Transcript PowerPoint 演示文稿 - Zhejiang University

Mitochondrial
Membrane
Potential
(MMP)
Contents
 Definition
of MMP
 Formation of MMP
 Measurement/estimation of MMP
Role of MMP in function of both
normal and pathophysiology
 Definition
of MMP
 Formation of MMP
 Measurement/estimation of MMP
Role of MMP in mitochondrial
function of both normal and
pathophysiology
The miotchondrial
membrane potential is a
highly negative (~ -180mV)
potential across the inner
membrane of mitochondria.
 Definition
of MMP
 Formation of MMP
 Measurement/estimation of MMP
Role of MMP in mitochondrial
function of both normal and
pathophysiology
Sketch map showing the structure of mitochondria (on the left ) and principle of
generation of MMP in mitochondria. Electron transport system (ETS) pumps protons
across the inner mitochondrial membrane (i.m) and thus generates membrane potential
(MMP)
 Definition
of MMP
 Formation of MMP
 Measurement/estimation of MMP
Role of MMP in mitochondrial
function of both normal and
pathophysiology
Principle of measurement
of MMP
Directly
measurement using
electrode
Indirectly measurement
using fluorescent indicators
Directly
measurement using
electrode
Indirectly measurement
using fluorescent indicators
Directly
measurement using
electrode
Indirectly measurement
using fluorescent indicators
Fluorescent indicators in
measurement of MMP
Rhodamine 123
Tetramethylrhodamine
methyl ester (TMRM)
Tetramethylrhodamine ethyl
ester (TMRE)
Carbocyanine JC-1
Characteristics of these
indicators

Accumulate in the mitochondrial matrix because
of the solubility in both the inner mitochondrial
membrane and matrix space.
 The distribution of these indicators across the
inner membrane has been shown to follow the
Nernst equation.
 Rhodamines are single-excitation, single emission
dyes, whereas JC-1 emits red light (590nm) at
high concentration (JC-aggregates form), and
emits green light (530nm) at low concentrations
(monomer form)
Principle of MMP
measurement/estimation in
isolated ventricular myocytes
(1)MMP can be calculated using the following
equation :
Vm =-(RT/zF) * ln(Fmitochondria/Fnucleus)
where Vm is membrane potential, R is the gas
constant, T is absolute temperature,F is
Faraday’s constant, and F is fluorescence.
(2) MMP can be estimated by fluorescence
changes in mitochondrial matrix.
Fluorescence detection

(1)Confocal fluorescence microscopy
Confocal imaging of
mitochondria in a guinea
pig ventricular
myocyte with use of
membrane potential
indicator
tetramethylrhodamine
methyl ester (TMRE).
Cells were loaded with
200 nM TMRE Bright
areas in image represent
mitochondria that have
preferentially
accumulated TMRE.
Brightest
mitochondria are those
centered within the focal
plane.
Laser scanning confocal microscopic images of
JC-1(1ug/ml) fluorescenceA: Control astrocytes.
B: NH4Cl treatment showing a significant degree
of depolarization. C:Cells treated with NH4Cl +
CsA(5 mM) are similar to control. FCCP (3 mM,
20 min) induced a total collapse of the Dcm
(inset in A).
Graph on the left shows the time-dependence of
ammonia (5 mM ) induced dissipation of the
MMP in atrocytes. Quantitation is expressed as
mean red/green fluorescence ratios and
compared to untreated controls that were set at
(2) Flow cytometre
Effects of 48hr with 5 mM NH4Cl on the MMP as determined by flow cytometry.Data
is plotted as cell counts vs. TMRE fluorescence.ammonia results in a spectral shift to
the left indicating mitochondrial depolarization. This effect was blocked by CsA (5
uM).A:Control. B: 5 mM NH4Cl .C: 5 uM CsA.D: NH4Cl+ CsA.
E: FCCP
 Definition
of MMP
 Formation of MMP
 Measurement/estimation of MMP
Role of MMP in mitochondrial
function of both normal and
pathophysiology
IN normal cell function of MMP

Maintenance of MMP is essential for ATP
synthesis, i.e. providing motive force for
translocatione of proton from mitochondrial
matrix into the intermembrane space.
 MMP provides driving force for Ca2+
uptake into mitochondria by the Ca2+
uniporter.

Maintenance of MMP is essential for ATP
synthesis, i.e. providing motive force for
translocatione of proton from mitochondrial
matrix into the intermembrane space.
 MMP provides driving force for Ca2+
uptake into mitochondria by the Ca2+
uniporter.
The MMP is necessary for conversion of ADP to ATP (ATP synthesis).

Maintenance of MMP is essential for ATP
synthesis, i.e. providing motive force for
translocatione of proton from mitochondrial
matrix into the intermembrane space.
 MMP provides driving force for Ca2+
uptake into mitochondria by the Ca2+
uniporter.
PTP = permeability transition pore, mNCE = mitochondrial
sodium calcium exchanger, F1F0 = ATP synthase, e.t.c = electron
transport chain, mCU = mitochondrial calcium uniporter, DpH is
the pH gradient (~0.5) and DY is the membrane potential (~180mV)
MMP in pathophysiology
 MMP in
oxygen stress
 MMP in ischemia/reperfusion
 MMP in IPC
MMP in
oxygen stress
MMP in ischemia/reperfusion
injury
MMP in IPC
Mitochondrial defence against
radical-induced oxidative stress.
A: under normal conditions,
respiratory chain (RC) produces
2~4% reactive oxygen species
(ROS)from an electron flux. B:
higher cellular oxygen causes
increase of ROS, and radical
scavengers is the first step to
defense.C:if scavengers are
exausted, the increased oxygen
stress result in an increase of
proton conductance and a small
dissipation of MMP.D: PTP
opening when a certain MMP
decline is reached. E:ongoing
oxidative stress resulted in
irreversible permeability
transition causing a breakdown
of MMP, cytochrome c
release,cell apoptosis.
MMP in
oxygen stress
MMP in
MMP in
ischemia/reperfusion
injury
MMP in IPC
Time course of changes in cell length and mitochondrial
membrane potential during anoxia and reoxygenation.
MMP in
oxygen stress
MMP in ischemia/reperfusion
injury
MMP in IPC
It has been reported that the mitochondrial ATP- sensitive
potassium channel play important role in the cardioprotection of IPC
Nicorandil, a selective mKATP opener partially depolarized the MMP
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