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Am J Physiol Heart Circ Physiol 294: H2701-H2711, 2008. First published April 18, 2008; doi:10.1152/ajpheart.00160.2007
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A novel mouse model of X-linked cardiac hypertrophy

L. Leatherbury,1,4 Q. Yu,1 B. Chatterjee,1 D. L. Walker,1 Z. Yu,2 X. Tian,3 and C. W. Lo1

1Laboratory of Developmental Biology, 2Pathology Core, and 3Office of Biostatistics Research, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland; and 4Pediatric Cardiology, Children's National Medical Center, Washington, District of Columbia

Submitted 7 February 2007 ; accepted in final form 15 April 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We recovered a novel mouse mutant exhibiting neonatal lethality associated with severe fetal cardiac hypertrophy and with some adult mice dying suddenly with left ventricular hypertrophic cardiomyopathy. Using Doppler echocardiography, we screened surviving adult mice in this mutant line for cardiac hypertrophy. Cardiac dimensions were obtained either from two-dimensional images collected using a novel ECG-gated ultra-high-frequency ultrasound system or by traditional M-mode imaging on a clinical ultrasound system. These analyses identified, among the littermates, two populations of mice: those with apparent cardiac hypertrophy with hypercontractile function characterized by ejection fraction of 75–80%, and normal littermates with ejection fraction of 53–55%. Analysis of the ECG-gated two-dimensional cines indicated that the hypertrophy was of the nonobstructive type. Further analysis of heart-to-body weight ratio confirmed the ultrasound diagnosis of left ventricular hypertrophic cardiomyopathy. Histopathology showed increased ventricular wall thickness, enlarged myocyte size, and mild myofiber disarray. Ultrastructural analysis by electron microscopy revealed mitochondria hyperproliferation and dilated sarcoplasmic reticulum. Genome scanning using microsatellite DNA markers mapped the mutation to the X chromosome. DNA sequencing showed no mutations in the coding regions of several candidate genes on the X chromosome, including several known to be associated with left ventricular hypertrophic cardiomyopathy. These findings suggest that this mouse line may harbor a mutation in a novel gene causing X-linked cardiomyopathy.

echocardiography; X-linked cardiomyopathy; hypertrophic cardiomyopathy; ultrasound

HYPERTROPHIC CARDIOMYOPATHY (HCM) is a major cardiovascular disease in human adults and children (17, 22), with a prevalence of 1:500 in the young and likely higher in older individuals due to age-dependent penetrance (23). It has varied presentations, from no symptoms to severe heart failure or sudden cardiac death, that do not correlate directly with the anatomic abnormality. It is a major cause of sudden cardiac death in apparently healthy, young individuals (24). Infants that present with cardiac hypertrophy may present with a murmur, but some also have severe congestive heart failure and a high mortality (38, 42). In children and adults, HCM may also be a major cause of morbidity and mortality (25, 28, 32).

HCM is characterized by thickening of the ventricular heart muscle peculiar to the ventricular myocardium and not secondary to another defect, such as aortic valve disease or systemic hypertension. There are clinical subcategories, with 25% of human adults having hypertrophic obstructive cardiomyopathy with asymmetric hypertrophy of the interventricular septum. This is usually associated with obstructive flow from the left ventricular (LV) outflow tract (LVOT). However, HCM is predominantly a nonobstructive disease, with 75% of patients showing no significant resting outflow gradient (22). These hearts exhibit symmetric or concentric hypertrophy associated with the interventricular septum and LV.

Over a dozen genes and 200 mutations have been identified in contractile sarcomeric proteins in association with HCM (21). However, it is noteworthy that cardiac hypertrophy also can arise from storage diseases. These exhibit similar echo presentations, but have different histological characteristics and may show ventricular preexcitation (1). Transgenic and knockout mouse models with mutations in genes encoding sarcomeric proteins have demonstrated parallels with human clinical disease (7, 12, 20). In addition, mouse models for human cardiomyopathies involving fatty acid oxidation or carnitine metabolic disorders also have been generated (11, 15, 26, 41). These studies show mouse models may be invaluable for elucidating the disease mechanism and progressive pathology associated with LV HCM.

