Expression of Constitutively Active Guanylate Cyclase in Cardiomyocytes Inhibits the Hypertrophic Effects of Isoproterenol and Aortic Constriction on Mouse Hearts

Evidence from several rodent models has suggested that a reduction of either atrial natriuretic peptide or its receptor in the heart affects cardiac remodeling by promoting the onset of cardiac hypertrophy. The atrial natriuretic peptide receptor mediates signaling at least in part via the generation of intracellular cyclic GMP. To directly test whether accumulation of intracellular cyclic GMP conveys protection against cardiac hypertrophy, we engineered transgenic mice that overexpress a catalytic fragment of constitutively active guanylate cyclase domain of the atrial natriuretic peptide receptor in a cardiomyocyte-specific manner. Expression of the transgene increased the intracellular concentration of cyclic GMP specifically within cardiomyocytes and had no detectable effect on cardiac performance under basal conditions. However, expression of the transgene attenuated the effects of the pharmacologic hypertrophic agent isoproterenol on cardiac wall thickness and prevented the onset of the fetal gene expression program normally associated with cardiac hypertrophy. Likewise, expression of the transgene inhibited the hypertrophic effects of abdominal aortic constriction, since it abolished its effects on ventricular wall thickness and greatly attenuated its effects on cardiomyocyte size. Altogether, our results suggest that cyclic GMP is a cardioprotective agent against hypertrophy that acts via a direct local effect on cardiomyocytes. Evidence from several rodent models has suggested that a reduction of either atrial natriuretic peptide or its receptor in the heart affects cardiac remodeling by promoting the onset of cardiac hypertrophy. The atrial natriuretic peptide receptor mediates signaling at least in part via the generation of intracellular cyclic GMP. To directly test whether accumulation of intracellular cyclic GMP conveys protection against cardiac hypertrophy, we engineered transgenic mice that overexpress a catalytic fragment of constitutively active guanylate cyclase domain of the atrial natriuretic peptide receptor in a cardiomyocyte-specific manner. Expression of the transgene increased the intracellular concentration of cyclic GMP specifically within cardiomyocytes and had no detectable effect on cardiac performance under basal conditions. However, expression of the transgene attenuated the effects of the pharmacologic hypertrophic agent isoproterenol on cardiac wall thickness and prevented the onset of the fetal gene expression program normally associated with cardiac hypertrophy. Likewise, expression of the transgene inhibited the hypertrophic effects of abdominal aortic constriction, since it abolished its effects on ventricular wall thickness and greatly attenuated its effects on cardiomyocyte size. Altogether, our results suggest that cyclic GMP is a cardioprotective agent against hypertrophy that acts via a direct local effect on cardiomyocytes. Left ventricular hypertrophy (LVH) 1The abbreviations used are: LVHleft ventricular hypertrophyANPatrial natriuretic peptideNPRAnatriuretic peptide receptor ATGtransgenicNTnontransgenicAACabdominal aortic constrictionBWbody weightHWheart weightLVPWleft ventricular posterior wallIVSWinterventricular septal wallLVIDdleft ventricular internal diameters at end-diastoleLVIDsleft ventricular internal diameters at end-systoleM-CPT-Imuscle isoform of carnitine palmitoyltransferase IMCADmedium-chain acyl-CoA dehydrogenaseLSDleast squares difference.1The abbreviations used are: LVHleft ventricular hypertrophyANPatrial natriuretic peptideNPRAnatriuretic peptide receptor ATGtransgenicNTnontransgenicAACabdominal aortic constrictionBWbody weightHWheart weightLVPWleft ventricular posterior wallIVSWinterventricular septal wallLVIDdleft ventricular internal diameters at end-diastoleLVIDsleft ventricular internal diameters at end-systoleM-CPT-Imuscle isoform of carnitine palmitoyltransferase IMCADmedium-chain acyl-CoA dehydrogenaseLSDleast squares difference. results from the activation of multiple signaling pathways by either mechanical or neurohumoral stimuli (1Sugden P.H. Clerk A. J. Mol. Med. 1998; 76: 725-746Crossref PubMed Scopus (338) Google Scholar, 2Molkentin J.D. Dorn G.W.I. Annu. Rev. Physiol. 