Auto/Paracrine C-Type Natriuretic Peptide/Cyclic GMP Signaling Prevents Endothelial Dysfunction.

Endothelial dysfunction is cause and consequence of cardiovascular diseases. The endothelial hormone C-type natriuretic peptide (CNP) regulates vascular tone and the vascular barrier. Its cGMP-synthesizing guanylyl cyclase-B (GC-B) receptor is expressed in endothelial cells themselves. To characterize the role of endothelial CNP/cGMP signaling, we studied mice with endothelial-selective GC-B deletion. Endothelial EC GC-B KO mice had thicker, stiffer aortae and isolated systolic hypertension. This was associated with increased proinflammatory E-selectin and VCAM-1 expression and impaired nitric oxide bioavailability. Atherosclerosis susceptibility was evaluated in such KO and control littermates on Ldlr (low-density lipoprotein receptor)-deficient background fed a Western diet for 10 weeks. Notably, the plaque areas and heights within the aortic roots were markedly increased in the double EC GC-B/Ldlr KO mice. This was accompanied by enhanced macrophage infiltration and greater necrotic cores, indicating unstable plaques. Finally, we found that EC GC-B KO mice had diminished vascular regeneration after critical hind-limb ischemia. Remarkably, all these genotype-dependent changes were only observed in female and not in male mice. Auto/paracrine endothelial CNP/GC-B/cGMP signaling protects from arterial stiffness, systolic hypertension, and atherosclerosis and improves reparative angiogenesis. Interestingly, our data indicate a sex disparity in the connection of diminished CNP/GC-B activity to endothelial dysfunction.

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.

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.

ß Cell-specific deletion of guanylyl cyclase A, the receptor for atrial natriuretic peptide, accelerates obesity-induced glucose intolerance in mice.

BACKGROUND: The cardiac hormones atrial (ANP) and B-type natriuretic peptides (BNP) moderate arterial blood pressure and improve energy metabolism as well as insulin sensitivity via their shared cGMP-producing guanylyl cyclase-A (GC-A) receptor. Obesity is associated with impaired NP/GC-A/cGMP signaling, which possibly contributes to the development of type 2 diabetes and its cardiometabolic complications. In vitro, synthetic ANP, via GC-A, stimulates glucose-dependent insulin release from cultured pancreatic islets and ß-cell proliferation. However, the relevance for systemic glucose homeostasis in vivo is not known. To dissect whether the endogenous cardiac hormones modulate the secretory function and/or proliferation of ß-cells under (patho)physiological conditions in vivo, here we generated a novel genetic mouse model with selective disruption of the GC-A receptor in ß-cells. METHODS: Mice with a floxed GC-A gene were bred to Rip-CreTG mice, thereby deleting GC-A selectively in ß-cells (ß GC-A KO). Weight gain, glucose tolerance, insulin sensitivity, and glucose-stimulated insulin secretion were monitored in normal diet (ND)- and high-fat diet (HFD)-fed mice. ß-cell size and number were measured by immunofluorescence-based islet morphometry. RESULTS: In vitro, the insulinotropic and proliferative actions of ANP were abolished in islets isolated from ß GC-A KO mice. Concordantly, in vivo, infusion of BNP mildly enhanced baseline plasma insulin levels and glucose-induced insulin secretion in control mice. This effect of exogenous BNP was abolished in ß GC-A KO mice, corroborating the efficient inactivation of the GC-A receptor in ß-cells. Despite this under physiological, ND conditions, fasted and fed insulin levels, glucose-induced insulin secretion, glucose tolerance and ß-cell morphology were similar in ß GC-A KO mice and control littermates. However, HFD-fed ß GC-A KO animals had accelerated glucose intolerance and diminished adaptative ß-cell proliferation. CONCLUSIONS: Our studies of ß GC-A KO mice demonstrate that the cardiac hormones ANP and BNP do not modulate ß-cell's growth and secretory functions under physiological, normal dietary conditions. However, endogenous NP/GC-A signaling improves the initial adaptative response of ß-cells to HFD-induced obesity. Impaired ß-cell NP/GC-A signaling in obese individuals might contribute to the development of type 2 diabetes.

Endothelial actions of atrial natriuretic peptide prevent pulmonary hypertension in mice.

