Figure 5. NO increases CaMKII-dependent SR Ca²⁺ leak.

A) NO-dependent DAF-2 fluorescence (n = 6). Spearman correlation = 1.0 for SNAP, 0.9 for ISO, and −0.05 for control. B) SNAP-dependent SR Ca²⁺ leak. The SR Ca²⁺ leak (right) in [Ca]_SRT matched data (left, n = 9–13). C) Data was matched such that leak was the same (left) with the [Ca]_SRT needed to induced that leak shown on the right (n = 12–17). D) Purified CaMKII pre-activated with 200 µM Ca²⁺ and CaM. H₂O₂ (Lane 2) or 500 µM SNAP (Lane 3) was added followed by EGTA. ATP³² was added along with purified β2a L-type Ca²⁺ channel subunit on nickel beads. Incorporation of P³² was measured as an indicator of Ca-independent sustained kinase activity. Lane 1 is CaMKII without Ca²⁺, CaM, or ATP; Lane 4 is CaMKII without Ca²⁺, CaM, or ATP plus the addition of SNAP (500 µM) alone. Lane 5 is P³² incorporation in the continued presence of Ca²⁺ and CaM. E) Cardiac myocytes were field stimulated at 0.5 Hz under the indicated conditions. CaMKII was then immunoprecipitated from cellular homogenates which were then blotted with antibody to S-NO. *different from ISO, **different from both ISO and control (t-test, p<0.05).