Differential protein kinase c isoform regulation and increased constitutive activity of acetylcholine-regulated potassium channels in atrial remodeling

Samy Makary, Niels Voigt, Ange Maguy, Reza Wakili, Kunihiro Nishida, Masahide Harada, Dobromir Dobrev, Stanley Nattel

Research output: Contribution to journalArticle

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Abstract

Rationale Atrial fibrillation (AF) causes atrial-tachycardia remodeling (ATR), with enhanced constitutive acetylcholine-regulated K+ current (IKAChC) contributing to action potential duration shortening and AF promotion. The underlying mechanisms are unknown. Objective: To evaluate the role of protein-kinase C (PKC) isoforms in ATR-induced IKAChC activation. Methods and Results: Cells from ATR-dogs (400-bpm atrial pacing for 1 week) were compared to control dog cells. In vitro tachypaced (TP; 3 Hz) canine atrial cardiomyocytes were compared to parallel 1-Hz paced cells. I KAChC single-channel activity was assessed in cell-attached and cell-free (inside-out) patches. Protein expression was assessed by immunoblot. In vitro TP activated IKAChC, mimicking effects of in vivo ATR. Discrepant effects of PKC activation and inhibition between control and ATR cells suggested isoform-selective effects and altered PKC isoform distribution. Conventional PKC isoforms (cPKC; including PKCα) inhibited, whereas novel isoforms (including PKCε) enhanced, acetylcholine-regulated K current (IKACh) in inside-out patches. TP and ATR downregulated PKCα (by 33% and 37%, respectively) and caused membrane translocation of PKCε, switching PKC predominance to the stimulatory novel isoform. TP increased [Ca2+]i at 2 hours by 30%, with return to baseline at 24 hours. Buffering [Ca 2+]i during TP with the cell-permeable Ca2+ chelator BAPTA-AM (1 μmol/L) or inhibiting the Ca-dependent protease calpain with PD150606 (20 μmol/L) prevented PKCα downregulation and TP enhancement of IKAChC. PKCε inhibition with a cell-permeable peptide inhibitor suppressed TP/ATR-induced IKAChC activation, whereas cPKC inhibition enhanced IKAChC activity in 1-Hz cells. Conclusions: PKC isoforms differentially modulate IKACh, with conventional Ca-dependent isoforms inhibiting and novel isoforms enhancing activity. ATR causes a rate-dependent PKC isoform switch, with Ca/calpain-dependent downregulation of inhibitory PKCα and membrane translocation of stimulatory PKCε, enhancing I KAChC. These findings provide novel insights into mechanisms underlying IKAChC dysregulation in AF.

Original languageEnglish
Pages (from-to)1031-1043
Number of pages13
JournalCirculation Research
Volume109
Issue number9
DOIs
Publication statusPublished - 14-10-2011

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Atrial Remodeling
Potassium Channels
Protein Kinases
Protein Kinase C
Acetylcholine
Protein Isoforms
Tachycardia
Atrial Fibrillation
Calpain
Down-Regulation
splenotritin
Dogs