The cardiac hormone atrial natriuretic peptide (ANP) regulates systemic and pulmonary arterial blood pressure by activation of its cyclic GMP-producing guanylyl cyclase-A (GC-A) receptor. In the lung, these hypotensive effects were mainly attributed to smooth muscle-mediated vasodilatation. It is unknown whether pulmonary endothelial cells participate in the homeostatic actions of ANP. Therefore, we analyzed GC-A/cGMP signalling in lung endothelial cells and the cause and functional impact of lung endothelial GC-A dysfunction. Western blot and cGMP determinations showed that cultured human and murine pulmonary endothelial cells exhibit prominent GC-A expression and activity which were markedly blunted by hypoxia, a condition known to trigger pulmonary hypertension (PH). To elucidate the consequences of impaired endothelial ANP signalling, we studied mice with genetic endothelial cell-restricted ablation of the GC-A receptor (EC GC-A KO). Notably, EC GC-A KO mice exhibit PH already under resting, normoxic conditions, with enhanced muscularization of small arteries and perivascular infiltration of inflammatory cells. These alterations were aggravated on exposure of mice to chronic hypoxia. Lung endothelial GC-A dysfunction was associated with enhanced expression of angiotensin converting enzyme (ACE) and increased pulmonary levels of Angiotensin II. Angiotensin II/AT1-blockade with losartan reversed pulmonary vascular remodelling and perivascular inflammation of EC GC-A KO mice, and prevented their increment by chronic hypoxia. This experimental study indicates that endothelial effects of ANP are critical to prevent pulmonary vascular remodelling and PH. Chronic endothelial ANP/GC-A dysfunction, e.g. provoked by hypoxia, is associated with activation of the ACE-angiotensin pathway in the lung and PH.

A cardiac pathway of cyclic GMP-independent signaling of guanylyl cyclase A, the receptor for atrial natriuretic peptide.

Cardiac atrial natriuretic peptide (ANP) regulates arterial blood pressure, moderates cardiomyocyte growth, and stimulates angiogenesis and metabolism. ANP binds to the transmembrane guanylyl cyclase (GC) receptor, GC-A, to exert its diverse functions. This process involves a cGMP-dependent signaling pathway preventing pathological [Ca(2+)](i) increases in myocytes. In chronic cardiac hypertrophy, however, ANP levels are markedly increased and GC-A/cGMP responses to ANP are blunted due to receptor desensitization. Here we show that, in this situation, ANP binding to GC-A stimulates a unique cGMP-independent signaling pathway in cardiac myocytes, resulting in pathologically elevated intracellular Ca(2+) levels. This pathway involves the activation of Ca(2+)-permeable transient receptor potential canonical 3/6 (TRPC3/C6) cation channels by GC-A, which forms a stable complex with TRPC3/C6 channels. Our results indicate that the resulting cation influx activates voltage-dependent L-type Ca(2+) channels and ultimately increases myocyte Ca(2)(+)(i) levels. These observations reveal a dual role of the ANP/GC-A-signaling pathway in the regulation of cardiac myocyte Ca(2+)(i) homeostasis. Under physiological conditions, activation of a cGMP-dependent pathway moderates the Ca(2+)(i)-enhancing action of hypertrophic factors such as angiotensin II. By contrast, a cGMP-independent pathway predominates under pathophysiological conditions when GC-A is desensitized by high ANP levels. The concomitant rise in [Ca(2+)](i) might increase the propensity to cardiac hypertrophy and arrhythmias.

Stress-dependent dilated cardiomyopathy in mice with cardiomyocyte-restricted inactivation of cyclic GMP-dependent protein kinase I.

