scholarly journals Smooth muscle-selective CPI-17 expression increases vascular smooth muscle contraction and blood pressure

2013 ◽  
Vol 305 (1) ◽  
pp. H104-H113 ◽  
Author(s):  
Wen Su ◽  
Zhongwen Xie ◽  
Shu Liu ◽  
Lindsay E. Calderon ◽  
Zhenheng Guo ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Protein Kinase ◽  
Vascular Smooth Muscle ◽  
Mesenteric Arteries ◽  
Myosin Phosphatase ◽  
Inhibitory Protein ◽  
Muscle Contractility ◽  
Smooth Muscle Contractility ◽  
High Potassium

Recent data revealed that protein kinase C-potentiated myosin phosphatase inhibitor of 17 kDa (CPI-17), a myosin phosphatase inhibitory protein preferentially expressed in smooth muscle, is upregulated/activated in several diseases but whether this CPI-17 increase plays a causal role in pathologically enhanced vascular smooth muscle contractility and blood pressure remains unclear. To address this possibility, we generated a smooth muscle-specific CPI-17 transgenic mouse model (CPI-17-Tg) and demonstrated that the CPI-17 transgene was selectively expressed in smooth muscle-enriched tissues, including mesenteric arteries. The isometric contractions in the isolated second-order branch of mesenteric artery helical strips from CPI-17-Tg mice were significantly enhanced compared with controls in response to phenylephrine, U-46619, serotonin, ANG II, high potassium, and calcium. The perfusion pressure increases in isolated perfused mesenteric vascular beds in response to norepinephrine were also enhanced in CPI-17-Tg mice. The hypercontractility was associated with increased phosphorylation of CPI-17 and 20-kDa myosin light chain under basal and stimulated conditions. Surprisingly, the protein levels of rho kinase 2 and protein kinase Cα/δ were significantly increased in CPI-17-Tg mouse mesenteric arteries. Radiotelemetry measurements demonstrated that blood pressure was significantly increased in CPI-17-Tg mice. However, no vascular remodeling was detected by morphometric analysis. Taken together, our results demonstrate that increased CPI-17 expression in smooth muscle promotes vascular smooth muscle contractility and increases blood pressure, implicating a pathological significant role of CPI-17 upregulation.

The regulation of smooth muscle contractility by zipper-interacting protein kinaseThis paper is one of a selection of papers published in this Special Issue, entitled Young Investigators' Forum.

2007 ◽  
Vol 85 (1) ◽  
pp. 79-87 ◽  
Author(s):  
Eikichi Ihara ◽  
Justin A. MacDonald
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Specific Inhibitor ◽  
Inhibitory Protein ◽  
Muscle Contractility ◽  
Smooth Muscle Contractility ◽  
Independent Manner ◽  
Interacting Protein

Smooth muscle contractility is mainly regulated by phosphorylation of the 20 kDa myosin light chains (LC20), a process that is controlled by the opposing activities of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Recently, intensive research has revealed that various protein kinase networks including Rho-kinase, integrin-linked kinase, zipper-interacting protein kinase (ZIPK), and protein kinase C (PKC) are involved in the regulation of LC20 phosphorylation and have important roles in modulating smooth muscle contractile responses to Ca2+ (i.e., Ca2+ sensitization and Ca2+ desensitization). Here, we review the general background and structure of ZIPK and summarize our current understanding of its involvement in a number of cell processes including cell death (apoptosis), cell motility, and smooth muscle contraction. ZIPK has been found to induce the diphosphorylation of LC20 at Ser-19 and Thr-18 in a Ca2+-independent manner and to regulate MLCP activity directly through its phosphorylation of the myosin-targeting subunit of MLCP or indirectly through its phosphorylation of the PKC-potentiated inhibitory protein of MLCP. Future investigations of ZIPK function in smooth muscle will undoubtably focus on determining the mechanisms that regulate its cellular activity, including the identification of upstream signaling pathways, the characterization of autoinhibitory domains and regulatory phosphorylation sites, and the development of specific inhibitor compounds.

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

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.

Smoothelin-B is essential for functional vascular smooth muscle contractility

2006 ◽  
Vol 45 (3) ◽  
pp. e104-e105
Author(s):  
P. Niessen ◽  
S. Rensen ◽  
J. van Deursen ◽  
M. Gijbels ◽  
E. Heijman ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Muscle Contractility ◽  
Smooth Muscle Contractility

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

scholarly journals Vascular smooth muscle cell contractile protein expression is increased through protein kinase G-dependent and -independent pathways by glucose-6-phosphate dehydrogenase inhibition and deficiency

2016 ◽  
Vol 311 (4) ◽  
pp. H904-H912 ◽  
Author(s):  
Sukrutha Chettimada ◽  
Sachindra Raj Joshi ◽  
Vidhi Dhagia ◽  
Alessandro Aiezza ◽  
Thomas M. Lincoln ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Protein Kinase ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cell ◽  
Muscle Cell ◽  
Vascular Smooth Muscle Cell ◽  
Contractile Proteins ◽  
Protein Kinase G ◽  
Glucose 6 Phosphate Dehydrogenase

Homeostatic control of vascular smooth muscle cell (VSMC) differentiation is critical for contractile activity and regulation of blood flow. Recently, we reported that precontracted blood vessels are relaxed and the phenotype of VSMC is regulated from a synthetic to contractile state by glucose-6-phosphate dehydrogenase (G6PD) inhibition. In the current study, we investigated whether the increase in the expression of VSMC contractile proteins by inhibition and knockdown of G6PD is mediated through a protein kinase G (PKG)-dependent pathway and whether it regulates blood pressure. We found that the expression of VSMC-restricted contractile proteins, myocardin (MYOCD), and miR-1 and miR-143 are increased by G6PD inhibition or knockdown. Importantly, RNA-sequence analysis of aortic tissue from G6PD-deficient mice revealed uniform increases in VSMC-restricted genes, particularly those regulated by the MYOCD-serum response factor (SRF) switch. Conversely, expression of Krüppel-like factor 4 (KLF4) is decreased by G6PD inhibition. Interestingly, the G6PD inhibition-induced expression of miR-1 and contractile proteins was blocked by Rp-β-phenyl-1, N2-etheno-8-bromo-guanosine-3′,5′-cyclic monophosphorothioate, a PKG inhibitor. On the other hand, MYOCD and miR-143 levels are increased by G6PD inhibition through a PKG-independent manner. Furthermore, blood pressure was lower in the G6PD-deficient compared with wild-type mice. Therefore, our results suggest that the expression of VSMC contractile proteins induced by G6PD inhibition occurs via PKG1α-dependent and -independent pathways.

Sign in / Sign up

Export Citation Format

Share Document