IP_3-dependent nuclear Ca~(2+) signalling in the mammalian heart

摘要

In cardiac myocytes the type-2 inositol 1,4,5-trisphosphate receptor (IP_3R2) is the predominant isoform expressed. The IP_3R2 channel is localized to the SR and to the nuclear envelope. We studied IP_3-dependent nuclear Ca~(2+) signals ([Ca~(2+)]_(Nuc)) in permeabilized atrial myocytes and in isolated cardiac nuclei. In permeabilized myocytes IP_3 (20 mum) and the more potent IP_3R agonist adenophostin (5 mum) caused an elevation of [Ca~(2+)]_(Nuc). An IP_3-dependent increase of [Ca~(2+)]_(Nuc) was still observed after pretreatment with tetracaine to block Ca~(2+) release from ryanodine receptors (RyRs), and the effect of IP_3 was partially reversed or prevented by the IP_3R blockers heparin and 2-APB. Isolated nuclei were superfused with an internal solution containing the Ca~(2+) indicator fluo-4 dextran. Exposure to IP_3 (10 mum) and adenophostin (0.5 mum) increased [Ca~(2+)]_(Nuc) by 25 and 27%, respectively. [Ca~(2+)]_(Nuc) increased to higher levels than [Ca~(2+)]_(Cyt) immediately adjacent to the outer membrane of the nuclear envelope, suggesting that a significant portion of nuclear IP_3 receptors are facing the nucleoplasm. When nuclei were pretreated with heparin or 2-APB, IP_3 failed to increase [Ca~(2+)]_(Nuc). Isolated nuclei were also loaded with the membrane-permeant low-affinity Ca~(2+) probe fluo-5N AM which compartmentalized into the nuclear envelope. Exposure to IP_3 and adenophostin resulted in a decrease of the fluo-5N signal that could be prevented by heparin. Stimulation of IP_3R caused depletion of the nuclear Ca~(2+) stores by approximately 60% relative to the maximum depletion produced by the ionophores ionomycin and A23187. The fluo-5N fluorescence decrease was particularly pronounced in the nuclear periphery, suggesting that the nuclear envelope may represent the predominant nuclear Ca~(2+) store. The data indicate that IP_3 can elicit Ca~(2+) release from cardiac nuclei resulting in localized nuclear Ca~(2+) signals.