To determine how O-GlcNAcylation increased SOICR independently of CaMKII at higher Ca
2+ concentrations, we next measured the threshold for SOICR using the intra-luminal Ca
2+ indicator CEPIA. Our previous work has shown that a common mechanism by which the propensity of SOICR is increased is due to a reduction in the release threshold for SOICR (F
SOICR) [
5,
7]. We also examined the termination threshold, the intracellular Ca
2+ store level (expressed as a percentage of the total store) at which SOICR events terminate (F
Termi). Measuring F
SOICR and F
Termi also allows us to determine whether O-GlcNAcylation alters the magnitude of SOICR (fractional release) [
15].
Figure
4 A shows representative CEPIA traces from cells expressing RyR2 treated with 25mM glucose in the presence or absence of Thm-G, with (KN-93) or without (KN-92) CaMKII inhibition. In Fig.
4, F
max represents the maximum Ca
2+ store in the presence of 2 mM tetracaine and F
min represents the minimum Ca
2+ store in the presence of 20 mM caffeine. The free Ca
2+ store size of each cell was obtained by F
max-F
min. F
SOICR was determined by the mean fluorescence immediately prior to each SOICR event (downward deflection), F
Termi was determined by the mean fluorescence of the nadir of SOICR. The release threshold (Fig.
4E) and fractional release (Fig.
4F) are reported as a percentage of free Ca
2+ store as previously described [
15]. As shown in Fig.
4 Thm-G (+ KN-92) significantly decreases the release threshold (F
SOICR) and fractional release compared to RyR2 in the absence of Thm-G (both p < 0.0001). Importantly, when treated with the CaMKII inhibitor KN-93, Thm-G retains a significant effect on SOICR (p < 0.0001 and 0.001 for F
SOICR and fractional release, respectively), indicating a CaMKII independent effect of O-GlcNAcylation on SOICR. Although the effect of Thm-G on SOICR was retained after CaMKII inhibition, +KN-93 abrogated the effect, increasing both F
SOICR and fractional release compared to cells treated with Thm-G without CaMKII inhibition (p = 0.0019 and p = 0.0084 for F
SOICR and fractional release, respectively). This indicates an additive effect of CaMKII and non-CaMKII O-GlcNAc mediated effects on SOICR. Interestingly, although it does not appear to translate to a change in the occurrence of SOICR (Fig.
3E), inhibition of CaMKII was still able to increase F
SOICR and fractional release in presence of Thm-G (p = 0.0019 and 0.0084 for F
SOICR and fractional release, respectively), suggesting CaMKII might still have a partial role under these conditions.