Assessment of Valvular Oxidative Stress in a Young Patient with Primary Severe Mitral Regurgitation

Mitral valve regurgitation (MR) is the most common valvular heart disease. Diagnosing and managing mitral regurgitation is often challenging and requires a structured approach, integrating ndings on history, physical examination and imaging. A common cardiac anomaly is myxomatous mitral valve prolapse. Excess myxomatous lea et tissue, bilea et prolapse or billowing, chordae elongation and annular dilatation are all features of Barlow’s disease. Currently, there is no successful pharmacological treatment available to prevent or slow its progression. Here, we report the case of a 30-year-old male patient, with no previous signi cant medical background and no medication at home, who was diagnosed with severe mitral regurgitation at a regular check-up and was admitted to our clinic with mild exertion dyspnea and fatigue. Transthoracic echocardiography showed intensely thickened mitral lea ets with a myxomatous appearance, prolapse of the middle scallop of the posterior lea et (P2), apparently with ruptured chordae, and severe mitral regurgitation with a holosystolic eccentric jet. While oxidative stress is a central pathomechanism of cardiovascular disease, information regarding valvular oxidative stress in the literature is rather scarce. In this respect, we assessed oxidative stress through confocal microscopy in a sample of a mitral valve harvested during valvular surgery. We found an increased production of reactive oxygen species in the mitral valve sample that was alleviated after incubation with the angiotensin 2 receptor type I (AT1) antagonist irbesartan. This case is worth mentioning as a starting point for a prospective study aimed at assessing the role of valvular oxidative stress and mitochondrial dysfunction in patients with various degrees of primary and secondary mitral regurgitation.


Introduction
Mitral regurgitation is the most common valvular disorder worldwide that usually requires surgical intervention, a ecting over two million people, with an alarming increase in the prevalence with advanced age [ ]. Mitral regurgitation may occur as a result of mitral valve lea et disease and/or defects in the mitral valve apparatus, or as a consequence of left ventricular dysfunction [ ]. This condition may be classi ed as either primary or secondary. Primary regurgitation, also known as degenerative or organic regurgitation, is caused by an intrinsic lesion of the mitral valve apparatus. Secondary mitral regurgitation, frequently called functional or ischemic, is a disease of the left ventricle [ ].
Degenerative mitral valve disease, most often mitral lea et prolapse and/or ail, is the most common cause of primary mitral regurgitation, with two major phenotypes: broelastic de ciency and Barlow's disease [ ].
Younger patients with a persistent murmur are more likely to have Barlow's disease, a condition distinguished by an abundance of lea et tissue. The chordae and lea ets appear thickened, redundant and elongated [ ]. During systole, multiple scallops of either anterior and posterior leaflets prolapse or flail into the left atrium [ ].
Prolapse is estimated to occur in approximately . % of the general population using existing echocardiographic diagnostic criteria. Just -% of patients with mitral valve prolapse have severe regurgitation. Most of the time, mitral valve prolapse is associated with chronic mitral regurgitation, but complications (i.e., chordae rupture, infective endocarditis) can lead to the onset of acute mitral regurgitation. The rate at which regurgitation progresses is an interindividual variable. Factors that influence progression are primarily anatomical and related to the etiology of the lesion, with patients with mitral valve flail and mitral ring dilation experiencing faster progression.
Despite the fact that Barlow's disease was rst described in the early s, no signi cant risk factors have been identi ed, and the mechanisms underlying mitral valve prolapse are still partially understood. Moreover, although Barlow's disease is the most common cause of non-ischemic mitral regurgitation in developed countries, currently, there is no successful pharmacological treatment available to prevent or slow its progression. Studies in humans aimed at examining the cellular and molecular mechanisms underlying the di erent stages of myxomatous degeneration development and progression are rather scarce. One potential contributor is the local, persistent oxidative stress [ ].
Here, we present the case of a young patient with primary severe mitral regurgitation that underwent surgical intervention, describing the molecular mechanisms underlying the treatment and his evolution.

