Investigation of factors affecting the sodium/calcium exchanger in a rabbit model of left ventricular dysfunction

The sodium-calcium exchanger (NCX) is the major calcium efflux pathway in the cardiomyocyte and thus is intrinsically involved in intracellular Ca2+ ([Ca2+]i) homeostasis. Previous work has identified that in a rabbit infarct model of left ventricular dysfunction (LVD), NCX protein is increased whilst NCX activity is decreased. This thesis examines the sub-cellular basis for this dichotomy. Investigation of the factors regulating NCX including the transverse-tubule (t- tubule) network, the proteins sorcin and phospholemman and NCX phosphorylation was performed and levels were quantified in LVD and control (sham) hearts using various techniques. The fluorophore di-8-ANNEPS and confocal microscopy were used in isolated rabbit cardiomyocytes to label the t-tubule network. Quantification of the mean t-tubule density revealed a 25% reduction in LVD compared to control. On permeabilisation, this difference was not apparent indicating that this percentage of t-tubules in LVD was uncoupled from the surface sarcolemma and disconnected from the extracellular space. NCX protein is reported to be concentrated in the t-tubule network; therefore, NCX protein contained in the internalised tubules would be isolated rendering it non-functional. Sorcin over-expression has been shown to stimulate NCX activity in the rabbit. Quantitative Western blotting of whole left ventricular homogenates showed that sorcin dimer expression was reduced by 33% in LVD vs. sham. This was independently confirmed using monoclonal and polyclonal primary antibodies. Biochemical analysis of the phosphorylation status of the membrane protein phospholemman (PLM) showed no significant difference at either of the two major phosphorylation sites. In vitro back-phosphorylation assessed endogenous levels of phosphorylation of NCX itself and revealed a 3-fold hyperphosphorylation in LVD. Problems associated with this technique and interpretation of these data are discussed in detail. In order to measure the functional effects of sorcin, single cell electrophysiology was carried out. Attempts to use recombinant sorcin (3 mumol/L) were unsuccessful as the low [Ca2+]i buffering required to measure cellular properties made the membranes' mechanical properties unstable. Finally, phosphorylation of sorcin as a modulator of NCX was investigated using constitutively phosphorylated and de-phosphorylated sorcin mutants. These were both ineffective indicating that the tertiary conformation of sorcin is critical for its effect on NCX function. In summary, the results showed that t-tubule density, sorcin dimer expression and NCX phosphorylation status all contributed to the modulation of NCX in LVD whilst phospholemman and phosphorylation of sorcin were eliminated. Simplified estimates and previously published values were used to relate the quantitative change in each of the three factors modulating NCX to the dichotomy and assess their relative contributions in the model. In doing so, it was determined (using previously published data) that the reduced t-tubule network would account for a 16% loss of functioning NCX protein in contact with the extracellular space. Sorcin down-regulation was calculated to result in an 8% reduction in NCX activity. The NCX hyperphosphorylation result requires further confirmation. Combining the effects on NCX activity of t-tubule and sorcin reduction, a 23% reduction in NCX activity was accounted for. As NCX activity was reduced by approximately 57% in the model of LVD additional factors must account for the remaining 20%.