Calcium sparks in intact skeletal muscle fibers of the frog

Abstract

Calcium sparks were studied in frog intact skeletal muscle fibers using a home-built confocal scanner whose point-spread function was estimated to be ∼0.21 μm in x and y and ∼0.51 μm in z. Observations were made at 17-20°C on fibers from Rana pipiens and Rana temporaria. Fibers were studied in two external solutions: normal Ringer's ([K+] = 2.5 mM; estimated membrane potential, -80 to -90 mV) and elevated [K+] Ringer's (most frequently, [K+] = 13 mM; estimated membrane potential, -60 to -65 mV). The frequency of sparks was 0.04-0.05 sarcomere-1 s-1 in normal Ringer's; the frequency increased approximately tenfold in 13 mM [K+] Ringer's. Spark properties in each solution were similar for the two species; they were also similar when scanned in the x and the y directions. From fits of standard functional forms to the temporal and spatial profiles of the sparks, the following mean values were estimated for the morphological parameters: rise time, sim;4 ms; peak amplitude, sim;1 ΔF/F (change in fluorescence divided by resting fluorescence); decay time constant, sim;5 ms; full duration at half maximum (FDHM), sim;6 ms; late offset, sim;0.01 ΔF/F; full width at half maximum (FWHM), sim;1.0 μm; mass (calculated as amplitude × 1.206 × FWHM3), 1.3-1.9 μm3. Although the rise time is similar to that measured previously in frog cut fibers (5-6 ms; 17-23°C), cut fiber sparks have a longer duration (FDHM, 9-15 ms), a wider extent (FWHM, 1.3-2.3 μm), and a strikingly larger mass (by 3-10-fold). Possible explanations for the increase in mass in cut fibers are a reduction in the Ca2+ buffering power of myoplasm in cut fibers and an increase in the flux of Ca2+ during release.