Total internal reflectance fluorescence imaging of genetically engineered ryanodine receptor-targeted Ca2+ probes in rat ventricular myocytes.

The details of cardiac Ca2+ signaling within the dyadic junction remain unclear because of limitations in rapid spatial imaging techniques, and availability of Ca2+ probes localized to dyadic junctions. To critically monitor ryanodine receptors' (RyR2) Ca2+ nano-domains, we combined the use of genetically engineered RyR2-targeted pericam probes, (FKBP-YCaMP, Kd=150nM, or FKBP-GCaMP6, Kd=240nM) with rapid total internal reflectance fluorescence (TIRF) microscopy (resolution, ∼80nm). The punctate z-line patterns of FKBP,2-targeted probes overlapped those of RyR2 antibodies and sharply contrasted to the images of probes targeted to sarcoplasmic reticulum (SERCA2a/PLB), or cytosolic Fluo-4 images. FKBP-YCaMP signals were too small (∼20%) and too slow (2-3s) to detect Ca2+ sparks, but the probe was effective in marking where Fluo-4 Ca2+ sparks developed. FKBP-GCaMP6, on the other hand, produced rapidly decaying Ca2+ signals that: a) had faster kinetics and activated synchronous with ICa3 but were of variable size at different z-lines and b) were accompanied by spatially confined spontaneous Ca2+ sparks, originating from a subset of eager sites. The frequency of spontaneously occurring sparks was lower in FKBP-GCaMP6 infected myocytes as compared to Fluo-4 dialyzed myocytes, but isoproterenol enhanced their frequency more effectively than in Fluo-4 dialyzed cells. Nevertheless, isoproterenol failed to dissociate FKBP-GCaMP6 from the z-lines. The data suggests that FKBP-GCaMP6 binds predominantly to junctional RyR2s and has sufficient on-rate efficiency as to monitor the released Ca2+ in individual dyadic clefts, and supports the idea that β-adrenergic agonists may modulate the stabilizing effects of native FKBP on RyR2.