As part of a high throughput ultrasound screen for mutations causing congenital heart defects in ethyl nitrosourea (ENU) mutagenized mice (46), we recovered a mutant mouse line, referred to as Family 78, exhibiting ventricular hypertrophy. Some of the offspring in this mutant line exhibited neonatal lethality associated with severe cardiac hypertrophy, while some surviving adult mice also exhibited LV hypertrophy (LVH) cardiomyopathy associated with sudden death. Using a combination of conventional echocardiography and ECG-gated ultrasound imaging, we assessed cardiac structure and function in adult mice from this mutant mouse line. Our studies showed hypercontractile function associated with cardiac hypertrophy of the nonobstructive type. Further analysis by histopathology and electron microscopy (EM) showed the LVH cardiomyopathy was characterized by mild myofiber disarray and mitochondrial hyperproliferation. Genome scanning using microsatellite markers mapped the mutation to the X chromosome. This new mouse model may harbor a novel mutation causing cardiac hypertrophy and will be invaluable for studying X-linked LVH cardiomyopathy.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mouse breeding and line maintenance. The mutant mouse line, Family 78, was recovered during an ENU mutagenesis screen (46). The ENU mutagenized mice were from a C57BL6/J in background, but were subsequently intercrossed into the C3H/Hej strain background to map the mutation. Ultrasound interrogations were carried out using five litters of all surviving 7-mo-old adult mice: two with a clinical ultrasound system, and three with an ultra-high-frequency-gated biomicroscope. All of the animal studies have been approved by the National Heart, Lung, and Blood Institute Animal Care and Use Committee.

Mouse echocardiography. Adult mice were ultrasound scanned, while sedated with 1.5% isoflurane, by two blinded pediatric cardiologists. The anesthetized animal was placed on a heating pad, hair on the thorax was removed with depilatory cream, and prewarmed ultrasonic gel was applied. Body temperature was tracked using a rectal probe, and ECG leads were taped to the paws for monitoring the heart rate and for ECG gating. The body temperature was maintained at 37 ± 1°C, and the heart rate at 400–550 beats/min. Ultrasound imaging was conducted using the clinical Acuson Sequoia C256 or Visualsonics Vevo 660 biomicroscope ultrasound systems. The Acuson Sequoia C256 system was used with a 15-MHz L8 phased linear array transducer with an imaging depth of 8–20 mm without a standoff. Two-dimensional (2D) imaging was done at 440-µm axial and 630-µm lateral resolution at 30 Hz. 2D cine pulse repetition frequency was between 75 and 105 Hz. The color Doppler preset was at a Nyquist limit of 0.34 m/s and a spectral Doppler sweep of 200 mm/s for a run of 8–12 s. The long-axis and five chamber views were used for 2D and color flow imaging and spectral Doppler interrogation of the aortic outflow tract. Short-axis M-mode tracings were obtained using the left anterior axillary line and obtained at 1,000 Hz at 150 mm/s for 6 s. M-mode measurements were carried out using the leading edge technique of the American Society for Echocardiography (13).

ECG-gated ultrasound imaging was carried out on the Visualsonics Vevo 660 ultrasound system using a 30-MHz mechanical sector transducer. The field of view is 12 mm2, with an ideal imaging depth of 12.5 mm without a standoff. In nongated mode, 2D imaging provided axial resolution of 30 µm and lateral resolution of 62 µm at 30 Hz. For ECG-gated imaging, the transducer was attached to a fixed arm, and, over 6 min, ECG data and M-mode images were collected and then processed for reconstruction of 2D cine spanning one cardiac cycle. The same short-axis view used for M-mode imaging was used to collect data for ECG-gated imaging. This provided 2D images of the LV at its maximal diameter just below the mitral valve and at the level of the papillary muscles. The ultrasound measurements were statistically analyzed using the two-sample t-test for equality of means without equal variances.

Histopathology, EM, and immunohistochemistry. After ultrasound scanning, the mice were euthanized, and their heart and body weights were obtained. To ensure the hearts arrested at diastole, the hearts were infused with KCl before fixation in 10% formalin. Standard histopathology, or analysis by episcopic fluorescence image capture (EFIC), was carried out as previously described (35). EFIC imaging on the chest was used to generate three-dimensional (3D) reconstructions to examine for cardiac hypertrophy. For three LVH and four normal hearts, serial paraffin sections were cut and mounted with three sections on each slide, resulting in 50 slides, or, at a minimum, 150 sections per heart. Every 10th slide was stained with hematoxylin and eosin, periodic acid Schiff, and Mason's trichrome. Myocyte hypertrophy was quantitated by measuring the diameter of 30 randomly selected longitudinal sectioned myocytes at the nuclear level from the histological sections. For EM, hearts were fixed in 2.5% gluteraldehyde and 1% osmium tetraoxide and embedded in Epon resin, and the ultrathin sections were poststained with uranyl acetate and citrate lead.

