In Situ Calibration of Nucleoplasmic versus Cytoplasmic Ca2+ Concentration in Adult Cardiomyocytes

Abstract

Quantification of subcellularly resolved Ca2+ signals in cardiomyocytes is essential for understanding Ca2+ fluxes in excitation-contraction and excitation-transcription coupling. The properties of fluorescent indicators in intracellular compartments may differ, thus affecting the translation of Ca2+-dependent fluorescence changes into [Ca2+] changes. Therefore, we determined the in situ characteristics of a frequently used Ca2+ indicator, Fluo-4, and a ratiometric Ca2+ indicator, Asante Calcium Red, and evaluated their use for reporting and quantifying cytoplasmic and nucleoplasmic Ca2+ signals in isolated cardiomyocytes. Ca2+ calibration curves revealed significant differences in the apparent Ca2+ 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 [Ca2+]. Resting and diastolic [Ca2+] were always higher in the nucleoplasm. Systolic [Ca2+] was usually higher in the cytoplasm, but some cells (15%) exhibited higher systolic [Ca2+] in the nucleoplasm. Ca2+ store depletion or blockade of Ca2+ leak pathways eliminated the resting [Ca2+] 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 [Ca2+] gradients in resting myocytes and during the cardiac cycle. Nucleoplasmic [Ca2+] in cardiomyocytes may be regulated via two mechanisms: diffusion from the cytoplasm and active Ca2+ release via inositol 1,4,5-trisphosphate receptors from perinuclear Ca2+ stores.