Preserved expression of GLUT4 prevents enhanced agonist-induced vascular reactivity and MYPT1 phosphorylation in hypertensive mouse aorta

2007 ◽  
Vol 293 (1) ◽  
pp. H402-H408 ◽  
Author(s):  
Kevin B. Atkins ◽  
Antoine Prezkop ◽  
James L. Park ◽  
Jharna Saha ◽  
Damon Duquaine ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Knockout Mice ◽  
Vascular Reactivity ◽  
Calcium Sensitivity ◽  
Negative Regulator ◽  
Myosin Phosphatase ◽  
Glut4 Expression ◽  
Arterial Reactivity

We previously showed that GLUT4 expression is decreased in arterial smooth muscle of deoxycorticosterone acetate (DOCA)-salt hypertensive rats and that GLUT4-knockout mice have enhanced arterial reactivity. Therefore, we hypothesized that increased GLUT4 expression in vascular smooth muscle in vivo would prevent enhanced arterial reactivity and possibly reduce blood pressure in DOCA-salt hypertensive mice. Adult wild-type (WT) and GLUT4 transgenic (TG) mice were subjected to DOCA-salt hypertension with uninephrectomy or underwent uninephrectomy and remained normotensive. GLUT4 expression was increased more than twofold in the aortas of GLUT4 TG mice compared with WT aortas. Eight weeks after implantation of the DOCA pellets, GLUT4 expression decreased by 75% in aortas of WT hypertensive mice, but not in GLUT4 TG hypertensive aortas. Systolic blood pressure was significantly and similarly increased in WT and GLUT4 TG DOCA-salt mice compared with their respective sham-treated controls (159 vs. 111 mmHg). Responsiveness to the contractile agonist 5-HT was significantly increased in aortic rings from WT DOCA-salt mice but remained normal in GLUT4 TG DOCA mice. Phosphorylation of the myosin phosphatase targeting subunit MYPT1 was significantly enhanced in aortas of WT DOCA-salt mice, and this increase was prevented in GLUT4 TG mice. MYPT1 phosphorylation was also increased in nonhypertensive GLUT4-knockout mice. Myosin phosphatase, a major negative regulator of calcium sensitivity, is itself negatively regulated by phosphorylation of MYPT1. Therefore, our results show that preservation of GLUT4 expression prevents enhanced arterial reactivity in hypertension, possibly via effects on myosin phosphatase activity.

Abstract P501: Editing of Myosin Phosphatase Gene as a Novel Approach for Vasodilator Sensitization and Lowering of Blood Pressure

Hypertension ◽  
2017 ◽  
Vol 70 (suppl_1) ◽  
Author(s):  
Mariam Meddeb ◽  
Jeanine Ursitti ◽  
John Reho ◽  
Steven A Fisher
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Leucine Zipper ◽  
Splice Variants ◽  
Species Conservation ◽  
Mesenteric Arteries ◽  
Hek293 Cells ◽  
Myosin Phosphatase ◽  
Novel Approach

Myosin Phosphatase (MP) is the primary effector of vascular smooth muscle (VSM) relaxation and a key end target of signaling pathways that regulate vessel tone. Regulated splicing of alternative Exon24 (E24) of Myosin Phosphatase Regulatory/ Targeting subunit (MYPT1) sets vasodilator sensitivity. Skipping E24 codes for a Mypt1 isoform that contains a C-terminal leucine zipper (LZ) motif required for cGK1α binding and NO/cGMP activation of MP resulting in vasodilation. Inclusion of 31 nt E24 shifts the reading frame coding for a Mypt1 isoform with a distinct C-terminus (LZ-) that is unresponsive to NO/cGMP. We are using two editing approaches to test the function of Mypt1 E24 splice variants in the control of BP in vivo. First, LoxP sites were inserted in introns flanking E24, crossed with smMHCCre ER , and treated with Tamoxifen to achieve smooth muscle-specific cKO of E24 (SMcKO E24), thereby converting Mypt1 to the LZ+ isoform. E24 cKO mice had mean BP that was 15 + 3 mmHg lower than control (n=3-5; p<0.05). Mesenteric arteries from these mice were significantly more sensitive to DEA/NO mediated relaxation (EC 50 : 2.1+0.5 nM vs 18.2+5.6 μM; n=5-6, p<0.05). We now are developing CRISPR/CAS9 editing of Mypt1 for translation into humans with hypertension. Guide(g)RNAs targeting E24 were designed using Benchling.com and selected for further study based on predicted efficacy, specificity (>10%,>60%) and cross-species conservation. Plasmids were generated by sub-cloning of oligonucleotides into the parent pX601 plasmid for the purpose of co-expression of gRNA and saCas9. These plasmids were transfected into HEK293 cells singly and in combinations and Mypt1 gene editing assayed by PCR, Surveyor nuclease assays and sequencing of genomic DNA. Single gRNAs yielded deletions of 1-3 nt. Combinations yielded deletions of 104-334 nt that removed >80% of E24 with an efficiency of editing that varied from 10% (gRNAs 6+9 and 5+9) to 40% (gRNAs 6+11 and 5+11). We have now generated AAVgE24 and are testing their efficiency of editing of VSM in vivo. These studies support that AAV mediated CRISPR/Cas9 editing of Mypt1 E24 could be a novel strategy for vasodilator sensitization and effective lowering of blood pressure in humans.

