Empagliflozin restores the deregulated cardiac endothelial cell transcriptome in vivo upon myocardial ischemia reperfusion injury and reduces the infiltration of immune cells
Authors: Nikolaou P E , Mylonas N , Miliotis M , Kostopoulos I , Georgoulis A , Orologas N , Hatzigeorgiou A , Zuurbier C , Pieper M P , Tsitsilonis O , Andreadou I 
Published: November 2023

Abstract

Background/Introduction
Empagliflozin (EMPA) is a sodium-glucose co-transporter 2 (SGLT-2) inhibitor with established cardioprotective effects in heart failure but its use in myocardial ischemia-reperfusion (IR) injury is still under investigation. We have shown that chronic administration of EMPA reduces infarct size in normoglycemic mice subjected to IR and that endothelial cells (ECs) are implicated in the protective mechanism.

Purpose
We aimed to determine EMPA’s distinct effect on three cell populations: cardiac ECs, fibroblasts (FBs) and cardiomyocytes (CMs) in vivo.

Methods
C57Bl6 male mice were randomized as follows: 1) Control-Sham, 2) Control-IR and 3) EMPA-IR. EMPA group received EMPA 10mg/kg/day for 6 weeks which is a clinically relevant dose and Control groups received vehicle (5% DMSO in normal saline). At the end of the administrations, mice were subjected to 30’I/2hR or sham operation and the ischemic heart was collected to isolate CMs. From the non-CMs populations the cardiac ECs and FBs were sorted. RNA was obtained from the distinct cell populations and 3’ RNA sequencing was applied to investigate the pathways affected by IR and EMPA treatment. An additional cohort of mice was employed to validate the results at mRNA level. In order to examine the effects of EMPA upon prolonged reperfusion, we repeated the experimental protocol, and the mice were sacrificed upon 7 days of R. Based on the RNA sequencing data, the infiltration of the heart with immune cells was examined.

Results
Comparison between IR and Sham groups revealed that IR alters the transcriptomic profile of ECs and FBs with 576 and 571 genes respectively being significantly deregulated (FDR&lt;0.05). CMs transcriptome was not altered by IR. The comparison between Control-IR and EMPA-IR groups revealed that EMPA does not affect the FBs’ gene expression profile while in CMs, EMPA affects only 3 genes expression (FDR&lt;0.05) related to mitochondrial metabolism. The ECs are mostly affected by EMPA treatment with 211 genes being significantly altered (FDR&lt;0.05). Pathway enrichment analysis in ECs revealed that the deregulated pathways by IR related to extracellular matrix organization, matrix degradation and immune cell adhesion are restored by EMPA treatment. The expression levels of several significant genes of our dataset including Mmp-2, Timp-1, Icam, Vecam, etc was validated in the independent cohort of mice. On the 7th day of R, EMPA reduced the infiltration of neutrophils and inflammatory (Ly6C high) monocytes in the myocardium compared to the Control group indicating that EMPA may affect the remodeling process upon IR.

Conclusions
We identified ECs and FBs as cell target populations for therapeutic interventions at 2h of R. EMPA pre-treatment reverses the deregulated transcriptome related to extracellular matrix organization, matrix degradation and immune cell adhesion in ECs and eventually reduces the infiltration of immune cells in the ischemic myocardium.