Myocardial Remodeling Is Controlled by Myocyte-Targeted Gene Regulation of Phosphodiesterase Type 5

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1 Journal of the American College of Cardiology Vol. 5, No., by the American College of Cardiology Foundation ISSN 75-97/$. Published by Elsevier Inc. doi:./j.jacc..8. PRE-CLINICAL RESEARCH Myocardial Remodeling Is Controlled by Myocyte-Targeted Gene Regulation of Phosphodiesterase Type 5 Manling Zhang, MD, Eiki Takimoto, MD, PHD, Steven Hsu, MD, Dong I. Lee, PHD, Takahiro Nagayama, PHD, Thomas Danner, Norimichi Koitabashi, MD, PHD, Andreas S. Barth, MD, Djahida Bedja, MS, Kathleen L. Gabrielson, PHD, Yibin Wang, PHD, David A. Kass, MD Baltimore, Maryland; and Los Angeles, California Objectives Background Methods Results Conclusions We tested the hypothesis that bi-directional, gene-targeted regulation of cardiomyocyte cyclic guanosine monophosphate selective phosphodiesterase type 5 (PDE5) influences maladaptive remodeling in hearts subjected to sustained pressure overload. PDE5 expression is up-regulated in human hypertrophied and failing hearts, and its inhibition (e.g., by sildenafil) stimulates protein kinase G activity, suppressing and reversing maladaptive hypertrophy, fibrosis, and contractile dysfunction. Sildenafil is currently being clinically tested for the treatment of heart failure. However, researchers of new studies have questioned the role of myocyte PDE5 and protein kinase G (PKG) to this process, proposing alternative targets and mechanisms. Mice with doxycycline-controllable myocyte-specific PDE5 gene expression were generated (medium transgenic [TG] and high TG expression lines) and subjected to sustained pressure overload. Rest myocyte and heart function, histology, and molecular profiling were normal in both TG lines versus controls at months of age. However, upon exposure to pressure overload (aortic banding), TG hearts developed more eccentric remodeling, maladaptive molecular signaling, depressed function, and amplified fibrosis with upregulation of tissue growth factor signaling pathways. PKG activation was inhibited in TG myocytes versus controls. After establishing a severe cardiomyopathic state, high-tg mice received doxycycline to suppress PDE5 expression/activity only in myocytes. This in turn enhanced PKG activity and reversed all previously amplified maladaptive responses, despite sustained pressure overload. Sildenafil was also effective in this regard. These data strongly support a primary role of myocyte PDE5 regulation to myocardial pathobiology and PDE5 targeting therapy in vivo and reveal a novel mechanism of myocyte-orchestrated extracellular matrix remodeling via PDE5/cyclic guanosine monophosphate PKG regulatory pathways (J Am Coll Cardiol ;5: ) by the American College of Cardiology Foundation Cardiac hypertrophy and associated maladaptive cellular and molecular changes often develop from sustained pressure overload and are a major worldwide cause of morbidity and mortality. Current treatments target load reduction From the Department of Medicine, Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland; Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, Maryland; and the Department of Medicine and Anesthesia, UCLA, Los Angeles, California. Supported by National Institutes of Health National Heart, Lung, and Blood Institute grants HL-8997, HL-89 and Fondation Leducq (Dr. Kass), HL-9 (Drs. Takimoto and Kass), and T-HL-77 (Drs. Zhang and Barth) and a fellowship grant to Dr. Zhang from the American Heart Association. All authors have reported that they have no relationships to disclose. Manuscript received April 8, ; revised manuscript received July,, accepted August,. with diuretics and vasodilators, yet the disease often is refractory to these approaches. One alternative is to stimulate intrinsic negative regulators of hypertrophy, such as cyclic guanosine monophosphate (cgmp) and its downstream protein kinase G (PKG) (). cgmp levels are enhanced by nitric oxide (NO) and natriuretic peptides or See page by inhibiting cgmp-targeted phosphodiesterases (PDE), such as PDE5. The latter is currently used to treat erectile dysfunction and pulmonary hypertension, yet recent experimental studies show that it also blunts cardiac disease induced by ischemia/re-oxygenation (), pressure-overload

