Comprehensive ultrasound imaging of right ventricular remodeling under surgically induced pressure overload in mice.

This study reports a new methodology for right heart imaging by ultrasound in mice under right ventricular (RV) pressure overload. Pulmonary artery constriction (PAC) or sham surgeries were performed on C57BL/6 male mice at 8 wk of age. Ultrasound imaging was conducted at 2, 4, and 8 wk postsurgery using both classical and advanced ultrasound imaging modalities including electrocardiogram (ECG)-based kilohertz visualization, anatomical M-mode, and strain imaging. Based on pulsed Doppler, the PAC group demonstrated dramatically enhanced pressure gradient in the main pulmonary artery (MPA) as compared with the sham group. By the application of advanced imaging modalities in novel short-axis views of the ventricles, the PAC group demonstrated increased thickness of RV free wall, enlarged RV chamber, and reduced RV fractional shortening compared with the sham group. The PAC group also showed prolonged RV contraction, asynchronous interplay between RV and left ventricle (LV), and passive leftward motion of the interventricular septum (IVS) at early diastole. Consequently, the PAC group exhibited prolongation of LV isovolumic relaxation time, without change in LV wall thickness or systolic function. Significant correlations were found between the maximal pressure gradient in MPA measured by Doppler and the RV systolic pressure by catheterization, as well as the morphological and functional parameters of RV by ultrasound.NEW & NOTEWORTHY The established protocol overcomes the challenges in right heart imaging in mice, thoroughly elucidating the changes of RV, the dynamics of IVS, and the impact on LV and provides new insights into the pathophysiological mechanism of RV remodeling.

RGS4 controls Gαi3-mediated regulation of Bcl-2 phosphorylation on TGN38-containing intracellular membranes.

Intracellular pools of the heterotrimeric G-protein α-subunit Gαi3 (encoded by GNAI3) have been shown to promote growth factor signaling, while at the same time inhibiting the activation of JNK and autophagic signaling following nutrient starvation. The precise molecular mechanisms linking Gαi3 to both stress and growth factor signaling remain poorly understood. Importantly, JNK-mediated phosphorylation of Bcl-2 was previously found to activate autophagic signaling following nutrient deprivation. Our data shows that activated Gαi3 decreases Bcl-2 phosphorylation, whereas inhibitors of Gαi3, such as RGS4 and AGS3 (also known as GPSM1), markedly increase the levels of phosphorylated Bcl-2. Manipulation of the palmitoylation status and intracellular localization of RGS4 suggests that Gαi3 modulates phosphorylated Bcl-2 levels and autophagic signaling from discreet TGN38 (also known as TGOLN2)-labeled vesicle pools. Consistent with an important role for these molecules in normal tissue responses to nutrient deprivation, increased Gαi signaling within nutrient-starved adrenal glands from RGS4-knockout mice resulted in a dramatic abrogation of autophagic flux, compared to wild-type tissues. Together, these data suggest that the activity of Gαi3 and RGS4 from discreet TGN38-labeled vesicle pools are critical regulators of autophagic signaling that act via their ability to modulate phosphorylation of Bcl-2.

Myocardial Infarction Induces Cardiac Fibroblast Transformation within Injured and Noninjured Regions of the Mouse Heart.

Heart failure (HF) is associated with pathological remodeling of the myocardium, including the initiation of fibrosis and scar formation by activated cardiac fibroblasts (CFs). Although early CF-dependent scar formation helps prevent cardiac rupture by maintaining the heart's structural integrity, ongoing deposition of the extracellular matrix in the remote and infarct regions can reduce tissue compliance, impair cardiac function, and accelerate progression to HF. In our study, we conducted mass spectrometry (MS) analysis to identify differentially altered proteins and signaling pathways between CFs isolated from 7 day sham and infarcted murine hearts. Surprisingly, CFs from both the remote and infarct regions of injured hearts had a wide number of similarly altered proteins and signaling pathways that were consistent with fibrosis and activation into pathological myofibroblasts. Specifically, proteins enriched in CFs isolated from MI hearts were involved in pathways pertaining to cell-cell and cell-matrix adhesion, chaperone-mediated protein folding, and collagen fibril organization. These results, together with principal component analyses, provided evidence of global CF activation postinjury. Interestingly, however, direct comparisons between CFs from the remote and infarct regions of injured hearts identified 15 differentially expressed proteins between MI remote and MI infarct CFs. Eleven of these proteins (Gpc1, Cthrc1, Vmac, Nexn, Znf185, Sprr1a, Specc1, Emb, Limd2, Pawr, and Mcam) were higher in MI infarct CFs, whereas four proteins (Gstt1, Gstm1, Tceal3, and Inmt) were higher in MI remote CFs. Collectively, our study shows that MI injury induced global changes to the CF proteome, with the magnitude of change reflecting their relative proximity to the site of injury.

