INTRODUCTION

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FAMILIAL HYPERTROPHIC CARDIOMYOPATHY (FHC) is an autosomal dominant disease, characterized by ventricular hypertrophy, myocellular disarray, fibrosis, arrhythmias, and increased occurrence of sudden cardiac death (8, 9). The disease is genetically heterogeneous, but all of the known mutant genes encode structural proteins of the sarcomere, including myosin heavy chain (MHC), essential myosin light chain, regulatory myosin light chain, myosin binding protein C, -tropomyosin, troponin T, and troponin I (14). In most patients with FHC, systolic function is normal or supranormal, whereas diastolic function is impaired (11). However, 10-15% of patients with symptomatic hypertrophic cardiomyopathy present with a progressive left ventricular (LV) wall thinning and chamber dilation that occur in conjunction with systolic dysfunction (10, 15, 17). Approximately 30% of patients with FHC have mutations in the cardiac -MHC gene (21). There are several missense mutations in this gene. Generally, patients with mutations in the -MHC gene exhibit uniform hypertrophy, but variable sudden death. One missense mutation in this gene [arginine to glutamine at position 403 (R403Q)] produces a severe form of FHC in humans (22).

A number of MHC transgenic (TG) animal models of FHC have been created in an attempt to understand the pathogenesis of FHC phenotypes typically seen in humans. Because the ventricular myocardium of mice is 100% -MHC, the appropriate MHC to express as a mutant is in the context of -MHC rather than -MHC. None of the residues implicated in FHC differ between - and -MHC. Our MHC TG mouse model consists of an -MHC R403Q mutation and a deletion in the actin binding domain of the -MHC. These mice exhibit many of the features seen with FHC in humans, including LV and right ventricular (RV) hypertrophy, cellular disarray, and fibrosis (20). Two molecular markers of compensatory hypertrophy, atrial natriuretic factor and -skeletal actin mRNA, were upregulated in 3-mo-old female TG mice (19), but this phenomenon occurred independently of ventricular hypertrophy. Interestingly, this FHC model displayed gender differences in which at 3 mo of age both male and female TG mice developed ventricular hypertrophy. However, by 8 mo of age the hearts of female TG mice continued to exhibit hypertrophy, whereas the male TG hearts began to show signs of LV dilation (20). Quantitative measurements of the internal LV chamber area using echocardiography demonstrated a significant increase in LV chamber area in 10-mo-old male TG mice compared with age-matched WT controls (4).

Another mouse model of FHC introduced the R403Q mutation into one allele of the -MHC by a "hit-and-run" homologous recombination. The hearts from this FHC mouse model (-MHC^403/+) showed myocyte disarray, fibrosis, diastolic dysfunction, and normal systolic function. At 4 mo the -MHC^403/+ hearts had enlarged atria but no ventricular hypertrophy (5, 16). However, subsequent to the original report of no hypertrophy in the -MHC^403/+ model (5, 16), LV hypertrophy was recently described (3). A myosin binding protein C (truncation) model has also been shown to exhibit hypertrophy (24). The -MHC^403/+ model had in vivo LV diastolic dysfunction, whereas the myosin binding protein C truncation model did not (3). Recently, a TG rabbit model of FHC with a R403Q mutation in the -MHC was developed. The rabbit model showed many similarities with human FHC, including LV hypertrophy, myocellular disarray, and fibrosis; however, to date, no functional parameters have been investigated in this model (7).

The present study was designed to investigate whether the functional characteristics of the -MHC TG mouse hearts resemble human FHC and to determine the interactive effects of age and gender on LV contractile performance. We hypothesized that diastolic function would be impaired in hearts from TG mice independent of gender or age and that older male mice would show signs of systolic dysfunction as a result of LV chamber dilation. We found that diastolic function was indeed impaired in this murine TG model of FHC and that the LV chamber dilation seen in older male TG mice was associated with systolic dysfunction.

	METHODS

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Animals. The TG mouse model of FHC used in this study expressed a mutant -MHC with expression driven by a rat -MHC promoter (20). The transgene coding region contained two mutations, a point mutation R403Q and a deletion of 59 amino acids in the actin binding site of the -MHC, bridged by an addition of 9 nonmyosin amino acids. The mutant MHC protein made up 10-12% of the total myosin in the TG (line no. 140) mice (20). The heterozygous TG mice and their non-TG wild-type (WT) littermates used in this study were generated by backcrossing TG mice to C57/B16 mice. PCR-amplified tail DNA and restriction enzyme digestion was used to genotype each mouse. The animals were maintained under specific pathogen-free conditions with food and water ad libidum. All animal protocols were approved by the institutional animal use and care committee at the University of Colorado at Boulder.

