Differentiation of EC coupling in stem-cell precursors for myocardial repair The possibility to use stem-cell derived precursors to replace dead myocardial tissue is currently receiving considerable attention. Our group is involved in a European project for the development of strategies to promote in vitro replication and differentiation of stem-cells precursors for therapeutic use. Our specific role within the project is to assess the functional counterpart of differentiation of precursors into cardiac myocytes.
Cardiac excitation-contraction (EC) coupling is based on a complex interplay between cell structure (T-tubules, sarcoplasmic reticulum etc.) and the function of specific proteins associated with the sarcolemma and the sarcoplasmic reticulum (SR) membrane. The development of EC coupling may require highly coordinated organization of such components. Thus, functional analysis of EC coupling may be a very sensitive tool in the assessment of cellular differentiation and is among the studies planned in the context of the european stem-cell project.
Mesenchimal precursor cells will be characterized under basal conditions and after exposure to the differentiation protocols, developed in collaboration with the cell biology units participating in the project.
Membrane currents will be studied by the patch-clamp technique. Intracellular Ca2+ concentration will be measured in single cells by fluorescence (Indo-1) detection under voltage-clamp conditions. By applying appropriate experimental protocols to intact myocytes, it is possible to functionally isolate individual components of the Ca2+ control system, such as the Na+/Ca2+ exchanger, the sarcoplasmic reticulum Ca-ATPase (SERCA), and the sarcoplasmic reticulum Ca2+ release channels (RyR). Their function will then be evaluated through the analysis of changes in the kinetics of Ca2+ transients induced by chemical (caffeine etc.) or electrical stimulation. To assess the relation between intracellular Ca2+ changes and mechanical activity, shortening of single myocytes will be quantified by imaging techniques.
In summary, a PhD student involved in the activities within this project will acquire expertise in the following areas:
Measurement of electrical activity and transmembrane currents in isolated cells by the patch-clamp technique.
Optical measurements of intracellular Ca2+-dynamics by fluorescent dyes.
Theoretical basis for the analysis of Ca2+ transport mechanisms in subcellular compartments.
Cell culture techniques
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