Left Ventricular Fibrosis and CMR Tissue Characterization of Papillary Muscles in Mitral Valve Prolapse Patients

Purpose. Mitral valve prolapse (MVP) is associated with left ventricle (LV) fibrosis, including the papillary muscles (PM), which is in turn linked to malignant arrhythmias. This study aims to evaluate comprehensive tissue characterization of the PM by cardiovascular magnetic resonance (CMR) imaging and its association with LV fibrosis observed by intraoperative biopsies. Methods. MVP patients with indication for surgery due to severe mitral regurgitation (n=19) underwent a preoperative CMR with characterization of the PM: dark-appearance on cine, T1 mapping, conventional bright blood (BB) and dark blood (DB) late gadolinium enhancement (LGE). CMR T1 mapping was performed on 21 healthy volunteers as controls. LV inferobasal myocardial biopsies were obtained in MVP patients and compared to CMR findings. Results. MVP patients (54±10 years old, 14 male) had a dark-appearance of the PM with higher native T1 and extracellular volume (ECV) values compared with healthy volunteers (1096±78ms vs 994±54ms and 33.9±5.6% vs 25.9±3.1%, respectively, p<0.001). Seventeen MVP patients (89.5%) had fibrosis by biopsy. BB-LGE+ in LV and PM was identified in 5 (26.3%) patients, while DB-LGE+ was observed in LV in 9 (47.4%) and in PM in 15 (78.9%) patients. DB-LGE+ in PM was the only technique that showed no difference with detection of LV fibrosis by biopsy. Posteromedial PM was more frequently affected than the anterolateral (73.7% vs 36.8%, p=0.039) and correlated with biopsy-proven LV fibrosis (Rho 0.529, p=0.029). Conclusions. CMR imaging in MVP patients referred for surgery shows a dark-appearance of the PM with higher T1 and ECV values compared with healthy volunteers. The presence of a positive DB-LGE at the posteromedial PM by CMR may serve as a better predictor of biopsy-proven LV inferobasal fibrosis than conventional CMR techniques.


Introduction
Mitral valve prolapse (MVP) is the most common valvular heart disease in high-income countries with a prevalence of 2-3% (1). MVP may lead to ventricular arrhythmias and sudden cardiac death (SCD), mitral regurgitation (MR), left ventricular (LV) remodeling, and heart failure (1). Given the negative prognostic impact of myocardial changes secondary to MVP, increasing interest has been focused on early identi cation of LV structural alterations.
In MVP patients, regional myocardial brosis, mainly in the basal inferolateral LV wall (i.e. inferobasal region) and at the level of the papillary muscles (PM), has been demonstrated (2-6). This regional brosis has been hypothesized to be in part secondary to abnormal PM traction / mechanical stress in response to increased chordal tension from a prolapsing MV (4,(7)(8)(9). Importantly, recent data indicate that PM brosis may act as an arrhythmogenic substrate in MVP patients (2,10,11).
While conventional "bright-blood" LGE (BB-LGE) has been commonly used to detect PM brosis in MVP patients (2,5,(12)(13)(14), more recently, Van De Heyning et al. showed that "dark-blood" LGE (DB-LGE) sequences with suppression of the blood pool signal were more sensitive for the detection of PM brosis (15). Furthermore, Scatteia et al. found a dark-appearance of the PM on cine images to be a typical feature of MVP (16). Finally, CMR T1 mapping is suitable to quantify diffuse interstitial brosis by increased extracellular volume (ECV) expansion and higher native T1 times (17)(18)(19), but has not been described at the level of the PM.
Hence, the aims of this prospective study involving MVP patients referred for MV surgery due to severe MR are: 1) to perform a comprehensive tissue characterization of the PM on CMR using healthy individuals as reference group for CMR T1 mapping-derived measurements; 2) to evaluate the diagnostic accuracy of conventional and DB-LGE compared to the results of intraoperative LV myocardial biopsy; and 3) to analyze the association between histological quanti cation of LV brosis and the comprehensive tissue characterization of the PM on CMR. We hypothesize that CMR tissue characterization of PM increases the identi cation of myocardial brosis in MVP patients as determined by myocardial biopsy.

Study Population
Patients were prospectively enrolled in the study between March 2020 and February 2022. The study was conducted in accordance with the Declaration of Helsinki, and was approved by the local research ethics committee (450-18-ek). All subjects gave written informed consent prior to study initiation. Patients with MVP referred for MV surgery were included in the analysis. Screening and assessment of MVP and MR severity were performed using echocardiography according to standard guidelines (20). Exclusion criteria were atrial brillation, previous heart valve surgery, more than mild disease of other heart valves, intracardiac shunts, other known causes of cardiomyopathy including coronary artery disease, or typical contraindications for CMR imaging. All patients underwent successful MV repair via a right mini-thoracotomy approach.
Patient demographics and clinical data were obtained at the time of hospital admission for MV repair surgery, followed by echocardiography and CMR including a comprehensive tissue characterization of the PM.
Healthy individuals without MVP, more than mild heart valve disease, diabetes, and LV hypertrophy or other known cardiomyopathies were enrolled as a control group for CMR T1 mapping values.

