observed in 50-week-old MyBP-C^t/+ mouse hearts. Histological sections obtained from >125-week-old MyBP-C^t/+ mouse hearts demonstrated myocyte hypertrophy, interstitial fibrosis, and myofibrillar disarray in 50% of mutant animals. However, similar histological abnormalities were observed in a comparable proportion of hearts from age-matched wild-type mice (Figure 2 and data not shown).

View larger version (154K): [in this window] [in a new window]   Figure 2. Myocardial histology in >125 weeks MyBP-Ct/+ (top) and age-matched wild-type (bottom) mice. Myocyte hypertrophy, disarray, and fibrosis are present in the left ventricle of both mouse hearts. A wide range of histological findings was observed within groups of MyBP-Ct/+ and wild-type mice; the mouse hearts shown have similar amounts of disarray, fibrosis, and hypertrophy. Sections were stained with H&E. Magnification x200.

Cardiac Morphology of Mutant and Wild-Type Mice
Cardiac hypertrophy in mutant and wild-type mice was assessed using transthoracic echocardiography (Table 1). Neither 10- to 20-week-old male MyBP-C^t/+ or MHC^403/+ mouse hearts demonstrated significant differences from age-matched wild-type mouse hearts (Table 1). By 30 to 50 weeks, both MyBP-C^t/+ mice and MHC^403/+ mice were beginning to demonstrate signs of their mutations. MyBP-C^t/+ mice displayed significantly enlarged atria and a slight increase in LV wall thickness compared with wild-type mice (0.96±0.08 versus 0.87±0.05 mm; P<0.05). However, MHC^403/+ mice demonstrated significantly increased left atrial dimensions and much greater LV wall thickness than either MyBP-C^t/+ or wild-type mice (compare left ventricular anterior wall thickness [LVAW] of MHC^403/+ mice, 1.12±0.07 mm versus LVAW of wild-type, 0.87±0.06 mm, and MyBP-C^t/+, 0.96±0.08 mm, mice; P<0.05; Table 1). In addition, only 32% (6 of 19) of the MyBP-C^t/+ mice had developed cardiac hypertrophy (>1.0 mm) by 50 weeks of age (Figure 3) compared with 92% (11 of 12) MHC^403/+ mice. However, LVAW of >125-week-old MyBP-C^t/+ mice was significantly greater than age-matched wild-type mice (1.39±0.10 versus 1.08±0.05 mm, P<0.001; Table 1), demonstrating that cardiac hypertrophy is late onset in heterozygous MyBP-C mutant mice. The fractional shortening was not significantly different in MyBP-C^t/+ mice (55±4%) compared with age-matched wild-type mice (52±5%) at >125 weeks. Heart rates during echocardiographic studies were similar in all groups of mice (Table 1 and data not shown). (The different heart rates of age- and strain-matched mice reported in Tables 1 and 2 are due to differences in anesthesia used in different studies.)

View this table: [in this window] [in a new window]   Table 1. Echocardiographic Characteristics of Wild-Type, MyBP-Ct/+, and MHC403/+ Mice Aged 10 to 20, 30 to 50, and >125 Weeks

View larger version (15K): [in this window] [in a new window]   Figure 3. LVAW of age-matched MyBP-Ct/+ and MHC403/+ mice. Wall thickness was evaluated in wild-type, MyBP-Ct/+, and MHC403/+ mice at 10 to 20 and 30 to 50 weeks of age and wild-type and MyBP-Ct/+ mice at >125 weeks of age using transthoracic echocardiography. LVAW >1.0 mm, in mice 10 to 50 weeks of age, is considered to be hypertrophied. , Wild-type mice; •, MHC403/+ mice; and , MyBP-Ct/+ mice.

View this table: [in this window] [in a new window]   Table 2. Left Ventricular Function of Wild-Type, MyBP-Ct/+, and MHC403/+ Mice Aged 30 to 50 Weeks and Wild-Type and MyBP-Ct/+ Mice Aged >125 Weeks

LV Pressure-Volume Analyses
Previous studies have demonstrated that 8- to 20-week-old MHC^403/+ mice have impaired cardiac function compared with age-matched wild-type mice.¹² We demonstrate in the present study that 30- to 50-week-old MHC^403/+ mice, like 20-week-old MHC^403/+ mice, exhibited altered LV diastolic kinetics with delayed pressure relaxation and chamber filling (Table 2). Thirty to 50-week-old MHC^403/+ mice also had altered elevated LV systolic pressure (Table 2).

