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 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.

Novel role of the protein phosphatase 1 regulatory subunit PPP1R3A in atrial fibrillation

2018 ◽  
Vol 124 ◽  
pp. 108
Author(s):  
Katherina Alsina ◽  
Mohit Hulsurkar ◽  
Chunxia Yao ◽  
Barbara Langer ◽  
David Chiang ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Protein Phosphatase ◽  
Protein Phosphatase 1 ◽  
Regulatory Subunit

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 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.

scholarly journals TIMAP inhibits endothelial myosin light chain phosphatase by competing with MYPT1 for the catalytic protein phosphatase 1 subunit PP1cβ

2019 ◽  
Vol 294 (36) ◽  
pp. 13280-13291
Author(s):  
Xin Wang ◽  
Marya Obeidat ◽  
Laiji Li ◽  
Phuwadet Pasarj ◽  
Salah Aburahess ◽  
...  
Keyword(s):  
Light Chain ◽  
Protein Phosphatase ◽  
Myosin Light Chain ◽  
Protein Phosphatase 1 ◽  
Myosin Light Chain Phosphatase

scholarly journals Nuclear organisation of NIPP1, a regulatory subunit of protein phosphatase 1 that associates with pre-mRNA splicing factors

1999 ◽  
Vol 112 (2) ◽  
pp. 157-168 ◽  
Author(s):  
L. Trinkle-Mulcahy ◽  
P. Ajuh ◽  
A. Prescott ◽  
F. Claverie-Martin ◽  
S. Cohen ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Mrna Splicing ◽  
Dominant Negative ◽  
Protein Phosphatase 1 ◽  
Regulatory Subunit ◽  
Dominant Negative Mutant ◽  
Splicing Factors ◽  
Nuclear Extracts ◽  
Nuclear Organisation

Protein phosphatase-1 (PP1) is complexed to many proteins that target it to particular subcellular locations and regulate its activity. Here, we show that ‘nuclear inhibitor of PP1’ (NIPP1), a major nuclear PP1-binding protein, shows a speckled nucleoplasmic distribution where it is colocalised with pre-mRNA splicing factors. One of these factors (Sm) is also shown to be complexed to NIPP1 in nuclear extracts. Immunodepletion of NIPP1 from nuclear extracts, or addition of a ‘dominant negative’ mutant lacking a functional PP1 binding site, greatly reduces pre-mRNA splicing activity in vitro. These findings implicate the NIPP1-PP1 complex in the control of pre-mRNA splicing.

scholarly journals Loss of Protein Phosphatase 1 Regulatory Subunit PPP1R3A Promotes Atrial Fibrillation

Circulation ◽  
2019 ◽  
Vol 140 (8) ◽  
pp. 681-693 ◽  
Author(s):  
Katherina M. Alsina ◽  
Mohit Hulsurkar ◽  
Sören Brandenburg ◽  
Daniel Kownatzki-Danger ◽  
Christof Lenz ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Endoplasmic Reticulum ◽  
Knockout Mice ◽  
Protein Phosphatase ◽  
Calcium Atpase ◽  
Stimulated Emission ◽  
Protein Phosphatase 1 ◽  
Regulatory Subunit ◽  
Wild Type ◽  
Endoplasmic Reticulum Calcium

