Regulatory Subunit Of Myosin Phosphatase Research Articles

An antisense oligonucleotide efficiently suppresses splicing of an alternative exon in vascular smooth muscle in vivo.

Targeting alternative exons for therapeutic gain has been achieved in a few instances and potentially could be applied more broadly. The myosin phosphatase (MP) enzyme is a critical hub upon which signals converge to regulate vessel tone. Alternative exon 24 of myosin phosphatase regulatory subunit (Mypt1 E24) is an ideal target as toggling between the two isoforms sets smooth muscle sensitivity to vasodilators such as nitric oxide (NO). This study aimed to develop a gene-based therapy to suppress splicing of Mypt1 E24 thereby switching MP enzyme to the NO-responsive isoform. CRISPR/Cas9 constructs were effective at editing of Mypt1 E24 in vitro; however, targeting of vascular smooth muscle in vivo with AAV9 was inefficient. In contrast, an octo-guanidine conjugated antisense oligonucleotide targeting the 5' splice site of Mypt1 E24 was highly efficient in vivo. It reduced the percent splicing inclusion of Mypt1 E24 from 80% to 10% in mesenteric arteries. The maximal and half-maximal effects occurred at 12.5 and 6.25 mg/kg, respectively. The effect persisted for at least 1 mo without toxicity. This highly effective splice-blocking antisense oligonucleotide could be developed as a novel therapy to reverse vascular dysfunction common to diseases such as hypertension and heart failure.NEW & NOTEWORTHY Alternative exon usage is a major driver of phenotypic diversity in all cell types including smooth muscle. However, the functional significance of most of the hundreds of thousands of alternative exons has not been defined, nor in most cases even tested. If their importance to vascular function were known these alternative exons could represent novel therapeutic targets. Here, we present injection of Vivo-morpholino splice-blocking antisense oligonucleotides as a simple, efficient, and cost-effective method for suppression of alternative exon usage in vascular smooth muscle in vivo.

Smoothelin-like protein 1 promotes insulin sensitivity and modulates the contractile properties of endometrial epithelial cells with insulin resistance.

The incidence of infertility is significantly higher in women with diseases linked to impaired glucose homeostasis, such as insulin resistance. Defective glucose metabolism interferes with fertilization; however, the molecular mechanism underlying this interference is unclear. Smoothelin-like protein 1 (SMTNL1) was isolated from muscle and steroid hormone-responsive tissues and regulates the contractile functions of various cell types through the inhibition of myosin phosphatase (MP) holoenzyme. In addition, SMTNL-1 after phosphorylation at Ser301 by protein kinase A translocates to the nucleus and functions as a transcriptional co-activator of the progesterone receptor-B. SMTNL1 null mice exhibit reduced reproductive fitness and are more prone to type 2 diabetes mellitus. However, the role of SMTNL1 in endometrial epithelial cells is not known. The effect of SMTNL1 overexpression was investigated in pregnancy and in gestational diabetic endometrial epithelial cell models by immunofluorescent staining, cell migration, and semi quantitative Western blot analysis and glucose uptake assay. We show that SMTNL1 promotes the differentiation of endometrial epithelial cells in a progesterone-dependent manner to attenuate insulin resistance. Furthermore, SMTNL1 hampers the migration capacity of epithelial cells in a gestational diabetes model by inhibiting the expression of MYPT1, the regulatory subunit of MP, and the activity of the holoenzyme, resulting in increased phosphorylation of the 20 kDa regulatory myosin light chain. SMTNL1 also acts as an insulin-sensitizing agent by increasing the gene expression of PP2A and DUPS9 protein phosphatases, resulting in decreased ERK1/2 activity and, hence, decreasing the phosphorylation of IRS-1 at Ser612 under gestational diabetes conditions. SMTNL1 may have therapeutic relevance to the progesterone-dependent inhibition of endometrial epithelial cell migration under hyperglycemic conditions and insulin sensitivity in the endometrium in gestational diabetes or other metabolic disorders.

