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.

Detection of differentially regulated subsarcolemmal calcium signals activated by vasoactive agonists in rat pulmonary artery smooth muscle cells

“…The higher apparent K d of Lyn-D3cpv is not due to the use of ␣-toxin for membrane permeabilization because K d values of the nucleoplasmictargeted 3NLS-D3cpv are similar when calibrated using digitonin or ␣-toxin. Our finding is congruent with previous observations that the same cameleon may show different K d values in different subcellular compartments (14,32). One potential limitation of D3cpv is the slower kinetics compared with the Ca 2ϩ fluorescent dyes (19), so it may miss the detection of very rapid transient events.…”

Asante Calcium Green and Asante Calcium Red—Novel Calcium Indicators for Two-Photon Fluorescence Lifetime Imaging

“…This is about 1.7 times larger than the measured in vitro K D,t value. The only available in situ K D values for ACR so far were published by Ljubojević et al [44]. In that study, K D,i values were determined in rat ventricular cardiomyocytes at 2183 nM in nucleoplasm and 1336 nM in cytoplasm, respectively.…”

Regulation of Phosphatidylinositol-specific Phospholipase C at the Nuclear Envelope in Cardiac Myocytes

“…Other components of the calcium handling machinery are present on the nuclear envelope such as the Serca pump, and Ryr2 [43]. The presence of IP 3 receptors on the nuclear envelope, if localized facing the nuclear matrix, suggests that IP 3 can regulate nuclear calcium levels.…”