P4477Editing of myosin phosphatase as a novel approach for sensitization of vascular smooth muscle to vasodilators (NO/cGMP/ROS) and lowering of blood pressure in hypertension

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
S Fisher ◽  
J J Reho ◽  
M Meddeb ◽  
J Ursitti ◽  
M Htet
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
World Wide ◽  
Mesenteric Arteries ◽  
Smooth Muscle Relaxation ◽  
Myosin Phosphatase ◽  
Substantial Impact

Abstract Background Despite the many drugs for treatment of hypertension, it remains inadequately treated in >50% of patients and the number one contributor to cardiovascular mortality world-wide. Thus new targets and treatment strategies are badly needed. Myosin Phosphatase (MP) is a viable target: it is the primary effector of vascular smooth muscle relaxation and a critical mediator of signaling pathways regulating vessel tone. Purpose We are using complementary/ translatable approaches to test the hypothesis: editing of the Myosin Phosphatase Regulatory (Targeting) subunit (MYPT1), by shifting the expression of naturally occurring isoforms, will sensitize vascular smooth muscle to NO/cGMP/ROS mediated vasorelaxation and thereby lower BP in models of hypertension. A further goal is to determine mechanisms by which these signals activate MP thereby causing vasorelaxation. Methods LoxP sites were inserted in introns flanking alternative Exon24 (E24) of Mypt1. Mice were crossed with smMHCCreER mice and treated with Tamoxifen for smooth muscle specific deletion of E24 (SMcKO E24).Skipping E24 codes for a Mypt1 isoform that contains a C-terminal leucine zipper (LZ) motif required for cGMP-dependent protein kinase (cGK1) binding and NO/cGMP/ROS activation of MP. Second, we developed and tested guide RNAs for the purpose of AAV-CRISPR/CAS9 editing of Mypt1 E24 as a treatment for hypertension. Effect of editing is tested in otherwise normal mice and in the AngII sub-pressor model of hypertension. Results SMcKO E24 mice had mean BP that was 15+3 mmHg lower than control (n=5; p<0.05). Mesenteric arteries from these mice were significantly more sensitive to DEA/NO mediated relaxation (EC50: 2.1+0.5 nM vs 18.2+5.6 mM; n=5–6, p<0.05). Experiments testing response to AngII infusion are in progress and will be presented at the meeting. Preliminary biochemical assays support a 2-pool model, in which NO/cGMP/ROS activates the LZ+ pool, while contractile agonists inhibit the LZ- pool of MP, in the control of BP/ blood flow. We have generated a number of AAV Crispr/Cas9 gRNAs and validated their efficacy of editing of Mypt1 E24 in vitro. Experiments are in progress to test their efficacy and effect on BP in vivo. Conclusion These studies support that editing of Mypt1 E24 could be a novel strategy for vasodilator sensitization and effective lowering of blood pressure in humans with hypertension, thereby having a substantial impact on CV mortality world-wide. Acknowledgement/Funding NIH

scholarly journals Estrogen and testosterone in concert with EFNB3 regulate vascular smooth muscle cell contractility and blood pressure

