Attenuated Mitral Leaflet Enlargement Contributes to Functional Mitral Regurgitation After Myocardial InfarctionOns Marsit, MSC,a Marie-Annick Clavel, DVM, PHD,a Claudia Côté-Laroche, MD,a Sandra Hadjadj, MSC,a

Marc-André Bouchard, MD,a Mark D. Handschumacher, BS,b Marine Clisson, MSC,a Marie-Claude Drolet, MSC,a Marie-Chloé Boulanger, PHD,a Dae-Hee Kim, MD, PHD,c J. Luis Guerrero, BS,d Philipp Emanuel Bartko, MD,d Jacques Couet, PHD,a Marie Arsenault, MD,a Patrick Mathieu, MD, MSC,a Philippe Pibarot, DVM, PHD,a

Elena Aïkawa, MD, PHD,d Joyce Bischoff, PHD,e Robert A. Levine, MD,d Jonathan Beaudoin, MDa

ABSTRACT

BACKGROUND Mitral leaflet enlargement has been identified as an adaptive mechanism to prevent mitral regurgita-tion in dilated left ventricles (LVs) caused by chronic aortic regurgitation (AR). This enlargement is deficient in patients with functional mitral regurgitation, which remains frequent in the population with ischemic cardiomyopathy.Maladaptive fibrotic changes have been identified in post-myocardial infarction (MI) mitral valves. It is unknown if these changes can interfere with valve growth and whether they are present in other valves.

OBJECTIVES This study sought to test the hypothesis that MI impairs leaflet growth, seen in AR, and induces fibrotic changes in mitral and tricuspid valves.

METHODS Sheep models of AR, AR þ MI, and controls were followed for 90 days. Cardiac magnetic resonance, echocardiography, and computed tomography were performed at baseline and 90 days to assess LV volume, LV function, mitral regurgitation and mitral leaflet size. Histopathology and molecular analyses were performed in excised valves.

RESULTS Both experimental groups developed similar LV dilatation and dysfunction. At 90 days, mitral valve leaflet size was smaller in the AR þ MI group (12.8 ± 1.3 cm2 vs. 15.1 ± 1.6 cm2,p ¼ 0.03). Mitral regurgitant fraction was 4% ± 7% in the AR group versus 19% ± 10% in the AR þ MI group (p ¼ 0.02). AR þ MI leaflets were thicker compared with AR and control valves. Increased expression of extracellular matrix remodeling genes was found in both the mitral and tricuspid leaflets in the AR þ MI group.

CONCLUSIONS In these animal models of AR, the presence of MI was associated with impaired adaptive valve growth and more functional mitral regurgitation, despite similar LV size and function. More pronounced extracellular remodeling was observed in mitral and tricuspid leaflets, suggesting systemic valvular remodeling after MI.(J Am Coll Cardiol 2020;75:395–405) © 2020 by the American College of Cardiology Foundation

From the aInstitut Universitaire de Cardiologie et de Pneumologie de Québec–Université Laval, Québec City, Quebec, Canada; bCenter for Excellence in Vascular Biology, Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts; cDivision of Cardiology, Asan Medical Center, College of Medicine, University of Ulsan, Seoul, Korea; dCardiac Ultrasound Lab, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts; and the eVascular Biology Program and Department of Surgery, Boston Children ’s Hospital and Department of Surgery, Harvard Medical School, Boston, Massachusetts. This work has been funded by the Heart and Stroke Foundation of Canada (GIA G-15- 0008860), Canadian Institutes for Health Research (399323), and Fonds de Recherche Santé-Québec (to Dr. Beaudoin). Dr. Clavel has had a Core Laboratory contract with Edwards Lifesciences; and has received a research grant from Medtronic. Dr. Pibarot has had Echo Core Laboratory contracts with Cardiac Phoenix and Edwards Lifesciences. Dr. Aïkawa has received grants from the National Institutes of Health. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.

Manuscript received October 15, 2019; revised manuscript received November 8, 2019, accepted November 13, 2019

Although functional mitral regurgitation (FMR) is primarily caused by left ventricular (LV) dilatation and dysfunction (1–8), intrinsic valvular changes in response to LV disease are also present. Three-dimensional (3D) imaging studies have shown that LV dilatation is associated with mitral valve (MV) enlargement, suggest- ing a potential mechanism for preventing FMR (9–13). However, this compensatory enlargement is variable, and FMR is predom- inantly seen in patients with smaller MVs (10,13,14). The factors influencing valve enlargement are not known. Clinical and post-mortem studies have suggested maximal MV enlargement in patients with chronic aortic regurgitation (AR) (15), whereas relatively smaller valve areas were found in patients with ischemic heart dis- ease, despite comparable LV volumes (13).

