High-speed digital imaging of cytosolic Ca²⁺ and contraction in single cardiomyocytes

A charge-coupled device (CCD) camera, with the capacity for simultaneous spatially resolved photon counting and rapid frame transfer, was utilized for high-speed digital image collection from an inverted epifluorescence microscope. The unique properties of the CCD detector were applied to an analysis of cell shortening and the Ca²⁺ transient from fluorescence images of fura-loaded cardiomyocytes. On electrical stimulation of the cell, a series of sequential subimages was collected and used to create images of Ca²⁺ within the cell during contraction. The high photosensitivity of the camera, combined with a detector-based frame storage technique, permitted collection of fluorescence images 10 ms apart. This rate of image collection was sufficient to resolve the rapid events of contraction, e.g., the upstroke of the Ca²⁺ transient (< 40 ms) and the time to peak shortening (< 80 ms). The technique was used to examine the effects of β-adrenoceptor activation, fura-2 load, and stimulus frequency on cytosolic Ca²⁺ transients and contractions of single cardiomyocytes. β-Adrenoceptor stimulation resulted in pronounced increases in peak Ca²⁺, maximal rates of rise and decay of Ca²⁺, extent of shortening, and maximal velocities of shortening and relaxation. Raising the intracellular load of fura-2 had little effect on the rising phase of Ca²⁺ or the extent of shortening but extended the duration of the Ca²⁺ transient and contraction. In related experiments utilizing differential-interference contrast microscopy, the same technique was applied to visualize sarcomere dynamics in contracting cells. This newly developed technique is a versatile tool for analyzing the Ca²⁺ transient and mechanical events in studies of excitation-contraction coupling in cardiomyocytes.