Published online before print January 12, 2009, doi: 10.1161/CIRCULATIONAHA.108.822072
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(Circulation. 2009;119:408-416.)
© 2009 American Heart Association, Inc.
Heart Failure |
Ventricular Phosphodiesterase-5 Expression Is Increased in Patients With Advanced Heart Failure and Contributes to Adverse Ventricular Remodeling After Myocardial Infarction in Mice
From the Vesalius Research Center (P.P., S.V., G.M., P.V., X.L., H.G., M.P., L.S., S.P.J.), VIB (Flanders Institute for Biotechnology), Leuven, Belgium; Division of Clinical Cardiology (J.V., S.P.J.), Experimental Cardiac Surgery (A.V.d.B., P.H.), Experimental Cardiology (P.P., V.B., I.L., L.B., K.S.), Imaging and Cardiovascular Dynamics, Department of Cardiovascular Medicine (O.G.), and Department of Medical Diagnostic Sciences (A.V.L., E.V.), KU Leuven, Leuven, Belgium; and Cardiovascular Research Center and Department of Anesthesia and Critical Care (C.B., E.B., K.D.B.), Massachusetts General Hospital and Harvard Medical School, Boston, Mass.
Correspondence to Stefan P. Janssens, MD, PhD, Division of Clinical Cardiology and Vesalius Research Center, University of Leuven, Campus Gasthuisberg, 49 Herestraat, B-3000 Leuven, Belgium. E-mail stefan.janssens{at}med.kuleuven.be
Received September 15, 2008; accepted November 6, 2008.
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Abstract |
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Background— Ventricular expression of phosphodiesterase-5 (PDE5), an enzyme responsible for cGMP catabolism, is increased in human right ventricular hypertrophy, but its role in left ventricular (LV) failure remains incompletely understood. We therefore measured LV PDE5 expression in patients with advanced systolic heart failure and characterized LV remodeling after myocardial infarction in transgenic mice with cardiomyocyte-specific overexpression of PDE5 (PDE5-TG).
Methods and Results— Immunoblot and immunohistochemistry techniques revealed that PDE5 expression was greater in explanted LVs from patients with dilated and ischemic cardiomyopathy than in control hearts. To evaluate the impact of increased ventricular PDE5 levels on cardiac function, PDE5-TG mice were generated. Confocal and immunoelectron microscopy revealed increased PDE5 expression in cardiomyocytes, predominantly localized to Z-bands. At baseline, myocardial cGMP levels, cell shortening, and calcium handling in isolated cardiomyocytes and LV hemodynamic measurements were similar in PDE5-TG and wild-type littermates. Ten days after myocardial infarction, LV cGMP levels had increased to a greater extent in wild-type mice than in PDE5-TG mice (P<0.05). Ten weeks after myocardial infarction, LV end-systolic and end-diastolic volumes were larger in PDE5-TG than in wild-type mice (57±5 versus 39±4 and 65±6 versus 48±4 µL, respectively; P<0.01 for both). LV systolic dysfunction and diastolic dysfunction were more marked in PDE5-TG than in wild-type mice, associated with enhanced hypertrophy and reduced contractile function in isolated cardiomyocytes from remote myocardium.
Conclusions— Increased PDE5 expression predisposes mice to adverse LV remodeling after myocardial infarction. Increased myocardial PDE5 expression in patients with advanced cardiomyopathy may contribute to the development of heart failure and represents an important therapeutic target.