Valve growth in AR has been studied experimentally, showing the activation of growth pathways in the valve, similar to what was found in models of stretched MV (11,16).

Conversely, other data suggest that adverse leaflet remodeling can contribute to FMR. Fibrotic leaflet changes induced by myocardial ischemia have been suggested as a potential contributor to post- myocardial infarction (MI) FMR (17,18), and abnor- mally stiff leaflets have been found in patients with heart failure with FMR (19,20). These changes include leaflet thickening, extracellular matrix remodeling with the presence of activated myofibroblasts, transforming growth factor (TGF)-b, and formation of microvessels (18). Current knowledge, therefore, suggests a complex role for leaflet remodeling in FMR: compensatory enlargement seems beneficial, whereas adverse fibrotic thickening is potentially harmful. The role of the underlying LV disease on mitral biology is still poorly explored. In particular, the link between the observed histologic/molecular MV changes after MI and the resulting lack of compensatory enlargement and occurrence of FMR have not been clearly demonstrated.

In this study, we hypothesized that fibrotic mitral changes associated with MI would negatively affect the compensatory leaflet growth seen in AR. Stan- dardized large-animal models of AR and AR þ MI have been created to assess leaflet enlargement and relate it to the occurrence of FMR and subsequent micro- scopic changes in the valve. To support the idea of post-MI systemic factors influencing valvular biology, the tricuspid valves (TVs) were also harvested for analyses.

METHODS

ANIMAL MODELS. Sixteen adult Dorset sheep (weight: 45 kg; 50% male/50% female) were divided equally in to AR and AR þ MI groups. The animals were loaded for 3 days with amiodarone (200 mg/day) before the intervention. All procedures and imaging studies were performed under general anesthesia. AR was achieved by adapting a method previously used in small animals (21). After carotid cannulation, a bioptome device was introduced to reach the aortic valve. Under transesophageal echocardiography guidance, the valve was grasped and perforated until severe AR was obtained (Figure 1) (22). The AR þ MI group had the same procedure followed by a limited left thoracotomy to access and ligate the distal left anterior descending coronary artery to produce a small apical MI (Figure 1) (17). Animals were observed for 90 days and killed. Three additional control sheep had no intervention and were kept for the same pro- tocol duration to provide normal valve tissue. MVs and TVs were dissected and divided for histopathol- ogy (frozen in optimal cutting

temperature compound and stored at –80o C), Western blots and quantitative polymerase chain reaction (PCR). This protocol was approved by the Laval University Animal Protection Committee according to the recommendations of the Canadian Council on Laboratory Animal Care.

FIGURE 1 Imaging Methodologies

(A) Procedural transesophageal echocardiography: long axis view showing the aortic valve with and without color Doppler. Out-of-plane imaging was required to visualize the eccentric aortic regurgitation jet (Online Videos 1 and 2). (B) Phase contrast magnetic resonance imaging in the ascending aorta, confirming moderate to severe aortic regurgitation (40% regurgitant fraction in the displayed case). (C) Late gadolinium enhancement sequence showing the apical myocardial infarction. (D) Computed tomography image with 3-dimensional mitral leaflet reconstruction. (E) Pathology specimen showing aortic valve perforation. (F) Pathology specimen showing the apical myocardial infarction.

TABLE 1 Ventricular and Regurgitant Volumes (MRI) at Baseline and 90 Days

Baseline

AR

Follow-Up

AR

Baseline

þ MI

Follow-Up

p Intergroups

Baseline Follow-Up

LVEDV, ml 87 ± 7 186 ± 54

84 ± 5 178 ± 53 0.555 0.809

LVESV, ml 39 ± 2 110 ± 44

36 ± 8 101 ± 43 0.414 0.692

LVEF, % 55 ± 3 42 ± 7 57 ± 10 45 ± 9 0.700

0.559

RVEDV, ml 72 ± 15

88 ± 10 71 ± 3 82 ± 7 0.742 0.290

RVESV, ml 23 ± 9 32 ± 10 25 ± 8 28 ± 7 0.347 0.434RVEF, % 69 ± 6 63 ± 10 65 ± 12 65 ± 9 0.132 0.710MR fraction, % 4 ± 5 4 ± 7 1 ± 2 19 ± 10 0.419 0.019AR fraction, % — 27 ± 8 — 26 ± 10 — 0.812

Values are mean ± SD.AR ¼ aortic regurgitation; CMR ¼ cardiac magnetic resonance; LVEDV ¼ left ventricular end-

diastolic volume; LVEF ¼ left ventricular ejection fraction; LVESV ¼ left ventricular end-systolic volume; MI ¼ myocardial infarction; MR ¼ mitral regurgitation; MRI ¼ magnetic resonance imaging; MV ¼ mitral valve; RVEDV ¼ right ventricular end-diastolic volume; RVEF ¼ right ventricular ejection fraction; RVESV ¼ right ventricular end- systolic volume.

