Abstract 1084: Myosin Light Chain Kinase Signaling Regulates Ectopic Atrial Activity
Objective: Determine whether calmodulin (CaM) acts through novel signaling pathways to elicit left atrial ectopic activity.
Methods: Rat left atria (n=6 –9 per group) were isolated and superfused at 30°C. Mechanical function was recorded using a Grass recorder. Left atrial proteins were analyzed using Western analysis.
Findings: Our rat left atria are quiescent in the absence of external pacing. However, in the presence of 2-aminoethoxydiphenyl borate (20μM; 2APB), a novel sarcoplasmic reticulum (SR) calcium leak inducer, left atria contract spontaneously at 67±12 contractions (min) − 1 in the absence of pacing. Pre-treating left atria with two irreversible CaM inhibitors, phenoxybenzamine and fluphenazine chloroethane (90μM each), decreases spontaneous contraction frequency to 5±3 and 2±1 (min) − 1, respectively, indicating CaM signaling is activated and required for this ectopic activity. Pre-treating left atria with 20μM KN62 did not decrease the frequency of 2APB-induced ectopic activity (72±8 contractions (min) − 1) indicating calmodulin-dependent protein kinase II (CAMKII) is not a downstream target here. In contrast, 20μM ML7, a specific inhibitor of myosin light chain kinase (MLCK), a second cardiac CaM-regulated kinase, decreases 2APB-induced spontaneous contractions to 7±3 min−1. Western analysis shows left atria contain smooth muscle MLCK. This kinase preferentially phosphorylates non-muscle myosin light chain 2 (nmMLC2). Western analyses also show left atria contain nmMLC2 which is not phosphorylated in untreated muscles. However, nmMLC2 phosphorylation increases significantly in left atria exposed to 2APB.
Conclusions: CaM and MLCK inhibitors prevent the ectopic atrial activity induced by 2APB. This suggests a unique SR calcium leak occurs under this condition which activates CaM signaling whose specific downstream target is MLCK, a major myocardial CaM-dependent kinase. MLCK signaling via its sole substrate, nmMLC2, is required for this novel ectopic atrial activity. Hyperphosphorylation of SR ryanodine receptors by CAMKII is known to induce an SR calcium leak that underlies triggered ectopic activity. Our data suggest multiple calcium leaks and CaM signaling pathways regulate the stability of intact atrial muscle.