Steven Hsu

Regulator of G protein signaling 2 mediates cardiac compensation to pressure overload and antihypertrophic effects of PDE5 inhibition in mice.

The heart initially compensates for hypertension-mediated pressure overload by enhancing its contractile force and developing hypertrophy without dilation. Gq protein-coupled receptor pathways become activated and can depress function, leading to cardiac failure. Initial adaptation mechanisms to reduce cardiac damage during such stimulation remain largely unknown. Here we have shown that this initial adaptation requires regulator of G protein signaling 2 (RGS2). Mice lacking RGS2 had a normal basal cardiac phenotype, yet responded rapidly to pressure overload, with increased myocardial Gq signaling, marked cardiac hypertrophy and failure, and early mortality. Swimming exercise, which is not accompanied by Gq activation, induced a normal cardiac response, while Rgs2 deletion in Galphaq-overexpressing hearts exacerbated hypertrophy and dilation. In vascular smooth muscle, RGS2 is activated by cGMP-dependent protein kinase (PKG), suppressing Gq-stimulated vascular contraction. In normal mice, but not Rgs2-/- mice, PKG activation by the chronic inhibition of cGMP-selective phosphodiesterase 5 (PDE5) suppressed maladaptive cardiac hypertrophy, inhibiting Gq-coupled stimuli. Importantly, PKG was similarly activated by PDE5 inhibition in myocardium from both genotypes, but PKG plasma membrane translocation was more transient in Rgs2-/- myocytes than in controls and was unaffected by PDE5 inhibition. Thus, RGS2 is required for early myocardial compensation to pressure overload and mediates the initial antihypertrophic and cardioprotective effects of PDE5 inhibitors.

Sildenafil Stops Progressive Chamber, Cellular, and Molecular Remodeling and Improves Calcium Handling and Function in Hearts With Pre-Existing Advanced Hypertrophy Caused by Pressure Overload

OBJECTIVE This study sought to test the efficacy of phosphodiesterase type 5A (PDE5A) inhibition for treating advanced hypertrophy/remodeling caused by pressure overload, and to elucidate cellular and molecular mechanisms for this response. BACKGROUND Sildenafil (SIL) inhibits cyclic guanosine monophosphate-specific PDE5A and can blunt the evolution of cardiac hypertrophy and dysfunction in mice subjected to pressure overload. Whether and how it ameliorates more established advanced disease and dysfunction is unknown. METHODS Mice were subjected to transverse aortic constriction (TAC) for 3 weeks to establish hypertrophy/dilation, and subsequently treated with SIL (100 mg/kg/day) or placebo for 6 weeks of additional TAC. RESULTS The SIL arrested further progressive chamber dilation, dysfunction, fibrosis, and molecular remodeling, increasing myocardial protein kinase G activity. Isolated myocytes from TAC-SIL hearts showed greater sarcomere shortening and relaxation, and enhanced Ca(2+) transients and decay compared with nontreated TAC hearts. The SIL treatment restored gene and protein expression of sarcoplasmic reticulum Ca(2+) uptake adenosine triphosphatase (SERCA2a), phospholamban (PLB), and increased PLB phosphorylation (S16), consistent with improved calcium handling. The phosphatase calcineurin (Cn) and/or protein kinase C-alpha (PKCalpha) can both lower phosphorylated phospholamban and depress myocyte calcium cycling. The Cn expression and PKCalpha activation (outer membrane translocation) were enhanced by chronic TAC and reduced by SIL treatment. Expression of PKCdelta and PKCepsilon also increased with TAC but were unaltered by SIL treatment. CONCLUSIONS SIL treatment applied to well-established hypertrophic cardiac disease can prevent further cardiac and myocyte dysfunction and progressive remodeling. This is associated with improved calcium cycling, and reduction of Cn and PKCalpha activation may be important to this improvement.

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

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.

Sustained soluble guanylate cyclase stimulation offsets nitric-oxide synthase inhibition to restore acute cardiac modulation by sildenafil.