AIMS: Cardiac hypertrophy is a common and often lethal complication of arterial hypertension. Elevation of myocyte cyclic GMP levels by local actions of endogenous atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) or by pharmacological inhibition of phosphodiesterase-5 was shown to counter-regulate pathological hypertrophy. It was suggested that cGMP-dependent protein kinase I (cGKI) mediates this protective effect, although the role in vivo is under debate. Here, we investigated whether cGKI modulates myocyte growth and/or function in the intact organism. METHODS AND RESULTS: To circumvent the systemic phenotype associated with germline ablation of cGKI, we inactivated the murine cGKI gene selectively in cardiomyocytes by Cre/loxP-mediated recombination. Mice with cardiomyocyte-restricted cGKI deletion exhibited unaltered cardiac morphology and function under resting conditions. Also, cardiac hypertrophic and contractile responses to ß-adrenoreceptor stimulation by isoprenaline (at 40 mg/kg/day during 1 week) were unaltered. However, angiotensin II (Ang II, at 1000 ng/kg/min for 2 weeks) or transverse aortic constriction (for 3 weeks) provoked dilated cardiomyopathy with marked deterioration of cardiac function. This was accompanied by diminished expression of the [Ca(2+)]i-regulating proteins SERCA2a and phospholamban (PLB) and a reduction in PLB phosphorylation at Ser16, the specific target site for cGKI, resulting in altered myocyte Ca(2+)i homeostasis. In isolated adult myocytes, CNP, but not ANP, stimulated PLB phosphorylation, Ca(2+)i-handling, and contractility via cGKI. CONCLUSION: These results indicate that the loss of cGKI in cardiac myocytes compromises the hypertrophic program to pathological stimulation, rendering the heart more susceptible to dysfunction. In particular, cGKI mediates stimulatory effects of CNP on myocyte Ca(2+)i handling and contractility.

Martin Heinrich Corten (1889-1962): Nazi victim or perpetrator?

At first glance, Martin Heinrich Corten's biography appears to be the classic story of a German physician persecuted by the Nazis. Because of his "Jewish descent," Corten lost his position as a pathologist in Berlin and later his license to practice medicine. Emigration failed. But Corten was not a typical Nazi victim. In 1933, he applied for membership in the Nazi Party, and during the last two years of the war he collaborated with the Gestapo as the Hamburg representative of the "Reich Association of Jews in Germany." Although he tried to provide basic welfare and health care for Hamburg's remaining Jewish population, he was heavily involved in the deportation of Jewish citizens to concentration and extermination camps. After the war, Corten thus came into conflict with the Jewish community and lost his position as medical director of the Jewish Hospital in Hamburg. He became a "double outcast" and was unable to re-establish himself in academia. His biography, however, is a vivid example of how the line between victim and perpetrator can be blurred.

C-type natriuretic peptide/cGMP/FoxO3 signaling attenuates hyperproliferation of pericytes from patients with pulmonary arterial hypertension.

Pericyte dysfunction, with excessive migration, hyperproliferation, and differentiation into smooth muscle-like cells contributes to vascular remodeling in Pulmonary Arterial Hypertension (PAH). Augmented expression and action of growth factors trigger these pathological changes. Endogenous factors opposing such alterations are barely known. Here, we examine whether and how the endothelial hormone C-type natriuretic peptide (CNP), signaling through the cyclic guanosine monophosphate (cGMP) -producing guanylyl cyclase B (GC-B) receptor, attenuates the pericyte dysfunction observed in PAH. The results demonstrate that CNP/GC-B/cGMP signaling is preserved in lung pericytes from patients with PAH and prevents their growth factor-induced proliferation, migration, and transdifferentiation. The anti-proliferative effect of CNP is mediated by cGMP-dependent protein kinase I and inhibition of the Phosphoinositide 3-kinase (PI3K)/AKT pathway, ultimately leading to the nuclear stabilization and activation of the Forkhead Box O 3 (FoxO3) transcription factor. Augmentation of the CNP/GC-B/cGMP/FoxO3 signaling pathway might be a target for novel therapeutics in the field of PAH.

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.

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.

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.

Novel insights into the mechanisms mediating the local antihypertrophic effects of cardiac atrial natriuretic peptide: role of cGMP-dependent protein kinase and RGS2.