2 Zhang et al. JACC Vol. 5, No., Cardiac Stress Response and Myocyte PDE5 December 7, : Abbreviations and Acronyms cgmp cyclic guanosine monophosphate DOX doxycycline ECM extracellular matrix LV left ventricular MHC myosin heavy chain PDE phosphodiesterase PKG protein kinase G TAC trans-aortic constriction TG transgenic TGF transforming growth factor (,), and doxorubicin toxicity (5). Such results helped foster the current National Institutes of Health sponsored multicenter clinical trial (the RELAX [Evaluating the Effectiveness of Sildenafil at Improving Health Outcomes and Exercise Ability in People With Diastolic Heart Failure] study; NCT787) testing sildenafil in patients with heart failure and a preserved ejection fraction. The mechanisms by which sildenafil suppresses and/or reverses experimental cardiac disease remain somewhat controversial. Recent studies have suggested that PDE5 is not the primary target, proposing the more abundant dual-substrate PDE as an alternative (,7). Furthermore, whether myocyte PDE5 and corresponding PKG regulation modulates cardiac hypertrophy/remodeling to counter cardiac stress has been recently questioned (). Clarification of this signaling is important because this could argue for pursuing alternative molecular targets. All prior studies in which PDE5 activity was suppressed employed inhibitors that are not perfectly selective and that influence multiple cell types. Global gene deletion is embryonically lethal, and successful attempts at conditional models have yet to be reported. One alternative model is to genetically modulate myocyte PDE5 in a bi-directional manner using a doxycycline (DOX)-responsive alpha myosin heavy chain (MHC) promoter-driven overexpression. Up-regulation is relevant as myocardial PDE5 expression/ activity rises several-fold in pressure-overload () and in human cardiac hypertrophy (8) and dilated heart failure (9). Importantly, the transgene can be selectively re-suppressed in myocytes after disease is established. Here, we used this strategy to test the hypothesis that myocyte PDE5 suppression and associated PKG activation is an important modulator of pressure-overload maladaptation. The results support this role and reveal novel cross-talk between myocyte cgmp/pkg and extracellular matrix (ECM) remodeling. Methods Cardiac-specific PDE5A-inducible transgenic mice. Tetracycline-controlled conditional PDE5 mice were generated by crossing mice expressing PDE5A (tagged with Flag at the N-terminus) with a modified alpha-mhc promoter tetracycline-off vector (gift from Jeffrey Robbins) () (Fig. A) with mice expressing tetracycline transactivator (alpha-mhc promoter). Two founders (sv9x C57BL/J background) were generated (medium transgenic [me-tg] and high transgenic [hi-tg]), and litter mates expressing tetracycline transactivator served as controls. Mice were born without DOX, so PDE5A was overexpressed after birth, but it could be subsequently repressed by adding DOX (.5 mg/ml) to drinking water. Cardiac and myocyte functional, biochemical, and molecular analyses. These methods and statistical analysis are provided in detail in the Online Appendix. Results Cardiac myocyte-specific PDE5 transgenic mice using tetracycline-off induction system. Two independent conditional PDE5 overexpression lines were generated (me- TG:.7 activity; hi-tg: activity) (Figs. B and C), both born in normal Mendelian ratios and surviving into adulthood (to months). PDE5 expression in the lung was unchanged (Online Fig. A). Expression of alternative cgmp-pdes (PDE, PDE9) was also unchanged in TG mice (Online Fig. B), consistent with unaltered non PDE5-dependent cgmp-pde activity (Online Fig. C). Transgene PDE5 localized to myocyte z-disks, similar to native protein () (Online Fig. ). Up-regulation of PDE5 activity in TG myocytes reduced resting PKG activity (Fig. D) 5% (p.5) and inhibited stimulated PKG activity (Fig. E). The latter was indexed by the lack of plasma membrane translocation of PKG-alpha and PKG-beta in TG myocytes when exposed to cgmp (). Basal myocyte cgmp was below detection in all models. Resting cardiac chamber and myocyte function is normal in young adult PDE5-TG mice. Resting phenotype was comprehensively assessed at age to months. Both me-tg and hi-tg hearts had normal structural morphology, myocardial histology, molecular signaling, and left ventricular (LV) function (Online Figs. and ). LV myocytes displayed similar resting sarcomere shortening and response to beta-adrenergic stimulation (isoproterenol) (Online Fig. 5). The latter was suppressed by sildenafil, consistent with prior reports (,) confirming functional regulation by the transgene protein. In me-tg mice, cardiac function and histology remained similar to that of controls over months of observation, whereas LV function declined after months in hi-tg, with hypertrophy and interstitial fibrosis documented at months (Online Fig. ). For subsequent studies of pressure overload with bi-directional myocyte-pde5 gene regulation, - to -month-old animals were used. Myocyte PDE5 up-regulation worsens cardiac response to pressure overload. To test the impact of PDE5 upregulation on pressure-overload stress, -month-old mice were subjected to weeks of trans-aortic constriction (TAC). TAC induced concentric and generally compensated hypertrophy in controls; however, both TG lines displayed substantial chamber dilation and reduced fractional shortening (Figs. A and B). Pressure volume relations confirmed dilation, particularly in hi-tg, and contractile depression reflected by load-independent indexes. Relaxation rate prolonged only in the TG lines