Echocardiography
Transthoracic echocardiography (TTE) was performed using standard commercially available ultrasound machines equipped with 2.25-4.25 MHz transducers. Evaluation of MVP was carried out by one cardiologist, expert in echocardiography and valvular heart diseases, blinded to the results of CMR exams. TTE was acquired using standard views, and Doppler measurements were evaluated as the average of three cycles. Color Doppler interrogation of the MR jet was performed in multiple views. A multimodal approach was used to determine MR severity, in concordance with recent guidelines (20,21).

Cardiovascular magnetic resonance imaging
All CMR examinations were performed on a dedicated 1.5 Tesla magnetic resonance scanner (Ingenia, Philips Healthcare, Best, The Netherlands) using a 28-element array coil. Image data acquisition (Additional le, Appendix) adhered to current recommendations (22). In brief, for cine CMR, steady-state free precession (SSFP) sequences were used during repetitive breath-holding. All standard cardiac geometries were acquired (multiple, gapless short-axis slices covering the entire left ventricle and 2-, 3-and 4-chamber views). Two-dimensional phase-contrast ow measurements were performed in the ascending aorta. Cine short axis images were used to measure left ventricular end-diastolic (LVEDV), end-systolic volume (LVESV), and left ventricular stroke volume (LVSV). With standard volumetric CMR regurgitant volume (RVol) and fraction (RF) were derived as previous described (23).
I. Papillary muscles signal (dark-appearance). For the measurement of PM signal, a mid-ventricular short-axis cine SSFP image in end-systolic phase was selected as it allowed for a better visualization of both PM. The PM signal was visually categorized as "dark-appearance" or "normal PM" when compared with LV myocardium, and quanti ed using the signal intensity ratio as previously described (16).
II. Late gadolinium enhancement. Both BB-LGE and DB-LGE were performed in succession 10-15 min after the application of intravenous contrast (gadolinium-DTPA, 0.2 mmol/kg) in the same three long-axis and short-axis views used for cine imaging (Additional le, Appendix). All LGE images were acquired during breath-holding, and dark-blood was acquired as the last sequence of each CMR study.
Images were evaluated o ine for the presence (LGE+) or absence (LGE-) of LGE in LV and both PM, LV pattern (subendocardial, mid-myocardial, subepicardial, transmural), and regional distribution of LGE areas using a standardized myocardial 17-segment model.
III. T1 and extracellular volume measurements. An ECG-gated modi ed Look-Locker inversion (MOLLI) sequence with SSFP image readout was acquired at a basal, mid, and apical LV short-axis geometry pre-and 15 min post-contrast, making efforts to obtain the maximal myocardial mass of PM. Native T1 and ECV values were determined for the 16 LV segments of the short axis and separately also for both PM: anterolateral (PMal) and posteromedial (PMpm).
Post-processing was carried out o ine (using IntelliSpace Portal 6, Philips Healthcare) with all readers fully blinded to clinical and echocardiographic data. T1-mapping, BB-LGE and DB-LGE sequences were analyzed by a cardiologist (RS) with over 7 years of experience in CMR imaging, separated and in random order at least 4 weeks apart from initial acquisitions, and blinded to the general imaging and histological data. In case of uncertainty, consensus was provided by a second level III observer (CJ).
Histological and immunohistochemical evaluation LV biopsies were obtained from each patient from the inferobasal LV wall using a surgical rongeur under thoracoscopic guidance. The biopsies were xed in 4% formaldehyde/phosphate-buffered saline and embedded in para n before cutting 3 µm sections. Histopathology and immunohistochemistry methods are described in the Appendix (Additional le). Brie y, Masson-Goldner's trichrome staining kit (Carl Roth GmbH, Karlsruhe, Germany) was used to stain connective tissue and collagen I was stained by immunohistochemistry using the mouse anti-collagen I antibody (Abcam, Cambridge, UK).
Documentation and quanti cation of connective tissue and collagen were performed using AxioPlan 2 microscope, Axio Cam camera system,  segments. There was insu cient evidence to support differences in the numbers of LV segments detected by each LGE methods.
LGE was detected in the PM in 5 subjects by BB-LGE and in 15 subjects by DB-LGE (26.3 % vs. 78.9 %, p = 0.001). All cases with BB-LGE+ at the level of the PM were con rmed on DB-LGE. Overall, brosis was found more frequently in the PMpm than the PMal (73.7 % vs. 36.8 %, p = 0.039).

