1 Center for Cardiovascular Research, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri
2 Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri
3 Division of Bio-Organic Chemistry, Washington University School of Medicine, St. Louis, Missouri
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ABSTRACT |
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Both insulin-dependent (type 1) and noninsulin-dependent (type 2) forms of diabetes in humans are accompanied by a greatly increased risk of cardiovascular death (13). Both types of diabetes are associated with cardiomyopathy that share many similar characteristics, including ventricular hypertrophy, decreased ventricular diastolic relaxation, and a reduced peak filling rate (47). Diabetic cardiomyopathy is distinct from ischemic cardiomyopathy because it is present in diabetic patients and animal models of diabetes in the absence of coronary artery disease.
Recently, rodent models of diabetes were used to uncover the pathogenesis of diabetic cardiomyopathy. One animal model of diabetic cardiomyopathy was developed that consists of cardiac-specific overexpression of the transcription factor myosin heavy chain (MHC)peroxisome proliferatoractivated receptor- (PPAR-
) (8). PPAR-
is a ligand-activated transcription factor that regulates genes involved in cardiac fatty acid uptake and oxidation. PPAR-
is activated in the diabetic heart (8). MHC-PPAR transgenic mice develop a metabolic phenotype that is similar to the diabetic state in the heart but not in other tissues, with increased lipid uptake and oxidation and reduced glucose uptake and oxidation (8). Furthermore, MHC-PPAR mice develop a cardiomyopathy with ventricular hypertrophy, activation of gene markers of pathological hypertrophic growth, and transgene expressiondependent alteration in systolic ventricular dysfunction. It is unclear whether the cardiomyopathy seen in MHC-PPAR mice develops as a direct consequence of altered myocardial metabolism or rather because of abnormal intracellular signal transduction, which may occur secondarily to these metabolic changes.
A second rodent model of type 1 diabetes is based on the systemic administration of streptozotocin (STZ), a compound that contains a glucose molecule with a nitrosourea side chain that has cytotoxic action (9). The glucose portion of STZ directs the agent to pancreatic ß-cells, which are then destroyed by the highly reactive nitrosourea side chain. STZ-induced diabetic rats develop a cardiomyopathy that is characterized by decreased left ventricular (LV) contractility, diminished ventricular compliance with markedly abnormal diastolic function, and decreased inotropic and chronotropic responses to certain ligands (9,10). The abnormalities in diastolic function in diabetic rats are manifested by prolonged isovolumic relaxation time, increased atrial contribution to diastolic filling, and elevated in vivo LV end-diastolic pressure (10). STZ-induced diabetic rats do not develop atherosclerosis or hypertension, so the cardiomyopathy is presumably caused by a direct effect of diabetes on the cardiac myocyte. The cardiomyopathy in STZ-induced diabetic rats may be caused, in part, by abnormalities in G-proteinmediated signal transduction. In particular, ß-adrenergic receptor density is decreased in STZ-induced diabetic rat hearts, and there is reduced inotropic and chronotropic responsiveness to ß-adrenergic agonists (11,12). In addition, Gq protein content and protein kinase C
activity is increased in STZ-induced diabetic rat cardiac tissue (1316). Interestingly, there is an increase in diacylglycerol levels in the myocardium of diabetic rats that presumably reflects increased phospholipase Cß-activity. Furthermore, treatment of diabetic rats with angiotensin type 1 receptor antagonists blocks myocardial protein kinase C activation (15).
Regulator of G-protein signaling (RGS) proteins are GTPase-activating proteins that promote the deactivation of heterotrimeric G-proteins (17,18). We previously demonstrated that RGS family members are expressed in the heart and that overexpression of RGS4, a GAP for Gq and Gi, inhibited ligand-induced cardiomyocyte hypertrophy in culture (19,20). Furthermore, we demonstrated that transgenic mice with cardiac-specific overexpression of RGS4 were resistant to pressure overloadinduced cardiac hypertrophy (21). Cardiac overexpression of RGS4 also reversed the cardiac dysfunction caused by overexpression of the Gq subunit (22). In the present study, we evaluated the role of Gq-mediated signal transduction in the pathogenesis of diabetic cardiomyopathy by use of transgenic mice with increased or decreased cardiac Gq activity.
