Intracellular Free Ca2+ Movements in Cultured Cardiac Myocytes as Shown by Rapid Scanning Confocal Microscopy

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Abstract
Two-dimensional images of intracellular free Ca2+ movements in cultured cardiac myocytes were obtained at 33-ms intervals with a Ca2+ -sensitive fluorescence probe, fluo-3, and a rapid scanning confocal laser microscope, a prototype of Nikon RCM8000. The cells used were isolated from the ventricular myocardium of neonatal mice, cultured for ≈72 h and loaded with fluo-3. One type of cytoplasmic Ca2+ movement observed was a simultaneous increase in [Ca2+] throughout the cytoplasm, termed a “spike”; another type was a local increase in [Ca2+] propagating in the cytoplasm, termed a “wave.” Cells with either spike or wave or both types of movements were observed. Tetrodotoxin (TTX) 10-5M, nicardipine 10-6M, and increased extracellular potassium concentration (40 mM) selectively inhibited spike, and ryanodine 10-6M and cyclopiazonic acid (CPA) 3 × 10-6M selectively inhibited wave. These results indicate that spike was triggered by depolarization-induced Ca2+ influx across the sarcolemma, whereas wave was a propagating local increase in Ca2+ due to Ca2+ release from the sarcoplasmic reticulum (SR). On spike, nuclear [Ca2+] was shown to increase and decrease synchronously with cytoplasmic [Ca2+], with a delay and slower time course.

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