Myocardial cGMP Research Articles

Endothelial estrogen - myocardial cGMP axis critically determines angiogenesis and cardiac performance during pressure-overload.

Estrogen exerts beneficial cardiovascular effects by binding to specific receptors on various cells to activate nuclear and non-nuclear actions. Estrogen receptor α (ERα) non-nuclear signaling confers protection against heart failure remodeling, involving myocardial cyclic guanosine monophosphate (cGMP) - cGMP-dependent protein kinase G (PKG) activation; however, its tissue-specific role remains elusive. Herein, we examined the cell type-specific role of ERα non-nuclear signaling in estrogen-conferred protection against heart failure. We first assessed the tissue-specific impacts of ERα in estrogen's cardiac benefits, utilizing endothelial ERα deletion (ERαf/f/Tie2Cre+) and myocyte ERα deletion (ERαf/f/αMHCCre+) female mice. Female mice were ovariectomized and the effect of estradiol (E2) was assessed in hearts exposed to 3week pressure-overload (TAC). E2 failed to improve cardiac function in ERαf/f/Tie2Cre+ TAC hearts but provided benefits in ERαf/f/αMHCCre+ TAC hearts, indicating that endothelial ERα is essential. We next assessed the role of non-nuclear signaling in endothelial cells, employing animals with endothelial-specific inactivation of ERα non-nuclear signaling (ERαKI/KI/Tie2Cre+). Female OVX mice were supplemented with E2 and subjected to 3-week TAC. ERαKI/KI/Tie2Cre+ TAC hearts revealed exacerbated cardiac dysfunction and reduced myocardial PKG activity as compared to littermate TAC hearts, which was associated with attenuated myocardial induction of vascular endothelial growth factor (VEGF) and angiogenesis as assessed with CD31-stained capillary density. This phenotype of ERαKI/KI/Tie2Cre+ was rescued by myocardial PKG activation from chronic treatment with soluble guanylate cyclase (sGC) stimulator. We performed co-culture experiments to determine endothelial-cardiomyocyte interactions. VEGF induction by E2 in cardiac myocytes required co-existence of intact endothelial ERα signaling in a NOS-dependent manner. On the other hand, VEGF was induced in myocytes directly with an sGC stimulator in the absence of endothelial cells. An endothelial estrogen - myocardial cGMP axis stimulates angiogenic response and improves cardiac performance during pressure-overload.

Abstract 14306: Sex Differences in GC1-cGMP Related Cardioprotective Mechanism for Right Heart Failure in Mice Subjected to Right Ventricular Pressure Overload

Introduction: Right ventricular dysfunction and failure are independently associated with worse clinical outcomes, regardless of left ventricular EF. The molecular pathophysiology of right ventricular failure and its sex differences are poorly understood. The recent success of cGMP enhancing drugs in HF clinical trials indicated potential right heart benefits by activating cGMP signaling. However, details how myocardial cGMP signaling regulates right heart failure remain to be elucidated. Hypothesis: GC1α-cGMP signaling confers anti-remodeling benefits to RV with improvement of LV diastolic function after RV pressure overload. Method: Adult male and female constitutive GC1α knockout mice (GC1α KO) were subjected to pulmonary artery banding (PAB, 25G needle) and serial echocardiography were performed at 3- and 6-weeks post-surgery. Age and sex-matched wild type C57B6/J mice were used as controls. Results: Survival at 6wks post-PAB was worse in GC1αKO compared to WT controls (100 % of WT vs 38.5 % of GC1αKO, p<0.05; Gehan-Breslow-Wilcoxon-test) indicating the exacerbated heart failure by GC1α ablation. RV systolic function at 3 weeks post-surgery was deteriorated in GC1αKO male, assessed with RV FAC (%) (WT vs GC1αKO: 58.0 vs 35.6 p<0.05; t-test) and TAPSE (mm) (WT vs GC1αKO: 0.94 vs 0.64 p<0.05; t-test). Female showed limited RV systolic dysfunction and worse LV diastolic dysfunction at 3 weeks post-surgery compared to male, assessed with TAPSE (mm) (WT vs GC1αKO: 0.85 vs 0.63, p<0.05; t-test) and mitral IVS E/E’ (WT vs GC1αKO: 18.3 vs 29.3, p<0.05; t-test). Interestingly, the impact of GC1α ablation to RV systolic function is bigger in male, but that to LV diastolic function is bigger in female, indicating sex differences of GC1-cGMP regulated cardio-protective effects to right heart failure. The fetal gene expressions( nppb and Myh7 ) in right heart also showed worse right heart remodeling by GC1α ablation especially in male at 6 weeks post-surgery. Conclusions: Inactivation of GC1α-cGMP signaling induced right heart remodeling after PAB. Female mice showed LV diastolic dysfunction with limited right heart remodeling compared to age-matched male, indicating sex differences in GC1-cGMP related right heart protective mechanism.