Key Words: phosphodiesterase-5 • cyclic GMP • myocardial infarction • heart failure
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Introduction |
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In the heart, cyclic adenosine and guanosine monophosphates (cAMP and cGMP) are important second–messenger molecules controlled by tightly regulated and compartmentalized synthesis and degradation processes. cAMP and cGMP have often opposing roles in cardiovascular homeostasis, because many of the inotropic and chronotropic effects of β-adrenergic stimulation in the mammalian heart are mediated by cAMP, whereas cGMP opposes these effects via activation of its intracellular target protein kinase G.1–3 Cardiac cGMP synthesis is stimulated by activation of soluble guanylate cyclase by nitric oxide (NO) and by the binding of natriuretic peptides to their receptors (NPR-1 and NPR-2).4–6 cGMP is catabolized by phosphodiesterases (PDEs), many of which are compartmentalized in cardiomyocytes.2,3,7 Of the 11 different known PDE isoenzymes, PDE5, PDE6, and PDE9 degrade cGMP specifically, whereas PDE1 through PDE3 and PDE 10 and 11 can hydrolyze both cAMP and cGMP.8 Cardiac cAMP hydrolysis, under normal conditions, is controlled predominantly by PDE3 and PDE4, and PDE4-knockout mice develop progressive cardiomyopathy.9,10 Increased cGMP concentrations can modulate cardiomyocyte contractility by inhibiting PDE3-mediated cAMP hydrolysis or stimulating PDE2-mediated cAMP hydrolysis, but PDE2 represents a minor part of cAMP hydrolysis in cardiomyocytes.2,8,11,12
Clinical Perspective p 416
The role of PDE5 in regulating smooth muscle tone in the systemic and pulmonary vasculature has long been recognized.13 Because PDE5 expression in the heart under normal physiological conditions is low, the importance of PDE5 has only recently begun to be appreciated in cardiac disease, when perturbations in NO and natriuretic peptide signaling occur.11,14,15 For example, Takimoto and colleagues16 reported that transverse aortic constriction increased left ventricular (LV) PDE5 expression and that treatment with sildenafil, a specific PDE5 inhibitor, prevented the development of LV hypertrophy and reversed already-established LV remodeling. Recently, Nagendran et al17 reported that PDE5 expression was increased in right ventricular (RV) biopsy samples from patients with RV hypertrophy and pulmonary hypertension. However, it remained to be determined whether PDE5 expression is increased in the LVs of patients with cardiomyopathy and whether increased PDE5 expression contributes to adverse LV remodeling or protects against it.
In the present study, we measured PDE5 expression in LVs of patients with end-stage cardiomyopathy and observed that PDE5 expression was markedly greater in LV tissues from these patients than in unused donor LV tissues. To evaluate the impact of increased ventricular PDE5 expression on cardiac function, we characterized LV structure and function in wild-type mice and mice with cardiomyocyte-specific overexpression of PDE5 at baseline and after myocardial infarction (MI). We report that increased ventricular PDE5 expression does not affect baseline cardiac function but predisposes mice to adverse LV remodeling after MI.
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Methods |
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Human Cardiac Samples
Cardiac tissue samples were obtained from patients with ischemic and dilated cardiomyopathy who were referred for cardiac transplantation to Gasthuisberg University Hospital (KU University of Leuven, Belgium) and Massachusetts General Hospital (Harvard University, Boston). Unused LV tissues from donors without documented prior cardiac disease were studied as controls. PDE5 expression and collagen deposition were analyzed with immunohistochemical and immunoblot techniques (n=10 cardiomyopathy patients and 5 control hearts; for details, see the online-only Data Supplement).
Experimental Animals
The bovine PDE5 cDNA (GenBank L16545, revised April 11, 1996), kindly provided by J. Corbin, was ligated into a plasmid containing the 
-myosin heavy chain promoter.18 A restriction fragment that contained the full expression cassette was microinjected into FVB mouse eggs (Charles River Laboratories, Wilmington, Mass). Oocytes were implanted into a pseudopregnant Swiss foster mother, and offspring were screened with polymerase chain reaction to confirm successful transgene integration. Transgenic founders were backcrossed for 5 generations onto a C57BL/6 background. Wild-type littermates served as controls in all experiments.
Measurement of PDE5 Expression With Immunoblot, Immunohistochemical, and Immunoelectron Microscopy Techniques
Levels of PDE5 protein in mouse LV were measured with immunoblot and immunohistochemical techniques and a rabbit anti-PDE5 polyclonal antibody. Subcellular localization of PDE5 was ascertained in cardiomyocytes isolated from wild-type (WT) and PDE5-transgenic (PDE5-TG) mice (online-only Data Supplement). Cardiomyocytes were stained with rabbit polyclonal PDE5 antiserum and a murine monoclonal antibody recognizing desmin (#M0760, DAKO, Glostrup, Denmark). Bound primary antibodies were revealed with Alexa 488– and Alexa 568–labeled goat anti-rabbit and anti-mouse polyclonal antibodies, respectively. Nuclei were stained with TOPRO-3 (Invitrogen, Merelbeke, Belgium). Cardiomyocytes were examined with confocal laser scanning microscopy (LSM510, Zeiss, Jena, Germany). For immunoelectron microscopy studies, 1-µm-thick sections from resin-embedded hearts were stained with a gold-labeled goat anti-rabbit antibody (online-only Data Supplement). All animal experiments were approved by the Animal Ethics Committee of KU Leuven and were in accordance with established guidelines.
