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Am J Physiol Heart Circ Physiol 292: H2119-H2130, 2007. First published December 15, 2006; doi:10.1152/ajpheart.00816.2006
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Cardiac response to pressure overload in 129S1/SvImJ and C57BL/6J mice: temporal- and background-dependent development of concentric left ventricular hypertrophy

Cordelia J. Barrick,1,2 Mauricio Rojas,3,4 Robert Schoonhoven,5 Susan S. Smyth,3,4 and David W. Threadgill1,2,3,5,6

1Curriculum in Toxicology, 2Department of Genetics, 3Carolina Cardiovascular Biology Center, 4Department of Medicine, 5Center for Environmental Health and Susceptibility, and 6Carolina Center for Genome Sciences, University of North Carolina, Chapel Hill, North Carolina

Submitted 29 July 2006 ; accepted in final form 12 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Left ventricular hypertrophy (LVH), a risk factor for cardiovascular morbidity and mortality, is commonly caused by essential hypertension. Three geometric patterns of LVH can be induced by hypertension: concentric remodeling, concentric hypertrophy, and eccentric hypertrophy. Clinical studies suggest that different underlying etiologies, genetic modifiers, and risk of mortality are associated with LVH geometric patterns. Since pressure overload-induced LVH can be modeled experimentally using transverse aortic constriction (TAC) and since C57BL/6J (B6) and 129S1/SvImJ (129S1) strains, which have different baseline cardiovascular phenotypes, are commonly used, we conducted serial echocardiographic studies to assess cardiac function up to 8 wk of post-TAC in male B6, 129S1, and B6129F1 (F1) mice. B6 mice had an earlier onset and more pronounced impairment in contractile function, with corresponding left and right ventricular dilatation, fibrosis, change in expression of hypertrophy marker, and increased liver weights at 5 wk of post-TAC. These observations suggest that B6 mice had eccentric hypertrophy with systolic dysfunction and right-sided heart failure. In contrast, we found that 129S1 and F1 mice delayed transition to decompensated heart failure, with 129S1 mice exhibiting preserved systolic function until 8 wk of post-TAC and relatively mild alterations in histology and markers of hypertrophy at 5 wk post-TAC. Consistent with concentric hypertrophy, our results show that these strains manifest different cardiac responses to pressure overload in a time-dependent manner and that genetic susceptibility to initial concentric hypertrophy is dominant to eccentric hypertrophy. These results also imply that genetic background differences can complicate interpretation of TAC studies when using mixed genetic backgrounds.

mouse model; cardiac hypertrophy; genetic background

LEFT VENTRICULAR (LV) hypertrophy (LVH), or increased LV mass, is associated with many cardiovascular disorders and is recognized as an independent risk factor for cardiac-related morbidity and mortality (5, 6, 23, 36, 43, 61). Although LVH can be associated with mechanical stress, growth factors, catecholamines, cytokines, and primary genetic abnormalities, a principal determinant of LVH is systemic hypertension. Of the nearly one in three adult Americans who have essential hypertension (EH), 20–60% will have evidence of LVH (17). Moreover, the beneficial effects of reversing LVH with antihypertensive therapy have been well documented in clinical trials (44, 46, 66).

Three patterns of LVH are observed in patients with EH and can be classified according to LV mass index (LVMI) and relative wall thickness [RWT, or ratio of diastolic LV posterior wall thickness (LVPWThD) to LV chamber cavity radius]: concentric hypertrophy, characterized by an increase in both LVMI and RWT; eccentric hypertrophy, characterized by an increase in LVMI with normal RWT; and concentric remodeling, characterized by normal LVMI in the setting of an increased RWT. Additionally, some individuals maintain normal LV geometry in the face of EH (19). The geometric pattern of LVH appears to be closely related to LV function and patient prognosis and may be a better predictor of outcome than traditional cardiovascular risk factors (15, 32). LV geometry and LV mass are highly variable among patients with comparable systolic blood pressure or environmental and other known predisposing factors, suggesting a significant heritable susceptibility to specific geometric patterns of LVH. Recent estimates attribute as much as 60% of the blood pressure-independent variation in cardiac mass to genetic factors; moreover, a significant correlation exists between adjusted relative risk for concentric LVH and elevated LVMI with race and ethnic background, suggesting the presence of genetic modifiers conferring differential susceptibility to pressure overload-induced LV remodeling and LVH (12, 25, 31, 38, 45). Recent studies identifying polymorphisms in genes encoding ghrelin, angiotensin-converting enzyme (ACE), and bradykinin B2 receptor and in genes involved with carnitine transport as candidate modifiers of LV remodeling and LV mass further support a strong genetic link (3, 13, 18, 24, 33, 42, 54, 65).

Surgical transverse aortic constriction (TAC) in mice causes chronic LV pressure overload, progressive LVH remodeling, and subsequent cardiac failure, providing an experimental model for human cardiac response to systemic hypertension (27, 37, 52, 64). Since its development, the TAC model has been used extensively on genetically engineered mice to investigate the role of specific genes during the development of LVH and cardiac failure in vivo. However, there is considerable variation in the degree of hypertrophy, LV geometry, and time to heart failure in this experimental model (40). Since many studies use engineered mouse models maintained on outbred or mixed genetic backgrounds (47, 55, 60, 63), genetic variability may contribute to observed phenotypic variability, including cardiac response to TAC.