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RESEARCH DESIGN AND METHODS |
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Generation of MHC-PPAR x MHC-RGS4 compound transgenic mice.
MHC- PPAR transgenic mice in the C57BL/6 x CBA/J genetic background were generated as previously described (8). Positive offspring in the highest expressing line (404-3) were bred over six generations with C57BL/6 nontransgenic mice to generate hemizygous offspring in the C57BL/6 strain.
MHC-PPAR (404-3) hemizygous transgenic mice in the C57BL/6 genetic background were bred with MHC-RGS4 hemizygous transgenic mice to generate compound transgenic mice. Compound and single transgenic mice were identified by amplification of -MHCPPAR and
-MHCRGS4-myc constructs from mouse tail DNA by PCR (8,21,22). Sex- and age-matched mice were compared for each genotype (nontransgenic, MHC-RGS4, MHC-PPAR, and MHC-PPAR x MHC-RGS4), and 50% of mice for each genotype were male.
Generation of MHC-G188S transgenic mice.
The G188S point mutant form of Gq, G
q-G188S, is unable to bind to RGS proteins (23). G
q-G188S, in contrast to the G
q-Q209L mutant form, is not constitutively active when expressed in cultured cells, but it is resistant to deactivation by RGS proteins after ligand-mediated activation. We generated transgenic mice with cardiac-specific expression of G
q-G188S. The
-MHC promoter was linkered to the coding region of G
q-G188S (24). The transgenic construct had nucleotide substitutions at codon 188: TCG instead of GGG, thereby encoding serine instead of glycine. Two transgenic lines were generated in the C57BL/6 strain: one with genomic integration of three copies of the transgene (MHCG188S-3x) and a second with genomic integration of seven copies of the transgene (MHCG188S-7x). Positive offspring were bred with C57BL/6 nontransgenic mice to generate hemizygous offspring. Transgenic mice were identified by amplification of the
-MHCG
q-G188S construct from mouse-tail DNA by PCR (21,22). The MHCG188S-7x transgenic line was used in all experiments in this study because it had higher G
q-G188S protein levels in cardiac tissue.
STZ injection.
The 12-week-old C57BL/6 nontransgenic, MHC-RGS4 transgenic, or MHCG188S-7x transgenic mice were injected with 200 mg/kg i.p. STZ or vehicle (8). Random tail blood glucose measurements were performed at 7-day intervals after STZ injection. Animals were considered to be diabetic if they had a random blood glucose measurement of >250 mg/dl (8). Mice with tail blood glucose concentrations of <250 mg/dl were reinjected with STZ (200 mg/kg), and the tail blood glucose was measured again 7 days later. All other physiological, anatomic, biochemical, and genetic studies were performed on animals 28 days after STZ injection. Mice were age matched in each case but were not sex matched.
Echocardiography.
Live anaesthetized mice were evaluated by echocardiography 28 days after injection with STZ or vehicle to determine cardiac mass and function (21,22). We used a Sequoia cardiac echocardiography system (Acuson, Mountain View, CA) that was equipped with a 15-MHz linear transducer.
Cardiac catheterization.
Live anesthetized mice were evaluated by cardiac catheterization 28 days after injection with STZ or vehicle to determine intracardiac pressures and cardiac function as previously described (21). A 1.4-F Millar catheter was used, and continuous aortic pressure and LV systolic and diastolic pressures were recorded.
Quantitative real-time PCR.
RNA was purified from quick-frozen cardiac tissue by use of Trizol reagent (Sigma). The TaqMan Gold RT-PCR kit (PE Biosystems) was used according to the manufacturers instructions. Quantitative PCR was performed by use of real-time detection technology and analyzed on a model 7700 Sequence Detector (Applied Biosystems) with specific primers and fluorescent probes for atrial natriuretic factor (ANF), ß-MHC, mitochondrial carnitine palmitoyltransferase 1 (mCPT-1), sarcoplasmic and endoplasmic reticulum calcium exchanger 2a (SERCA2a), and GLUT4. mRNA levels were compared at various time points after correction by use of concurrent glyceraldehyde-3-phosphate dehydrogenase message amplification.
Protein analysis.