THE INHIBITORY EFFECTS OF VALPROIC ACID ON RHOA-MEDIATED VASCULAR SMOOTH MUSCLE CELL CONTRACTION

Objective: Valproic acid (VPA) has been used to treat epilepsy and bipolar disorder. Dysfunction of vascular smooth muscle cells (VSMCs) is well-established to contribute to the pathogenesis of various vascular diseases. Growing evidence indicates that increased VSMC contractility plays a primary role in the development of pathological artery spasms. Nevertheless, effect of VPA on VSMC contractility, and its mechanism of action remain unknown. Here, we investigated the mechanism by which VPA inhibits VSMC contractility and vessel contraction in rat VSMCs and endothelium-deprived aortas. Design and method: We performed quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), RhoA activity assay, transfection of constitutively active (CA)-RhoA gene, and western blot analyses. We also conducted ex vivo studies using isolated rat aortas. Results: VPA inhibited phenylephrine (PE)-induced phosphorylation of myosin light chain (MLC) at Ser19 (p-MLC-Ser19) in VSMCs. VPA did not alter expressions of MLC kinase and Rho-associated kinase 1, the upstream kinases responsible for MLC phosphorylation. In addition, VPA did not affect phosphorylation of myosin phosphatase target subunit 1, a regulatory subunit of myosin phosphatase. VPA decreased RhoA mRNA expression and its protein levels in a time- and dose-dependent manner, and as expected, VPA decreased amount of active GTP-bound RhoA in a dose-dependent manner. Ectopic expression of CA-RhoA gene significantly reversed VPA-inhibited p-MLC-Ser19 and p-CPI-17-Thr38. Finally, VPA significantly attenuated PE-induced vessel contraction in endothelium-deprived rat aortas, and similar to the in vitro results, VPA decreased RhoA expression and levels of p-MLC-Ser19 and p-CPI-17-Thr38 in rat aortas. Conclusions: We demonstrate that VPA mitigates PE-induced VSMC contractility and vessel contraction by decreasing expressions of RhoA and p-MLC-Ser19 in rat VSMCs and aortas. These results suggest that VPA may be useful in the treatment of vasospastic diseases including coronary artery vasospasm and post-subarachnoid hemorrhage cerebral vasospasm.

Persistent Müllerian duct syndrome associated with genetic defects in the regulatory subunit of myosin phosphatase.

Can mutations of genes other than AMH or AMHR2, namely PPP1R12A coding myosin phosphatase, lead to persistent Müllerian duct syndrome (PMDS)? The detection of PPP1R12A truncation mutations in five cases of PMDS suggests that myosin phosphatase is involved in Müllerian regression, independently of the anti-Müllerian hormone (AMH) signaling cascade. Mutations of AMH and AMHR2 are detectable in an overwhelming majority of PMDS patients but in 10% of cases, both genes are apparently normal, suggesting that other genes may be involved. DNA samples from 39 PMDS patients collected from 1990 to present, in which Sanger sequencing had failed to detect biallelic AMH or AMHR2 mutations, were screened by massive parallel sequencing. To rule out the possibility that AMH or AMHR2 mutations could have been missed, all DNA samples of good quality were analyzed by targeted next-generation sequencing. Twenty-four samples in which the absence of AMH or AMHR2 biallelic mutations was confirmed were subjected to whole-exome sequencing with the aim of detecting variants of other genes potentially involved in PMDS. Five patients out of 24 (21%) harbored deleterious truncation mutations of PP1R12A, the gene coding for the regulatory subunit of myosin phosphatase, were detected. In addition to PMDS, three of these patients presented with ileal and one with esophageal atresia. The congenital abnormalities associated with PMDS in our patients are consistent with those described in the literature for PPP1R12A variants and have never been described in cases of AMH or AMHR2 mutations. The role of chance is therefore extremely unlikely. The main limitation of the study is the lack of experimental validation of the role of PPP1R12A in Müllerian regression. Only circumstantial evidence is available, myosin phosphatase is required for cell mobility, which plays a major role in Müllerian regression. Alternatively, PPP1R12A mutations could affect the AMH transduction pathway. The study supports the conclusion that failure of Müllerian regression in males is not necessarily associated with a defect in AMH signaling. Extending the scope of molecular analysis should shed light upon the mechanism of the initial steps of male sex differentiation. The study was funded by la Fondation Maladies Rares, GenOmics 2021_0404 and la Fondation pour la Recherche Médicale, grant EQU201903007868. The authors report no conflict of interest. N/A.

Starving a Cell Promotes Airway Smooth Muscle Relaxation: Inhibition of Glycolysis Attenuates Excitation-Contraction Coupling.