Cardiac atrial natriuretic peptide (ANP) locally counteracts cardiac hypertrophy via the guanylyl cyclase-A (GC-A) receptor and cGMP production, but the downstream signalling pathways are unknown. Here, we examined the influence of ANP on beta-adrenergic versus Angiotensin II (Ang II)-dependent (G(s) vs. G(alphaq) mediated) modulation of Ca(2+) (i)-handling in cardiomyocytes and of hypertrophy in intact hearts. L-type Ca(2+) currents and Ca(2+) (i) transients in adult isolated murine ventricular myocytes were studied by voltage-clamp recordings and fluorescence microscopy. ANP suppressed Ang II-stimulated Ca(2+) currents and transients, but had no effect on isoproterenol stimulation. Ang II suppression by ANP was abolished in cardiomyocytes of mice deficient in GC-A, in cyclic GMP-dependent protein kinase I (PKG I) or in the regulator of G protein signalling (RGS) 2, a target of PKG I. Cardiac hypertrophy in response to exogenous Ang II was significantly exacerbated in mice with conditional, cardiomyocyte-restricted GC-A deletion (CM GC-A KO). This was concomitant to increased activation of the Ca(2+)/calmodulin-dependent prohypertrophic signal transducer CaMKII. In contrast, beta-adrenoreceptor-induced hypertrophy was not enhanced in CM GC-A KO mice. Lastly, while the stimulatory effects of Ang II on Ca(2+)-handling were absent in myocytes of mice deficient in TRPC3/TRPC6, the effects of isoproterenol were unchanged. Our data demonstrate a direct myocardial role for ANP/GC-A/cGMP to antagonize the Ca(2+) (i)-dependent hypertrophic growth response to Ang II, but not to beta-adrenergic stimulation. The selectivity of this interaction is determined by PKG I and RGS2-dependent modulation of Ang II/AT(1) signalling. Furthermore, they strengthen published observations in neonatal cardiomyocytes showing that TRPC3/TRPC6 channels are essential for Ang II, but not for beta-adrenergic Ca(2+) (i)-stimulation in adult myocytes.

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.

The heart communicates with the endothelium through the guanylyl cyclase-A receptor: acute handling of intravascular volume in response to volume expansion.

Atrial natriuretic peptide (ANP) regulates arterial blood pressure and volume. Its guanylyl cyclase-A (GC-A) receptor is expressed in vascular endothelium and mediates increases in cGMP, but the functional relevance is controversial. Notably, mice with endothelial-restricted GC-A deletion [EC GC-A knockout (KO) mice] exhibit significant chronic hypervolemic hypertension. The present study aimed to characterize the endothelial effects of ANP and their relevance for the acute regulation of intravascular fluid volume. We studied the effect of ANP on microvascular permeability to fluorescein isothiocyanate-labeled albumin (BSA) using intravital microscopy on mouse dorsal skinfold chambers. Local superfusion of ANP (100 nm) increased microvascular fluorescein isothiocyanate-BSA extravasation in control but not EC GC-A KO mice. Intravenous infusion of synthetic ANP (500 ng/kg x min) caused immediate increases in hematocrit in control mice, indicating intravascular volume contraction. In EC GC-A KO mice, the hematocrit responses were not only abolished but even reversed. Furthermore, acute vascular volume expansion, which caused release of endogenous cardiac ANP, did not affect resting central venous pressure of control mice but rapidly and significantly increased central venous pressure of EC GC-A KO mice. In cultured lung endothelial cells, ANP provoked cGMP-dependent protein kinase I-mediated phosphorylation of vasodilator-stimulated phosphoprotein. We conclude that ANP, via GC-A, enhances microvascular endothelial macromolecule permeability in vivo. This effect might be mediated by cGMP-dependent protein kinase I-dependent phosphorylation of vasodilator-stimulated phosphoprotein. Modulation of transcapillary protein and fluid transport may represent one of the most important hypovolemic actions of ANP.

CaMKII-mediated increased lusitropic responses to beta-adrenoreceptor stimulation in ANP-receptor deficient mice.

OBJECTIVE: Mice with genetic disruption of the guanylyl cyclase-A (GC-A) receptor for atrial natriuretic peptide (ANP), have chronic arterial hypertension and marked cardiac hypertrophy. Intriguingly, despite pronounced remodeling, cardiac contractile functions and cardiomyocyte Ca(2+)-handling are preserved and even enhanced. The present study aimed to characterize the specific molecular mechanisms preventing cardiac failure. METHODS AND RESULTS: Contractile function and expression as well as phosphorylation of regulatory proteins were evaluated in isolated perfused working hearts from wild-type and GC-A KO mice under baseline conditions and during beta(1)-adrenergic stimulation. Ca(i)(2+)-transients were monitored in Indo-1 loaded isolated adult cardiomyocytes. Cardiac contractile, especially lusitropic responsiveness to beta-adrenergic stimulation was significantly increased in GC-A KO mice. This was concomitant to enhanced expression and activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), increased dual-site phosphorylation of phospholamban (PLB) at Ser(16) and Thr(17), enhanced amplitude of Ca(i)(2+) transients, and accelerated Ca(i)(2+) decay. In contrast, the expression of cardiac ryanodine receptors and phosphorylation at Ser(2809) and Ser(2815) was not altered. Pharmacological inhibition of CaMKII-but not of protein kinase A-mediated PLB phosphorylation totally abolished the increased effects of beta-adrenergic stimulation on cardiac contractility and Ca(i)(2+)-handling. Thus, acceleration of sarcoplasmic reticulum Ca(2+)-uptake and increased availability of Ca(2+) for contraction, both secondary to increased CaMKII-mediated PLB phosphorylation, seem to mediate the augmented responsiveness of GC-A KO hearts to catecholamines. CONCLUSION: Our observations show that increased CaMKII activity enhances the contractile relaxation response of hypertrophic GC-A KO hearts to beta-adrenergic stimulation and emphasize the critical role of CaMKII-dependent pathways in beta(1)-adrenoreceptor modulation of myocardial Ca(2+)-homeostasis and contractility.

