Molecular Determinants of Altered Ca ²⁺ Handling in Human Chronic Atrial Fibrillation

Abstract

Background— Abnormal Ca 2+ handling may contribute to impaired atrial contractility and arrhythmogenesis in human chronic atrial fibrillation (cAF). Here, we assessed the phosphorylation levels of key proteins involved in altered Ca 2+ handling and contractility in cAF patients. Methods and Results— Total and phosphorylation levels of Ca 2+ -handling and myofilament proteins were analyzed by Western blotting in right atrial appendages of 49 patients in sinus rhythm and 52 cAF patients. We found a higher total activity of type 1 (PP1) and type 2A phosphatases in cAF, which was associated with inhomogeneous changes of protein phosphorylation in the cellular compartments, ie, lower protein kinase A (PKA) phosphorylation of myosin binding protein-C (Ser-282 site) at the thick myofilaments but preserved PKA phosphorylation of troponin I at the thin myofilaments and enhanced PKA (Ser-16 site) and Ca 2+ -calmodulin protein kinase (Thr-17 site) phosphorylation of phospholamban. PP1 activity at sarcoplasmic reticulum is controlled by inhibitor-1 (I-1), which blocks PP1 in its PKA-phosphorylated form only. In cAF, the ratio of Thr-35–phosphorylated to total I-1 was 10-fold higher, which suggests that the enhanced phosphorylation of phospholamban may result from a stronger PP1 inhibition by PKA-hyperphosphorylated (activated) I-1. Conclusions— Altered Ca 2+ handling in cAF is associated with impaired phosphorylation of myosin binding protein-C, which may contribute to the contractile dysfunction after cardioversion. The hyperphosphorylation of phospholamban probably results from enhanced inhibition of sarcoplasmic PP1 by hyperphosphorylated I-1 and may reinforce the leakiness of ryanodine channels in cAF. Restoration of sarcoplasmic reticulum–associated PP1 function may represent a new therapeutic option for treatment of atrial fibrillation.

Arrhythmias, elicited by catecholamines and serotonin, vanish in human chronic atrial fibrillation

“…S6 and S7). Our data are at variance with a report of increased phosphorylation of PLB-Ser-16 and decreased phosphorylation of Prot-C-Ser-282 in atria from patients with AF, frozen in the operating theater (11). Thus, we compared the phosphorylation of PLB at Ser-16 in trabeculae frozen in the operating theater with trabeculae from the same patient that was contracted for 1 h in an organ bath.…”

“…In contrast to a previous report (11), here the phosphorylation of PLB at Ser-16 and Thr-17 was consistently lower in trabeculae frozen in the operating theater from patients with AF than in those from patients with SR. The high basal phosphorylation levels in trabeculae frozen in the operating theater likely reflect catecholamine surges during surgery.…”

“…S7B). Increased phosphorylation of RyR2 channels at Ser-2808, catalyzed by PKA (11,12), and Ser-2814, catalyzed by CaMKII, has been implicated in the arrhythmogenic release of Ca 2+ through leaky RyR2 channels (13,20); however, in contrast to previous reports, neither basal phosphorylation (SI Appendix, Figs. S6 and S7) nor phosphorylation stimulated by agonists of RyR2 at Ser-2808 ( Fig.…”

“…Increased CaMKII-catalyzed phosphorylation of PLB and RyR2 in the absence of agonists have been reported in AF and implicated in the triggering and/or maintenance of this arrhythmia in human atrium (11,13,20). The reports of CaMKII-catalyzed hyperphosphorylation have suggested the therapeutic use of CaMKII inhibitors in AF (13,30); however, our evidence indicates that CaMKII-dependent phosphorylation is either reduced or unchanged in human AF.…”

“…Spontaneous impulse generation could be related to increased activity of PKA and/or CaMKII, with subsequent uncoordinated release of Ca 2+ from the sarcoplasmic reticulum. Such a concept is attractive, because Ca 2+ released from the "leaky" sarcoplasmic reticulum would activate the Na + -Ca 2+ exchanger to extrude Ca 2+ and to produce a arrhythmogenic depolarizing current, thereby explaining both the contractile dysfunction and the high recurrence rate (11)(12)(13).…”