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Page 4 of 12 Sabe et al. Vessel Plus 2024;8:2 https://dx.doi.org/10.20517/2574-1209.2023.95

150 bpm were done by injecting 5 mL of isotope-labeled microspheres into the left atrium while
simultaneously withdrawing 10 mL of blood from the femoral artery catheter. Hemodynamic measurements
were obtained by placing a pressure-volume (PV) catheter via a 6F sheath to the left ventricular apex. The
heart was removed at the end of the procedure, and heart tissue was quickly separated into 16 different
segments corresponding to the distribution of the left cirumflex artery and left anterior descending artery.
Myocardial tissue segments were either dried in a warm oven and then stored for microsphere-based studies
or submerged in liquid nitrogen and then frozen at -80 °C for western blot experiments and frozen sections.

Myocardial perfusion measurements
Using isotope-labeled microspheres given during ameroid and harvest procedures, myocardial perfusion
was assessed. To define the left ventricle's perfusion territory by the LCxA, 5 mL of gold-labeled
microspheres were injected into the left atrial appendage while employing a vessel loop to occlude LCxA.
During the harvesting procedure, 5 mL of Lutetium-labeled microspheres were introduced into the left
atrium, and concurrently, 10 mL of blood was withdrawn from the femoral artery at a fixed rate via a
withdrawal pump. Samarium-labeled microspheres were used for the same protocol during pacing at
150 bpm. Samples of blood and myocardial tissue from 10 distinct sections were collected, and selected
based on their proximity to both the LAD and LCxA arteries. The weight of these samples was obtained,
and then they were subjected to oven-drying, and subsequently forwarded to the Biophysics Assay
Laboratory for analysis of microsphere density and blood flow calculations.

Cardiac functional measurements
To collect cardiac functional assessments while conducting the harvest procedure, a PV catheter was
directly introduced into the left ventricle's apex. Throughout breath holds, load-dependent information was
gathered to mitigate the influence of respiratory fluctuations, while during respiratory holds and vessel loop
occlusion of the IVC using a vessel loop, load-independent data were acquired. Hemodynamic parameters
were documented and processed using “LabChart” software. Measurements collected included stroke
volume (SV), stroke work (SW), cardiac output (CO), left ventricular stiffness (ß), preload recruitable stroke
work (PRSW), and dP/dt max.

Microvessel quantification
[21]
Immunofluorescence staining was used to determine microvessel density, as described previously .
Primary and secondary antibodies used in this protocol are listed in Supplementary Table 1. Images were
examined at 20× magnification using an Olympus VS200 Slide Scanner. Image analysis was conducted using
QuPath software . Capillary density was assessed by determining the percentage of tissue area stained
[22]
through the thresholding of positive isolectin B4 staining. Arteriolar density was analyzed by defining
positive SMA staining through thresholding and calculating the object number per tissue section area.

Immunoblotting studies
Immunoblotting studies were performed as previously described . Primary and secondary antibodies used
[9]
in this protocol are listed in Supplementary Table 1. Material sources are listed in Supplementary Table 2.
NIH Image J software was used for band density densitometry.

Data analysis
Median values with interquartile ranges are used to present all data. The statistical analysis was performed
using the Wilcoxon rank-sum test, and Holm correction was applied for multiple comparisons, utilizing R
software. Probability values less than 0.05 were deemed statistically significant.