Author + information
- Received April 8, 2010
- Revision received July 22, 2010
- Accepted August 3, 2010
- Published online December 7, 2010.
- 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‡ and
- David A. Kass, MD*,* ()
- ↵*Reprint requests and correspondence:
Dr. David A. Kass, Division of Cardiology, Johns Hopkins University School of Medicine, Ross Building 858, 720 Rutland Avenue, Baltimore, Maryland 21205
Objectives 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.
Background 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.
Methods 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.
Results Rest myocyte and heart function, histology, and molecular profiling were normal in both TG lines versus controls at 2 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 up-regulation 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.
Conclusions 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
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 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) (1). cGMP levels are enhanced by nitric oxide (NO) and natriuretic peptides or 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 (2), pressure-overload (3,4), 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; NCT00763867) 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 PDE1 as an alternative (6,7). Furthermore, whether myocyte PDE5 and corresponding PKG regulation modulates cardiac hypertrophy/remodeling to counter cardiac stress has been recently questioned (6). 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 (4) 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.
Cardiac-specific PDE5A-inducible transgenic mice
Tetracycline-controlled conditional PDE5 mice were generated by crossing mice expressing PDE5A (tagged with 3×Flag at the N-terminus) with a modified alpha-MHC promoter tetracycline-off vector (gift from Jeffrey Robbins) (10) (Fig. 1A)with mice expressing tetracycline transactivator (alpha-MHC promoter). Two founders (sv129xC57BL/6J 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 (0.5 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.
Cardiac myocyte-specific PDE5 transgenic mice using tetracycline-off induction system
Two independent conditional PDE5 overexpression lines were generated (me-TG: 6.7× activity; hi-TG: 10× activity) (Figs. 1B and 1C), both born in normal Mendelian ratios and surviving into adulthood (to 16 months). PDE5 expression in the lung was unchanged (Online Fig. 1A). Expression of alternative cGMP-PDEs (PDE1, PDE9) was also unchanged in TG mice (Online Fig. 1B), consistent with unaltered non–PDE5-dependent cGMP-PDE activity (Online Fig. 1C). Transgene PDE5 localized to myocyte z-disks, similar to native protein (11) (Online Fig. 2). Up-regulation of PDE5 activity in TG myocytes reduced resting PKG activity (Fig. 1D) ∼35% (p < 0.05) and inhibited stimulated PKG activity (Fig. 1E). The latter was indexed by the lack of plasma membrane translocation of PKG1-alpha and PKG1-beta in TG myocytes when exposed to cGMP (12). 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 2 to 3 months. Both me-TG and hi-TG hearts had normal structural morphology, myocardial histology, molecular signaling, and left ventricular (LV) function (Online Figs. 3 and 4). 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 (11,13) confirming functional regulation by the transgene protein. In me-TG mice, cardiac function and histology remained similar to that of controls over 16 months of observation, whereas LV function declined after 6 months in hi-TG, with hypertrophy and interstitial fibrosis documented at 16 months (Online Fig. 3). For subsequent studies of pressure overload with bi-directional myocyte-PDE5 gene regulation, 2- to 3-month-old animals were used.
Myocyte PDE5 up-regulation worsens cardiac response to pressure overload
To test the impact of PDE5 up-regulation on pressure-overload stress, 2-month-old mice were subjected to 6 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. 2Aand 2B). 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 (Figs. 2C and 2D). 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. 3).Apoptosis (activated caspase 3) 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 ∼80% over baseline in controls (about rest level in me-TG) (Fig. 4A).PDE1a-c expression was unaltered after TAC (Online Fig. 6). Basal myocardial PKG activity was similar, reflecting low basal cGMP synthesis; however, TAC stimulated PKG activity in controls by 70%, but not at all in TG myocardium and isolated myocytes (Fig. 4B). Increased PKG activity despite PDE5 up-regulation after TAC in controls likely reflects a balance and compartmentation of cGMP synthesis/hydrolysis (4). These data demonstrate that this balance can tilt toward PKG suppression with sufficient PDE5 activity. Protein expression of PKG1-alpha and -beta was less in TG at rest, the latter at a slightly lower weight, suggesting post-translational change. Myocyte gene expression for both proteins did not change with TAC (Online Fig. 7A); however, PKG1-alpha protein levels rose similarly in both groups, and PKG1-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 post-translational modulation.