Immunohistochemistry was performed using indirect immunoperoxidase staining, which was also done in an additional three LVH and four normal hearts. The staining was done in triplicate to ensure consistent results. Negative controls were obtained by omission of the primary antibody, and these gave only low background staining. Primary antibodies used are as follows: rabbit anti-atrial natriuretic factor (ANF) (natriuretic peptide precursor type A, Peninsula Laboratories, San Carlos, CA); rabbit anti-carnitine palmitoyl transferase-1a, muscle (Alpha Diagnostic, San Antonio, TX); mouse anti-acyl-coenzyme A dehydrogenase, long chain (Novus Biologicals, Littleton, CO); mouse anti-calsequestrin 2, cardiac muscle (Novus); rabbit anti-peroxisome proliferator-activated receptor-{gamma} coactivator 1 (PGC-1) (Chemicon, Temecula, CA); and mouse anti-sarco(endo)plasmic reticulum Ca2+-ATPase 2 (SERCA2) ATPase (Novus Biologicals, Littleton, CO). Briefly, deparaffinized sections were treated by microwaving in citrate buffer (Dako, Carpenteria, CA) for 10 min at 1,000 W. Endogenous peroxidase activity was quenched with 0.6% hydrogen peroxide in methanol for 20 min at room temperature, and nonspecific binding of IgG was suppressed by treatment with 5% normal horse or goat serum for 20 min. The sections were then incubated with the primary antibodies overnight at 4°C. After washing with phosphate-buffered saline, the sections were incubated with biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) for 1 h and avidin-biotinylated horseradish peroxidase (ABC kit, Vector) for 30 min. The color of the peroxidase reaction was developed with diaminobenzidine tetrahydrochloride (Vector) for 5 min. The sections were counterstained with hematoxylin. Negative control immunohistochemical procedure was obtained by omission of the primary antibody and showed the negative results.

Genome scan analysis. To map the mutation in this family, DNA collected from B6/C3H hybrid mice ultrasound diagnosed as having HCM were PCR amplified using primers for 48 B6/C3H polymorphic microsatellite markers (46). The resultant PCR products were pooled and separated by capillary electrophoresis on the Avant 3100 Genetic Analyzer (Applied Biosystems), and the data generated were analyzed using recombinant interval haplotype analysis (31).


   RESULTS
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Family 78 was initially identified from phenotyping conducted using in utero fetal echocardiography, which showed some fetuses with cardiac hypertrophy. Such fetuses died at birth and were confirmed by necropsy and histology to have severe cardiac hypertrophy (Fig. 1, A and B). Some adult mice in this mutant line also died suddenly and were found to have concentric LVH (Fig. 1, C and E; compare with age-matched adult heart in Fig. 1, D and F). These phenotypes were heritable through the germ line, with severe cardiac hypertrophy associated with neonatal lethality, while some surviving adult animals also showed characteristics of HCM.


Figure 1
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  		Fig. 1. Neonatal and adult mice show left ventricular (LV) hypertrophy (LVH) cardiomyopathy. A: episcopic fluorescence image capture (EFIC) histology sections show markedly concentrically thickened LV walls associated with a newborn mouse from Family 78 that died at birth. B: for comparison is a section from a normal newborn mouse. C: a 7-mo-old mouse from Family 78 ultrasound diagnosed with LVH cardiomyopathy was necropsied after ultrasound scanning and shown to have an enlarged heart. D: the heart from a normal littermate. EFIC short-axis sections are from this LVH cardiomyopathy heart (E) and the littermate normal heart (F). The interventricular septum (white lines) and the LV free wall are concentrically thickened in the nonobstructive LVH cardiomyopathy heart. P, LV papillary muscles. Scale bars: 0.5 mm (A and B), 1.5 mm (C and D), and 0.8 mm (E and F).

 
Echocardiography. To further analyze the cardiac phenotype in this mutant mouse line, we used echocardiography to screen and identify adult mice with concentric LVH cardiomyopathy. This was conducted by comparing a new ultra-high-frequency ultrasound system equipped with ECG-gated 2D imaging (Fig. 2), or a conventional clinical ultrasound system (Fig. 3). Similar results were obtained using either ultrasound system verifying that the novel ECG-gated 2D imaging data were accurate compared with the previous gold standard of echo measurements by M-mode (Table 1 and 2). Both echocardiographic systems showed two distinct groups of mice in Family 78: one group with increased ventricular wall thickness, decreased cavity size, and increased contractility indexes diagnostic of LVH cardiomyopathy; and another group that are presumed to be normal littermates (Tables 1 and 2).


