title: 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.
authors.affiliation: Division of Cardiology, Medical University of Graz, Graz, Austria.
authors.forename: Senka
authors.initials: S
authors.lastname: Ljubojević
authors.forename: Stefanie
authors.initials: S
authors.lastname: Walther
authors.forename: Mojib
authors.initials: M
authors.lastname: Asgarzoei
authors.forename: Simon
authors.initials: S
authors.lastname: Sedej
authors.forename: Burkert
authors.initials: B
authors.lastname: Pieske
authors.forename: Jens
authors.initials: J
authors.lastname: Kockskämper
mesh_terms: D000375:Aging / Q000187:drug effects / Q000378:metabolism*; D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D001896:Boron Compounds / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002138:Calibration; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D003971:Diastole / Q000187:drug effects; D004558:Electric Stimulation; D005453:Fluorescence; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D013599:Systole / Q000187:drug effects; D013748:Tetracaine / Q000494:pharmacology; D014966:Xanthenes / Q000378:metabolism
chemical_list: D000814:Aniline Compounds; D001896:Boron Compounds; C409648:Fluo 4; D014966:Xanthenes; D013748:Tetracaine; C109986:2-aminoethoxydiphenyl borate; D002118:Calcium