C57BL/6J (B6) and 129S1/SvImJ (129S1) inbred mouse strains are some of the most widely used strains to generate genetically engineered mice (1, 2, 16), with many subsequent analyses being done on a mixed B6 and 129S1 genetic background. Since B6 and 129S1 mice have different baseline cardiovascular phenotypes (14, 28) and dissimilar responses to various cardiovascular stressors (21, 67), it is likely that varied combinations of B6 and 129S1 alleles will cause divergent cardiac responses to chronic pressure overload.

Although several studies have investigated the B6 response to TAC, no comparative studies exist using 129S1 and B6129F1 (F1) mice. Because of the strain-based variation in baseline cardiac phenotypes, we hypothesized that the cardiac response to TAC would also vary by genetic background and that strain-specific genetic modifiers may be a source of variability in previous studies on LVH, LV geometry, and cardiac failure. Furthermore, interstrain variability in response to TAC may provide experimental models for different geometric patterns of LVH response to hypertension observed in humans. Therefore, to identify potential strain-specific differences in response to TAC, we quantified the effects of pressure overload induced by TAC on cardiac morphology, histology, hypertrophy-associated gene expression, and LV function in B6, 129S1, and F1 male mice. We found that these strains manifested very different cardiac responses to pressure overload, suggesting that genetic background modifiers are important determinants for the response to pressure overload, possibly complicating the interpretation of TAC studies using genetically engineered mice on mixed genetic backgrounds. Our results also identify the 129S1 strain as a model for concentric LVH and indicate that concentric LVH is dominant to the eccentric LVH observed in B6 mice. These models will be useful in determining how similar cardiac insults can result in very different responses among individuals and for the identification of therapeutic strategies for specific geometric patterns of LVH. The results also emphasize the importance of using proper genetic controls for studies investigating gene function using engineered mouse models.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Mice were obtained from Jackson Laboratory (Bar Harbor, ME). Ten-week old 129S1, B6, and F1 males were acclimated to the local environment for 1 wk, which included housing in ventilated cages with high-efficiency particulate, air-filtered air and Purina 5058 rodent chow provided ad libitum.

Aortic banding. Pressure overload of the LV was induced by TAC of nine mice each from B6, 129S1, and F1 genetic backgrounds as described (27). The aorta was ligated between the innominate and left common carotid arteries by tying a 7-0 silk suture around a tapered 27-gauge needle placed on top of the aorta. The tapered needle was removed, leaving the suture to produce a defined stenosis of the vessel. The skin was closed with separate sutures (6-0 silk), and buprenorphine was administered for analgesia. To estimate the load produced by TAC, Doppler velocities were measure in the right and left carotid arteries before and after ligation using a handheld 20-mHz Doppler probe (Indus Instruments). Work by others has demonstrated that the right coronary artery-to-left coronary artery peak velocity ratio significantly correlates with heart weight (HW) after TAC and with peak jet across the banding site and can thus be used to estimate the pressure drop across the banding site (24). Peak systolic gradients post-TAC were similar among the genetic backgrounds [43.5 ± 10 (B6) vs. 40 ± 12 (F1) vs. 48 ± 12 (129S1) mmHg]. Six mice of each genetic background received a sham operation in which the aortic arch was isolated and a band was twined around the aorta, but not ligated, and subsequently removed. Surgery was also performed on a smaller cohort of mice from each genetic background (n = 2 to 3 sham-operated and 3–5 TAC mice) to assess cardiac function past 8 wk.

Echocardiography. Transthoracic echocardiography (TTE) was performed at baseline and at 5 wk post-TAC using a 30-MHz probe and the Vevo 660 Ultrasonograph (VisualSonics). Mice were lightly anesthetized with 1–1.5% isoflurane, maintaining heart rate at 400–450 beats/min, and a topical depilatory agent was applied before mice were placed in the left lateral decubitus position under a heat lamp to maintain body temperature at 37°C. The heart was imaged in the two-dimensional mode in the parasternal long-axis view. From this view, an M-mode cursor was positioned perpendicular to the interventricular septum and the posterior wall of the LV at the level of the papillary muscles. Diastolic and systolic LV wall thickness, LV end-diastolic dimensions, and LV end-systolic chamber dimensions were measured. All measurements were done from leading edge to leading edge according to the American Society of Echocardiography guidelines (41). Two-dimensional short- and long-axis views of the LV were obtained. M-mode tracings were recorded and used to determine LV end-diastolic diameter (LVEDD), LV end-systolic diameter (LVESD), LVPWThD, and LVPWTh systole over three cardiac cycles. LV fractional shortening (FS) was calculated using the formula %FS = (LVEDD – LVESD)/(LVEDD). LV mass was calculated according to uncorrected cube assumptions with some modifications using the equation LV mass (in mg) = 1.055[(LVEDD + LVPWThD + LVIVSThD)3 – (LVEDD)3] (10), where 1.055 is the gravity of myocardium and LVIVSThD is diastolic intraventricular septal wall thickness. Stroke volume (SV) was estimated by the formula SV = (LVvolume,d – LVvolume,s) (where LVvolume,d and LVvolume,s are LV diastolic and systolic volumes, respectively) and was multiplied by mean heart rate to estimate cardiac output. Percent ejection fraction (%EF) was calculated by using the formula %EF = (LVvolume,d – LVvolume,s)/(LVvolume,d). RWT was approximated by using the formula RWT = (LVPWThD + LVIVSThD)/(LVEDD).

