Question: 

Nucleoplasmic Ca²⁺ concentration ([Ca²⁺]_nuc) plays a key role in cardiac excitation-transcription coupling. Its regulation, however, is only poorly understood largely because reliable quantification is difficult. Here we used a novel in situ calibration approach to quantify [Ca²⁺]_nuc in electrically stimulated and quiescent cardiomyocytes.

Methods: 

Cytoplasmic [Ca²⁺] ([Ca²⁺]_cyto) and [Ca²⁺]_nuc were imaged simultaneously in isolated ventricular myocytes (mouse, rat) loaded with a non-ratiometric Ca²⁺ dye, Fluo-4 (8mM), or a ratiometric Ca²⁺ dye, Asante Calcium Red (10mM), by means of confocal microscopy. In situ calibration of the dyes was performed using calibration solutions with known [Ca²⁺], a Ca²⁺ ionophore, metabolic inhibitors, and an inhibitor of myofilaments.

Results: 

Both Ca²⁺ dyes exhibited distinct properties in the cytoplasm versus nucleoplasm of cardiomyocytes. The apparent K_d for Ca²⁺ binding was higher in the nucleoplasm. Using the obtained calibration parameters, fluorescence was transformed into calibrated [Ca²⁺]. In electrically stimulated myocytes, diastolic [Ca²⁺] was higher whereas systolic [Ca²⁺] was lower in the nucleoplasm. Systolic [Ca²⁺]_nuc correlated with systolic [Ca²⁺]_cyto (r=0.63). In quiescent cells, [Ca²⁺]_nuc was always higher than [Ca²⁺]_cyto. Depletion of intracellular Ca²⁺ stores (5mM CPA) or inhibition of intracellular Ca²⁺ release channels (3mM 2-APB and 1mM tetracaine) abolished the nucleo-to-cytoplasmic [Ca²⁺] gradient. Inhibition of IP₃ receptors (3mM 2-APB) caused a selective decrease of [Ca²⁺]_nuc leading to a reversal of the nucleo-to-cytoplasmic [Ca²⁺] gradient.

Conclusions: 

[Ca²⁺]_nuc differs from [Ca²⁺]_cyto during the cardiac cycle and in quiescent myocytes. [Ca²⁺]_nuc is regulated passively by [Ca²⁺]_cyto and actively by Ca²⁺ release mainly through IP₃ receptors from perinuclear Ca²⁺ stores.