scholarly journals The Role of Actin Filament Dynamics in the Myogenic Response of Cerebral Resistance Arteries

2012 ◽  
Vol 33 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Michael P Walsh ◽  
William C Cole
Keyword(s):  
Light Chain ◽  
Actin Polymerization ◽  
Myosin Light Chain ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Intraluminal Pressure ◽  
Regulatory Subunit ◽  
Myogenic Response ◽  
Force Transmission ◽  
Resistance Arteries

The myogenic response has a critical role in regulation of blood flow to the brain. Increased intraluminal pressure elicits vasoconstriction, whereas decreased intraluminal pressure induces vasodilatation, thereby maintaining flow constant over the normal physiologic blood pressure range. Improved understanding of the molecular mechanisms underlying the myogenic response is crucial to identify deficiencies with pathologic consequences, such as cerebral vasospasm, hypertension, and stroke, and to identify potential therapeutic targets. Three mechanisms have been suggested to be involved in the myogenic response: (1) membrane depolarization, which induces Ca2+ entry, activation of myosin light chain kinase, phosphorylation of the myosin regulatory light chains (LC20), increased actomyosin MgATPase activity, cross-bridge cycling, and vasoconstriction; (2) activation of the RhoA/Rho-associated kinase (ROCK) pathway, leading to inhibition of myosin light chain phosphatase by phosphorylation of MYPT1, the myosin targeting regulatory subunit of the phosphatase, and increased LC20 phosphorylation; and (3) activation of the ROCK and protein kinase C pathways, leading to actin polymerization and the formation of enhanced connections between the actin cytoskeleton, plasma membrane, and extracellular matrix to augment force transmission. This review describes these three mechanisms, emphasizing recent developments regarding the importance of dynamic actin polymerization in the myogenic response of the cerebral vasculature.

scholarly journals Abnormal myosin phosphatase targeting subunit 1 phosphorylation and actin polymerization contribute to impaired myogenic regulation of cerebral arterial diameter in the type 2 diabetic Goto-Kakizaki rat

2016 ◽  
Vol 37 (1) ◽  
pp. 227-240 ◽  
Author(s):  
Khaled S Abd-Elrahman ◽  
Olaia Colinas ◽  
Emma J Walsh ◽  
Hai-Lei Zhu ◽  
Christine M Campbell ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Light Chain ◽  
Actin Polymerization ◽  
Myosin Light Chain ◽  
Intraluminal Pressure ◽  
Myogenic Response ◽  
Myosin Phosphatase ◽  
Myosin Light Chain Phosphatase ◽  
Myogenic Regulation

The myogenic response of cerebral resistance arterial smooth muscle to intraluminal pressure elevation is a key physiological mechanism regulating blood flow to the brain. Rho-associated kinase plays a critical role in the myogenic response by activating Ca2+ sensitization mechanisms: (i) Rho-associated kinase inhibits myosin light chain phosphatase by phosphorylating its targeting subunit myosin phosphatase targeting subunit 1 (at T855), augmenting 20 kDa myosin regulatory light chain (LC20) phosphorylation and force generation; and (ii) Rho-associated kinase stimulates cytoskeletal actin polymerization, enhancing force transmission to the cell membrane. Here, we tested the hypothesis that abnormal Rho-associated kinase-mediated myosin light chain phosphatase regulation underlies the dysfunctional cerebral myogenic response of the Goto-Kakizaki rat model of type 2 diabetes. Basal levels of myogenic tone, LC20, and MYPT1-T855 phosphorylation were elevated and G-actin content was reduced in arteries of pre-diabetic 8–10 weeks Goto-Kakizaki rats with normal serum insulin and glucose levels. Pressure-dependent myogenic constriction, LC20, and myosin phosphatase targeting subunit 1 phosphorylation and actin polymerization were suppressed in both pre-diabetic Goto-Kakizaki and diabetic (18–20 weeks) Goto-Kakizaki rats, whereas RhoA, ROK2, and MYPT1 expression were unaffected. We conclude that abnormal Rho-associated kinase-mediated Ca2+ sensitization contributes to the dysfunctional cerebral myogenic response in the Goto-Kakizaki model of type 2 diabetes.

scholarly journals Increased degradation of MYPT1 contributes to the development of tolerance to nitric oxide in porcine pulmonary artery