2001; 63: 391-426Crossref PubMed Scopus (574) Google Scholar). A great number of studies have used animal models of transgenesis or gene inactivation to test the possible roles of these pathways in the induction of LVH in vivo. However, the contributions of possible negative regulators of LVH have so far received much less attention. Recently, several lines of evidence have suggested that atrial natriuretic peptide (ANP) and endothelial nitric-oxide synthase may act as such negative regulators, since 1) blood-pressure-independent LVH is present in mice with either general (3Knowles J.W. Esposito J.W. Mao L. Hagaman J.R. Fox J.E. Smithies O. Rockman H.A. Maeda N. Proc. Natl. Acad. Sci. U. S. A. 2001; 107: 975-984Google Scholar) or cardiomyocyte-restricted (4Holtwick R. van Eickels M. Skryabin B.V. Baba H.A. Bubikat A. Begrow F. Scheider M.D. Garbers D.L. Kuhn M. J. Clin. Invest. 2003; 111: 1399-1407Crossref PubMed Scopus (295) Google Scholar) inactivation of natriuretic peptide receptor A (NPRA); 2) cardiomyocyte-specific expression of NPRA partially rescues the cardiac hypertrophic phenotype seen in NPRA-null mice (5Kishimoto I. Rossi K. Garbers D.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2703-2706Crossref PubMed Scopus (231) Google Scholar); 3) we have shown that cardiac mass and ventricular expression of ANP were both associated (in an inverse fashion) with a naturally occurring allele of natriuretic peptide precursor A (i.e. the gene that codes for ANP) (6Masciotra S. Picard S. Deschepper C.F. Circ. Res. 1999; 84: 1453-1458Crossref PubMed Scopus (37) Google Scholar, 7Deschepper C.F. Masciotra S. Zahabi A. Boutin-Ganache I. Picard S. Reudelhuber T. Circ. Res. 2001; 88: 222-227Crossref Scopus (41) Google Scholar, 8Boutin-Ganache I. Picard S. Deschepper C.F. Physiol. Genomics. 2002; 12: 61-67Crossref PubMed Scopus (12) Google Scholar); and 4) overexpression of an endothelial nitric-oxide synthase transgene attenuates isoproterenol-induced LVH (9Ozaki M. Kawashima S. Yamashita T. Hirase T. Ohashi Y. Inoue N. Hirata K.-I. Yokohama M. Circ. J. 2002; 66: 851-856Crossref PubMed Scopus (75) Google Scholar). The common denominator between ANP and endothelial nitric-oxide synthase is that many (if not most) of their biologic effects are mediated by cGMP (10Brenner B.M. Ballerman B.J. Gunning M.E. Zeidel M.L. Physiol. Rev. 1990; 70: 665-699Crossref PubMed Scopus (843) Google Scholar, 11Denninger J.W. Marletta M.A. Biochim. Biophys. Acta. 1999; 1411: 334-350Crossref PubMed Scopus (853) Google Scholar). Interestingly, it has also been shown that cGMP protects cultured neonatal cardiomyocytes against the effects of hypertrophic agents in vitro (12Calderone A. Thaik C.M. Takahashi N. Chang D.L.F. Colucci W.S. J. Clin. Invest. 1998; 101: 812-818Crossref PubMed Scopus (403) Google Scholar, 13Silberbach M. Gorenc T. Hershberger R.E. Stork P.J.S. Steyger P.S. Roberts Jr., C.T. J. Biol. Chem. 1999; 274: 24858-24864Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 14Wollert K.C. Fiedler B. Gambaryan S. Smolenski A. Heineke J. Butt E. Trautwien C. Lohmann S.M. Drexler H. Hypertension. 2002; 39: 87-92Crossref PubMed Scopus (116) Google Scholar). However, it remains to be proven that cGMP has similar actions in vivo. left ventricular hypertrophy atrial natriuretic peptide natriuretic peptide receptor A transgenic nontransgenic abdominal aortic constriction body weight heart weight left ventricular posterior wall interventricular septal wall left ventricular internal diameters at end-diastole left ventricular internal diameters at end-systole muscle isoform of carnitine palmitoyltransferase I medium-chain acyl-CoA dehydrogenase least squares difference. left ventricular hypertrophy atrial natriuretic peptide natriuretic peptide receptor A transgenic nontransgenic abdominal aortic constriction body weight heart weight left ventricular posterior wall interventricular septal wall left ventricular internal diameters at end-diastole left ventricular internal diameters at end-systole muscle isoform of carnitine palmitoyltransferase I medium-chain acyl-CoA dehydrogenase least squares difference. To address this central question, we have engineered transgenic (TG) mice that express a constitutively activated guanylate cyclase domain of the NPRA receptor in a cardiomyocyte-specific manner and tested whether expression of the transgene inhibited the effects of exogenous hypertrophic stimuli. DNA Constructs and TG Animals—A portion of the rat cDNA coding for NPRA (15Chinkers M. Garbers D.L. Chang M.-S. Lowe D.G. Chin H. Goeddel D.V. Schulz S. Nature. 1989; 338: 78-83Crossref PubMed Scopus (877) Google Scholar) (generous gift from D. G. Garbers, University of Texas Southwestern Medical Center) ranging from nt 2547 to 3478 was amplified by PCR. This generated a cDNA fragment coding for a cytoplasmic domain of NPRA containing the catalytic fragment of the guanylate cyclase domain. It has been shown that this fragment codes for a soluble cytoplasmic protein that has constitutive guanylate cyclase activity in transfected COS cells (16Thompson D.K. Garbers D.L. J. Biol. Chem. 1995; 270: 425-430Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 17Kohno M. Horio T. Yokokawa K. Kurihara N. Takeda T. Hypertension. 