Murine ventricular cytosolic extracts were generated as previously described, and proteins were separated by SDS-PAGE (21). Proteins were electrophoretically transferred to nitrocellulose. Filters were blocked in Tris-buffered saline containing 1% Tween 20 and 2% nonfat dried milk. Filters were washed and incubated with primary antibody. Primary antibodies used included murine monoclonal (M5) anti-FLAG antibody (Sigma), rabbit polyclonal anti-p38 mitogen-activated protein kinase antibody, and rabbit polyclonal Gq/G
11 (C-19; Santa Cruz Biotechnology). Filters were extensively washed in Tris-buffered saline containing 1% Tween 20 and then incubated with horseradish peroxidaseconjugated anti-mouse or anti-rabbit secondary antibody (Amersham, Piscataway, NJ). Bands were visualized by use of the enhanced chemiluminescence system (Amersham) (21).
Histological analysis.
Ventricular tissue was fixed in formalin and embedded in paraffin (21). Embedded mid-LV tissue was sectioned with a microtome, deparaffinized, stained with Massons trichrome or hematoxylin and eosin, and examined by compound microscopy.
Statistical analysis.
All data are reported as the means ± SD. Differences between values were evaluated for statistical significance by use of a nonpaired Students t test or one-way ANOVA with the Fishers post hoc procedure when appropriate. Significance was defined as P < 0.05.
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RESULTS |
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The cardiac function and gene expression phenotype of MHC-PPAR x MHC-RGS4 compound transgenic mice were compared with MHC-PPAR and MHC-RGS4 single transgenic mice and with nontransgenic mice at 12 weeks of age. MHC-PPAR x MHC-RGS4 compound transgenic and MHC-PPAR single transgenic mice had identical cardiac PPAR- protein levels, as measured by immunoblotting of ventricular cytosolic lysates (Fig. 1A).
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MHC-PPAR single transgenic mice had LV hypertrophy at 12 weeks of age by echocardiography with a calculated LV mass of 83.5 ± 13.8 mg compared with 57.4 ± 5.8 mg for nontransgenic mice (P < 0.05). MHC-PPAR x MHC-RGS4 compound transgenic mice exhibited a trend toward less LV mass by echocardiographic analysis than MHC-PPAR single transgenic mice (67.6 ± 9.8 mg, P = 0.1) (Fig. 1C).
Morphometric analysis confirmed that MHC-PPAR single transgenic mice, but not MHC-PPAR x MHC-RGS4 compound transgenic mice, had cardiac hypertrophy at 12 weeks of age. The biventricular weighttobody weight ratio in MHC-PPAR single transgenic mice was 6.5 ± 0.62 mg/g, but it was only 5.3 ± 0.31 mg/g in MHC-PPAR x MHC-RGS4 compound transgenic mice (P = 0.024) (Fig. 1D). Similarly, the LV weighttobody weight ratio was 4.9 ± 0.35 mg/g in MHC-PPAR single transgenic mice, but it was only 4.2 ± 0.36 in MHC-PPAR x MHC-RGS4 compound transgenic mice (P = 0.027) (Fig. 1E).
Analysis of gene expression by quantitative real-time RT-PCR revealed that MHC-PPAR single transgenic mice had elevated cardiac mCPT-1 gene expression, a PPAR- target gene involved in fatty acid uptake and utilization known to be upregulated in the diabetic heart (8). MHC-PPAR x MHC-RGS4 compound transgenic mice had a similar elevation in mCPT-1 mRNA (Fig. 2). MHC-PPAR single transgenic mice had reduced cardiac glucose transporter GLUT4 gene expression, a gene inhibited by PPAR-
(8), and MHC-PPAR x MHC-RGS4 mice had a similar reduction in GLUT4 mRNA (Fig. 2). MHC-PPAR single transgenic mice had reduced SERCA2a gene expression, a marker of cardiac hypertrophy. In contrast, SERCA2a gene expression was normalized in MHC-PPAR x MHC-RGS4 compound transgenic mice (Fig. 2). Furthermore, MHC-PPAR single transgenic mice had increased ANF and ß-MHC gene expression that was normalized in MHC-PPAR x MHC-RGS4 compound transgenic mice (Fig. 2).