Resveratrol Inhibits Neointimal Growth after Arterial Injury in High-Fat-Fed Rodents: The Roles of SIRT1 and AMPK.

We have shown that both insulin and resveratrol (RSV) decrease neointimal hyperplasia in chow-fed rodents via mechanisms that are in part overlapping and involve the activation of endothelial nitric oxide synthase (eNOS). However, this vasculoprotective effect of insulin is abolished in high-fat-fed insulin-resistant rats. Since RSV, in addition to increasing insulin sensitivity, can activate eNOS via pathways that are independent of insulin signaling, such as the activation of sirtuin 1 (SIRT1) and AMP-activated kinase (AMPK), we speculated that unlike insulin, the vasculoprotective effect of RSV would be retained in high-fat-fed rats. We found that high-fat feeding decreased insulin sensitivity and increased neointimal area and that RSV improved insulin sensitivity (p < 0.05) and decreased neointimal area in high-fat-fed rats (p < 0.05). We investigated the role of SIRT1 in the effect of RSV using two genetic mouse models. We found that RSV decreased neointimal area in high-fat-fed wild-type mice (p < 0.05), an effect that was retained in mice with catalytically inactive SIRT1 (p < 0.05) and in heterozygous SIRT1-null mice. In contrast, the effect of RSV was abolished in AMKPα2-null mice. Thus, RSV decreased neointimal hyperplasia after arterial injury in both high-fat-fed rats and mice, an effect likely not mediated by SIRT1 but by AMPKα2.

Deficiency of Natriuretic Peptide Receptor 2 Promotes Bicuspid Aortic Valves, Aortic Valve Disease, Left Ventricular Dysfunction, and Ascending Aortic Dilatations in Mice.

RATIONALE: Aortic valve disease is a cell-mediated process without effective pharmacotherapy. CNP (C-type natriuretic peptide) inhibits myofibrogenesis and osteogenesis of cultured valve interstitial cells and is downregulated in stenotic aortic valves. However, it is unknown whether CNP signaling regulates aortic valve health in vivo. OBJECTIVE: The aim of this study is to determine whether a deficient CNP signaling axis in mice causes accelerated progression of aortic valve disease. METHODS AND RESULTS: In cultured porcine valve interstitial cells, CNP inhibited pathological differentiation via the guanylate cyclase NPR2 (natriuretic peptide receptor 2) and not the G-protein-coupled clearance receptor NPR3 (natriuretic peptide receptor 3). We used Npr2+/- and Npr2+/-;Ldlr-/- mice and wild-type littermate controls to examine the valvular effects of deficient CNP/NPR2 signaling in vivo, in the context of both moderate and advanced aortic valve disease. Myofibrogenesis in cultured Npr2+/- fibroblasts was insensitive to CNP treatment, whereas aged Npr2+/- and Npr2+/-;Ldlr-/- mice developed cardiac dysfunction and ventricular fibrosis. Aortic valve function was significantly impaired in Npr2+/- and Npr2+/-;Ldlr-/- mice versus wild-type littermates, with increased valve thickening, myofibrogenesis, osteogenesis, proteoglycan synthesis, collagen accumulation, and calcification. 9.4% of mice heterozygous for Npr2 had congenital bicuspid aortic valves, with worse aortic valve function, fibrosis, and calcification than those Npr2+/- with typical tricuspid aortic valves or all wild-type littermate controls. Moreover, cGK (cGMP-dependent protein kinase) activity was downregulated in Npr2+/- valves, and CNP triggered synthesis of cGMP and activation of cGK1 (cGMP-dependent protein kinase 1) in cultured porcine valve interstitial cells. Finally, aged Npr2+/-;Ldlr-/- mice developed dilatation of the ascending aortic, with greater aneurysmal progression in Npr2+/- mice with bicuspid aortic valves than those with tricuspid valves. CONCLUSIONS: Our data establish CNP/NPR2 signaling as a novel regulator of aortic valve development and disease and elucidate the therapeutic potential of targeting this pathway to arrest disease progression.

