INTRACELLULAR FREE CA2-SCANNING CONFOCAL MICROSCOPY( MOVEMENTS IN CULTURED CARDIAC MYOCYTES AS SHOWN BY RAPID)

Two-dimensional images of intracellular free Ca2+ movements in culture d cardiac myocytes were obtained at 33-ms intervals with a Ca2+-sensit ive fluorescence probe, fluo-3, and a rapid scanning confocal laser mi croscope, a prototype of Nikon RCM8000. The cells used were isolated f rom the ventricular myocardium of neonatal mice, cultured for similar to 72 h and loaded with fluo-3. One type of cytoplasmic Ca2+ movement observed was a simultaneous increase in [Ca2+] throughout the cytoplas m, termed a ''spike''; another type was a local increase in [Ca2+] pro pagating in the cytoplasm, termed a ''wave.'' Cells with either spike or wave or both types of movements were observed. Tetrodotoxin (TTX) 1 0(-5) M, nicardipine 10(-6) M, and increased extracellular potassium c oncentration (40 mM) selectively inhibited spike, and ryanodine 10(-6) M and cyclopiazonic acid (CPA) 3x10(-6) M selectively inhibited wave. These results indicate that spike was triggered by depolarization-ind uced Ca2+ influx across the sarcolemma, whereas wave was a propagating local increase in Ca2+ due to Ca2+ release from the sarcoplasmic reti culum (SR), On spike, nuclear [Ca2+] was shown to increase and decreas e synchronously with cytoplasmic [Ca2+], with a delay and slower time course.