IMAGING PROTOCOLS AND ANALYSES. Transesophageal echocardiography (Philips EPIC 7C ultra- sound system, X8-2t transducer; Philips Healthcare, Andover, Massachusetts) was used to guide the pro- cedure and document AR severity and LV wall motion abnormality for the AR þ MI group. Contrast- enhanced cardiac computed tomography (CT) (Phi- lips iCT 256 slices) and magnetic resonance imaging (MRI) (Philips Achieva 3T) under general anesthesia were performed at baseline and repeated 90 days later. CT acquisitions were retrospectively gated with intravenous beta-blockers as needed to achieve a target heart rate of <80 beats/min. CT and MRI datasets were anonymized for blinded analyses. CT images were processed in dedicated software (Omni4D, MD Handschumacher, Boston, Massachu- setts) as described previously (14) by using zoomed views on the mitral apparatus (excluding the apex). MV area was measured in diastole (Figure 1), and the closure area (3D surface produced by the closed leaflets) was traced in midsystole. The ratio of total leaflet area/closure area was calculated to assess the adequacy of leaflet enlargement. The annulus area (projected on its least-square plane) was also measured in its maximal and minimal dimensions to compute the annular contraction. Other FMR key parameters, such as tenting area, tenting volume, and tethering distances (distance between each papillary muscle and contralateral annulus), were measured. The MRI protocol included T1- and T2-weighted sequences, balanced steady-state free precession (SSFP) sequences in standard planes (complete short axis stack, long axis views) for global morphology and function, phase contrast sequence in the proximal aorta, and late gadolinium enhancement for MI size and location. The MI size was quantified on the late gadolinium enhancement short axis images by using a threshold for enhancement of 5 standard deviations compared with a normal reference zone (23). Left and right ventricle volume and function were derived from the short axis SSFP sequences. AR severity was assessed by using the regurgitant flow from the phase contrast sequence in the proximal aorta (Figure 1). The mitral regurgitation mechanism was assessed visually by echocardiography, and regurgitant vol- ume was computed from the MRI sequences as the difference between LV stroke volume and proximal aorta systolic flow (24). MRI images were processed with CVI42 (Circle Cardiovascular Imaging Inc., Cal gary, Canada).

EXCISED VALVE ANALYSES. A portion of each excised MV and TV leaflet was frozen in optimal cutting temperature compound. Serial transversal sections of MV and TV leaflets were obtained for Masson trichrome coloration and immunohisto- chemistry. Activated valvular interstitial cell pheno- type was achieved with a recognized myofibroblast marker anti–a-smooth muscle actin antibody (clone 1A4, catalog no. A5228, Sigma, St. Louis, Missouri). Other primary antibodies, including matrix metal- loproteinase (MMP)-2 (catalog no. IM51, Millipore, Ontario, Canada) and TGF-b1 (catalog no. orb214661, Biorbyt, St. Louis, Missouri), were used to document extracellular remodeling, and anti-ki67 antibody (catalog no. ab15580, Abcam, Cambridge, United Kingdom) was used to determine cell proliferation.Two blinded observers independently measured mitral and tricuspid leaflet thickness using image processing software (Image J, version 1.49, National Institutes of Health, Bethesda, Maryland). Leaflet thickness was determined by measuring the 10 thickest areas across the leaflets on the Masson tri- chrome coloration. Microvessel count was obtained on the central section of the anterior leaflet.

WESTERN BLOT ANALYSIS. Immunoblotting was performed as described (Online Appendix). All pri- mary antibodies were used at a 1:1,000 dilution and were purchased from Cell Signaling Technology (Danvers, Massachusetts) or Millipore.

REAL-TIME PCR. The analysis of MV and TV messenger RNA levels by quantitative real-time PCR is described in the Online Appendix. IDT (Coralville, Iowa) primer assays (pre-optimized specific primer pairs) and SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, California) were used.

