Estimation of Diffusion
Restrictions in Cardiomyocytes
Using Kinetic Measurements


Mervi Sepp  Supervisor: Marko Vendelin, PhD Laboratory of Systems Biology, Institute of Cybernetics TUT

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



  1. Sepp,  Vendelin, Vija, Birkedal ADP compartmentation analysis reveals coupling between pyruvate kinase and ATPases in heart muscle. Biophysical Journal,  2010.
  2. Jepihhina, Beraud, Sepp, Birkedal, Vendelin Permeabilized rat cardiomyocyte response demonstrates intracellular origin of diffusion obstacles. Biophysical Journal,  2011.
  3. Illaste, Kalda, Schryer, Sepp Life of mice - development of cardiac energetics. Journal of Physiology, 2010.
  4. Branovets, Sepp, Kotlyarova, Jepihhina, Sokolova, Aksentijevic, Lygate, Neubauer, Vendelin, Birkedal Unchanged mitochondrial organization and compartmentation in creatine deficient GAMT-/- mouse heart. AJP-Heart and Circulatory Physiology,  2013.
  5. Sepp, Sokolova, Jugai, Mandel, Peterson, Vendelin Glycolysis-coupled 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)

Restriction in cytosol

Saks 2001:

Skinned rat soleus fibers: "A powerful ADP-trapping 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?


  Confirmed





 

Experimental setup



  • ATP production through respiration
  • ATP utilization through cytosolic ATPase activity

Mathematical Modeling


   

Mathematical Modeling


JATP=Csol-cyt(ATPsol-ATPcyt) JMOM ATP=CMOM(ATPcyt-ATPIMS)

Diffusion: Fick's Law


Mathematical Modeling

VATPsyn=VmaxATPsynADPIMSADPIMS+KmATPsyn VATPase=VmaxATPaseATPcytoATPcyto+KmATPase(1+ADPcytKiATPase)

Enzyme kinetics: Michaelis-Menten


Results:

  • Series of characteristic experiments to study compartmentalization

  • Collection of mathematical models to analyze the data

  • some ATPases coupled to endPK




Which ATPase?





SERCA

SR Ca pump

NKA

cell membrane Na/K pump

Intracellular Na 10mM

Extracellular Na 135-145mM

14:1

Intracellular K 155mM

Extracellular K 3.5-5mM

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  GAMT-deficient 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 GAMT-deficient 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
    • NKA was found to be coupled to endPK
    • Demonstration of intracellular origin of diffusion restrictions

  • Approach applied on mice
    • Wildtype
    • GAMT-deficient
    • 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 0.75-1 mg/ml

collagenase A/P (Roche). pH was adjusted to 7.1 with NaOH at 25

Mitomed solution (mM): KH2PO4 PO2 3, MgCl2 sucrose 110, K-lactobionate 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 1-2mM, free10-50uM

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

Stimulation of mitochondrial ATP synthesis by ADP in sceletal muscle and isolated mitochondria (inset)

Jeneson et al 1996 JBC


thesis

By Mervi Sepp

thesis

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