Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase G

Autores
Gonano, Luis Alberto; Aitken-Buck, Hamish M.; Chakraborty, Akash D.; Worthington, Luke P. I.; Cully, Tanya R.; Lamberts, Regis R.; Vila Petroff, Martín Gerardo; Jones, Peter P.
Año de publicación
2022
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Background: The cGMP-dependent protein kinase G (PKG) phosphorylates the cardiac ryanodine receptor (RyR2) in vitro. We aimed to determine whether modulation of endogenous PKG alters RyR2-mediated spontaneous Ca²⁺ release and whether this effect is linked to a change in RyR2 phosphorylation. Methods: & Results: Human embryonic kidney (HEK293) cells with inducible RyR2 expression were treated with the cGMP analogue 8-Br-cGMP (100 μM) to activate endogenous PKG. In cells transfected with luminal Ca²⁺ sensor, D1ER, PKG activation significantly reduced the threshold for RyR2-mediated spontaneous Ca²⁺ release (93.9 ± 0.4% of store size with vehicle vs. 91.7 ± 0.8% with 8-Br-cGMP, P = 0.04). Mutation of the proposed PKG phosphorylation sites, S2808 and S2030, either individually or as a combination, prevented the decrease in Ca²⁺ release threshold induced by endogenous PKG activation. Interestingly, despite a functional dependence on expression of RyR2 phosphorylation sites, 8-Br-cGMP activation of PKG did not promote a detectable change in S2808 phosphorylation (P = 0.9). Paradoxically, pharmacological inhibition of PKG with KT 5823 (1 μM) also reduced the threshold for spontaneous Ca²⁺ release through RyR2 without affecting S2808 phosphorylation. Silencing RNA knockdown of endogenous PKG expression also had no quantifiable effect on RyR2 S2808 phosphorylation (P = 0.9). However, unlike PKG inhibition with KT 5823, PKG knockdown did not alter spontaneous Ca²⁺ release propensity or luminal Ca²⁺ handling. Conclusion: In an intact cell model, activation of endogenous PKG reduces the threshold for RyR2-mediated spontaneous Ca²⁺ release in a manner dependent on the RyR2 phosphorylation sites S2808 and S2030. This study clarifies the regulation of RyR2 Ca²⁺ release by endogenous PKG and functionally implicates the role of RyR2 phosphorylation.
Facultad de Ciencias Médicas
Centro de Investigaciones Cardiovasculares
Materia
Medicina
Protein kinase G
Cardiac ryanodine receptor
Phosphorylation
Calcium
Store overload-induced calcium release
KT 5823
Nivel de accesibilidad
acceso abierto
Condiciones de uso
http://creativecommons.org/licenses/by-nc-nd/4.0/
Repositorio
SEDICI (UNLP)
Institución
Universidad Nacional de La Plata
OAI Identificador
oai:sedici.unlp.edu.ar:10915/154468

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repository_id_str 1329
network_name_str SEDICI (UNLP)
spelling Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase GGonano, Luis AlbertoAitken-Buck, Hamish M.Chakraborty, Akash D.Worthington, Luke P. I.Cully, Tanya R.Lamberts, Regis R.Vila Petroff, Martín GerardoJones, Peter P.MedicinaProtein kinase GCardiac ryanodine receptorPhosphorylationCalciumStore overload-induced calcium releaseKT 5823Background: The cGMP-dependent protein kinase G (PKG) phosphorylates the cardiac ryanodine receptor (RyR2) in vitro. We aimed to determine whether modulation of endogenous PKG alters RyR2-mediated spontaneous Ca²⁺ release and whether this effect is linked to a change in RyR2 phosphorylation. Methods: & Results: Human embryonic kidney (HEK293) cells with inducible RyR2 expression were treated with the cGMP analogue 8-Br-cGMP (100 μM) to activate endogenous PKG. In cells transfected with luminal