Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase G
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. Method...
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2022
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| Acceso en línea: | http://sedici.unlp.edu.ar/handle/10915/154468 |
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I19-R120-10915-1544682023-06-16T20:07:38Z http://sedici.unlp.edu.ar/handle/10915/154468 issn:2665-9441 Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase G 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. 2022 2023-06-16T18:12:09Z en Medicina Protein kinase G Cardiac ryanodine receptor Phosphorylation Calcium Store overload-induced calcium release KT 5823 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 Articulo Articulo http://creativecommons.org/licenses/by-nc-nd/4.0/ Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) application/pdf 171-178 |
| institution |
Universidad Nacional de La Plata |
| institution_str |
I-19 |
| repository_str |
R-120 |
| collection |
SEDICI (UNLP) |
| language |
Inglés |
| topic |
Medicina Protein kinase G Cardiac ryanodine receptor Phosphorylation Calcium Store overload-induced calcium release KT 5823 |
| spellingShingle |
Medicina Protein kinase G Cardiac ryanodine receptor Phosphorylation Calcium Store overload-induced calcium release KT 5823 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. Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase G |
| topic_facet |
Medicina Protein kinase G Cardiac ryanodine receptor Phosphorylation Calcium Store overload-induced calcium release KT 5823 |
| 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. |
| format |
Articulo Articulo |
| author |
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_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_sort |
Gonano, Luis Alberto |
| title |
Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase G |
| 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 |
| publishDate |
2022 |
| url |
http://sedici.unlp.edu.ar/handle/10915/154468 |
| work_keys_str_mv |
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