Cardiac natriuretic peptide deficiency sensitizes the heart to stress-induced ventricular arrhythmias via impaired CREB signalling

The cardiac natriuretic peptides [atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP)] are important regulators of cardiovascular physiology, with reduced natriuretic peptide (NP) activity linked to multiple human cardiovascular diseases. We hypothesized that deficiency of either ANP or BNP would lead to similar changes in left ventricular structure and function given their shared receptor affinities. We directly compared murine models deficient of ANP or BNP in the same genetic backgrounds (C57BL6/J) and environments. We evaluated control, ANP-deficient (Nppa-/-) or BNP-deficient (Nppb-/-) mice under unstressed conditions and multiple forms of pathological myocardial stress. Survival, myocardial structure, function and electrophysiology, tissue histology, and biochemical analyses were evaluated in the groups. In vitro validation of our findings was performed using human-derived induced pluripotent stem cell cardiomyocytes (iPS-CMs). In the unstressed state, both ANP- and BNP-deficient mice displayed mild ventricular hypertrophy which did not increase up to 1 year of life. NP-deficient mice exposed to acute myocardial stress secondary to thoracic aortic constriction (TAC) had similar pathological myocardial remodelling but a significant increase in sudden death. We discovered that the NP-deficient mice are more susceptible to stress-induced ventricular arrhythmias using both in vivo and ex vivo models. Mechanistically, deficiency of either ANP or BNP led to reduced myocardial cGMP levels and reduced phosphorylation of the cAMP response element-binding protein (CREBS133) transcriptional regulator. Selective CREB inhibition sensitized wild-type hearts to stress-induced ventricular arrhythmias. ANP and BNP regulate cardiomyocyte CREBS133 phosphorylation through a cGMP-dependent protein kinase 1 (PKG1) and p38 mitogen-activated protein kinase (p38 MAPK) signalling cascade. Our data show that ANP and BNP act in a non-redundant fashion to maintain myocardial cGMP levels to regulate cardiomyocyte p38 MAPK and CREB activity. Cardiac natriuretic peptide deficiency leads to a reduction in CREB signalling which sensitizes the heart to stress-induced ventricular arrhythmias.

Impact of Syndecan-2-Selected Mesenchymal Stromal Cells on the Early Onset of Diabetic Cardiomyopathy in Diabetic db/db Mice

Background: Mesenchymal stromal cells (MSCs) are an attractive cell type for cell therapy given their immunomodulatory, anti-fibrotic, and endothelial-protective features. The heparin sulfate proteoglycan, syndecan-2/CD362, has been identified as a functional marker for MSC isolation, allowing one to obtain a homogeneous cell product that meets regulatory requirements for clinical use. We previously assessed the impact of wild-type (WT), CD362−, and CD362+ MSCs on local changes in protein distribution in left ventricular (LV) tissue and on LV function in an experimental model of early-onset diabetic cardiomyopathy. The present study aimed to further explore their impact on mechanisms underlying diastolic dysfunction in this model.Materials: For this purpose, 1 × 106 WT, CD362−, or CD362+ MSCs were intravenously (i.v.) injected into 20-week-old diabetic BKS.Cg-m+/+Leprdb/BomTac, i.e., db/db mice. Control animals (db+/db) were injected with the equivalent volume of phosphate-buffered saline (PBS) alone. After 4 weeks, mice were sacrificed for further analysis.Results: Treatment with all three MSC populations had no impact on blood glucose levels in db/db mice. WT, CD362−, and CD362+ MSC application restored LV nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) levels in db/db mice, which correlated with a reduction in cardiomyocyte stiffness. Furthermore, all stromal cells were able to increase arteriole density in db/db mice. The effect of CD362+ MSCs on NO and cGMP levels, cardiomyocyte stiffness, and arteriole density was less pronounced than in mice treated with WT or CD362− MSCs. Analysis of collagen I and III protein expression revealed that fibrosis had not yet developed at this stage of experimental diabetic cardiomyopathy. All MSCs reduced the number of cardiac CD3+ and CD68+ cells in db/db mice, whereas only splenocytes from CD362−- and CD362+-db/db mice exhibited a lower pro-fibrotic potential compared to splenocytes from db/db mice.Conclusion: CD362+ MSC application decreased cardiomyocyte stiffness, increased myocardial NO and cGMP levels, and increased arteriole density, although to a lesser extent than WT and CD362− MSCs in an experimental model of early-onset diabetic cardiomyopathy without cardiac fibrosis. These findings suggest that the degree in improvement of cardiomyocyte stiffness following CD362+ MSC application was insufficient to improve diastolic function.

Swimming exercise inhibits myocardial ER stress in the hearts of aged mice by enhancing cGMP‑PKG signaling.