1992; 19: 320-325Crossref PubMed Scopus (64) Google Scholar). This cDNA fragment was cloned downstream of a 5.8-kb fragment of the α-myosin heavy chain gene promoter (generous gift from J. Robbins, University of Cincinnati) and upstream of a portion of the rabbit β-globin gene containing an intron and a polyadenylation signal. The excised construct was microinjected in hybrid F1 C3H-C57Bl/6 embryos according to standard protocols. Three TG founder lines (named TG9, TG19, and TG41) with germ line integration were generated in this fashion. Results shown hereafter are from experiments performed with TG19, but similar results have been obtained with animals from the other two lines (data not shown). All of the founder lines were back-crossed into the C57Bl/6 mouse strain. For experimental purposes, all TG animals had been back-crossed for at least five generations and were compared with non-TG (NT) littermates. To avoid the possible artifactual influence of gene inactivation by insertion, all TG animals used for experiments were heterozygous for the transgene. The institutional Institut de Recherches Cliniques review board approved all animal protocols used in this study. Transgene Expression—Tissue-specific expression of the transgene was verified by Northern blot analysis of total RNA extracted from either heart, kidney, liver, striated muscle, lung, stomach, spleen, or brain. In some additional mice, adult mouse cardiomyocytes were isolated from the ventricles of 12-week-old mice (either NT or TG) by Langendorff perfusion of sequential solutions containing various concentrations of proteases and/or Ca2+, as described previously (18Zhou Y.-Y. Wang S.-Q. Zhu W.-Z. Chruscinski A. Kobilka B.K. Ziman B. Wang S. Lakatta E.G. Cheng H. Xiao R.-P. Am. J. Physiol. 2000; 279: H429-H436Crossref PubMed Google Scholar). This preparation yielded mostly viable and noncontracting rod-shaped cells along with minor amounts of cellular debris. Cell preparations were used to measure guanylate cyclase activity in soluble and particulate fractions. Freshly prepared cells were disrupted in homogenization buffer at 4 °C with a glass-glass homogenizer. The cytosolic fraction was prepared by recovering the supernatant after centrifuging the homogenates at 100,000 × g for 60 min at 4 °C. For preparation of the particulate fraction, the pellet was resuspended in homogenization buffer containing 1% Triton X-100 and incubated for 15 min at 4 °C, and the supernatant was recovered after centrifugation at 100,000 × g for 60 min at 4 °C. Guanylate cyclase activity was measured in aliquots of both fractions (containing 12 μg of protein) as described previously (19Deschepper C.F. Picard S. J. Neurochem. 1994; 62: 1974-1982Crossref PubMed Scopus (35) Google Scholar). Isolated adult mouse cardiomyocytes were also used to measure intracellular cGMP concentration, as described previously (20Mazzetti L. Ruocco C. Giovannelli L. Ciuffi M. Franchi-Micheli S. Marra F. Zilletti L. Failli P. Br. J. Pharmacol. 2001; 134: 596-602Crossref PubMed Scopus (14) Google Scholar). Immediately after isolation, the cells were incubated for 10 min at 37 °C in fetal bovine serum-supplemented Dulbecco's modified Eagle's medium in the presence of 200 μm isobutylmethylxanthine (to inhibit phosphodiesterases). After centrifugation, the pellets were extracted two times with 65% ethanol, the extracts were evaporated, and extracts were resuspended in assay buffer to assay cGMP by radioimmune assay. The remaining pellets were dissolved in 0.1 n NaOH for protein determination. Adenylyl Cyclase Stimulation—Freshly isolated cardiomyocytes from the hearts of 3-month-old TG and NT mice were disrupted at 4 °C in buffer containing 50 mm Tris, pH 7.4, 250 mm sucrose, 1 mm EDTA, 1 mm dithiothreitol, and protease inhibitors. Plasma membranes were then recovered by centrifugation at 2000 rpm at 4 °C and further resuspended in 500 μl of buffer containing 50 mm Tris, pH 7.4, 4 mm MgCl2, and protease inhibitors. Aliquots of 25 μl of plasma membranes were then added to 200 μl of assay buffer containing 50 mm Tris, pH 7.5, 5 mm MgCl2, 1 mm ATP, 15 mm creatinine phosphate, 1 mg/ml creatine kinase, 10 μm GTP, 1 mm EGTA, 1 mm dithiothreitol, and 1 mm isobutylmethylxanthine, either with or without 10–7m isoproterenol. The membranes were further incubated for 4 min at 37 °C, and the reaction was stopped by the addition of 200 μl of cold 0.2 m HCl and quick freezing at –70 °C. After thawing, proteins were precipitated by centrifugation, and the amount of cAMP generated in the supernatants was assayed by radioimmune assay. The results were normalized for protein content. Animal Procedures—LVH was induced in TG male mice and their NT littermates by two different experimental maneuvers. The first method involved the subcutaneous implantation of an Alzet osmotic minipump (Durect, Cupertino, CA) delivering isoproterenol (30 mg/kg/day) for 4 days to 12-week-old male mice, as reported previously (21Saadane N. Alpert L. Br. J. Pharmacol. 