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(Circulation. 2003;108:2926.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Common Genomic Response in Different Mouse Models of ß-Adrenergic–Induced Cardiomyopathy

Vinciane Gaussin, PhD; James E. Tomlinson, PhD; Christophe Depre, MD, PhD; Stefan Engelhardt, MD; Christopher L. Antos, PhD; Gen Takagi, MD, PhD; Lutz Hein, MD; James N. Topper, MD, PhD; Stephen B. Liggett, MD; Eric N. Olson, PhD; Martin J. Lohse, MD; Stephen F. Vatner, MD; Dorothy E. Vatner, MD

From the University of Medicine and Dentistry of New Jersey and New Jersey Medical School (V.G., C.D., G.T., S.F.V., D.E.V.), Newark, NJ; Millennium Pharmaceuticals Inc (J.E.T., J.N.T.), South San Francisco, Calif; University of Wurzburg (S.E., L.H., M.J.L.), Wurzburg, Germany; UT Southwestern Medical Center (C.L.A., E.N.O.), Dallas, Tex; and University of Cincinnati Medical Center (S.B.L.), Cincinnati, Ohio.

Correspondence to Dr Stephen F. Vatner, UMDNJ, Department of Cell Biology and Molecular Medicine, Cardiovascular Research Institute, 185 S Orange Ave, MSB-G609, Newark, NJ 07101. E-mail vatnersf{at}umdnj.edu

Received March 24, 2003; de novo received June 6, 2003; accepted August 12, 2003.

	Abstract

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Background— Although ß-adrenergic receptor (AR) blockade therapy is beneficial in the treatment of heart failure, little is known regarding the transcriptional mechanisms underlying this salutary action.

Methods and Results— In the present study, we screened mice overexpressing Gs, ß₁AR, ß₂AR, or protein kinase A to test if a common genomic pathway exists in different models with enhanced ß-adrenergic signaling. In mice overexpressing Gs, differentially expressed genes were identified by mRNA profiling. In addition to well-known markers of cardiac hypertrophy (atrial natriuretic factor, CARP, and ß-myosin heavy chain), uncoupling protein 2 (UCP2), a protein involved in the control of mitochondrial membrane potential, and four-and-a-half LIM domain protein-1 (FHL1), a member of the LIM protein family, were predicted to be upregulated. Upregulation of these genes was confirmed by quantitative reverse transcriptase–polymerase chain reaction at all time points tested during the development of cardiomyopathy in mice overexpressing Gs. In mice overexpressing ß₁AR, ß₂AR, or protein kinase A, increased UCP2 and FHL1 expression was also observed at the onset of cardiomyopathy. ßAR blockade treatment reversed the cardiomyopathy and suppressed the increased expression of UCP2 and FHL1 in mice overexpressing Gs.

Conclusions— UCP2 and FHL1 are important candidate genes that correlate with the development of ßAR-induced cardiomyopathy in different mouse models with enhanced ßAR signaling. In addition to preserving cardiac function, ßAR blockade treatment also prevents the genomic regulation that correlates with the onset of heart failure.

Key Words: receptors, adrenergic, beta • cardiomyopathy • genomics

	Introduction

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The deleterious effects of chronic ß-adrenergic receptor (AR) stimulation in the heart have been documented in several mouse models. Transgenic mice overexpressing Gs selectively in the myocardium develop cardiomyopathy with age, characterized by left ventricular (LV) dilation and hypertrophy, reduced LV function, fibrosis, apoptosis, and premature mortality.^1,2 Mice overexpressing ß₁AR,^3,4 high levels of ß₂AR,^5,6 or protein kinase A (PKA)⁷ also develop cardiomyopathy with aging. In patients with heart failure, ßAR blockade therapy has been proven to be beneficial.^8–11 However, little is known regarding the transcriptional mechanisms underlying this salutary action. In the present study, we screened different mouse models with enhanced ßAR signaling to identify potential genomic targets of ßAR blockade therapy. Using a sensitive, differential gene expression technology¹² to study gene expression profiles in Gs-overexpressing mice, we identified unexpected genes that participate in the development of cardiomyopathy in these mice. Furthermore, we show that these candidate genes are involved in the development of cardiomyopathy in mice overexpressing ß₁AR, ß₂AR, or PKA. Most interestingly, the preservation of cardiac function brought by ßAR blockade treatment in mice overexpressing Gs normalizes the expression of uncoupling protein 2 (UCP2) and four-and-a-half LIM domain protein-1 (FHL1), showing that ß-blockers alleviate the genomic dysregulation that accompanies heart failure.

