Differential Protein Kinase C Isoform Regulation and Increased Constitutive Activity of Acetylcholine-Regulated Potassium Channels in Atrial Remodeling

Rationale: Atrial fibrillation (AF) causes atrial-tachycardia remodeling (ATR), with enhanced constitutive acetylcholine-regulated K + current (I KAChC ) 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 I KAChC 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 I KAChC , 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 (I KACh ) 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 [Ca 2+ ] i at 2 hours by 30%, with return to baseline at 24 hours. Buffering [Ca 2+ ] i during TP with the cell-permeable Ca 2+ chelator BAPTA-AM (1 μmol/L) or inhibiting the Ca 2+ -dependent protease calpain with PD150606 (20 μmol/L) prevented PKCα downregulation and TP enhancement of I KAChC . PKCε inhibition with a cell-permeable peptide inhibitor suppressed TP/ATR-induced I KAChC activation, whereas cPKC inhibition enhanced I KAChC activity in 1-Hz cells. Conclusions: PKC isoforms differentially modulate I KACh , with conventional Ca 2+ -dependent isoforms inhibiting and novel isoforms enhancing activity. ATR causes a rate-dependent PKC isoform switch, with Ca 2+ /calpain-dependent downregulation of inhibitory PKCα and membrane translocation of stimulatory PKCε, enhancing I KAChC . These findings provide novel insights into mechanisms underlying I KAChC dysregulation in AF.

Rationale: Atrial fibrillation (AF) causes atrial-tachycardia remodeling (ATR), with enhanced constitutive acetylcholine-regulated K + current (I KAChC ) 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 I KAChC 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 I KAChC , 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 (I KACh ) 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 [Ca 2+ ] i at 2 hours by 30%, with return to baseline at 24 hours. Buffering [Ca 2+ ] i during TP with the cell-permeable Ca 2+ chelator BAPTA-AM (1 μmol/L) or inhibiting the Ca 2+ -dependent protease calpain with PD150606 (20 μmol/L) prevented PKCα downregulation and TP enhancement of I KAChC . PKCε inhibition with a cell-permeable peptide inhibitor suppressed TP/ATR-induced I KAChC activation, whereas cPKC inhibition enhanced I KAChC activity in 1-Hz cells. Conclusions: PKC isoforms differentially modulate I KACh , with conventional Ca 2+ -dependent isoforms inhibiting and novel isoforms enhancing activity. ATR causes a rate-dependent PKC isoform switch, with Ca 2+ /calpain-dependent downregulation of inhibitory PKCα and membrane translocation of stimulatory PKCε, enhancing I KAChC . These findings provide novel insights into mechanisms underlying I KAChC dysregulation in AF.