The Concise Guide to PHARMACOLOGY 2023/24: Catalytic receptors.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and nearly 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org/), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16180. Catalytic receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Ablation of C-type natriuretic peptide/cGMP signaling in fibroblasts exacerbates adverse cardiac remodeling in mice.

Excessive activation of cardiac fibroblasts (CFs) in response to injury provokes cardiac fibrosis, stiffness, and failure. The local mediators counterregulating this response remain unclear. Exogenous C-type natriuretic peptide (CNP) exerts antifibrotic effects in preclinical models. To unravel the role of the endogenous hormone, we generated mice with fibroblast-restricted deletion (KO) of guanylyl cyclase-B (GC-B), the cGMP-synthesizing CNP receptor. CNP activated GC-B/cGMP signaling in human and murine CFs, preventing proliferative and promigratory effects of angiotensin II (Ang II) and TGF-ß. Fibroblast-specific GC-B-KO mice showed enhanced fibrosis in response to Ang II infusions. Moreover, after 2 weeks of mild pressure overload induced by transverse aortic constriction (TAC), such KO mice had augmented cardiac fibrosis and hypertrophy, together with systolic and diastolic contractile dysfunction. This was associated with increased expression of the profibrotic genes encoding collagen I, III, and periostin. Notably, such responses to Ang II and TAC were greater in female as compared with male KO mice. Enhanced Ang II-induced CNP expression in female hearts and augmented GC-B expression and activity in female CFs may contribute to this sex disparity. The results show that paracrine CNP signaling in CFs has antifibrotic and antihypertrophic effects. The CNP/GC-B/cGMP pathway might be a target for therapies combating pathological cardiac remodeling.

Natriuretic peptide pathways in heart failure: further therapeutic possibilities.

The discovery of the heart as an endocrine organ resulted in a remarkable recognition of the natriuretic peptide system (NPS). Specifically, research has established the production of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) from the heart, which exert pleiotropic cardiovascular, endocrine, renal, and metabolic actions via the particulate guanylyl cyclase A receptor (GC-A) and the second messenger, cGMP. C-type natriuretic peptide (CNP) is produced in the endothelium and kidney and mediates important protective auto/paracrine actions via GC-B and cGMP. These actions, in part, participate in the efficacy of sacubitril/valsartan in heart failure (HF) due to the augmentation of the NPS. Here, we will review important insights into the biology of the NPS, the role of precision medicine, and focus on the phenotypes of human genetic variants of ANP and BNP in the general population and the relevance to HF. We will also provide an update of the existence of NP deficiency states, including in HF, which provide the rationale for further therapeutics for the NPS. Finally, we will review the field of peptide engineering and the development of novel designer NPs for the treatment of HF. Notably, the recent discovery of a first-in-class small molecule GC-A enhancer, which is orally deliverable, will be highlighted. These innovative designer NPs and small molecule possess enhanced and novel properties for the treatment of HF and cardiovascular diseases.

Skeletal muscle derived Musclin protects the heart during pathological overload.

Cachexia is associated with poor prognosis in chronic heart failure patients, but the underlying mechanisms of cachexia triggered disease progression remain poorly understood. Here, we investigate whether the dysregulation of myokine expression from wasting skeletal muscle exaggerates heart failure. RNA sequencing from wasting skeletal muscles of mice with heart failure reveals a reduced expression of Ostn, which encodes the secreted myokine Musclin, previously implicated in the enhancement of natriuretic peptide signaling. By generating skeletal muscle specific Ostn knock-out and overexpressing mice, we demonstrate that reduced skeletal muscle Musclin levels exaggerate, while its overexpression in muscle attenuates cardiac dysfunction and myocardial fibrosis during pressure overload. Mechanistically, Musclin enhances the abundance of C-type natriuretic peptide (CNP), thereby promoting cardiomyocyte contractility through protein kinase A and inhibiting fibroblast activation through protein kinase G signaling. Because we also find reduced OSTN expression in skeletal muscle of heart failure patients, augmentation of Musclin might serve as therapeutic strategy.