All Science Journal Classification (ASJC) codes

  • Physiology
  • Cardiology and Cardiovascular Medicine

Cite this

Makary, Samy ; Voigt, Niels ; Maguy, Ange ; Wakili, Reza ; Nishida, Kunihiro ; Harada, Masahide ; Dobrev, Dobromir ; Nattel, Stanley. / Differential protein kinase c isoform regulation and increased constitutive activity of acetylcholine-regulated potassium channels in atrial remodeling. In: Circulation Research. 2011 ; Vol. 109, No. 9. pp. 1031-1043.
@article{0c32c5ff611946958c0b6b5a67cf22ac,
title = "Differential protein kinase c isoform regulation and increased constitutive activity of acetylcholine-regulated potassium channels in atrial remodeling",
abstract = "Rationale Atrial fibrillation (AF) causes atrial-tachycardia remodeling (ATR), with enhanced constitutive acetylcholine-regulated K+ current (IKAChC) contributing to action potential duration shortening and AF promotion. The underlying mechanisms are unknown. Objective: To evaluate the role of protein-kinase C (PKC) isoforms in ATR-induced IKAChC activation. Methods and Results: Cells from ATR-dogs (400-bpm atrial pacing for 1 week) were compared to control dog cells. In vitro tachypaced (TP; 3 Hz) canine atrial cardiomyocytes were compared to parallel 1-Hz paced cells. I KAChC single-channel activity was assessed in cell-attached and cell-free (inside-out) patches. Protein expression was assessed by immunoblot. In vitro TP activated IKAChC, mimicking effects of in vivo ATR. Discrepant effects of PKC activation and inhibition between control and ATR cells suggested isoform-selective effects and altered PKC isoform distribution. Conventional PKC isoforms (cPKC; including PKCα) inhibited, whereas novel isoforms (including PKCε) enhanced, acetylcholine-regulated K current (IKACh) in inside-out patches. TP and ATR downregulated PKCα (by 33{\%} and 37{\%}, respectively) and caused membrane translocation of PKCε, switching PKC predominance to the stimulatory novel isoform. TP increased [Ca2+]i at 2 hours by 30{\%}, with return to baseline at 24 hours. Buffering [Ca 2+]i during TP with the cell-permeable Ca2+ chelator BAPTA-AM (1 μmol/L) or inhibiting the Ca-dependent protease calpain with PD150606 (20 μmol/L) prevented PKCα downregulation and TP enhancement of IKAChC. PKCε inhibition with a cell-permeable peptide inhibitor suppressed TP/ATR-induced IKAChC activation, whereas cPKC inhibition enhanced IKAChC activity in 1-Hz cells. Conclusions: PKC isoforms differentially modulate IKACh, with conventional Ca-dependent isoforms inhibiting and novel isoforms enhancing activity. ATR causes a rate-dependent PKC isoform switch, with Ca/calpain-dependent downregulation of inhibitory PKCα and membrane translocation of stimulatory PKCε, enhancing I KAChC. These findings provide novel insights into mechanisms underlying IKAChC dysregulation in AF.",
author = "Samy Makary and Niels Voigt and Ange Maguy and Reza Wakili and Kunihiro Nishida and Masahide Harada and Dobromir Dobrev and Stanley Nattel",
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Differential protein kinase c isoform regulation and increased constitutive activity of acetylcholine-regulated potassium channels in atrial remodeling. / Makary, Samy; Voigt, Niels; Maguy, Ange; Wakili, Reza; Nishida, Kunihiro; Harada, Masahide; Dobrev, Dobromir; Nattel, Stanley.

In: Circulation Research, Vol. 109, No. 9, 14.10.2011, p. 1031-1043.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Differential protein kinase c isoform regulation and increased constitutive activity of acetylcholine-regulated potassium channels in atrial remodeling

