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Page 12 of 14 Broadwin et al. Vessel Plus 2023;7:25 https://dx.doi.org/10.20517/2574-1209.2023.103

Limitations
Our study was not without its limitations. Given the small sample size of each arm (n = 3), we may have
been underpowered to demonstrate significance. Given this lack of power, we may have also been
vulnerable to type II error after the Bonferroni correction. This lack of power may have contributed to the
similarity in the scope of detectable changes by our experimental conditions, and prompts further
repeatability studies that could test this premise. The animal model itself also represents the obvious
limitation of being a surrogate used to examine a chronic human condition and can only approximate the
pathophysiology and physiologic response to disease seen in patients. However, this large animal model
does allow for a closer examination of these processes and is likely more relevant than small mammal
studies.

Conclusion
Examination of the effect of diet and EV treatment on the methylome of chronically ischemic cardiac tissue
has raised intriguing possibilities regarding the epigenetic response to ischemic stress. This investigation
further demonstrates that not all tissue has the same epigenetic response to a stressing event, meaning that
blood may not be an accurate surrogate for events occurring in the myocardial methylome. Although our
study did not reveal a global change or singular response by the entire methylome, this is likely due to a lack
of statistical power. Nevertheless, it is noteworthy that responses in specific methylation patterns may
represent a subtler, yet still impactful, tissue response to ischemic stress.

DECLARATIONS
Author’s contribution
Data analysis, project design, and manuscript preparation: Broadwin M
Manuscript preparation: Aghagoli G, Sabe SA, Harris DD
Data collection and analysis: Wallace J, Lawson J, Ragayendran A
Project design: Fedulov AV, Sellke FW

Availability of data and materials
All methylome data has been submitted to GRA/GEO, and all additional data will be made available upon
reasonable request to the authors.

Financial support and sponsorship
This work was supported financially by the following grants: National Heart, Lung, and Blood Institute
(NHLBI) 1F32HL160063-01 (Sabe SA); NIH T32HL160517 (Sellke FW, Harris DD, Broadwin M);
R01HL46716 and R01HL128831 (Sellke FW).

Conflicts of interest
All authors declared that there are no conflicts of interest.

Ethical approval and consent to participate
All animal procedures were conducted in accordance with protocols approved by the Institutional Animal
Care and Use Committee of Rhode Island Hospital (#505821).

Consent for publication
Not applicable

Copyright
© The Author(s) 2023.