Rise of cGMP by partial phosphodiesterase-3A degradation enhances cardioprotection during hypoxia.

3',5'-cyclic guanosine monophosphate (cGMP) is a druggable second messenger regulating cell growth and survival in a plethora of cells and disease states, many of which are associated with hypoxia. For example, in myocardial infarction and heart failure (HF), clinical use of cGMP-elevating drugs improves disease outcomes. Although they protect mice from ischemia/reperfusion (I/R) injury, the exact mechanism how cardiac cGMP signaling is regulated in response to hypoxia is still largely unknown. By monitoring real-time cGMP dynamics in murine and human cardiomyocytes using in vitro and in vivo models of hypoxia/reoxygenation (H/R) and I/R injury combined with biochemical methods, we show that hypoxia causes rapid but partial degradation of cGMP-hydrolyzing phosphodiesterase-3A (PDE3A) protein via the autophagosomal-lysosomal pathway. While increasing cGMP in hypoxia prevents cell death, partially reduced PDE3A does not change the pro-apoptotic second messenger 3',5'-cyclic adenosine monophosphate (cAMP). However, it leads to significantly enhanced protective effects of clinically relevant activators of nitric oxide-sensitive guanylyl cyclase (NO-GC). Collectively, our mouse and human data unravel a new mechanism by which cardiac cGMP improves hypoxia-associated disease conditions.

Loss of Mitochondrial Ca2+ Uniporter Limits Inotropic Reserve and Provides Trigger and Substrate for Arrhythmias in Barth Syndrome Cardiomyopathy.

BACKGROUND: Barth syndrome (BTHS) is caused by mutations of the gene encoding tafazzin, which catalyzes maturation of mitochondrial cardiolipin and often manifests with systolic dysfunction during early infancy. Beyond the first months of life, BTHS cardiomyopathy typically transitions to a phenotype of diastolic dysfunction with preserved ejection fraction, blunted contractile reserve during exercise, and arrhythmic vulnerability. Previous studies traced BTHS cardiomyopathy to mitochondrial formation of reactive oxygen species (ROS). Because mitochondrial function and ROS formation are regulated by excitation-contraction coupling, integrated analysis of mechano-energetic coupling is required to delineate the pathomechanisms of BTHS cardiomyopathy. METHODS: We analyzed cardiac function and structure in a mouse model with global knockdown of tafazzin (Taz-KD) compared with wild-type littermates. Respiratory chain assembly and function, ROS emission, and Ca2+ uptake were determined in isolated mitochondria. Excitation-contraction coupling was integrated with mitochondrial redox state, ROS, and Ca2+ uptake in isolated, unloaded or preloaded cardiac myocytes, and cardiac hemodynamics analyzed in vivo. RESULTS: Taz-KD mice develop heart failure with preserved ejection fraction (>50%) and age-dependent progression of diastolic dysfunction in the absence of fibrosis. Increased myofilament Ca2+ affinity and slowed cross-bridge cycling caused diastolic dysfunction, in part, compensated by accelerated diastolic Ca2+ decay through preactivated sarcoplasmic reticulum Ca2+-ATPase. Taz deficiency provoked heart-specific loss of mitochondrial Ca2+ uniporter protein that prevented Ca2+-induced activation of the Krebs cycle during ß-adrenergic stimulation, oxidizing pyridine nucleotides and triggering arrhythmias in cardiac myocytes. In vivo, Taz-KD mice displayed prolonged QRS duration as a substrate for arrhythmias, and a lack of inotropic response to ß-adrenergic stimulation. Cellular arrhythmias and QRS prolongation, but not the defective inotropic reserve, were restored by inhibiting Ca2+ export through the mitochondrial Na+/Ca2+ exchanger. All alterations occurred in the absence of excess mitochondrial ROS in vitro or in vivo. CONCLUSIONS: Downregulation of mitochondrial Ca2+ uniporter, increased myofilament Ca2+ affinity, and preactivated sarcoplasmic reticulum Ca2+-ATPase provoke mechano-energetic uncoupling that explains diastolic dysfunction and the lack of inotropic reserve in BTHS cardiomyopathy. Furthermore, defective mitochondrial Ca2+ uptake provides a trigger and a substrate for ventricular arrhythmias. These insights can guide the ongoing search for a cure of this orphaned disease.