	Methods

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Animals
Mice overexpressing Gs,¹ ß₁AR,³ ß₂AR,⁵ or PKA⁷ have been previously described. Mice overexpressing ß₂AR were originally described by Milano et al⁵ and purchased from Jackson Laboratories (Bar Harbor, Maine). Genotyping^1,3,5,7 and ßAR blockade treatment¹³ were carried out as described. Three to 9 animals have been studied in each group.

Differential Gene Expression Analysis by GeneCalling
Total RNA was prepared using Tri reagent (Sigma) from the hearts of 3-, 6-, 9-, 12-, and 15-month-old Gs-overexpressing mice or control littermates. Three animals per age group and genotype were analyzed individually. GeneCalling reactions¹⁴ were performed as described in Figure 1. Briefly, double-stranded DNA was generated from the pools of mRNA and digested with 96 pairs of restriction enzymes. The fragments were amplified by polymerase chain reaction (PCR) in the presence of fluorescent primers and separated according to their size by capillary electrophoresis. The different fragmentation profiles were compared to identify fragments with differential fluorescence, ie, expression level, between 2 samples. The identification of the gene corresponding to the fragment of interest relied on the size of the fragment and the 6-nucleotide sequence in 5' and 3' that corresponded to the restriction sites used to generate the fragment. Confirmation of the identity of a gene was done by competitive PCR.

View larger version (31K): [in this window] [in a new window]   Figure 1. GeneCalling technology.14 RNA samples are reverse transcribed into cDNAs, which, in turn, are digested by 96 pairs of restriction enzymes (RE). cDNA fragments are amplified by PCR in the presence of fluorescent primers and separated according to their size by capillary electrophoresis. Differential fluorescence of the same fragment in 2 different samples suggests differential level of expression of the corresponding gene. Identification of the gene is based on the size of the fragment and the 6-nucleotide sequence in 5' and 3' that corresponds to the restriction sites used to generate the fragment.14 Confirmation of a gene is done by competitive PCR. If correctly identified, the peak corresponding to the confirmed fragment is blunted (dotted line).

Quantitative Reverse Transcriptase-PCR
Total RNA from transgenic mice and control littermates was prepared from frozen heart tissues using Tri reagent (Sigma). The mRNA of interest was reverse transcribed according to standard protocol. Real-time quantitative PCR (qPCR) (7700 Prizm, Perkin-Elmer/Applied Biosystems) was performed with specific primers and fluorogenic probes derived with FAM and TAMRA (Table 1).^15,16 Internal standards were prepared for each transcript from its PCR-amplified cDNA after ligation of the T7 promoter.¹⁶ Results were normalized to 36B4¹⁶ or cyclophilin.¹⁷

View this table: [in this window] [in a new window]   TABLE 1. Characteristics of the qPCR Assays Used in This Study

Statistics
Results obtained for transgenic mice were compared with those obtained for their age-matched control littermates using the unpaired Student t test.

	Results

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Genomic Profiling
We compared the gene expression profiles in mice overexpressing Gs¹ and control littermates at 3, 6, 9, 12, and 15 months of age. Candidate genes with at least 2.5-fold predicted differential expression were identified (Table 2). As expected, the transgene, Gs, was predicted to be high in Gs-overexpressing mice at all ages analyzed. In addition, genes of the following categories were predicted to have a modified expression pattern: extracellular matrix deposition (collagen III), metabolism (UCP2), transcription factors (FHL1 and cardiac ankyrin repeat protein [CARP]), stress (atrial natriuretic factor [ANF]), and myofilament (myosin heavy chain [MHC] and M-protein). Of particular interest, UCP2, a protein involved in the control of mitochondrial membrane potential,¹⁸ and FHL1, a new member of the LIM protein family,¹⁹ are known genes with unknown function in the heart. Interestingly, UCP2 and FHL1 expression were also found to increase during heart failure in different mouse models^20,21 and in patients.^22,23 These genes might therefore bring new insights into the understanding of the development of cardiomyopathy induced by chronic stimulation of ßAR signaling.

