Estimation of Diffusion
Restrictions in Cardiomyocytes
Using Kinetic Measurements
Opponents: Jeroen Jeneson, PhD
BioModeling and Bioinformatics, Eindhoven University of Technology
Agu Laisk, DSc, Prof
Institute of Molecular and Cell Biology, University of Tartu
PhD Defense
Overview

Sepp, Vendelin, Vija, Birkedal
ADP compartmentation analysis reveals coupling between pyruvate kinase and ATPases in heart muscle.
Biophysical Journal, 2010.
 Jepihhina, Beraud, Sepp, Birkedal, Vendelin Permeabilized rat cardiomyocyte response demonstrates intracellular origin of diffusion obstacles. Biophysical Journal, 2011.
 Illaste, Kalda, Schryer, Sepp Life of mice  development of cardiac energetics. Journal of Physiology, 2010.
 Branovets, Sepp, Kotlyarova, Jepihhina, Sokolova, Aksentijevic, Lygate, Neubauer, Vendelin, Birkedal Unchanged mitochondrial organization and compartmentation in creatine deficient GAMT/ mouse heart. AJPHeart and Circulatory Physiology, 2013.
 Sepp, Sokolova, Jugai, Mandel, Peterson, Vendelin Glycolysiscoupled Na+/K+ATPase has high activity in permeabilized rat cardiomyocytes. Submitted 2013.
Cellular energetics
AIM
characterize intracellular compartmentation in a relaxed heart cell
Evidence of diffusion restrictions
Km
fibers CM 0.3  0.5mM
mitochondria 0.015mM
Saks et al (Biochim Biophys Acta 1991)
Kümmel (Cardiovascular research 1988)
Restriction on MoM
Evidence of diffusion restrictions
Km
Cardiomyocytes 0.3  0.5mM
Isolated mitochondria 0.015mM
Saks et al (Biochim Biophys Acta 1991)
Restriction on MoM
...there is more
Seppet et al (Biochimica et Biophysica Acta 2001)
Saks et al (Biochem. J. 2001)
Saks 2001:
Skinned rat soleus fibers: "A powerful ADPtrapping system (PK+PEP) decreased the rate of respiration initiated by the addition of 2mM ATP not more than 20%. ...total activity of PK (20 units/ml) exceeded the combined rates of the ATPase and oxidative phosphorylation in oxygraphic cells (maximally 0.2umol/min per ml) by two orders of magnitude"
Skinned rat cardiac fibers:"...2mM MgATP, was inhibited not more than 40% by PK+PEP"
Why are diffusion restrictions important?
Boudina et al (Am J Physiol Heart Circ Physiol 2002)
Overall design
Competition for ADP
PEP in solution activates
endogenous PK?
Experimental setup
 ATP production through respiration
 ATP utilization through cytosolic ATPase activity
Mathematical Modeling
Mathematical Modeling
Diffusion: Fick's Law
Mathematical Modeling
Enzyme kinetics: MichaelisMenten
Results:

Series of characteristic experiments to study compartmentalization

Collection of mathematical models to analyze the data

some ATPases coupled to endPK
SERCA
SR Ca pumpNKA
cell membrane Na/K pumpIntracellular Na 10mM
Extracellular Na 135145mM
14:1
Intracellular K 155mM
Extracellular K 3.55mM
1:30
Is SERCA active?
RESULT: SERCA is inactive in our conditions
Is NKA active?
RESULT: NKA is active in our conditions
Application of the approach showed NKA tightly coupled to endogenousPK
Applications of approach
Diffusion restrictions  Alternative explanation
Diffusion restrictions not intracellular but caused by clumping of cells and change in effective diffusion distances
Kongas et al (Am J Physiol Cell Physiol, 2002)
vs
Assessment of cell respiration using autofluorescence
Experiments performed by Natalja Jepihhina
NADH florescence
Experiments performed by Natalja Jepihhina
Hypothesis: ADP accumulation in solution in population studies?
Single cell vs population
Analysis of the experimental setup
Dashed  fast diffusion
Dotted  ADP supplied by just diffusion
Black solid  flow + ADP diffusion
Red  DR Km=0.015mM, Green  0.3mM, Blue  0.45mM
Application of the approach on mice
WT vs GAMTdeficient mice
GAMT (Guanidinoacetate ethyltransferase) one of the enzymes on Cr synthesis
pathway
Experiments performed by Jelena Branovets
Analysis of kinetic data with mathematical models showed no differences in kinetic parameters WT vs GAMTdeficient mice
Summary
 Experimental and numerical approach to study intracellular compartmentation was developed
 Approach applied on rat
 Analysis demonstrated the presence of coupling between endPK and a fraction of ATPases
 Analysis demonstrated the presence of coupling between endPK and a fraction of ATPases
 NKA was found to be coupled to endPK
 Demonstration of intracellular origin of diffusion restrictions
 Approach applied on mice
 Wildtype
 GAMTdeficient
 No adaptational changes in the compared animal models
KmATPsyn=0.015mM
KmATPsyn=0.056mM Jacobus 1982
Sigmoidal relationship for VATPsyn Jeneson 1996
Wash solution (mM): NaCl 117, KCl 5.7, NaHCO3 4.4, MgCl2 1.7, KH2PO4 1.5, sucrose
120, BES 21, taurine 20, glucose 11.7 and creatine 10. pH was adjusted to 7.1 with NaOH at 25
Digestion solution (mM): NaCl 117, KCl 5.7, NaHCO3 4.4, MgCl2 1.7, KH2PO4 1.5, HEPES 21.1, taurine 20, glucose 11.7, creatine 11, phoshocreatine 10, pyruvate 2, 2 mg/ml BSA and 0.751 mg/ml
Mitomed solution (mM): KH2PO4 PO2 3, MgCl2 sucrose 110, Klactobionate 60, taurine 20, HEPES 20, EGTA 0.5, DTT 0.5, malate 2, glutamate 5 and 5mg/ml BSA. pH adjusted to 7.1 with KOH at 25C
Chemical composition of cytosol
ATP 0 in vivo 5  10
ADP 0 in vivo measured 12mM, free1050uM
Pi 3 in vivo < 1mM
pH 7.1
Na ca 4 (storage + Mitomed + pH adjustment)
K 3
Ca ~ 0
Mg 3
PEP 0
pyruvate 0
lactate 0
ATP inhibition of respiration
Jacobus et al, JBC 1982
Isolated mitochondria, VO2(ADP)
Jacobus et al, JBC 1982
Jeneson et al 1996 JBC
thesis
By Mervi Sepp