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Sabe et al. Vessel Plus 2024;8:2 https://dx.doi.org/10.20517/2574-1209.2023.95 Page 3 of 12

current literature create major knowledge gaps in our understanding of the comparative effects of SGLT2i
and DPP4i, which have yet to be addressed adequately.

In order to address these knowledge gaps, we sought to examine the comparative actions of SGLT2i and
DPP4i in the context of longstanding coronary disease by utilization of our swine model of ameroid
constrictor-induced myocardial ischemia. In this study, we compare the effects of SGLT2i canagliflozin and
DPP4i sitagliptin on myocardial functional parameters, coronary perfusion, coronary microvascular density,
and molecular signaling in a non-diabetic porcine model of CMI.

METHODS
Overview of study design
21 pigs (Yorkshire breed) that were 11 weeks old received a thoracotomy incision for placement of an
ameroid constrictor to the left circumflex artery (LCxA). The ameroid constrictor produces CMI by
absorption of heat and moisture and expansion inwardly to slowly occlude the artery, as reported by
others [18-20] . Following a two-week period, the pigs were given either vehicle (CON, 8 pigs of which 3 were
female and 5 were male), 300 mg oral canagliflozin daily (CANA, 8 pigs of which 4 were female and 4 were
male), or 100 mg oral sitagliptin daily (SIT, 5 pigs of which 4 were male and 1 was female). Doses of
medication were based on recommended doses for adult patients. Medications were administered orally by
trained veterinary and research team staff. After five weeks of treatment, swine underwent euthanasia with
myocardial tissue harvest for analysis. Per data from our prior experiments, we estimated the minimum
sample size for significance using a 2 tailed α level of 0.01, ß error level of 0.10, and standard deviation for
perfusion analysis of 0.150 mL/[mg·min], resulting in a minimum sample size of 7 per group. Of note,
sitagliptin treatment was held for three weeks in two animals per the “RIH Institutional Animal Care and
Use Committee” with early halting of this arm of the protocol due to mortality as described previously
[13]
resulting in a smaller sample size in the SIT group . This study was approved by the RIH IACUC
(#505821).

Ameroid constrictor placement
An ameroid constrictor was placed on the proximal LCxA of all pigs in the study. The pigs received oral
30 mg/kg cephalexin and 10 mg/kg aspirin one day before and five days after the surgery. A fentanyl patch
of 4 ug/kg was applied just before the surgery and kept for 72 h. The pigs were given an intramuscular
injection of 4.4 mg/kg telazol and 2.2 mg/kg xylazine to induce anesthesia. The pigs underwent intubation
and mechanical ventilation, while isoflurane inhalation was employed for anesthesia maintenance.
Intravenous administration of normal saline at a rate of 5 mL/kg/h was initiated. Supine positioning of the
pigs followed, with sterile preparation and draping preceding a mini-thoracotomy on the left side.
Subsequently, sharp entry was made into the pericardium for visualization of the left atrium, allowing for
retraction and visualization of the LCxA close to its branching off from the left main coronary artery. To
temporarily obstruct blood flow through the LCxA, a loop was placed around it after systemic
heparinization. The LCxA was effectively blocked for a duration of 22 min by elevating the loop, verified by
observable changes in ST and/or T waves on the electrocardiogram (ECG). This period of LCxA occlusion
coincided with the injection of 5 mL of gold-labeled microspheres into the left atrial cavity. Upon releasing
the vessel loop and restoring LCxA blood flow, ECG alterations were monitored until they reverted to
normal. A hygroscopic casein ameroid constrictor containing a titanium case was placed circumferentially
around the LCxA. The surgical incision was sutured in multiple layers for proper reapproximation.

Terminal harvest
The terminal harvest procedure was performed five weeks after treatment. The pigs received systemic
heparinization (80 IU/kg). Midline sternotomy was performed. Resting and pacing blood flow analyses at