View this table: [in this window] [in a new window]   TABLE 2. Differential Gene Expression Analysis: Candidate Genes Predicted to Have Increased (+) or Decreased (-) Expression in Gs-Overexpressing Mice Compared With Control Littermates at the Indicated Ages

Uncoupling Protein 2
After capillary electrophoresis, the peak corresponding to UCP2 was found to increase in 3-month-old Gs-overexpressing mice (Figure 2A) and in 6-, 9-, 12-, and 15-month-old transgenic mice (not shown) compared with age-matched control mice. Competitive PCR was performed to confirm that the peak indeed corresponds to UCP2 (Figure 2A). Quantification of the changes in UCP2 expression during aging of Gs-overexpressing mice was done by qPCR. From 3 months of age onward, UCP2 expression was doubled in Gs-overexpressing mice compared with control littermates (Figure 2B).

View larger version (35K): [in this window] [in a new window]   Figure 2. UCP2 expression is increased in Gs-overexpressing mice with aging. A, Capillary electrophoresis traces are shown for 3-month-old control littermates (top) and Gs mice before (middle) and after (bottom) competitive PCR. Traces from 3 different animals in each group are shown. Arrow indicates the peak corresponding to UCP2. B, Quantification of UCP2 expression by qPCR in the heart of Gs-overexpressing mice (•) and control littermates () harvested at the indicated ages. *P<0.05 between transgenic mice and respective control littermates.

Four-and-a-Half LIM Domain Protein-1
After capillary electrophoresis, the peak corresponding to FHL1 was increased in 3-month-old Gs-overexpressing mice (Figure 3A). This was also observed in 6, 9, 12, and 15-month-old transgenic mice (not shown). After competitive PCR, the corresponding peak was blunted, confirming the identity of the gene (Figure 3A). Quantification of the changes in FHL1 expression during aging in Gs-overexpressing mice was done by qPCR. From 3 months of age onward, a 2- to 3-fold increase in FHL1 expression was seen in Gs-overexpressing mice compared with control littermates (Figure 3B). In adult striated muscle, 3 isoforms of FHL have been described, FHL-1, -2, and -3.²⁴ We found increased expression of FHL1 in Gs-overexpressing mice, but no change in expression of FHL-2 or FHL-3 could be detected (Figures 3B through 3D).

View larger version (42K): [in this window] [in a new window]   Figure 3. FHL1 expression is increased in Gs-overexpressing mice with aging. A, Capillary electrophoresis traces are shown for 3-month-old control littermates (top) and Gs mice before (middle) and after (bottom) competitive PCR. Traces from 3 different animals in each group are shown. Arrow indicates the peak corresponding to FHL1. B through D, Quantification of level of expression of FHL1 (B), FHL2 (C), and FHL3 (D) by qPCR in the heart of Gs-overexpressing mice (•) and control littermates () harvested at the indicated ages. *P<0.05 between transgenic mice and respective control littermates.

Other Genes
Additional candidate genes were validated by qPCR during aging in Gs-overexpressing mice. As expected, expression of the transgene was high in Gs-overexpressing mice at all ages analyzed compared with control littermates (Figure 4A). The expression of markers of cardiac hypertrophy, ie, ANF, ß-MHC, and CARP, were increased in 15-month-old Gs-overexpressing mice (Figures 4D through 4F). Because changes in the expression of these genes occurred in old transgenic mice, they are likely to be a consequence and not the cause of the cardiomyopathy developed in these mice. The prediction of an upregulation of -MHC (Table 2) did not reach a statistical significance after validation by qPCR (not shown). An upregulation of collagen type III was seen in Gs-overexpressing mice throughout aging and is related to the fibrosis seen in these mice² (Figure 4B). M-protein, which is part of the M-band, is the only gene found so far with decreased expression in mice overexpressing Gs (Figure 4C).

View larger version (28K): [in this window] [in a new window]   Figure 4. Confirmation of candidate gene expression changes by qPCR. Expression of the Gs transgene (A), collagen III (B), M-protein (C), ß-MHC (D), ANF (E), and CARP (F) was measured by qPCR in the heart of Gs-overexpressing mice (•) and control littermates () harvested at the indicated ages. *P<0.05 between transgenic mice and respective control littermates.