Isovolumic heart preparation. Isovolumic LV pressure dynamics were recorded from eight different groups of mice: 4-mo-old WT (4WT) males (n = 9) and females (n = 6); 4-mo-old TG (4TG) males (n = 6) and females (n = 8); 10-mo-old WT (10WT) males (n = 6) and females (n = 7); and 10-mo-old TG (10TG) males (n = 8) and females (n = 9). All animals were heparinized (250 units ip) 15 min before administration of an anesthetic dose of pentobarbital sodium (35 mg/kg body wt ip). Under deep anesthesia, hearts were exposed by midline thoracomoty, rapidly excised, and arrested in ice-cold saline solution. Excess tissue was removed, and the aorta fixed to a 21-g stainless steel cannula with 6-O surgical silk suture. Each heart was retrogradely perfused at a constant pressure of 85 mmHg with a Krebs-Henseleit buffer containing (in mM) 117.4 NaCl, 4.7 KCl, 1.2 MgSO₄, 2.5 CaCl₂, 1.2 KH₂PO₄, 25.0 NaHCO₃, 11.0 glucose, 5.0 pyruvate, and 0.5 EDTA. The buffer was continuously bubbled with 95% O₂-5% CO₂ at 37°C to yield a pH of 7.4.

Statistical analyses. Data are presented as means ± SE. Statistics were performed with SPSS 6.1 (SPSS, Chicago, IL), and male and female groups were analyzed separately. Using ANOVA, comparisons of morphometric data were made between the two mutation groups and the two age groups. To test for the effects of mutation, age, and different minimum pressures, repeated measures ANOVA were performed between the two mutation groups and the two age groups and across the four groups (P_min). For all variables, simple effects were examined to determine significant differences between the WT and TG subgroups for both 4- and 10-mo-old mice. To reduce the possibility of committing a type II interpretive error (a false negative) significance was reported at both the P  0.05 and P  0.10 levels (23).

	RESULTS

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Heart mass and coronary flow measurements. Morphological and coronary flow data from the isovolumically perfused hearts are presented in Table 1. No body weight differences existed between the WT and TG mice compared with their own age-matched controls. In the male mice, LV mass tended to be greater in the TG groups, with 5 and 7% increases in the 4- and 10-mo-groups, respectively (mutation main effect, P = 0.10). In the female mice, LV mass was greater in both 4TG and 10TG groups relative to their WT counterparts (8 and 28% increases, respectively). In our study, there was no WT versus TG difference in RV mass between the male groups, whereas the 10TG females had a significantly increased RV mass compared with hearts from the 10WT group (34% increase). When normalizing heart weight (LV and RV weights combined) to body weight, we found that 4-mo-old male TG mice had an increased heart weight-to-body weight ratio (simple effect, P = 0.04) compared with age-matched WT control. At 10 mo of age, however, the heart weight-to-body weight ratio was not different between the male TG and WT mice (P = 0.15). A lack of hypertrophy is consistent with previous reports of chamber dilation in this TG model (4, 20). Among the females, both 4- and 10-mo-old TG mice had larger heart weight-to-body weight ratios relative to WT mice (mutation main effect, P < 0.001). In males, relative to WT controls, coronary flow-to-heart weight ratios were significantly reduced by 16 and 28% in the 4TG and 10TG groups, respectively. Coronary flow-to-heart weight ratios in female 4TG and 10TG groups were decreased relative to WT controls by 4 and 34%, respectively (mutation main effect, P = 0.05).