Serial TTE under anesthesia was performed on a smaller cohort of mice at additional time points (0, 2, 4, 5, 6, and 8 wk postsurgery; n = 2 to 3 sham-operated and 3 to 5 TAC mice). Conscious TTE was also performed on a smaller cohort of mice (n = 3 sham-operated and 5 TAC mice) at 5 wk post-TAC. All measurements were made by an independent observer with no knowledge of treatment or genetic background.

Histology. Mice were weighed, and hearts, lungs, livers, and kidneys were dissected 5 wk after TAC (n = 6) or sham-operated surgery (n = 4) from mice of each genetic background, rinsed in PBS, and weighed. Hearts were cut in a cross section just below the level of the papillary muscle. The top half of the heart was formalin fixed and embedded in paraffin. Sections (5 µm) were prepared at 200-µm intervals. The sections were stained with hematoxylin and eosin for examination of gross appearance, whereas Masson's Trichrome or periodic acid-Schiff counterstained with hematoxylin (PAS-H) was employed to facilitate quantification of fibrosis and cardiomyocyte size, respectively. Cardiomyocyte hypertrophy was assessed by measuring cross-sectional area of 100 cardiomyocytes per PAS-H-stained section in 10 randomly selected fields having nearly circular capillary profiles and centered nuclei in the LV free wall. Cardiac fibrosis was determined by calculating the percentage of Masson's Trichrome-stained area of interstitial fibrosis per total area of cardiac tissue. Inflammatory cells were detected by using the pan rat anti-mouse monoclonal IgG macrophage/monocyte marker MCA519G (clone number MOMA-2; Accurate Chemical) followed by biotinylated goat anti-rat IgG (Jackson ImmunoResearch) with visualization using the ABC Elite kit (Vector). Positively stained cells per cross section were manually counted in three sections per heart. Histological images were analyzed by using Nova Prime 6.75.10 software (BioQuant Image Analysis). Blinded measurements were made by two independent observers.

Gene expression. Total RNA was extracted from the lower half of the LV by using TRIzol (Invitrogen). After DNAse treatment, 500 ng of total RNA were reverse transcribed using the High-Capacity cDNA Archive Kit (Applied Biosystems). The expression of {alpha}-myosin heavy chain (Myh6), beta-myosin heavy chain (Myh7), atrial nautriuretic peptide (Nppa), brain nautriuretic peptide (Nppb), and medium chain acyl dehydrogenase (Acadm) was determined by real-time quantitaive PCR using Taqman Univeral Master Mix and Assays-on Demand primers and probes (Applied Biosystems). beta-actin (Actb) was used as an internal control for the results presented here; similar results were obtained using beta-glucuronidase, (Gusb) as an internal control. There were no significant differences by genetic background or treatment in Actb expression levels. Reactions were run on a Stratagene MX3000P machine with analysis software. Threshold cycles (CT) were determined by an in-program algorithm assigning a fluorescence baseline based on readings before exponential amplification. Fold change in expression was calculated using the 2–{delta}{delta}CFormula method (38a) using Actb as the endogenous control. Results are represented as mean fold changes relative to sham-operated B6 LV expression.

Statistical analysis. All results were expressed as means ± SE. Statistical analysis was performed using StatView version 5 (SAS Institute). The Kolmogorov-Smirnov test was used to test for normal distribution. Statistical significance within genetic backgrounds was determined by using the two-tailed unpaired Student's t-test or nonparametric Mann-Whitney test, whereas a two-way ANOVA or the Kruskal-Wallis test was used to determine statistical significance within treatment groups. A P value of <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Previous studies have suggested that many cardiovascular phenotypes vary by genetic background. Consequently, strain-specific genetic modifiers may have been a source of variability in previous studies using engineered models of LVH, LV geometry, and cardiac failure. To evaluate and characterize genetic background-specific responses, the effects of pressure overload induced by TAC were used to identify experimental models for different geometric patterns of LVH response to hypertension that is observed in humans.

Baseline strain-specific differences in cardiovascular phenotypes. Sham-treated B6 and 129S1 control mice had natural baseline variation in cardiac morphology (Fig. 1, A–D). Sections taken through the hearts showed that B6 hearts were rounded at the apex with a larger LV diameter, whereas 129S1 hearts were longer and narrower with a thicker septal wall and with a smaller LV diameter. Histological analysis indicated that the 129S1 hearts had more tightly packed cardiomyocytes in the septal and free LV wall (Fig. 1B compared with 1D). The hearts of F1 sham-operated mice were similar to those of 129S1 mice in morphology and cardiomyocyte density (data not shown).