Cardiac function of wild-type and MyBP-C^t/+ mice at 30 to 50 weeks and >125 weeks were assessed by in vivo cardiac catheterization (Figure 4). Thirty to 50-week-old MyBP-C^t/+ mice had normal systolic and diastolic LV function when compared with age-matched wild-type mice (Table 2 and data not shown). Similarly, cardiac function of >125-week-old MyBP-C^t/+ mice was indistinguishable from that of age-matched wild-type mice but significantly better than cardiac function of 30- to 50-week-old MHC^403/+ mice (Figure 4, Table 2, and data not shown).

View larger version (30K): [in this window] [in a new window]   Figure 4. LV pressure-volume relations and the first derivative of ventricular pressure (dP/dt) in 125-week wild-type (A and D), 125-week MyBP-Ct/+ mice (B and E), and MHC403/+ mice (C and F) aged 30 to 50 weeks.

Electrophysiology
Baseline recordings in 30- to >125-week-old MyBP-C^t/+ mice showed the same electrocardiographic interval conduction times and axes as observed in age-matched wild-type mice (data not shown). Neither MyBP-C^t/+ nor MHC^403/+ mice exhibited PR prolongation, AV block, or abnormally long atrioventricular conduction block coupling intervals. Normal atrial and ventricular conduction parameters and refractory periods were also demonstrated in both strains of mice (data not shown and Reference 20²⁰ ). Using a standard murine pacing and programmed electrical stimulation protocol,^{15 18 19} inducible arrhythmias were elicited in significantly more (8 of 15) 30- to 50-week-old MHC^403/+ mice than age-matched wild-type (0 of 10) or MyBP-C^t/+ (1 of 9) mice. Similar proportions of >125-week-old MyBP-C^t/+ (2 of 6) and wild-type (2 of 4) mice demonstrated inducible arrhythmias.

RNA Expression Associated With Cardiomyopathy
Atrial natriuretic factor, brain natriuretic factor, and -skeletal actin mRNAs, which are induced in other models of cardiac hypertrophy, were measured in mutant mice at 10 to 20, 30 to 50, and >125 weeks of age by Northern blot analyses. The amounts of atrial natriuretic factor, brain natriuretic factor, and -skeletal actin RNA transcripts in 10- to 20-week-old MHC^403/+ and MyBP-C^t/+ hearts were the same as in age-matched wild-type hearts (data not shown). However, by 50 weeks, MHC^403/+ left ventricles demonstrated increases in atrial natriuretic factor (4.9±1.0-fold), brain natriuretic factor (2.5±0.3-fold), and -skeletal actin (1.9±0.2-fold) RNAs compared with 50-week-old wild-type left ventricles (Figure 5). Left ventricles from 50-week-old MyBP-C^t/+ mice had the same amounts of these RNAs as left ventricles from age-matched wild-type mice. However, left ventricles of >125-week-old MyBP-C^t/+ mice demonstrated significant increases in atrial natriuretic factor (3.1±0.4-fold), brain natriuretic factor (2.3±0.2-fold), and -skeletal actin (3.3±0.7-fold) RNAs compared with left ventricles from age-matched wild-type hearts (Figure 5).

View larger version (96K): [in this window] [in a new window]   Figure 5. RNA expression in hearts from MHC403/+ (40 to 50 weeks) and MyBP-Ct/+ (>125 weeks) mice compared with expression in age-matched wild-type hearts. Northern blot shows increased expression of atrial natriuretic factor (ANF), brain natriuretic factor (BNP), and -skeletal actin (-sk-actin) at >125 weeks of age. Signals were normalized based on hybridization to GAPDH.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We demonstrate that both MHC^403/+ and MyBP-C^t/+ mice, two strains of mice bearing different FHC-causing mutations, develop LV hypertrophy, but age-matched MHC^403/+ mice have much more LV hypertrophy than MyBP-C^t/+ mice. Mice bearing the Arg403Gln missense mutation in the -cardiac MHC gene have impaired cardiac function (Figure 4 and References 11 and 12^{11 12} ) and some have electrophysiological abnormalities. However, no difference in cardiac function of mice expressing the truncated cardiac MyBP-C gene and wild-type mice was detected (Figure 4 and Table 2) and neither demonstrated inducible arrhythmias. These observations may explain the different clinical features observed in humans bearing different sarcomere protein gene mutations. Although the mechanisms by which these mutations cause hypertrophy remain uncertain, some insights into this process can be gained by considering the effects of these different mutations on cardiac function and LV morphology.