Rab family proteins regulate the endosomal trafficking and function of RGS4.

RGS4, a heterotrimeric G-protein inhibitor, localizes to plasma membrane (PM) and endosomal compartments. Here, we examined Rab-mediated control of RGS4 internalization and recycling. Wild type and constitutively active Rab5 decreased RGS4 PM levels while increasing its endosomal targeting. Rab5, however, did not appreciably affect the PM localization or function of the M1 muscarinic receptor (M1R)/Gq signaling cascade. RGS4-containing endosomes co-localized with subsets of Rab5-, transferrin receptor-, and Lamp1/Lysotracker-marked compartments suggesting RGS4 traffics through PM recycling or acidified endosome pathways. Rab7 activity promoted TGN association, whereas Rab7(dominant negative) trapped RGS4 in late endosomes. Furthermore, RGS4 was found to co-localize with an endosomal pool marked by Rab11, the protein that mediates recycling/sorting of proteins to the PM. The Cys-12 residue in RGS4 appeared important for its Rab11-mediated trafficking to the PM. Rab11(dominant negative) decreased RGS4 PM levels and increased the number of RGS4-containing endosomes. Inhibition of Rab11 activity decreased RGS4 function as an inhibitor of M1R activity without affecting localization and function of the M1R/Gq signaling complex. Thus, both Rab5 activation and Rab11 inhibition decreased RGS4 function in a manner that is independent from their effects on the localization and function of the M1R/Gq signaling complex. This is the first study to implicate Rab GTPases in the intracellular trafficking of an RGS protein. Thus, Rab GTPases may be novel molecular targets for the selective regulation of M1R-mediated signaling via their specific effects on RGS4 trafficking and function.

Enhanced proliferation and altered calcium handling in RGS2-deficient vascular smooth muscle cells.

CONTEXT: Regulator of G-protein signaling-2 (RGS2) inhibits Gq-mediated regulation of Ca(2+) signalling in vascular smooth muscle cells (VSMC). OBJECTIVE: RGS2 knockout (RGS2KO) mice are hypertensive and show arteriolar remodeling. VSMC proliferation modulates intracellular Ca(2+) concentration [Ca(2+)]i. RGS2 involvement in VSMC proliferation had not been examined. METHODS: Thymidine incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion assays measured cell proliferation. Fura-2 ratiometric imaging quantified [Ca(2+)]i before and after UTP and thapsigargin. [(3)H]-labeled inositol was used for phosphoinositide hydrolysis. Quantitative RT-PCR and confocal immunofluorescence of select Ca(2+) transporters was performed in primary aortic VSMC. RESULTS AND DISCUSSION: Platelet-derived growth factor (PDGF) increased S-phase entry and proliferation in VSMC from RGS2KO mice to a greater extent than in VSMC from wild-type (WT) controls. Consistent with differential PDGF-induced changes in Ca(2+) homeostasis, RGS2KO VSMC showed lower resting [Ca(2+)]i but higher thapsigargin-induced [Ca(2+)]i as compared with WT. RGS2KO VSMC expressed lower mRNA levels of plasma membrane Ca(2+) ATPase-4 (PMCA4) and Na(+) Ca(2+) Exchanger (NCX), but higher levels of sarco-endoplasmic reticulum Ca(2+) ATPase-2 (SERCA2). Western blot and immunofluorescence revealed similar differences in PMCA4 and SERCA2 protein, while levels of NCX protein were not reduced in RGS2KO VSMC. Consistent with decreased Ca(2+) efflux activity, (45)Ca-extrusion rates were lower in RGS2KO VSMC. These differences were reversed by the PMCA inhibitor La(3+), but not by replacing extracellular Na(+) with choline, implicating differences in the activity of PMCA and not NCX. CONCLUSION: RGS2-deficient VSMC exhibit higher rates of proliferation and coordinate plasticity of Ca(2+)-handling mechanisms in response to PDGF stimulation.

