Impact of time-of-the-day of aortic valve replacement on peri-operative inflammation and subsequent cardiac reverse remodeling. – TOMIS-leukocyte

This study was conducted on the Lille POMI-AF prospective cohort (“Post-Operative Myocardial Incident & Atrial Fibrillation,” NCT03376165). Patients scheduled for cardiac surgery at Lille University Hospital were included in this study. Blood samples and a cardiac biopsy were taken at the time of surgery (D0, patients fasting), then blood samples were taken 1 and 7 days after the procedure (D1 and D7) in order to perform various assays and isolate circulating leukocytes. Patients were followed up in the short term (7 days) and long term (3 to 5 years).

WP1a - Association between the amplitude of the perioperative inflammatory response and the timing of cardiac surgery

The perioperative inflammatory response was studied by measuring C-reactive protein (CRP) levels, the exploration of inflammatory proteins through gene expression studies and blood tests on days 0, 1, and 7 in a total of 87 patients (n=51 operated on in the morning between 8 a.m. and 11 a.m. and n=36 in the afternoon between 2 p.m. and 5 p.m.)

WP1b - Association between preoperative immunological phenotype and postoperative morbidity and mortality

WP1c - Association between perioperative inflammatory response and postoperative morbidity and mortality

The preoperative immunological phenotype was studied by mass cytometry (CyTOF) on blood leukocytes and cardiac biopsies from patients in the POMI-AF cohort. Blood DNA isolation enabled genotyping (n=104) to identify patients carrying a genetic mutation in leukocytes (HSM for hematopoietic somatic mosaicism). Postoperative major cardiovascular events were monitored over a period of 3 to 5 years.

WP2 - Identify transcriptional alterations in circulating and myocardial leukocytes associated with postoperative complications

Gene expression analysis was performed on immune cells (monocyte-derived macrophages) from patients, isolated from blood samples.

WP3 - Study the role of the transcription pathways identified in patient leukocytes in WP2 in regulating the immune response induced in a mouse model of cardiac remodeling and during its disappearance (reverse remodeling)

A mouse model of abdominal aortic constriction (AAC, diameter 27G) was developed in the laboratory on mice aged 8 to 14 weeks. We compared AAC mice operated on during the sleep/wakefulness transition phase with AAC mice in the resting phase and characterized the immune cells in the blood and heart by flow cytometry.

WP1a :

Cardiac surgery promotes inflammation with a significant increase in the amount of inflammatory proteins on D1 vs. D0 in the blood. Patients who underwent surgery in the afternoon had a lower peak inflammatory response with lower CRP values (177 ± 82 mg/dL vs. 142 ± 52 mg/dL, p=0.02) compared to those who underwent surgery in the morning. No other significant changes in the concentration or gene expression of the inflammatory proteins tested were found in blood leukocytes on D0, D1, and D7 post-surgery. However, we did note a difference in the metabolic profile of patients, with a significant decrease in blood glucose (132 ± 46 vs. 105 ± 19 mg/dL, p=0.01) and a significant increase in beta-hydroxybutyrate concentration, a ketone body, (0.14 +/- 0.14 mmol/L vs 0.32 +/- 0.25 mmol/L, p=0.004), reflecting prolonged fasting in patients operated on in the afternoon, without any alteration in the lipid profile. Since the metabolic status of patients operated on in the afternoon is different, this could be a factor influencing their post-surgical outcome.

WP1b et 1c :

Patients with a somatic genetic mutation in leukocytes (HSM) had an increased postoperative inflammation, marked by an elevation in peak CRP within 72 hours after surgery, with a 3.5-fold higher risk of postoperative atrial fibrillation (age-adjusted OR: 3.5; 95% CI: 1.52-8.03; p=0.003). In addition, we have shown that HSM status is independently associated with a higher risk of major cardiovascular events in the long term (HR 1.8, 95% CI 1.05-3.41, n=314 patients). Finally, patients with HSM have a higher number of monocytes in the circulation and a higher number of monocyte-derived macrophages in the heart at the time of cardiac surgery.

Published data : Ninni et al. J Am Coll Card 2023 & Ninni et al. J Am Heart Assoc 2024

WP2 :

The exploration of transcriptomic data on macrophages derived from monocytes in patients is still ongoing.