Bronchomotor tone modulated by airway smooth muscle shortening represents a key mechanism that increases airway resistance in asthma. Altered glucose metabolism in inflammatory and airway structural cells is associated with asthma. Although these observations suggest a causal link between glucose metabolism and airway hyperresponsiveness, the mechanisms are unclear. We hypothesized that glycolysis modulates excitation-contraction coupling in human airway smooth muscle (HASM) cells. Cultured HASM cells from human lung donors were subject to metabolic screenings using Seahorse XF cell assay. HASM cell monolayers were treated with vehicle or PFK15 (1-(Pyridin-4-yl)-3-(quinolin-2-yl)prop-2-en-1-one), an inhibitor of PFKFB3 (PFK-1,6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3) that generates an allosteric activator for glycolysis rate-limiting enzyme PFK1 (phosphofructokinase 1), for 5-240 minutes, and baseline and agonist-induced phosphorylation of MLC (myosin light chain), MYPT1 (myosin phosphatase regulatory subunit 1), Akt, RhoA, and cytosolic Ca2+ were determined. PFK15 effects on metabolic activity and contractile agonist-induced bronchoconstriction were determined in human precision-cut lung slices. Inhibition of glycolysis attenuated carbachol-induced excitation-contraction coupling in HASM cells. ATP production and bronchodilator-induced cAMP concentrations were also attenuated by glycolysis inhibition in HASM cells. In human small airways, glycolysis inhibition decreased mitochondrial respiration and ATP production and attenuated carbachol-induced bronchoconstriction. The findings suggest that energy depletion resulting from glycolysis inhibition is a novel strategy for ameliorating HASM cell shortening and bronchoprotection of human small airways.

Senescent murine femoral arteries undergo vascular remodelling associated with accelerated stress-induced contractility and reactivity to nitric oxide.

This work explored the mechanism of augmented stress-induced vascular reactivity of senescent murine femoral arteries (FAs). Mechanical and pharmacological reactivity of young (12-25 weeks, y-FA) and senescent (>104 weeks, s-FAs) femoral arteries was measured by wire myography. Expression and protein phosphorylation of selected regulatory proteins were studied by western blotting. Expression ratio of the Exon24 in/out splice isoforms of the regulatory subunit of myosin phosphatase, MYPT1 (MYPT1-Exon24 in/out), was determined by polymerase chain reaction (PCR). While the resting length-tension relationship showed no alteration, the stretch-induced-tone increased to 8.3±0.9mN in s-FA versus only 4.6±0.3mN in y-FAs. Under basal conditions, phosphorylation of the regulatory light chain of myosin at S19 was 19.2 ± 5.8% in y-FA versus 49.2 ± 12.6% in s-FA. Inhibition of endogenous NO release raised tone additionally to 10.4±1.2mN in s-FA, whereas this treatment had a negligible effect in y-FAs (4.8±0.3 mN). In s-FAs, reactivity to NO donor was augmented (pD2 = -4.5 ± 0.3 in y-FA vs. -5.2 ± 0.1 in senescent). Accordingly, in s-FAs, MYPT1-Exon24-out-mRNA, which is responsible for expression of the more sensitive to protein-kinase G, leucine-zipper-positive MYPT1 isoform, was increased. The present work provides evidence that senescent murine s-FA undergoes vascular remodelling associated with increases in stretch-activated contractility and sensitivity to NO/cGMP/PKG system.

Editing of the myosin phosphatase regulatory subunit suppresses angiotensin II induced hypertension via sensitization to nitric oxide mediated vasodilation.

Alternative splicing of exon 24 (E24) of the myosin phosphatase regulatory subunit (Mypt1) tunes smooth muscle sensitivity to NO/cGMP-mediated vasorelaxation and thereby controls blood pressure (BP) in otherwise normal mice. This occurs via the toggling in or out of a C-terminal leucine zipper (LZ) motif required for hetero-dimerization with and activation by cGMP-dependent protein kinase cGK1α. Here we tested the hypothesis that editing (deletion) of E24, by shifting to the LZ positive isoform of Mypt1, would suppress the hypertensive response to angiotensin II (AngII). To test this, mice underwent tamoxifen-inducible and smooth muscle-specific deletion of E24 (E24 cKO) at age 6 weeks followed by a chronic slow-pressor dose of AngII (400 ng/kg/min) plus additional stressors. E24 cKO suppressed the hypertensive response to AngII alone or with the addition of a high salt diet. This effect was not a function of altered salt balance as there were no differences in intake or renal excretion of sodium. This effect was NO dependent as L-NAME in the drinking water caused an exaggerated hypertensive response in the E24cKO mice. E24cKO mouse mesenteric arteries were more sensitive to DEA/NO-induced vasorelaxation and less responsive to AngII- and α-adrenergic-induced vasoconstriction at baseline. Only the latter two effects were still present after 2 weeks of chronic AngII treatment. We conclude that editing of Mypt1 E24, by shifting the expression of naturally occurring isoforms and sensitizing to NO-mediated vasodilation, could be a novel approach to the treatment of human hypertension.

