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Title: Spontaneous Ca2+ waves in rabbit cardiac myocytes : a modelling study
Author: MacQuaide, Niall
ISNI:       0000 0001 3615 9839
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2004
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Propagating intracellular Ca2+ waves in cardiac myocytes occur as a consequence of the overloaded state of the sarcoplasmic reticulum (SR). To examine these events in detail, ventricular cardiomyocytes were isolated from rabbit hearts and permeabilised with beta-escin. Cytosolic Ca2+ signals were monitored using Fluo-5F (10muM) in combination with laser-scanning confocal microscopy. Through careful calibration of the intracellular Ca2+ signals and construction of analysis programs, the fluxes which underlie the Ca2+ wave were derived and subsequently incorporated into a mathematical model. The decline in cytosolic Ca2+ subsequent to rapid application of caffeine was used to quantify cellular Ca2+ diffusional loss (diffusional constant = 31.2+/-0.9 s-1). binding to cellular proteins was then calculated and the sum of the free Ca2+, bound Ca2+ and Ca2+ lost by diffusion was used as the integral of the Ca2+ flux across the SR. The first derivative of this was taken as the trans-SR flux rate. From the analysis of these signals it was apparent that the released from the SR during a wave was not significantly different from that released on application of lOmM caffeine (0.149 +/-0.10 mM vs. 0.154+/-0.10 mM)). This information, coupled with values of intra-SR buffering allowed calculation of intra-SR [Ca2+]. This in turn allowed the trans SR [Ca2+] gradient to be estimated and the subsequent calculation of RyR and SERCA mediated Ca2+ flux . These measurements were used to derive parameters for construction of a 3- compartment model of Ca2+ flux using existing models of Ca2+ buffering, SERCA activity and leak. Three experimental interventions were used to study changes in Ca2+ wave properties and assess the effectiveness of the model in predicting wave frequency, minimum and maximum [Ca2+]. These were: (i) changing extracellular Ca2+, (ii) inhibiting the RyR using tetracaine and (iii) inhibiting SERCA using 2',5'-di(tert- butyl)-l ,4-benzohydroquinone (TBQ). As cytosolic Ca2+ was increased from 300 to 900nM, so frequency and systolic Ca2+ were shown to increase nonlinearly, whilst diastolic [Ca2+] increased linearly. Calculated SR release threshold was found not to change. SERCA Vmax and KD both increased, with Vmax rising from 160 to 380 muMs-1 and KD rising from 239+/-48 to 354+/-18nM as extracellular [Ca2+] was increased from 300nM to 900nM. The calculated peak permeability of RyR mediated flux also increased from 41.1 +/-6.5 to 61.2+/-3.6 s-1 over this range. These changes, when included in the model, subsequently provided acceptable predictions of experimental results. Tetracaine caused frequency of the Ca2+ waves to decrease from 0.59+/-0.03 Hz to 0.35+/-0.02 Hz, systolic [Ca2+] to increase from 2.06+/-0.11 muM to 3.16+/-0.24 muM and diastolic [Ca2+] to decrease from 185+/-9 nM to 157+/-10 nM. Flux analysis indicated that these changes were associated with an increase in the SR release threshold from 1.16+/-0.04 mM to 1.58+/-0.08 mM (n=6). Implementation of this threshold change in the computational model predicted a decrease in Ca2+ wave frequency to a similar value to that observed experimentally. The increased systolic [Ca2+] was comparable to but greater than that observed experimentally. In contrast, the model predicted diastolic [Ca2+] to increase while a decrease diastolic was observed experimentally. Application of the SERCA inhibitor TBQ (1muM) decreased SR Ca2+ content, the amplitude and frequency of Ca2+ waves. Analysis of the underlying fluxes suggested that TBQ caused a 43% reduction in SERCA Vmax, with no significant change in KD. Analysis also suggested that this reduction in Vmax was accompanied by a 25% reduction in SR release threshold. While the reduced SERCA Vmax is consistent with TBQ's known action on SERCA, the effect on Ca2+ wave amplitude was unexpected and cannot be easily explained with the current Ca2+ wave model.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available