WP3 :

Initial analyses of the mouse model of abdominal aortic ligation, used to mimic heart failure, show that seven days after the procedure, the heart becomes hypertrophied, exhibits increased fibrosis, and we found an elevation in plasma NT-proBNP levels, a marker of heart failure. Immunophenotyping revealed an increase in the cell number of monocyte-derived macrophages in mice operated on during the sleep/wakefulness transition. These data remain to be confirmed.

To date, it has been difficult for clinicians to predict the trajectory of patients undergoing cardiac surgery. For the same type of surgery, some will experience serious cardiovascular complications, while others will recover perfectly. We believe it is essential to identify markers or develop tests that enable us to screen patients with the highest risk of post-operative cardiovascular complications and to strengthen their medical follow-up.

Our data have shown that the patients with highest risk are those who undergo surgery in the morning, suggesting that the biological clock influences cardiovascular outcomes, with potential mechanisms including a different inflammatory response depending on the time of day when the procedure is performed. This inflammatory signature is still being explored in our mouse model.

We have shown that patients with genetic mutations in leukocytes (HSM) are at greater risk of short- and long-term cardiovascular complications. Although it is difficult to sequence the genome of each patient before surgery, these same patients show slightly higher leukocyte activation in the preoperative period. Optimizing in vitro tests to identify patients with more active leukocytes in the preoperative period could enable us to move toward precision medicine and prevent the onset of postoperative cardiovascular complications

Background. Aortic valve replacement is the sole therapeutic solution to improve outcomes in patients with aortic stenosis. AVR is commonly performed during on-pump cardiac surgery, whereas trans-catheter aortic valve implantation is increasingly performed in patients with high-risk for surgery complications. Whatever the technical approach, AVR is expected to improve not only survival, but also functional status as a result of cardiac reverse remodeling, i.e. regression of the left ventricular hypertrophy. No medication is currently available to specifically improve and/or accelerate reverse remodeling after AVR.
We have recently demonstrated that afternoon (vs. morning) AVR results in decreased incidence of both immediate perioperative myocardial infarction and acute heart failure within 500 days after surgery. Interestingly, disruption of diurnal rhythms by modulating molecular clock genes immediately after myocardial infarction in mice impaired healing and exacerbated maladaptive cardiac remodeling.
Working hypothesis. We postulate that the circadian clock plays a key role in immune cell recruitment and peri-operative myocardial injury healing and remodeling after AVR, i.e. differences in pre- and post-operative immune-inflammatory responses and subsequent cardiac repair and remodeling are responsible for the lower frequency of heart failure development months after afternoon (vs. morning) AVR.
Objectives and methods. The project will be organized to determine: (i) the impact of peri-operative inflammation on surgery complications and subsequent cardiac remodeling after AVR, (ii) the impact of the time-of-day on peri-operative inflammatory responses and their role in post-operative outcomes.
We will perform a bi-centric prospective randomized study at the University Hospital CHU de Lille (France) and CHU Rennes (France) in patients undergoing first AVR for severe aortic stenosis with LVEF>50% to determine whether the pre-operative leukocyte immune-phenotype and their post-operative activation display a morning-afternoon variation, and whether this activation is associated with reverse cardiac remodeling during the year following AVR. Deep immuno-phenotyping of peripheral blood leukocytes will be performed by mass cytometry (CYTOF). Second, the transcriptional pathways associated with the time-of-the-day variation and AVR-induced immune-phenotype will be explored in cells identified in previous WP by RNAseq analysis. Third, based on our previous studies, we will determine the role of the clock gene REV-ERB? in the regulation of the time-of-the-day variation of the immune-inflammatory response and its impact on cardiac remodeling. We will perform functional analysis on freshly isolated human monocyte sub-populations and study cardiac remodeling after abdominal aortic constriction (AAC) and reverse remodelling after AAC relief in mouse models deficient for REV-ERB? (cell type to be chosen based on the results from other WP). Finally, studies using novel REV-ERB? ligands will be done to validate this target as a pharmacological approach.
Expected results and relevance of the project. Our translational project including patients and preclinical models will identify the contribution of the immune-inflammatory response in maladaptive cardiac healing and reverse remodeling after AVR., as well as proof-of-concept studies for feasible therapeutic strategies interfering with clock gene signaling in immune-inflammatory cells to prevent maladaptive cardiac remodeling for which virtually no drugs exist to date.