Methods
Right atrial tissue sampling - Samples of right atria were taken
immediately prior to and following cardioplegic cardiac arrest using a
double purse-string approach, which is a well-established model of human
myocardial ischemia-reperfusion injury15–17. The
heart was arrested with high potassium (27mEq/L) blood cardioplegia
solution and was maintained with a low potassium formulation at 4°C.
After weaning from bypass and a brief period of reperfusion (5-15
minutes), the tissue between the two purse-string sutures was harvested.
The pre-cardioplegic arrest samples wre taken pre-cardiopulmonary bypass
(CPB) and pre-arrest at the time of venous cannulation. All tissues were
flash frozen in liquid nitrogen. The average age of the young
participants was 42.6 years, and all were male (n=5). The average age of
the aged participants was 73.8 years, and 10 of 11 were male (n=11).
Patients were matched for co-morbidities including dyslipidemia and
hypertension, as well as for prescribed medications. Participants were
excluded if they had diabetes, kidney disease, cancer or any other
underlying issues, and were excluded if they currently identified as
smokers. Use of patient samples from the St. Michael’s BioBank was
approved by the Office of Research Ethics at York University
(Certificate #2018-114), as well as the Research and Ethics board at
St. Michael’s Hospital (REB 15-385).
Protein Extraction- Atrial samples were weighed and diluted in
10x Sakamoto buffer with protease and phosphatase inhibitors and lysed
with a Tissue Lyser. Lysates were centrifuged at 14,000g for 10 minutes,
and the supernate was stored at -80º C.
Immunoblotting- Atrial proteins were separated on polyacrylamide
SDS-PAGE gels at 120mV. Proteins were transferred onto nitrocellulose
membranes and incubated in 5% milk in wash buffer. Membranes were
incubated with primary antibody overnight at 4°C, and then with
HRP-conjugated secondary antibodies for 1 hr at room temperature the
following day. Membranes were visualized with enhanced chemiluminesence.
Image J software was used for quantifications.
Statistical Analyses- Unpaired t-tests were used to detect
differences between young and aged subjects using GraphPad Prism 6.0.
When comparing the effect ischemia-reperfusion (pre- and post-CABG) in
the young and aged atria, a two-way ANOVA with repeated measures was
used. The critical value was set at p<0.05. All error bars
represent the SEM.