Figure 1
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  		Fig. 1. C57BL/B6 (B6) and 129S1/SvImJ (129S1 or 129) male mice have innate differences in cardiac morphology. Representative hematoxylin and eosin staining of cardiac sections from B6 and 129S1 sham-operated (A–D) and transverse aortic constriction (TAC; E and F) hearts 5 wk postsurgery (A, C, E, and F, magnification, x1.6; B and D, magnification, x10; bar = 100 µm). G: representative M-mode tracings taken from the short-axis 5-wk postsurgery from B6 and 129S1 mice. LVEDD, left ventricular (LV) end-diastolic diameter; LVESD, LV end-systolic diameter; LVPWThD, LV posterior wall thickness diastole, LVPWThS, LV posterior wall thickness systole.

 
These baseline differences among the strains were also observed by TTE. 129S1 mice had the smallest LVEDD and LVESD but longer LV length than B6 mice (Fig. 1G and supplemental Table 1; all supplemental tables are available with the online version of this article). Baseline-calculated values of %FS and %EF were significantly lower in F1 mice compared with 129S1 and B6 mice, whereas LVvolume,d and LVvolume,s were smaller in 129S1 mice compared with B6 and F1 mice, suggesting an intrinsic strain-dependent variation in cardiac contractility and LV geometry (Table 1 and supplemental Table 1).


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  		Table 1. Percent change in echocardiographic parameters compared to baseline 5 wk postsurgery

 
Differences in body and organ weights across the strains were also observed (Table 2). 129S1 sham-operated mice were lighter and had smaller wet organ weights compared with those in B6 and F1 mice; however, when normalized to body weight (BW), there was no significant difference in organ weights by genetic background. There was also no significant difference in mean aortic diameter measured at the level of the aortic root at systole or diastole by genetic background at baseline. As has been previously reported, 129S1 mice had higher systolic blood pressures than did B6 mice (122 ± 5.3 vs. 116 ± 2.9 mmHg; supplemental Table 2).


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  		Table 2. Comparison of organ weights between sham-operated and TAC-treated mice

 
Pressure overload induces strain-specific differences in cardiac function. Because it is possible that baseline differences could impact cardiovascular pathophysiological responses, we subjected B6, 129S1, and F1 hybrid mice to TAC to identify strain-dependent cardiac responses to elevated pressure overload (Fig. 1, E and F). No significant differences in heart rate were detected at baseline or after treatment or by genetic background. Following TAC, peak systolic gradients across the left and right carotid arteries were similar in B6, F1, and 129S1 mice (43.5 ± 10, 40 ± 12, and 48 ± 12 mmHg, respectively), indicating a similar initial pressure gradient. Approximately 20% of B6 mice died between 1- to 3-wk post-TAC, similar to published mortality rates for this strain, with only 40% of B6 TAC mice surviving to 8 wk postsurgery (37). No mortality occurred in 129S1, F1, or sham-operated mice followed up to 8 wk post-TAC.

To determine whether the increased mortality in B6 mice could reflect more severe LV dysfunction, changes in LV geometry and function were noninvasively measured by TTE. At 5 wk post-TAC, B6 mice had LV chamber dilatation, reduced %FS, and reduced %EF with no significant change in RWT compared with those values in sham-operated B6 mice (Table 1), consistent with eccentric hypertrophy and depressed systolic function. Both 129S1 and F1 mice developed concentric hypertrophy at 5 wk post-TAC, as marked by significantly reduced chamber size, increased wall thickness, increased RWT, and increased %FS and %EF compared with those values in controls (Table 1 and supplemental Table 1). Although LVPWTh increased in all three genetic backgrounds with chronic pressure overload, 129S1 TAC mice manifested the greatest percent increase in LVPWTh compared with sham-operated controls (Table 1). In contrast, calculated LV mass also increased in all TAC mice compared with sham-operated mice, but a significantly greater increase was observed in B6 TAC mice compared with other genetic backgrounds [percent increase LV mass over baseline: 89.9 ± 14.9 (B6) vs. 66.3 ± 16.3 (F1) vs. 56.1 ± 7.1 (129S1); P < 0.05]. Sections taken through the hearts confirmed that B6 TAC mice developed a dilated cardiomyopathy with RV enlargement, whereas 129S1 TAC mice had concentric hypertrophy (Fig. 1, E and F). Interestingly, measurements of mean aortic diameter proximal to the constriction site taken at 5 wk postsurgery were significantly greater in B6 TAC mice compared with sham-operated mice as well as F1 and 129S1 TAC mice, suggesting possible strain differences in the aortic response to load (supplemental Table 1).