Ca²⁺ sensor, D1ER, PKG activation significantly reduced the threshold for RyR2-mediated spontaneous Ca²⁺ release (93.9 ± 0.4% of store size with vehicle vs. 91.7 ± 0.8% with 8-Br-cGMP, P = 0.04). Mutation of the proposed PKG phosphorylation sites, S2808 and S2030, either individually or as a combination, prevented the decrease in Ca²⁺ release threshold induced by endogenous PKG activation. Interestingly, despite a functional dependence on expression of RyR2 phosphorylation sites, 8-Br-cGMP activation of PKG did not promote a detectable change in S2808 phosphorylation (P = 0.9). Paradoxically, pharmacological inhibition of PKG with KT 5823 (1 μM) also reduced the threshold for spontaneous Ca²⁺ release through RyR2 without affecting S2808 phosphorylation. Silencing RNA knockdown of endogenous PKG expression also had no quantifiable effect on RyR2 S2808 phosphorylation (P = 0.9). However, unlike PKG inhibition with KT 5823, PKG knockdown did not alter spontaneous Ca²⁺ release propensity or luminal Ca²⁺ handling. Conclusion: In an intact cell model, activation of endogenous PKG reduces the threshold for RyR2-mediated spontaneous Ca²⁺ release in a manner dependent on the RyR2 phosphorylation sites S2808 and S2030. This study clarifies the regulation of RyR2 Ca²⁺ release by endogenous PKG and functionally implicates the role of RyR2 phosphorylation.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculares2022info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionArticulohttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdf171-178http://sedici.unlp.edu.ar/handle/10915/154468enginfo:eu-repo/semantics/altIdentifier/issn/2665-9441info:eu-repo/semantics/altIdentifier/doi/10.1016/j.crphys.2022.03.004info:eu-repo/semantics/openAccesshttp://creativecommons.org/licenses/by-nc-nd/4.0/Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)reponame:SEDICI (UNLP)instname:Universidad Nacional de La Platainstacron:UNLP2025-09-29T11:40:03Zoai:sedici.unlp.edu.ar:10915/154468Institucionalhttp://sedici.unlp.edu.ar/Universidad públicaNo correspondehttp://sedici.unlp.edu.ar/oai/snrdalira@sedici.unlp.edu.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:13292025-09-29 11:40:03.391SEDICI (UNLP) - Universidad Nacional de La Platafalse
dc.title.none.fl_str_mv Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase G
title Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase G
spellingShingle Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase G
Gonano, Luis Alberto
Medicina
Protein kinase G
Cardiac ryanodine receptor
Phosphorylation
Calcium
Store overload-induced calcium release
KT 5823
title_short Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase G
title_full Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase G
title_fullStr Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase G
title_full_unstemmed Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase G
title_sort Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase G
dc.creator.none.fl_str_mv Gonano, Luis Alberto
Aitken-Buck, Hamish M.
Chakraborty, Akash D.
Worthington, Luke P. I.
Cully, Tanya R.
Lamberts, Regis R.
Vila Petroff, Martín Gerardo
Jones, Peter P.
author Gonano, Luis Alberto
author_facet Gonano, Luis Alberto
Aitken-Buck, Hamish M.
Chakraborty, Akash D.
Worthington, Luke P. I.
Cully, Tanya R.
Lamberts, Regis R.
Vila Petroff, Martín Gerardo
Jones, Peter P.
author_role author
author2 Aitken-Buck, Hamish M.
Chakraborty, Akash D.
Worthington, Luke P. I.
Cully, Tanya R.
Lamberts, Regis R.
Vila Petroff, Martín Gerardo
Jones, Peter P.
