Page 86 - 79_02

A.
Gómez
et
col.

(mtROSp),
mitochondrial
oxygen
consumption
in
states
4
(resting)
and
3

(phosphorylating),
the
amounts
of
the
respiratory
complex
I,
II,
III
and
IV
proteins,

the
apoptosis-­-inducing
factor
(AIF)
which
is
also
required
for
the

assembly/maintenance
of
complex
I
(24),
the
marker
of
oxidative
damage
to

mitochondrial
DNA
(mtDNA)
8-­-oxo-­-7,8-­-dihydro-­-2´-­-deoxyguanosine
(8-­-oxodG),
five

different
specific
markers
of
protein
oxidative
modification:
the
specific
protein

carbonyls
glutamic
and
aminoadipic
semialdehydes
(GSA
and
AASA)
indicating

purely
protein
oxidative
modification,
the
protein
glycoxidation
markers

carboxyethyl
lysine
(CEL)
and
carboxymethyl
lysine
(CML),
and
the
protein

lipoxidation
markers
CML
and
malondialdehyde
lysine
(MDAL).
We
also
measured

the
full
fatty
acid
composition
of
heart
and
SKM
mitochondrial
membranes
which

allowed
us
to
calculate
their
global
unsaturation
indexes
(the
DBI,
and
the

peroxidizability
index,
PI).
The
mitochondrial
biogenesis
indicators
peroxisome

proliferator-­-activated
receptor-­-?
coactivator
(PGC)-­-1a
(PGC1),
the
mitochondrial

transcription
factor
(TFAM),
antioxidant
regulation
transcription
factor
Nrf2

(Nuclear
factor
erythroid
2-­-related
factor
2),
and
the
p-­-ERK
and
SIRT1
signalling

proteins
related
to
longevity
(25),
as
well
as
the
basal
metabolic
rate
of
the
whole

animal,
the
rectal
temperature,
and
the
heart
rate,
the
systolic
and
diastolic
and
mean

arterial
pressures
were
also
measured.

2.
MATERIALS
AND
METHODS

2.1.
Animals
and
study
design

128
C57BL/6
male
mice
were
maintained
in
a
separate
room
of
Complutense

University
animal
house
at
the
Faculty
of
Medicine
under
SPF
conditions
during
their

whole
life
or
until
some
of
them
were
sacrificed
to
measure
physiological
or

biochemical
relevant
parameters.
86
of
these
animals
(43
Old
control
and
43
Old-­-AT-­-
treated)
were
used
only
to
obtain
the
survival
curves.
These
animals
were

maintained
under
optimum
conditions
(12:12
(light-­-dark)
cycle,
22°C
±
2°C
and

50%
±
10%
relative
humidity),
were
left
intact
during
their
whole
lifespan
(except

for
the
AT-­-treatment)
and
their
day
of
spontaneous
death
was
recorded.
The

treatment
with
atenolol
was
started
at
2
months
of
age.
A
separate
group
of
42

animals
(21
controls
and
21
AT-­-treated)
was
established
at
the
beginning
of
the

experiment
in
order
to
measure
the
different
physiological
and
biochemical

parameters
when
reaching
old
age
(after
16
months
of
experimentation).
Among
the

21
reporter
animals
of
each
of
these
two
groups
(control
and
AT-­-treated),
7
animals

were
used
for
the
ROS
and
oxygen
consumption
measurements
in
isolated

mitochondria,
7
animals
were
used
for
the
measurement
of
8-­-oxodG,
and
7
animals

were
used
to
assay
the
rest
of
the
biochemical
parameters
after
being
sacrificed
by

cervical
dislocation.
At
the
time
of
sacrifice
these
reporter
animals
had
18
months
of

age.
The
animals
in
the
atenolol
group
had
free
access
to
a
solution
of
0.1
g/L
of

256