Both heterozygous MHC^403/+ and MyBP-C^t/+ mice develop LV hypertrophy, whereas homozygous MHC^403/+ and MyBP-C^t/t mice develop dilated cardiomyopathy.^{14 16} Homozygous MHC^403/+ mice develop severe dilated cardiomyopathy and die by day 10 after birth¹⁶ whereas MyBP-C^t/t mice develop a milder dilated cardiomyopathy and survive for up to 2 years.¹⁴ Thus, the severity of dilated cardiomyopathy in homozygous mice bearing sarcomere protein mutations paralleled the severity of hypertrophic cardiomyopathy observed in heterozygous mice bearing the same mutations. Perhaps the signal that determines the severity of dilated cardiomyopathy in homozygous mutant mice is the same signal that determines the severity of LV hypertrophy in heterozygous mutant mice.

Young MHC^403/+ mice have cardiac dysfunction¹² and this dysfunction is also found in older MHC^403/+ mice (Table 2). Thus, cardiac dysfunction precedes cardiac hypertrophy in these mice (Table 1 and Figures 3 and 4). We conclude that cardiac dysfunction is not secondary to the cardiac hypertrophy, myocyte disarray, myocyte hypertrophy, and fibrosis observed in older MHC^403/+ mice. Previous studies^{21 22 23} have suggested that sarcomere dysfunction per se can lead to cardiac hypertrophy. Are the findings demonstrated in the present study consistent with this model? MHC^403/+ mice who develop cardiac hypertrophy by age 30 weeks (Table 1) have defective sarcomere function^{24 25} and significant cardiac dysfunction^{11 12 13} (see Table 2). However, cardiac function of the MyBP-C^t/+ mice (Table 2 and Figure 4) appears normal, and we anticipate that sarcomere dysfunction will be minimal. These animals do not develop cardiac hypertrophy until much later in life. Two hypotheses can be proposed: (1) that cardiac hypertrophy arises in MyBP-C^t/+ mice in the absence of cardiac dysfunction and (2) that MyBP-C^t/+ mice have very mild cardiac dysfunction that is not detectable by the methods used in the present study (see Materials and Methods). We strongly favor the latter model. We have demonstrated that these mice express a truncated cardiac MyBP-C in their myocardium and that homozygous mice expressing this mutation have significant cardiac dysfunction. Therefore, we suggest that cardiac dysfunction induces hypertrophy in these FHC models.

Electrophysiological abnormalities might contribute to the more severe disease process observed in MHC^403/+ mice. However, we do not know whether these abnormalities precede cardiac hypertrophy and its associated histopathological changes. Characterization of MyBP-C^t/+ mice suggests that cardiac hypertrophy can occur in mice without causing electrophysiological abnormalities because >125-week-old mutant animals were no more susceptible to inducible arrhythmias than >125-week-old wild-type mice. Previous studies of individuals with hypertrophic cardiomyopathy have suggested that electrophysiological abnormalities are not a good prognostic indicator in this disease.^{26 27} The findings reported in the present study suggest that some FHC-causing mutations cause more electrophysiological abnormalities than other FHC-causing mutations. Why one sarcomere gene mutation causes such abnormalities and not another sarcomere gene mutation remains uncertain.

The prognostic value derived from identification of FHC-causing mutations has been debated for the past several years.² Physicians have recognized for the past two decades that some FHC-causing mutations cause more severe disease than other mutations. However, when a new mutation is identified in an individual, predicting whether this mutation will cause severe or mild disease in other family members is difficult. Characterization of murine FHC models suggests that one approach to this problem may be to introduce mutations into otherwise genetically identical mice and then study the phenotype of these murine models. We suggest that the strong correlation between murine phenotypes and clinical features observed in humans bearing analogous FHC-causing mutations will be observed in mice bearing other FHC-causing mutations. Further production and evaluation of other mouse strains bearing FHC-causing mutations should help to define the predictive value of these murine FHC models. Eventually, characterization of other murine FHC models will provide important prognostic information to patients and physicians. Another approach to the problem of predicting the severity of an FHC-causing mutation in humans is suggested by the results in the present study. Based on the two murine models, mutations that cause significant deficits in cardiac function cause severe disease whereas mutations that cause less impairment of cardiac function cause milder disease. Cardiac function of genetically affected family members should provide a good indicator of the clinical consequences of an FHC-causing mutation.

	Acknowledgments

 
The Howard Hughes Medical Institute supported these studies. J.M. and M.H. were recipients of Sarnoff Foundation fellowships. C.S. and C.B. received National Heart Foundation of Australia and National Institutes of Health Grant K08-03607, respectively.

	Footnotes

 
Original received October 23, 2000; resubmission received January 3, 2001; accepted January 23, 2001.

¹ These authors contributed equally to this work.

This manuscript was sent to James T. Willerson, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

	References

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