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.

Mypt1 regulates Bmp signaling to promote embryonic exocrine pancreas growth in zebrafish.

Myosin phosphatase targeting subunit 1 (Mypt1) is the regulatory subunit of myosin phosphatase which dephosphorylates the light chain of myosin II to inhibit its contraction. Although biochemical properties of Mypt1 have been characterized in detail, its biological functions in organisms are not well understood. The zebrafish mypt1 sq181 allele was found defective in the ventral pancreatic bud and extrapancreatic duct development, resulting in dysplasia of exocrine pancreas. In mypt1 sq181 mutant, the early growth of the ventral pancreatic bud was initiated but failed to expand due to impaired cell proliferation and increased cell apoptosis. As Mypt1 is essential for cell migration, the loss-of-function of Mypt1 in the mutant disrupted the lateral plate mesoderm migration during gut looping, therefore, altering the Bmp2a expression pattern within it, and eventually leading to impaired Bmp signaling in the adjacent exocrine pancreas. Overexpression of bmp2a could rescue the development of exocrine pancreas, suggesting that the impaired Bmp2a signaling is responsible for the pancreatic development defects. Bmp2a has been reported to promote the early specification of the ventral pancreatic bud, and our study reveals that it continues to serve as a cell proliferation/survival signal to ensure pancreatic bud growth properly in zebrafish.

Notch signaling regulates arterial vasoreactivity through opposing functions of Jagged1 and Dll4 in the vessel wall.

Functional interactions between endothelial cells (ECs) and smooth muscle cells (SMCs) in the arterial wall are necessary for controlling vasoreactivity that underlies vascular resistance and tone. Key signaling pathways converge on the phosphorylation of myosin light chain (p-MLC), the molecular signature of force production in SMCs, through coordinating the relative activities of myosin light chain kinase (MLCK) and myosin phosphatase (MP). Notch signaling in the vessel wall serves critical roles in arterial formation and maturation and has been implicated in arterial vasoregulation. In this report, we hypothesized that Notch signaling through ligands Jagged1 (in SMCs) and delta-like protein-4 (Dll4; in ECs) regulates vasoreactivity via homotypic (SMC-SMC) and heterotypic (EC-SMC) cell interactions. Using ligand induction assays, we demonstrated that Jagged1 selectively induced smooth muscle MLCK gene expression and p-MLC content while inhibiting MP function (i.e., increased Ca2+ sensitization) in a Rho kinase II-dependent manner. Likewise, selective deficiency of smooth muscle Jagged1 in mice resulted in MLCK and p-MLC loss, reduced Ca2+ sensitization, and impaired arterial force generation measured by myography. In contrast, smooth muscle Notch signaling triggered by Dll4 increased expression of MP-targeting subunit 1 (MYPT1; the MP regulatory subunit), whereas arteries from endothelial Dll4-deficient mice featured reduced MYPT1 levels, enhanced force production, and impaired relaxation independent of endothelium-derived nitric oxide signaling. Taken together, this study identifies novel opposing vasoregulatory functions for ligand-specific Notch signaling in the vessel wall, underscoring instructional signaling between ECs and SMCs and suggesting that Notch signals might behave as a "rheostat" in arterial tone control. NEW & NOTEWORTHY The present study unveils novel roles for ligand-specific Notch signaling in arterial function. Smooth muscle Jagged1 and endothelial cell delta-like protein-4 ligands exhibit selective regulation of myosin light chain kinase and myosin phosphatase-targeting subunit 1/myosin phosphatase, respectively, providing a mechanistic link through which Notch signals modulate contractile activities in vascular smooth muscle. These findings may inform vascular derangements observed in human syndromes of Notch signaling deficiency while offering fundamental molecular insights into arterial physiological function.