The purpose of the present study was to explore aging-associated cardiac dysfunction and the possible mechanism by which swimming exercise modulates cardiac dysfunction in aged mice. Aged mice were divided into two groups: i) Aged mice; and ii) aged mice subjected to swimming exercises. Another cohort of 4-month-old male mice served as the control group. Cardiac structure and function in mice were analyzed using hematoxylin and eosin staining, and echocardiography. The levels of oxidative stress were determined by measuring the levels of superoxide dismutase, malondialdehyde and reactive oxygen species (ROS). Levels of the endoplasmic reticulum (ER) stress-related protein PKR-like ER kinase, glucose-regulated protein 78 and C/EBP homologous protein were determined to evaluate the level of ER stress. The aged group exhibited an abnormal cardiac structure and decreased cardiac function, both of which were ameliorated by swimming exercise. The hearts of the aged mice exhibited pronounced oxidative and ER stress, which were ameliorated by exercise, and was accompanied by the reactivation of myocardial cGMP and suppression of cGMP-specific phosphodiesterase type 5 (PDE5). The inhibition of PDE5 attenuated age-induced cardiac dysfunction, blocked ROS production and suppressed ER stress. An ER stress inducer abolished the beneficial effects of the swimming exercise on cardiac function and increased ROS production. The present study suggested that exercise restored cardiac function in mice with age-induced cardiac dysfunction by inhibiting oxidative stress and ER stress, and increasing cGMP-protein kinase G signaling.

Phosphodiesterase 5 inhibition improves contractile function and restores transverse tubule loss and catecholamine responsiveness in heart failure

Heart failure (HF) is characterized by poor survival, a loss of catecholamine reserve and cellular structural remodeling in the form of disorganization and loss of the transverse tubule network. Indeed, survival rates for HF are worse than many common cancers and have not improved over time. Tadalafil is a clinically relevant drug that blocks phosphodiesterase 5 with high specificity and is used to treat erectile dysfunction. Using a sheep model of advanced HF, we show that tadalafil treatment improves contractile function, reverses transverse tubule loss, restores calcium transient amplitude and the heart’s response to catecholamines. Accompanying these effects, tadalafil treatment normalized BNP mRNA and prevented development of subjective signs of HF. These effects were independent of changes in myocardial cGMP content and were associated with upregulation of both monomeric and dimerized forms of protein kinase G and of the cGMP hydrolyzing phosphodiesterases 2 and 3. We propose that the molecular switch for the loss of transverse tubules in HF and their restoration following tadalafil treatment involves the BAR domain protein Amphiphysin II (BIN1) and the restoration of catecholamine sensitivity is through reductions in G-protein receptor kinase 2, protein phosphatase 1 and protein phosphatase 2 A abundance following phosphodiesterase 5 inhibition.

Abstract 16677: Protein Kinase G I Alpha Attenuates Inducible Ventricular Tachycardia and QRS/T Wave Alternans in the Type II Diabetic Heart

Introduction: Diabetes increases ventricular tachycardia (VT) and sudden cardiac death risk in humans. We previously found the type I diabetic (DMI) heart displays: reduced responsiveness to parasympathetic stimulation; increased QRS/T Wave alternans (marker of proarrhythmic calcium dyshomeostasis); and hyperactive GSK3beta. Cardiac parasympathetic stimulation promotes production of intracellular cyclic GMP (cGMP). The effects of cGMP and its downstream effector PKG, on these indices of VT in type II diabetes (DMII) remain poorly understood. Hypothesis: PKGIa modulates inducibility of VT and QRS/TWA in the DMII heart and mediates the cGMP effect on VT through inhibition of myocardial GSK3beta. Methods: Using an established protocol of programmed ventricular stimulation we measured: VT incidence, duration, and QRS/T Wave alternans. We studied the following mice: wild type; Db/Db model of DMII; high fat high sucrose (HFHS) model of insulin resistance; and the PKGIa leucine zipper mutant (LZM) mouse, which has no DM but has PKGIa disrupting mutations. Mice were treated with or without the cGMP-augmenting phosphodiesterase inhibitor sildenafil 10 mg/kg, or the GSK3b inhibitor TWS119 20 mg/kg. Results: LVs of HFHS mice displayed 30 ± 8% reduction of cGMP compared with control (p<0.05, n=3 per group), while myocardial cGMP in LZM mice did not differ from control. In failing LVs from humans, there was also a 46% reduction in myocardial cGMP in DM compared with nondiabetic (p= .058, n=3 per group). Db/Db and HFHS mice had increased inducible VT (0.25 +/- 0.1 s in Db/Db, 2.2 +/- 1 s HFHS, vs 0 s in WT control, p <0.05), as well as TWA. Treatment with sildenafil reduced VT incidence, duration, and TWA in Db/Db and HFHS mice. LZM mice also had increased VT duration and TWA incidence (0 of 4 in WT vs 5 of 6 in LZM, p<0.05). Inhibition of GSK3β rescued inducibility of VT in Db/Db, HFHS, and in LZM mice. Conclusions: These findings support that in the DMII heart reduced cGMP promotes VT, whereas pharmacological augmentation of cGMP inhibits VT. Inducible VT and TWA in the PKG LZM mouse provides direct support that PKGIa opposes VT in the DMII heart. These support that cGMP activation of PKG inhibits GSK3beta, leading to reduced incidence of inducible VT.

Cardiac Specific Gene Delivery of the Natriuretic Peptide Receptor Improves Diastolic Function in a Canine Model of HFpEF.

