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(Circulation Research. 2000;86:802.)
© 2000 American Heart Association, Inc.

Integrative Physiology

Determinants of the Cardiomyopathic Phenotype in Chimeric Mice Overexpressing Cardiac Gs

Dorothy E. Vatner¹, Gui-Ping Yang¹, Yong-Jian Geng, Kuniya Asai, Jeung S. Yun, Thomas E. Wagner, Yoshihiro Ishikawa, Sanford P. Bishop, Charles J. Homcy, Stephen F. Vatner

From the Weis Center for Research (D.E.V., G.-P.Y., Y.-J.G., K.A., Y.I., S.P.B., S.F.V.), Pennsylvania State University College of Medicine, Danville, Pa, and Allegheny University of the Health Sciences, Pittsburgh, Pa; Oncology Research Institute (J.S.Y., T.E.W.), Greenville Hospital System and Clemson University, Greenville, SC; and COR Therapeutics, Inc (C.J.H.), South San Francisco, Calif.

Correspondence to Stephen F. Vatner, MD, Director of the Henry Hood Research Program, Charles B. Degenstein Professor, Weis Center for Research, Pennsylvania State University College of Medicine, 100 N Academy Ave, Danville, PA 17822-2601. E-mail svatner{at}psghs.edu

	Abstract

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Abstract—Mice with overexpressed cardiac Gs develop cardiomyopathy, characterized by myocyte hypertrophy and extensive myocardial fibrosis. The cardiomyopathy likely involves chronically enhanced ß-adrenergic signaling, because it can be blocked with long-term propranolol treatment. It remains unknown whether the genotype of the myocyte is solely responsible for the progressive pathological changes. A chimeric population in the heart should answer this question. Accordingly, we developed a chimeric animal, which combined cells from a transgenic overexpressed Gs parent and a Rosa mouse containing the LacZ reporter gene, facilitating identification of the non–Gs cells, which express a blue color with exposure to ß-galactosidase. We studied these animals at 14 to 17 months of age (when cardiomyopathy should have been present), with the proportion of Gs cells in the myocardium ranging from 5% to 88%. ß-Galactosidase staining of the hearts demonstrated Gs and Rosa cells, exhibiting a mosaic pattern. The fibrosis and hypertrophy, characteristic of the cardiomyopathy, were not distributed randomly. There was a direct correlation (r=0.85) between the extent of myocyte hypertrophy (determined by computer imaging) and the quantity of Gs cells. The fibrosis, determined by picric acid Sirius red, was also more prominent in areas with the greatest Gs cell density, with a correlation of r=0.88. Thus, the overexpressed Gs can exert its action over the life of the animal, resulting in a local picture of cardiomyopathic damage in discrete regions of the heart, where clusters of the overexpressed Gs cells reside, sparing the clusters of normal cells derived from the normal Rosa parent.

Key Words: hypertrophy • cardiomyopathy • heart failure • ß-adrenergic receptor • sympathetic nervous system

	Introduction

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With the advent of molecular biology and genetic transformation of animal models and with the potential for gene therapy in patients, an important question arises, ie, does the resulting phenotype reflect precisely the altered genotype, or have other compensatory changes modified the original phenotype? For example, the mouse with overexpressed cardiac Gs responds to sympathetic stimulation with enhanced ß-adrenergic signal transduction as a young adult.^{1 2 3} However, over the life of this animal, ß-adrenergic receptor desensitization mechanisms are ineffective in the presence of overexpressed Gs,⁴ and the prolonged exposure to enhanced ß-adrenergic receptor signaling exerts a toll, resulting in a picture of cardiomyopathy.^{2 5} This cardiomyopathy is reflected by depressed left ventricular (LV) function with a dilated heart, and the architecture of the heart is characterized by extensive interstitial fibrosis and hypertrophy of myocytes. The question is posed again more specifically: Is the cardiomyopathy observed in older mice with overexpressed Gs due to chronically enhanced ß-adrenergic signaling, resulting in reduced subendocardial coronary reserve, an imbalance between myocardial oxygen demand and supply, with consequent myocardial ischemia and consequent myocyte necrosis and apoptosis? Or, does the overexpressed Gs exert an effect locally or in the microenvironment that leads to hypertrophy, myocyte necrosis, and fibrosis?