Abstract 063: Role of Ribosomal S6 Kinase 2 in Vascular Myogenic Tone and Blood Pressure Regulation

Smooth muscle (SM) contractile force is generated through phosphorylation of the myosin regulatory light chain (RLC20) by the myosin light chain kinase. However, emerging evidence also suggests that additional Ser/Thr kinases may contribute to the pathways that regulate SM contractility. Recently, we published that the 90 kDa ribosomal S6 kinase (RSK), extensively studied in cancers of epithelial origin, is a major new player in the regulation of SM contractility. RSKs are unusual kinases having two catalytic domains separated by a linker having multiple phosphorylation sites and are activated by a cascade of ERK phosphorylations and by PDK, which phosphorylates RSK at Ser227 resulting in transition to a fully active enzyme. The thromboxane agonist, U46619 leads to activation of ERK1/2 and PDK1 in SM. We find that 4 th order mesenteric arteries are significantly more dilated at a pressure of 80 mm Hg in Rsk2 -/- mice compared to wild-type (WT) (2.0±0.8 vs 12.8±4.5 %, p <0.05, respectively). The contractile response to dose-dependent phenylephrine stimulation is significantly smaller in the absence of RSK2, p <0.0001 two-way ANOVA, while EC50 for phenylephrine contractions did not differ. Using isolated arcades of mesenteric vessels, phosphorylation of myosin RLC20 and myosin phosphatase regulatory subunit and RSK Ser227 (PDK site) and Thr 577 (ERK site) increased with in 30secs following an increase in intraluminal pressure to 80 mm Hg to induce myogenic tone development. Mean baseline systolic blood pressure (SBP, mmHg) was significantly lower in Rsk2 -/- animals (WT=107.4±1.6 and RSK2 -/- =95.8±2.2, n=23, p< 0.0002). In view of the lower SBP in the Rsk2 -/- animals, we examined the potential role for RSK signaling in the kidney, since RSK2 is known to phosphorylate NHE3. By immunohistochemistry, we show for the first time the expression of RSK2 in human kidney tubules. Lithium excretion, moles/kg/day, was increased in Rsk2-/- mice compared to WT (21.7±3.7 vs 48.6±7.9, respectively), indicative of defective renal Na + reabsorption. Taken together, RSK2 signaling in vascular SM and the kidney is a novel mechanism for the regulation of SM contractility and of BP. RSK2 may be a potential selective target in hypertension.

A splice variant of the myosin phosphatase regulatory subunit tunes arterial reactivity and suppresses response to salt loading.

The cGMP activated kinase cGK1α is targeted to its substrates via leucine zipper (LZ)-mediated heterodimerization and thereby mediates vascular smooth muscle (VSM) relaxation. One target is myosin phosphatase (MP), which when activated by cGK1α results in VSM relaxation even in the presence of activating calcium. Variants of MP regulatory subunit Mypt1 are generated by alternative splicing of the 31 nt exon 24 (E24), which, by changing the reading frame, codes for isoforms that contain or lack the COOH-terminal LZ motif (E24+/LZ-; E24-/LZ+). Expression of these isoforms is vessel specific and developmentally regulated, modulates in disease, and is proposed to confer sensitivity to nitric oxide (NO)/cGMP-mediated vasorelaxation. To test this, mice underwent Tamoxifen-inducible and smooth muscle-specific knockout of E24 (E24 cKO) after weaning. Deletion of a single allele of E24 (shift to Mypt1 LZ+) enhanced vasorelaxation of first-order mesenteric arteries (MA1) to diethylamine-NONOate (DEA/NO) and to cGMP in permeabilized and calcium-clamped arteries and lowered blood pressure. There was no further effect of deletion of both E24 alleles, indicating high sensitivity to shift of Mypt1 isoforms. However, a unique property of MA1s from homozygous E24 cKOs was significantly reduced force generation to α-adrenergic activation. Furthermore 2 wk of high-salt (4% NaCl) diet increased MA1 force generation to phenylephrine in control mice, a response that was markedly suppressed in the E24 cKO homozygotes. Thus Mypt1 E24 splice variants tune arterial reactivity and could be worthy targets for lowering vascular resistance in disease states.

A splice variant of smooth muscle myosin phosphatase regulatory subunit tunes arterial reactivity and suppresses response to salt loading