3 JACC Vol. 5, No., December 7, : Zhang et al. Cardiac Stress Response and Myocyte PDE5 Figure Inducible Cardiac-Specific PDE5 Transgenic Mouse Model (A) Schematic of cardiac specific tetracycline (Tet)-off phosphodiesterase-5 (PDE5) transgenic (TG) system. Two founder lines were developed. (B) Western blot analysis of PDE5 expression in myocytes. (C) Myocyte PDE5 activity from founder lines (referred as medium transgenic [me-tg] and high transgenic [hi-tg] based on expression level; p. vs. control, n to per group). (D) Myocyte protein kinase G (PKG) activity of control and TG group at resting condition (p.5 vs. control, n to 8 per group). (E) Activation of PKG alpha (left) and PKG beta (right), stained green in cardiomyocyte exposed to 8Br cyclic guanosine monophosphate (cgmp), is reflected by rapid translocation to the plasma membrane. This occurred in control () but not TG myocytes (original magnification ). MHC myosin heavy chain. (Figs. C and D). Importantly, afterload increase (arterial elastance) was similar in all TAC groups. Worsened function was accompanied by hypertrophy at chamber and myocyte levels, increased heart and lung weight, and interstitial fibrosis (Fig. ). Apoptosis (activated caspase ) was minimal and unaltered after TAC in all groups, and rare positive cells were interstitial and never myocytes (data not shown). Lack of PKG activation and enhanced maladaptive signaling pathways in TG-TAC hearts. Enhanced myocardial PDE5 activity was unaltered in TG myocardium before and after TAC, whereas it rose 8% over baseline in controls (about rest level in me-tg) (Fig. A). PDEa-c expression was unaltered after TAC (Online Fig. ). Basal myocardial PKG activity was similar, reflecting low basal cgmp synthesis; however, TAC stimulated PKG activity in controls by 7%, but not at all in TG myocardium and isolated myocytes (Fig. B). Increased PKG activity despite PDE5 up-regulation after TAC in controls likely reflects a balance and compartmentation of cgmp synthesis/hydrolysis (). These data demonstrate that this balance can tilt toward PKG suppression with sufficient PDE5 activity. Protein expression of PKG-alpha and -beta was less in TG at rest, the latter at a slightly lower weight, suggesting posttranslational change. Myocyte gene expression for both proteins did not change with TAC (Online Fig. 7A); however, PKG-alpha protein levels rose similarly in both groups, and PKG-beta increased in TG. Thus reduced PKG activity in TG-TAC hearts could not be attributed to depressed PKG alpha/beta expression, but rather to posttranslational modulation.

4 Zhang et al. JACC Vol. 5, No., Cardiac Stress Response and Myocyte PDE5 December 7, : A Sham TAC wks me-tg hi-tg B C 8 FS (%) me-tg hi-tg w w w w LV- EDD (mm) LV- ESD (mm) w w w w w w w w sham Con TAC w me-tg TAC w hi-tg TAC w LV Pressure (mm Hg) D Ea (mmhg/μl) LVVed (μl) p<. p=. 8 8 LV volume (μl) Ees (mmhg/μl.g) p=. 8 8 p=. PWRmax/EDV (mmhg/s) p<. PRSW (mmhg) p=.5 Tau (ms) Sham TAC w me-tg TAC w hi-tg TAC w Figure Exacerbated LV Chamber Dysfunction and Dilation in me-tg and hi-tg Mice Subjected to Weeks of TAC (A) Representative echocardiograms and (B) Summary data for fractional shortening (FS%), left ventricular (LV) end-diastolic and end-systolic dimension (LV-EDD, LV-ESD) (n to 5/group; -way analysis of variance p.5 for interaction of group time for each; p.5 vs. -TAC weeks; p. vs. -TAC weeks). (C) Representative LV pressure-volume (PV) loops and relations show a right shift and contractile depression, particularly in hi-tg mice versus controls. Slope of the end-systolic PV (left upper line) relationship (Ees) reflects contractile function. (D) Summary data from PV loop analysis. Effective arterial elastance (LV afterload, Ea) increased similarly in all groups. Ees end-systolic elastance; LVVed end-diastolic left ventricular volume; PRSW pre-load recruitable stroke work; PWRmax/EDV maximal LV power divided by EDV; TAC trans-aortic constriction; Tau time constant of relaxation. The first are indexes of contractile function (n toper group; p value for -way analysis of variance shown for each). p. versus control TAC weeks; p.5 versus control TAC weeks; p. versus control sham; p.5 versus control sham, Kruskal-Wallis test; p. versus control TAC weeks. Upon exposure to TAC, both TG models developed worsened molecular abnormalities consistent with maladaptive hypertrophy over controls. Fetal gene recapitulation (Nppa and Myh7) was more prominent and regulator of calcineurin- (Rcan, reflecting calcineurin activity) expression rose more, whereas sarcoplasmic reticulum ATPase (Atpa) and phospholamban (Pln), both key calciumhandling proteins, declined more (Fig. D). Respective changes in protein expression/activation were also observed (data not shown). Given worsened fibrosis in TG mice, we determined expression of ECM-regulating genes (Tgfb, and Ctgf). All increased more in TG over controls. Before TAC, their expression was similar (Online Fig. ).