Phosphodiesterase type 5 (PDE5) inhibitors are used to treat erectile dysfunction, and growing evidence supports potential cardiovascular utility. Their efficacy declines with reduced nitric-oxide synthase (NOS) activity common to various diseases. We tested whether direct soluble guanylate cyclase (sGC) stimulation restores in vivo cardiovascular modulation by PDE5 inhibition despite acute or chronically suppressed NOS activity. Mice (C57/Bl6; n = 62) were studied by in vivo pressure-volume analysis to assess acute modulation by the PDE5 inhibitor sildenafil (SIL; 100 μg/kg/min) of the cardiac response to isoproterenol (ISO) with or without NOS inhibition [Nω-nitro-l-arginine methyl ester (l-NAME)] and cotreatment by the sGC stimulator 2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-(4-morpholinyl)pyrimidine-4,6-diamine (BAY 41-8543). SIL induced mild vasodilation but no basal cardiac effects and markedly blunted ISO-stimulated contractility. Acute BAY 41-8543 at a dose lacking cardiovascular effects did not alter ISO responses. However, after acute l-NAME, SIL ceased to influence cardiovascular function, but adding BAY 41-8543 fully restored SIL effects. After 1 week of l-NAME, neither SIL nor SIL + BAY 41-8543 acutely induced vasodilation or blunted ISO responses. However, sustained BAY 41-8543 despite concurrent NOS inhibition restored the cardiovascular efficacy of SIL. The disparity between acute and chronic NOS inhibition related to diffusion of PDE5 away from myocyte z-bands coupled with reduced protein kinase G activation. Both were restored by sustained sGC costimulation. Thus, PDE5 regulation of adrenergic reserve and systemic vasodilation depends upon NOS-induced cGMP/protein kinase G and can be enhanced by sustained low-level stimulation of sGC. This may prove beneficial for enhancing the efficacy of PDE5 inhibitors in conditions with chronically reduced NOS activity.

Phosphodiesterase 5 inhibition blocks pressure overload-induced cardiac hypertrophy independent of the calcineurin pathway.

AIMS Cyclic GMP (cGMP)-specific phosphodiesterase 5 (PDE5) inhibition by sildenafil (SIL) activates myocardial cGMP-dependent protein kinase G (PKG) and blunts cardiac hypertrophy. To date, the only documented target of PKG in myocardium is the serine-threonine phosphatase calcineurin (Cn), which is central to pathological cardiac hypertrophy. We tested whether Cn suppression is necessary in order to observe anti-hypertrophic effects of SIL. METHODS AND RESULTS Mice lacking the Cn-Abeta subunit (CnAbeta(-/-)) and wild-type (WT) controls were subjected to transverse aorta constriction (TAC) with or without SIL (200 mg/kg/day, p.o.) for 3 weeks. TAC-induced elevation of Cn expression and activity in WT was absent in CnAbeta(-/-) hearts, and the latter accordingly developed less cardiac hypertrophy (50 vs. 100% increase in heart weight/tibia length, P < 0.03) and chamber dilation. SIL remained effective in CnAbeta(-/-) mice, increasing PKG activity similarly as in WT, suppressing hypertrophy and fetal gene expression, and enhancing heart function without altering afterload. TAC-stimulated calcium-calmodulin kinase II, Akt, and glycogen synthase kinase 3beta in both groups (the first rising more in CnAbeta(-/-) hearts), and SIL also suppressed these similarly. Activation of extracellular signal-regulated kinase observed in WT-TAC but not CnAbeta(-/-) hearts was also suppressed by SIL. CONCLUSION PDE5A inhibition and its accompanying PKG activation blunt hypertrophy and improve heart function even without Cn activation. This occurs by its modulation of several alternative pathways which may result from concomitant distal targeting, or activity against a common proximal node.

Pressure-overload magnitude-dependence of the anti-hypertrophic efficacy of PDE5A inhibition.