2010 ◽  
Vol 299 (1) ◽  
pp. L117-L123 ◽  
Author(s):  
Huijuan Ma ◽  
Qiong He ◽  
Dou Dou ◽  
Xiaoxu Zheng ◽  
Lei Ying ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Pulmonary Artery ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Leucine Zipper ◽  
Critical Role ◽  
Pulmonary Arteries ◽  
Regulatory Subunit ◽  
Inhibitor Of Protein Synthesis ◽  
Development Of Tolerance

Myosin phosphatase target subunit 1 (MYPT1) is the regulatory subunit of myosin light chain phosphatase (MLCP). It plays a critical role in vasodilatation induced by cGMP-elevating agents such as nitric oxide (NO). The present study was performed to determine the role of MYPT1 in the development of tolerance of the pulmonary artery to NO. Incubation of isolated porcine pulmonary arteries for 24 or 48 h with DETA NONOate (DETA NO) significantly reduced protein levels of MYPT1 and the leucine zipper-positive (LZ+) isoform of MYPT1 but not that of PP1cδ. The extent of reduction in total MYPT1 protein level was comparable to that of MYPT1 (LZ+). The decrease in MYPT1 protein caused by 48-h DETA NO incubation was prevented by ODQ, an inhibitor of guanylyl cyclase, and by inhibitors of proteasomes (MG-132 and lactacystin) but was not affected by the inhibitor of protein synthesis, cycloheximide. A reduction in MYPT1 protein was also obtained with 8-bromo-cGMP, but this was prevented by Rp-8-bromo-PET-cGMP [inhibitor of cGMP-dependent protein kinase (PKG)]. Incubation for 48 h with DETA NO also reduced dephosphorylation of myosin light chain and relaxation of the artery in response to DETA NO, which was prevented by MG-132. These results suggest that the reduction in MYPT1 protein contributes to the development of tolerance of pulmonary arteries to NO. This may result from increased degradation of MYPT1 after prolonged PKG activation.

scholarly journals A Novel Regulatory Mechanism of Myosin Light Chain Phosphorylation via Binding of 14-3-3 to Myosin Phosphatase

2008 ◽  
Vol 19 (3) ◽  
pp. 1062-1071 ◽  
Author(s):  
Yasuhiko Koga ◽  
Mitsuo Ikebe
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Regulatory Mechanism ◽  
Kinase Inhibitor ◽  
Myosin Ii ◽  
Critical Role ◽  
Rho Kinase ◽  
Myosin Phosphatase ◽  
Dependent Cell ◽  
Direct Binding

Myosin II phosphorylation–dependent cell motile events are regulated by myosin light-chain (MLC) kinase and MLC phosphatase (MLCP). Recent studies have revealed myosin phosphatase targeting subunit (MYPT1), a myosin-binding subunit of MLCP, plays a critical role in MLCP regulation. Here we report the new regulatory mechanism of MLCP via the interaction between 14-3-3 and MYPT1. The binding of 14-3-3β to MYPT1 diminished the direct binding between MYPT1 and myosin II, and 14-3-3β overexpression abolished MYPT1 localization at stress fiber. Furthermore, 14-3-3β inhibited MLCP holoenzyme activity via the interaction with MYPT1. Consistently, 14-3-3β overexpression increased myosin II phosphorylation in cells. We found that MYPT1 phosphorylation at Ser472 was critical for the binding to 14-3-3. Epidermal growth factor (EGF) stimulation increased both Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase inhibitor inhibited the EGF-induced Ser472 phosphorylation and the binding of MYPT1-14-3-3. Rho-kinase specific siRNA also decreased EGF-induced Ser472 phosphorylation correlated with the decrease in MLC phosphorylation. The present study revealed a new RhoA/Rho-kinase–dependent regulatory mechanism of myosin II phosphorylation by 14-3-3 that dissociates MLCP from myosin II and attenuates MLCP activity.

Abstract P780: Myosin Light Chain Dephosphorylation by Ppp1r12c Promotes Atrial Hypocontractility in Atrial Fibrillation

Stroke ◽  
2021 ◽  
Vol 52 (Suppl_1) ◽  
Author(s):  
Francisco J Gonzalez-Gonzalez ◽  
Perike Srikanth ◽  
Andrielle E Capote ◽  
Alsina Katherina M ◽  
Benjamin Levin ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Molecular Mechanisms ◽  
Contractile Function ◽  
Right Atrial ◽  
Western Blots