ß2-Adrenoceptor agonist-induced RGS2 expression is a genomic mechanism of bronchoprotection that is enhanced by glucocorticoids.

In asthma and chronic obstructive pulmonary disease, activation of G(q)-protein-coupled receptors causes bronchoconstriction. In each case, the management of moderate-to-severe disease uses inhaled corticosteroid (glucocorticoid)/long-acting ß(2)-adrenoceptor agonist (LABA) combination therapies, which are more efficacious than either monotherapy alone. In primary human airway smooth muscle cells, glucocorticoid/LABA combinations synergistically induce the expression of regulator of G-protein signaling 2 (RGS2), a GTPase-activating protein that attenuates G(q) signaling. Functionally, RGS2 reduced intracellular free calcium flux elicited by histamine, methacholine, leukotrienes, and other spasmogens. Furthermore, protection against spasmogen-increased intracellular free calcium, following treatment for 6 h with LABA plus corticosteroid, was dependent on RGS2. Finally, Rgs2-deficient mice revealed enhanced bronchoconstriction to spasmogens and an absence of LABA-induced bronchoprotection. These data identify RGS2 gene expression as a genomic mechanism of bronchoprotection that is induced by glucocorticoids plus LABAs in human airway smooth muscle and provide a rational explanation for the clinical efficacy of inhaled corticosteroid (glucocorticoid)/LABA combinations in obstructive airways diseases.

Priming of hypoxia-inducible factor by neuronal nitric oxide synthase is essential for adaptive responses to severe anemia.

Cells sense and respond to changes in oxygen concentration through gene regulatory processes that are fundamental to survival. Surprisingly, little is known about how anemia affects hypoxia signaling. Because nitric oxide synthases (NOSs) figure prominently in the cellular responses to acute hypoxia, we defined the effects of NOS deficiency in acute anemia. In contrast to endothelial NOS or inducible NOS deficiency, neuronal NOS (nNOS)(-/-) mice demonstrated increased mortality during anemia. Unlike wild-type (WT) animals, anemia did not increase cardiac output (CO) or reduce systemic vascular resistance (SVR) in nNOS(-/-) mice. At the cellular level, anemia increased expression of HIF-1α protein and HIF-responsive mRNA levels (EPO, VEGF, GLUT1, PDK1) in the brain of WT, but not nNOS(-/-) mice, despite comparable reductions in tissue PO(2). Paradoxically, nNOS(-/-) mice survived longer during hypoxia, retained the ability to regulate CO and SVR, and increased brain HIF-α protein levels and HIF-responsive mRNA transcripts. Real-time imaging of transgenic animals expressing a reporter HIF-α(ODD)-luciferase chimeric protein confirmed that nNOS was essential for anemia-mediated increases in HIF-α protein stability in vivo. S-nitrosylation effects the functional interaction between HIF and pVHL. We found that anemia led to nNOS-dependent S-nitrosylation of pVHL in vivo and, of interest, led to decreased expression of GSNO reductase. These findings identify nNOS effects on the HIF/pVHL signaling pathway as critically important in the physiological responses to anemia in vivo and provide essential mechanistic insight into the differences between anemia and hypoxia.

Amino-terminal cysteine residues differentially influence RGS4 protein plasma membrane targeting, intracellular trafficking, and function.

