Mervi Sepp

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 purpose, an interdisciplinary approach combining experimental measurements and mathematical modeling was used. Specifically, kinetic analysis was applied to rat heart cells, whereas experimental data was systematically collected from relaxed cardiomyocytes at the cell-population level. The results confirm significant diffusion restrictions at the mitochondrial outer membrane and demonstrate the existence of diffusion obstacles in the cytosolic compartment. The conclusion that diffusion restrictions indeed have an intracellular origin is in accordance with single-cell kinetics studies, providing stronger evidence against theories explaining these results as probable experimental artifacts.

The work revealed the 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 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 a minor role for SERCA in our preparation. NKA, however, was shown to account for up to about 45% of the total ATPase activity under our conditions and to be strongly coupled to glycolysis via pyruvate kinase.

The developed kinetic approach was applied to different animal models. Mathematical models were used to compare the compartmentalization of cellular energetics in wild-type mice and 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 an effectively inactive CK system. This is contrary to widespread acknowledgment of the importance of the 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 a preference for energy production routes: glycolysis or oxidative phosphorylation. This finding proves the importance of glycolysis even in highly oxidative tissue such as heart muscle. The main result of this dissertation is an interdisciplinary method and its application to unravel intracellular compartmentation in the heart, which widens our understanding of energy transfer in this organ.

Supervisors

  • 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 at http://digi.lib.ttu.ee/i/?930.

The defense presentation can be viewed here.