5 JACC Vol. 5, No., December 7, : Zhang et al. Cardiac Stress Response and Myocyte PDE5 5 A HE WGA µm MT µm B sham Cell size (µm ) 8 me-tg hi-tg Interstitial fibrosis(%) C TAC w HW/TL (mg/cm) LW/TL (mg/cm) Sham TAC w me-tg TAC w hi-tg TAC w Figure Chamber, Myocyte, and Molecular Remodeling Induced by -Week TAC Are Exacerbated in me-tg and hi-tg Mice (A) Representative cardiac cross-sections (top: hematoxylin and eosin [HE] stain; middle: wheat germ agglutinin [WGA] stain; bottom: Masson-trichrome [MT] stain). (B) Summary data for myocyte cross-sectional area and interstitial fibrosis (analysis and sample size in Online Appendix). (C) Heart weight/tibia length(hw/tl) and lung weight/tibia length (LW/TL); p.5 for -way analysis of variance, p. versus control () Sham; p.5 versus trans-aortic constriction (TAC) weeks; p. versus TAC weeks. Re-suppression of PDE5 in myocytes reverses maladaptive remodeling. TAC-induced cardiac abnormalities and early lethality were observed within week in hi-tg mice (Online Figs. 7B and 7C), but not in me-tg mice. This provided a model to test the impact of subsequently downregulating only cardiomyocyte PDE5. Both controls and hi-tg mice were exposed to weeks of TAC, with DOX added to the drinking water after day 7, inhibiting PDE5 transgene expression in TG mice thereafter. DOX eliminated the disparity in PDE5 activity between groups (Fig. 5A) and PKG activity rose in hi-tg plus DOX, matching control levels (Fig. 5B). Figure 5C shows echocardiography summary data. After 7 days of TAC, hi-tg mice displayed worsened function, dilation, and hypertrophy. Without subsequent DOX, this progressed further over the ensuing 7 days, whereas DOX-treated TG animals displayed recovery to behavior in non-tg controls. DOX treatment did not alter controls. At weeks of TAC, DOX-treated hi-tg mice had similar LV hypertrophy, fetal gene expression, interstitial fibrosis, and corresponding expression of connective tissue genes as observed in controls (Figs. 5D, 5E, and 5F). We also examined genome-wide modifications in these models using microarray analysis. Controls and hi-tg mice had generally similar profiles pre-tac, but transcriptional changes after TAC were more pronounced in hi-tg mice. KEGG pathway analysis found greater up-regulation of transforming growth factor (TGF) beta, focal adhesion, and ECM receptor interaction genes and reduced oxidative phosphorylation and metabolic pathway genes (Online Fig. 8). These disparities were largely reversed when PDE5 expression was reduced by DOX treatment. Lastly, we compared these results with non cell-specific and less selective inhibition with sildenafil ( mg/kg/day; yielding free plasma concentration of to nmol/l, similar to humans with standard doses). TAC was instituted and sildenafil started on day 8, by which time hi-tg mice already displayed much more hypertrophy and dysfunction, and was then continued for weeks. LV function improved and chamber dilation diminished in hi-tg TAC plus sildenafil