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Analysis of gene expression revealed that MHC-RGS4 transgenic animals were resistant to STZ-stimulated ANF and ß-MHC induction. ANF gene expression was induced by 14.1-fold in STZ-injected nontransgenic C57BL/6 animals, but it actually declined by 50 ± 40% in STZ-injected MHC-RGS4 animals when compared with vehicle-injected MHC-RGS4 mice (Fig. 3). ß-MHC gene expression was induced by 13.5-fold in STZ-injected nontransgenic C57BL/6 animals, but it declined by 16% in STZ-injected MHC-RGS4 animals when compared with vehicle-injected MHC-RGS4 animals (Fig. 3).
Generation and preliminary characterization of MHC-G188S transgenic mice.
RGS proteins deactivate heterotrimeric G-proteins by stabilizing the transition state between the GTP- and GDP-bound forms of the -subunit (17,18). We generated a point mutant form of G
q, G
q-G188S, that is unable to bind to RGS proteins (23). The G188S point mutant form of G
q, in contrast to the Q209L mutant form, is not constitutively active when expressed in cultured cells, but it is resistant to deactivation by RGS proteins after ligand-mediated activation. We generated transgenic mice with cardiac-specific expression of G
q-G188S. The
-MHC promoter was linked to the coding region of G
q-G188S. Two transgenic lines were generated in the C57BL/6 strain: one with genomic integration of three copies of the transgene (MHCG188S-3x) and a second with genomic integration of seven copies of the transgene (MHCG188S-7x). Both lines have increased total G
q protein in ventricular lysates (Fig. 4A). Mice from both lines appear normal and live normal life spans. The basal cardiac structure and function of these mice is normal at 12 weeks of age in the MHCG188S-3x (data not shown) and MHCG188S-7x lines, as determined by echocardiographic and histological examination (Figs. 4BC).
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Response of MHC-G188S transgenic mice to STZ injection.
MHCG188S-7x animals were evaluated by echocardiography 28 days after STZ or vehicle injection. MHCG188S-7x diabetic mice had preserved systolic function when compared with vehicle-injected controls. The fractional shortening was 57 ± 3% in MHCG188S-7x animals 28 days after STZ injection, and it was 53 ± 4% in vehicle-injected MHCG188S-7x control animals. Interestingly, MHCG188S-7x mice became bradycardic in response to STZ-injection. The heart rate was 606 ± 68 bpm in STZ-injected MHCG188S-7x mice, but it was 680 ± 31 bpm in vehicle-injected animals (P = 0.01) (Fig. 4E). Although both the biventricular weight and the whole body weight was reduced in STZ-injected MHCG188S-7x animals, the biventricular weighttobody weight ratio was higher in diabetic mice (0.0043 ± 0.0002) than in vehicle-injected transgenic mice (0.0037 ± 0.0004) (Fig. 4D).
Analysis of gene expression revealed that MHCG188S-7x transgenic animals were highly sensitized to STZ-stimulated ANF and ß-MHC induction. ANF and ß-MHC gene expression levels were similar in nondiabetic C57BL/6 and MHCG188S-7x transgenic animals (data not shown). ANF gene expression was induced by 33-fold in STZ-injected MHCG188S-7x animals, but it was induced by 14.1-fold in STZ-injected nontransgenic animals when compared with vehicle-injected controls (Fig. 3). ß-MHC gene expression was induced by 218.7-fold in STZ-injected MHCG188S-7x animals, but it was induced by 13.5-fold in STZ-injected nontransgenic animals when compared with vehicle-injected controls (Fig. 3).
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DISCUSSION |
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In addition to angiotensin II, it is possible that additional secreted ligands, such as endothelin-1, are involved in the pathogenesis of diabetic cardiomyopathy (31). Diabetic patients have elevated serum endothelin-1 levels (31). In addition, treatment of STZ-injected rats with bosentan, an endothelin-1 receptor antagonist, resulted in improved cardiac diastolic and systolic function (32). These findings suggest that angiotensin II inhibition may not completely block the development of diabetic cardiomyopathy.