Case Report
A -year-old male patient, with no previous signi cant medical background and no medication at home, was diagnosed with severe mitral regurgitation at a regular check-up, about three weeks prior to presentation at our clinic. He presented with mild exertion dyspnea and fatigue and was admitted to the hospital for further investigations and treatment. Clinical examination revealed regular heart sounds and a late systolic murmur with a mid-to-late systolic click.
The patient's blood pressure at arrival was / mmHg. The ECG at admission showed a sinus rhythm, HR = bpm, and standard blood tests were within normal limits.
Transthoracic echocardiography showed intensely thickened mitral lea ets with a myxomatous appearance, prolapse of the middle scallop of the posterior lea et (P ), apparently with ruptured chordae, severe mitral regurgitation with a holosystolic eccentric jet (reaching the anterior wall of the left atrium) and left atrial enlargement. Left ventricular systolic function was within normal limits, with a left ventricular ejection fraction (LVEF) of %, left ventricular end-diastolic volume (LVEDV) of mL and left ventricular end-systolic volume (LVESV) of mL. There was also ultrasound evidence of mild tricuspid regurgitation and mild secondary pulmonary hypertension, with a normal systolic function and normal right ventricle size.
The transesophageal echocardiography with D reconstruction revealed a suggestive aspect for Barlow's disease ( Figure ): P scallop ail with chordal rupture (surgical view), and in the color Doppler section ( Figure ), we visualized severe mitral regurgitation with an eccentric jet adjacent to the anterior mitral valve. The next step was to initiate treatment with a loop diuretic carefully administered to relieve the pulmonary pressures.
Given the presence of heart failure symptoms, minimally invasive mitral valve repair was performed, with triangular P resection and ring annuloplasty. Postoperatively, the patient's evolution was favorable, without complications. He was discharged after days, with a signi cantly improved clinical status.
Identi cation of pathways leading to the development of di use myxomatous degeneration of lea et tissue is of major clinical relevance, given the fact that myxomatous mitral valve disease a ects young adult patients. Therefore, our purpose was to investigate valvular oxidative stress using a sample of a mitral valve harvested during valvular surgery, but after rst receiving the patient's permission in a signed consent document.
We assessed reactive oxygen species (ROS) production after sample incubation with angiotensin ( nM, h) with and without the angiotensin receptor type (AT ) antagonist (irbesartan microM, h), by confocal microscopy using the dihydroethidium (DHE) marker. Our choice was irbesartan, a strong and selective antagonist of AT receptors that is often used to treat mild to moderate hypertension. Previous research has shown that irbesartan possesses anti-in ammatory properties at the endothelium level by inhibiting TNF alpha and IL , as well as a strong antioxidant e ect on many body tissues. Brie y, the valve sample was frozen, cut in cryosections ( µm thick) and incubated with DHE for min. The slides were mounted and analyzed using a confocal microscope (Olympus Fluoview FV ). Images were obtained using laser excitation at nm and emission at nm. Image analysis was performed using ImageJ software.
We observed that the level of ROS in the mitral valve sample prelevated from our patient was alleviated after incubation with the angiotensin receptor type I (AT ) antagonist (irbesartan microM), especially after angiotensin incubation (Figure ).