An initial assessment of LV geometry and function by TTE was performed on mice anethestized with inhaled isoflurane, which in published studies has been found to have mild effects on cardiac function and has been proposed to be the best anesthetic for repeated measurement in the same animal (8, 9, 29, 30, 53, 56). To exclude the possibility that the differences we were observing in the cardiac geometry and function in B6, F1, and 129S1 mice by TTE could be ascribed to differential strain suspectibility to anesthesia, we performed echocardiography on conscious mice and compared the results to those obtained under isoflurane. TTE on conscious mice yielded similar patterns to those observed with isoflurane-anesthetized animals with 129S1 and F1 TAC mice having reduced LVEDD, LVESD, and increased %FS and %EF consistent with concentric hypertrophy, whereas B6 TAC mice had increased LVEDD, LVESD, and reduced %FS and %EF consistent with LV dilatation (supplemental Table 3).

At 5 wk post-TAC, significant strain differences were observed in BW, organ weights, and organ weight-to-BW ratios (Table 2). Pressure overload significantly increased heart weight (HW) and HW-to-BW ratios on all three genetic backgrounds relative to controls. However, wet lung weight (LuW) and LuW-to-BW ratios were significantly increased in F1 and 129S1 TAC mice (P < 0.02 vs. shams), whereas wet liver weight (LiW) was significantly higher in TAC B6 mice (P < 0.03 vs. shams) but, when corrected for BW, did not reach significance (P < 0.06 vs. shams). There were no differences in wet kidney weight (KiW) or KiW-to-BW ratios between banded and sham-operated mice (data not shown). When wet organ weights were expressed as percent increase over sham-operated mice, B6 TAC mice had the largest relative increase in HW and LiW, whereas 129S1 mice had the largest relative increase in LuW. Since B6 mice also have evidence of RV dilatation (Fig. 1E), it is likely that they manifest passive liver congestion and right-sided heart failure in response to chronic pressure overload, whereas 129S1 mice have significant pulmonary congestion.

B6 mice have heightened cellular responses to pressure overload. Cardiomyocyte hypertrophy was assessed by histomorphometry in mice from all three genetic backgrounds. Changes in cardiomyocyte size varied with background, approximately doubling in B6 TAC hearts whereas only increasing 1.6-fold in F1 TAC hearts and 1.3-fold in 129S1 TAC hearts relative to sham-operated controls (Fig. 2A). Within the TAC group, B6 cardiomyocytes had the largest cross-sectional area (Fig. 2, B and C).


Figure 2
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  		Fig. 2. Comparison of mean cardiomyocyte area across treatment group and genetic background. A: cardiomyocyte size was significantly increased in TAC mice relative to mice receiving sham-operated surgery on all genetic backgrounds (*P < 0.05 vs. sham controls). Within the TAC treatment group, the hearts of B6 mice had the greatest increase in cardiomyocyte area [P < 0.0001 vs. B6129F1 (F1) and 129S1 TAC]. Representative histology of periodic acid-Schiff counterstained with hematoxylin-stained cardiac sections from the LV septal wall of B6 TAC (B) and sham (C) and 129S1 TAC (D) and sham (E) hearts (magnification, x40; bar = 50 µm). Cardiomyocyte area is represented as mean ± SE.

 
The hypertrophic response to pathological stimuli often involves fibrosis (68). Quantitative analysis of Masson's Trichrome-stained sections, in which collagen stains a bright blue color, indicated increased interstitial fibrosis in B6 and 129S1 TAC mice compared with sham-operated mice (Fig. 3A). B6 TAC mice had significantly greater interstitial fibrosis than respective TAC mice on other genetic backgrounds (Fig. 3, C and E). Additionally, only B6 TAC mice tended to have extensive perivascular fibrosis (Fig. 3, B and D). B6 TAC mice also had inflammatory infiltrate found in regions of fibrosis (Fig. 4A). Comparably fewer inflammatory cells were detected in the hearts of sham-operated mice on all genetic backgrounds as well as in hearts of F1 and 129S1 TAC mice (Fig. 4C) and were not associated with fibrosis (Fig. 4B).


Figure 3
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  		Fig. 3. Comparison of severity and location of cardiac fibrosis by treatment and genetic background. A: interstitial fibrosis was significantly increased in B6 and 129S1 TAC mice compared with mice receiving sham-operated surgery (**P < 0.01 vs. sham). B6 sham-operated and TAC mice had more interstitial fibrosis compared with F1 and 129S1 mice in their respective groups (P < 0.05 vs. F1 and 129S1 shams; P < 0.01 vs. F1 and 129S1 TAC). B–E: Masson's Trichrome (MT)-stained sections demonstrated that B6 TAC mice have extensive perivascular fibrosis associated with intimal hyperplasia (arrow in B) as well as interstitial fibrosis (C). In comparison, 129S1 TAC mice had very little fibrosis (D and E) (B–E, magnification, x20; bar = 50 µm). MT cardiac section (in %interstitial fibrosis/mm2) is represented as mean ± SE.

 

Figure 4
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  		Fig. 4. Inflammatory infiltrate is associated with fibrosis in B6 TAC hearts. A and B: staining for the pan macrophage/monocyte marker MOMA-2 revealed inflammatory infiltrate in regions of perivascular and interstitial fibrosis in B6 TAC hearts (A, brown staining at arrows), whereas little to no infiltrate was detected in fibrotic regions of F1 (not shown) or 129 TAC hearts (B) (magnification, x40; bar = 25 µm). C: a low number of positively staining cells were detected in the hearts of all sham-operated mice as well as F1 and 129S1 TAC hearts. Mean cell count (per mm2 of tissue) is represented as mean ± SE. **P < 0.01.