Figure 2
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  		Fig. 2. Gated two-dimensional (2D) images in long-axis views show nonobstructive LVH cardiomyopathy. Gated 2D images in the long-axis view at end diastole (A) and end systole (B) show concentrically thickened LV walls, hypertrophied papillary muscles, and decreased chamber size, indicative of LVH cardiomyopathy (a = 0.99 mm, b = 4.04 mm, c = 1.08 mm, d = 1.22 mm, e = 3.00 mm, f = 1.03 mm). C: in comparison, note thinner wall and barely detectable papillary muscles seen in long-axis view of a normal littermate (in diastole) (a = 0.52 mm, b = 3.04 mm, c = 0.60 mm). Quantitative measurements in the short-axis view obtained with M-mode imaging (D) for the mutant animal confirmed concentric LVH (a = 1.09 mm, b = 2.50 mm, c = 0.94 mm, d = 1.16 mm, e = 0.94 mm, f = 1.05 mm), compared with similar measurements obtained by ECG-gated 2D imaging (E and F) in the same short-axis view for the normal littermate (a = 1.03 mm, b = 3.09 mm, c = 0.80 mm, d = 1.00 mm, e = 2.77 mm, f = 1.00 mm). Measurements are in end diastole (a, b, c) vs. end systole (d, e, f).

 

Figure 3
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  		Fig. 3. Analysis of LVH cardiomyopathy using clinical ultrasound M-mode and color flow spectral Doppler analyses. Clinical M-mode imaging showed one animal with LVH cardiomyopathy (A), as evident with measurements of cardiac dimensions in end diastole (a, b, c) and end systole (d, e, f) using the M-mode tracings compared with a normal littermate (C). Measurements of the concentric LVH cardiomyopathy mouse show a = 1.11 mm, b = 3.60 mm, c = 1.04 mm, d = 1.65 mm, e = 1.96 mm, f = 1.62 mm, whereas normal littermate shows a = 0.75 mm, b = 4.54 mm, c = 0.92 mm, d = 0.97 mm, e = 3.49 mm, f = 1.08 mm. These measurements show the LVH cardiomyopathy mouse with 46% shortening fraction (SF) and 89% ejection fraction (EF), compared with the normal littermate with 21% SF and 49% EF. B: color flow spectral Doppler in the long-axis view showed the mouse with LVH cardiomyopathy had nonturbulent flow in the LV outflow tract (LVOT; green arrow) and a normal aortic velocity of 0.44 M/s, which is evidence against LVOT obstruction. D: this is similar to the normal littermate, which exhibited an aortic velocity of 0.5 M/s.

 

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  		Table 1. Cardiac measurements from ECG-gated two-dimensional images

 

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  		Table 2. Cardiac measurements from M-mode images

 
Two litters of mice were scanned using the new ECG-gated 2D imaging system. With its improved 2D and temporal resolution, cardiac measurements could be made directly from images captured from the 2D cine clips in the short-axis views. These measurements showed increased contractility in the LVH cardiomyopathy mice, with 51% shortening fraction (SF) and 80% ejection fraction (EF) (Table 1). In comparison, normal littermates exhibited 27% SF and 55% EF, which is similar to normal C3H wild-type mice (28% SF and 53% EF; Table 1). The high-resolution, slow-motion 2D cine clips generated by gated ECG-based kilohertz visualization (EKV) imaging in the long-axis view showed concentrically thickened LV walls, hypertrophied papillary muscles, decreased chamber size, and no LVOT, i.e., neither asymmetric septal hypertrophy (diagnostic of obstructive HCM), thickened aortic valve leaflets with poor opening (diagnostic of valvar aortic stenosis), or supravalvar aortic obstruction caused the LVH (Fig. 2, A and B). We further investigated for LVOT obstruction, wall dyskinesis, and valve abnormalities. In comparison, thinner walls and barely detectable papillary muscles were seen in normal littermates (Fig. 2C).

Three other litters of Family 78 mice scanned using a clinical ultrasound system also showed two distinct groups of mice, with one group exhibiting evidence of LVH cardiomyopathy and the other showing normal littermates similar to the mice studied with gated ultrasound imaging. Measurements obtained using M-mode images indicated increased wall thickness and decreased LV internal dimensions that corresponded to an increased contractility of 38 ± 5% SF and 75 ± 7% EF. In comparison, normal littermates showed 24 ± 3% SF and 53 ± 5% EF (Table 2). We also used spectral Doppler color flow analysis on the clinical ultrasound system to look for turbulent color flow and increased aortic outflow velocity (6, 14). Laminar flow with normal outflow velocities was observed in the LVH cardiomyopathy mice and normal littermates (Fig. 3, B and D). Furthermore, the spectral Doppler did not suggest an increased afterload. Spectral Doppler did not suggest that the LVH was secondary. Follow-up analysis of heart and body weight measurements confirmed these ultrasound findings. They showed a heart-to-body weight ratio of 5.8 x 10–3 for the LVH cardiomyopathy mice compared with 4.15 x 10–3 for the normal littermates (Table 3).