1999; PubMed Scopus Google Scholar). For the LVH was induced by of an abdominal aortic constriction on mice under A was on of the abdominal to the a was both the and the with a and the was The hearts were from 12-week-old mice 4 after All of mice were by body weight was the hearts were the were and heart weight was to the of TG and NT mice were measured by with the according to previously Hagaman J.R. Smithies O. Hypertension. 1995; PubMed Scopus Google Scholar). mice were to the for a total of 10 were the of performed the hypertrophy and cardiac were by at the of in mice with body was to that heart was that 500 in mouse the was performed a with a were obtained in both and at the of and used for the of ventricular All of the were performed according to the by the of A. J. A. 2001; Scopus Google Scholar). the of posterior wall and interventricular septal wall were measured internal diameters were measured and and fraction were with the standard was by performed at the of the The of was by heart to cardiac and the was by body weight to cardiac mouse cardiomyocytes were isolated from the ventricles of 12-week-old mice (either NT or with either or performed at of as described The cells were and their were by previously C.F. Picard S. G. R. Am. J. Physiol. 2002; Scholar). Northern RNA was extracted from cardiac ventricles obtained from NT and TG mice with either a or an osmotic and aliquots of 10 μg were on an to a The cDNA used for to the membranes were the of the 1) natriuretic peptide precursor A of D. G. 2) natriuretic peptide precursor of M. 3) the muscle isoform of rat carnitine palmitoyltransferase I gift from H. and 4) rat medium-chain acyl-CoA dehydrogenase of D. P. were to a The were and and normalized to the of the in between were performed by analysis of by The of transgene expression was by Northern blot analysis Transgene expression was in 10 μg of total RNA from heart of TG mice but not in RNA from other the protein we that soluble guanylate cyclase activity was 4 times in extracts of isolated cardiomyocytes from TG as compared with from NT littermates In particulate guanylate cyclase activity was not in cardiomyocytes isolated from TG mice as compared with that from NT animals results are with the that the protein be cGMP concentration was in cardiomyocytes isolated from TG mice of protein) in from NT mice of Expression of the transgene had no effect on in basal since it was and mm n in NT and TG mice, basal was no between TG and NT mice for either or thickness of and in was no either in basal cardiac performance since was no in either or left ventricular performance left ventricular fraction, and cardiac increased as as the thickness of both and in NT mice in its effects on and was in TG in NT mice, and the had no effect on in TG increased cardiac and heart to the in both NT and TG was no of cardiac since were in both of mice To the that expression of the transgene with the presence of at the of we tested the effects of isoproterenol on cAMP generation by plasma membranes prepared from cardiomyocytes from either NT or TG For TG the amounts of cAMP generated by cardiomyocyte preparations as of were and n 3) in either the or presence of 10–7m For NT the amounts of cAMP generated by the preparations were and of min in either the or presence of isoproterenol results that 1) cardiomyocytes from TG mice in their plasma that are to cyclase and 2) that the of the cyclase in TG mice was similar to that in NT in and fraction in a we performed Northern blot to test the effect of transgene expression on of increased the of the of the coding for the of ANP and natriuretic peptide in NT mice but had no effects in TG mice To the that some of these from the of these by cGMP of the effects of the transgene on we also measured the of expression of additional (i.e. and for in and their expression is in to hypertrophic stimuli of fetal D.L. 