Dataset Information

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

ABSTRACT: 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 ?-adrenergic agonists may modulate the stabilizing effects of native FKBP on RyR2.

SUBMITTER: Pahlavan S

PROVIDER: S-EPMC5599148 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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

Pahlavan Sara S Morad Marin M

Cell calcium 20170719

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 fluorescen ...[more]

PMID: 28807154

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Project description:Spontaneous Ca2+-release events (SCaEs) from the sarcoplasmic reticulum play crucial roles in the initiation of cardiac arrhythmias by promoting triggered activity. However, the subcellular determinants of these SCaEs remain incompletely understood. Structural differences between atrial and ventricular cardiomyocytes, e.g., regarding the density of T-tubular membrane invaginations, may influence cardiomyocyte Ca2+-handling and the distribution of cardiac ryanodine receptors (RyR2) has recently been shown to undergo remodeling in atrial fibrillation. These data suggest that the subcellular distribution of Ca2+-handling proteins influences proarrhythmic Ca2+-handling abnormalities. Here, we employ computational modeling to provide an in-depth analysis of the impact of variations in subcellular RyR2 and L-type Ca2+-channel distributions on Ca2+-transient properties and SCaEs in a human atrial cardiomyocyte model. We incorporate experimentally observed RyR2 expression patterns and various configurations of axial tubules in a previously published model of the human atrial cardiomyocyte. We identify an increased SCaE incidence for larger heterogeneity in RyR2 expression, in which SCaEs preferentially arise from regions of high local RyR2 expression. Furthermore, we show that the propagation of Ca2+ waves is modulated by the distance between RyR2 bands, as well as the presence of experimentally observed RyR2 clusters between bands near the lateral membranes. We also show that incorporation of axial tubules in various amounts and locations reduces Ca2+-transient time to peak. Furthermore, selective hyperphosphorylation of RyR2 around axial tubules increases the number of spontaneous waves. Finally, we present a novel model of the human atrial cardiomyocyte with physiological RyR2 and L-type Ca2+-channel distributions that reproduces experimentally observed Ca2+-handling properties. Taken together, these results significantly enhance our understanding of the structure-function relationship in cardiomyocytes, identifying that RyR2 and L-type Ca2+-channel distributions have a major impact on systolic Ca2+ transients and SCaEs.

Dataset Information

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

Publications

Total internal reflectance fluorescence imaging of genetically engineered ryanodine receptor-targeted Ca<sup>2+</sup> probes in rat ventricular myocytes.

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