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: Catalytic receptors.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15541. Catalytic receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

The ß2-Subunit of Voltage-Gated Calcium Channels Regulates Cardiomyocyte Hypertrophy.

L-type voltage-gated calcium channels (LTCCs) regulate crucial physiological processes in the heart. They are composed of the Cavα1 pore-forming subunit and the accessory subunits Cavß, Cavα2δ, and Cavγ. Cavß is a cytosolic protein that regulates channel trafficking and activity, but it also exerts other LTCC-independent functions. Cardiac hypertrophy, a relevant risk factor for the development of congestive heart failure, depends on the activation of calcium-dependent pro-hypertrophic signaling cascades. Here, by using shRNA-mediated Cavß silencing, we demonstrate that Cavß2 downregulation enhances α1-adrenergic receptor agonist-induced cardiomyocyte hypertrophy. We report that a pool of Cavß2 is targeted to the nucleus in cardiomyocytes and that the expression of this nuclear fraction decreases during in vitro and in vivo induction of cardiac hypertrophy. Moreover, the overexpression of nucleus-targeted Cavß2 in cardiomyocytes inhibits in vitro-induced hypertrophy. Quantitative proteomic analyses showed that Cavß2 knockdown leads to changes in the expression of diverse myocyte proteins, including reduction of calpastatin, an endogenous inhibitor of the calcium-dependent protease calpain. Accordingly, Cavß2-downregulated cardiomyocytes had a 2-fold increase in calpain activity as compared to control cells. Furthermore, inhibition of calpain activity in Cavß2-downregulated cells abolished the enhanced α1-adrenergic receptor agonist-induced hypertrophy observed in these cells. Our findings indicate that in cardiomyocytes, a nuclear pool of Cavß2 participates in cellular functions that are independent of LTCC activity. They also indicate that a downregulation of nuclear Cavß2 during cardiomyocyte hypertrophy promotes the activation of calpain-dependent hypertrophic pathways.

Nanoenviroments of the ß-Subunit of L-Type Voltage-Gated Calcium Channels in Adult Cardiomyocytes.

In cardiomyocytes, Ca2+ influx through L-type voltage-gated calcium channels (LTCCs) following membrane depolarization regulates crucial Ca2+-dependent processes including duration and amplitude of the action potentials and excitation-contraction coupling. LTCCs are heteromultimeric proteins composed of the Cavα1, Cavß, Cavα2δ and Cavγ subunits. Here, using ascorbate peroxidase (APEX2)-mediated proximity labeling and quantitative proteomics, we identified 61 proteins in the nanoenvironments of Cavß2 in cardiomyocytes. These proteins are involved in diverse cellular functions such as cellular trafficking, cardiac contraction, sarcomere organization and excitation-contraction coupling. Moreover, pull-down assays and co-immunoprecipitation analyses revealed that Cavß2 interacts with the ryanodine receptor 2 (RyR2) in adult cardiomyocytes, probably coupling LTCCs and the RyR2 into a supramolecular complex at the dyads. This interaction is mediated by the Src-homology 3 domain of Cavß2 and is necessary for an effective pacing frequency-dependent increase of the Ca2+-induced Ca2+ release mechanism in cardiomyocytes.

Empagliflozin Reduces Renal Hyperfiltration in Response to Uninephrectomy, but Is Not Nephroprotective in UNx/DOCA/Salt Mouse Models.