Nitric oxide (NO) and reactive oxygen species (ROS) through activation of cGMP‐dependent protein kinase (cGK1a) activate smooth muscle Myosin Phosphatase (MP), thereby causing de‐phosphorylation of myosin and vasorelaxation. cGK1a is targeted to the MP regulatory subunit (Mypt1) via Leucine Zipper (LZ)‐mediated hetero‐dimerization. Isoforms of Mypt1 with variable presence of the C‐terminal LZ motif are generated by alternative splicing of the 31 nt Exon24 (E24). We have demonstrated that the expression of these isoforms is vessel‐specific, developmentally regulated and modulates in disease. Based on correlations of expression and function and biochemical assays we have proposed a model in which Mypt1 E24 splicing tunes arterial sensitivity to NO/ROS mediated vasorelaxation. To directly test this model LoxP sites were inserted in introns flanking E24 and bred into smMHCCreER for Tamoxifen‐inducible and smooth muscle‐specific deletion of E24 from Mypt1 (E24 cKO). This converts Mypt1 to the E24‐ splice variant coding for the LZ+ isoform. We generated an allelic series of mice: Cre+ with 0, 1 or 2 floxed alleles and measured blood pressure (BP) by telemetry and arterial function by wire myography. E24 cKO had significantly lower systemic BP with no difference between hetero‐ and homo‐zygotes. There was no effect on the circadian variation in BP. Vasorelaxation of 1st order mesenteric arteries (MA1) to the NO donor DEA/NO, and its 2nd second messenger cGMP in permeabilized and calcium clamped arteries, was markedly enhanced again with no difference between hetero‐ and homo‐zygotes. The only parameter in which these mice differed was a marked suppression of force generation to the a‐adrenergic agonist phenylephrine (PE) in the homozygotes. This was specific to this signaling pathway as there were no changes in force generation to the thromboxane analogue U46619, Angiotensin II, KCl depolarization or calcium. Two weeks of salt loading (4% sodium diet) increased MA1 force generation to PE in wild type mice. This response was abrogated y in the homozygous Mypt1 E24 cKO mice. We conclude that Mypt1 E24 splice variants tune arterial sensitivity to vasodilators such as NO/ROS signaling though cGK1 activation of MP. Targeting of therapies to the contractile apparatus of microvascular smooth muscle may have salutary effects in the treatment of hypertension and other cardiovascular diseases in which increased vascular resistance has a dominant role.Support or Funding InformationNIH R01‐HL066171, 7R37‐HL023081, T32‐HL072751

Unique gene program of rat small resistance mesenteric arteries as revealed by deep RNA sequencing

Deep sequencing of RNA samples from rat small mesenteric arteries (MA) and aorta (AO) identified common and unique features of their gene programs. ∼5% of mRNAs were quantitatively differentially expressed in MA versus AO. Unique transcriptional control in MA smooth muscle is suggested by the selective or enriched expression of transcription factors Nkx2-3, HAND2, and Tcf21 (Capsulin). Enrichment in AO of PPAR transcription factors and their target genes of mitochondrial function, lipid metabolism, and oxidative phosphorylation is consistent with slow (oxidative) tonic smooth muscle. In contrast MA was enriched in contractile and calcium channel mRNAs suggestive of components of fast (glycolytic) phasic smooth muscle. Myosin phosphatase regulatory subunit paralogs Mypt1 and p85 were expressed at similar levels, while smooth muscle MLCK was the only such kinase expressed, suggesting functional redundancy of the former but not the latter in accordance with mouse knockout studies. With regard to vaso-regulatory signals, purinergic receptors P2rx1 and P2rx5 were reciprocally expressed in MA versus AO, while the olfactory receptor Olr59 was enriched in MA. Alox15, which generates the EDHF HPETE, was enriched in MA while eNOS was equally expressed, consistent with the greater role of EDHF in the smaller arteries. mRNAs that were not expressed at a level consistent with impugned function include skeletal myogenic factors, IKK2, nonmuscle myosin, and Gnb3. This screening analysis of gene expression in the small mesenteric resistance arteries suggests testable hypotheses regarding unique aspects of small artery function in the regional control of blood flow.

TRA2β controls Mypt1 exon 24 splicing in the developmental maturation of mouse mesenteric artery smooth muscle.

