Characteristics of Oxalate Supported Calcium Uptake and Release from the SR of Permeabilised Rabbit Ventricular Myocytes

This thesis investigated the characteristics of sarcoplasmic reticulum (SR) calcium uptake and release from permeabilised cardiac myocytes: focusing on adenosine trisphosphate calcium pump (Ca2+ -ATPase pump), the ryanodine receptor (RyR) and the non-specific SR leak. In order to do this, the following aims were investigated. 1. Cell viability: do balled-up cells contribute to total cell protein? 2. Is the rate of calcium uptake and leak affected by SR calcium load, RuR and by varying the oxalate concentration? 3. How sensitive is this preparation to stimulatory and inhibitory concentrations of ryanodine? 4. To what extent is the caffeine-mediated calcium leak blocked by ryanodine and ruthenium red (RuR)? 5. What are the characteristics of the RuR/ryanodine insensitive SR leak? Method Cardiac myocytes from New Zealand White, male rabbits were dissociated using the Langendorff retrograde perfusion system. The cells were permeabilized with lOOj..lglml 13escin, and measurements of SR calcium uptake were made using the fluorescent indicator Fura-2 (1OJ.lM). Calcium Uptake Versus protein Measurements The protein measurements of permeabilised cardiac myocytes. indicated that the haemocytometer was an accurate and consistent method of cell counting and that rod shaped myocytes did not influence the total protein concentration to a significant degree. Therefore, balled-up cells contribute to total cellular protein and may retain functional calcium pumps as a greater number of cells were observed to induce an increase in the calcium uptake rate constant, i.e. a greater number of cells give rise to more functional pumps available to sequester calcium. Characteristics of Calcium Leak A significant leak, referred to as the non specific leak, exists through the RyR at IJ.lM free calcium concentration and is independent of conventional RyR activity. In the absence of a calcium challenge, an SR calcium leak was not observed suggesting that the SR calcium content is negligible at the start of the experiment but with a single calcium challenge, a sufficient SR load was generated to sustain a leak. It was noted that 3 calcium challenges produced reproducible calcium transients, induced a large SR leak gradient in the presence of 5J.lM thapsigargin, as well as generating a large steady state calcium level in the presence of 10J.lM ionomycin. This suggests that the sequential additions of calcium to the cuvette system caused a progressive increase in the luminal free calcium concentration and SR calcium load. In this study, 10 or 20mM oxalate effectively clamped intracellular calcium concentration in order to facilitate calcium uptake into the SR. This was noted by a faster rate of decay, a lower SR leak gradient and a reduction in the amount of calcium available for release. Ryanodine Mediated Calciwn leak In response to repeated calcium challenges in the absence of RuR or ryanodine, a gradual increase in the rate of uptake of the calcium transient was observed which could arise from the gradual phosphorylation of phospholamban. Low concentrations of ryanOdine (0.3J.lM and 3J.lM) were observed to induce a sustained increase in the SR calcium leak from the RyR. High concentrations of ryanodine (30J.lM, 300J.lM and 3mM) induced an increase in the calcium leak from the SR for a short period before a reduction in the SR leak was noted and, during the given time frame, oxalate supported cuvette based experiments favour a block with 3mM ryanodine. This is consistent with the literature that suggest high concentrations of ryanodine are required to decrease the opening probability of the RyR and that RyR possess two binding sites for ryanodine (a high affinity and a low affinity site). The leak produced in the presence of 0.3J.lm ryanodine was not inhibited with 5J.lM RuR and a slight slowing of the rate of uptake was observed with the addition of 5J.lM RuR in the presence of 3mM ryanodine. Therefore, 5J.lM RuR in the presence of 3mM ryanodine or repeated calcium challenges may enhance the detrimental effects of SERCA2A noted by Kargacin & Kargacin (1998). Caffeine Mediated Calcium Leak In this study it was observed that caffeine mediated calcium release from the SR of permeabilised cardiac myocytes occurred in a dose dependent manner and that even in the presence of high concentrations of caffeine (up to 20mM), the SR'accumulated a significant amount of calcium but only in the presence of ATP. At caffeine concentrations above 20mM, calcium reuptake was observed to be incomplete. At low intracellular calcium concentration (in the absence of calcium challenges) 40mM was required to induce a significant release of calcium from the SR, a release that was greater than that produced with 20mM caffeine. Howev~r, in the presence of activating calcium concentrations, as low as 2mM caffeine was observed to induce an SR leak. 3mM ryanodine as well as 5J.IM RuR inhibited caffeine mediated calcium release and, varying the oxalate concentration did not· significantly affect the ryanodine block of the caffeine mediated leak. SR calcium Leak and Bastadin Mix In isolated cardiac muscle preparations thapsigargin unmasks an SR calcium leak. A portion of this leak was blocked with the addition of 5J.IM RuR and 3mM ryanodine but only when the free calcium concentration ex~eeded 500nM. This indicates that the RyR c~ntributes greatly to the SR leak under these conditions. Interestingly, the fraction of the SR leak that remained in the presence of blocking concentrations of RuR or ryanodine was inhibited by 20f1M Bastadin mix (5-10 Bastadin Mix), therefore a component of the SR leak may be due to the RyR being in an open configuration that can be blocked by Bastadin. In conclusion, balled-up cells were included in the cell count for the cuvette based uptake studies as they were deemed as viable cells and may retain functional pumps. The gradual increase in the rate of calcium uptake of the calcium transient observed in response to repeated calcium challenges could arise from the gradual phosphorylation of phospholamban. A substantial leak exists through the RyR under control conditions as 5J.IM was observed to induce a significant increase in the rate of calcium uptake. The non specific leak unmasked by 5JlM thapsigargin is RuRiryanodine insensitive ~nd is influen'ced by SR calcium load and oxalate concentration. The oxalate supported cuvette based experiments favour 3mM ryanodine and 5JlM RuR in order to block the SR calcium leak observed in the presence of a calcium challenge as well as blocking caffeine mediated SR leak. The inhibition by ~OJlM Bastadin mix of the SR leak that remained in the presence of blocking concentrations of RuR or ryanodine indicated that a component of the SR leak may be due to the RyR being in an open configuration. /EThOS - Electronic Theses Online ServiceGBUnited Kingdo