Regulator of G-protein signaling (RGS) proteins are potent inhibitors of heterotrimeric G-protein signaling. RGS4 attenuates G-protein activity in several tissues. Previous work demonstrated that cysteine palmitoylation on residues in the amino-terminal (Cys-2 and Cys-12) and core domains (Cys-95) of RGS4 is important for protein stability, plasma membrane targeting, and GTPase activating function. To date Cys-2 has been the priority target for RGS4 regulation by palmitoylation based on its putative role in stabilizing the RGS4 protein. Here, we investigate differences in the contribution of Cys-2 and Cys-12 to the intracellular localization and function of RGS4. Inhibition of RGS4 palmitoylation with 2-bromopalmitate dramatically reduced its localization to the plasma membrane. Similarly, mutation of the RGS4 amphipathic helix (L23D) prevented membrane localization and its G(q) inhibitory function. Together, these data suggest that both RGS4 palmitoylation and the amphipathic helix domain are required for optimal plasma membrane targeting and function of RGS4. Mutation of Cys-12 decreased RGS4 membrane targeting to a similar extent as 2-bromopalmitate, resulting in complete loss of its G(q) inhibitory function. Mutation of Cys-2 did not impair plasma membrane targeting but did partially impair its function as a G(q) inhibitor. Comparison of the endosomal distribution pattern of wild type and mutant RGS4 proteins with TGN38 indicated that palmitoylation of these two cysteines contributes differentially to the intracellular trafficking of RGS4. These data show for the first time that Cys-2 and Cys-12 play markedly different roles in the regulation of RGS4 membrane localization, intracellular trafficking, and G(q) inhibitory function via mechanisms that are unrelated to RGS4 protein stabilization.

Proximal cerebral arteries develop myogenic responsiveness in heart failure via tumor necrosis factor-α-dependent activation of sphingosine-1-phosphate signaling.

BACKGROUND: Heart failure is associated with neurological deficits, including cognitive dysfunction. However, the molecular mechanisms underlying reduced cerebral blood flow in the early stages of heart failure, particularly when blood pressure is minimally affected, are not known. METHODS AND RESULTS: Using a myocardial infarction model in mice, we demonstrate a tumor necrosis factor-α (TNFα)-dependent enhancement of posterior cerebral artery tone that reduces cerebral blood flow before any overt changes in brain structure and function. TNFα expression is increased in mouse posterior cerebral artery smooth muscle cells at 6 weeks after myocardial infarction. Coordinately, isolated posterior cerebral arteries display augmented myogenic tone, which can be fully reversed in vitro by the competitive TNFα antagonist etanercept. TNFα mediates its effect via a sphingosine-1-phosphate (S1P)-dependent mechanism, requiring sphingosine kinase 1 and the S1P(2) receptor. In vivo, sphingosine kinase 1 deletion prevents and etanercept (2-week treatment initiated 6 weeks after myocardial infarction) reverses the reduction of cerebral blood flow, without improving cardiac function. CONCLUSIONS: Cerebral artery vasoconstriction and decreased cerebral blood flow occur early in an animal model of heart failure; these perturbations are reversed by interrupting TNFα/S1P signaling. This signaling pathway may represent a potential therapeutic target to improve cognitive function in heart failure.

Origin-specific epigenetic program correlates with vascular bed-specific differences in Rgs5 expression.

Cells from multiple origins contribute to vascular smooth muscle cell (VSMC) development. Phenotypic heterogeneity of VSMCs is associated with their point of developmental origin; however, the mechanisms driving such differences are unknown. We here examined the mechanisms controlling vascular bed-specific differences in Rgs5 expression during development. Rgs5 levels were similar across different regions of the vasculature in neonatal animals but were >15-fold higher in descending aortas compared with carotid arteries of adult mice. Thus, vessel bed-specific changes in regulation of Rgs5 expression occurred during vessel maturation. Examination of adult Rgs5-LacZ reporter mice revealed lower Rgs5 expression in VSMCs originating from the third (carotid artery) branchial arch compared with those originating in the fourth and sixth (aortic B segment, right subclavian, and ductus arteriosus) branchial arches. Indeed, a mosaic Rgs5 expression pattern, with discreet LacZ boundaries between VSMCs derived from different developmental origins, was observed. Furthermore, Rgs5-LacZ expression was correlated with the site of VSMC origin (splanchic mesoderm ≈ local mesenchyme > somites > proepicardium > mesothelium). Surprisingly, Rgs5 reporter activity in cultured carotid artery- and descending aorta-derived cells did not recapitulate the differences observed in vivo. Consistent with a developmental origin-specific epigenetic mechanism driving the observed expression differences in vivo, the Rgs5 promoter showed increased methylation on CpG dinucleotides in carotid arteries compared with that in descending aortas in adult but not in neonatal mice. In vitro methylation of the Rgs5 promoter confirmed that its activity is sensitive to transcriptional down-regulation by CpG methylation. These data suggest that an origin-dependent epigenetic program regulates vascular bed- and maturation state-dependent regulation of VSMC-specific gene transcription.