One approach to address this question is to study a chimeric animal with a heart composed of both normal myocytes and myocytes overexpressing cardiac Gs. Our goal was to study these chimeras, when the cardiomyopathy should have been fully manifest (14 to 17 months of age). By using a Rosa parent, whose cells are tagged with the LacZ reporter gene, the origin of the cells, ie, from the normal Rosa or Gs parent, could be identified by exposing the cells to ß-galactosidase, which results in the Rosa cells expressing a blue color. The major goal was to quantitate the extent of fibrosis and the extent of myocyte hypertrophy in cells of overexpressed Gs origin and to compare this with normal Rosa cells, to determine whether the characteristic features of cardiomyopathy, fibrosis and hypertrophy, exhibit a predilection for the overexpressed Gs cells or are expressed in areas of normal Rosa cells as well.

	Materials and Methods

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Chimeric mice were generated by the investigators by the fusion of morulae from which the zona pellucida had been removed by pronase treatment. The morulae from a line of transgenic mice expressing the LacZ gene controlled by a promoter, which directs expression to essentially all tissues (Rosa 26, Jackson Laboratories), on a Balb C background with an albino phenotype were fused with the morulae from the Gs transgenic mice on a C57/SJL background with an agouti phenotype. Of 386 fusions, 338 fusions were successful. The fused embryos were transferred to 42 pseudopregnant female recipients. Of these, 7 chimeric offspring originating from different litters survived 14 to 17 months so that they could be studied at a time when the cardiomyopathy should have been manifest. The numbers of animals studied included 7 chimeric, 3 Rosa controls, and 6 Gs wild-type controls. Animals used in this study were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (DHHS publication No. [NIH] 83-23, revised 1996).

Physiology
Heart rate was measured using telemetry techniques in the conscious, unrestrained state⁶ in 7 chimeric mice and 6 age-matched Gs wild-type controls. Arterial pressure was measured in 6 chimeric mice using a 1.4F micromonometer catheter (Millar Instruments). Echocardiographic assessment was possible in 6 of 7 chimeric mice. One animal did not tolerate the anesthesia. After determination of body weight, mice were anesthetized with ketamine (0.065 mg/g), acepromazine (0.002 mg/g), and xylazine (0.013 mg/g), which was injected intraperitoneally. The procedure for echocardiography (Apogee X-200; Interspec, Inc) has been described in previous reports from our laboratory.^{2 5}

Tissue Preparation and Histochemical Examination
After the echocardiography, the atria and great vessels were removed, and the combined left and right ventricles were weighed. Cryosections cut at 10 µm were incubated overnight at 37°C in ß-galactosidase, which results in expression of a blue color in the cells from the Rosa parent but does not affect the color of Gs cells. One entire cross section from each mouse was examined to determine the fraction of Gs versus Rosa cells. Cryosections stained with ß-galactosidase were subsequently stained with picric acid Sirius red for collagen. Picric acid Sirius red–stained collagen, highlighted by the Metamorph system, was expressed as volume percent collagen for each region, which was classified according to the percentage of Gs cells. On average, 7 areas were analyzed from each mouse. Staining was adequate in 6 of 7 chimeric animals. Myocyte cross-sectional area was measured on 10-µm-thick cryosections stained in ß-galactosidase solution overnight followed by laminin immunostaining (Sigma) to outline the basement membrane of cardiac myocytes. This procedure used a biotinylated secondary antibody and ExtrAvidin peroxidase for colorization. Individual regions within the ventricular wall were analyzed by parental origin based on ß-galactosidase staining and classified according to the percentage of Gs cells. On average, 7 (range 5 to 9) areas were analyzed from each mouse. Staining was adequate in 6 of 7 animals.

Statistics
The myocyte cross-sectional area and the volume percent of collagen were correlated using linear regression analysis versus the percentage of Gs cells in the chimeric animals. All data are expressed as mean±SEM. All statistical data were analyzed with ANOVA using a computer with appropriate software (StatView).