Atrial fibrillation (AF) is the most common sustained arrhythmia, with an estimated prevalence in the U.S.of 6.1 million. AF increases the risk of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. We have recently identified protein phosphatase 1 subunit 12c (PPP1R12C) as a key molecule targeting myosin light-chain phosphorylation in AF. Objective: We hypothesize that the overexpression of PPP1R12C causes hypophosphorylation of atrial myosin light-chain 2 (MLC2a), thereby decreasing atrial contractility in AF. Methods and Results: Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluate the role of the PP1c-PPP1R12C interaction in MLC2a de-phosphorylation, we utilized Western blots, co-immunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A), PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion: The overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, associated with a reduction in atrial contractility and an increase in AF inducibility. All these discoveries suggest that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

Abstract 70: Protein Phosphatase 1 Contributes to Atrial Stunning in Atrial Fibrillation

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Srikanth Perike ◽  
Xander Wehrens ◽  
Dawood Darbar ◽  
Mark McCauley
Keyword(s):  
Atrial Fibrillation ◽  
Light Chain ◽  
Protein Phosphatase ◽  
Myosin Light Chain ◽  
Stroke Risk ◽  
Protein Phosphatase 1 ◽  
Regulatory Subunit ◽  
Future Studies ◽  
Hek Cells

Background: Atrial fibrillation (AF) is the most common cardiac arrhythmia, and increases a patient’s stroke risk five-fold. Reduced atrial contractility (stunning) is observed in AF and contributes to stroke risk; however, the mechanisms responsible for atrial stunning in AF are unknown. Recent data from our laboratory indicate that protein phosphatase 1 (PP1) dephosphorylation of myosin light chain 2a (MLC2a) may contribute to atrial stunning in AF. Objective: To determine how the PP1 regulatory subunit 12C (PPP1R12C) and catalytic (PPP1c) subunits modify atrial sarcomere phosphorylation in AF. Methods: We evaluated the protein expression, binding and phosphorylation among PPP1R12C, PPP1c, and MLC2a in transfected HL-1 cells, murine atrial tissue (Pitx2null +/– mice, with a genetic predisposition AF), and in HEK cells. An inhibitor of PPP1R12C phosphorylation, BDP5290, was used to enhance the PPP1R12C-PPP1C interaction. Results: In Pitx2 null +/– mice, PPP1R12C was increased by 2-fold ( P <0.01) and associated with a 40% reduction in S-19-MLC2a phosphorylation versus WT mice ( P <0.058). BDP5290 increased PPP1R12C-PPP1C binding by >3-fold in HL-1 cells ( P <0.01). BDP5290 reduced MLC2a phosphorylation by 40% through an enhanced interaction with PPP1R12C by >3-fold in HEK cells ( P <0.01). Conclusion: In Pitx2 null+/- mice, increased expression of PPP1R12C is associated with PP1 holoenzyme targeting to sarcomeric MLC2a, and is associated with reduced S19-MLC2a phosphorylation. Additionally, BDP5290 enhances the PPP1R12C-PPP1C interaction and models PP1 activity in AF. Future studies will examine the effects of both AF and BDP5290 upon atrial contractility in vitro.

Abstract P458: Myosin Light Chain Dephosphorylation By Ppp1r12c Promotes Atrial Hypocontractility In Atrial Fibrillation

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Francisco J Gonzalez-Gonzalez ◽  
Srikanth Perike ◽  
Frederick Damen ◽  
Andrielle Capote ◽  
Katherina M Alsina ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Molecular Mechanisms ◽  
Contractile Function ◽  
Right Atrial ◽  
Western Blots

Introduction: Atrial fibrillation (AF), is the most common sustained arrhythmia, with an estimated prevalence in the U.S. of 2.7 million to 6.1 million and is predictive to increase to 12.1 million in 2030. AF increases the chances of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. Objective: The overexpression of PPP1R12C, causes hypophosphorylation of atrial myosin light chain 2 (MLC2a), decreasing atrial contractility. Methods and Results: Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluated the role of PP1c-PPP1R12C interaction in MLC2a de-phosphorylation we used Western blots, coimmunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A) PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion: The Overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, that cause a reduction in atrial contractility and increases AF inducibility. All these discoveries advocate that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

Abstract WMP39: Protein Phosphatase 1 Regulatory Subunit 12C Contributes to Atrial Myosin Light Chain Dephosphorylation in Atrial Fibrillation

Stroke ◽  
2020 ◽  
Vol 51 (Suppl_1) ◽  
Author(s):  
Srikanth Perike ◽  
Katherina M Alsina ◽  
Arvind Sridhar ◽  
Dawood Darbar ◽  
Xander Wehrens ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Light Chain ◽  
Protein Phosphatase ◽  
Myosin Light Chain ◽  
Stroke Risk ◽  
Contractile Function ◽  
Protein Phosphatase 1 ◽  
Regulatory Subunit ◽  
Western Blots ◽  
Atrial Myosin