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Med. 1998; PubMed Scopus Google Scholar). that isoproterenol the of and in NT as as in TG However, the of both was in TG in NT both under basal and blot of the of the and performed with total RNA from the hearts of TG mice and their NT with either or isoproterenol The results are shown at the along with the of the The by To whether cGMP had similar effects in of induced we measured the of cardiac hypertrophy in animals to The increased as as the thickness of not that of in NT mice but not in TG mice In to isoproterenol effect on of was attenuated in TG these effects of on were abolished in TG increased cardiac to the in both NT and TG mice with was no of cardiac since were in both of mice mice with either or abdominal aortic fraction in a to test whether expression of the transgene the hypertrophic phenotype at the cellular we measured cardiomyocytes isolated from either adult NT or TG mice either or experimental hypertrophy For these was to isoproterenol since the may in of cardiomyocytes and their of its hypertrophic effects J.E. K. Circ. Res. 1989; PubMed Scopus Google Scholar). In NT increased cardiomyocyte by in NT mice but by in TG mice of similar were for of cardiomyocyte Expression of the α-myosin heavy chain gene promoter has been used to expression of the transgene in the is activated in cardiac ventricles after and in mice a at of A. J. Circ. Res. PubMed Scopus Google Scholar, A. J. S. J. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the first from our is that overexpression of constitutively active guanylate cyclase within ventricular cardiomyocytes from to to increased of cGMP within adult cardiomyocytes but has no effect on either the thickness of ventricular cardiomyocyte or cardiac performance under basal conditions. This is in with cardiomyocyte-restricted inactivation of and cardiomyocyte are increased in the of other experimental (4Holtwick R. van Eickels M. Skryabin B.V. Baba H.A. Bubikat A. Begrow F. Scheider M.D. Garbers D.L. Kuhn M. J. Clin. 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In isoproterenol-induced our that expression of the transgene 1) attenuated the effects of the on either or ventricular wall 2) the effects of the on ventricular ANP and natriuretic peptide of and 3) increased the expression of two to be hypertrophy by both in basal and conditions. effects not be on the of the of in the plasma of cardiomyocytes of TG mice and that are downstream of the isoproterenol is a of experimental hypertrophy, its effects are much and normally be in since cardiac mass by in a of For this we tested whether in cardiomyocyte cGMP also the hypertrophic effects of a LVH is less and it the of that expression of the transgene the effects of on and ventricular wall thickness the that was no evidence that it cardiac in the of a we that the effect of cardiomyocyte cGMP was at the cellular since it greatly attenuated the effects of on the and of cardiomyocytes. had shown that inactivation of codes for the NPRA cardiac hypertrophy in a manner (3Knowles J.W. Esposito J.W. Mao L. Hagaman J.R. Fox J.E. Smithies O. Rockman H.A. Maeda N. Proc. Natl. Acad. Sci. U. S. A. 2001; 107: 975-984Google Scholar, R. van Eickels M. Skryabin B.V. Baba H.A. Bubikat A. Begrow F. Scheider M.D. Garbers D.L. Kuhn M. J. Clin. Invest. 2003; 111: 1399-1407Crossref PubMed Scopus (295) Google Scholar). results these by that the is also and that cGMP is the of such TG or animals have been used mostly to and the effects of agents or pathways that LVH 2Molkentin J.D. Dorn G.W.I. Annu. Rev. Physiol. 2001; 63: 391-426Crossref PubMed Scopus (574) Google and B.J. Garbers D.L. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). However, it be to the effects of cardiac that protection against hypertrophy. such are the protein A. J.W. D. J. Clin. Invest. 1998; PubMed Scopus Google Scholar) and N. J. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar, S. G. H. T. A. J.D. A. J. R. A. T. J. Cell Biol. 2000; PubMed Scopus Google have been from experiments with TG mice as possible negative of the hypertrophic suggest that intracellular cGMP may such negative and evidence that it LVH in via a direct local on cardiomyocytes. some in vitro experiments in neonatal cardiomyocytes have suggested that the effects of cGMP be mediated via protein I K.C. Fiedler B. Gambaryan S. Smolenski A. Heineke J. Butt E. Trautwien C. Lohmann S.M. Drexler H. Hypertension. 2002; 39: 87-92Crossref PubMed Scopus (116) Google Scholar, B. Lohmann S.M. Smolenski A. S. B. F. J.D. Drexler H. K.C. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google via with the signaling B. Lohmann S.M. Smolenski A. S. B. F. J.D. Drexler H. K.C. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). experiments are to test whether cGMP its effects in by with the or with other signaling the Animal and The at the Institut de Recherches Cliniques de

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