A New Ca2+ Probe, Calstabi-Cam, Targeted to Ryanodine Receptors of Cardiomyocytes

Abstract

The contractile force of cardiomyocytes is controlled by Ca2+ cross-signaling between L-type Ca2+ channels and ryanodine receptors (RyR2) across the narrow dyadic cleft. To detect the junctional Ca2+ signal, we designed a peptide probe (Calstabi-Cam) with calmodulin as its Ca2+ sensor, yellow fluorescent protein (EYFP) as reporting fluorophor, and FKBP12.6 (calstabin2) as subunit of RyR2. Effective adenoviral expression in cultured adult rat cardiomyocytes was verified after 48 hours when Calstabi-Cam co-localized with fluorescent RyR antibodies in a sarcomeric z-lines pattern. Dissociation constant (kd) of Calstabi-Cam for Ca2+ measured in permeablized myocytes was 80 nM. The kinetic of Ca2+ signals was measured in voltage-clamped cells with a Leica TIRF microscope which allowed comparison of rapidly interlaced images of cytosolic Ca2+ probes (fura-2 or fluo-4) and Calstabi-Cam. Fluo-4 Ca2+ sparks were detected superimposed on the sarcomeric fluorescence patterns of Calstabi-Cam. On activation of Ca2+ release by caffeine or membrane depolarization, Calstabi-Cam fluorescence signals had slower rise times compared to fura-2, but had much slower decay kinetics. Scans of focal Calstabi-Cam signals at different sites, occurring spontaneously or at the onset of evoked Ca2+ releases, appeared to have a significant distribution of magnitudes and latencies. We conclude that Ca2+-sensing biological peptides may be targeted to the cleft-space occupied by DHPR/RyRs complex as to make it possible to record the variance of Ca2+ signals at different dyadic junctions.