Large-scale clinical outcome studies demonstrated the efficacy of SGLT2 inhibitors in patients with type II diabetes. Besides their therapeutic efficacy in diabetes, significant renoprotection was observed in non-diabetic patients with chronic kidney disease (CKD), suggesting the existence of glucose-independent beneficial effects of SGLT2 inhibitors. However, the relevant mechanisms by which SGLT2 inhibition delays the progression of renal injury are still largely unknown and speculative. Previous studies showed that SGLT2 inhibitors reduce diabetic hyperfiltration, which is likely a key element in renoprotection. In line with this hypothesis, this study aimed to investigate the nephroprotective effects of the SGLT2 inhibitor empagliflozin (EMPA) in different mouse models with non-diabetic hyperfiltration and progressing CKD to identify the underlying diabetes-independent cellular mechanisms. Non-diabetic hyperfiltration was induced by unilateral nephrectomy (UNx). Since UNx alone does not result in renal damage, renal disease models with varying degrees of glomerular damage and albuminuria were generated by combining UNx with high NaCl diets ± deoxycorticosterone acetate (DOCA) in different mouse strains with and without genetic predisposition for glomerular injury. Renal parameters (GFR, albuminuria, urine volume) were monitored for 4-6 weeks. Application of EMPA via the drinking water resulted in sufficient EMPA plasma concentration and caused glucosuria, diuresis and in some models renal hypertrophy. EMPA had no effect on GFR in untreated wildtype animals, but significantly reduced hyperfiltration after UNx by 36%. In contrast, EMPA did not reduce UNx induced hyperfiltration in any of our kidney disease models, regardless of their degree of glomerular damage caused by DOCA/salt treatment. Consistent with the lack of reduction in glomerular hyperfiltration, EMPA-treated animals developed albuminuria and renal fibrosis to a similar extent as H2O control animals. Taken together, the data clearly indicate that blockade of SGLT2 has the potential to reduce non-diabetic hyperfiltration in otherwise untreated mice. However, no effects on hyperfiltration or progression of renal injury were observed in hypervolemic kidney disease models, suggesting that high salt intake and extracellular volume might attenuate the protective effects of SGLT2 blockers.

C-type natriuretic peptide moderates titin-based cardiomyocyte stiffness.

Heart failure is often accompanied by titin-dependent myocardial stiffness. Phosphorylation of titin by cGMP-dependent protein kinase I (PKGI) increases cardiomyocyte distensibility. The upstream pathways stimulating PKGI-mediated titin phosphorylation are unclear. We studied whether C-type natriuretic peptide (CNP), via its guanylyl cyclase-B (GC-B) receptor and cGMP/PKGI signaling, modulates titin-based ventricular compliance. To dissect GC-B-mediated effects of endogenous CNP in cardiomyocytes, we generated mice with cardiomyocyte-restricted GC-B deletion (CM GC-B-KO mice). The impact on heart morphology and function, myocyte passive tension, and titin isoform expression and phosphorylation was studied at baseline and after increased afterload induced by transverse aortic constriction (TAC). Pressure overload increased left ventricular endothelial CNP expression, with an early peak after 3 days. Concomitantly, titin phosphorylation at Ser4080, the site phosphorylated by PKGI, was augmented. Notably, in CM GC-B-KO mice this titin response was abolished. TAC-induced hypertrophy and fibrosis were not different between genotypes. However, the KO mice presented mild systolic and diastolic dysfunction together with myocyte stiffness, which were not observed in control littermates. In vitro, recombinant PKGI rescued reduced titin-Ser4080 phosphorylation and reverted passive stiffness of GC-B-deficient cardiomyocytes. CNP-induced activation of GC-B/cGMP/PKGI signaling in cardiomyocytes provides a protecting regulatory circuit preventing titin-based myocyte stiffening during early phases of pressure overload.

Heart-Microcirculation Connection: Effects of ANP (Atrial Natriuretic Peptide) on Pericytes Participate in the Acute and Chronic Regulation of Arterial Blood Pressure.