6 Zhang et al. JACC Vol. 5, No., Cardiac Stress Response and Myocyte PDE5 December 7, : A PDE5 activity (normalized) B PKG activity (normalized) 5 Myocardial Myocardial Myocyte Baseline TAC w Baseline TAC w Sham me-tg TAC w C PKGα GAPDH PKGβ Myocyte Sham Sham TAC w hi-tg TAC w...8. PKGα/GAPDH..8. PKGβ/GAPDH GAPDH Baseline TAC w Baseline TAC w D Rcan..8. Atpa..8. Pln Nppa 8 Myh7 Sham TAC w me-tg TAC w hi-tg TAC w Tgfb Tgfb Ctgf 9 Figure Effect of PDE5 Upregulation on TAC-Stimulated PKG Activity, Calcineurin, and Calcium Cycling Proteins (A) Myocardial phosphodiesterase-5 (PDE5) activity in controls () versus high-transgenic (hi-tg) mice before and after trans-aortic constriction (TAC) (p. vs., same condition). (B) Myocardial protein kinase G (PKG) activity in versus hi-tg mice before and after weeks of TAC (p.5 vs. baseline; p.5 vs. TAC weeks); right: isolated myocyte PKG activity determined after week of TAC (p. vs., n to 5 in each group. (C) PKG-alpha and PKG-beta protein expression at baseline and after TAC in and hi-tg mice. (-way analysis of variance, p.5 vs. baseline; p.5 vs. baseline; p.5 vs. hi-tg baseline; p. vs. same group baseline). (D) Gene expression assessed by quantitative real-time polymerase chain reaction for regulator of calcineurin (Rcan), SERCAA (Atpa), phospholamban (Pln), A-type natriuretic peptide (Nppa), beta-myosin heavy chain (Myh7), tissue growth factor beta- and beta- (Tgfb, Tgfb), and connective tissue growth factor (Ctgf), each normalized to Gapdh. p. versus control TAC weeks; p.5 versus control TAC weeks; p. versus control sham; p.5 versus control sham (n to in each group).

7 JACC Vol. 5, No., December 7, : Zhang et al. Cardiac Stress Response and Myocyte PDE5 7 A 5 PDE5 activity N.S B PKG activity hi-tg C Baseline TAC+Dox Baseline TAC+DOX +DOX +DOX FS (%) LV-EDD(mm) LV mass (mg) 5 DOX.5 9 Base D7 D7 D.5.5 DOX Base D7 D7 D 7 DOX Base D7 D7 D D HW / TL (mg/cm) N.S E Nppa. NS. Myh7 NS hi-tg.. TAC TAC + DOX F -TAC hitg-tac Interstitial fibrosis(%).5 Ctgf NS NS.5 TAC TAC+ DOX TAC+ DOX.5 Tgfβ NS.5 Tgfβ NS μm.5.5 -TAC+DOX hitg-tac+dox TAC+ DOX TAC+ DOX Figure 5 Re-Suppression of Myocyte PDE5 Expression Reverses Established Maladaptive Cardiac Remodeling Despite Sustained Pressure Overload Control () and high-transgenic (hi-tg) mice were subjected to 7 days of trans-aortic constriction (TAC), then randomized to either doxycycline (DOX) to suppress phosphodiesterase-5 (PDE5) transgene expression or no treatment, whereas TAC continued for 7 more days. (A) Myocardial PDE5 and (B) protein kinase G (PKG) activity measured in and hi-tg at baseline and at end of study. DOX restored both to levels observed in ; p. versus hi-tg pre-dox treatment; p. versus ; p. versus same genotype at pre-dox treatment (n to /group). (C) Echocardiogram data for DOX reversal study. LV mass left ventricle mass; other abbreviations are as in Figure. All indexes improved in hi-tg with DOX treatment and becoming indistinguishable from values by day. p. versus at same time point and treatment; p.5 versus at same time point and treatment (n 5to).(D) Cardiac hypertrophy assessed by heart weight/tibia length (HW/TL) rose in TG-TAC (end of study), but declined to same level as in with DOX treatment (-way analysis of variance, p.; p. versus -TAC; p. versus hi-tg-tac. (E) Fetal gene expression (normalized to Gapdh) is similar in and hi-tg TAC groups with DOX treatment. (F) Myocardial fibrosis was exacerbated in hi-tg after TAC without DOX, but fell to levels with DOX. Summary data for histological analysis and connective tissue genes (expression shown normalized to -TAC, determined at final time point; p. vs. -TAC; p. vs. hi-tg TAC).