ßAR Blockade
To test whether the increased expression of UCP2 and FHL1 seen in Gs-overexpressing mice is attributable directly to the chronic stimulation of ßAR signaling in these mice, a ßAR-blockade experiment was designed. We previously showed that ßAR blockade was sufficient to arrest myocyte damage and preserve cardiac function in mice overexpressing Gs.¹³ The increased expression of UCP2 and FHL1 seen in Gs-overexpressing mice in the absence of treatment was decreased significantly by the ßAR-blockade treatment, as measured by qPCR (Figures 5A and 5B). Because ßAR blockade can decrease -MHC expression,²⁵ we verified that the treatment did not affect the expression of the Gs transgene, which is under the control of the -MHC promoter¹ (Figure 5C).

View larger version (12K): [in this window] [in a new window]   Figure 5. Increased expression of UCP2 and FHL1 seen in Gs-overexpressing mice is reversed by ßAR-blockade treatment. Expression of UPC2 (A), FHL1 (B), and the transgene Gs (C) was measured by qPCR in the heart of 9-month-old Gs-overexpressing mice (closed bars) and control littermates (open bars) that were treated (+ propranolol) or not (- propranolol) for 6 weeks with ßAR blockade. *P<0.05 between transgenic mice and respective control littermates. P<0.05 between treated mice and respective untreated mice. ND indicates not detectable.

Common Genomic Pathway
Increased expression of UCP2 and FHL1 in Gs-overexpressing mice suggests that these genes might play a role in the development of the cardiomyopathy seen in these mice. To test if a common transcriptional pathway leads to cardiomyopathy in other models of enhanced ßAR signaling, we studied the expression of UCP2 and FHL1 in mice overexpressing ß₁AR,^3,4 ß₂AR,^5,6 or PKA.⁷

In ß₁AR transgenic mice, overexpression at 15 times endogenous ßAR levels is sufficient to lead to increased contractility at a young age (7 weeks) and to result in cardiomyopathy with aging (30 weeks).³ Expression of UPC2 (Figure 6A) and FHL1 (Figure 6B) was significantly increased at both time points in ß₁AR-overexpressing mice compared with control littermates.

View larger version (27K): [in this window] [in a new window]   Figure 6. Expression of UCP2 and FHL1 in mouse models of chronic stimulation of ßAR signaling. UCP2 (A, C, and E) and FHL1 (B, D, and F) expression were measured by qPCR in the heart of mice overexpressing ß1AR (A and B), ß2AR (C and D), and PKA (E and F) harvested at the indicated ages. Open bars indicate control littermates; closed bars, transgenic mice. *P<0.05 between transgenic mice and respective control littermates.

In a thorough study analyzing several lines of ß₂AR-overexpressing mice, Liggett et al⁶ reported that cardiomyopathy is seen only with high (>100-fold) levels of ß₂AR overexpression and develops more slowly than in mice overexpressing ß₁AR. The severity of the cardiomyopathy and mortality correlates with the expression level of ß₂AR. Milano et al⁵ reported another line of ß₂AR-overexpressing mice that we obtained through Jackson Laboratories for this study. These mice have a 200-fold overexpression of ß₂AR in the heart.⁵ At 3 months of age, they displayed enhanced contractility (ejection fraction: wild-type, 69±2%; transgenic, 75±1%; P<0.05; fraction shortening: wild-type, 33±1%; transgenic, 37±1%; P<0.05) and no evidence of cell death.⁵ At 12 months of age, however, these ß₂AR-overexpressing mice showed a significant decrease in left ventricular function (ejection fraction: young transgenic, 75±1%; old transgenic, 59±2%; P<0.05; fraction shortening: young transgenic, 37±1%; old transgenic, 26±2%; P<0.05), whereas no significant change was seen with aging in control littermates. UCP2 expression was unchanged in 3-month-old ß₂AR-overexpressing mice compared with control littermates but dramatically increased with aging in the transgenic mice (Figure 6C). FHL1 was significantly increased at both ages (Figure 6D).

Mice overexpressing PKA show dilated cardiomyopathy and sudden death independent of more proximal events in ßAR signaling.⁷ Expression of UCP2 and FHL1 were significantly increased in 3-month-old PKA-overexpressing mice compared with control littermates (Figures 6E and 6F).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present study, we used different mouse models with enhanced ßAR signaling to identify potential genomic targets of ßAR blockade therapy and found that UCP2 and FHL1, two genes with unknown function in the heart, might play an important role in the development of ßAR-induced cardiomyopathy.