2016 ◽  
Vol 310 (7) ◽  
pp. H861-H872 ◽  
Author(s):  
Yujia Wang ◽  
Zenghui Wu ◽  
Eric Thorin ◽  
Johanne Tremblay ◽  
Julie L. Lavoie ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Gene Knockout ◽  
Target Cells ◽  
Wild Type ◽  
Cell Contractility ◽  
Risk Gene ◽  
Kinase Phosphorylation

EPH kinases and their ligands, ephrins (EFNs), have vital and diverse biological functions, although their function in blood pressure (BP) control has not been studied in detail. In the present study, we report that Efnb3 gene knockout (KO) led to increased BP in female but not male mice. Vascular smooth muscle cells (VSMCs) were target cells for EFNB3 function in BP regulation. The deletion of EFNB3 augmented contractility of VSMCs from female but not male KO mice, compared with their wild-type (WT) counterparts. Estrogen augmented VSMC contractility while testosterone reduced it in the absence of EFNB3, although these sex hormones had no effect on the contractility of VSMCs from WT mice. The effect of estrogen on KO VSMC contractility was via a nongenomic pathway involving GPER, while that of testosterone was likely via a genomic pathway, according to VSMC contractility assays and GPER knockdown assays. The sex hormone-dependent contraction phenotypes in KO VSMCs were reflected in BP in vivo. Ovariectomy rendered female KO mice normotensive. At the molecular level, EFNB3 KO in VSMCs resulted in reduced myosin light chain kinase phosphorylation, an event enhancing sensitivity to Ca2+ flux in VSMCs. Our investigation has revealed previously unknown EFNB3 functions in BP regulation and show that EFNB3 might be a hypertension risk gene in certain individuals.

scholarly journals Rgs5 Targeting Leads to Chronic Low Blood Pressure and a Lean Body Habitus

2008 ◽  
Vol 28 (8) ◽  
pp. 2590-2597 ◽  
Author(s):  
Hyeseon Cho ◽  
Chung Park ◽  
Il-Young Hwang ◽  
Sang-Bae Han ◽  
Dan Schimel ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
G Protein ◽  
Muscle Cells ◽  
Cardiovascular Development ◽  
Protein Signaling ◽  
Body Habitus ◽  
Low Blood Pressure

ABSTRACT RGS5 is a potent GTPase-activating protein for Giα and Gqα that is expressed strongly in pericytes and is present in vascular smooth muscle cells. To study the role of RGS5 in blood vessel physiology, we generated Rgs5-deficient mice. The Rgs5 −/− mice developed normally, without obvious defects in cardiovascular development or function. Surprisingly, Rgs5 −/− mice had persistently low blood pressure, lower in female mice than in male mice, without concomitant cardiac dysfunction, and a lean body habitus. The examination of the major blood vessels revealed that the aortas of Rgs5 −/− mice were dilated compared to those of control mice, without altered wall thickness. Isolated aortic smooth muscle cells from the Rgs5 −/− mice exhibited exaggerated levels of phosphorylation of vasodilator-stimulated phosphoprotein and extracellular signal-regulated kinase in response to stimulation with either sodium nitroprusside or sphingosine 1-phosphate. The results of this study, along with those of previous studies demonstrating that RGS5 stability is under the control of nitric oxide via the N-end rule pathway, suggest that RGS5 may balance vascular tone by attenuating vasodilatory signaling in vivo in opposition to RGS2, another RGS (regulator of G protein signaling) family member known to inhibit G protein-coupled receptor-mediated vasoconstrictor signaling. Blocking the function or the expression of RGS5 may provide an alternative approach to treat hypertension.