The natriuretic peptide/guanylyl cyclase--a system functions as a stress-responsive regulator of angiogenesis in mice.

Cardiac atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) modulate blood pressure and volume by activation of the receptor guanylyl cyclase-A (GC-A) and subsequent intracellular cGMP formation. Here we report what we believe to be a novel function of these peptides as paracrine regulators of vascular regeneration. In mice with systemic deletion of the GC-A gene, vascular regeneration in response to critical hind limb ischemia was severely impaired. Similar attenuation of ischemic angiogenesis was observed in mice with conditional, endothelial cell-restricted GC-A deletion (here termed EC GC-A KO mice). In contrast, smooth muscle cell-restricted GC-A ablation did not affect ischemic neovascularization. Immunohistochemistry and RT-PCR revealed BNP expression in activated satellite cells within the ischemic muscle, suggesting that local BNP elicits protective endothelial effects. Since within the heart, BNP is mainly induced in cardiomyocytes by mechanical load, we investigated whether the natriuretic peptide/GC-A system also regulates angiogenesis accompanying load-induced cardiac hypertrophy. EC GC-A KO hearts showed diminished angiogenesis, mild fibrosis, and diastolic dysfunction. In vitro BNP/GC-A stimulated proliferation and migration of cultured microvascular endothelia by activating cGMP-dependent protein kinase I and phosphorylating vasodilator-stimulated phosphoprotein and p38 MAPK. We therefore conclude that BNP, produced by activated satellite cells within ischemic skeletal muscle or by cardiomyocytes in response to pressure load, regulates the regeneration of neighboring endothelia via GC-A. This paracrine communication might be critically involved in coordinating muscle regeneration/hypertrophy and angiogenesis.

Why is D-serine nephrotoxic and alpha-aminoisobutyric acid protective?

D-Serine selectively causes necrosis of S(3) segments of proximal tubules in rats. This leads to aminoaciduria and glucosuria. Coinjection of the nonmetabolizable amino acid alpha-aminoisobutyric acid (AIB) prevents the tubulopathy. D-serine is selectively reabsorbed in S(3), thereby gaining access to peroxisomal D-amino acid oxidase (D-AAO). D-AAO-mediated metabolism produces reactive oxygen species. We determined the fractional excretion of amino acids and glucose in rats after intraperitoneal injection of d-serine alone or together with reduced glutathione (GSH) or AIB. Both compounds prevented the hyperaminoaciduria. We measured GSH concentrations in renal tissue before (control) and after D-serine injection and found that GSH levels decreased to approximately 30% of control. This decrease was prevented when equimolar GSH was coinjected with D-serine. To find out why AIB protected the tubule from D-serine toxicity, we microinfused D-[(14)C]serine or [(14)C]AIB (0.36 mmol/l) together with [(3)H]inulin in late proximal tubules in vivo and measured the radioactivity in the final urine. Fractional reabsorption of D-[(14)C]serine and [(14)C]AIB amounted to 55 and 70%, respectively, and 80 mmol/l of AIB or D-serine mutually prevented reabsorption to a great extent. D-AAO activity measured in vitro (using D-serine as substrate) was not influenced by a 10-fold higher AIB concentration. We conclude from these results that 1) D-AAO-mediated d-serine metabolism lowers renal GSH concentrations and thereby provokes tubular damage because reduction of reactive oxygen species by GSH is diminished and 2) AIB prevents d-serine-induced tubulopathy by inhibition of D-serine uptake in S(3) segments rather than by interfering with intracellular D-AAO-mediated D-serine metabolism.