Impaired Intracellular Ca²⁺Dynamics in Live Cardiomyocytes Revealed by Rapid Line Scan Confocal Microscopy

Altered intracellular Ca²⁺dynamics are characteristically observed in cardiomyocytes from failing hearts. Studies of Ca²⁺handling in myocytes predominantly use Fluo-3 AM, a visible light excitable Ca²⁺chelating fluorescent dye in conjunction with rapid line-scanning confocal microscopy. However, Fluo-3 AM does not allow for traditional ratiometric determination of intracellular Ca²⁺concentration and has required the use of mathematic correction factors with values obtained from separate procedures to convert Fluo-3 AM fluorescence to appropriate Ca²⁺concentrations. This study describes methodology to directly measure intracellular Ca²⁺levels using inactivated, Fluo-3-AM-loaded cardiomyocytes equilibrated with Ca²⁺concentration standards. Titration of Ca²⁺concentration exhibits a linear relationship to increasing Fluo-3 AM fluorescence intensity. Images obtained from individual myocyte confocal scans were recorded, average pixel intensity values were calculated, and a plot is generated relating the average pixel intensity to known Ca²⁺concentrations. These standard plots can be used to convert transient Ca²⁺fluorescence obtained with experimental cells to Ca²⁺concentrations by linear regression analysis. Standards are determined on the same microscope used for acquisition of unknown Ca²⁺concentrations, simplifying data interpretation and assuring accuracy of conversion values. This procedure eliminates additional equipment, ratiometric imaging, and mathematic correction factors and should be useful to investigators requiring a straightforward method for measuring Ca²⁺concentrations in live cells using Ca²⁺-chelating dyes exhibiting variable fluorescence intensity.