Regulator of G protein signaling 5 protects against cardiac hypertrophy and fibrosis during biomechanical stress of pressure overload.

The development of cardiac hypertrophy in response to increased hemodynamic load and neurohormonal stress is initially a compensatory response that may eventually lead to ventricular dilation and heart failure. Regulator of G protein signaling 5 (Rgs5) is a negative regulator of G protein-mediated signaling by inactivating Galphaq and Galphai, which mediate actions of most known vasoconstrictors. Previous studies have demonstrated that Rgs5 expresses among various cell types within mature heart and showed high levels of Rgs5 mRNA in monkey and human heart tissue by Northern blot analysis. However, the critical role of Rgs5 on cardiac remodeling remains unclear. To specifically determine the role of Rgs5 in pathological cardiac remodeling, we used transgenic mice with cardiac-specific overexpression of human Rgs5 gene and Rgs5-/- mice. Our results demonstrated that the transgenic mice were resistant to cardiac hypertrophy and fibrosis through inhibition of MEK-ERK1/2 signaling, whereas the Rgs5-/- mice displayed the opposite phenotype in response to pressure overload. These studies indicate that Rgs5 protein is a crucial component of the signaling pathway involved in cardiac remodeling and heart failure.

Disrupting circadian control of peripheral myogenic reactivity mitigates cardiac injury following myocardial infarction.

AIMS: Circadian rhythms orchestrate important functions in the cardiovascular system: the contribution of microvascular rhythms to cardiovascular disease progression/severity is unknown. This study hypothesized that (i) myogenic reactivity in skeletal muscle resistance arteries is rhythmic and (ii) disrupting this rhythmicity would alter cardiac injury post-myocardial infarction (MI). METHODS AND RESULTS: Cremaster skeletal muscle resistance arteries were isolated and assessed using standard pressure myography. Circadian rhythmicity was globally disrupted with the ClockΔ19/Δ19 mutation or discretely through smooth muscle cell-specific Bmal1 deletion (Sm-Bmal1 KO). Cardiac structure and function were determined by echocardiographic, hemodynamic and histological assessments. Myogenic reactivity in cremaster muscle resistance arteries is rhythmic. This rhythm is putatively mediated by the circadian modulation of a mechanosensitive signalosome incorporating tumour necrosis factor and casein kinase 1. Following left anterior descending coronary artery ligation, myogenic responsiveness is locked at the circadian maximum, although circadian molecular clock gene expression cycles normally. Disrupting the molecular clock abolishes myogenic rhythmicity: myogenic tone is suspended at the circadian minimum and is no longer augmented by MI. The reduced myogenic tone in ClockΔ19/Δ19 mice and Sm-Bmal1 KO mice associates with reduced total peripheral resistance (TPR), improved cardiac function and reduced infarct expansion post-MI. CONCLUSIONS: Augmented microvascular constriction aggravates cardiac injury post-MI. Following MI, skeletal muscle resistance artery myogenic reactivity increases specifically within the rest phase, when TPR would normally decline. Disrupting the circadian clock interrupts the MI-induced augmentation in myogenic reactivity: therapeutics targeting the molecular clock, therefore, may be useful for improving MI outcomes.

Genetic deletion of regulator of G-protein signaling 4 (RGS4) rescues a subset of fragile X related phenotypes in the FMR1 knockout mouse.