STATISTICS. Imaging data are expressed by mean ± SD to summarize the characteristics of the animals. Continuous measurements obtained at baseline and follow-up were analyzed with a linear mixed model to compare sheep models of AR and AR þ MI (fixed factor group) measured at 2 periods (fixed factor time) with an interaction term between fixed factors. The dependence between measurements was modeled with an unstructured covariance matrix of correla- tion. Because data are correlated, a transformation (Cholesky factorization) was performed on the error distribution from the statistical model to verify the normality assumption with the Shapiro-Wilk tests. The Brown and Forsythe variation of Levene’s test statistic was used to verify the homogeneity of vari- ances. Some variables (LV end diastolic volume and LV end systolic volume) were log-transformed to fulfill the normality and variance assumptions from the statistical model. Explanted valve analyses (microscopic thickness, microvessels count, Western blot, and real-time PCR results) were compared in both experimental groups and with the control animals using analysis of variance and Student’s t-test versus Kruskal-Wallis and Mann-Whitney U analyses according to data distribution. The results were considered significant with p values <0.05.

FIGURE 2 Left Ventricle and Mitral Valve Metrics by Cardiac MRI and CT

A

B300 p = 0.80

200 p = 0.69

200

100

150

100

50

0Baseline

AR

CFollow-Up

ARBaseline AR+MI

Follow-Up AR+MI

0Baseline

AR

D

Follow-Up AR

Baseline AR+MI

Follow-Up AR+MI

20 p = 0.03

15

40 p < 0.01

30

20

1010

0

5

E1.6

1.4

1.2

1.0

Baseline AR

Follow-Up AR

Baseline AR+MI

p = 0.02

Follow-Up AR+MI

–10

F

1.6

1.4

1.2

1.0

AR AR+MI

Leaflet Area/Closure Area Ratio and Infarct Size

r = –0.939p = 0.002

0.8

Baseline AR

Follow-Up AR

Baseline AR+MI

Follow-Up AR+MI

0.80 5 10

Infarct Size (g)

CTL AR+MI

15 20

(A, B) LV dilatation was comparable in sheep with AR alone and AR þ MI. (C) Mitral valve area increased in the AR group, with smaller values in the AR þ MI group. (D) Mitral regurgitation was more important in AR þ MI group. (E) Mitral leaflet area adjusted for closure area. (F) There was a negative correlation between the adequacy of valve adaptation (ratio of leaflet area/closure area) and infarct size. Whiskers represent the minimal and maximal values for each group. AR ¼ aortic regurgitation; CT ¼ computed tomography; CTL ¼ control; LV ¼ left ventricle; MI ¼ myocardial infarction; MRI ¼ magnetic resonance imaging.

Mitr

al V

alve

Lea

flet A

rea

(cm

2 )Le

afle

t Are

a/Cl

osur

e Ar

eaLV

End

-Dia

stol

ic V

olum

e (m

l)

Leaf

let A

rea/

Clos

ure

Area

%

MR

LV E

nd-S

ysto

lic V

olum

e (m

l)

(A) Representative images showing an increased number of microvessels in AR þ MI mitral valves, also seen by (B) quantitative analysis.(C) Quantitative comparison of leaflet thickness: leaflets were thicker in the AR þ MI sheep. Values are mean ± SEM. *p < 0.05 between the AR and AR þ MI groups. Abbreviations as in .

Control AR AR+MI0.0

ARAR+MIControl0

31.020.51

*p < 0.01*

1.5p < 0.01

C4

B

Microvessels

AR+MIARControlA

FIGURE 3 Microscopic Morphologic Analyses

TABLE 2 Mitral Geometry (CT) at Baseline and 90 Days

Baseline

AR

Follow-Up Baseline

AR

þ MI

Follow-Up

p

Baseline

Intergroups

Follow-Up

MV leaflet area, cm2 9.7 ± 0.6 15.1 ± 1.6 9.4 ± 1.2 12.8 ± 1.3 0.760 0.030Closure area, cm2 7.8 ± 0.5 12.0 ± 1.4 7.5 ±

0.912.0 ± 1.5 0.554 0.961

Annulus area, cm2 6.9 ± 0.2 10.1 ± 1.2 6.7 ± 1.0

10.4 ± 1.6 0.745 0.734

Leaflet area/closure area 1.27 ± 0.04 1.26 ± 0.07 1.24 ± 0.08

1.06 ± 0.13 0.707 0.023

Annulus contraction, % 25.0 ± 4.9 15.5 ± 5 21.7 ± 4.1

14.0 ± 8 0.347 0.753

Tethering distance MP, mm 43.3 ± 2.2 49.9 ± 4.1 41.1 ± 1.9

51.7 ± 3.9 0.132 0.505

Tethering distance LP, mm 35.5 ± 1.8 43.4 ± 4.8 36.2 ± 1.7

43.7 ± 3.1 0.574 0.920

Values are mean ± SD.CT ¼ computed tomography; LP ¼ lateral papillary muscle; MP ¼ medial papillary muscle; other abbreviations as in Table 1.