8 8 Zhang et al. JACC Vol. 5, No., Cardiac Stress Response and Myocyte PDE5 December 7, : A FS (%) -TAC 5 HITG-TAC HITG-TAC+SIL ESD (mm) D D7 D7 D D D7 D7 D B PDE5 activity n.s.5. PKG activity n.s TAC hitg-tactac -TAC hitg-tactac +SIL +SIL Figure Improvement in Cardiac Function With PDE5 Inhibitor Treatment in hi-tg Mice (A) Data for sham-control, high-transgenic (hi-tg; untreated), and hi-tg plus sildenafil (SIL) treated is shown at baseline (-month-old mice), 7 days of trans-aortic constriction (TAC; pre-treatment), and at days 7 and ( and 7 days after starting treatment during continued TAC). SIL blocked progressive dilation and depressed function observed in untreated hi-tg mice. p. by -way analysis of variance for fractional shortening (FS) and end-systolic dimension (ESD). p. versus same time point control ()-TAC group; p. versus same time point hi-tg-tac group; p.5 versus same time point hi-tg-tac group. (B) SIL treatment reduced phosphodiesterase-5 (PDE5) activity to levels observed in and enhanced protein kinase G (PKG) activity also to levels comparable to that of controls. (Figs. A and B), lowering PDE5 activity and enhanced PKG activity (Fig. B), all matching levels observed in the control TAC group. Importantly, sildenafil was applied when there was already far more cardiac disease in the TG group, so it indeed reversed severity as observed before, and this occurred despite continued myocyte PDE5 upregulation and sustained TAC. Discussion The present study addressed the question of whether bi-directional regulation of myocyte PDE5 influences myocardial function and remodeling in hearts subjected to stress. Enhancing PDE5 expression/activity suppressed PKG activation and worsened responses to pressure overload in a dose-dependent manner. Subsequent gene-targeted downregulation of myocyte PDE5 reversed pre-established chamber remodeling and dysfunction despite continued pressure overload and raised PKG activity to control levels. Similar reversal was obtained with sildenafil. These data support a role for myocyte PDE5 in heart disease and demonstrate that reduction of such activity in myocytes is sufficient to observe profound reversal of maladaptive cardiac stress responses, including cross-talk between myocytes and the ECM. Resting myocyte PDE5 expression and activity is low; indeed some laboratories have detected neither and attributed the sildenafil effect to its off-target suppression of PDE (,7). PDE is a dual-substrate esterase requiring Ca calmodulin for activation, and although PDE blockade inhibited cell and organ hypertrophy induced by isoproterenol in a PKG-dependent manner, this was additive to effects from PDE5 inhibition, suggesting they regulate different cgmp pools (). Ours and other laboratories have observed myocyte PDE5 expression (8,9,,5), and gene-silencing studies further support the specificity and selectivity of these results (5). Importantly, PDE5 is up-regulated - to -fold in experimental mice and human heart disease (,7 9), potentially increasing its influence and pharmacologic impact from its subsequent inhibition. In contrast, PDE changes were small and did not reach statistical significance. PDE5 gene up-regulation worsens cardiac responses to myocardial infarction in experimental animals (9), though reversibility was not tested in this prior study. Here we show that myocyte-targeted PDE5 gene upand down-regulation potently re-orients cardiac stress responses, with the latter indicating that myocyte PDE5 suppression itself confers potent cardiac protective effects. In addition to expression and activity of PDE5 protein itself, its regulatory effects critically depend on cgmp-

9 JACC Vol. 5, No., December 7, : Zhang et al. Cardiac Stress Response and Myocyte PDE5 9 cyclase activity that provides it substrate, and co-stimulation of pathways targeted by PKG (or cgmp) signaling. The cyclase most directly involved is soluble guanylate cyclase coupled to nitric oxide stimulation, as mice with nitric oxide synthase (NOS) inhibited or endothelial NOS genetically deleted lost myocardial modulation by PDE5 inhibitors (). Both cgmp synthesis and cgmp-pkg targeted signaling (such as calcineurin) are stimulated by pathological stress coupled to G-alpha-q cascades (, 8), even as PDE5 is up-regulated, setting the stage whereby subsequent PDE5 inhibition provides greater impact. On the basis of such data, we and others have proposed that PDE5 inhibition acts like a targeted intracellular brake, with minimal impact in normal hearts but an enhanced impact in those under stress (,7). Although sustained PKG activation is traditionally thought to