In this work, we investigated the role of Gq proteins in the pathogenesis of diabetic cardiomyopathy. Both angiotensin II and endothelin-1 activated seven transmembrane receptors that are coupled to Gq. We first tested a robust genetic model of diabetic cardiomyopathy, MHC-PPAR transgenic mice (8). These mice exhibit abnormal cardiac metabolism, although they are systemically euglycemic, and they spontaneously develop ventricular hypertrophy, systolic contractile dysfunction, and "fetal" gene induction. The observation that MHC-PPAR mice are not hyperglycemic is a limitation that makes them an imperfect model of human diabetic cardiomyopathy. We hypothesized that G-protein signaling might play a role in the pathogenesis of MHC-PPAR cardiomyopathy. MHC-PPAR mice were bred with MHC-RGS4 transgenic mice because RGS4 antagonizes Gq signaling in the heart (22). MHC-PPAR x MHC-RGS4 compound transgenic mice exhibited an intermediate phenotype with near-complete rescue of the ventricular hypertrophy and partial rescue of the contractile dysfunction. In addition, embryonic gene induction was markedly reduced in compound transgenic mice when compared with MHC-PPAR single transgenic animals. However, RGS4 did not reverse the direct transcriptional effects of PPAR- on the regulation of fatty acid metabolism gene expression. These results suggest that the effects of PPAR-
overexpression on metabolic gene expression do not inexorably lead to cardiac hypertrophy and dysfunction.
We next used a murine model of type 1 diabetes that involved the intraperitoneal injection of the pancreatic ß-cell toxin STZ (8). We induced diabetes in nontransgenic animals and in transgenic animals with decreased (MHC-RGS4) and increased (MHCG188S-7x) Gq signaling. Nontransgenic animals developed diabetes after a single injection of STZ, and when analyzed at 28 days, they exhibited mild bradycardia, intact systolic contractile function, and marked induction of embryonic marker genes, including ANF and ß-MHC. In the present study, we demonstrated that MHC-RGS4 mice were resistant to STZ-induced bradycardia and embryonic gene induction.
We used MHCG188S-7x transgenic mice as a model system of mildly increased Gq-mediated cardiac signaling. We thought that these mice would be superior to Gq-40 mice for this purpose because they have a less dramatic cardiac phenotype in the absence of provocative stimulation (26,27). As predicted, MHCG188S-7x mice were sensitized to STZ-induced bradycardia and embryonic gene induction. However, MHCG188S-7x mice did not develop ventricular systolic dysfunction 28 days after STZ injection.
The cardiac phenotype observed in nontransgenic mice after STZ injection was much milder than that observed in rats, and this difference may be a result of the profound cachexia and dehydration that develops in diabetic mice. Indeed, STZ-injected mice did not develop the diastolic dysfunction that is characteristic of human diabetic cardiomyopathy and that is also observed in STZ-injected rats. Whether the profound cachexia and dehydration observed in STZ-injected mice prevented the development of diastolic dysfunction is not known, and further study is required to address this issue. It is possible that subtherapeutic insulin administration to STZ-injected miceby reducing cachexia and volume depletion without completely restoring euglycemiamay better model human diabetic cardiomyopathy. Taken together, these experiments demonstrate that increased or decreased Gq-mediated signaling affect the development of diabetic cardiomyopathy, and they suggest that medical therapy targeting this signaling pathway may benefit patients with diabetic cardiomyopathy and may prevent the development of this condition in patients with diabetes.
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ACKNOWLEDGMENTS |
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The authors thank Attila Kovacs and Mike Courtois of the Mouse Cardiovascular Phenotyping Core Facility of the Center for Cardiovascular Research at Washington University for assistance with the echocardiography and cardiac catheterization studies.
Address correspondence and reprint requests to Dr. Anthony J. Muslin, Box 8086, 660 South Euclid Ave., St. Louis, MO 63110. E-mail: amuslin{at}imgate.wustl.edu
Received for publication May 30, 2004 and accepted in revised form August 25, 2004
ANF, atrial natriuretic factor; LV, left ventricular; mCPT-1, mitochondrial carnitine palmitoyltransferase 1; MHC, myosin heavy chain; PPAR, peroxisome proliferatoractivated receptor; RGS, regulator of G-protein signaling; SERCA2a, sarcoplasmic and endoplasmic reticulum calcium exchanger 2a; STZ, streptozotocin
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REFERENCES |
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