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  		Table 3. Heart and body weight analysis

 
Histological and ultrastructural analysis. Necropsy showed mice ultrasound phenotyped as having LVH cardiomyopathy exhibited enlarged hearts with ventricles that appeared to have thickened walls (Fig. 1, C and D). EFIC histology showed concentric LVH when viewed in the transverse plane (Fig. 1, E and F). Detailed examination of the EFIC 2D serial image stacks and 3D reconstructed volumes of the chest confirmed the cardiac hypertrophy was of the nonobstructive type, and there were no abnormalities of the chest organs or blood vessels (data not shown). The heart-to-body weight ratios were obtained for all of the mice that were ultrasound scanned. Mice phenotyped by ultrasound to have LVH cardiomyopathy showed significant increased heart weights, as well as increased heart-to-body weight ratios, and these were correlated with increased EF (Fig. 4). Analysis by standard histopathology showed some myofiber disarray, bizarre enlarged nuclei, and abnormal vacuolization (Fig. 5). No evidence was found for inflammation or of white blood cell infiltrates or necrosis. We did not observe myofiber degeneration or atrophy. There was a mild interstitial fibrosis, which was confirmed with further analysis by Mason's trichrome staining (Fig. 5, C and D). Periodic acid Schiff staining showed no abnormal accumulation of glycogen (Fig. 5, E and F), nor did we find thickening of the basement membrane (Fig. 5, C and D). Measurement of myocyte size showed a mean diameter of 20 ± 3.0 µm in the hypertrophied hearts compared with 13.7 ± 2.5 µm in the hearts of normal littermates. Ultrastructural analysis by EM showed normal sarcomeric structure with no abnormal myofibrillar branching (Fig. 6). However, there was a marked enlargement of the sarcoplasmic reticulum, and large perinuclear vacuoles were observed (Fig. 6, B and C). Immunohistochemistry showed increased expression of ANF, SERCA2, and calsequestrin (Fig. 7). Real-time PCR analysis also showed an increase in SERCA2 transcripts. Scanning electron microscopy analysis revealed a marked increase in the abundance of mitochondria in the hearts with LVH cardiomyopathy, which suggested possible alteration in fatty acid metabolism (Fig. 6) (10, 11, 15, 26, 41). Consistent with this, we found an abnormal accumulation of lipid droplets (Fig. 6), and immunohistochemistry showed elevated expression of the fatty acid oxidation enzymes muscle carnitine palmitoyltransferase-1 and long-chain acyl-CoA dehydrogenase, as well as the upstream regulator PGC-1 (Fig. 8).


Figure 4
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  		Fig. 4. Analysis of heart-to-body weight ratio confirms ultrasound diagnosis of LVH cardiomyopathy. A: heart-to-body weight ratios (left) and EFs (right) are plotted for mice with LVH cardiomyopathy vs. normal littermates. Individual measurements are shown by the open (normal) or solid (LVH cardiomyopathy) triangles, with the mean of the measurements denoted by the solid line. P values for comparison between LVH cardiomyopathy vs. normal mice are also indicated. B: EFs are plotted against heart-to-body weight ratios just for mice with LVH cardiomyopathy, with the least squares line superimposed to show the correlation trend. Kendall's rank correlation coefficient is 0.42, with a P value = 0.045, indicating indeed changes in heart-to-body weight ratio are correlated significantly with changes in EF.

 

Figure 5
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  		Fig. 5. Cardiac hypertrophy indicated by histopathology. Histological sections are shown from the interventricular septum of normal littermate (B, D, and F) vs. Family 78 mouse with LVH cardiomyopathy (A, C, and E). Sections were stained with hematoxylin and eosin (A and B), Mason's trichrome (C and D), or periodic acid Schiff (E and F). Note enlarged size of myocyte (white arrowheads in A and E), bizarre nuclei (white asterisk in A), myofiber disarray (A, C, E), and abnormal vacuolization (black arrows in A and E) in affected animal. C: trichrome stain shows mild fibrosis associated with the cardiac hypertrophy. Magnification is the same for all panels.