	Results

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Physiology
In conscious, unrestrained mice, we observed no differences in heart rates between the chimeric mice (624±24 bpm, n=7) and wild-type mice (611±18 bpm, n=6) (Figure 1).

View larger version (13K): [in this window] [in a new window]   Figure 1. Average±SEM LV fractional shortening was reduced in chimeric mice (top). Heart rates, derived using the telemetry system in conscious chimeric mice, were not different (bottom).

Echocardiography (n=6) demonstrated that fractional shortening was significantly less in the chimeric mice (23±2%, n=7) compared with the control Gs wild-type littermates (32±1%; n=6) (Figure 1). One control Rosa mouse was studied and found to have normal LV fractional shortening (38.1%). Other than fractional shortening, which was reduced, and arterial pressure, which was elevated, there were no differences in hemodynamics or LV mass between the chimerics and controls (Table).

View this table: [in this window] [in a new window]   Table 1. Hemodynamics and LV Mass

Morphology
The chimeric mice exhibited a variegated coat color, reflecting the expression of the different parental genotypes. The myocytes in the hearts also reflected the origin of both parents (Figure 2), with the blue-stained cells from the normal Rosa parent and the unstained cells reflecting the overexpressed Gs parent. Note that the two different cell types were not distributed randomly but rather were clustered (Figure 2), as might be predicted from the techniques used to produce the chimeras. Histopathological evaluations at higher power revealed a pattern of larger cells and enhanced interstitial fibrosis clustered around the Gs cells (Figure 2). When this was quantitated for the 6 chimeric animals, there was a direct correlation between the extent of myocyte hypertrophy and the percentage of Gs cells for the cells overexpressing Gs (r=0.85) (Figure 3). There was no correlation between Rosa myocyte size and percentage of Gs cells (r=0.32). The y-intercept for the cell size in the Rosa chimerics (152 µm²) was not different from the average Gs wild-type control (154±2 µm²) or the Rosa control (155±7 µm²). The extent of fibrosis in the 6 chimerics was correlated to the density of Gs cells (r=0.88) (Figure 4). The y-intercept for the volume percent collagen in the chimerics (2.6%) was not different from the Gs wild-type controls (2.4±0.2%) or the Rosa controls (2.3±0.4%). Body weights and LV/body weights were not different between groups.

View larger version (85K): [in this window] [in a new window]   Figure 2. A, Low-power micrograph of a chimeric mouse heart showing the clusters of normal Rosa cells in blue and Gs cells not staining blue. B, High-power micrograph of a section from a chimeric heart showing hypertrophy of Gs myocytes, which do not stain blue. C, High-power micrograph of a section from a chimeric heart showing increased fibrosis (red color indicates picric acid Sirius red staining for collagen) in areas of Gs myocytes not staining blue.

View larger version (17K): [in this window] [in a new window]   Figure 3. Regression analysis comparing myocyte cross-sectional area versus percentage of Gs cells, ranging from 0% to 100% Gs cells. The relationship was significant for Gs cell size (P<0.01, r=0.85 [•]), but there was no significant relationship for Rosa cell size []). These data were collected from 6 chimeric hearts. No control hearts are included.

View larger version (12K): [in this window] [in a new window]   Figure 4. Regression analysis comparing the percentage of fibrosis versus Gs cells, ranging from 0% to 100%. There was significantly more fibrosis in the areas with greater concentrations of Gs cells, with a significant correlation (P<0.01, r=0.88). These data were collected from 6 chimeric hearts. No control hearts are included.

Immunoblotting
To determine the extent of overexpression of Gs in the chimeric heart, we performed immunoblotting using total protein extracts from the chimeric, Gs, Rosa, and wild-type control mice. As expected from the histology, there was a graded increase in cardiac Gs from wild-type controls to the chimeras to the pure, overexpressed Gs hearts. The Gs hearts showed a 3- to 5-fold overexpression, as demonstrated previously,⁴ whereas the chimeric demonstrated an intermediate amount of cardiac Gs.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, the key features of cardiomyopathy, interstitial fibrosis and myocyte hypertrophy, were evident in these unique chimeric transgenic animals. We asked the question: Is the resultant phenotype of the altered genotype solely the consequence of the altered gene, or are there other effects in vivo that alter the phenotype secondarily?

