- Title
- Regulation of calcium release channels (RyR2) in healthy and failing human hearts
- Creator
- Walweel, Kafa
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2014
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Heart failure (HF) is a complex disorder that involves changes in Ca2+ handling protein expression, Ca2+ homeostasis and tissue remodelling. A general feature of heart dysfunction associated with HF is aberrant Ca2+ flux across the sarcolemma and the sarcoplasmic reticulum (SR) of cardiac cells. The Ca2+ release channel (RyR2) activates and modulates heart function by controlling the Ca2+ release from the SR. The RyR2 forms a complex with many accessory proteins that regulate channel activity. The regulation of the RyR2 from human heart by intracellular Ca2+ and Mg2+ is still poorly defined. In this study single channel recordings of RyR2s were used to compare Ca2+ and Mg2+ regulation of RyR2s from patients with healthy, cystic fibrosis (CF) and failing hearts, specifically, Ischaemic cardiomyopathy (ICM) and Emery Dreifuss muscular dystrophy (EDMD, a rare inherited disorder that affects skeletal and cardiac muscle). We also compared these functional changes with r emodelling of the macromolecular structural properties using Western blot. RyR2s from healthy human hearts (n=4) were incorporated into lipid bilayers and the channel gating was measured at diastolic [Ca2+]. Under these conditions, at least 90% of the channels had open probabilities (Po) that comprised a normal distribution on a logarithmic scale with a mean Po= 0.02 and a standard deviation of 6.3 fold. Grouping the data according to the four donor hearts revealed no significant difference between these groups in the Ca2+ and Mg2+ regulation of RyRs. Initially, a CF heart was the only source of non-failing samples. However, as the study progressed and hearts became available from healthy donors, it became clear that RyR2 from the CF heart differed from those obtained from four healthy hearts and these are now reported as a separate group. This thesis is the first to report on the regulation by Ca2+ and Mg2+ of native RyR2 receptor activity from healthy human hearts. Human RyR2s displayed cytoplasmic Ca2+ activation (Ka = 6 μM) and inhibition by cytoplasmic Mg2+ (Ki = 10 μM at 100 nM cytoplasmic Ca2+). The Ka for luminal Ca2+ activation was 35 μM and the Ki for luminal Mg2+ inhibition was 550 μM (cytoplasmic [Ca2+] = 100 nM). Modulation of RyR2 gating by luminal Ca2+ and Mg2+ only occurred when the cytoplasmic [Ca2+] was less than 1 μM. In this range, luminal and cytoplasmic Ca2+have a synergistic action on the RyR2 opening rate where cytoplasmic Ca2+ increased the luminal Ca2+ response. The activation response of RyR2 to luminal and cytoplasmic Ca2+ was strongly dependent on the Mg2+ concentration. Addition of physiological levels (1 mM) of Mg2+ raised the Ka for cytoplasmic Ca2+ to 25 μM and raised the Ka for luminal Ca2+ from below 60 μM to ~1 mM. Single channel studies showed that RyRs from CF and failing human hearts exhibited higher activity (4-fold for CF, 5-fold for ICM, 13-fold for EDMD, and 35-fold for ICM trabecule) where cytoplasmic solutions contained diastolic concentrations of Ca2+ (100 nM Ca2+) and 2 mM ATP and luminal solutions contained 0.1 mM Ca2+, at -40mV. At high cytoplasmic Ca2+ concentrations (systolic [Ca2+], 10-100 μM), RyR2s from healthy and failing human hearts showed similar channel activity. RyR2s from CF hearts showed similar inhibition by intracellular Mg2+ to healthy hearts, whereas RyR2s from failing hearts exhibited less sensitivity (10-1000 fold attenuation) to intracellular Mg2+ inhibition. The attenuation of Mg2+ inhibition and increased RyR2 activity at low [Ca2+] would cause these channels to leak Ca2+ from the SR during diastole. The distribution of RyR2 activity from CF, EDMD and ICM hearts compared to healthy heart was also examined. RyR2s from healthy heart showed a large variation in open probability, ranging from 0.001 up to 0.75. RyR2s from CF heart showed a similar range of open probability values but the distribution was skewed to higher open probability in CF hearts. This preliminary finding suggests that pulmonary stress associated with cystic fibrosis induces a form of HF in this group that has not previously been identified. However, RyR2s from failing hearts showed greater scatter than those from healthy hearts. Distributions of the opening rate from failing hearts showed two groups of activity. One group (low activity group consisting of 85% of RyR2 in ICM hearts and 50% of RyR2 in EDMD hearts and none from ICM trabecule) had a similar distribution to healthy hearts whilst the other high activity group, exhibited more than a 10-fold higher mean value. These differences in the proportion of high activity RyR2s compared to those from healthy hearts suggest that a sub population of RyR2 are altered during HF. Western Blots of RyR2s showed higher phosphorylation at PS2808 and PS2814 in both ICM and EDMD hearts. They also exhibited decreased expression levels of RyR2s and dephosphorylating enzymes, PP1 and PP2A. SERCA2a was decreased in ICM heart. CSQ2 and triadin-1 were reduced in EDMD heart. Finally, both FKBP12 and FKBP12.6 were found to be dissociated from RyR2s in failing hearts. These findings indicate that RyR2 remodelling in HF may have a role in RyR2 hyperactivity at diastolic [Ca2+] and reduced sensitivity to intracellular Mg2+ inhibition. It has been shown that calmodulin (CaM) binds to healthy human RyR2 with high affinity, while in HF RyR2 shows decreased CaM affinity. Binding of CaM to RyR2s from mice cardiomyocytes inhibits SR Ca2+ release. However, no one has examined the effect of CaM on the gating of human RyR2s and how it may be affected by RyR2 remodeling in failing hearts. Importantly, this study showed that the regulatory action of CaM depends on whether RyR2s were obtained from healthy or failing hearts. Addition of CaM (0.5 μM) to the cytoplasmic bath caused a reduction (~ 35%) in the open probability of RyR2s from failing hearts. Surprisingly, RyR2s from healthy human heart were not affected by CaM addition which was also different to what is seen in commonly used animal models for RyR2 function in HF, dog and sheep. These findings suggest that animals may not provide accurate models for the RyR2 function in humans. These data also indicate that the effect of CaM binding to RyR2s depended on the phosphorylation state of the RyR2, which is different in healthy and failing hearts.Regulation by intracellular Ca2+ and Mg2+ from human RyR2s was also compared to that seen in two commonly used animal models for RyR function, rat and sheep. Human RyR2s displayed the same regulation by cytoplasmic Ca2+/ Mg2+ as seen in rat and sheep. However, RyR2 sensitivity to luminal Ca2+ and Mg2+ varied between species. The Ka’s for luminal Ca2+ activation were 35 μM for human, 60 μM for sheep and 10 μM for rat whilst peak activation for human RyR2s and sheep were similar but 10-fold higher than rat RyR2s. Furthermore, human RyR2s were 10-fold less sensitive to luminal Mg2+ than RyR2s from rat and sheep. Together with CaM results, these differences highlighted the importance of using RyR2s from healthy human tissues in lieu of using animal models to understand how RyR2 function is altered in HF.
- Subject
- heart failure; Ca2+ release channels
- Identifier
- http://hdl.handle.net/1959.13/1055964
- Identifier
- uon:15961
- Rights
- Copyright 2014 Kafa Walweel
- Language
- eng
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