Upon exposure to TAC, both TG models developed worsened molecular abnormalities consistent with maladaptive hypertrophy over controls. Fetal gene recapitulation (Nppaand Myh7) was more prominent and regulator of calcineurin-1 (Rcan1, reflecting calcineurin activity) expression rose more, whereas sarcoplasmic reticulum ATPase (Atp2a2) and phospholamban (Pln), both key calcium-handling proteins, declined more (Fig. 4D). 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 (Tgfb1,2and Ctgf). All increased more in TG over controls. Before TAC, their expression was similar (Online Fig. 4).
Re-suppression of PDE5 in myocytes reverses maladaptive remodeling
TAC-induced cardiac abnormalities and early lethality were observed within 1 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 down-regulating only cardiomyocyte PDE5. Both controls and hi-TG mice were exposed to 4 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 17 days, whereas DOX-treated TG animals displayed recovery to behavior in non-TG controls. DOX treatment did not alter controls.
At 4 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 (100 mg/kg/day; yielding free plasma concentration of 10 to 30 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 3 weeks. LV function improved and chamber dilation diminished in hi-TG–TAC plus sildenafil (Figs. 6Aand 6B), lowering PDE5 activity and enhanced PKG activity (Fig. 6B), 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 up-regulation and sustained TAC.
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 down-regulation 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 PDE1 (6,7). PDE1 is a dual-substrate esterase requiring Ca2+calmodulin for activation, and although PDE1 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 (14). Ours and other laboratories have observed myocyte PDE5 expression (8,9,11,15), and gene-silencing studies further support the specificity and selectivity of these results (15). Importantly, PDE5 is up-regulated 2- to 6-fold in experimental mice and human heart disease (4,7–9), potentially increasing its influence and pharmacologic impact from its subsequent inhibition. In contrast, PDE1 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 up- and 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-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 (11). Both cGMP synthesis and cGMP-PKG targeted signaling (such as calcineurin) are stimulated by pathological stress coupled to G-alpha-q cascades (12,16–18), 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 (16,17).
Although sustained PKG activation is traditionally thought to benefit stressed hearts, direct proof remains lacking (19). Genetic models involving global PKG-1 deletion induce early lethality coupled to intestinal dilation and malabsorption (20). Recently, Lukowski et al. (6) studied mice in which PKG1-beta was overexpressed under control of the smooth muscle promoter sm22 in mice globally lacking both PKG1 (alpha and beta). PKG was lacking in myocytes, yet pressure overload or isoproterenol- induced 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. (6) 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 (21). 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 (22). Atrial natriuretic peptide and nitric oxide block TGF-beta (23,24) and myocyte-secreted connective tissue growth factor (25), 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 (6). However, our data reveal that targeted genetic bi-directional control of myocyte PDE5 is sufficient to regulate interstitial fibrosis and associated signaling cascades.
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, 2-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, TAC-induced 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.
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 myocyte-orchestrated 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 bi-directionally impact maladaptive remodeling responses provide new insight into the role of this pathway and support ongoing clinical therapeutic efforts to modulate it.
The authors thank Marissa Hildebrandt for assistance with wheat germ agglutinin analysis and Dr. Carla Ellis for histology assistance.
For an expanded Methods section and supplementary figures and their legends, please see the online version of this article.
Myocardial Remodeling Is Controlled by Myocyte-Targeted Gene Regulation of Phosphodiesterase Type-5
Supported by National Institutes of Health–National Heart, Lung, and Blood Institutegrants HL-089297, HL-084946and Fondation Leducq(Dr. Kass), HL-093432(Drs. Takimoto and Kass), and T32-HL-07227(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.
- Abbreviations and Acronyms
- cyclic guanosine monophosphate
- extracellular matrix
- left ventricular
- myosin heavy chain
- protein kinase G
- trans-aortic constriction
- transforming growth factor
- Received April 8, 2010.
- Revision received July 22, 2010.
- Accepted August 3, 2010.
- American College of Cardiology Foundation
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