It is possible that the enhanced ß-adrenergic signaling with increased heart rate, LV wall stress, and contraction in the face of limited coronary reserve is, in part, the cause of hypertrophy and cardiomyopathy in the overexpressed Gs mouse.^{2 5} If this is the mechanism, then all myocytes in the chimeric heart would be hypertrophied. In contrast, the fibrosis and hypertrophy were limited to the Gs cells. This suggests that the adverse effects of overexpressed Gs are expressed locally and are not due to the secondary changes of enhanced global LV contractility, wall stress, tachycardia, and reduced coronary reserve in the subendocardium. Both the extent of hypertrophy and fibrosis increased proportionally to the concentration of Gs cells. This indicates that simple expression of the gene was not sufficient to induce the full cardiomyopathic phenotype, because the hypertrophy and fibrosis were not evenly distributed among the Gs cells, regardless of their location. In areas of low concentration of Gs cells, little fibrosis and hypertrophy were observed. Rather, it was in areas of larger concentrations of clustered Gs cells where hypertrophy and/or fibrosis were most prominent, suggesting that local areas of enhanced contraction or neurohormonal signaling were responsible for the cardiomyopathic phenotype. These observations, in combination with a recent study from our laboratory demonstrating that chronic propranolol rescued the cardiomyopathic phenotype, implicate ß-adrenergic signaling.⁷ Thus, the effects of locally amplified ß-adrenergic signaling are sufficient to induce phenotypic characteristics of cardiomyopathy. Moreover, these changes are due to the chronically enhanced contraction or to the altered microenvironment created in the Gs clusters, and this effect is proportional to the numbers of cells that exhibit enhanced ß-adrenergic signaling.

Another feature of the overexpressed Gs model is chronic tachycardia.⁶ Interestingly, the tachycardia in the chimeric transgenic mice was attenuated, ie, the heart rates were not different, in the chimeras compared with the wild-type controls. Why the heart rates were not different is not clear. Given that the Gs and Rosa cells were not distributed homogeneously, it is possible that the sinoatrial nodal environment is not influenced in a linear fashion by the overall percentage of Gs cells in the entire heart. However, the lack of relationship between heart rate and fibrosis or hypertrophy suggests that the mechanism of hypertrophy and interstitial fibrosis in this model is not exclusively due to the more rapid rate of contraction that is characteristic of Gs mice. Even if heart rate had been significantly elevated, a heart rate mechanism would not be responsible for the hypertrophy and fibrosis, because all cells in the chimeric heart experience the same frequency of contraction. It is conceivable that the enhanced ß-adrenergic signaling increases the local force of contraction in isolated parts of the heart containing the overexpressed Gs cells. However, it is equally plausible that other signaling pathways that play a key role in hypertrophy and cell death are responsible for the pathological changes, independent from global or local hemodynamics. In either event, it is important that the altered genotype is responsible for the altered phenotype on a local level. It is interesting to speculate that the genotypic alteration may be the reason that this Gs model does not desensitize and consequently permits the development of myocyte hypertrophy and cardiomyopathy.⁴ These results also have implications for understanding the effects of ß-blockade therapy in heart failure where chronically enhanced sympathetic signaling to the heart occurs. In view of these findings, ß-adrenergic receptor blockade clearly has effects that reach beyond simple reduction in heart rate.

Communication among cells in the heart (myocytes, fibroblasts, endothelial cells) through electrical or paracrine pathways is important in determining the overall progression of cardiac architecture and function.⁸ With communication between Gs and Rosa cells, there would have been less of a clear-cut distinction between the normal and Gs cells in terms of hypertrophy and fibrosis. The data from the present study support the position that the effects of the overexpressed Gs are predominantly local, because adjacent clusters of Gs or Rosa cells demonstrated markedly different phenotypes. The present data support the position that the genotype in combination with the microenvironment is responsible for the hypertrophy and fibrosis characteristic of the cardiomyopathic Gs phenotype.