Background: Abnormal calcium (Ca 2+ ) release from the sarcoplasmic reticulum (SR) contributes to the pathogenesis of atrial fibrillation (AF). Increased phosphorylation of 2 proteins essential for normal SR-Ca 2+ cycling, the type-2 ryanodine receptor (RyR2) and phospholamban (PLN), enhances the susceptibility to AF, but the underlying mechanisms remain unclear. Protein phosphatase 1 (PP1) limits steady-state phosphorylation of both RyR2 and PLN. Proteomic analysis uncovered a novel PP1-regulatory subunit (PPP1R3A [PP1 regulatory subunit type 3A]) in the RyR2 macromolecular channel complex that has been previously shown to mediate PP1 targeting to PLN. We tested the hypothesis that reduced PPP1R3A levels contribute to AF pathogenesis by reducing PP1 binding to both RyR2 and PLN. Methods: Immunoprecipitation, mass spectrometry, and complexome profiling were performed from the atrial tissue of patients with AF and from cardiac lysates of wild-type and Pln -knockout mice. Ppp1r3a -knockout mice were generated by CRISPR-mediated deletion of exons 2 to 3. Ppp1r3a -knockout mice and wild-type littermates were subjected to in vivo programmed electrical stimulation to determine AF susceptibility. Isolated atrial cardiomyocytes were used for Stimulated Emission Depletion superresolution microscopy and confocal Ca 2+ imaging. Results: Proteomics identified the PP1-regulatory subunit PPP1R3A as a novel RyR2-binding partner, and coimmunoprecipitation confirmed PPP1R3A binding to RyR2 and PLN. Complexome profiling and Stimulated Emission Depletion imaging revealed that PLN is present in the PPP1R3A-RyR2 interaction, suggesting the existence of a previously unknown SR nanodomain composed of both RyR2 and PLN/sarco/endoplasmic reticulum calcium ATPase-2a macromolecular complexes. This novel RyR2/PLN/sarco/endoplasmic reticulum calcium ATPase-2a complex was also identified in human atria. Genetic ablation of Ppp1r3a in mice impaired binding of PP1 to both RyR2 and PLN. Reduced PP1 targeting was associated with increased phosphorylation of RyR2 and PLN, aberrant SR-Ca 2+ release in atrial cardiomyocytes, and enhanced susceptibility to pacing-induced AF. Finally, PPP1R3A was progressively downregulated in the atria of patients with paroxysmal and persistent (chronic) AF. Conclusions: PPP1R3A is a novel PP1-regulatory subunit within the RyR2 channel complex. Reduced PPP1R3A levels impair PP1 targeting and increase phosphorylation of both RyR2 and PLN. PPP1R3A deficiency promotes abnormal SR-Ca 2+ release and increases AF susceptibility in mice. Given that PPP1R3A is downregulated in patients with AF, this regulatory subunit may represent a new target for AF therapeutic strategies.

scholarly journals The Evolution of Duplicated Genes of the Cpi-17/Phi-1 (ppp1r14) Family of Protein Phosphatase 1 Inhibitors in Teleosts

2020 ◽  
Vol 21 (16) ◽  
pp. 5709
Author(s):  
Irene Lang ◽  
Guneet Virk ◽  
Dale C. Zheng ◽  
Jason Young ◽  
Michael J. Nguyen ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Phosphatase ◽  
Smooth Muscle Contraction ◽  
Cellular Systems ◽  
Protein Phosphatase 1 ◽  
Regulatory Subunit ◽  
Myosin Phosphatase ◽  
Phosphatase Inhibitors ◽  
Regulatory Myosin Light Chain

The Cpi-17 (ppp1r14) gene family is an evolutionarily conserved, vertebrate specific group of protein phosphatase 1 (PP1) inhibitors. When phosphorylated, Cpi-17 is a potent inhibitor of myosin phosphatase (MP), a holoenzyme complex of the regulatory subunit Mypt1 and the catalytic subunit PP1. Myosin phosphatase dephosphorylates the regulatory myosin light chain (Mlc2) and promotes actomyosin relaxation, which in turn, regulates numerous cellular processes including smooth muscle contraction, cytokinesis, cell motility, and tumor cell invasion. We analyzed zebrafish homologs of the Cpi-17 family, to better understand the mechanisms of myosin phosphatase regulation. We found single homologs of both Kepi (ppp1r14c) and Gbpi (ppp1r14d) in silico, but we detected no expression of these genes during early embryonic development. Cpi-17 (ppp1r14a) and Phi-1 (ppp1r14b) each had two duplicate paralogs, (ppp1r14aa and ppp1r14ab) and (ppp1r14ba and ppp1r14bb), which were each expressed during early development. The spatial expression pattern of these genes has diverged, with ppp1r14aa and ppp1r14bb expressed primarily in smooth muscle and skeletal muscle, respectively, while ppp1r14ab and ppp1r14ba are primarily expressed in neural tissue. We observed that, in in vitro and heterologous cellular systems, the Cpi-17 paralogs both acted as potent myosin phosphatase inhibitors, and were indistinguishable from one another. In contrast, the two Phi-1 paralogs displayed weak myosin phosphatase inhibitory activity in vitro, and did not alter myosin phosphorylation in cells. Through deletion and chimeric analysis, we identified that the difference in specificity for myosin phosphatase between Cpi-17 and Phi-1 was encoded by the highly conserved PHIN (phosphatase holoenzyme inhibitory) domain, and not the more divergent N- and C- termini. We also showed that either Cpi-17 paralog can rescue the knockdown phenotype, but neither Phi-1 paralog could do so. Thus, we provide new evidence about the biochemical and developmental distinctions of the zebrafish Cpi-17 protein family.

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