Cardiac ANP (atrial natriuretic peptide) moderates arterial blood pressure. The mechanisms mediating its hypotensive effects are complex and involve inhibition of the renin-angiotensin-aldosterone system, increased natriuresis, endothelial permeability, and vasodilatation. The contribution of the direct vasodilating effects of ANP to blood pressure homeostasis is controversial because variable levels of the ANP receptor, GC-A (guanylyl cyclase-A), are expressed among vascular beds. Here, we show that ANP stimulates GC-A/cyclic GMP signaling in cultured microvascular pericytes and thereby the phosphorylation of the regulatory subunit of myosin phosphatase 1 by cGMP-dependent protein kinase I. Moreover, ANP prevents the calcium and contractile responses of pericytes to endothelin-1 as well as microvascular constrictions. In mice with conditional inactivation (knock-out) of GC-A in microcirculatory pericytes, such vasodilating effects of ANP on precapillary arterioles and capillaries were fully abolished. Concordantly, these mice have increased blood pressure despite preserved renal excretory function. Furthermore, acute intravascular volume expansion, which caused release of cardiac ANP, did not affect blood pressure of control mice but provoked hypertensive reactions in pericyte GC-A knock-out littermates. We conclude that GC-A/cGMP-dependent modulation of pericytes and microcirculatory tone contributes to the acute and chronic moderation of arterial blood pressure by ANP. Graphic Abstract A graphic abstract is available for this article.

Guanylyl Cyclase A/cGMP Signaling Slows Hidden, Age- and Acoustic Trauma-Induced Hearing Loss.

In the inner ear, cyclic guanosine monophosphate (cGMP) signaling has been described as facilitating otoprotection, which was previously observed through elevated cGMP levels achieved by phosphodiesterase 5 inhibition. However, to date, the upstream guanylyl cyclase (GC) subtype eliciting cGMP production is unknown. Here, we show that mice with a genetic disruption of the gene encoding the cGMP generator GC-A, the receptor for atrial and B-type natriuretic peptides, display a greater vulnerability of hair cells to hidden hearing loss and noise- and age-dependent hearing loss. This vulnerability was associated with GC-A expression in spiral ganglia and outer hair cells (OHCs) but not in inner hair cells (IHCs). GC-A knockout mice exhibited elevated hearing thresholds, most pronounced for the detection of high-frequency tones. Deficits in OHC input-output functions in high-frequency regions were already present in young GC-A-deficient mice, with no signs of an accelerated progression of age-related hearing loss or higher vulnerability to acoustic trauma. OHCs in these frequency regions in young GC-A knockout mice exhibited diminished levels of KCNQ4 expression, which is the dominant K+ channel in OHCs, and decreased activation of poly (ADP-ribose) polymerase-1, an enzyme involved in DNA repair. Further, GC-A knockout mice had IHC synapse impairments and reduced amplitudes of auditory brainstem responses that progressed with age and with acoustic trauma, in contrast to OHCs, when compared to GC-A wild-type littermates. We conclude that GC-A/cGMP-dependent signaling pathways have otoprotective functions and GC-A gene disruption differentially contributes to hair-cell damage in a healthy, aged, or injured system. Thus, augmentation of natriuretic peptide GC-A signaling likely has potential to overcome hidden and noise-induced hearing loss, as well as presbycusis.

An Activating Deletion Variant in the Submembrane Region of Natriuretic Peptide Receptor-B Causes Tall Stature.

