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Regulation of human RYR2 by intracellular Ca2+ and Mg2+

K. Walweel,1 N.A. Beard,2 J. Li,1 D.F. van Helden,1 M.S. Imtiaz,1 P. Molenaar3 and D.R. Laver,1 1School of Biomedical Sciences, Univ of Newcastle and Hunter Medical Research Institute, Callaghan, NSW 2308, Australia, 2John Curtin School of Medical Research, Australian National University, ACT 0200, Australia and 3School of Medicine, University of Queensland, QLD 4032, Australia.

Heart failure is a complex disorder thah involves changes in Ca2+ handling protein expression, Ca2+ homeostasis, and tissue remodelling. The Ca2+ release channel (RyR2) activates and modulates heart function by controlling the Ca2+ release from SR. RyR2 is controlled by four different Ca2+/Mg2+-dependent mechanisms (Laver & Honen, 2008). Release of Ca2+ from the SR is stimulated by Ca2+ activation of RyR2 as a result of Ca2+ binding to either the cytoplasmic side (A-site, in the case of Ca2+ influx through voltage-dependent L-type channels) or luminal side of the channel (L-site, in the case of SR overload). In addition, there are two Ca2+ inactivation sites (I1 and I2) located on its cytoplasmic face. Intracellular Mg2+ (∼1 mmol/l) inhibits RyRs and acts as a ‘brake’ for Ca2+ release. In diastole, Mg2+ is a competitive antagonist for Ca2+ at the A- and L-sites and also inactivates RyR2 via the I1-site, which has similar affinity for both Ca2+ and Mg2+ (Laver, Baynes & Dulhunty, 1997). During systole, Mg2+ inhibition occurs mainly via the I1-site (Laver, Baynes & Dulhunty, 1997). This model is used here as a framework in which to understand how Ca2+ and Mg2+ regulate RyR2 in human heart and how it may differ from that of established animal models such as sheep and rat hearts.

RyR2 was isolated from failing human (Emery Dreifuss Muscular Dystrophy with cardiomyopathy, ischemic cardiomyopathy, and dilated cardiomyopathy), non-failing human, rat and sheep heart muscle as described previously for sheep RyRs (Laver et al., 1995). Human tissues were obtained with approval from the Ethics Committee, while animal tissues obtained with approval from the Animal Care and Ethics Committee of the University of Newcastle Australia. Channel gating was measured by single channel recording in the presence of ATP (2 mmol/l) and varying concentrations of Ca2+ and Mg2+. Initially, we compared the activity of RyRs from four non-failing human hearts in bilayer experiments using 100 nmol/l Ca2+ in the cytoplasm (diastolic [Ca2+]), and 0.1 mmol/l Ca2+ in the lumen. Under these conditions, RyRs from all hearts showed similar gating activity. RyRs from failing hearts were significantly higher in activity compared to healthy heart. Western blots of RyR2 showed higher phosphorylation at PS2808 and PS2814 in failing hearts, consistent with the proposal that in failing hearts, RyR2 activity is increased due to hyperphosphorylation as a result of upregulation of CaMKII and PKA (Marx et al., 2000).

Regulation by intracellular Ca2+ and Mg2+ from human RyR2 was also compared to that seen in two commonly used animal models for RyR function, rat and sheep. We found that cytoplasmic Ca2+ dependence of RyRs Po from sheep, rat, and human showed similar bell-shaped responses to cytoplasmic Ca2+ with half-activating concentrations (Ka) of 1-3 μmol/l Ca2+ and half-inhibiting concentration (Ki) of ∼1 mmol/l (Ca2+ binding to the A and I1-site, respectively). All species were similarly inhibited by cytoplasmic Mg2+ in the presence of 100 nmol/l Ca2+ in the cytoplasm (diastolic [Ca2+]). However, at high cytoplasmic Ca2+, RyR2 from rat was 10-fold more sensitive to cytoplasmic Mg2+ than sheep and human. RyRs from the three species could be activated by luminal Ca2+. RyRs showed a single, hyperbolic dependence on luminal Ca2+ with maximum opening rate of 2/s, 4/s, and 18 for rat, sheep, and human, respectively. Human hearts were 10-fold more sensitive to luminal Ca2+ than those from rat and sheep, corresponding to the Ca2+ affinity of the L-site. However, human RyR2 is 4-5 fold less sensitive to luminal Mg2+ at low luminal [Ca2+] in comparison to rat and sheep. These differences in the regulation between human, sheep and rat RyRs may reflect differences in excitation-contraction coupling in species with very different basal heart rates.

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