LV PDE Activity and Cyclic Nucleotide Levels
cGMP-hydrolyzing PDE enzyme activity in hearts from WT and PDE5-TG mice was measured in the presence and absence of sildenafil (2 µmol/L; Pfizer, Groton, Conn) with an [8-3H]cGMP conversion assay according to a modification of the method described by Turko et al19 (online-only Data Supplement). Sildenafil-inhibitable cGMP catabolism was measured over a wide range of substrate concentrations (0.06 to 100 µmol/L), and Km (cGMP concentration at which activity was half-maximal) was determined. Cyclic nucleotide concentrations were measured in lyophilized extracts prepared from LVs at baseline and at 10 days and 10 weeks after MI with an enzyme immunoassay (online-only Data Supplement).
Quantitative Polymerase Chain Reaction Analysis
Expression of genes encoding proteins involved in cGMP metabolism was measured with quantitative reverse-transcriptase polymerase chain reaction analysis on ventricular samples obtained from hearts of WT and PDE5-TG mice with SYBR Green (Applied Biosystems, Foster City, Calif). Specific primers recognizing sequences of cDNAs encoding murine PDE2, PDE3, PDE5, soluble guanylate cyclase subunits (sGC1 and sGCβ1), cGMP-dependent protein kinase (PKG-I), NPR-1, and NPR-2 were used (online-only Data Supplement Table I). Transcript levels were normalized to levels of mRNA encoding hypoxanthine phosphoribosyl transferase or 18S ribosomal RNA.
Contractility and Calcium Studies in Isolated Cardiomyocytes
Cardiomyocytes were isolated from WT and PDE5-TG mice at baseline and at 10 weeks after MI, as described previously, and examined in a perfusion chamber mounted on the stage of an inverted microscope (Nikon Diaphot, Nikon Benelux, Brussels, Belgium).20 Cell length and width were determined in a random sample of 40 cardiomyocytes per heart with 3 to 5 animals examined per group and condition. Cell shortening during field stimulation at 0.5, 1, 2, and 4 Hz was measured with a video edge detector (Crescent Electronics, Sandy, Utah). After stabilization at 1 Hz, the effect of β-adrenergic stimulation (isoproterenol 50 nmol/L) on cell shortening was recorded in the presence or absence of PDE5 inhibitor II {30 nmol/L 2-butyl-5-(4-methoxyphenyl)-5,6,11,11a-tetrahydro-1H-imidazo[1',5':1,6]pyrido[3,4-b]indole-1,3(2H)-dione, #524714, Calbiochem, San Diego, Calif}. Percent cell shortening was calculated as the ratio of twitch amplitude to diastolic length, and time to peak shortening and half-time of relaxation were determined. L-type calcium currents (ICaL) and intracellular calcium concentrations were measured with the whole-cell ruptured patch-clamp technique (online-only Data Supplement).20
Hemodynamic Measurements at Baseline and After MI
MI was induced in 2- to 4-month-old PDE5-TG and WT mice by permanent ligation of the left anterior descending coronary artery, as described previously.21 For invasive, closed-chest hemodynamic measurements, mice were anesthetized with urethane and -chloralose (1200 and 50 mg/g body weight IP) and mechanically ventilated. Instantaneous LV pressures and volumes with and without preload reduction were recorded in unoperated mice (WT n=16, PDE5-TG n=11) and in mice 10 weeks after MI (n=15 for both genotypes) with a 1.4F Millar SPR839 pressure-conductance catheter (Millar Instruments, Houston, Tex) as described previously.22 We also performed separate hemodynamic measurements in unoperated 44-week-old mice of both genotypes (n=10). Indices of systolic and diastolic function were calculated, including ejection fraction, preload-recruitable stroke work, arterial elastance, preload-adjusted maximal power, and the time constant of isovolumic relaxation (
). The response to β-adrenergic stimulation was studied in additional unoperated mice and in mice 10 weeks after MI during infusion of dobutamine (1, 3, and 10 ng · g–1 · min–1, n=7 for each genotype and condition).
