Comparison of IP3R and RyR Expression and Ca2+ Release Characteristics in Isolated Cardiac Nuclei

Abstract

In cardiac muscle, the role of the inositol trisphosphate receptor (IP3R) and its regulation is not fully understood. A contribution to nuclear Ca2+ signalling has been proposed. This study compares expression and Ca2+ release characteristics of the IP3R and the ryanodine receptor (RyR) in purified functional cardiac nuclei. It also examines whether the IP3R may exist as a multi-protein complex in these preparations. Quantitative immunoblotting of IP3R and RyR protein levels in isolated nuclei demonstrated greater expression of the IP3R; nucleolin was used as an internal control for quantification. Ca2+ release in response to IP3 and caffeine from single isolated nuclei was used to compare IP3R and RyR activity. Changes in nuclear [Ca2+] were measured as fluorescence signals from nuclei loaded with 10μM Fluo 5N-AM. IP3 or caffeine was applied by hydrostatic pressure ejection and signals expressed as ratios (F/F0) of fluorescence counts relative to baseline. Ca2+ release in response to IP3 (10μM) was significantly greater than that released in response to caffeine (10mM) (0.12 ± 0.02 v's 0.017 ± 0.002 [Ca2+]Nuc (F/F0) for IP3 and caffeine respectively, n=6). When tetracaine (100μM) was applied to the nuclei, IP3-mediated Ca2+ release was unaffected but the response to caffeine was abolished, suggesting RyR activation does not contribute to IP3-mediated nuclear Ca2+ release. The potential for other nuclear proteins interacting with the nuclear IP3R was also investigated. Immunoblot analysis demonstrated expression of both FKBP12 and calcineurin in cardiac nuclei. These proteins are known to interact with the IP3R in other tissue types. Co-immunoprecipitation experiments using an anti-IP3R (type II) antibody suggest IP3R/calcineurin/FKBP12 interaction specifically at the nucleus. These results highlight the existence of a nuclear multi-protein IP3R complex, providing further scope for regulation of cardiac nuclear Ca2+ release.