Increased myocardial cGMP, achieved by enhancing cyclase activity or impeding cGMP hydrolysis by phosphodiesterase type-5 (PDE5A), suppresses cellular and whole organ hypertrophy. The efficacy of the latter also requires cyclase stimulation and may depend upon co-activation of maladaptive signaling suppressible by cGMP-stimulated kinase (cGK-1). Thus, PDE5A inhibitors could paradoxically be more effective against higher than lower magnitudes of pressure-overload stress. To test this, mice were subjected to severe or moderate trans-aortic constriction (sTAC, mTAC) for 6 wks +/-co-treatment with oral sildenafil (SIL 200 mg/kg/d). LV mass (LVM) rose 130% after 3-wks sTAC and SIL blunted this by 50%. With mTAC, LVM rose 56% at 3 wks but was unaffected by SIL, whereas a 90% increase in LVM after 6 wks was suppressed by SIL. SIL minimally altered LV function and remodeling with mTAC until later stages that stimulated more hypertrophy and remodeling. SIL stimulated cGK-1 activity similarly at 3 and 6 wks of mTAC. However, pathologic stress signaling (e.g. calcineurin, ERK-MAPkinase) was little activated after 3-wk mTAC, unlike sTAC or later stage mTAC when activity increased and SIL suppressed it. With modest hypertrophy (3-wk mTAC), GSK3beta and Akt phosphorylation were unaltered but SIL enhanced it. However, with more severe hypertrophy (6-wk mTAC and 3-wk sTAC), both kinases were highly phosphorylated and SIL treatment reduced it. Thus, PDE5A-inhibition counters cardiac pressure-overload stress remodeling more effectively at higher than lower magnitude stress, coupled to pathologic signaling activation targetable by cGK-1 stimulation. Such regulation could impact responses of varying disease models to PDE5A inhibitors.

Right Ventricular Functional Reserve in Pulmonary Arterial Hypertension

Background— Right ventricular (RV) functional reserve affects functional capacity and prognosis in patients with pulmonary arterial hypertension (PAH). PAH associated with systemic sclerosis (SSc-PAH) has a substantially worse prognosis than idiopathic PAH (IPAH), even though many measures of resting RV function and pulmonary vascular load are similar. We therefore tested the hypothesis that RV functional reserve is depressed in SSc-PAH patients. Methods and Results— RV pressure-volume relations were prospectively measured in IPAH (n=9) and SSc-PAH (n=15) patients at rest and during incremental atrial pacing or supine bicycle ergometry. Systolic and lusitropic function increased at faster heart rates in IPAH patients, but were markedly blunted in SSc-PAH. The recirculation fraction, which indexes intracellular calcium recycling, was also depressed in SSc-PAH (0.32±0.05 versus 0.50±0.05; P=0.039). At matched exercise (25 W), SSc-PAH patients did not augment contractility (end-systolic elastance) whereas IPAH did (P<0.001). RV afterload assessed by effective arterial elastance rose similarly in both groups; thus, ventricular-vascular coupling declined in SSc-PAH. Both end-systolic and end-diastolic RV volumes increased in SSc-PAH patients to offset contractile deficits, whereas chamber dilation was absent in IPAH (+37±10% versus +1±8%, P=0.004, and +19±4% versus –1±6%, P<0.001, respectively). Exercise-associated RV dilation also strongly correlated with resting ventricular-vascular coupling in a larger cohort. Conclusions— RV contractile reserve is depressed in SSc-PAH versus IPAH subjects, associated with reduced calcium recycling. During exercise, this results in ventricular-pulmonary vascular uncoupling and acute RV dilation. RV dilation during exercise can predict adverse ventricular-vascular coupling in PAH patients.