Histological Analysis of LV Remodeling After MI
After completion of hemodynamic measurements 10 weeks after MI, LVs were weighed, and cardiomyocyte width was measured in the remote, noninfarcted area of laminin-stained, 5-µm-thick LV sections. To assess the degree of fibrosis in the LV, the area of collagen deposition was traced on Sirius red–stained sections with polarized light and spectral thresholding (online-only Data Supplement). Infarct size was determined in additional WT and PDE5-TG mice 3 days after left anterior descending coronary artery ligation on consecutive Sirius red–stained sections encompassing the entire LV free wall, according to the midline infarct length method validated by Takagawa et al.23
Statistical Analysis
All data are expressed as mean±SEM unless otherwise indicated. Differences between groups were determined with Student t test, ANOVA, or 2-way ANOVA for repeated measurements where indicated. When applicable, significant differences were further analyzed with Bonferroni and Student-Newman-Keuls post hoc tests. A value of P<0.05 was considered significant.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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Increased PDE5 Expression in LV Tissues From Patients With End-Stage Heart Failure
In LVs from patients without cardiac disease, PDE5 expression was minimal in cardiomyocytes, as well as in myocardial extracts (Figure 1A and 1J), but PDE5 was readily detectable in vascular smooth muscle (Figure 1A, inset). In contrast, we detected markedly increased PDE5 immunoreactivity in cardiomyocytes from patients with dilated and ischemic cardiomyopathy (Figure 1D and 1G) and in myocardial extracts from patients with dilated cardiomyopathy (Figure 1J). Densitometric analysis confirmed an almost 5-fold increase in PDE5 expression levels (Figure 1K; P<0.01). In heart failure patients, the PDE5 expression pattern was nonuniform, and PDE5 immunoreactivity was scattered between areas of fibrosis, as indicated by the Sirius red stain (Figure 1F and 1I).
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PDE5 Localizes to Sarcomeric Z-Bands and Is Abundantly Expressed in Cardiomyocytes From PDE5-TG Mice
PDE5 immunoreactivity was much more prominent in LV tissue and in cardiomyocytes from PDE5-TG mice (Figure 2A and 2B) than in WT mice (Figure 2C and 2D). The intensity and distribution of recombinant PDE5 were comparable in newborn and 2- to 6-month-old PDE5-TG mice (data not shown). Immunoblot analysis revealed abundant 98-kDa PDE5 protein in hearts of PDE5-TG mice: PDE5 expression was almost 9-fold greater in PDE5-TG than in WT LV extracts (Figure 2E and 2F; P=0.001).
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To investigate the intracellular localization of PDE5 in WT and PDE5-TG mice, confocal laser scanning microscopy was performed on isolated cardiomyocytes with fluorescently labeled antibodies against PDE5 (green in Figure 3A and 3D) and desmin (red in Figure 3B and 3E). In WT cardiomyocytes, low levels of PDE5 immunoreactivity were predominantly localized around Z-bands. In contrast, high PDE5 levels were detected in PDE5-TG cardiomyocytes. The merged images (yellow in Figure 3C and 3F) and the immunoelectron microscopy analysis (online-only Data Supplement Figure I) confirmed that recombinant PDE5 was predominantly localized to sarcomeric Z-bands.
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Enhanced PDE5 Enzymatic Activity Does Not Affect Myocardial cGMP Levels at Baseline or Expression of cGMP-Metabolizing Enzymes
Sildenafil-inhibitable cGMP hydrolysis, a measure of PDE5 enzyme activity, was 10-fold greater in PDE5-TG than WT LV extracts. The Km for PDE5 in transgenic mice (4.9±0.8 µmol/L) did not differ from values reported for purified bovine PDE5 (online-only Data Supplement Figure II).24 At baseline, concentrations of cGMP in LV extracts were very low and did not differ between PDE5-TG and WT mice (0.013±0.004 versus 0.013±0.008 pmol/mg protein, respectively; n=8 for both). Moreover, baseline cAMP nucleotide levels were similar in LV extracts from PDE5-TG and WT mice (1.76±0.08 and 1.58±0.04 pmol/mg protein; n=12 for both). Cardiomyocyte-specific overexpression of PDE5 did not affect LV expression of endogenous PDE isoforms (including PDE2, PDE3, and PDE5), PKG-I, soluble guanylate cyclase subunits, or natriuretic peptide receptors (WT n=9, PDE5-TG n=10; online-only Data Supplement Figure III).