CONTEXT: C-type natriuretic peptide (CNP) is critically involved in endochondral bone growth. Variants in the genes encoding CNP or its cyclic guanosine monophosphate (cGMP)-forming receptor (natriuretic peptide receptor-B [NPR-B], gene NPR2) cause monogenic growth disorders. Here we describe a novel gain-of-function variant of NPR-B associated with tall stature and macrodactyly of the great toes (epiphyseal chondrodysplasia, Miura type). DESIGN: History and clinical characteristics of 3 family members were collected. NPR2 was selected for sequencing. Skin fibroblasts and transfected HEK-293 cells were used to compare mutant versus wild-type NPR-B activities. Homology modeling was applied to understand the molecular consequences of the variant. RESULTS: Mother's height was +2.77 standard deviation scores (SDS). The heights of her 2 daughters were +1.96 SDS at 7 years and +1.30 SDS at 4 years of age. Skeletal surveys showed macrodactyly of the great toes and pseudo-epiphyses of the mid- and proximal phalanges. Sequencing identified a novel heterozygous variant c.1444_1449delATGCTG in exon 8 of NPR2, predicted to result in deletion of 2 amino acids Met482-Leu483 within the submembrane region of NPR-B. In proband's skin fibroblasts, basal cGMP levels and CNP-stimulated cGMP production were markedly increased compared with controls. Consistently, assays with transfected HEK-293 cells showed markedly augmented baseline and ligand-dependent activity of mutant NPR-B. CONCLUSIONS: We report the second activating variant within the intracellular submembrane region of NPR-B resulting in tall stature and macrodactyly. Our functional and modeling studies suggest that this domain plays a critical role in the baseline conformation and ligand-dependent structural rearrangement of NPR-B required for cGMP production.

Stabilization of Perivascular Mast Cells by Endothelial CNP (C-Type Natriuretic Peptide).

OBJECTIVE: Activated perivascular mast cells (MCs) participate in different cardiovascular diseases. Many factors provoking MC degranulation have been described, while physiological counterregulators are barely known. Endothelial CNP (C-type natriuretic peptide) participates in the maintenance of vascular barrier integrity, but the target cells and mechanisms are unclear. Here, we studied whether MCs are regulated by CNP. Approach and Results: In cultured human and murine MCs, CNP activated its specific GC (guanylyl cyclase)-B receptor and cyclic GMP signaling. This enhanced cyclic GMP-dependent phosphorylation of the cytoskeleton-associated VASP (vasodilator-stimulated phosphoprotein) and inhibited ATP-evoked degranulation. To elucidate the relevance in vivo, mice with a floxed GC-B (Npr2) gene were interbred with a Mcpt5-CreTG line to generate mice lacking GC-B in connective tissue MCs (MC GC-B knockout). In anesthetized mice, acute ischemia-reperfusion of the cremaster muscle microcirculation provoked extensive MC degranulation and macromolecule extravasation. Superfusion of CNP markedly prevented MC activation and endothelial barrier disruption in control but not in MC GC-B knockout mice. Notably, already under resting conditions, such knockout mice had increased numbers of degranulated MCs in different tissues, together with elevated plasma chymase levels. After transient coronary occlusion, their myocardial areas at risk and with infarction were enlarged. Moreover, MC GC-B knockout mice showed augmented perivascular neutrophil infiltration and deep vein thrombosis in a model of inferior vena cava ligation. CONCLUSIONS: CNP, via GC-B/cyclic GMP signaling, stabilizes resident perivascular MCs at baseline and prevents their excessive activation under pathological conditions. Thereby CNP contributes to the maintenance of vascular integrity in physiology and disease.

Natriuretic Peptides Attenuate Retinal Pathological Neovascularization Via Cyclic Guanosine Monophosphate Signaling in Pericytes and Astrocytes.