Angiotensin II-induced hypertrophy is potentiated in mice overexpressing p22phox in vascular smooth muscle

2005 ◽  
Vol 288 (1) ◽  
pp. H37-H42 ◽  
Author(s):  
David S. Weber ◽  
Petra Rocic ◽  
Adamantios M. Mellis ◽  
Karine Laude ◽  
Alicia N. Lyle ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Angiotensin Ii ◽  
Vascular Smooth Muscle ◽  
In Vivo Model ◽  
Ros Generation ◽  
Ang Ii ◽  
Vascular Hypertrophy ◽  
Wall Area

Increased reactive oxygen species (ROS) are implicated in several vascular pathologies associated with vascular smooth muscle hypertrophy. In the current studies, we utilized transgenic (Tg) mice (Tg p22smc) that overexpress the p22 phox subunit of NAD(P)H oxidase selectively in smooth muscle. These mice have a twofold increase in aortic p22 phox expression and H2O2 production and thus provide an excellent in vivo model in which to assess the effects of increased ROS generation on vascular smooth muscle cell (VSMC) function. We tested the hypothesis that overexpression of VSMC p22 phox potentiates angiotensin II (ANG II)-induced vascular hypertrophy. Male Tg p22smc mice and negative littermate controls were infused with either ANG II or saline for 13 days. Baseline blood pressure was not different between control and Tg p22smc mice. ANG II significantly increased blood pressure in both groups, with this increase being slightly exacerbated in the Tg p22smc mice. Baseline aortic wall thickness and cross-sectional wall area were not different between control and Tg p22smc mice. Importantly, the ANG II-induced increase in both parameters was significantly greater in the Tg p22smc mice compared with control mice. To confirm that this potentiation of vascular hypertrophy was due to increased ROS levels, additional groups of mice were coinfused with ebselen. This treatment prevented the exacerbation of hypertrophy in Tg p22smc mice receiving ANG II. These data suggest that although increased availability of NAD(P)H oxidase-derived ROS is not a sufficient stimulus for hypertrophy, it does potentiate ANG II-induced vascular hypertrophy, making ROS an excellent target for intervention aimed at reducing medial thickening in vivo.

scholarly journals Myosin Phosphatase Target Subunit 1 (MYPT1) Regulates the Contraction and Relaxation of Vascular Smooth Muscle and Maintains Blood Pressure

2014 ◽  
Vol 289 (32) ◽  
pp. 22512-22523 ◽  
Author(s):  
Yan-Ning Qiao ◽  
Wei-Qi He ◽  
Cai-Ping Chen ◽  
Cheng-Hai Zhang ◽  
Wei Zhao ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Myosin Phosphatase ◽  
Contraction And Relaxation

Dynamic changes in expression of myosin phosphatase in a model of portal hypertension

2004 ◽  
Vol 286 (5) ◽  
pp. H1801-H1810 ◽  
Author(s):  
Michael C. Payne ◽  
Hai-Ying Zhang ◽  
Yuichi Shirasawa ◽  
Yasuhiko Koga ◽  
Mitsuo Ikebe ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Portal Hypertension ◽  
Leucine Zipper ◽  
Splice Variants ◽  
Phenotypic Diversity ◽  
Vena Cava ◽  
Force Production ◽  
Calcium Sensitivity ◽  
Myosin Phosphatase ◽  
Minor Variation

Myosin phosphatase is a target for signaling pathways that modulate calcium sensitivity of force production in smooth muscle. Myosin phosphatase targeting subunit 1 (MYPT1) isoforms are generated by cassette-type alternative splicing of exons in the central and 3′ portion of the transcript. Exclusion of the 3′ alternative exon, coding for the leucine zipper (LZ)-positive MYPT1 isoform, is associated with the ability to desensitize to calcium (relax) in response to NO/cGMP-dependent signaling. We examined expression of MYPT1 isoforms and smooth muscle phenotype in normal rat vessels and in a prehepatic model of portal hypertension characterized by arteriolar dilation. The large capacitance vessels, aorta, pulmonary artery, and inferior vena cava expressed predominantly the 3′ exon-out/LZ-positive MYPT1 isoform. The first-order mesenteric resistance artery (MA1) and portal vein (PV) expressed severalfold higher levels of MYPT1 with predominance of the 3′ exon-included/LZ-negative isoform. There was minor variation in the presence of the MYPT1 central alternative exons. Myosin heavy and light chain splice variants in part cosegregated with MYPT1 isoforms. In response to portal hypertension induced by PV ligature, abundance of MYPT1 in PV and MA1 was significantly reduced and switched to the LZ-positive isoform. These changes were evident within 1 day of PV ligature and were maintained for up to 10 days before reverting to control values at day 14. Alteration of MYPT1 expression was part of a complex change in protein expression that can be generalized as a modulation from a phasic (fast) to a tonic (slow) contractile phenotype. Implications of vascular smooth muscle phenotypic diversity and reversible phenotypic modulation in portal hypertension with regards to regulation of blood flow are discussed.