An important limitation to the interpretation of the data from the present study is derived from the nature of the chimeric model. As noted above, the cells from the two lines were not distributed randomly or homogeneously but were clustered. Therefore, clusters of Gs cells developing hypertrophy and fibrosis in one part of the left ventricle could affect LV function out of proportion to the total number of cells present. However, this limitation in experimental design does not have an impact on the major conclusion of this study, demonstrating hypertrophy and fibrosis in one cell type (Gs) to the exclusion of the other. One final point needs to be mentioned. This chimeric approach is useful not only to address questions raised in this study relevant to this transgenic animal model but also for other genetically altered models. For example, some altered genotypes will be toxic (ie, embryonically lethal) when expressed confluently in the heart and lead to premature death, particularly in homozygous models. However, if these altered cells could be expressed in a mosaic fashion, the remainder of the normal cells could permit the animal to survive to adulthood and allow examination of the local action of either gene overexpression or gene deletion in the heart.⁹

	Acknowledgments

 
This study was supported in part by United States Public Health Service Grants HL59139, R01 HL33107, R01 AG14121, R01 HL33065, R01 HL62716, and R01 HL59874.

	Footnotes

 
¹ Both authors contributed equally to this study.

Received January 3, 2000; accepted January 13, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Gaudin C, Ishikawa Y, Wight DC, Vatner DE, Mahdavi V, Nadal-Ginard B, Wagner TE, Homcy CJ. Overexpression of Gs protein in the hearts of transgenic mice. J Clin Invest. 1995;95:1676–1686.
2. Iwase M, Bishop SP, Uechi M, Vatner DE, Shannon RP, Kudej R, Wight DC, Wagner TE, Ishikawa Y, Homcy CJ, Vatner SF. Adverse effects of chronic endogenous sympathetic drive induced by cardiac Gs overexpression. Circ Res. 1996;78:517–524.[Abstract/Free Full Text]
3. Kim S-J, Yatani A, Vatner DE, Yamamoto S, Ishikawa Y, Wagner TE, Shannon RP, Kim Y-K, Takagi G, Asai K, Homcy CJ, Vatner SF. Differential regulation of inotropy and lusitropy in overexpressed Gs myocytes through cAMP and Ca²⁺ channel pathways. J Clin Invest. 1999;103:1089–1097.[Medline] [Order article via Infotrieve]
4. Vatner DE, Asai K, Iwase M, Ishikawa Y, Wagner TE, Shannon RP, Homcy CJ, Vatner SF. Overexpression of myocardial Gs prevents full expression of catecholamine desensitization despite increased ß-ARK. J Clin Invest. 1998;101:1916–1922.[Medline] [Order article via Infotrieve]
5. Iwase M, Uechi M, Vatner DE, Asai K, Shannon RP, Kudej RK, Wagner TE, Wight DC, Patrick TA, Ishikawa Y, Homcy CJ, Vatner SF. Cardiomyopathy induced by cardiac Gs overexpression. Am J Physiol. 1997;272:H585–H589.[Abstract/Free Full Text]
6. Uechi M, Asai K, Osaka M, Smith A, Sato N, Wagner TE, Ishikawa Y, Vatner DE, Shannon RP, Homcy CJ, Vatner SF. Depressed heart rate variability and arterial baroreflex in conscious transgenic mice with overexpression of cardiac Gs. Circ Res. 1998;82:416–423.[Abstract/Free Full Text]
7. Asai K, Yang G-P, Geng Y-J, Takagi G, Bishop SP, Ishikawa Y, Shannon RP, Wagner TE, Vatner DE, Homcy CJ, Vatner SF. ß-Adrenergic receptor blockage arrests myocyte damage and preserves cardiac function in the transgenic Gs mouse. J Clin Invest. 1999;104:551–558.[Medline] [Order article via Infotrieve]
8. Matsusaka T, Katori H, Inagami T, Fogo A, Ichikawa I. Communication between myocytes and fibroblasts in cardiac remodeling in angiotensin chimeric mice. J Clin Invest. 1999;103:1451–1458.[Medline] [Order article via Infotrieve]
9. Cichowski K, Shih TS, Schmitt E, Santiago S, Reilly K, McLaughlin ME, Bronson RT, Jacks T. Mouse models of tumor development in neurofibromatosis type 1. Science. 1999;286:2172–2176.[Abstract/Free Full Text]

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