Abstract-A neuronal isoform of nitric oxide synthase (nNOS) has recently been located to the cardiac sarcoplasmic reticulum (SR). Subcellular localization of a constitutive NOS in the proximity of an activating source of Ca 2ϩ suggests that cardiac nNOS-derived NO may regulate contraction by exerting a highly specific and localized action on ion channels/transporters involved in Ca 2ϩ cycling. To test this hypothesis, we have investigated myocardial Ca 2ϩ handling and contractility in nNOS knockout mice (nNOS Ϫ/Ϫ ) and in control mice (C) after acute nNOS inhibition with 100 mol/L L-VNIO. nNOS gene disruption or L-VNIO increased basal contraction both in left ventricular (LV)

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Na + -Ca 2+ exchange (NCX) current has been suggested to play a role in cardiac pacemaking, particularly in association with Ca 2+ release from the sarcoplasmic reticulum (SR) that occurs just before the action potential upstroke. The present experiments explore in more detail the contribution of NCX to pacemaking. Na + -Ca 2+ exchange current was inhibited by rapid switch to low-Na + solution (with Li + replacing Na + ) within the time course of a single cardiac cycle to avoid slow secondary effects. Rapid switch to low-Na + solution caused immediate cessation of spontaneous action potentials. ZD7288 (3 μM), to block I f (funny current) channels, slowed but did not stop the spontaneous activity, and tetrodotoxin (10 μM), to block Na + channels, had little effect, but in the presence of either of these agents, rapid switch to low-Na + solution again caused immediate cessation of spontaneous action potentials. Spontaneous electrical activity was also stopped following loading of the cells with the Ca 2+ chelators BAPTA and EGTA, and by exposure to the NCX inhibitor KB-R7943 (5 μM). When rapid switch to low-Na + solution caused cessation of spontaneous activity, this was found (using confocal microscopy, with fluo-4 as the

These observations are consistent with the hypothesis that endogenous cADPR plays an important role during normal contraction of cardiac myocytes. One possibility is that cADPR sensitizes the CICR mechanism to Ca2+, an action antagonized by 8-amino-cADPR (leading to reduced Ca2+ transients and contractions). A direct effect of 8-amino-cADPR on CICR cannot be excluded, but observations with caffeine are not consistent with a non-selective block of release channels.