Introduction Natriuretic peptides stimulate guanylyl cyclase-A (GCA) to produce cGMP. Deficient myocardial cGMP signaling has been implicated in the pathogenesis of diastolic dysfunction and heart failure (HF) with preserved ejection fraction (HFpEF). We hypothesized that cardiac specific over-expression of GCA via gene-delivery with an adeno-associated virus-9 (AAV9-GCA) vector will enhance response to endogenous natriuretic peptides and improve diastolic dysfunction in experimental HFpEF. Methods The HFpEF model included unilateral renal wrapping (RW) to induce hypertension plus DOCA-salt exposure in aged beagles. Trans-catheter (C-cath®) endocardial injections (≈ 14 per dog) of AAV9-GCA or placebo were performed 2 weeks prior to RW. HFpEF+Placebo (n=8) and HFpEF+AAV9-GCA (n=8) underwent echo and invasive pressure-volume analysis at 11 weeks post-RW. Aged Control (n=6) dogs (no RW) were also studied. In HFpEF dogs, systemic blood pressure (BP) was monitored weekly with an implanted CardioMEMS sensor. All dogs underwent a bio-assay to exclude anti-AAV9 antibodies prior to treatment. Results At end study, as compared to Control dogs, HFpEF+Placebo dogs had higher EF (67% vs 63%, p=0.03), relative wall thickness (RWT; 0.55 vs 0.47, p Conclusion Catheter based, cardiac specific gene delivery of GCA in experimental HFpEF was feasible, resulted in persistent GCA over-expression at 13 weeks post-delivery and was associated with improved LV diastolic stiffness.

Phosphodiesterase 2 inhibition preferentially promotes NO/guanylyl cyclase/cGMP signaling to reverse the development of heart failure

Heart failure (HF) is a shared manifestation of several cardiovascular pathologies, including hypertension and myocardial infarction, and a limited repertoire of treatment modalities entails that the associated morbidity and mortality remain high. Impaired nitric oxide (NO)/guanylyl cyclase (GC)/cyclic guanosine-3',5'-monophosphate (cGMP) signaling, underpinned, in part, by up-regulation of cyclic nucleotide-hydrolyzing phosphodiesterase (PDE) isozymes, contributes to the pathogenesis of HF, and interventions targeted to enhancing cGMP have proven effective in preclinical models and patients. Numerous PDE isozymes coordinate the regulation of cardiac cGMP in the context of HF; PDE2 expression and activity are up-regulated in experimental and human HF, but a well-defined role for this isoform in pathogenesis has yet to be established, certainly in terms of cGMP signaling. Herein, using a selective pharmacological inhibitor of PDE2, BAY 60-7550, and transgenic mice lacking either NO-sensitive GC-1α (GC-1α-/-) or natriuretic peptide-responsive GC-A (GC-A-/-), we demonstrate that the blockade of PDE2 promotes cGMP signaling to offset the pathogenesis of experimental HF (induced by pressure overload or sympathetic hyperactivation), reversing the development of left ventricular hypertrophy, compromised contractility, and cardiac fibrosis. Moreover, we show that this beneficial pharmacodynamic profile is maintained in GC-A-/- mice but is absent in animals null for GC-1α or treated with a NO synthase inhibitor, revealing that PDE2 inhibition preferentially enhances NO/GC/cGMP signaling in the setting of HF to exert wide-ranging protection to preserve cardiac structure and function. These data substantiate the targeting of PDE2 in HF as a tangible approach to maximize myocardial cGMP signaling and enhancing therapy.

Cyclic Guanosine Monophosphate Enhancing Therapeutic Strategy for HFpEF (cGETS study): effects on cardiac dysfunction and remodeling

Background: Most trials testing phosphodiesterase 5 inhibitors (PDE5I) in heart failure with preserved ejection fraction (HFpEF) yielded a neutral outcome possibly because of low myocardial cyclic guanosine monophosphate (cGMP) content, which reduces effectiveness of PDE5I. Angiotensin converting enzyme inhibitors (ACEI), angiotensin II receptor blockers (ARB) and statins (STAT) increase myocardial cGMP content through upregulated endothelial nitric oxide synthase. The acute and chronic effects on cardiac remodeling and dysfunction of PDE5I after optimized ACEI-ARB-STAT therapy were therefore evaluated.

Acute stimulation of the soluble guanylate cyclase does not impact on left ventricular capacitance in normal and hypertrophied porcine hearts in vivo.