Fragile X syndrome (FXS), the most common cause of inherited mental retardation, is caused by the loss of the mRNA binding protein, FMRP. Persons with FXS also display epileptic seizures, social anxiety, hyperactivity, and autistic behaviors. The metabotropic glutamate receptor theory of FXS postulates that in the absence of FMRP, enhanced signaling though G-protein coupled group I metabotropic glutamate receptors in the brain contributes to many of the abnormalities observed in the disorder. However, recent evidence suggests that alterations in cellular signaling through additional G-protein coupled receptors may also be involved in the pathogenesis of FXS, thus providing impetus for examining downstream molecules. One group of signaling molecules situated downstream of the receptors is the regulator of G-protein signaling (RGS) proteins. Notably, RGS4 is highly expressed in brain and has been shown to negatively regulate signaling through Group I mGluRs and GABA(B) receptors. To examine the potential role for RGS4 in the pathogenesis of FXS, we generated FXS/RGS4 double knockout mice. Characterization of these mice revealed that a subset of FXS related phenotypes, including increased body weight, altered synaptic protein expression, and abnormal social behaviors, were rescued in the double knockout mice. Other phenotypes, such as hyperactivity and macroorchidism, were not affected by the loss of RGS4. These findings suggest that tissue and cell-type specific differences in GPCR signaling and RGS function may contribute to the spectrum of phenotypic differences observed in FXS.

Intracellular regulation of heterotrimeric G-protein signaling modulates vascular smooth muscle cell contraction.

The contractile function of vascular smooth muscle cells within the media of resistance arterioles is tightly connected to the role of these blood vessels in the maintenance of blood pressure homeostasis. Thus, much effort has been made to understand the intracellular signaling pathways that control vascular smooth muscle cell contractility with the aim that this knowledge will provide important clues for reducing the impact of uncontrolled blood pressure in our society. A key set of surface receptors, the G-protein coupled receptors, has been widely associated with the regulation of vascular smooth muscle cell contractility. Indeed, many of the current treatments for hypertension involve selective inhibition of these receptors. More recently, we have begun to understand the cellular mechanisms whereby G-protein coupled pathways are connected to the contractile machinery of the vascular smooth muscle cells. What has emerged is a view where there are multiple intracellular control points for G-protein signaling that coordinate and focus the extracellular stimuli into meaningful physiologic responses. This work will examine some of the recent advances in our understanding of G-protein signaling and its regulation of contractile function in vascular smooth muscle cells.

Regulator of G-protein signaling-2 mediates vascular smooth muscle relaxation and blood pressure.

Nitric oxide (NO) inhibits vascular contraction by activating cGMP-dependent protein kinase I-alpha (PKGI-alpha), which causes dephosphorylation of myosin light chain (MLC) and vascular smooth muscle relaxation. Here we show that PKGI-alpha attenuates signaling by the thrombin receptor protease-activated receptor-1 (PAR-1) through direct activation of regulator of G-protein signaling-2 (RGS-2). NO donors and cGMP cause cGMP-mediated inhibition of PAR-1 and membrane localization of RGS-2. PKGI-alpha binds directly to and phosphorylates RGS-2, which significantly increases GTPase activity of G(q), terminating PAR-1 signaling. Disruption of the RGS-2-PKGI-alpha interaction reverses inhibition of PAR-1 signaling by nitrovasodilators and cGMP. Rgs2-/- mice develop marked hypertension, and their blood vessels show enhanced contraction and decreased cGMP-mediated relaxation. Thus, PKGI-alpha binds to, phosphorylates and activates RGS-2, attenuating receptor-mediated vascular contraction. Our study shows that RGS-2 is required for normal vascular function and blood pressure and is a new drug development target for hypertension.

RGS4-Deficiency Alters Intracellular Calcium and PKA-Mediated Control of Insulin Secretion in Glucose-Stimulated Beta Islets.