 
Strain-specific hypertrophic response is supported by expression of molecular markers. Pressure overload and other cardiac stressors typically initiate induction of the fetal hypertrophic gene program, which is associated with pathological ventricular remodeling (59). The expression of atrial natriuretic peptide (Nppa), brain natriuretic peptide (Nppa), medium chain acyl dehydrogenase (Acadm), {alpha}-myosin heavy chain (Myh6), and beta-myosin heavy chain (Myh7), signature markers for cardiac hypertrophy, was measured by real-time quantitaive PCR using LV total RNA (Fig. 5). The reexpression of ventricular Nppb and Nppa, both cardioprotective hormones, is recognized as a marker for the induction of the embryonic gene program in ventricular hypertrophy (7). Although Nppa and Nppb expression was elevated in the LV of mice on all backgrounds after TAC relative to sham-treated mice of the same background, only LV from B6 and F1 mice showed significant changes. (Fig. 5, A and B). Interestingly, B6 mice (both sham-operated and TAC) had nearly threefold greater Nppb LV expression in the LV compared with F1 and 129S1 mice in respective treatment groups.


Figure 5
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  		Fig. 5. Comparison of relative expression of cardiac hypertrophy markers in LV by treatment and genetic background. A–E: expression of Nppa (atrial natriuretic peptide), Nppb (brain natriuretic peptide), and Myh7 [beta-myosin heavy chain (MHC)] were all significantly increased in B6 and F1 TAC mice, whereas Myh6 ({alpha}-MHC) and Acadm (medium chain acyl dehydrogenase) were significantly decreased in B6 TAC mice (*P < 0.05 vs. sham controls). Additionally, there were significant differences in the gene expression of some markers (Nppa, Nppb, and Acadm) among the sham groups. Fold change is relative to mean B6 sham values and is expressed as mean ± SE.

 
In failing mouse and human hearts, a shift in myosin heavy chain isoform expression from the adult form (Myh6) to the fetal form (Myh7) can occur, and a change in the ratio of these isoforms is associated with pathological hypertrophy. Pressure overload of C57BL/6J x SJL/J mice was previously reported to suppress the expression of Myh6 by more than 50% after 10 wk of banding (20). We also found decreased Myh6 expression (by ~75%), whereas Mhy7 expression was dramatically increased in B6 TAC LVs compared with that in controls (Fig. 5, C and D). Milder alterations in the expression of these markers were also observed in F1 mice. Additionally, within the TAC cohort, B6 mice had significantly lower Myh6 expression and higher Myh7 expression than F1 and 129S1 mice (P < 0.05).

During the transition to heart failure, the chief myocardial energy source switches from fatty-acid beta-oxidation to glycolyis, indicating a reversion to the fetal energy substrate preference pattern. Expression of Acadm, coding for a rate-limiting fatty-acid beta-oxidation enzyme, is reported to be downregulated during the progression from cardiac hypertrophy to failure (4). Consistent with this expression, Acadm expression was significantly decreased in B6 TAC mice compared with sham-operated mice as well other TAC mice (Fig. 5E; P < 0.05). Together with the echocardiographic data, altered gene expression of these markers may reflect a transition in the B6 mice to decompensated heart failure.

129S1 genetic modifiers delay transition to decompensated heart failure. To determine whether the 129S1 mice would eventually progress to an eccentric, decompensated phenotype, cardiac geometry and function were assessed serially by TTE for up to 8 wk post-TAC in all three strains (n = 3 to 4 TAC and 2 to 3 sham-operated mice; Fig. 6, A–D). By week 8 postsurgery, LVEDD, LVESD, and LVPWTh were significantly increased and %FS was significantly decreased in all TAC mice relative to the respective sham-operated controls, demonstrating that pressure overload ultimately leads to dilatation and decreased systolic function irrespective of genetic background. However, there were both a blunting and a temporal delay in the transition to decompensated heart failure in 129S1 and F1 mice. Interestingly, both 129S1 and B6 mice went through an initial stage characterized by pronounced concentric hypertrophy. In contrast, the B6 TAC mice developed characteristics of decompensated heart failure by 5 wk; the F1 and 129S1 TAC mice did not have significant evidence of LV dilation or systolic dysfunction until week 6 and 8 post-TAC, respectively. Even after 8 wk, surviving B6 TAC mice retained the largest percent increase in LV diameter and the greatest reduction in %FS relative to baseline values. Together, these data suggest that the 129S1 strain harbors protective genetic modifers that enable prolonged compensation to chronic pressure overload.


