IP3-dependent nuclear Ca2+ signalling in the mammalian heart

Abstract

In cardiac myocytes the type-2 inositol 1,4,5-trisphosphate receptor (IP(3)R2) is the predominant isoform expressed. The IP(3)R2 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 microm) and the more potent IP(3)R agonist adenophostin (5 microm) 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(3)R 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 microm) and adenophostin (0.5 microm) 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(3)R 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.

Figure 5. Differential IP₃-dependent changes of [Ca²⁺]_Cyt and [Ca²⁺]_Nuc in isolated nuclei

Isolated cardiac nuclei were loaded with fluo-4 dextran and bathed in an internal solution containing 20 μm fluo-4 dextran. A, confocal images of an isolated cardiac nucleus (top). Bottom, changes of [Ca²⁺]_Nuc (average fluorescence recorded from the ROI marked by the dashed black line) and [Ca²⁺]_Cyt (average fluorescence recorded from the ring-shaped ROI delimited by the nuclear border and the white dashed line) induced by stimulation with IP₃ (10 μm). B, average peak amplitude and time to peak (TTP) of [Ca²⁺]_Nuc and [Ca²⁺]_Cyt. The numbers in parentheses indicate the number of individual nuclei tested. *P < 0.001.

Figure 1. IP₃-dependent nuclear and cytoplasmic Ca²⁺ signals in permeabilized myocytes

A, confocal linescan images and [Ca²⁺] (F/F₀) recordings from nuclear ([Ca²⁺]_Nuc; upper trace) and cytoplasmic ([Ca²⁺]_Cyt; lower trace) regions in a permeabilized atrial myocyte. Individual frames were recorded at times indicated. IP₃ (20 μm) caused elevations of [Ca²⁺]_Nuc and [Ca²⁺]_Cyt with distinctly different spatial and temporal patterns. B, mean Ca²⁺ spark frequencies recorded under control (Ctrl) conditions and after 30 and 120 s exposure to IP₃. Percentage increase of basal [Ca²⁺]_Cyt (C) and [Ca²⁺]_Nuc (D) after 30 and 120 s exposure to IP₃, respectively. *P < 0.05 and **P < 0.01 compared to control. n= 7 cells.

Figure 2. IP₃-dependent nuclear Ca²⁺ signals during RyR inhibition

A, [Ca²⁺]_Nuc during inhibition of RyRs with tetracaine (0.7 mm). IP₃ (20 μm) caused an increase of [Ca²⁺]_Nuc. The effect of IP₃ was partially reversed by the IP₃R blocker heparin (Hep; 0.5 mg ml⁻¹). The letters a–c indicate when confocal images (top row) were recorded. Right panel: relative changes of [Ca²⁺]_Nuc (%) induced by IP₃ and IP₃+ Hep (n= 7 cells). B, increase of [Ca²⁺]_Nuc induced by the IP₃R agonist adenophostin (Aden; 5 μm). Right panel: relative changes of [Ca²⁺]_Nuc (%) induced by adenophostin (n= 8 cells). *P < 0.05, **P < 0.01.

Figure 4. Heparin and 2-APB inhibit IP₃-dependent Ca²⁺ signals from isolated nuclei

A, isolated cardiac nuclei with entrapped fluo-4 dextran were exposed to the IP₃R blockers heparin (Hep; 0.5 mg ml⁻¹; top) and 2-APB (10 μm; bottom) followed by exposure to IP₃ (10 μm). B, average percentage increases of [Ca²⁺]_Nuc elicited with IP₃, IP₃+ Hep, and IP₃+ 2-APB. The numbers in parentheses indicate the number of individual nuclei tested. *P < 0.001 compared to IP₃ alone.

Figure 3. IP₃-dependent Ca²⁺ signals from isolated nuclei

A, isolated cardiac nuclei were loaded with the high molecular weight Ca²⁺ indicator fluo-4 dextran. Isolated nuclei with entrapped fluo-4 dextran were exposed to IP₃ (10 μm; top), adenophostin (Adeno; 0.5 μm; middle) and caffeine (Caff; 10 mm; bottom). The numbers 1–3 indicate time when confocal images (top panel) were recorded. B, average percentage increases of [Ca²⁺]_Nuc elicited with IP₃, adenophostin and caffeine. The numbers in parentheses indicate the number of individual nuclei tested. *P < 0.001 compared to the effect of IP₃ and Adeno.

Figure 7. Heparin prevents nuclear Ca²⁺ depletion by IP₃

A, [Ca²⁺]_NE recorded from single cardiac nuclei loaded with fluo-5N AM. Nuclei were pretreated with the IP₃R blocker heparin (Hep; 0.5 mg ml⁻¹), followed by exposure to IP₃ (10 μm). B, normalized average [Ca²⁺]_NE levels under control conditions (100%), and after exposure to IP₃ in the presence and absence of heparin. The numbers in parentheses indicate the number of individual nuclei tested. *P < 0.001.

Figure 6. IP₃-induced Ca²⁺ depletion of the nuclear envelope of isolated nuclei

A, isolated cardiac nuclei were loaded with the membrane-permeant low-affinity Ca²⁺ probe fluo-5N AM. Fluo-5N signal changes (changes of [Ca²⁺] in the nuclear envelope, [Ca²⁺]_NE) in response to IP₃ (10 μm; top), adenophostin (Adeno; 0.5 μm; middle) and caffeine (Caff; 10 mm; bottom). The Ca²⁺ ionophore A23187 was used to determine the remaining fluo-5N signal after maximal depletion of the nuclear envelope. B, normalized average [Ca²⁺]_NE levels under control conditions (100%), and after exposure to IP₃, adenophostin, caffeine, A23187 and ionomycin (Iono). The numbers in parentheses indicate the number of individual nuclei tested. *P < 0.01 and **P < 0.001 compared to control.