benefit stressed hearts, direct proof remains lacking (9). Genetic models involving global PKG- deletion induce early lethality coupled to intestinal dilation and malabsorption (). Recently, Lukowski et al. () studied mice in which PKG-beta was overexpressed under control of the smooth muscle promoter sm in mice globally lacking both PKG (alpha and beta). PKG was lacking in myocytes, yet pressure overload or isoproterenolinduced hypertrophy was similar to that of controls, leading the authors to conclude that myocyte PKG is unimportant to hypertrophic regulation. The current study provides a counterargument in that myocardial (and myocyte) PKG activity was inversely correlated with myocyte PDE5 activation and maladaptive remodeling after TAC. Several factors could explain these differences. The model used by Lukowski et al. () exhibits striking premature mortality, whereas even our hi-tg mice had a normal lifespan. In addition, disparities in the severity of the hypertrophic stress and associated PKG activation, and as well as the nature of targeted intracellular cgmp/pkg regulation, may have contributed. Cardiac remodeling in response to pressure overload involves complex communication among myocytes, myofibroblasts, and vascular cells. Each component informs the other of changes in the stress environment, and targeted signaling in one compartment can potently impact the others (). One prominent communicator is the cytokine TGF-beta, which is synthesized and has potent activity on multiple cell types. Importantly, TGF-beta signaling can be blunted by PKG by its phosphorylation of Smads at unique sites to inhibit their nuclear translocation and thereby transcriptional activity (). Atrial natriuretic peptide and nitric oxide block TGF-beta (,) and myocyte-secreted connective tissue growth factor (5), likely underlying antifibrotic effects. PDE5 is more highly expressed in myofibroblast and vascular cells, and its inhibition in such cells rather than myocytes has been suggested to underlie the cardiac effects of sildenafil (). However, our data reveal that targeted genetic bi-directional control of myocyte PDE5 is sufficient to regulate interstitial fibrosis and associated signaling cascades. Study limitations. Several potential study limitations should be noted. First, TG models can be subject to nonspecific effects from excessive protein expression. However, normal cardiac PDE5 expression is low, and its overexpression still resulted in low levels of protein displaying normal subcellular localization and activity in a pathophysiologically relevant range. Second, -month-old hi-tg mice had no abnormal molecular profiles, cardiac morphology, or functional changes, but responded markedly and adversely to TAC within only a few days. Last, TACinduced changes were reversible with either subsequent transgene silencing or sildenafil treatment (maintaining PDE5 overexpression), both despite persistent pressure overload. Another potential limitation is that our analysis relied on reversing an overexpression model rather than using conditional gene deletion. However, we contend that TAC responses in hi-tg mice rapidly invoked multiple cascades, and the capacity to broadly reverse these by targeted gene down-regulation supports the role of myocyte PDE5 and potential role for PDE5 inhibitors in heart failure patients. Conclusions We provide strong support for regulatory control of cardiac stress remodeling and PKG activity by myocyte PDE5. The data counter recent suggestions that neither play a role in cardiac hypertrophy. Furthermore, we show that myocyte PDE5 modulation can itself suppress fibrosis-related genes and fibrosis, highlighting a novel mechanism for myocyteorchestrated extracellular matrix remodeling via PDE5/ cgmp-pkg regulatory pathways. Reports that PDE5 expression rises in human heart diseases coupled with the present findings that myocyte gene targeting can bidirectionally impact maladaptive remodeling responses provide new insight into the role of this pathway and support ongoing clinical therapeutic efforts to modulate it. Acknowledgments The authors thank Marissa Hildebrandt for assistance with wheat germ agglutinin analysis and Dr. Carla Ellis for histology assistance. Reprint requests and correspondence: Dr. David A. Kass, Division of Cardiology, Johns Hopkins University School of Medicine, Ross Building 858, 7 Rutland Avenue, Baltimore, Maryland 5. dkass@jhmi.edu. REFERENCES. Tsai EJ, Kass DA. Cyclic GMP signaling in cardiovascular pathophysiology and therapeutics. Pharmacol Ther 9;: 8.. Das A, Xi L, Kukreja RC. Protein kinase G-dependent cardioprotective mechanism of phosphodiesterase-5 inhibition involves phosphorylation of ERK and GSKbeta. J Biol Chem 8;8:

10 Zhang et al. JACC Vol. 5, No., Cardiac Stress Response and Myocyte PDE5 December 7, :. Nagayama T, Hsu S, Zhang M, et al. Sildenafil stops progressive chamber, cellular, and molecular remodeling and improves calcium handling and function in hearts with pre-existing advanced hypertrophy due to pressure-overload. J Am Coll Cardiol 9:5:7 5.. Takimoto E, Champion HC, Li M, et al. Chronic inhibition of cyclic GMP phosphodiesterase 5A prevents and reverses cardiac hypertrophy. Nat Med 5::. 5. Fisher PW, Salloum F, Das A, Hyder H, Kukreja RC. Phosphodiesterase-5 inhibition with sildenafil attenuates cardiomyocyte apoptosis and left ventricular dysfunction in a chronic model of doxorubicin cardiotoxicity. Circulation 5;:.. Lukowski R, Rybalkin SD, Loga F, et al. Cardiac hypertrophy is not amplified by deletion of cgmp-dependent protein kinase I in cardiomyocytes. Proc Natl Acad Sci U S A ;7: Vandeput F, Krall J, Ockaili R, et al. cgmp-hydrolytic activity and its inhibition by sildenafil in normal and failing human and mouse myocardium. J Pharmacol Exp Ther 9;: Nagendran J, Archer SL, Soliman D, et al. Phosphodiesterase type 5 is highly expressed in the hypertrophied human right ventricle, and acute inhibition of phosphodiesterase type 5 improves contractility. Circulation 7;: Pokreisz P, Vandenwijngaert S, Bito V, et al. Ventricular phosphodiesterase-5 expression is increased in patients with advanced heart failure and contributes to adverse ventricular remodeling after myocardial infarction in mice. Circulation 9;9:8.. Sanbe A, Gulick J, Hanks MC, et al. Reengineering inducible cardiac-specific transgenesis with an attenuated myosin heavy chain promoter. Circ Res ;9:9.. Takimoto E, Champion HC, Belardi D, et al. cgmp catabolism by phosphodiesterase 5A regulates cardiac adrenergic stimulation by NOS-dependent mechanism. Circ Res 5;9: 9.. Takimoto E, Koitabashi N, Hsu S, et al. Regulator of G protein signaling mediates cardiac compensation to pressure overload and antihypertrophic effects of PDE5 inhibition in mice. J Clin Invest 9;9:8.. Lee DI, Vahebi S, Tocchetti CG, et al. PDE5A suppression of acute beta-adrenergic activation requires modulation of myocyte beta- signaling coupled to PKG-mediated troponin I phosphorylation. Basic Res Cardiol ;5:7 7.. Miller CL, Oikawa M, Cai Y, et al. Role of Ca /calmodulinstimulated cyclic nucleotide phosphodiesterase in mediating cardiomyocyte hypertrophy. Circ Res 9;5: Zhang M, Koitabashi N, Nagayama T, et al. Expression, activity, and pro-hypertrophic effects of PDE5A in cardiac myocytes. Cell Signal 8;:.. Nagayama T, Hsu S, Zhang M, et al. Pressure-overload magnitudedependence of the anti-hypertrophic efficacy of PDE5A inhibition. J Mol Cell Cardiol 9;: Tokudome T, Kishimoto I, Horio T, et al. Regulator of G-protein signaling subtype mediates antihypertrophic effect of locally secreted natriuretic peptides in the heart. Circulation 8;7: Koitabashi N, Aiba T, Hesketh GG, et al. Cyclic GMP/PKGdependent inhibition of TRPC channel activity and expression negatively regulates cardiomyocyte NFAT activation: Novel mechanism of cardiac stress modulation by PDE5 inhibition. J Mol Cell Cardiol 9;8:7. 9. Hofmann F, Feil R, Kleppisch T, Schlossmann J. Function of cgmp-dependent protein kinases as revealed by gene deletion. Physiol Rev ;8:.. Weber S, Bernhard D, Lukowski R, et al. Rescue of cgmp kinase I knockout mice by smooth muscle specific expression of either isozyme. Circ Res 7;:9.. Kakkar R, Lee RT. Intramyocardial fibroblast myocyte communication. Circ Res ;: Li P, Wang D, Lucas J, et al. Atrial natriuretic peptide inhibits transforming growth factor beta-induced Smad signaling and myofibroblast transformation in mouse cardiac fibroblasts. Circ Res 8; : Li P, Oparil S, Novak L, et al. ANP signaling inhibits TGF-betainduced Smad and Smad nuclear translocation and extracellular matrix expression in rat pulmonary arterial smooth muscle cells. J Appl Physiol 7;:9 8.. Saura M, Zaragoza C, Herranz B, et al. Nitric oxide regulates transforming growth factor-beta signaling in endothelial cells. Circ Res 5;97:5. 5. Koitabashi N, Arai M, Kogure S, et al. Increased connective tissue growth factor relative to brain natriuretic peptide as a determinant of myocardial fibrosis. Hypertension 7;9: 7. Key Words: cyclic GMP y heart failure y phosphodiesterase-5 y pressure overload y protein kinase G y transgenic mouse models. APPENDIX For an expanded Methods section and supplementary figures and their legends, please see the online version of this article.