Diversity of smooth muscle within the vascular system is generated by alternative splicing of exons, yet there is limited understanding of its timing or control mechanisms. We examined splicing of myosin phosphatase regulatory subunit (Mypt1) exon 24 (E24) in relation to smooth muscle myosin heavy chain (Smmhc) and smoothelin (Smtn) alternative exons (Smmhc E6 and Smtn E20) during maturation of mouse mesenteric artery (MA) smooth muscle. The role of transformer 2β (Tra2β), a master regulator of splicing in flies, in maturation of arterial smooth muscle was tested through gene inactivation. Splicing of alternative exons in bladder smooth muscle was examined for comparative purposes. MA smooth muscle maturation began after postnatal week 2 and was complete at maturity, as indicated by switching to Mypt1 E24+ and Smtn E20- splice variants and 11-fold induction of Smmhc. Similar changes in bladder were complete by postnatal day 3. Splicing of Smmhc E6 was temporally dissociated from Mypt1 E24 and Smtn E20 and discordant between arteries and bladder. Tamoxifen-induced smooth muscle-specific inactivation of Tra2β within the first week of life but not in maturity reduced splicing of Mypt1 E24 in MAs. Inactivation of Tra2β causing a switch to the isoform of MYPT1 containing the COOH-terminal leucine zipper motif (E24-) increased arterial sensitivity to cGMP-mediated relaxation. In conclusion, maturation of mouse MA smooth muscle begins postnatally and continues until sexual maturity. TRA2β is required for specification during this period of maturation, and its inactivation alters the contractile properties of mature arterial smooth muscle.

Neural programming of mesenteric and renal arteries.

There is evidence for developmental origins of vascular dysfunction yet little understanding of maturation of vascular smooth muscle (VSM) of regional circulations. We measured maturational changes in expression of myosin phosphatase (MP) and the broader VSM gene program in relation to mesenteric small resistance artery (SRA) function. We then tested the role of the sympathetic nervous system (SNS) in programming of SRAs and used genetically engineered mice to define the role of MP isoforms in the functional maturation of the mesenteric circulation. Maturation of rat mesenteric SRAs as measured by qPCR and immunoblotting begins after the second postnatal week and is not complete until maturity. It is characterized by induction of markers of VSM differentiation (smMHC, γ-, α-actin), CPI-17, an inhibitory subunit of MP and a key target of α-adrenergic vasoconstriction, α1-adrenergic, purinergic X1, and neuropeptide Y1 receptors of sympathetic signaling. Functional correlates include maturational increases in α-adrenergic-mediated force and calcium sensitization of force production (MP inhibition) measured in first-order mesenteric arteries ex vivo. The MP regulatory subunit Mypt1 E24+/LZ- isoform is specifically upregulated in SRAs during maturation. Conditional deletion of mouse Mypt1 E24 demonstrates that splicing of E24 causes the maturational reduction in sensitivity to cGMP-mediated vasorelaxation (MP activation). Neonatal chemical sympathectomy (6-hydroxydopamine) suppresses maturation of SRAs with minimal effect on a conduit artery. Mechanical denervation of the mature rat renal artery causes a reversion to the immature gene program. We conclude that the SNS captures control of the mesenteric circulation by programming maturation of the SRA smooth muscle.

Increased Activation of the Rho-A/Rho-Kinase Pathway in the Renal Vascular System Is Responsible for the Enhanced Reactivity to Exogenous Vasopressin in Endotoxemic Rats*

We evaluated the role of the renal vascular system and the Rho-A/Rho-kinase pathway in the maintenance of the pressor effects of vasopressin in endotoxemic rats. In vitro and in vivo animal study. University research laboratory. Male Wistar rats (200-300 g). Rats received either saline or lipopolysaccharide (10 mg/kg, intraperitoneal) 6 or 24 hours before the experiments. The effects of vasopressin on isolated aortic rings, cardiac function, mean arterial pressure, and both the renal vascular perfusion pressure of perfused kidneys in vitro and renal blood flow in situ were evaluated. The role of Rho-kinase in the renal and systemic effects of vasopressin was investigated through administration of the selective inhibitor Y-27632 and Western blot analysis. The effect of vasopressin on mean arterial pressure was unaltered and that on renal vascular perfusion pressure enhanced in endotoxemic rats at both 6 and 24 hours after lipopolysaccharide, despite reduced contractile responses in aortic rings and the lack of effect on cardiac function. Vasopressin (3, 10, and 30 pmol/kg, IV) produced increased reduction in renal blood flow in endotoxemic rats. In perfused kidneys from lipopolysaccharide groups, administration of Y-27632 reverted the hyperreactivity to vasopressin. Treatment with Y-27632 partially inhibited the effects of vasopressin on mean arterial pressure and significantly reduced the effects of vasopressin on renal blood flow in control but not in endotoxemic rats. Although the protein levels of Rho-A and Rho-kinase I and II had not been impaired, the levels of phosphorylated myosin phosphatase-targeting subunit 1, the regulatory subunit of myosin phosphatase that is inhibited by Rho-kinase, were increased in both the renal cortex and the renal medulla of endotoxemic rats. Our data suggest that activation of Rho-kinase potentiates the vascular effects of vasopressin in the kidneys, contributing to the maintenance of the hypertensive effects of this agent during septic shock.

