Genetic manipulation of calcium handling proteins in cardiac myocytes: Experiments and mathematical models.

Abstract

This thesis is focused on diastolic dysfunction (DD), a pathological condition characterized by slow myocardial muscle relaxation. Two genetic approaches have been proposed to correct DD, over-expression of the sarcoplasmic reticulum calcium ATPase (Serca2a) and expression of a specialized calcium buffer, not normally present in the heart, parvalbumin (Parv). The aim of this thesis is to perform an in-depth analysis and comparison between these two genetic approaches. Using gene transfer in isolated rat cardiac myocytes, a dose-response was established by examining myocyte function at different time points after gene transfer. We found that there is an optimal range of Parv concentration (0.01--0.1 mM) for which sarcomere shortening relaxation kinetics are accelerated without compromising the contractile amplitude. Above this range, the contractile amplitude is attenuated. Serca2a and Parv myocytes were then compared under several experimental conditions. Serca2a over-expressing myocytes had a blunted beta-adrenergic response when compared to Parv expressing myocytes or controls, but the Serca2a over-expressing myocytes were the least sensitive to variation in pacing rates as assessed during post rest potentiation tests. In addition to this comparison, we established that Parv corrected slow relaxation in two myocyte models of DD, one due to impaired calcium removal, and one due to mutation in a myofilament protein. Finally, a mathematical model specific to the rat myocyte, including ionic channels, ionic homeostasis, force development, and sarcomere movement was developed. The mathematical model fitted the majority of the experimental data and was used to gain mechanistic insights into cardiac myocyte function. In addition, the model was used to make predictions on topics such as the possibility of modifying Parv binding characteristics to optimize its function as a relaxing agent, and the effect of Parv/Serca2a on diastolic calcium levels or on cellular energy consumption.