Experimental data indicate that stimulation of the nitric oxide-soluble guanylate cyclase(sGC)-cGMP-PKG pathway can increase left ventricular (LV) capacitance via phosphorylation of the myofilamental protein titin. We aimed to test whether acute pharmacological sGC stimulation with BAY 41-8543 would increase LV capacitance via titin phosphorylation in healthy and deoxycorticosteroneacetate (DOCA)-induced hypertensive pigs. Nine healthy Landrace pigs and 7 pigs with DOCA-induced hypertension and LV concentric hypertrophy were acutely instrumented to measure LV end-diastolic pressure-volume relationships (EDPVRs) at baseline and during intravenous infusion of BAY 41-8543 (1 and 3 μg·kg-1·min-1 for 30 min, respectively). Separately, in seven healthy and six DOCA pigs, transmural LV biopsies were harvested from the beating heart to measure titin phosphorylation during BAY 41-8543 infusion. LV EDPVRs before and during BAY 41-8543 infusion were superimposable in both healthy and DOCA-treated pigs, whereas mean aortic pressure decreased by 20-30 mmHg in both groups. Myocardial titin phosphorylation was unchanged in healthy pigs, but total and site-specific (Pro-Glu-Val-Lys and N2-Bus domains) titin phosphorylation was increased in DOCA-treated pigs. Bicoronary nitroglycerin infusion in healthy pigs ( n = 5) induced a rightward shift of the LV EDPVR, demonstrating the responsiveness of the pathway in this model. Acute systemic sGC stimulation with the sGC stimulator BAY 41-8543 did not recruit an LV preload reserve in both healthy and hypertrophied LV porcine myocardium, although it increased titin phosphorylation in the latter group. Thus, increased titin phosphorylation is not indicative of increased in vivo LV capacitance. NEW & NOTEWORTHY We demonstrate that acute pharmacological stimulation of soluble guanylate cyclase does not increase left ventricular compliance in normal and hypertrophied porcine hearts. Effects of long-term soluble guanylate cyclase stimulation with oral compounds in disease conditions associated with lowered myocardial cGMP levels, i.e., heart failure with preserved ejection fraction, remain to be investigated.

Stretch-induced compliance: a novel adaptive biological mechanism following acute cardiac load.

The heart is constantly challenged with acute bouts of stretching or overload. Systolic adaptations to these challenges are known but adaptations in diastolic stiffness remain unknown. We evaluated adaptations in myocardial stiffness due to acute stretching and characterized the underlying mechanisms. Left ventricles (LVs) of intact rat hearts, rabbit papillary muscles and myocardial strips from cardiac surgery patients were stretched. After stretching, there was a sustained >40% decrease in end-diastolic pressure (EDP) or passive tension (PT) for 15 min in all species and experimental preparations. Stretching by volume loading in volunteers and cardiac surgery patients resulted in E/E' and EDP decreases, respectively, after sustained stretching. Stretched samples had increased myocardial cGMP levels, increased phosphorylated vasodilator-stimulated phosphoprotein phosphorylation, as well as, increased titin phosphorylation, which was reduced by prior protein kinase G (PKG) inhibition (PKGi). Skinned cardiomyocytes from stretched and non-stretched myocardia were studied. Skinned cardiomyocytes from stretched hearts showed decreased PT, which was abrogated by protein phosphatase incubation; whereas those from non-stretched hearts decreased PT after PKG incubation. Pharmacological studies assessed the role of nitric oxide (NO) and natriuretic peptides (NPs). PT decay after stretching was significantly reduced by combined NP antagonism, NO synthase inhibition and NO scavenging, or by PKGi. Response to stretching was remarkably reduced in a rat model of LV hypertrophy, which also failed to increase titin phosphorylation. We describe and translate to human physiology a novel adaptive mechanism, partly mediated by titin phosphorylation through cGMP-PKG signalling, whereby myocardial compliance increases in response to acute stretching. This mechanism may not function in the hypertrophic heart.

Prevention of the development of heart failure with preserved ejection fraction by the phosphodiesterase-5A inhibitor vardenafil in rats with type 2 diabetes.

AimsHeart failure with preserved ejection fraction (HFpEF) has a great epidemiological burden. The pathophysiological role of cyclic guanosine monophosphate (cGMP) signalling has been intensively investigated in HFpEF. Elevated levels of cGMP have been shown to exert cardioprotective effects in various cardiovascular diseases, including diabetic cardiomyopathy. We investigated the effect of long‐term preventive application of the phosphodiesterase‐5A (PDE5A) inhibitor vardenafil in diabetic cardiomyopathy‐associated HFpEF.Methods and resultsZucker diabetic fatty (ZDF) rats were used as a model of HFpEF and ZDF lean rats served as controls. Animals received vehicle or 10 mg/kg body weight vardenafil per os from weeks 7 to 32 of age. Cardiac function, morphology was assessed by left ventricular (LV) pressure–volume analysis and echocardiography at week 32. Cardiomyocyte force measurements were performed. The key markers of cGMP signalling, nitro‐oxidative stress, apoptosis, myocardial hypertrophy and fibrosis were examined. The ZDF animals showed diastolic dysfunction (increased LV/cardiomyocyte stiffness, prolonged LV relaxation time), preserved systolic performance, decreased myocardial cGMP level coupled with impaired protein kinase G (PKG) activity, increased nitro‐oxidative stress, enhanced cardiomyocyte apoptosis, and hypertrophic and fibrotic remodelling of the myocardium. Vardenafil effectively prevented the development of HFpEF by maintaining diastolic function (decreased LV/cardiomyocyte stiffness and LV relaxation time), by restoring cGMP levels and PKG activation, by lowering apoptosis and by alleviating nitro‐oxidative stress, myocardial hypertrophy and fibrotic remodelling.ConclusionsWe report that vardenafil successfully prevented the development of diabetes mellitus‐associated HFpEF. Thus, PDE5A inhibition as a preventive approach might be a promising option in the management of HFpEF patients with diabetes mellitus.

