Functional and proteomic investigation of potential role of steroid hormones in regulating early cardiac recovery in a rat model of isoproterenol induced intermittent myocardial ischemia
Authors: Barta B A , Ruppert M , Froehlich K E , Cosenza-Contreras M J , Olah A , Sayour A A , Karvaly G B , Kovacs K , Werner M , Merkely B , Schilling O , Radovits T 
Published: October 2021

Abstract

Introduction
Women benefit from higher levels of protection from cardiovascular diseases until menopause, after which they gradually lose their privileged status. Pivotal role of sex hormones, primarily estrogens were the focus of interest in explaining this clinical observation. Estradiol (E2) was a prime target of these investigations showing promising results. Nonetheless the potential influence of other estrogens and numerous estrogen metabolites have so far been neglected.

Purpose
The aim of our study was to investigate the influence of circulating steroid hormones on early functional and proteomic changes following repeated ischemic periods in female rats by applying the highly unbiased methods of in vivo pressure-volume analysis and mass spectrometry based proteomics.

Methods
Diffuse subendocardial ischemia was induced in female (F-Isch) Wistar rats with sc. injection of isoproterenol (ISO, 85mg/kg) daily for two consecutive days, while the control group (F-Co) received an equivalent volume of sc. saline solution. 48 hours after the first injection pressure-volume analysis (P-V) was carried out to assess left ventricular function. FFPE tissue slides were scanned and analyzed digitally, while peptides from the snap frozen left ventricular myocardium were measured by liquid chromatography-tandem mass spectrometry using isobaric labeling (TMT11plex). Serum and plasma samples were taken to measure circulating steroid hormone levels with mass spectrometry.

Results
Two day induction of ischemia resulted in 17% mortality in F-Isch. ISO treatment resulted in significant myocardial tissue damage compared to controls as assessed by histology. Ischemia led to a prominent impairment of diastolic aspects (active relaxation and myocardial stiffness) of left ventricular function (Tau: 11.1±0.7 vs. 16.1±1.1 F-Co vs. F-Isch; EDPVR: 0.05±0.005 vs. 0.131±0.016 F-Co vs. F-Isch.). Unsupervised hierarchical clustering performed on P-V parameters identified two distinct subgroups of F-Isch with severe or mild functional impairment. Supervised PLS-DA analysis of P-V and hormone datasets found three estrogens that might play a role in determining functional outcomes (2-Hydroxyestrone: 2-OHE1, 4-Hydroxyestrone: 4-OHE1, 4-Methoxyestrone: 4-MeE2). PLS analysis followed by gene ontology enrichment associated E2 and 2-OHE1 concentrations with mitochondrial protein expressions, while 4-OHE1 seemed to influence the reassembly of contractile structures after ischemia.

Conclusions
Our study has highlighted 2-OHE1 and 4-OHE1 as well as E2 as potentially influential estrogens on early post-ischemic recovery both on functional and on proteomic level.

Funding Acknowledgement
Type of funding sources: Public grant(s) – EU funding. Main funding source(s): New National Excellence Program of the Ministry of Human Capacities of Hungary (ÚNKP-20-3-I-SE-1 to BA. B.) National Research, Development and Innovation Office (NKFIH) of Hungary (K134939 to T. R.)