Abstract 5533: Interactions Between Inward Rectifier K + Current And Abnormal Calcium Homeostasis Contribute To Altered Bathmotropic Threshold And Increased Spontaneous Activity In Nos3 −/− Mice

Abstract

BACKGROUND: We have previously shown that there is prolonged action potential (AP) duration, greater L-type Ca 2+ current (I Ca ), and enhanced spontaneous activity (early and delayed afterdepolarizations) during β-adrenergic (β-AR) stimulation in endothelial nitric oxide synthase knockout (NOS3 −/− ) myocytes. Altered repolarization and/or diastolic depolarization may contribute to spontaneous activity and arrhythmogenesis and earlier studies suggested a role for NOS3 modulation of K + channels. HYPOTHESIS: We tested the hypothesis that NOS3 −/− myocytes have altered K + currents. METHODS: Ventricular myocytes were isolated from NOS3 −/− and wildtype (WT) mice. APs; transient outward (I to ), sustained (I Ksus ) and the inward rectifier (I K1 ) K + currents were measured at 36±1°C. RESULTS: No change in baseline AP duration or resting membrane potential was seen in NOS3 −/− vs WT myocytes (p = NS). AP duration at 90% repolarization during β-AR stimulation was longer in NOS3 −/− vs WT myocytes (102±11 msec vs 73±17 msec) consistent with our previous observations at room temperature. No differences in I to or I Ksus densities were seen between WT and NOS3 −/− . The peak outward I K1 current was not different between the two groups. Interestingly, NOS3 −/− myocytes had a reduced I K1 inward slope conductance vs. WT (0.27±0.005 vs. 0.34±0.001 mS/cm 2 ; p<0.05). To address spontaneous activity in the setting of reduced inward I K1 , further experiments to assess intra-sarcoplasmic reticulum (SR) calcium abnormalities were performed. A significant increase in β-AR stimulated SR Ca 2+ load (measured via caffeine) was found in NOS3 −/− vs WT myocytes (56±18 vs 28±8 % of without isoproterenol, p<0.05). CONCLUSION: We suggest that in NOS3 −/− greater SR Ca 2+ load can cause spontaneous Ca 2+ release from SR, activating reverse mode Na + /Ca 2+ exchanger, an inward transient current. The combination of increased cytosolic Ca 2+ resulting from higher β-AR stimulated I Ca and SR Ca 2+ load, in concert with decreased inward I K1 , provides a substrate for altered bathmotropic (excitability) threshold and arrhythmias in NOS3 −/− myocytes. This may have important implications for arrhythmias and sudden cardiac death in heart failure, where NOS3 expression and I K1 current are decreased. This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).