RESUMO
Quantification of subcellularly resolved Ca²âº signals in cardiomyocytes is essential for understanding Ca²âº fluxes in excitation-contraction and excitation-transcription coupling. The properties of fluorescent indicators in intracellular compartments may differ, thus affecting the translation of Ca²âº-dependent fluorescence changes into [Ca²âº] changes. Therefore, we determined the in situ characteristics of a frequently used Ca²âº indicator, Fluo-4, and a ratiometric Ca²âº indicator, Asante Calcium Red, and evaluated their use for reporting and quantifying cytoplasmic and nucleoplasmic Ca²âº signals in isolated cardiomyocytes. Ca²âº calibration curves revealed significant differences in the apparent Ca²âº dissociation constants of Fluo-4 and Asante Calcium Red between cytoplasm and nucleoplasm. These parameters were used for transformation of fluorescence into nucleoplasmic and cytoplasmic [Ca²âº]. Resting and diastolic [Ca²âº] were always higher in the nucleoplasm. Systolic [Ca²âº] was usually higher in the cytoplasm, but some cells (15%) exhibited higher systolic [Ca²âº] in the nucleoplasm. Ca²âº store depletion or blockade of Ca²âº leak pathways eliminated the resting [Ca²âº] gradient between nucleoplasm and cytoplasm, whereas inhibition of inositol 1,4,5-trisphosphate receptors by 2-APB reversed it. The results suggest the presence of significant nucleoplasmic-to-cytoplasmic [Ca²âº] gradients in resting myocytes and during the cardiac cycle. Nucleoplasmic [Ca²âº] in cardiomyocytes may be regulated via two mechanisms: diffusion from the cytoplasm and active Ca²âº release via inositol 1,4,5-trisphosphate receptors from perinuclear Ca²âº stores.