Pix-1 controls early elongation in parallel with mel-11 and let-502 in Caenorhabditis elegans.

Cell shape changes are crucial for metazoan development. During Caenorhabditis elegans embryogenesis, epidermal cell shape changes transform ovoid embryos into vermiform larvae. This process is divided into two phases: early and late elongation. Early elongation involves the contraction of filamentous actin bundles by phosphorylated non-muscle myosin in a subset of epidermal (hypodermal) cells. The genes controlling early elongation are associated with two parallel pathways. The first one involves the rho-1/RHOA-specific effector let-502/Rho-kinase and mel-11/myosin phosphatase regulatory subunit. The second pathway involves the CDC42/RAC-specific effector pak-1. Late elongation is driven by mechanotransduction in ventral and dorsal hypodermal cells in response to body-wall muscle contractions, and involves the CDC42/RAC-specific Guanine-nucleotide Exchange Factor (GEF) pix-1, the GTPase ced-10/RAC and pak-1.In this study, pix-1 is shown to control early elongation in parallel with let-502/mel-11, as previously shown for pak-1. We show that pix-1, pak-1 and let-502 control the rate of elongation, and the antero-posterior morphology of the embryos. In particular, pix-1 and pak-1 are shown to control head, but not tail width, while let-502 controls both head and tail width. This suggests that let-502 function is required throughout the antero-posterior axis of the embryo during early elongation, while pix-1/pak-1 function may be mostly required in the anterior part of the embryo. Supporting this hypothesis we show that low pix-1 expression level in the dorsal-posterior hypodermal cells is required to ensure high elongation rate during early elongation.

The minus‐end actin capping protein, UNC‐94/tropomodulin, regulates development of the Caenorhabditis elegans intestine

Tropomodulins are actin-capping proteins that regulate the stability of the slow-growing, minus-ends of actin filaments. The C. elegans tropomodulin homolog, UNC-94, has sequence and functional similarity to vertebrate tropomodulins. We investigated the role of UNC-94 in C. elegans intestinal morphogenesis. In the embryonic C. elegans intestine, UNC-94 localizes to the terminal web, an actin- and intermediate filament-rich structure that underlies the apical membrane. Loss of UNC-94 function results in areas of flattened intestinal lumen. In worms homozygous for the strong loss-of-function allele, unc-94(tm724), the terminal web is thinner and the amount of F-actin is reduced, pointing to a role for UNC-94 in regulating the structure of the terminal web. The non-muscle myosin, NMY-1, also localizes to the terminal web, and we present evidence that increasing actomyosin contractility by depleting the myosin phosphatase regulatory subunit, mel-11, can rescue the flattened lumen phenotype of unc-94 mutants. The data support a model in which minus-end actin capping by UNC-94 promotes proper F-actin structure and contraction in the terminal web, yielding proper shape of the intestinal lumen. This establishes a new role for a tropomodulin in regulating lumen shape during tubulogenesis.

Microvascular Smooth Muscle Gene Program is Highly Sensitive to Blood Flow

Microvascular smooth muscle (MVSM) is phenotypically and functionally distinct from that of the large arteries yet the deterministic input signals are not known. We use Myosin Phosphatase as a model to address this question as it is a primary mediator of vasomotor responses. Isoforms of the myosin phosphatase regulatory subunit (Mypt1) are generated by alternative splicing of exon 24 (E24) and determine MVSM responses to NO/cGMP. Mesenteric artery MVSM expresses the E24+ (fast) isoform; large arteries express the E24‐ (slow) isoform. Ligation of the downstream MA2s causes switching of the upstream MA1s to the E24‐ isoform suggesting the hypothesis that the unique (fast) contractile phenotype of MVSM is determined by blood flow. To test this we developed a model of reduced perfusion pressure in which placement of an occluder around the abdominal aorta allows for transient reductions in blood flow within the rat mesenteric arcade. Brief (20 & 60min) and repetitive (60min; 5days) perfusion pressure reduction via the balloon occluder causes a switch in the splicing of E24 in Mypt1 of mesenteric arteries from the E24+ to E24‐ isoform 6 hours after pressure reduction in both brief (20% E24+ vs 80% control; p<0.05) and repetitive (45% E24+; p<0.05) protocols. Given these effects of reduced perfusion pressure we propose that the unique properties of the MVSM are highly dependent upon the pulsatile blood flow generated by the heart