Restricted Propagation of Cytoplasmic Ca2+ Oscillation into the Nucleus in Guinea Pig Cardiac Myocytes as Revealed by Rapid Scanning Confocal Microscopy and Indo-1

Abstract

ABSTRACT-Two-dimensional images of cytoplasmic and nuclear free Ca2+ movements in cardiac myo cytes were obtained at 67-msec intervals using a Ca2+-sensitive fluorescence probe, indo-1, and a rapid scanning confocal laser microscope, Nikon RCM8000. Isolated guinea pig ventricular cells were loaded with indo-1 and stimulated at 0.5 Hz through patch pipettes. On stimulation, nuclear Ca2+ concentration ([Ca2+]) was observed to rise and fall following cytoplasmic [Ca2+] with an obvious delay. Application of isoproterenol significantly increased the peak [Ca2+] on stimulation in both the cytoplasm and nucleus with no substantial change in the basal [Ca2+]; the increase in peak [Ca2+]produced by application of isopro terenol was larger in the cytoplasm than in the nucleus. Under a low [Na+] condition, the basal [Ca2+] was increased from the control values in both the cytoplasm and nucleus; no difference in basal [Ca2+] was ob served between the two regions. The increase in peak [Ca2+] by low [Na+] in the cytoplasm was significantly larger than that in the nucleus. When the cells were voltage clamped at 0 mV for 3 sec, no difference in the steady state [Ca2+] was observed between the cytoplasm and nucleus. Nuclear [Ca2+] was also observed to increase following a Ca2+ wave, a local increase in [Ca2+] propagating within the cytoplasm, with a delay. Thus, we demonstrated in isolated myocardial cells that cytoplasmic Ca2+ movements, although hampered by the nuclear envelope, are propagated into the nucleus, a mechanism through which factors affecting cytoplasmic Ca2+ may influence intranuclear events. Keywords:Confocal microscopy, Calcium ion, Indo-1, Myocardium, Nucleus Intracellular Ca2+ is known to be involved in regula tion of various cellular activities such as cell contraction, secretion, differentiation and proliferation. An increase in intracellular Ca2+ concentration ([Ca2+]) was also shown to stimulate DNA synthesis and gene expression, suggesting that nuclear Ca2+ may play important roles in these cellular functions (reviewed in ref. 1). Investigating this subject would be difficult without direct measurement of nuclear [Ca 2+] under various conditions. Development of Ca 2+-sensitive fluoroprobes and their use in combina tion with confocal microscopy have been shown by many biologists to be remarkably powerful for obtaining two dimensional high spatial resolution images of intra cellular [Ca2+] in various cell types (1-7). Several reports have dealt with nuclear and cytoplasmic [Ca2+]. Some workers postulated that the regulation of nuclear [Ca2+] is separate from that of the cytoplasm (5, 6, 8), while others attribute the difference to the nuclear envelope restricting passive diffusion of cytoplasmic Ca 2+ into the nucleus (9, 10).Even fewer studies have dealt with nuclear Ca2+ in myo cardial cells. As the nuclei of isolated myocardial cells are cylindrically shaped with a diameter of a few tm, confocal microscopic observations are desirable for ac curate discrimination of cytoplasmic and nuclear Ca2+...

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