Biopsy derived myocardial brosis and correlations with CMR parameters
In all MVP patients, a myocardial biopsy was obtained from the inferobasal LV region. The extent of histological brosis was quantifed by CVF with a mean value of 25.1 ±16% and a collagen I fraction of 13.7 ±11%. In the entire cohort, a total of 17 MVP patients (89.5 %) had an abnormal histological nding (CVF >10.9 %). There were no sex-related differences in brosis (CVF male 22.8 ±15% vs. female 31.2 ±17%, p = 0.369).
Compared with histologic ndings, only PM DB-LGE+ showed no statistical differences in detection of brosis (89.5 % vs. 78.9 %; Figure 4). Figure 5 shows three examples of MVP patients with and without LGE.
The extent of biopsy-derived CVF and the presence of DB-LGE+ at the PM had both a signi cant inverse correlation with CMR LV volumes, and CVF likewise with MR severity (Table 3). From all CMR parameters for PM tissue characterization, only a DB-LGE+ at the level of the PMpm was directly correlated with both biopsy derived parameters: CVF and collagen I fraction from the inferobasal LV region (Figure 3, part II). Finally, CMR derived PM ECV signi cantly correlated with the extent of mitral annular disjunction (MAD), with PM native T1 values, and with the presence of PM DB-LGE+, particularly at the PMpm (Table 3).

Discussion
This is the rst study of CMR derived tissue characterization of the PM combining CMR cine images, T1 mapping, conventional and DB-LGE sequences and comparing its results with histological assessment of myocardial brosis of the inferobasal LV wall in MVP patients.
The main ndings of this study are: 1) only MVP patients showed a dark-appearance of the PM on CMR cine compared with healthy volunteers, with higher native T1 and ECV values at the level of the LV myocardium and even more pronounced at the level of the PM; 2) with the use of a DB-LGE technique there was only a tendency to detect a higher prevalence of LGE within LV myocardium, but a signi cantly higher rate of LGE detection at the level of the PM when compared with BB-LGE; 3) compared with histology, only PM DB-LGE showed no statistical differences in detection of brosis; 4) the presence of DB-LGE + at the PMpm correlated with biopsy-proven brosis of the inferobasal LV wall, while other CMR sequences did not; and 5) there was an inverse correlation between extent of brosis and CMR derived LV volumes and MR severity.
CMR tissue characterization of the papillary muscles Scatteia and coworkers have previously described a dark-appearance of the PM on standard short axis cine CMR images as a typical feature of MVP, which was not linked to myocardial brosis by LGE or to the occurrence of malignant arrhythmias (16). In accordance with their ndings, we also failed to demonstrate a correlation between dark-appearance of the PM on CMR cine and PM T1 mapping parameters, with LGE at the level of the PM or LV myocardium, or with histologically-proven LV brosis. This might suggest that dark-appearance of the PM in CMR cine constitutes an early sign related to increased mechanical stress (9).
While altered T1 mapping parameters at the level of the LV in MVP patients (12,19) and its association with worse remodeling after mitral valve repair (25)  the PMal, suggesting more pronounced mechanical stress of the posteromedial than the anterolateral PM.
We did not nd a correlation between LV LGE and inferobasal LV myocardial biopsy. This is in line with ndings by Liu and colleagues (26), and might suggest an underestimation of brosis even with the use of DB-LGE at the level of LV myocardium.
Finally, we found an inverse correlation between CVF by biopsy and CMR LV volumes and MR severity ( LGE developed a ail lea et in an earlier phase of the disease. Moreover, LV volume overload in MVP has been associated with increased interstitial space but progressive collagen degradation (33). Loss of extracellular matrix may allow for a more compliant LV.

Clinical implications
Regionalized inferobasal LV and PM brosis have been identi ed as an arrhythmogenic substrate and plausible explanation for SCD even in asymptomatic patients with hemodynamically uncomplicated MVP (2, 14, 34, 35). An increasing understanding of these mechanisms raises the question of whether surgical treatment or other medical interventions in a subgroup of high-risk selected MVP patients should occur earlier than currently indicated (36, 37). The major challenge is the early identi cation of patients at risk for developing severe ventricular arrhythmias or irreversible LV dysfunction within a large population of patients with asymptomatic hemodynamically uncomplicated MVP (33,35). The proposed CMR tissue characterization of the PM may serve as a supplementary diagnostic tool with potential application in this area.

Study limitations
The present study has some limitations. First, the study was not powered or designed to assess the relationship of the results with MVP subtypes or with clinical characteristics like ventricular arrhythmias. Second, the results should be interpreted with caution since a small sample size could lead to type II errors and correlations could not be adjusted for type I errors. Finally, histological ndings might be limited by sampling error. Thus, further studies utilizing a longitudinal design are required to better evaluate the in uence of MVP subtypes and disease progression on unfavorable myocardial structural changes and cardiac arrhythmias.        Detection of brosis with CMR and histology.