Abstract 5533: Interactions Between Inward Rectifier K+ Current And Abnormal Calcium Homeostasis Contribute To Altered Bathmotropic Threshold And Increased Spontaneous Activity In Nos3−/− Mice
BACKGROUND: We have previously shown that there is prolonged action potential (AP) duration, greater L-type Ca2+ current (ICa), 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 (Ito), sustained (IKsus) and the inward rectifier (IK1) 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 Ito or IKsus densities were seen between WT and NOS3−/− . The peak outward IK1 current was not different between the two groups. Interestingly, NOS3−/− myocytes had a reduced IK1 inward slope conductance vs. WT (0.27±0.005 vs. 0.34±0.001 mS/cm2; p<0.05). To address spontaneous activity in the setting of reduced inward IK1, further experiments to assess intra-sarcoplasmic reticulum (SR) calcium abnormalities were performed. A significant increase in β-AR stimulated SR Ca2+ 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 Ca2+ load can cause spontaneous Ca2+ release from SR, activating reverse mode Na+/Ca2+ exchanger, an inward transient current. The combination of increased cytosolic Ca2+ resulting from higher β-AR stimulated ICa and SR Ca2+ load, in concert with decreased inward IK1, 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 IK1 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).