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Long-­-life
supplementation
with
atenolol…

atenolol
(Sigma,
A7655)
in
drinking
water.
The
diet
(Panlab,
Spain)
was
offered
ad

libitum
to
all
animals.
Just
after
cervical
dislocation,
hearts
and
SKM
(total
hind
limb

muscle)
were
immediately
processed
to
isolate
functional
mitochondria,
which
were

used
to
measure
mitochondrial
respiration
and
rates
of
mtROSp.
Whole
hearts
and

SKM
tissue
samples
(total
hind
limb
muscle)
were
stored
at
-­-80°C
for
the
posterior

analyses
of
the
rest
of
the
biochemical
parameters.
The
experimental
animal
subject

review
committee
from
the
Complutense
University
of
Madrid
approved
all
the

experiments
in
C57BL/6
mice.

2.2.
Physiological
parameters

Rectal
temperature
was
measured
using
a
rectal
probe
(Thermocouple

thermometer
model
8112-­-20,
Cole-­-Parmer
Instrument
Company).
The

measurements
were
performed
three
times
in
each
mouse,
always
at
11:00,
on

three
different
days
separated
15
days
from
each
other,
during
the
last
2
months
of

experimentation.
To
estimate
the
basal
metabolic
rate,
individual
mice
we
placed

inside
a
closed-­-system
respirometer
(total
volume
2,600
ml)
and
the
carbon

dioxide
produced
was
captured
with
a
10%
KCl
solution.
The
rate
of
oxygen

consumption
of
each
animal
was
measured
at
rest
with
an
oxygen
analyzer
and

probe
(Model
600
Can
1691,
Engineered
Systems
&
Designs)
at
23±1°C.
The

measurements
were
realized
at
the
end
of
the
pharmacological
treatment.
Heart

rate
and
blood
pressure
were
measured
in
conscious
mice
with
a
noninvasive
tail-­-
cuff
manometry
system
(LE5001
Panlab
Harvard
Apparatus).
Each
animal
was

acclimatized
for
at
least
three
practice
sessions
in
the
three
consecutive
weeks

before
the
final
measurements
were
recorded.
In
each
session
8
consecutive

readings
were
recorded
and
their
average
was
used
to
obtain
systolic,
diastolic,

and
mean
blood
pressure.
These
measurements
were
performed
during
the
last
2

months
of
experimentation.

2.3.
Isolation
of
functional
mitochondria,
oxygen
consumption
and
ROS

production

Mitochondria
were
obtained
from
fresh
tissue
by
the
procedure
of
Mela
and

Seitz
(26)
with
modifications.
After
checking
the
functionality
and
phosphorylation

capacity
of
the
mitochondria
(high
respiratory
control
ratios)
the
rate
of
mtROSp

was
measured
by
the
fluorometric
method
established
at
our
laboratory
(27).
The

rate
of
oxygen
consumption
of
heart
and
SKM
mitochondria
was
measured
at
37°C

in
a
water-­-thermostatized
incubation
chamber
with
a
computer-­-controlled
Clark-­-
type
O2
electrode
(Oxygraph,
Hansatech,
UK)
as
previously
described
(28).

2.4.
Oxidative
damage
to
mtDNA
(8-­-oxodG)

Isolation
of
mtDNA
was
performed
by
the
method
of
Latorre
and
cols
(29)

adapted
to
mammals
(30).
The
isolated
mitochondrial
DNA
was
digested
to

deoxynucleoside
level
and
the
level
of
oxidative
damage
in
mtDNA
was
estimated

by
measuring
the
amount
of
8-­-oxo-­-7,8-­-dihydro-­-2’deoxyguanosine
(8-­-oxodG)

257