Long-‐life supplementation with atenolol…
257
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 para eters
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 O
2
electrode (Oxygraph, Hansatech, UK) as previously described (28).
2.4. Oxidative amage 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)