author2_role author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Medicina
Protein kinase G
Cardiac ryanodine receptor
Phosphorylation
Calcium
Store overload-induced calcium release
KT 5823
topic Medicina
Protein kinase G
Cardiac ryanodine receptor
Phosphorylation
Calcium
Store overload-induced calcium release
KT 5823
dc.description.none.fl_txt_mv Background: The cGMP-dependent protein kinase G (PKG) phosphorylates the cardiac ryanodine receptor (RyR2) in vitro. We aimed to determine whether modulation of endogenous PKG alters RyR2-mediated spontaneous Ca²⁺ release and whether this effect is linked to a change in RyR2 phosphorylation. Methods: & Results: Human embryonic kidney (HEK293) cells with inducible RyR2 expression were treated with the cGMP analogue 8-Br-cGMP (100 μM) to activate endogenous PKG. In cells transfected with luminal Ca²⁺ sensor, D1ER, PKG activation significantly reduced the threshold for RyR2-mediated spontaneous Ca²⁺ release (93.9 ± 0.4% of store size with vehicle vs. 91.7 ± 0.8% with 8-Br-cGMP, P = 0.04). Mutation of the proposed PKG phosphorylation sites, S2808 and S2030, either individually or as a combination, prevented the decrease in Ca²⁺ release threshold induced by endogenous PKG activation. Interestingly, despite a functional dependence on expression of RyR2 phosphorylation sites, 8-Br-cGMP activation of PKG did not promote a detectable change in S2808 phosphorylation (P = 0.9). Paradoxically, pharmacological inhibition of PKG with KT 5823 (1 μM) also reduced the threshold for spontaneous Ca²⁺ release through RyR2 without affecting S2808 phosphorylation. Silencing RNA knockdown of endogenous PKG expression also had no quantifiable effect on RyR2 S2808 phosphorylation (P = 0.9). However, unlike PKG inhibition with KT 5823, PKG knockdown did not alter spontaneous Ca²⁺ release propensity or luminal Ca²⁺ handling. Conclusion: In an intact cell model, activation of endogenous PKG reduces the threshold for RyR2-mediated spontaneous Ca²⁺ release in a manner dependent on the RyR2 phosphorylation sites S2808 and S2030. This study clarifies the regulation of RyR2 Ca²⁺ release by endogenous PKG and functionally implicates the role of RyR2 phosphorylation.
Facultad de Ciencias Médicas
Centro de Investigaciones Cardiovasculares
description Background: The cGMP-dependent protein kinase G (PKG) phosphorylates the cardiac ryanodine receptor (RyR2) in vitro. We aimed to determine whether modulation of endogenous PKG alters RyR2-mediated spontaneous Ca²⁺ release and whether this effect is linked to a change in RyR2 phosphorylation. Methods: & Results: Human embryonic kidney (HEK293) cells with inducible RyR2 expression were treated with the cGMP analogue 8-Br-cGMP (100 μM) to activate endogenous PKG. In cells transfected with luminal Ca²⁺ sensor, D1ER, PKG activation significantly reduced the threshold for RyR2-mediated spontaneous Ca²⁺ release (93.9 ± 0.4% of store size with vehicle vs. 91.7 ± 0.8% with 8-Br-cGMP, P = 0.04). Mutation of the proposed PKG phosphorylation sites, S2808 and S2030, either individually or as a combination, prevented the decrease in Ca²⁺ release threshold induced by endogenous PKG activation. Interestingly, despite a functional dependence on expression of RyR2 phosphorylation sites, 8-Br-cGMP activation of PKG did not promote a detectable change in S2808 phosphorylation (P = 0.9). Paradoxically, pharmacological inhibition of PKG with KT 5823 (1 μM) also reduced the threshold for spontaneous Ca²⁺ release through RyR2 without affecting S2808 phosphorylation. Silencing RNA knockdown of endogenous PKG expression also had no quantifiable effect on RyR2 S2808 phosphorylation (P = 0.9). However, unlike PKG inhibition with KT 5823, PKG knockdown did not alter spontaneous Ca²⁺ release propensity or luminal Ca²⁺ handling. Conclusion: In an intact cell model, activation of endogenous PKG reduces the threshold for RyR2-mediated spontaneous Ca²⁺ release in a manner dependent on the RyR2 phosphorylation sites S2808 and S2030. This study clarifies the regulation of RyR2 Ca²⁺ release by endogenous PKG and functionally implicates the role of RyR2 phosphorylation.
publishDate 2022
dc.date.none.fl_str_mv 2022
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
Articulo
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://sedici.unlp.edu.ar/handle/10915/154468
url http://sedici.unlp.edu.ar/handle/10915/154468
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/issn/2665-9441
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.crphys.2022.03.004
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
http://creativecommons.org/licenses/by-nc-nd/4.0/
Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)
eu_rights_str_mv openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by-nc-nd/4.0/
Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)
dc.format.none.fl_str_mv application/pdf
171-178
dc.source.none.fl_str_mv reponame:SEDICI (UNLP)
instname:Universidad Nacional de La Plata
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