Background: Atrial fibrillation (AF) increases stroke risk five-fold. Atrial hypocontractility from atrial myosin light chain (MLC2a) dephosphorylation contributes to stroke risk in AF. Recent proteomic data has shown increased protein phosphatase 1 subunit 12C (PPP1R12C) targeting to MLC2a in AF. However, it is unclear whether PPP1R12C causes MLC2a dephosphorylation in AF. Objective: Determine whether increased PPP1R12C expression causes MLC2a dephosphorylation and increases AF risk. Methods: Western blots and co-IPs were performed to evaluate the relationship among PPP1R12C, PP1c and MLC2a in human atrial tissues (AF vs SR). Mice with either a knockout (KO) or lentiviral (LV) cardiac overexpression of PPP1R12C were evaluated with invasive EP studies for AF inducibility vs WT controls. Results: In human AF, PPP1R12C was increased 4-fold ( P <0.005, n=6) with an 88% reduction in S-19-MLC2a phosphorylation ( P <0.05, n=4). PPP1R12C-PP1c and PPP1R12C-MLC2a binding was increased 2-fold in AF ( P <0.05, n=6). AF burden in LV-12C mice increased nearly tenfold vs. KO and WT mice ( P <0.05, n=6). Conclusion: In human AF, increased PPP1R12C expression is associated with reduced P-MLC2a through enhanced binding with the PP1c catalytic subunit. This dephosphorylation is a likely contributor to atrial hypocontractility and stroke risk in AF. Additionally, increased PPP1R12C expression in mice increases AF risk. Future studies will examine the effects of increased PPP1R12C expression upon atrial contractile function in mice.

scholarly journals Capn4 Enhances Osteopontin Expression through Activation of the Wnt/β-Catenin Pathway to Promote Epithelial Ovarian Carcinoma Metastasis

2017 ◽  
Vol 42 (1) ◽  
pp. 185-197 ◽  
Author(s):  
Xiaoming Yang ◽  
Jing Sun ◽  
Dandan Xia ◽  
Xupei Can ◽  
Lei Liu ◽  
...  
Keyword(s):  
Ovarian Carcinoma ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Western Blotting ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Epithelial Ovarian Carcinoma ◽  
Regulatory Subunit ◽  
Epithelial Tissue

Background and Aim: Increasing evidence shows that the calpain regulatory subunit Capn4 can modulate the proliferation and metastasis of cancer cells, and plays an important role in the development of malignant tumors. However, there is no information on the clinical significance of Capn4 in epithelial ovarian carcinoma (EOC) or the molecular mechanisms by which Capn4 promotes the growth and metastasis of EOC. Therefore, the aim of this study was to clarify the role of Capn4 in EOC. Methods: We evaluated Capn4 and osteopontin (OPN) expression in EOC cell lines and tissues from patients with ovarian cancer by western blotting and immunohistochemical analysis. We then created cell lines with downregulated and upregulated Capn4 expression, using Capn4-targeting small interfering RNA and a pcDNA3.1-Capn4 overexpression vector, respectively, to investigate its function in EOC in vitro. In addition, we investigated the potential mechanism underlying the function of Capn4 by examining the effect of modifying Capn4 expression on Wnt/β-catenin signaling pathway-related genes by western blotting. Results: Capn4 was overexpressed in clinical EOC tissues compared with that in normal ovarian epithelial tissue, and was associated with poor clinical outcomes. Upon silencing or overexpressing Capn4 in EOC cells, we concluded that Capn4 promotes cell proliferation and migration in vitro. Furthermore, Capn4 promoted EOC metastasis by interacting with the Wnt/β-catenin signaling pathway to upregulate OPN expression. Conclusion: Our study indicates that Capn4 plays a critical role in the progression and metastasis of EOC, and could be a potential therapeutic target for EOC management.

scholarly journals L-Type Calcium Channels Play a Critical Role in Maintaining Lens Transparency by Regulating Phosphorylation of Aquaporin-0 and Myosin Light Chain and Expression of Connexins

PLoS ONE ◽  
2013 ◽  
Vol 8 (5) ◽  
pp. e64676 ◽  
Author(s):  
Rupalatha Maddala ◽  
Tharkika Nagendran ◽  
Gustaaf G. de Ridder ◽  
Kevin L. Schey ◽  
Ponugoti Vasantha Rao
Keyword(s):  
Calcium Channels ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Critical Role ◽  
Lens Transparency
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