Isovolumic contractile response to varying minimum pressure. To assess the effect of the -MHC transgene expression on ventricular contractile function, various indexes of contraction and relaxation were measured. LV isovolumic contractile performance was measured over 4 (0, 5, 10, 20 mmHg) different P_min, chosen to span normal physiological to heart failure ranges. Figure 1 depicts changes in LVP in response to changes in P_min. In hearts from 4-mo-old males (Fig. 1A), LVP was significantly higher in the TG hearts relative to WT controls. However, by 10 mo of age the TG hearts developed significantly less LVP compared with age-matched WT hearts (Fig. 1B; mutation × age interaction, P = 0.009). Moreover, a differential response by the male TG hearts to changes in minimum pressure was observed (Fig. 1, A and B; mutation × P_min interaction, P = 0.01). At both 4 mo and 10 mo of age, the TG hearts had a blunted LVP response to increases in P_min compared with WT hearts. In the female hearts (Fig. 1, C and D), there was no WT versus TG difference in LVP at either 4 or 10 mo of age. However, similar to the male hearts, the female 4TG and 10TG hearts also exhibited a blunted LVP response to increases in P_min compared with WT hearts (mutation × P_min interaction, P = 0.001).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Developed left ventricular pressure (LVP) in response to 4 different minimal pressure (Pmin) values in hearts from male and female mice. LVP was increased in transgenic (TG) 4-mo-old male mice (A) compared with the wild-type (WT) mice, whereas LVP was significantly reduced in the TG male hearts at 10 mo of age (B). There was no difference in LVP between the TG and WT female hearts (C, D). In both female and male hearts, the mutation had a differential effect on LVP, whereas the TG mice showed less of a response to changes in minimum pressure. Simple effect differences are the following: *different from age-matched WT mice at P  0.05 level and different from age-matched WT mice at P 0.10 level. Sample sizes are the following: 4-mo-old TG mice (male, n = 6; female, n = 8); 4-mo-old WT mice (male, n = 9; female, n = 6); 10-mo-old TG mice (male, n = 8; female, n = 9); 10-mo-old WT mice (male, n = 6; female, n = 7).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Changes in the rate constant describing LVP pressure development (+dP/dt/LVP) in response to 4 different Pmin values in hearts from male and female mice. Compared with 4-mo-old WT controls +dP/dt/LVP was greater for the 4-mo-old TG male (A) and female (C) mice. In hearts from female mice, the effect of age on the mutation status was more pronounced in the TG group (C, D). Simple effect differences are the following: *different from age-matched WT mice at P  0.05 level and different from age-matched WT mice at P 0.10 level. Sample sizes were per legend of Fig. 1.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Changes in the rate constant describing LV relaxation (dP/dt/LVP) in response to 4 different Pmin values in hearts from male and female mice. dP/dt/LVP was smaller in the TG male (A, B) compared with the WT mice independent of age. There was no difference in hearts from female mice (C, D). Sample sizes were per legend of Fig. 1.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Time to 50 and 90% relaxation in response to 4 different Pmin values in hearts from 4- and 10-mo-old male mice. It took longer for the TG hearts from male mice to reach 50% (A, B) and 90% (C, D) of maximal relaxation compared with the WT mice. Simple effect differences are the following: *different from age-matched WT mice at P  0.05 level and different from age-matched WT mice at P 0.10 level. Sample sizes were per legend of Fig. 1.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Time to 50 and 90% relaxation in response to 4 different Pmin values in hearts from 4- and 10-mo-old female mice. It took longer for the TG hearts from female mice to reach 50% (A, B) and 90% (C, D) of maximal relaxation compared with the WT mice. Simple effect differences are the following: *different from age-matched WT mice at P  0.05 level and different from age-matched WT mice at P 0.10 level. Sample sizes were per legend of Fig. 1.

	DISCUSSION

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In the present study, our -MHC TG mouse model of FHC displayed many age- and gender-dependent morphological and functional differences in the myocardium (Table 3). These changes were strikingly similar to those seen in the human disease. To date, this is the only animal model with a R403Q mutation in the cardiac MHC that exhibits cardiac hypertrophy along with diastolic dysfunction and coronary perfusion abnormalities commonly seen in humans with FHC. Furthermore, our model displays some distinct phenotypic gender differences, which may have relevance to the heterogeneous nature of FHC and heart disease in humans (see below). These results indicate that the -MHC TG mice used in this study may show promise as a model for investigating the cellular and molecular defects underlying human FHC.

View this table: [in this window] [in a new window]   Table 3.   Summary of gender and age differences in the -MHC transgenic mouse model of FHC

Our morphological data were consistent with data from Vikstrom et al. (20), who found that by 3 mo of age, LV mass was increased more in the female TG hearts compared with age-matched TG males and that LV hypertrophy had more than doubled in the females by the time they were 8 mo old. This hypertrophic response was likely representative of a compensatory mechanism that allowed for the preservation of systolic function in TG female mice at all ages. In 10-mo-old TG males, LV mass was similar to that seen in their WT littermates. On the basis of prior morphological and echocardiographic studies, this absence of increase in LV mass in older TG males was likely associated with LV chamber dilation (4, 20). Although we did not directly assess the degree of dilation in the male 10TG hearts, we found that, in preliminary studies, normal-sized balloons used successfully to study hearts from the other groups (including male 10WT hearts) were not large enough to fill the LV cavity in the 10TG hearts to satisfy a flat pressure-volume balloon criterion. To do so, we had to increase the balloon size and volume to fit the dilated hearts (see METHODS). In addition, the diminution in LV systolic function that we observed in 10TG mice is consistent with the idea that LV dilation was present.