Figure 6
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  		Fig. 6. 129S1 genetic modifiers delay transition to decompensated heart failure. Temporal echocardiographic analysis of B6, F1, and 129S1 TAC mice postsurgery. Percent changes in LVEDD (A), LVESD (B), and LVPWThD (C) and percent fractional shortening (%FS; D) over time are shown. Echocardiographic measurements are represented as mean percent changes over baseline ± SE; n = 3–5 mice/group. *P < 0.05 or **P < 0.01 vs. sham controls.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
B6 and 129 strains are known to differ in baseline cardiovascular phenotypes such as blood pressure, morphological traits (i.e., atrial and ventricular size), heart rate variability, and cardiac metabolism, as well as in cardiac response to stressors (14, 21, 28, 67). Extending these findings, we present evidence that B6 and 129S1 mice manifest different cardiac phenotypes in response to TAC; moreover, genetic background influences the ability to maintain cardiac function in the face of prolonged cardiac stress as modeled by TAC-induced pressure overload.

Whereas B6 and 129S1 genetic backgrounds have a hypertrophic response, as indicated by increased heart weight and cardiomyocyte area over controls, they differ significantly in LV function over time, LVH geometric pattern, and pathological LV remodeling. By 5 wk postsurgery, B6 TAC mice have depressed contractile function, LV dilatation proportional to LV wall thickness, extensive fibrosis associated with inflammatory infiltrate, and significantly altered expression of classic hypertrophy markers. B6 mice also have the largest increase in LiWs and RV dilatation, suggesting passive liver congestion. Collectively, these observations suggest that B6 mice have LV remodeling consistent with eccentric hypertrophy (heart mass increases, but RWT does not change), accompanied by systolic dysfunction and right-sided heart failure. Although 129S1 TAC mice eventually progress to a "decompensated" phenotype, this transition is significantly delayed compared with B6 TAC mice. At 5 wk postsurgery, 129S1 mice have a phenotype characteristic of concentric hypertrophy in response to TAC, with increased cardiac mass and RWT and decreased LVEDD. Systolic function, estimated by FS and EF, is also enhanced, which is evidence of an increased contractile status, whereas cardiac output is significantly decreased, probably due to reduced LV chamber size and SV. Additionally, 129S1 mice have more pronounced increases in lung volumes after pressure overload, suggesting pulmonary congestion, possibly due to elevated LV end-diastolic pressure. These findings are consistent with clinical features observed in patients with diastolic dysfunction in the setting of concentric LVH. In response to TAC, F1 mice have a cardiac phenotype intermediate to B6 and 129S1 parental strains in many parameters but appear to be protected from early systolic dysfunction and cardiac fibrosis, responding similarly to parental 129S1 mice. This result implies that 129S1 genetic modifiers may protect against the more severe pathological changes seen in the hearts of B6 mice in response to TAC at earlier time points as well as delay transition to decompensated heart failure. This protective effect is significant, since, when all studies were combined, ~20% of all B6 TAC mice died by 3 wk post-TAC, with ~60% dying by 8 wk post-TAC. In contrast, no mortality was observed in 129S1 or F1 TAC mice.

Our echocardiographic and morphological data are comparable with prior studies investigating B6 response to pressure overload at similar time points (20, 37, 50). Less data are available for 129S1 mice, and no studies have reported a direct comparison between these widely used strains. Schmitt et al. (57) performed TAC on wild-type 129SvEv mice (substrain not specified) as controls for coisogenic mice carrying a mutation in {alpha}-cardiac myosin heavy chain (Myh403/+). Interestingly, they report that TAC-treated 129SvEv mice had significantly thickened LV walls and reduced LV diameter and were able to maintain normal contractile function for over 30 wk of postbanding (57). Data from our laboratory and others demonstrate that, when compared with B6 mice, 129S1 mice have a higher systolic pressure at baseline and in response to cardiovascular stressors (14, 26, 39, 71). Although there are no publications directly comparing B6 to 129S1 blood pressure response after TAC, a comparison of independently published values suggests that 129SvEv mice likely continue to maintain higher systolic pressures than B6 mice post-TAC (10, 22). Thus 129S1 TAC mice chronically maintain systolic function despite slightly higher afterload than B6 TAC mice.

Our data, together with reports in the literature, consistently indicate that the B6 and 129S1 strains differ greatly in their ability to adapt to acute pressure overload. Since many TAC studies use transgenic mice that have not been backcrossed to congenicity, it is possible that a combination of B6 and 129S1 modifiers would influence the response to pressure overload, contributing to variability. Thus the significance of an individual gene effect on pressure overload-induced LVH and remodeling could be masked or diluted by unidentified genetic modifiers in studies that do not adequately control for genetic background. Moreover, many published studies examine the cardiac response to TAC from 4–6 wk postsurgery, time points wherein our studies would predict that there would be more pronounced differences in response.

We also found significant differences in gene expression, fibrosis, organ weights, and cardiac morphology among control and sham-treated mice, underscoring the inherent cardiovascular differences of the parental strains. Similarly, the phenotype of transgenic mice overexpressing the sarcoplasmic reticulum Ca2+-binding protein calsequestrin (Casq1) are highly strain specific, showing wide variability when mice carrying the transgene are crossed with different inbred mouse strains (34, 35, 62, 69). In contrast to our results using the TAC model, which showed a dominant 129S1 modifier protecting against dilated cardiomyopathy, reciprocal backcrosses between DBA/2 and B6 mice revealed that the B6 background contributes a dominant susceptibility allele to the cardiomyopathic phenotype in the Casq1 transgene model, leading to dramatically reduced survival. Thus the same genetic insult may be significantly modified by genetic background, leading to variable cardiovascular compensation, even in the absence of additional stimuli.

