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Page 2 of 14 Broadwin et al. Vessel Plus 2023;7:25 https://dx.doi.org/10.20517/2574-1209.2023.103

EV treatment in ischemic tissue in subjects on a normal diet, there were 574 loci with nominal P value < 0.05 with
two loci Fructosamine 3 kinase related protein [(FN3KRP) (P < 0.001)] and SNTG1 (P = 0.03) significant after
Bonferroni correction. When examining the effect of EV treatment in ischemic tissue in HFD, there were 511 loci
with nominal P values < 0.05. After Bonferroni correction, two loci had P values less than 0.05, betacellulin [(BTC)
(P = 0.008)] and [proprotein convertase subtilisin/kexin type 7 (PCSK7) (P = 0.01)].

Conclusions: Alterations in DNA methylation were identified in pig myocardium after ischemic insult, change in
diet, and treatment with EVs. Hundreds of differentially methylated loci were detected, but the magnitude of the
effects was low. These changes represent significant alterations in DNA methylation and merit further
investigation.
Keywords: Methylation, methylome, myocardial ischemia, cardiovascular disease, epigenetics

INTRODUCTION
Cardiovascular disease is a major source of morbidity and mortality across the globe . Within the United
[1]
[2]
States, it is the leading cause of death, with an individual dying from cardiovascular disease every 33 s .
Given the burden of myocardial ischemia (MI) on society, a thorough understanding of the factors that
contribute to its pathogenesis is essential to prevention, diagnosis, and early intervention.

The incidence of cardiovascular diseases is highly associated with genetic factors and environmental
[3]
variables, such as diet . More recently, antidiabetic medications, such as SGLT2 inhibitors, have
demonstrated benefit in the treatment of heart failure in non-diabetic patients, resulting in a level 1A
recommendation for their use in heart failure irrespective of diabetic status . Given the number of patients
[4]
suffering from both metabolic syndrome (MetS) and cardiovascular disease, elucidating the interplay
between these two conditions is of the utmost importance. With the emergence of the field of epigenetics,
recent studies have shown that the cardiovascular impact of environmental factors such as diabetes,
malnutrition, smoking, and hypertension can be mediated by epigenetic changes, particularly through DNA
[5]
methylation, histone modification, and alterations in microRNA . Although the effects of a high-fat diet on
cardiovascular outcomes have been reported , the mechanism by which diet may influence the epigenome
[6]
is not well understood.
In addition to hypertension and other cardiovascular diseases, a large number of studies have reported the
[6,7]
role of epigenetic regulation in MI . One animal study investigating genome-wide DNA methylation in a
mouse model identified significantly different methylation at various 5′-C-phosphate-G-3′ (CpG) sites at
10 min, 1 h, 6 h, 24 h, and 72 h post acute MI compared to the sham group . Several human studies have
[8]
reported differential DNA methylation in blood cells or circulating EVs associated with cardiac
ischemia [9-11] . Notably, one study reported a relationship between acute coronary syndrome (ACS) and
blood DNA methylation at 47 CpG sites through a genome-wide analysis of DNA methylation of whole
blood in ACS patients and matched controls . Another study investigating DNA methylation with MI and
[9]
matched controls found significant changes in DNA methylation at 34 CpG sites in gene foci related to the
[10]
pathogenesis of MI, including lipid metabolism and inflammation . However, this group did not
investigate alterations to the epigenome of the myocardium itself.

Extracellular vesicles (EV) are small membrane-bound structures derived from cells that have been
demonstrated to carry cellular products such as miRNA, cytokines, proteins, and growth factors . Previous
[12]
research from our lab has shown that treatment of ischemic myocardium with EVs derived from
mesenchymal stem cells results in improved myocardial perfusion, increased vascular density, and