PDE5 inhibitor efficacy is estrogen dependent in female heart disease

Inhibition of cGMP-specific phosphodiesterase 5 (PDE5) ameliorates pathological cardiac remodeling and has been gaining attention as a potential therapy for heart failure. Despite promising results in males, the efficacy of the PDE5 inhibitor sildenafil in female cardiac pathologies has not been determined and might be affected by estrogen levels, given the hormones involvement in cGMP synthesis. Here, we determined that the heart-protective effect of sildenafil in female mice depends on the presence of estrogen via a mechanism that involves myocyte eNOS-dependent cGMP synthesis and the cGMP-dependent protein kinase Iα (PKGIα). Sildenafil treatment failed to exert antiremodeling properties in female pathological hearts from Gαq-overexpressing or pressure-overloaded mice after ovary removal; however, estrogen replacement restored the effectiveness of sildenafil in these animals. In females, sildenafil-elicited myocardial PKG activity required estrogen, which stimulated tonic cardiomyocyte cGMP synthesis via an eNOS/soluble guanylate cyclase pathway. In contrast, eNOS activation, cGMP synthesis, and sildenafil efficacy were not estrogen dependent in male hearts. Estrogen and sildenafil had no impact on pressure-overloaded hearts from animals expressing dysfunctional PKGIα, indicating that PKGIα mediates antiremodeling effects. These results support the importance of sex differences in the use of PDE5 inhibitors for treating heart disease and the critical role of estrogen status when these agents are used in females.

A Clinician's Guide to the ABCs of Cardiovascular Disease Prevention: The Johns Hopkins Ciccarone Center for the Prevention of Heart Disease and American College of Cardiology Cardiosource Approach to the Million Hearts Initiative

Atherosclerotic cardiovascular disease (CVD) is the leading cause of death in the United States and worldwide. Fortunately, it is often preventable with early adoption of lifestyle modification, prevention of risk factor onset, and aggressive treatment of existing risk factors. The Million Hearts Initiative is an effort by the Centers for Disease Control that aims to prevent 1 million myocardial infarctions and strokes over the next 5 years. As part of this initiative, we present a simply organized “ABCDE” approach for guiding a consistent comprehensive approach to managing cardiovascular risk in daily clinical practice. ABCDE stands for assessment of risk, antiplatelet therapy, blood pressure management, cholesterol management, cigarette/tobacco cessation, diet and weight management, diabetes prevention and treatment, and exercise, interventions regularly used to reduce cardiovascular (CV) risk. Throughout this article we summarize recommendations related to each topic and reference landmark trials and data that support our approach. We believe that the ABCDE approach will be the core framework for addressing CV risk in our effort to prevent CVD.

Pathological Cardiac Hypertrophy Alters Intracellular Targeting of Phosphodiesterase Type 5 From Nitric Oxide Synthase-3 to Natriuretic Peptide Signaling

Background In the normal heart, phosphodiesterase type 5 (PDE5) hydrolyzes cGMP coupled to nitric oxide– (specifically from nitric oxide synthase 3) but not natriuretic peptide (NP)–stimulated guanylyl cyclase. PDE5 is upregulated in hypertrophied and failing hearts and is thought to contribute to their pathophysiology. Because nitric oxide signaling declines whereas NP-derived cGMP rises in such diseases, we hypothesized that PDE5 substrate selectivity is retargeted to blunt NP-derived signaling. Methods and Results Mice with cardiac myocyte inducible PDE5 overexpression (P5+) were crossed to those lacking nitric oxide synthase 3 (N3−), and each model, the double cross, and controls were subjected to transaortic constriction. P5+ mice developed worse dysfunction and hypertrophy and enhanced NP stimulation, whereas N3− mice were protected. However, P5+/N3− mice behaved similarly to P5+ mice despite the lack of nitric oxide synthase 3–coupled cGMP generation, with protein kinase G activity suppressed in both models. PDE5 inhibition did not alter atrial natriuretic peptide–stimulated cGMP in the resting heart but augmented it in the transaortic constriction heart. This functional retargeting was associated with PDE5 translocation from sarcomeres to a dispersed distribution. P5+ hearts exhibited higher oxidative stress, whereas P5+/N3− hearts had low levels (likely owing to the absence of nitric oxide synthase 3 uncoupling). This highlights the importance of myocyte protein kinase G activity as a protection for pathological remodeling. Conclusions These data provide the first evidence for functional retargeting of PDE5 from one compartment to another, revealing a role for natriuretic peptide–derived cGMP hydrolysis by this esterase in diseased heart myocardium. Retargeting likely affects the pathophysiological consequence and the therapeutic impact of PDE5 modulation in heart disease.