AU - Makary, Samy

AU - Voigt, Niels

AU - Maguy, Ange

AU - Wakili, Reza

AU - Nishida, Kunihiro

AU - Harada, Masahide

AU - Dobrev, Dobromir

AU - Nattel, Stanley

PY - 2011/10/14

Y1 - 2011/10/14

N2 - Rationale Atrial fibrillation (AF) causes atrial-tachycardia remodeling (ATR), with enhanced constitutive acetylcholine-regulated K+ current (IKAChC) contributing to action potential duration shortening and AF promotion. The underlying mechanisms are unknown. Objective: To evaluate the role of protein-kinase C (PKC) isoforms in ATR-induced IKAChC activation. Methods and Results: Cells from ATR-dogs (400-bpm atrial pacing for 1 week) were compared to control dog cells. In vitro tachypaced (TP; 3 Hz) canine atrial cardiomyocytes were compared to parallel 1-Hz paced cells. I KAChC single-channel activity was assessed in cell-attached and cell-free (inside-out) patches. Protein expression was assessed by immunoblot. In vitro TP activated IKAChC, mimicking effects of in vivo ATR. Discrepant effects of PKC activation and inhibition between control and ATR cells suggested isoform-selective effects and altered PKC isoform distribution. Conventional PKC isoforms (cPKC; including PKCα) inhibited, whereas novel isoforms (including PKCε) enhanced, acetylcholine-regulated K current (IKACh) in inside-out patches. TP and ATR downregulated PKCα (by 33% and 37%, respectively) and caused membrane translocation of PKCε, switching PKC predominance to the stimulatory novel isoform. TP increased [Ca2+]i at 2 hours by 30%, with return to baseline at 24 hours. Buffering [Ca 2+]i during TP with the cell-permeable Ca2+ chelator BAPTA-AM (1 μmol/L) or inhibiting the Ca-dependent protease calpain with PD150606 (20 μmol/L) prevented PKCα downregulation and TP enhancement of IKAChC. PKCε inhibition with a cell-permeable peptide inhibitor suppressed TP/ATR-induced IKAChC activation, whereas cPKC inhibition enhanced IKAChC activity in 1-Hz cells. Conclusions: PKC isoforms differentially modulate IKACh, with conventional Ca-dependent isoforms inhibiting and novel isoforms enhancing activity. ATR causes a rate-dependent PKC isoform switch, with Ca/calpain-dependent downregulation of inhibitory PKCα and membrane translocation of stimulatory PKCε, enhancing I KAChC. These findings provide novel insights into mechanisms underlying IKAChC dysregulation in AF.

AB - Rationale Atrial fibrillation (AF) causes atrial-tachycardia remodeling (ATR), with enhanced constitutive acetylcholine-regulated K+ current (IKAChC) contributing to action potential duration shortening and AF promotion. The underlying mechanisms are unknown. Objective: To evaluate the role of protein-kinase C (PKC) isoforms in ATR-induced IKAChC activation. Methods and Results: Cells from ATR-dogs (400-bpm atrial pacing for 1 week) were compared to control dog cells. In vitro tachypaced (TP; 3 Hz) canine atrial cardiomyocytes were compared to parallel 1-Hz paced cells. I KAChC single-channel activity was assessed in cell-attached and cell-free (inside-out) patches. Protein expression was assessed by immunoblot. In vitro TP activated IKAChC, mimicking effects of in vivo ATR. Discrepant effects of PKC activation and inhibition between control and ATR cells suggested isoform-selective effects and altered PKC isoform distribution. Conventional PKC isoforms (cPKC; including PKCα) inhibited, whereas novel isoforms (including PKCε) enhanced, acetylcholine-regulated K current (IKACh) in inside-out patches. TP and ATR downregulated PKCα (by 33% and 37%, respectively) and caused membrane translocation of PKCε, switching PKC predominance to the stimulatory novel isoform. TP increased [Ca2+]i at 2 hours by 30%, with return to baseline at 24 hours. Buffering [Ca 2+]i during TP with the cell-permeable Ca2+ chelator BAPTA-AM (1 μmol/L) or inhibiting the Ca-dependent protease calpain with PD150606 (20 μmol/L) prevented PKCα downregulation and TP enhancement of IKAChC. PKCε inhibition with a cell-permeable peptide inhibitor suppressed TP/ATR-induced IKAChC activation, whereas cPKC inhibition enhanced IKAChC activity in 1-Hz cells. Conclusions: PKC isoforms differentially modulate IKACh, with conventional Ca-dependent isoforms inhibiting and novel isoforms enhancing activity. ATR causes a rate-dependent PKC isoform switch, with Ca/calpain-dependent downregulation of inhibitory PKCα and membrane translocation of stimulatory PKCε, enhancing I KAChC. These findings provide novel insights into mechanisms underlying IKAChC dysregulation in AF.

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