P399A view from inside: simultaneous live cell imaging of free SR and cytosolic calcium concentrations

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Purpose: Understanding of cardiac excitation-contraction-coupling has been greatly facilitated by measurements of the free Ca2+ concentration in the myoplasm ([Ca2+]i). However, only very few methods to measure the free Ca2+ concentration in the sarcoplasmic reticulum (SR) ([Ca2+]lu) have been published. Since a direct and live examination of [Ca2+]lu is necessary to better understand cardiac myocyte Ca2+ signalling, a method was established to measure [Ca2+]lu and [Ca2+]i simultaneously in the same cell.

Nicotinic acid adenine dinucleotide phosphate (NAADP) releases Ca2+ from intracellular stores in cardiac myocytes. Antagonism of NAADP abolished spontaneous diastolic Ca2+ transients and decreased arrythmogenic events evoked by β-adrenergic stimulation in awake mice. Aim of this study was to further analyse the role of NAADP in regulation of [Ca2+]lu and [Ca2+]i during ventricular arrhythmia.

Methods: Ca2+ transients were recorded both as increases of [Ca2+]i and as decreases of the [Ca2+]lu using mag-fura-2/AM and fluo-8/AM. Continuous Ca2+imaging was performed using a PerkinElmer imaging system built around a Leica microscope (type DM IRBE).

Results and conclusion: Selection of an adequate indicator and optimisation of measurements considered aspects of signal-to-noise ratio and bleaching-stability as well as detection of the typical SR staining pattern and appropriate changes in fluorescence upon electrical stimulation. In electrically paced ventricular cardiac myocytes characteristic trains of cytosolic and SR-Ca2+ transients were recorded. Under control conditions typical long Ca2+ transients were observed both in the cytosol and in the SR lumen. Strong β-adrenergic stimulation influenced both the shape and number of Ca2+ transients. The recovery back to baseline values of [Ca2+]lu transients proceeded more rapidly as compared to controls, resembling the changes in shape of [Ca2+]i transients evoked by isoprenaline (Iso). Importantly, spontaneous diastolic Ca2+ transients were observed upon strong β-adrenergic stimulation; this was mirrored by parallel spontaneous diastolic decreases of [Ca2+]lu indicating that the major source for the cytosolic Ca2+ transients evoked by Iso was the SR. NAADP antagonist BZ194 blocked spontaneous diastolic Ca2+ transients caused by high concentrations of Iso; again, this was mirrored by the kinetics of [Ca2+]lu.

In conclusion, a method for simultaneous analyses of [Ca2+]lu and [Ca2+]i was developed and applied to image arrhythmic events in ventricular myocytes involving the second messenger NAADP.

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