Calcium/calmodulin-dependent protein kinase II regulation of IKs during sustained β-adrenergic receptor stimulation

Tyler Shugg, Derrick E. Johnson, Minghai Shao, Xianyin Lai, Frank Witzmann, Theodore Cummins, Michael Rubart-von der Lohe, Andy Hudmon, Brian R. Overholser

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Background: Sustained β-adrenergic receptor (β-AR) stimulation causes pathophysiological changes during heart failure (HF), including inhibition of the slow component of the delayed rectifier potassium current (IKs). Aberrant calcium handling, including increased activation of calcium/calmodulin-dependent protein kinase II (CaMKII), contributes to arrhythmia development during HF. Objective: The purpose of this study was to investigate CaMKII regulation of KCNQ1 (pore-forming subunit of IKs) during sustained β-AR stimulation and associated functional implications on IKs. Methods: KCNQ1 phosphorylation was assessed using liquid chromatography-tandem mass spectrometry after sustained β-AR stimulation with isoproterenol (ISO). Peptide fragments corresponding to KCNQ1 residues were synthesized to identify CaMKII phosphorylation at the identified sites. Dephosphorylated (alanine) and phosphorylated (aspartic acid) mimics were introduced at identified residues. Whole-cell, voltage-clamp experiments were performed in human endothelial kidney 293 cells coexpressing wild-type or mutant KCNQ1 and KCNE1 (auxiliary subunit) during ISO treatment or lentiviral δCaMKII overexpression. Results: Novel KCNQ1 carboxy-terminal sites were identified with enhanced phosphorylation during sustained β-AR stimulation at T482 and S484. S484 peptides demonstrated the strongest δCaMKII phosphorylation. Sustained β-AR stimulation reduced IKs activation (P =.02 vs control) similar to the phosphorylated mimic (P =.62 vs sustained β-AR). Individual phosphorylated mimics at S484 (P =.04) but not at T482 (P =.17) reduced IKs function. Treatment with CN21 (CaMKII inhibitor) reversed the reductions in IKs vs CN21-Alanine control (P <.01). δCaMKII overexpression reduced IKs similar to ISO treatment in wild type (P <.01) but not in the dephosphorylated S484 mimic (P =.99). Conclusion: CaMKII regulates KCNQ1 at S484 during sustained β-AR stimulation to inhibit IKs. The ability of CaMKII to inhibit IKs may contribute to arrhythmogenicity during HF.

Original languageEnglish (US)
Pages (from-to)895-904
Number of pages10
JournalHeart Rhythm
Volume15
Issue number6
DOIs
StatePublished - Jun 1 2018

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Calcium-Calmodulin-Dependent Protein Kinase Type 2
Calcium-Calmodulin-Dependent Protein Kinases
Adrenergic Receptors
Isoproterenol
Phosphorylation
Heart Failure
Alanine
Peptide Fragments
Protein Kinase Inhibitors
Tandem Mass Spectrometry
Aspartic Acid
Liquid Chromatography
Cardiac Arrhythmias
Potassium
Calcium
Kidney

Keywords

  • Calcium/calmodulin-dependent protein kinase II
  • CaMKII
  • Delayed rectifier
  • Heart failure
  • I
  • KCNQ1
  • β-Adrenergic receptor

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)

Cite this

Calcium/calmodulin-dependent protein kinase II regulation of IKs during sustained β-adrenergic receptor stimulation. / Shugg, Tyler; Johnson, Derrick E.; Shao, Minghai; Lai, Xianyin; Witzmann, Frank; Cummins, Theodore; Rubart-von der Lohe, Michael; Hudmon, Andy; Overholser, Brian R.

In: Heart Rhythm, Vol. 15, No. 6, 01.06.2018, p. 895-904.

Research output: Contribution to journalArticle

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abstract = "Background: Sustained β-adrenergic receptor (β-AR) stimulation causes pathophysiological changes during heart failure (HF), including inhibition of the slow component of the delayed rectifier potassium current (IKs). Aberrant calcium handling, including increased activation of calcium/calmodulin-dependent protein kinase II (CaMKII), contributes to arrhythmia development during HF. Objective: The purpose of this study was to investigate CaMKII regulation of KCNQ1 (pore-forming subunit of IKs) during sustained β-AR stimulation and associated functional implications on IKs. Methods: KCNQ1 phosphorylation was assessed using liquid chromatography-tandem mass spectrometry after sustained β-AR stimulation with isoproterenol (ISO). Peptide fragments corresponding to KCNQ1 residues were synthesized to identify CaMKII phosphorylation at the identified sites. Dephosphorylated (alanine) and phosphorylated (aspartic acid) mimics were introduced at identified residues. Whole-cell, voltage-clamp experiments were performed in human endothelial kidney 293 cells coexpressing wild-type or mutant KCNQ1 and KCNE1 (auxiliary subunit) during ISO treatment or lentiviral δCaMKII overexpression. Results: Novel KCNQ1 carboxy-terminal sites were identified with enhanced phosphorylation during sustained β-AR stimulation at T482 and S484. S484 peptides demonstrated the strongest δCaMKII phosphorylation. Sustained β-AR stimulation reduced IKs activation (P =.02 vs control) similar to the phosphorylated mimic (P =.62 vs sustained β-AR). Individual phosphorylated mimics at S484 (P =.04) but not at T482 (P =.17) reduced IKs function. Treatment with CN21 (CaMKII inhibitor) reversed the reductions in IKs vs CN21-Alanine control (P <.01). δCaMKII overexpression reduced IKs similar to ISO treatment in wild type (P <.01) but not in the dephosphorylated S484 mimic (P =.99). Conclusion: CaMKII regulates KCNQ1 at S484 during sustained β-AR stimulation to inhibit IKs. The ability of CaMKII to inhibit IKs may contribute to arrhythmogenicity during HF.",
keywords = "Calcium/calmodulin-dependent protein kinase II, CaMKII, Delayed rectifier, Heart failure, I, KCNQ1, β-Adrenergic receptor",
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T1 - Calcium/calmodulin-dependent protein kinase II regulation of IKs during sustained β-adrenergic receptor stimulation

AU - Shugg, Tyler

AU - Johnson, Derrick E.