A number of diverse G-protein signaling pathways have been shown to regulate insulin secretion from pancreatic ß-cells. Accordingly, regulator of G-protein signaling (RGS) proteins have also been implicated in coordinating this process. One such protein, RGS4, is reported to show both positive and negative effects on insulin secretion from ß-cells depending on the physiologic context under which it was studied. We here use an RGS4-deficient mouse model to characterize previously unknown G-protein signaling pathways that are regulated by RGS4 during glucose-stimulated insulin secretion from the pancreatic islets. Our data show that loss of RGS4 results in a marked deficiency in glucose-stimulated insulin secretion during both phase I and phase II of insulin release in intact mice and isolated islets. These deficiencies are associated with lower cAMP/PKA activity and a loss of normal calcium surge (phase I) and oscillatory (phase II) kinetics behavior in the RGS4-deficient ß-cells, suggesting RGS4 may be important for regulation of both Gαi and Gαq signaling control during glucose-stimulated insulin secretion. Together, these studies add to the known list of G-protein coupled signaling events that are controlled by RGS4 during glucose-stimulated insulin secretion and highlight the importance of maintaining normal levels of RGS4 function in healthy pancreatic tissues.

Deletion of type VIII collagen reduces blood pressure, increases carotid artery functional distensibility and promotes elastin deposition.

Arterial stiffening is a significant predictor of cardiovascular disease development and mortality. In elastic arteries, stiffening refers to the loss and fragmentation of elastic fibers, with a progressive increase in collagen fibers. Type VIII collagen (Col-8) is highly expressed developmentally, and then once again dramatically upregulated in aged and diseased vessels characterized by arterial stiffening. Yet its biophysical impact on the vessel wall remains unknown. The purpose of this study was to test the hypothesis that Col-8 functions as a matrix scaffold to maintain vessel integrity during extracellular matrix (ECM) development. These changes are predicted to persist into the adult vasculature, and we have tested this in our investigation. Through our in vivo and in vitro studies, we have determined a novel interaction between Col-8 and elastin. Mice deficient in Col-8 (Col8-/-) had reduced baseline blood pressure and increased arterial compliance, indicating an enhanced Windkessel effect in conducting arteries. Differences in both the ECM composition and VSMC activity resulted in Col8-/- carotid arteries that displayed increased crosslinked elastin and functional distensibility, but enhanced catecholamine-induced VSMC contractility. In vitro studies revealed that the absence of Col-8 dramatically increased tropoelastin mRNA and elastic fiber deposition in the ECM, which was decreased with exogenous Col-8 treatment. These findings suggest a causative role for Col-8 in reducing mRNA levels of tropoelastin and the presence of elastic fibers in the matrix. Moreover, we also found that Col-8 and elastin have opposing effects on VSMC phenotype, the former promoting a synthetic phenotype, whereas the latter confers quiescence. These studies further our understanding of Col-8 function and open a promising new area of investigation related to elastin biology.

RGS4 regulates parasympathetic signaling and heart rate control in the sinoatrial node.

Heart rate is controlled by the opposing activities of sympathetic and parasympathetic inputs to pacemaker myocytes in the sinoatrial node (SAN). Parasympathetic activity on nodal myocytes is mediated by acetylcholine-dependent stimulation of M(2) muscarinic receptors and activation of Galpha(i/o) signaling. Although regulators of G protein signaling (RGS) proteins are potent inhibitors of Galpha(i/o) signaling in many tissues, the RGS protein(s) that regulate parasympathetic tone in the SAN are unknown. Our results demonstrate that RGS4 mRNA levels are higher in the SAN compared to right atrium. Conscious freely moving RGS4-null mice showed increased bradycardic responses to parasympathetic agonists compared to wild-type animals. Moreover, anesthetized RGS4-null mice had lower baseline heart rates and greater heart rate increases following atropine administration. Retrograde-perfused hearts from RGS4-null mice showed enhanced negative chronotropic responses to carbachol, whereas SAN myocytes showed greater sensitivity to carbachol-mediated reduction in the action potential firing rate. Finally, RGS4-null SAN cells showed decreased levels of G protein-coupled inward rectifying potassium (GIRK) channel desensitization and altered modulation of acetylcholine-sensitive potassium current (I(KACh)) kinetics following carbachol stimulation. Taken together, our studies establish that RGS4 plays an important role in regulating sinus rhythm by inhibiting parasympathetic signaling and I(KACh) activity.