 

Figure 6
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  		Fig. 6. Analysis by electron microscopy showed abnormal ultrastructure of a metabolic defect. Analysis by electron microscopy showed sarcomeric myofilament ultrastructure in the heart with LVH cardiomyopathy (B and D) was similar to that of the littermate control heart (A). However, associated with the cardiac hypertrophy was an abnormal increase in mitochondria (white asterisks in B–D) and lipid droplets (white arrows in B–D), vacuolar changes in the cellular membranes (black arrow in B), and perinuclear vacuoles (V in C), which were associated with abnormally shaped nuclei (N in C).

 

Figure 7
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  		Fig. 7. Immunohistochemistry showed increased expression of atrial natriuretic factor (ANF), calsiquestrin 2 (CASQ), and sarco(endo)plasmic reticulum Ca2+-ATPase 2 (SERCA2) associated with cardiac hypertrophy. Immunohistochemistry was used to examine expression of ANF (A and B), CASQ (C and D), and SERCA2 (E and F) in paraffin sections of hearts obtained from a mouse exhibiting cardiac hypertrophy (A, C, and E) vs. normal littermate control (B, D, and F). In the control heart, there was little or no ANF immunostaining (B), whereas, with cardiac hypertrophy, strong cytoplasmic granular staining was observed (A), indicating high levels of ANF expression. Similarly for CASQ, the control heart showed only a low level of diffuse staining (D), whereas the heart with hypertrophy showed strong immunostaining (C). SERCA2 immunostaining showed SERCA expression in the control (F) and hypertrophied (E) hearts, but much higher level of expression was seen with hypertrophy (E). All images are at the same magnification.

 

Figure 8
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  		Fig. 8. Alteration in fatty acid metabolism indicated by increased expression of muscle carnitine palmitoyltransferase-1 (MCPT-1), long-chain acyl-CoA dehydrogenase (LCAD), and peroxisome proliferator-activated receptor-{gamma} coactivator 1 (PGC-1). Immunohistochemical staining for MCPT-1 (A and B), LCAD (C and D), and PGC-1 (E and F) showed very weak staining in the heart of a normal littermate animal (B, D, and E), whereas strong staining was observed in the heart of an animal exhibiting LVH cardiomyopathy (A, C, and E). All images are at the same magnification.

 
Mutation maps to the X chromosome. To map the mutation in this mutant mouse line, a full genome scan was conducted using C57BL6/J:C3H/HeJ hybrid offspring of Family 78 exhibiting cardiac hypertrophy. This included 10 adult males and 3 newborn fetuses that died at birth with severe hypertrophy. With this method, DNA markers located near the ends of each mouse chromosome were used to demarcate intervals that were treated as haplotypes for the purpose of linkage analysis (31). The frequency with which recombinant haplotypes were found across the entire genome in the affected mice was tracked. The mutation is expected to lie in a chromosome interval that is nonrecombinant, i.e., consistently homozygous for the B6 strain in most or all of the affected fetuses. Using 48 polymorphic microsatellite markers and analysis of {chi}2 maximum percentage values showed linkage of the mutation to the distal region of the X chromosome, between markers DXMit81 and DXMit223 (Table 4). Several candidate genes in this chromosome interval were sequenced, including tafazzin (Taz) or G4.5 (8, 45). Taz was of particular interest, since it is associated with Barth syndrome, an infantile lethal subtype of cardiomyopathies associated with alterations in lipid metabolism (2–4). However, sequencing of the coding region of Taz showed no mutation in this mutant mouse line. Also sequenced was the gene encoding angiotension II type 2 receptor (Agtr2) and the gene for lysosomal membrane glycoprotein 2 (Lamp2). Lamp2 has been shown to cause cardiac hypertrophy associated with glycogen accumulation (1, 30, 33). Agtr2 has been implicated in cardiac hypertrophic process (9, 18). No mutation was found in either Lamp2 or Agtr2 coding sequences.


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  		Table 4. Mutation mapping by recombinant haplotype analysis

 
Analysis of sex linkage of LVH cardiomyopathy. In light of the genome scan data showing the mutation in Family 78 causing cardiac hypertrophy was localized to the X chromosome, further analysis was carried out to confirm if the LVH cardiomyopathy was transmitted in a sex-linked manner. This entailed examining the incidence of cardiac hypertrophy in progeny obtained from intercrosses between wild-type C3H mice and Family 78 male or female mice carrying the mutation (referred to as carriers). From the mating of male carriers with C3H wild-type females, we obtained 47 offspring, and, among these, 8 died at birth with severe cardiac hypertrophy, with all 8 being females. With this cross, as the mutation is being transmitted via the male parent, no affected male would be expected, if the mutation is indeed localized on the X chromosome. Statistical analysis showed this result is highly significant for sex linkage of the affected trait (Table 5).


