Contents

πŸ“’ Wei 2012

Dynamics of matrix-free Ca2+ in cardiac mitochondria: two components of Ca2+ uptake and role of phosphate buffering1

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INTRODUCTION

  • Ca2+ is the central signaling molecule for cardiac excitation-contraction coupling
  • Mitochondrial matrix free Ca2+ ([Ca2+]mito) is determined by the balance between Ca2+ uptake, extrusion, and buffering
    • uptake: MCU (mainly, Ru360–sensitive), rapid mode (RaM), mRyR
    • extrusion: mNCE, mPTP
    • buffering: Pi
  • the dynamics and quantitative determinants of changes in [Ca2+]mito are incompletely understood

MATERIALS AND METHODS

  • adult guinea pig hearts as Aon et al.
  • ΔΨm: TMRM
  • extramitochondrial Ca: Calcium green 5N
  • Matrix Ca: fura-FF, requires calibration https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3362519/bin/JGP_201210784_Fig1.jpg

RESULTS

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3362519/bin/JGP_201210784_Fig2.jpg
Response to Ca additions

  • ΔΨm decreased by only 3 mV (188 to 185mV) after the first Ca2+ addition but did not change for subsequent additions
  • For both small and large Ca additions, steady-state [Ca2+]mito settled into a range between 15 and 20 Β΅M
  • the ratio of bound/free Ca2+ spanned from ∼3 to 30,000

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3362519/bin/JGP_201210784_Fig4.jpg
Response to MCU inhibitor

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3362519/bin/JGP_201210784_Fig5.jpg
Resetting of [Ca2+]mito (black) for two Ca2+ additions

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3362519/bin/JGP_201210784_Fig6.jpg
Effect of Mg2+ on the regulation of mitochondrial Ca2+

  • The slowing of the Ca2+ uptake rate with MgATP could be attributed to the increased Mg2+ present in the solution

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3362519/bin/JGP_201210784_Fig7.jpg
Inorganic phosphate (Pi) dependence of mitochondrial Ca2+ uptake

  • varying Pi over the range of 0.1 to 10 mM for both large and small Ca2+ additions
  • the Ca2+ buffering action of Pi facilitates the regulation of [Ca2+]mito down to low levels in the face of very high mitochondrial Ca2+ loads and preserves the energy state of the mitochondria

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3362519/bin/JGP_201210784_Fig8.jpg
Effects of Pi, (black), arsenate (Asi, blue), or vanadate (Vi, red) anions on the regulation of mitochondrial Ca2+

  • Pi analogues: vanadate (Vi), or arsenate
  • the total Ca2+ load at which PTP was triggered was markedly lower with Vi or Asi

DISCUSSION

  • MCU(mode1) required at least 1 Β΅M Ru360 to be completely inhibited: esponded to small Ca2+ additions, faster => For Ca signaling

  • MCU(mode2) was blocked by <100 nM Ru360: bulk of total Ca2+ uptake for large Ca2+ additions, slower => For Ca buffering

  • small amounts of Ca2+ uptake lead to large changes in [Ca2+]mito, whereas large Ca2+ additions give only a transient increase in [Ca2+]mito with no change or an even a lower steady-state Ca2+

  • Substitution of Pi with Asi or Vi had striking effects on [Ca2+]mito and the threshold for PTP activation. Interestingly, Asi and Vi had opposite effects on [Ca2+]mito for multiple pulses but they both sensitized the mitochondria to PTP activation

    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3362519/bin/JGP_201210784_Fig9.jpg
    Summary of the mechanisms governing mitochondrial Ca2+ dynamics

  • In conclusion, understanding mitochondrial Ca2+ dynamics requires quantitative assessment of not only Ca2+ influx and efflux rates across the mitochondria, but rates of matrix Ca2+ buffering at different Ca2+ loads and rates of entry

References


  1. Wei AC, Liu T, Winslow RL, O’Rourke B. Dynamics of matrix-free Ca2+ in cardiac mitochondria: two components of Ca2+ uptake and role of phosphate buffering. J Gen Physiol. 2012;139(6):465-78. PMC3362519 ↩︎