Many patients with FHC have small vessel coronary disease (12, 18) and myocardial perfusion abnormalities (13) that can contribute to myocardial ischemia. Histological evidence of small vessel coronary disease was found previously in our TG mouse model (20). We extended those results by finding that normalized coronary flow was decreased substantially in hearts from 10-mo-old males (28%) and females (34%). A decrease in coronary flow may result from one or more of several mechanisms, including a decreased capillary density (6), small vessel coronary disease (12, 18), and impaired LV relaxation (11). Such chronic hypoperfusion, independent of mechanism, can lead to myocardial ischemia and eventually result in necrosis, infarction, and LV dysfunction. From a methodological perspective, it is worth considering the issue of whether or not the reduction in coronary flow observed in our TG hearts may have caused an ischemic state that adversely impacted our assessment of LV contractile function. A reduction in coronary flow would be expected to expose TG hearts to greater risk of ischemic injury, particularly in situations where cardiac work was markedly increased. However, in our preparations we do not believe this critical point was achieved for two reasons. First, under the conditions of our experimental protocol, the contractile function of both TG and WT control hearts were similarly stable over time. Second, as was recently demonstrated by our lab (4), the TG and WT preparations used under our baseline conditions were far below their contractile performance maxima because an isoproterenol challenge elicited an ~80-100% increase in LVP in both WT and TG preparations.

We used the isovolumic isolated left heart technique to investigate systolic and diastolic function in hearts from TG mice. A similar approach was used in another -MHC mutated mouse model of FHC in which one allele is WT and the other allele is the single amino acid substitution R403Q. Spindler et al. (16) measured LV isovolumic contractile performance over a range of perfusate calcium concentrations. In their -MHC^403/+ mice they found diastolic dysfunction but normal systolic function in hearts from 5- and 6-mo-old males. However, this particular mouse model did not develop ventricular hypertrophy but instead had atrial enlargement (5). Moreover, Spindler et al. (16) found no coronary flow abnormalities in the -MHC^403/+ hearts. Functional studies of the -MHC TG rabbit model of FHC have been limited to echocardiography (7). Neither LV end-diastolic dimensions nor LV end-systolic dimensions were different between the TG and WT rabbits as measured by echocardiography.

We observed in our model that normal or supranormal systolic function developed into mild systolic dysfunction, and that diastolic function worsened over the same period of time. The dilated hearts in the 10TG male mice had an impaired systolic function, similar to humans with dilated cardiomyopathy (15, 17). Ventricular dilation occurs in ~10% of patients with hypertrophic cardiomyopathy; however, no known studies have investigated the genetic background of these patients (15, 17). In addition, men have a higher incidence of dilated cardiomyopathy compared with women, especially at a younger age (2). In contrast to our 10-mo-old male mice hearts, the female hearts at the same age continued to hypertrophy, had diastolic dysfunction, and had normal systolic function. Human studies of gender differences in the hypertrophied heart found that women often develop a more severe LV hypertrophy in response to hypertrophic stimuli compared with men (1). Studies of men and women with aortic stenosis or long-standing hypertension found that older women developed a more marked concentric hypertrophy, lower levels of wall stress, and normal or supranormal systolic function compared with men with similar disease severity (1).

In summary, the hearts from young TG mice in this study exhibited cardiac hypertrophy along with LV diastolic dysfunction, but had normal and supranormal LV systolic function in females and males, respectively. In female TG hearts, concentric LV hypertrophy progressed with age and impaired diastolic function persisted in the absence of systolic dysfunction. In contrast, in male TG mice with advancing age, mild concentric hypertrophy progressed to ventricular dilation, diastolic dysfunction worsened, and systolic performance was impaired. Additionally, at 10 mo of age, coronary flow was diminished in both male and female TG hearts compared with WT hearts. Thus the -MHC TG mouse model of FHC used in this study may have great potential for use in the study of the pathogenesis of human FHC.