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  		Table 5. Breeding analysis showing sex-linked transmission

 
The converse mating with the mutation-carrying X chromosome being derived from the female parent yielded five male and three female neonates that died at birth with severe cardiac hypertrophy. As the mutation-carrying X chromosome in the latter cross was derived from the mother, it is expected that there is 50% probability for any offspring, male or female, to inherit the mutation-carrying X chromosome. However, with random X inactivation, more affected males would be expected than females. This is consistent with our finding of five male and three female neonates dying at birth with severe cardiac hypertrophy. We note, given the small sample size, that this difference was not statistically significant (Table 5).

To further evaluate the sex-linked transmission associated with intercrosses involving female carriers mated with wild-type C3H males, we examined 11 additional litters to determine the sex distribution among live offspring surviving to weaning (Table 5). In the absence of any deleterious mutation on the X chromosome, one would expect 50% males and females at weaning. Instead, at weaning, we observed 40 females and 24 males. Statistical analysis showed this difference was also highly significant (P < 0.00036), further confirming sex-linked transmission of a deleterious trait on the X chromosome. We also noted among the 64 offspring obtained from this last set of intercrosses that 4 males and 7 females had cardiac hypertrophy when examined at 7 mo of age. Overall, these findings are consistent with a genetic lesion on the X chromosome causing cardiac hypertrophy associated with neonatal lethality and adult cardiac hypertrophy in Family 78.


   DISCUSSION
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Analysis by echocardiography showed LVH cardiomyopathy not secondary to another cardiovascular etiology in some of the mice in Family 78. This was observed with cardiac measurements obtained by conventional M-mode imaging and 2D images generated from ECG-gated ultra-high-frequency echo imaging (supplemental movies 1 and 2; the online version of this article contains supplemental data). These measurements showed hypercontractile function with EF of 75–80% and SF of 38–51%. In contrast, normal littermates exhibited an EF of 53–55% and a SF of 24–27%, similar to measurements obtained in wild-type C3H mice. This increased contractility was associated in the EM with dilated sarcoplasmic reticulum. By immunohistochemistry, we showed increased expression of both SERCA and calsequestrin. Together, these results are consistent with the echocardiographic findings showing a marked increase in contractility.

Cardiac hypertrophy was independently confirmed with heart and body weight measurements. Analysis by histopathology also showed increased myofiber size and further confirmed the diagnosis of concentric LVH indicated by echocardiography. Immunohistochemical staining showed an increase in ANF expression. Increases in ANF expression have been reported previously in two mouse models of familial HCM with increased EFs (27). As seen by others, we found increased ANF expression in ventricular myocytes in conjunction with fibrosis and myofiber disarray (44).

We mapped the mutation causing LVH cardiomyopathy to the X chromosome. Sex-linked transmission was confirmed with the finding that male carriers mated with wild-type C3H female mice yielded neonates with severe cardiac hypertrophy that were all females. Moreover, analysis of mice surviving to weaning showed many fewer males than females in litters obtained from matings of C3H males with carrier females. This sex-linked transmission likely accounts for the survival of some of the adult females with less severe cardiac hypertrophy. As some adult males also survive with cardiac hypertrophy, this phenotype has variable expressivity and may be subject to genetic modifier effects in the C3H background.

ECG-gated echocardiography. This study represents the first report comparing a new EKV software on an ECG-gated biomicroscope to a clinical ultrasound system for routine echocardiographic measurements of adult mouse cardiac functions. Although noninvasive echocardiography is routinely used for cardiac assessments in mice, the high heart rates (400–550 beats/min) of adult mice, together with the slow frame rate (30 Hz) of 2D imaging in clinical ultrasound systems, had largely precluded the use of 2D imaging for mouse cardiac measurements. Thus a 30-Hz frame rate affords only four frames per cardiac cycle, making it highly problematic for 2D images to be captured reliably in true end diastole and end systole in adult mice. Instead, M-mode imaging with its 1-kHz frame rate (1,000 frames/s) has become the method of choice for quantitative mouse cardiac assessments. Cherin et al. (5) presented the new technology of ECG-gated 1,000 frames/s (1 kHz) Visualsonics software for excellent 2D cine resolution by EKV imaging of the heart. This paper also focused on "retrospective color flow imaging" in carotid arteries, but did not make any measurements of the heart structures or function (5). The Li et al. 2007 reference (19) acquired the gated EKV 2D cines with the Vevo 770 and used these very-high-resolution images for speckle-tracking quantification of regional myocardial motion compared with MRI in mice, but does not discuss conventional echocardiographic measurements.