Impact of Mitochondrial Ca2+-Sensitive Potassium (mBKCa) Channels in Sildenafil-Induced Cardioprotection in Rats.

BackgroundMitochondrial large-conductance Ca2+-sensitive potassium (mBKCa) channels are involved in myocardial ischemic preconditioning. Their role in sildenafil-induced cardioprotection is unknown. We investigated whether sildenafil-induced acute cardioprotection is mediated by activation of mBKCa channels in the rat heart in vitro.MethodsMale Wistar rats (n = 8 per group) were randomized and anesthetized with pentobarbital (90 mg/kg). Hearts were isolated, mounted on a Langendorff system and perfused with Krebs-Henseleit buffer at a constant pressure of 80 mmHg. Hearts underwent 30 min of global ischemia followed by 60 min of reperfusion. At the end of the experiments infarct size was determined by TTC staining. In the control group rats were not further treated. Sildenafil (3 μM) was administered over 10 min before the beginning of ischemia. The mBKCa channel inhibitor paxilline (1 μM) was administered with and without sildenafil before the onset of ischemia. The pathway underlying sildenafil-induced cardioprotection was further investigated with the protein kinase G blocker KT5823 (1 μM). Myocardial cGMP concentration was measured by ELISA. Data (mean±SD) were analysed with a one and two-way analysis of variance as appropriate.ResultsIn control animals infarct size was 52±8%. Sildenafil increased cGMP concentration and reduced infarct size to 35±6% (P<0.05 vs. control). Paxilline and KT5823 completely blocked sildenafil-induced cardioprotection (paxilline+sildenafil: 50±8%, KT5823+sildenafil: 45±8%; both P<0.05 vs. sildenafil). Functional heart parameters and coronary flow were not different between the study groups.ConclusionThis study shows that in male rats protein kinase G-dependent opening of mBKCa channels plays a pivotal role in sildenafil-induced cardioprotection.

Effect of Corin Deficiency and Mutation on Cardiac Natriuretic Peptide Processing in the Intact Heart Under Cardiac Pressure-Overload Conditions

Natriuretic peptides ProANP and Pro BNP are translated as biologically-inactive pro-hormones that require proteolytic cleavage into biologically active carboxyl-terminal fragments by the serine protease Corin. Corin gene mutations are associated with risk for hypertension and left-ventricular hypertrophy in Blacks. However, the relative importance of Corin and the Corin T555I/Q568P mutations to proBNP processing in the heart is lacking. We adapted the Langendorff ex vivo cardiac perfusion model to allow dynamic assessment of cardiac processing of cardiac-derived proBNP using immunoassays specific for the murine molecular forms of unprocessed BNP1-95 and the active, carboxyl-terminus fragment murine BNP45. We also developed an inducible, cardiomyocyte-restricted Corin knockout mouse and a Corin T623I/Q636P homozygous knockin mouse to study the importance of cardiac Corin and the human Corin T555I/Q568P mutations to cardiac processing of proBNP. Results. As compared to WT controls, cardiomyocyte-restricted Corin knockout mice demonstrated significantly decreased BNP processing, and reduced myocardial cGMP and PKG kinase activity. The Corin623I(636P) knockin mice also demonstrated less severe but significant impairment in proBNP processing and reduced cardiac cGMP and PKG activity consistent with reduced GC-A activity. Cardiomyocyte Corin is essential to adequate processing of cardiac-derived proBNP and the autocrine/paracrine NPS. The murine Corin 623I(636P) heterozygous knockin model demonstrates impaired processing and reduced NPS autocrine/paracrine signaling during acute cardiac distention suggesting that Blacks heterozygous for the Corin I555(P568) haplotype may have reduced cardiac protection form the influence of chronic hypertension.

Abstract 191: Phosphodiesterase 9a Modulates Cardiomyocyte Natriuretic Peptide-Stimulated cGMP Pool and Cardiac Hypertrophy