scholarly journals NO-induced vasodilation correlates directly with BP in smooth muscle-Na/Ca exchanger-1-engineered mice: elevated BP does not attenuate endothelial function

2021 ◽  
Vol 320 (1) ◽  
pp. H221-H237
Author(s):  
Youhua Wang ◽  
Jin Zhang ◽  
W.Gil Wier ◽  
Ling Chen ◽  
Mordecai P. Blaustein
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Transgenic Mice ◽  
Endothelial Function ◽  
Knockout Mice

Endothelium-dependent, ACh-induced vasodilation (EDV) is attenuated, and arterial myocyte Na+/Ca2+ exchangers (NCX1) are upregulated in many forms of hypertension. Surprisingly, mildly hypertensive smooth muscle-specific (SM)-NCX1 transgenic mice exhibited modestly enhanced EDV and augmented endothelium-independent vasodilation (EIV). Conversely, mildly hypotensive SM-NCX1-knockout mice had greatly attenuated EIV. These adaptations help compensate for NCX1 expression-induced alterations in cytosolic Ca2+ and blood pressure (BP) and belie the view that elevated BP, itself, causes the endothelial dysregulation in hypertension.

Abstract 1066: Cardiac-restricted Disruption of Supressor of Cytokine Signaling-3 Leads to Dilated Cardiomyopathy in a gp130 Independent Manner

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Toshitaka Yajima ◽  
Hideo Yasukawa ◽  
Hanbing Zhou ◽  
Tomoko Yajima ◽  
Byung-Kwan Lim ◽  
...  
Keyword(s):  
Dilated Cardiomyopathy ◽  
Knockout Mice ◽  
Cardiac Myocyte ◽  
Physiological Role ◽  
Calcium Sensitivity ◽  
Cytokine Signaling ◽  
Independent Manner ◽  
Downstream Signaling ◽  
Intercalated Discs

We recently reported that cardiac-restricted overexpression of suppressor of cytokine signaling-3 (SOCS3) markedly increases cardiac myocyte susceptibility to virus infection. This suggests that endogenous SOCS3 also has significant biological effect in the cardiac myocyte. To understand physiological role of SOCS3 in the cardiac myocyte, we generated cardiac-specific SOCS3 knockout mice. The mice were born at the expected Mendelian ratio, but developed cardiac dysfunction (%FS; wt: 45±2.6 vs. ko: 31±4.5, mean±SE, n=8, p=0.017, 5-month-old) estimated by echocardiogram from 5 months of age and died with signs of dyspnea by 7 months of age. Postmortem analysis revealed significant pleural effusions and ascites, consistent with the presence of heart failure. Histological analysis showed that the heart has a thin left and right ventricular walls with chamber dilatation; however, typical findings of a myopathic heart such as myocyte disarray, inflammation and fibrosis were only rarely observed. Ultrastructure analysis of the dilated heart samples showed intact myofibrils, mitochondria, intercalated discs and gap junctions, suggesting that the myocyte had non-structural abnormalities that led to functional impairment. Unexpectedly calcium transients in the isolated adult myocytes from the failing hearts were significantly increased as compared to those from age-matched normal hearts (Fura-2 intensity ratio 340/380 nm; wt: 0.44±0.01 vs. ko: 0.54±0.01, mean±SE, n=3, p<0.0002), suggesting the decreased calcium sensitivity of SOCS3 deficient myocytes. We also observed spontaneous ventricular tachycardia in the knockout mice by telemetry analysis. Since SOCS3 has been reported to be a crucial negative-feedback regulator of gp130 signaling in vivo, we sought to determine whether the phenotype is due to overactivation of cardiac gp130 signaling. While gp130 downstream signaling such as STAT3, ERK and AKT pathways were significantly activated in the knockout heart, cardiac-specific knockout of both gp130 and SOCS3 did not rescue the dilated cardiomyopathy phenotype. Conclusion: These results indicate that a gp130-independent mechanism is responsible for the dilated cardiomyopathy that occurs in SOCS3 deficient hearts.

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