Enhanced PDE5 Enzymatic Activity Does Not Affect Baseline Excitation-Contraction Coupling of Isolated Cardiomyocytes
At baseline, contraction amplitude and kinetics were similar in WT and PDE5-TG cardiomyocytes (Figure 4A; mean cell shortening, normalized to resting cell length, was 7.6% and 7.3%, respectively; >60 cells per genotype). The infusion of isoproterenol increased absolute cell shortening in both genotypes (Figure 4B), whereas preincubation with PDE5 inhibitors modestly but significantly reduced isoproterenol-mediated fractional cell shortening (Figure 4C). The amplitude and voltage dependence of calcium currents were similar in both genotypes (Figure 4D), as were calcium transients at various stimulation frequencies (online-only Data Supplement Figure IV).
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PDE5 Overexpression Does Not Affect Baseline Cardiac Function
Baseline LV systolic function (stroke work and preload-adjusted maximal power) and relaxation () did not differ between PDE5-TG and WT mice (Table 1). In both genotypes, infusion of sildenafil reduced LV afterload by 
10% and attenuated the dobutamine-induced rise in preload-recruitable stroke work, a load-independent index of contractile function (data not shown).
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PDE5 Overexpression Predisposes Mice to Adverse LV Remodeling After MI
Three days after left anterior descending coronary artery ligation, infarct size as a percentage of LV was similar in WT and PDE5-TG mice (36±5% versus 35±9%; online-only Data Supplement Figure V); however, 10 weeks after MI, LV end-systolic and -diastolic volumes were larger in PDE5-TG than in WT mice (Figure 5A and 5B). In PDE5-TG mice, LV ejection fraction and preload-recruitable stroke work were less than in WT mice (Figure 5C and 5D). Diastolic function was also impaired to a greater extent in PDE5-TG mice, as indicated by the more marked prolongation of (Table 1).
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P<0.05 vs WT). The dobutamine-induced increase in heart rate was similar in both genotypes (E), whereas the increase in preload-recruitable stroke work was blunted in PDE5-TG mice (F). Myocardial contractile reserve, 10 weeks after MI, was evaluated with graded β-adrenergic stimulation with dobutamine. Infusion of dobutamine increased the heart rate in a dose-dependent manner similarly in both genotypes (Figure 5E). In contrast, the ability of dobutamine to increase preload-recruitable stroke work after MI was impaired in PDE5-TG mice, which suggests a genotype-dependent reduction in myocardial contractile reserve (Figure 5F). Overall survival rates at 10 weeks after MI were not different in WT and PDE5-TG mice, although mortality was higher in male than in female mice, irrespective of genotype.
Whereas at baseline, the heart-to-body weight ratio did not differ between genotypes, at 10 weeks after MI, it was greater in PDE5-TG than in WT mice (P<0.05; Table 2). Ten weeks after MI, cardiomyocyte width in remote myocardium was greater in PDE5-TG than in WT mice (16.6±0.1 versus 15.1±0.3 µm, n=7 for both, P<0.001; Figure 6), which suggests greater hypertrophy in PDE5-TG mice. Deposition of thick, tightly packed type I collagen fibers and thin, loosely assembled type III collagen fiber did not differ between genotypes after MI (Table 2).
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Enhanced PDE5 Expression Reduces Myofilament Calcium Sensitivity After MI
To investigate the mechanisms responsible for greater impairment of myocardial contractile function after MI in PDE5-TG mice, cell shortening and intracellular calcium transients were studied in cardiomyocytes isolated from WT and PDE5-TG mice at 10 weeks after MI. Cardiomyocyte length tended to be greater in PDE5-TG than in WT cells (198±22 versus 162±6 µm, P=0.07; >120 cells in each group). Cell capacitance, a measure of total membrane surface area, was significantly greater in PDE5-TG than in WT cells (292±26 versus 200±9 pF; n=18 and 25 cells, respectively; P<0.05). The amplitude of cell shortening was decreased in both genotypes after MI compared with baseline values, particularly at higher stimulation frequencies (Figure 7A; baseline values >8% shortening; online-only Data Supplement Figure IV). The rates of contraction and relaxation were less in PDE5-TG than in WT cells (Figure 7B and 7C). In addition, resting diastolic calcium levels and amplitude of calcium transients were significantly greater at all stimulation frequencies in PDE5-TG cardiomyocytes (Figure 7D and 7E), but kinetics were not different from WT cardiomyocytes (data not shown). Calcium currents and sarcoplasmic reticulum calcium content did not differ between genotypes (data not shown). Increased calcium transients in the presence of slower contractions suggest that myofilament responsiveness to calcium was less in PDE5-TG than in WT cardiomyocytes after MI.