Discussion
Prolapse of the myxomatous mitral valve is a typical cardiac abnormality. Barlow's disease is characterized by an abundance of myxomatous lea et tissue, prolapse or billowing of bilea et tissue, chordae elongation and annular dilatation with or without calci cation [ ].
Besides echocardiography, up to now, there are no reliable biomarkers available for the identi cation of this pathology. Early therapies aimed to reduce lea et stress in genetically susceptible individuals may theoretically delay the progression of myxomatous mitral valve degeneration and prevent late valvular complications such as severe mitral valve regurgitation, arrhythmia or infective endocarditis.
Following a routine check-up, the patient was diagnosed with mitral valve prolapse without signi cant regurgitation, being asymptomatic at the time. He developed symptoms of heart failure after the onset of a complication: chordae rupture, which culminated in severe mitral regurgitation as a secondary complication.
Interestingly, there is one in vitro study that used surgically excised mitral lea et specimens and reported (poster presentation) that myxomatous mitral valve changes might be pharmacologically modi able by an angiotensin receptor blocker [ ].
Since the patient lacked anyrenin-angiotensin-aldosterone system medication, we thought to incubate ex vivo the mitral valve sample with angiotensin ( h, nM). This led to increased ROS production that was mitigated by the incubation with the angiotensin receptor type (AT ) antagonist irbesartan ( microM). These ndings add new information about the role of the renin-angiotensin system in the control of the oxidative stress response in our patient's case, highlighting the protective e ect of the AT receptor antagonist irbesartan. Certainly, a number of questions remain unsolved, and further study will be needed to understand the pathomechanisms underlying this phenomenon. Modulation of various blood proteins' expression was examined in patients su ering from di erent grades of mitral regurgitation (mild, moderate and severe) vs. healthy controls. To this end, several routine clinical assays and the multi-analyte pro le technology targeting proteins were used. High-density lipoprotein, apolipoprotein-A , haptoglobin and haptoglobin-α chain levels signi cantly decreased proportionally to the degree of mitral regurgitation as compared to the controls. High-density lipoprotein and apolipoprotein-A levels were associated with an e ective regurgitant ori ce area and regurgitant volume.
Apolipoprotein-A was an independent predictor of severe mitral regurgitation. Moreover, myxomatous mitral valves were studied by immunocytochemistry, and the authors observed an increase in LC , the marker of autophagy, in myxomatous mitral valves compared with healthy mitral valves.
Hagler et al. tested the hypothesis that increased transforming growth factor-β (TGF-β) signaling and ROS are major contributors to pro-brotic gene expression in human mitral valves. Studies were performed on samples of human mitral valve tissue collected during surgery at the Mayo Clinic, Rochester ( controls were non-myxomatous mitral valves explanted from cardiac transplant recipients, and samples were collected from individuals with myxomatous mitral valve degeneration during mitral valve repair procedures). Using qRT-PCR, the authors found that increased expression of TGF-β in mitral valves from humans with myxomatous degeneration was associated with increased expression of connective tissue growth factor (CTGF). They concluded that activation of TGF-βsignaling is a major contributor to brosis and matrix remodeling in myxomatous mitral valve degeneration and is ampli ed by increased oxidative stress. Treatments aimed at reducing TGF-β activation and mitigating oxidative stress in early myxomatous mitral valve degeneration may slow its progression [ ].
Gladden et al. identi ed increased xanthine oxidase as a source of oxidative stress, as well as clusters of small mitochondria as a hallmark of a bioenergetic defect, in the left ventricle of patients with isolated mitral regurgitation and an LVEF of > % [ ].
More recently, in patients, Sogia et al. investigated the osteoprotegerin levels, a molecule that has been associated with cardiometabolic disorders and increased cardiovascular mortality and also linked to oxidative stress, in patients who underwent mitral valve repair due to mitral valve prolapse [ ]. They assessed the levels of oxidized and reduced glutathione by means of liquid chromatography-tandem mass spectrometry. They reported increased oxidative stress and osteoprotegerin concentrations in patients with mitral valve prolapse as compared to the controls.

Conclusions
This case is worth mentioning as a starting point for a prospective study aimed at assessing the role of valvular oxidative stress and mitochondrial dysfunction in patients with various degrees of primary and secondary mitral regurgitation. Here, we rstly reported an increase in local oxidative stress in the presence of angiotensin in a mitral valve harvested from a young patient with primary severe mitral regurgitation. This e ect was mitigated after treating the sample with an angiotensin receptor type (AT ) antagonist. The sources and types of ROS will be further investigated in the presence vs. absence of other cardiovascular drugs as well.