In this study, we showed echocardiographic measurements obtained using ECG-gated 2D images using the new EKV technology were similar to conventional M-mode measurements, indicating 2D images were captured at true end diastole and end systole. In addition, with the high 2D spatial resolution provided by the 40-MHz transducer used for ECG-gated imaging, ventricular wall motion can be imaged for detecting regional wall motion abnormalities (supplemental movies 1 and 2). Valvular abnormalities also can be detected, although none was noted in this mutant mouse line. Overall, these studies showed Family 78 mice had concentric LVH cardiomyopathy that was not secondary. These studies demonstrate ECG-gated ultrasound imaging can be used to acquire cardiac dimensions directly from the 2D images, similar to the routine clinical use of 2D imaging for human cardiac assessments.

X-linked cardiac hypertrophy. The mutation causing cardiac hypertrophy in Family 78 was mapped to the distal region of the X chromosome. This map position includes the dystropin gene associated with X-linked Duchene muscular dystrophy. However, the cardiac hypertrophy exhibited by our mutant does not resemble the typical cardiomyopathy seen in the mdx mouse model of X-linked Duchenne dystrophy (34). Our EM analysis showed no obvious defect in the structure or organization of the myofilaments, thus indicating the mutation is likely a nonsarcomeric protein. We considered several nonsarcomeric candidate genes on the X chromosome, including Lamp2. Lamp2 mutations are associated with X-linked cardiac hypertrophy and had been shown to cause glycogen storage disease and conduction abnormalities (1, 33). However, examination of ECG records obtained during ECG-gated 2D imaging showed no conduction abnormalities. Thus there was no evidence of a short PR interval nor preexcitation pattern, as have been seen in patients with Lamp2 mutations. Histopathology and EM ultrastructural analyses also showed no evidence of glycogen storage disease. Finally, DNA sequencing showed no coding sequence changes in Lamp2 (1).

Our finding of the accumulation of lipid droplets and mitochondria hyperproliferation would suggest abnormalities in mitochondrial lipid metabolism. A mouse model of very-long-chain acyl-coenzyme A dehydrogenase deficiency in mice shows myocytes with microvesicular lipid accumulation and marked mitochondrial proliferation similar to our LVH cardiomyopathy mice (11). Indeed, our analyses indicated a marked increase in the protein level of fatty acid oxidation enzymes, such as muscle carnitine palmitoyltransferase-1 and LCAD and also the upstream regulator PGC-1. Mouse models and techniques of many defects in fatty acid metabolism that result in cardiomyopathy and perinatal death have become available (37, 43). New genetic defects in mitochondrial fatty acid oxidation have continued to be identified in infants and children that involve almost all the possible enzyme steps in the pathway (39). Barth syndrome, an X-linked human disorder characterized by infantile cardiomyopathy, is associated with mitochondrial defects that include the abnormal metabolism of cardiolipins (16, 29, 36). Recent studies show Barth syndrome is associated with mutations in the gene Taz, which encodes a protein with homology to acyl-transferases (4, 8, 45). However, DNA sequencing showed no changes in the coding region of Taz in Family 78. Other genes on the X chromosome analyzed included the gene for the angiotension II type 2 receptor (Atgr2) (9) and the gene for Fabry's syndrome, a disorder in which cardiac hypertrophy is associated with deficiency in lysosomal hydrolase {alpha}-galactosidase (40). We failed to detect any sequence changes in the coding regions of either gene. Even though we did not find any coding differences in the sequences of Taz/Atgr2/Lamp2, we cannot exclude the possibility of noncoding mutations. With this caveat, we suggest Family 78 may have a novel mutation in a gene not previously associated with X-linked cardiac hypertrophy.

Conclusion.

Using echocardiography, we identified asymptomatic adult mice in Family 78 as having LVH cardiomyopathy. Analysis by genome scanning using microsatellite markers mapped the mutation to the X chromosome. We excluded defects in the coding regions of several genes known to cause X-linked cardiac hypertrophy. This new mouse model may provide novel insights into X-linked cardiomyopathies.


   GRANTS
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Division of Intramural Research Grant ZO1-HL005701.


   ACKNOWLEDGMENTS
 
We acknowledge Julie Rosenthal and Ginger Slack for helping prepare the manuscript, and VisualSonics for providing a 1-year loan of the Vevo660 Ultrasound Biomicroscope equipped with a beta version of the gated EKV used for this study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. Leatherbury, LDB/NHLBI/NIH, 10 Center/MSC 1583/6C103, Bethesda, MD 20892-8001 (e-mail: lleather{at}cnmc.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
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