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Chronic loss of inhibitor-1 diminishes cardiac RyR2 phosphorylation despite exaggerated CaMKII activity

Abstract

Inhibitor-1 (I-1) modulates protein phosphatase 1 (PP1) activity and thereby counteracts the phosphorylation by kinases. I-1 is downregulated and deactivated in failing hearts, but whether its role is beneficial or detrimental remains controversial, and opposing therapeutic strategies have been proposed. Overactivity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) with hyperphosphorylation of ryanodine receptors (RyR2) at the CaMKII-site is recognized to be central for heart failure and arrhythmias. Using an I-1-deficient mouse line as well as transfected cell lines, we investigated the effects of acute and chronic modulation of I-1 on CaMKII activity and RyR2 phosphorylation. We demonstrate that I-1 acutely modulates CaMKII by regulating PP1 activity. However, while ablation of I-1 should thus limit CaMKII-activation, we unexpectedly found exaggerated CaMKII-activation under β-adrenergic stress upon chronic loss of I-1 in knockout mice. We unraveled that this is due to chronic upregulation of the exchange protein activated by cAMP (EPAC) leading to augmented CaMKII activation, and using computational modeling validated that an increase in EPAC expression can indeed explain our experimental findings. Interestingly, at the level of RyR2, the increase in PP1 activity more than outweighed the increase in CaMKII activity, resulting in reduced RyR phosphorylation at Ser-2814. Exaggerated CaMKII activation due to counterregulatory mechanisms upon loss of I-1 is an important caveat with respect to suggested therapeutic I-1-inhibition, as CaMKII overactivity has been heavily implicated in several cardiac pathologies.

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Abbreviations

CaMKII:

Ca2+/calmodulin-dependent protein kinase II

EPAC:

Exchange factor directly activated by cAMP

I-1:

Protein phosphatase type-1 inhibitor 1

I-1c:

Constitutive active I-1

KO:

Knockout

NRCM:

neonatal rat cardiomyocytes

PKA:

Protein kinase A

PLB:

Phospholamban

PP1:

Protein phosphatase type-1

PP2a:

Protein phosphatase type-2a

RyR2:

Ryanodine receptor type 2

SERCA2a:

Sarco/endoplasmic reticulum Ca2+-ATPase isotype 2a

SR:

Sarcoplasmic reticulum

WT:

Wild type

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Acknowledgements

This work was supported by Regensburg Medical Faculty intramural funding (ReForM B-grant, to S.N.), German Centre for Cardiovascular Research (DZHK) (DZHK B 15-014 Extern, to S.N.), the German Research Foundation (DFG) (grants TP A02 SFB 1002, EL 270/5-1 and EL 270/7-1 to A.E.A.; grants MA 1982/4-2 and TP A03 SFB 1002, to L.S.M.; grant WA 2586/4-1, to M.W.), by the Fondation Leducq (Alliance for Calmodulin Kinase II Signaling in Heart Failure and Arrhythmias, to L.S.M., and European-North American Atrial Fibrillation Research Alliance (grant 07CVD03), to D.D.), by the German Federal Ministry of Education and Research (DZHK (German Center for Cardiovascular Research), to D.D., L.S.M. and A.E.A.), and by the Netherlands Organization for Health Research and Development (grant ZonMW Veni 91616057, to J.H.).

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Correspondence to Ali El-Armouche.

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Neef, S., Heijman, J., Otte, K. et al. Chronic loss of inhibitor-1 diminishes cardiac RyR2 phosphorylation despite exaggerated CaMKII activity. Naunyn-Schmiedeberg's Arch Pharmacol 390, 857–862 (2017). https://doi.org/10.1007/s00210-017-1376-1

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Keywords

  • Inhibitor-1
  • Ca2+/calmodulin-dependent protein kinase II
  • Protein phosphatase 1
  • Ryanodine receptor