ESTIMATION OF DIFFUSION RESTRICTIONS IN CARDIOMYOCYTES USING KINETIC MEASUREMENTS

ABSTRACT

The aim of this dissertation is to study diffusion restrictions in the energetic pathways of heart muscle cells. For that interdisciplinary approach that combines experimental measurements and mathematical modeling, was used. Specifically, kinetic analysis is applied to rat heart cells whereas experimental data is systematically collected from relaxed cardiomyocytes at cell population level. The results confirm significant diffusion restrictions on mitochondrial outer membrane and demonstrate the existence of diffusion obstacles in cytosolic compartment. The conclusion that diffusion restrictions indeed have intracellular origin, is in accordance with single cell kinetics studies leading to stronger evidence against theories explaining these results with probable experimental artifacts.
The work revealed existence of coupling between a fraction of ATPases and endogenous pyruvate kinase (PK). This finding evoked a new course in the research project – the renewed target was to establish which of the cellular ATPases are linked to endogenous PK. The role of two membrane ATPases – sarcoplasmic reticulum Ca2+ ATPase (SERCA) and sarcolemma Na+/K+ ATPase (NKA), was investigated. The results showed minor role of SERCA in our preparation. NKA however, evidenced to makes up to about 45% of the total ATPase activity in our conditions, was shown to be strongly coupled to glycolysis via pyruvate kinase.
The developed kinetic approach was applied on different animal models. Mathematical models were used to compare the compartmentalization of cellular energetics in wild-type mice and in transgenic mice lacking the GAMT enzyme that produces creatine. The analysis showed no difference in energetic compartmentalization and mitochondrial functioning between GAMT-deficient and wild-type mice. No major structural adaptations were identified in mice with effectively inactive CK system. This is contrary to wide acknowledgment of the importance of CK shuttle in energy transfer.
This thesis demonstrates intracellular compartmentation in rat cardiomyocytes revealing that in this strongly compartmentalized environment a fraction of ATPases have preference over energy production routes – glycolysis or oxydative phosphorylation. Thus proving the importance of glycolysis even in highly oxydative tissue such as heart muscle. The main result of this dissertation is an interdisciplinary method and its application to unravel intracellular compartmentation in heart that widens our understanding in energy transfer of this organ.

SUPERVISOR: Marko Vendelin

OPPONENTS

• Jeroen Jeneson, BioModeling and Bioinformatics, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands
• Agu Laisk, Institute of Molecular and Cell Biology, Chair of Biophysics and Plant Physiology, Faculty of Science and Technology, University of Tartu

TIME OF DEFENSE

22 August 2013 at 13:00 in the Institute of Cybernetics, room B101

THESIS

You can download PDF of the thesis “Estimation of Diffusion Restrictions in Cardiomyocytes Using Kinetic Measurements” here. The defense presentation can be viewed here.