Clinical studies suggest that screening for LVH geometric pattern in patients with EH may have prognostic value in stratifying patients based on cardiovascular risk. Yet, relatively little is known about the natural history of LV geometric remodeling in human hypertension, for example, whether the remodeling patterns are temporal stages in the development of the hypertensive heart from normal geometry through compensated hypertrophy to dilation and heart failure or whether every pattern is genetically or hemodynamically predisposed. A recent study followed changes in LVM and LV geometry in 100 hypertensive patients for 5 yr and concluded that LV geometry is a rather conservative entity since transformation from one pattern to another was rarely observed, even in untreated hypertensive patients. In particular, concentric LVH was fairly stable, without transformation to dilatation over the course of the study; however, the development of eccentric hypertrophy in patients with normal baseline geometry was observed (11). This suggests that independent determinants of LV geometry exist despite similar arterial hypertension.

Consistent with these clinical observations, polymorphisms in key components of the renin-angiotensin system (RAS) have been shown to be associated with LVH and LV geometry in hypertensive patients. An insertion/deletion polymorphism in intron 16 of the ACE gene has been associated with multiple cardiovascular disorders. Patients homozygous for the ACE deletion allele have higher serum and cardiac ACE, increased cardiac mass, elevated RWT (characteristic of concentric hypertrophy), and an overall increased risk for cardiovascular disease (22, 48, 54). Evidence from several studies indicates that B6 and 129 mice also have genetic differences in RAS components, which translate into variable RAS activity at baseline and upon perturbation (39, 58). For example, treatment of deoxycorticosterone acetate/salt-induced hypertension with ramipril, an ACE inhibitor, abrogated the development of hypertension and LVH in 129S6/SvEvTac mice (51), but in B6 mice, LVH was only partially prevented without normalizing systolic pressure (49), reminiscent of the variable beneficial response in human hypertensive patients to RAS blockers. Interestingly, B6 and 129 mouse strains also harbor single nucleotide polymorphisms at the Agtr1a receptor locus, which was found to be highly polymorphic in the promoter region and linked to blood pressure variation (70). Together, these observations suggest that B6 and 129 mice have innate differences in RAS activity as well as RAS modulation in response to cardiovascular stress, highlighting the relevance of our models pertaining to the human cardiovascular responses to similar pressure overload.

In light of the unique patterns of LV remodeling in humans, continued exploration and comparison of inbred strain-specific responses to pressure overload will lead to better preclinical models for partitioning LVH by LV geometry. These mouse models will provide tools to investigate changes in LV remodeling and LV geometry over time, to decipher genetic networks that may contribute to different LV geometric patterns, and to compare benefits of pharmaceutical intervention based on LV geometry subtype. It is now clear that similar genes and signaling pathways regulate the development of the heart and vasculature, as well as cardiovascular response to stressors, in mice and humans. Molecular genetics will play an important role in discovering novel methods of diagnosing and treating patients with cardiovascular diseases (2, 43, 64), and mouse models for defined subsets of patients will be essential for preclinical therapeutic studies. Ultimately, these studies will contribute to understanding why some patients with chronic pressure overload manifest concentric LVH, whereas others progress to LV dilatation and systolic dysfunction.

As exemplified in our studies, the selection of appropriate genetic background controls is essential when performing gene function studies using engineered mouse models to study the response to TAC to ensure interpretable results. Although the vast majority of mutations are produced in 129 embryonic stem cells before generating chimeras, the resulting chimeras are typically bred with B6 mice to produce F1 hybrids followed by intercrossing of the offspring to generate homozygous mutants on mixed B6 and 129 genetic backgrounds for analysis. Frequently, viable homozygous mutant mice are maintained as closed colonies for study. Our results show that F1 hybrids from B6 and 129S1 mice are not appropriate controls for either of these experiments since the F1 response to TAC will not reflect either the B6 or 129 strains. If mutations are maintained as a closed breeding colony, there are no appropriate controls for genetic background since the composition of the background will be variable and unpredictable and the genetic background effects described here may incorrectly be associated with the mutation under study. Rather, the only adequate controls are littermates with an equivalently random distribution of the B6 and 129 genomes. However, because of the strong genetic background influence in response to TAC-induced pressure overload, significantly larger groups of mice will be required due to high intragroup variation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This research was supported by National Institutes of Health Grants K08-HL-070304 and R01-HL-074219 (to S. S. Smyth) and HD-039896 (to D. W. Threadgill). This research was also supported in part by center (P30-ES-10126) and training (T32-ES-007126) grants from the National Institute of Environmental Health Sciences.


   ACKNOWLEDGMENTS
 
We thank Kirk McNaughton and Carolyn Suitt for excellent histological assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. W. Threadgill, Dept. of Genetics, CB#7264, Univ. of North Carolina, Chapel Hill, NC 27599 (e-mail: dwt{at}med.unc.edu)

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.


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