Uncoupling proteins received their name from their role in uncoupling respiration from ATP production, generating heat in brown adipose tissue (UCP1) and skeletal muscle (UCP3).¹⁸ A potential role for UCP2, supported by the corresponding knockout mice,²⁶ is the regulation of reactive oxygen species (ROS) production. Reduction of ROS would be protective but would come at the cost of increased uncoupling activity that might compromise the mitochondrial membrane potential, eventually leading to cell death.^18,27 Enhanced UCP2 expression was found in the heart of rats after prolonged thyroid hormone treatment and is thought to have a detrimental effect on cardiac function in that model.²¹ In mice overexpressing Gs (Figure 2B) or ß₁AR (Figure 6A), UCP2 is upregulated before the development of cardiomyopathy and throughout aging, suggesting that it can play a role in the transition of phenotype between young and old transgenic mice. In young transgenic mice, increased expression of UCP2 might decrease the amount of ROS and therefore play a protective role. As mice age, sustained uncoupling might damage mitochondrial membrane, leading to apoptosis. No change in UCP2 expression was detected in young ß₂AR-overexpressing mice, suggesting that in this model, UCP2 might not be a cause of the cardiomyopathy developed with aging (Figure 6C).

FHL proteins are new members of the LIM-only protein family. The pattern of expression of FHL1 suggests an important role for FHL1 in the heart during development²⁰ and cardiomyopathy.^20,23 In this study, we show that FHL1, but not FHL2 or FHL3, is upregulated in mice overexpressing Gs (Figure 3). FHL1 expression, but not that of FHL2 or FHL3, also was found to increase in 2 mouse models with cardiac hypertrophy and dilated cardiomyopathy²⁰ and in hypertrophic and failing hearts from patients.^22,23 FHL1 is upregulated in all the models tested with chronic stimulation of ßAR signaling before the development of cardiomyopathy and throughout aging, suggesting that it might play a role in the transition of phenotype between young and old transgenic mice.

Interestingly, the time course of UCP2 upregulation is different in mice overexpressing ß₁AR and ß₂AR. Although both ß₁AR and ß₂AR couple to the Gs/adenylyl cyclase pathway, major differences exist between these 2 receptors,^28,29 reflected by differential actions on cardiac Ca²⁺ handling, contractility, cAMP accumulation (reviewed in Xiao et al³⁰), and apoptosis.³¹ In mice overexpressing ß₁AR and ß₂AR, there are also differences in the time and dosage required to develop cardiomyopathy. ß₁AR-overexpressed mice develop cardiomyopathy early, whereas cardiomyopathy in ß₂AR-overexpressed mice develops late, unless the receptors are overexpressed orders of magnitude greater than ß₁AR.⁶ This timing difference is reflected by the different time course of upregulation of UCP2 in both models: UCP2 expression is increased as early as at 7 weeks of age in ß₁AR-overexpressed mice, whereas in ß₂AR-overexpressed mice, it is unchanged at 3-months of age but increases at 12 months. This suggests that UCP2 is an important marker of the development of cardiomyopathy in these models. FHL1 expression is increased at both ages in ß₂AR-overexpressing mice, suggesting that modification of the expression of transcription factors precedes the modification of the expression of target proteins, such as UCP2.

In conclusion, we used genomic profiling to identify unexpected genes that might play a role in the development of cardiomyopathy induced by chronic ßAR signaling. We identified UCP2 and FHL1 as important candidate genes that correlate with the development of ßAR-induced cardiomyopathy in different animal models. Increased UCP2 expression results specifically from a ß₁AR-Gs-PKA pathway. The upregulation of UCP2 and FHL1 in mice overexpressing Gs was reversed by ßAR-blockade treatment, suggesting that these 2 genes could be direct targets for the treatment of heart failure.

	Acknowledgments

 
This work was supported in part by National Institutes of Health grants (grants AG14121, HL59139, HL33107, and HL69020 to Drs Stephen and Dorothy Vatner) and the American Heart Association (grant 0130100N to Dr Gaussin). We acknowledge Jill Thaisz-Warden for breeding and genotyping the mice, Jing Liu for echocardiography, and Qian Wang for technical help.

	Footnotes

 
The online Table is available in the online-only Data Supplement at http://www.circulationaha.org.

	References

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