AU - Shao, Minghai

AU - Lai, Xianyin

AU - Witzmann, Frank

AU - Cummins, Theodore

AU - Rubart-von der Lohe, Michael

AU - Hudmon, Andy

AU - Overholser, Brian R.

PY - 2018/6/1

Y1 - 2018/6/1

N2 - Background: Sustained β-adrenergic receptor (β-AR) stimulation causes pathophysiological changes during heart failure (HF), including inhibition of the slow component of the delayed rectifier potassium current (IKs). Aberrant calcium handling, including increased activation of calcium/calmodulin-dependent protein kinase II (CaMKII), contributes to arrhythmia development during HF. Objective: The purpose of this study was to investigate CaMKII regulation of KCNQ1 (pore-forming subunit of IKs) during sustained β-AR stimulation and associated functional implications on IKs. Methods: KCNQ1 phosphorylation was assessed using liquid chromatography-tandem mass spectrometry after sustained β-AR stimulation with isoproterenol (ISO). Peptide fragments corresponding to KCNQ1 residues were synthesized to identify CaMKII phosphorylation at the identified sites. Dephosphorylated (alanine) and phosphorylated (aspartic acid) mimics were introduced at identified residues. Whole-cell, voltage-clamp experiments were performed in human endothelial kidney 293 cells coexpressing wild-type or mutant KCNQ1 and KCNE1 (auxiliary subunit) during ISO treatment or lentiviral δCaMKII overexpression. Results: Novel KCNQ1 carboxy-terminal sites were identified with enhanced phosphorylation during sustained β-AR stimulation at T482 and S484. S484 peptides demonstrated the strongest δCaMKII phosphorylation. Sustained β-AR stimulation reduced IKs activation (P =.02 vs control) similar to the phosphorylated mimic (P =.62 vs sustained β-AR). Individual phosphorylated mimics at S484 (P =.04) but not at T482 (P =.17) reduced IKs function. Treatment with CN21 (CaMKII inhibitor) reversed the reductions in IKs vs CN21-Alanine control (P <.01). δCaMKII overexpression reduced IKs similar to ISO treatment in wild type (P <.01) but not in the dephosphorylated S484 mimic (P =.99). Conclusion: CaMKII regulates KCNQ1 at S484 during sustained β-AR stimulation to inhibit IKs. The ability of CaMKII to inhibit IKs may contribute to arrhythmogenicity during HF.

AB - Background: Sustained β-adrenergic receptor (β-AR) stimulation causes pathophysiological changes during heart failure (HF), including inhibition of the slow component of the delayed rectifier potassium current (IKs). Aberrant calcium handling, including increased activation of calcium/calmodulin-dependent protein kinase II (CaMKII), contributes to arrhythmia development during HF. Objective: The purpose of this study was to investigate CaMKII regulation of KCNQ1 (pore-forming subunit of IKs) during sustained β-AR stimulation and associated functional implications on IKs. Methods: KCNQ1 phosphorylation was assessed using liquid chromatography-tandem mass spectrometry after sustained β-AR stimulation with isoproterenol (ISO). Peptide fragments corresponding to KCNQ1 residues were synthesized to identify CaMKII phosphorylation at the identified sites. Dephosphorylated (alanine) and phosphorylated (aspartic acid) mimics were introduced at identified residues. Whole-cell, voltage-clamp experiments were performed in human endothelial kidney 293 cells coexpressing wild-type or mutant KCNQ1 and KCNE1 (auxiliary subunit) during ISO treatment or lentiviral δCaMKII overexpression. Results: Novel KCNQ1 carboxy-terminal sites were identified with enhanced phosphorylation during sustained β-AR stimulation at T482 and S484. S484 peptides demonstrated the strongest δCaMKII phosphorylation. Sustained β-AR stimulation reduced IKs activation (P =.02 vs control) similar to the phosphorylated mimic (P =.62 vs sustained β-AR). Individual phosphorylated mimics at S484 (P =.04) but not at T482 (P =.17) reduced IKs function. Treatment with CN21 (CaMKII inhibitor) reversed the reductions in IKs vs CN21-Alanine control (P <.01). δCaMKII overexpression reduced IKs similar to ISO treatment in wild type (P <.01) but not in the dephosphorylated S484 mimic (P =.99). Conclusion: CaMKII regulates KCNQ1 at S484 during sustained β-AR stimulation to inhibit IKs. The ability of CaMKII to inhibit IKs may contribute to arrhythmogenicity during HF.

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KW - CaMKII

KW - Delayed rectifier

KW - Heart failure

KW - I

KW - KCNQ1

KW - β-Adrenergic receptor

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