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Enhanced PDE5 Enzymatic Activity Reduces cGMP Levels in Infarcted Hearts
Ten days after MI, myocardial cGMP and cAMP concentrations increased in both genotypes compared with baseline (P<0.01 and P<0.05, respectively); however, cGMP concentrations increased >10-fold in WT mice (0.17±0.02 pmol/mg protein, n=7), whereas this increase was attenuated in PDE5-TG mice (0.09±0.02 pmol/mg protein, n=9; P<0.05 versus WT). In contrast, the rise in cAMP levels 10 days after MI was not different between WT and PDE5-TG mice (7.79±2.80 pmol/mg protein, n=8, versus 8.99±1.90 pmol/mg protein, n=9, respectively; P=0.72). Ten weeks after MI, myocardial cGMP levels were less and were no longer different between genotypes (0.06±0.02 pmol/mg protein in WT, n=8, and 0.04±0.03 pmol/mg protein in PDE5-TG, n=7).
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Discussion |
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In the present study, we report that PDE5 was markedly expressed in LVs from patients with end-stage ischemic and dilated cardiomyopathy, whereas PDE5 expression was barely detectable in hearts from patients without cardiac disease (Figure 1). In patients with congestive heart failure, PDE5 immunoreactivity was markedly increased in cardiomyocytes between areas of fibrosis. To investigate whether enhanced expression of PDE in the failing LV was protective or deleterious, we characterized the LV remodeling response to MI in transgenic mice with cardiomyocyte-selective PDE5 overexpression. In cardiomyocytes from PDE5-TG mice, we observed abundant PDE5 expression, with a marked clustering to Z-bands, as confirmed by confocal fluorescence imaging and immunoelectron microscopy. At baseline, total myocardial cGMP levels were very low in PDE5-TG and WT mice. In addition, cardiomyocyte-specific overexpression of PDE5 did not alter cardiac structure or function at baseline and did not affect sarcomere shortening, calcium transients, or peak calcium currents. After MI, however, myocardial cGMP levels increased >10-fold in WT mice, but this increase was significantly reduced in PDE5-TG mice. After MI, PDE5 overexpression led to a more marked impairment of LV systolic and diastolic function associated with an inability to mount a contractile response to β-adrenergic stimulation. Isolated cardiomyocytes from PDE5-TG mice after MI also had impaired contraction and relaxation, with reduced myofilament responsiveness to calcium. Taken together, these results suggest cGMP signaling has an important role in limiting the acute stress response after MI, as well as the long-term adverse LV remodeling and functional impairment.
In the mammalian heart, PDE5 is expressed at low levels, and because of its subcellular localization to Z-bands, it is thought to regulate intracellular cGMP microdomains that modulate β-adrenergic contractility.3 Inhibition of PDE5 has little effect on myocardial cGMP levels at baseline but enhances cGMP concentrations in these microdomains under conditions associated with hemodynamic and neurohumoral stress.3,16 Elevated cGMP levels counteract cAMP-mediated inotropic effects of catecholamines in mouse, dog, and human heart25–28 and pressure-overload–induced hypertrophic remodeling in mouse hearts.15 The present studies confirm that PDE5 expression levels are very low in normal human hearts and are the first to demonstrate markedly enhanced expression in residual cardiomyocytes of patients with end-stage ischemic or idiopathic dilated cardiomyopathy. Because the role of enhanced PDE5 expression in advanced heart failure is incompletely understood, we generated mice with cardiomyocyte-specific overexpression of PDE5 and studied functional and structural characteristics at baseline and in response to MI.