OBJECTIVE: In proliferative retinopathies, complications derived from neovascularization cause blindness. During early disease, pericyte's apoptosis contributes to endothelial dysfunction and leakage. Hypoxia then drives VEGF (vascular endothelial growth factor) secretion and pathological neoangiogenesis. Cardiac ANP (atrial natriuretic peptide) contributes to systemic microcirculatory homeostasis. ANP is also formed in the retina, with unclear functions. Here, we characterized whether endogenously formed ANP regulates retinal (neo)angiogenesis. Approach and Results: Retinal vascular development and ischemia-driven neovascularization were studied in mice with global deletion of GC-A (guanylyl cyclase-A), the cGMP (cyclic guanosine monophosphate)-forming ANP receptor. Mice with a floxed GC-A gene were interbred with Tie2-Cre, GFAP-Cre, or PDGF-Rß-CreERT2 lines to dissect the endothelial, astrocyte versus pericyte-mediated actions of ANP in vivo. In neonates with global GC-A deletion (KO), vascular development was mildly delayed. Moreover, such KO mice showed augmented vascular regression and exacerbated ischemia-driven neovascularization in the model of oxygen-induced retinopathy. Notably, absence of GC-A in endothelial cells did not impact retinal vascular development or pathological neovascularization. In vitro ANP/GC-A/cGMP signaling, via activation of cGMP-dependent protein kinase I, inhibited hypoxia-driven astrocyte's VEGF secretion and TGF-ß (transforming growth factor beta)-induced pericyte apoptosis. In neonates lacking ANP/GC-A signaling in astrocytes, vascular development and hyperoxia-driven vascular regression were unaltered; ischemia-induced neovascularization was modestly increased. Remarkably, inactivation of GC-A in pericytes retarded physiological retinal vascularization and markedly enhanced cell apoptosis, vascular regression, and subsequent neovascularization in oxygen-induced retinopathy. CONCLUSIONS: Protective pericyte effects of the ANP/GC-A/cGMP pathway counterregulate the initiation and progression of experimental proliferative retinopathy. Our observations indicate augmentation of endogenous pericyte ANP signaling as target for treatment of retinopathies associated with neovascularization.

SPRED2 deficiency elicits cardiac arrhythmias and premature death via impaired autophagy.

Cardiac functionality is dependent on a balanced protein turnover. Accordingly, regulated protein decay is critical to maintain cardiac function. Here we demonstrate that deficiency of SPRED2, an intracellular repressor of ERK-MAPK signaling markedly expressed in human heart, resulted in impaired autophagy, heart failure, and shortened lifespan. SPRED2-/- mice showed cardiomyocyte hypertrophy, cardiac fibrosis, impaired electrical excitability, and severe arrhythmias. Mechanistically, cardiomyocyte dysfunction resulted from ERK hyperactivation and dysregulated autophagy, observed as accumulation of vesicles, vacuolar structures, and degenerated mitochondria. The diminished autophagic flux in SPRED2-/- hearts was reflected by a reduced LC3-II/LC3-I ratio and by decreased Atg7, Atg4B and Atg16L expression. Furthermore, the autophagosomal adaptors p62/SQSTM1 and NBR1 and lysosomal Cathepsin D accumulated in SPRED2-/- hearts. In wild-type hearts, SPRED2 interacted physically with p62/SQSTM1, NBR1, and Cathepsin D, indicating that SPRED2 is required for autophagolysosome formation in regular autophagy. Restored inhibition of MAPK signaling by selumetinib led to an increase in autophagic flux in vivo. Therefore, our study identifies SPRED2 as a novel, indispensable regulator of cardiac autophagy. Vice versa, SPRED2 deficiency impairs autophagy, leading to cardiac dysfunction and life-threatening arrhythmias.

Marked disparity of microRNA modulation by cGMP-selective PDE5 versus PDE9 inhibitors in heart disease.

MicroRNAs (miRs) posttranscriptionally regulate mRNA and its translation into protein, and are considered master controllers of genes modulating normal physiology and disease. There is growing interest in how miRs change with drug treatment, and leveraging this for precision guided therapy. Here we contrast 2 closely related therapies, inhibitors of phosphodiesterase type 5 or type 9 (PDE5-I, PDE9-I), given to mice subjected to sustained cardiac pressure overload (PO). Both inhibitors augment cyclic guanosine monophosphate (cGMP) to activate protein kinase G, with PDE5-I regulating nitric oxide (NO) and PDE9-I natriuretic peptide-dependent signaling. While both produced strong phenotypic improvement of PO pathobiology, they surprisingly showed binary differences in miR profiles; PDE5-I broadly reduces more than 120 miRs, including nearly half those increased by PO, whereas PDE9-I has minimal impact on any miR (P < 0.0001). The disparity evolves after pre-miR processing and is organ specific. Lastly, even enhancing NO-coupled cGMP by different methods leads to altered miR regulation. Thus, seemingly similar therapeutic interventions can be barcoded by profound differences in miR signatures, and reversing disease-associated miR changes is not required for therapy success.