Cardiac hypertrophy in response to pathologic stress is a major contributor to heart disease. Many studies have revealed that compartmentalized cyclic guanosine monophosphate (cGMP) regulation by phosphodiesterases (PDEs) can potently modify acute and chronic cardiac stress responses. PDE5a normally regulates cGMP generated from nitric oxide (NO)-stimulated soluble guanylate cyclase (sGC) but not natriuretic peptide (NP)-stimulated cGMP. Its inhibition blunts maladaptive hypertrophy and remodeling. Regulators of NP-cGMP remain uncertain. Here we reveal that PDE9a, a highly cGMP-specific PDE (×100 affinity vs PDE5a) that is expressed predominantly in the brain modulates myocyte cGMP as well, targeting NP-stimulated pools. PDE9a gene expression is observed in rat neonatal cardiac myocytes (RNCM) and adult mouse myocytes. Protein expression was identified in myocytes by immunohistochemistry, using gene silencing models as a negative control. PDE9a expression is upregulated by various hypertrophic stimuli (phenylephrine (PE) or endothelin-1 (ET-1), and observed in myocytes from pressure-overloaded whole myocardium. PDE9a inhibition with a selective antagonist (PF-9613) or gene silencing significantly abrogated PE- or ET-1-dependent upregulation of pathological-hypertrophy fetal genes and NFAT activity in RNCM, and PDE9a inhibition attenuated ET-1-induced hypertrophy in adult mouse myocytes. PF-9613 had no impact in cells lacking PDE9a (from knockout mice) or after gene silencing. Biochemical and cGMP-sensitive fluorescent probe studies shows PDE9a inhibition augments ANP- but not NO-dependent cGMP stimuli in RNCM and adult myocytes. PDE9a knockout (KO) mice were protected against hypertrophy/remodeling after pressure-overload, accompanied by a rise in myocardial cGMP. In PDE9a KO mice also exhibited less fibrosis and reduced expression of fibrotsis-related genes over wild-type mice. Together, these data identify PDE9a as a novel regulator of myocyte cGMP and hypertrophy that impacts a different compartmentalized cGMP pool to that by PDE5a. Our observations may provide a novel therapeutic approach in the treatment of heart failure.

Increased nitrosative/oxidative stress lowers myocardial protein kinase G activity in heart failure with preserved ejection fraction

Results Patients with HFPEF (n=36), AS (n=67), and HFREF (n=43) were free of coronary artery disease. More HFPEF patients were obese (P<0.05) or had diabetes mellitus (P<0.05). Left ventricular myocardial biopsies were procured transvascularly in HFPEF and HFREF and perioperatively in aortic stenosis. Fpassive was measured in cardiomyocytes before and after PKG administration. Myocardial homogenates were used for assessment of PKG activity, cGMP concentration, proBNP-108 expression, and nitrotyrosine expression, a measure of nitrosative/oxidative stress. Additional quantitative immunohistochemical analysis was performed for PKG activity and nitrotyrosine expression. Cardiomyocyte Fpassive was higher in HFPEF (7.6±0.4 kN/m 2 ) than in AS (3.4±0.2 kN/m 2 ; P<0.001) and in HFREF (5.1±0.2 kN/m 2 ; P<0.001). In-vitro administration of PKG acutely lowered cardiomyocyte stiffness in all groups with the largest decrement in Fpassive in DHF patients. PKG activity in myocardial tissue homogenates was significantly lower in HFPEF (5.11±0.62 pmol/min/ mg) than in both AS (9.18±0.64 pmol/min/mg; P<0.01) and HFREF (11.51±2.0 pmol/min/mg; P<0.001). Immunohistochemical determination of myocardial PKG activity by pVASP/VASP ratio provided confirmatory evidence as it was also significantly lower in HFPEF (0.70±0.03) than in both AS (0.84±0.02; P<0.001) and HFREF (0.85±0.03; P<0.001). Lower PKG activity in HFPEF than in aortic stenosis or HFREF was associated with higher cardiomyocyte Fpassive (P<0.001) and related to lower cGMP concentration (P<0.001) and higher nitrosative/ oxidative stress (P<0.05). Reduced PKG activity and lower myocardial cGMP concentration in HFPEF did not result from altered myocardial sGC or PDE5A expression, which was similar in all groups nor from unequal BNP expression, which was comparable in HFPEF and AS. The downregulated cGMP-PKG signaling in HFPEF was therefore related to low myocardial nitric oxide bioavailability because of high nitrosative/ oxidative stress. Conclusion Low myocardial PKG activity in HFPEF was associated with raised cardiomyocyte Fpassive and was related to increased myocardial nitrosative/oxidative stress. The latter was probably induced by the high prevalence in HFPEF of metabolic comorbidities. Correction of myocardial PKG activity could be a target for specific HFPEF treatment.

Increased Cardiac Myocyte PDE5 Levels in Human and Murine Pressure Overload Hypertrophy Contribute to Adverse LV Remodeling

BackgroundThe intracellular second messenger cGMP protects the heart under pathological conditions. We examined expression of phosphodiesterase 5 (PDE5), an enzyme that hydrolyzes cGMP, in human and mouse hearts subjected to sustained left ventricular (LV) pressure overload. We also determined the role of cardiac myocyte-specific PDE5 expression in adverse LV remodeling in mice after transverse aortic constriction (TAC).Methodology/Principal FindingsIn patients with severe aortic stenosis (AS) undergoing valve replacement, we detected greater myocardial PDE5 expression than in control hearts. We observed robust expression in scattered cardiac myocytes of those AS patients with higher LV filling pressures and BNP serum levels. Following TAC, we detected similar, focal PDE5 expression in cardiac myocytes of C57BL/6NTac mice exhibiting the most pronounced LV remodeling. To examine the effect of cell-specific PDE5 expression, we subjected transgenic mice with cardiac myocyte-specific PDE5 overexpression (PDE5-TG) to TAC. LV hypertrophy and fibrosis were similar as in WT, but PDE5-TG had increased cardiac dimensions, and decreased dP/dtmax and dP/dtmin with prolonged tau (P<0.05 for all). Greater cardiac dysfunction in PDE5-TG was associated with reduced myocardial cGMP and SERCA2 levels, and higher passive force in cardiac myocytes in vitro.Conclusions/SignificanceMyocardial PDE5 expression is increased in the hearts of humans and mice with chronic pressure overload. Increased cardiac myocyte-specific PDE5 expression is a molecular hallmark in hypertrophic hearts with contractile failure, and represents an important therapeutic target.