In PDE5-TG mice, the recombinant protein has the expected Km of bovine PDE5 and is predominantly localized within the cells to Z-bands. At baseline, we did not detect a difference in cardiac cGMP levels between genotypes. Moreover, hemodynamic measurements in intact animals and electrophysiological studies in isolated cardiomyocytes suggested that overexpression of PDE5 did not affect baseline cardiac function (Figure 4C). This is consistent with limited activation of both soluble and particulate guanylate cyclases under normal physiological circumstances, when NO and natriuretic peptides levels are low.15,29 In contrast, we observed that MI induces a 10-fold increase in myocardial cGMP levels in WT mice, but the increase was significantly reduced in PDE5-TG mice. The present experiments do not allow identification of the mechanisms responsible for this increase, which may be related to increased atrial or brain natriuretic peptide production or induction of cardiac NO synthesis after MI.29,30 Increased cGMP levels may protect against the acute β-adrenergic stress that occurs in the first 10 days after MI and against adverse LV remodeling and hypertrophy in remote myocardium thereafter. Failure to increase myocardial cGMP levels could then account for the greater functional impairment and increased cardiomyocyte hypertrophy in PDE5-TG mice. Accentuated hypertrophy and impaired function of isolated cardiomyocytes from PDE5-TG mice further indicate a more profound maladaptive remodeling at the myocyte level.
How does the impaired ability to increase cGMP levels after MI contribute to excess LV dilatation and hypertrophy in transgenic mice? One possibility is that increased cGMP levels reduce MI size and thereby the stimulus to LV remodeling. This is unlikely, because MI size 3 days after left anterior descending coronary artery ligation was similar in both genotypes (online-only Data Supplement Figure V). A second possibility is that increased LV cGMP levels after MI counteract the adverse remodeling effects of excessive β-adrenergic signaling induced by hemodynamic and neurohumoral stress. Unopposed catecholamine signaling has been shown to contribute to cardiac dysfunction in paced animals and in heart failure patients deprived of β-blocker therapy.31 A third possibility is that increased cGMP levels may inhibit cAMP hydrolysis by PDE3, augmenting LV cAMP levels and contractile function after MI and thereby limiting the stimulus to adverse LV remodeling. Nagendran and colleagues17 reported that pharmacological PDE5 inhibition increased contractility of RV myocytes by inhibiting PDE3-mediated cAMP catabolism. Finally, increased cGMP levels may counteract adverse LV remodeling after MI via cAMP-independent mechanisms, for instance, via protein kinase G–dependent pathways. The observation that the increase in cardiac cAMP levels 10 days after MI did not differ in WT and PDE5-TG mice suggests that increased cardiac cGMP levels protect against adverse LV remodeling via a cAMP-independent mechanism.
We recognize the limitations of the present study. First, lifelong myocardial overexpression of the transgene may trigger counterregulatory or compensatory changes in cyclic nucleotide signaling, despite similar expression of other PDEs and cGMP-generating enzymes (online-only Data Supplement Figure III). In addition, our observations in end-stage heart failure patients do not permit us to conclusively establish the pathophysiological role for PDE5; this requires further prospective studies.
The present data suggest that PDE5 may have an important role in heart failure and that PDE5 inhibitors may be a valuable new treatment strategy. Initial studies of patients with systolic heart failure and RV dysfunction/pulmonary hypertension suggest that sildenafil can improve exercise tolerance and hemodynamics.32 The present findings add additional impetus to studies using PDE5 inhibitors to prevent or treat adverse LV remodeling.
In summary, PDE5 is markedly upregulated in cardiomyocytes of patients with end-stage heart failure. Cardiac-restricted overexpression of PDE5 does not affect baseline myocardial structure or function but significantly reduces systolic and diastolic function after MI and exacerbates adverse LV remodeling. These observations identify increased PDE5 expression in the LV as a novel target for heart failure treatment.
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Acknowledgments |
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The authors thank Federica del Monte, MD, PhD, Thomas MacGillivray, MD, and Judith Gwathmey, MD, for providing myocardial tissue samples from patients.
Sources of Funding
This work was supported by the Fund for Scientific Research-Flanders G.0381.05 (Dr Herijgers), EWM-B4361-G.0442.06 (Dr Janssens), and the Flanders Institute for Biotechnology (VIB, to Dr Janssens); National Institutes of Health grant HL070896 (Dr Bloch); Research Fund K.U. Leuven OT/05/55 (Dr Herijgers) and GOA/2007/13 (Drs Sipido, Pokreisz, and Janssens); and European Union LSHM-CT-2005-018833, EUGeneHeart (Dr Bito). Dr Janssens is a Principal Investigator of Flanders Institute of Technology and is holder of a chair supported by Astra-Zeneca.
Disclosures
None.
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The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.822072/DC1.
Guest Editor for this article was Douglas L. Mann, MD.
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Circulation 2009 119: 359-361.
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