Abstract 18928: Cardiac Soluble Guanylate Cyclase Protects Against Cardiac Dysfunction Induced by Chronic Doxorubicin Treatment in Mice

Background - The chemotherapeutic use of doxorubicin (DOX) is limited by cardiotoxicity. Growing evidence suggests a protective role for cyclic guanosine monophosphate (cGMP) in pathological LV remodeling. We therefore tested the hypothesis that reduced cGMP bioavailability in cardiac myocytes worsens myocardial dysfunction in mice after chronic DOX treatment. Methods and Results - We generated mice with an inducible (Tet-Off) cardiac myocyte-specific expression of a dominant-negative soluble guanylate cyclase (dnsGC) mutant, which inhibits sGC signaling selectively in the heart. Cardiac cGMP levels, measured using an enzyme immunoassay, were lower in dnsGC mice (n=10) than in WT littermates (n=20) 5 minutes after administration of the NO-donor GSNO (1mg/kg) (0.99±0.34 vs 1.85±0.17 nmol/mg protein, respectively, P&lt;0.05). At baseline, transthoracic echocardiography (TTE) demonstrated similar LV dimensions and function in dnsGC and WT (data not shown). Heart to body weight ratio was also comparable (HW/BW; 4.3±0.2 mg/g in dnsGC, n=13, vs 3.9±0.1 mg/g in WT, n=21). After 12 weeks DOX (2 mg/kg weekly), LV function was significantly reduced and LV diameters significantly enlarged as compared to saline-treated WT (Table). The impact of DOX on cardiac function and dimensions was more marked in dnsGC than in WT (P&lt;0.05 for both, Table). Survival was similarly impaired in DOX-treated dnsGC (67%, n=30) and WT (59%, n=24) compared with saline-treated WT (100%, n=25). HW/BW was similar in DOX-treated dnsGC and WT (4.3±0.1 vs 4.2±0.1 mg/g), as was the % bromodeoxyuridine (BrdU)-positive cardiac myocytes or cardiac progenitor cells (0.8±0.4%, n=7 vs 1.6±0.7%, n=9, respectively, P=NS). Conclusions - Reduced myocardial cGMP results in increased LV dysfunction and dilatation in doxorubicin-treated mice. Stimulation of sGC activity in cardiac myocytes may represent a promising therapeutic approach for doxorubicin cardiotoxicity.

Protein kinase G type I in cardiac myocytes: unmasked at last?

This editorial refers to ‘Stress-dependent dilated cardiomyopathy in mice with cardiomyocyte-restricted inactivation of cyclic GMP-dependent protein kinase I’[†][1], by S. Frantz et al. , on page 1233 Increasing evidence over the past decade has suggested that elevations in myocardial cGMP may protect against adverse ventricular remodelling. cGMP is produced in different cell types comprising cardiac muscle, including vascular smooth muscle and endothelial cells, fibroblasts, monocytes/macrophages, and cardiac myocytes (CMs) themselves, all of which participate in remodelling. In the latter, cGMP is produced either from particulate guanylate cyclase (pGC) contained within GC-A [natriuretic peptide receptor type A (NPRA) activated by atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP)] and GC-B [(NPRB) activated by C-type natriuretic peptide (CNP)], or from soluble GC (sGC) activated by NO and CO. Given the diffusible nature of NO, CM sGC can theoretically be activated by NO from autocrine sources [endogenously expressed nitric oxide synthases (NOS)], paracrine sources (i.e. mainly from endothelial and inflammatory cells), and, possibly, exogenous NO. cGMP-dependent protein kinase type I (cGKI) is part of a family of cGMP-activated protein kinases (PKs) encoded by two different genes; one encoding cGKIα and cGKIβ (through alternative splicing), and the other encoding cGKII, a membrane-bound PK classically detected in the intestinal brush border.1 Together with cGMP-modulated phosphodiesterases (PDEs), cGKs represent the main effectors of cGMP signalling in cardiovascular (and other) tissues. As such they modulate the function of key proteins involved in excitation–contraction (EC) coupling ( Figure 1 ), contractility, cell survival, metabolism, and CM hypertrophic remodelling ( Figure 2 ). Recent studies have highlighted the functional compartmentation of cGMP pools within CM, each susceptible to activate a specific subset of cGK and effectors; this would be in line with the identification of cGK anchoring proteins2 (akin to the PKA-associated A kinase anchoring proteins family) that … [1]: #fn-2