The p values presented in this report have not been adjusted for multiplicity, and therefore inferences drawn from these p values may not be reproducible. All analyses were conducted with the statistical packages R, version 3.0.2 (R Foundation for Statistical Computing, Vienna, Austria) and SAS, version 9.4 (SAS Institute, Cary, North Carolina).

RESULTS

Eight animals in each group underwent the planned procedures. The first 2 AR animals died during the

procedure, and AR could not be induced in another animal. In the AR þ MI group, AR could not be induced in 2 animals. Therefore, the final analysis includes 5 AR and 6 AR þ MI animals. All animals surviving the procedure remained alive until the end of protocol.

MYOCARDIAL FUNCTION AND VALVE REGURGITATION.Baseline values of LV volume and function were normal and comparable between groups (Table 1). There was no significant valve regurgitation before the procedure. At 90 days, both groups had similar AR

# of

Mic

rove

ssel

s /

Leaf

let (

Ant

erio

r)

Mitr

al L

eafle

t Th

ickn

ess

(mm

)

FIGURE 4 Mitral Valve Immunohistochemistry

a-Smooth muscle actin–positive cells are more frequent along the endothelium in AR þ MI valves versus AR-alone and control valves; staining for TGF-b1 is more apparent in AR þ MI valves with increased cellular proliferation indicated by Ki67 staining; MMP-2 staining is greatest in AR þ MI valves versus AR-alone and control valves. MMP ¼ matrix metalloproteinase; TGF ¼ transforming growth factor; other abbreviations as in Figure 2.

fraction (27% ± 8% vs. 26% ± 10%; p ¼ 0.81), similar regurgitant volume (20 ± 10 ml vs. 17 ± 10 ml; p ¼ 0.56), severe LV dilatation (LV volume was 2.2 times larger in both groups vs. baseline; p ¼ 0.80 between groups) (Figure 2), and mild systolic dysfunction (LVEF: 42% ± 7% vs. 45% ± 9%; p ¼ 0.55). Similar results for volumes and LVEF were obtained by using CT datasets (Online Figure 1). MRI-derived infarct size was 7.9 ± 4.0 g in the AR þ MI group. Right ventricular function remained normal and similar in both groups. By MRI, mitral regurgitant fraction at 90 days was 4% ± 7% in the AR group versus 19% ± 10% in the AR þ MI group (p ¼ 0.01) (Figure 2).MITRAL APPARATUS MORPHOLOGY. Midsystolic mitral closure area and tethering distances increased, whereas annulus contraction decreased at 90 days versus baseline. Those changes were similar in both groups (Table 2). Mitral leaflet area increased versus baseline in both groups, but enlargement was smaller in the AR þ MI group compared with the AR group (12.8 ± 1.3 cm2 vs. 15.1 ± 1.6 cm2; p ¼ 0.03) (Figure 2).At 90 days, the leaflet size/closure area ratio was stable in the AR group but was significantly reduced in the AR þ MI group (p ¼ 0.023) (Table 2, Figure 2). In the AR þ MI group, there was a negative correlation (R2 ¼ 0.88) between infarct size and this ra- tio (Figure 2).EXPLANTED VALVE ANALYSES. MV leaflets in the AR þ MI group were 1.4-fold thicker versus the AR group and 2.1-fold thicker versus the control group (1.12 ± 0.16 mm vs. 0.81 ± 0.08 mm vs. 0.53 ± mm in the AR þ MI, AR, and control groups, respectively; p ¼ 0.002) (Figures 3 and 4). There was no significant difference in TV leaflet thickness (0.71 ± 0.13 mm vs. 0.71 ± 0.21 mm vs. 0.70 ± 0.15 mm in the AR þ MI, AR, and control groups, respectively) (p ¼ 0.99). Immunohistochemistry showed qualita- tively increased a-smooth muscle actin, TGF-b, Ki67, and MMP-2 staining in the AR þ MI group versus AR and control valves. Finally, the AR þ MI group howed numerous microvessels in the MV, unlike the other groups. Western blot in mitral leaflets showed an increased protein level of collagen in the AR þ MI group compared with the AR group (p < 0.01) (Figure 5). A similar trend was found in the TVs but without a statistically significant difference between the AR and AR þ MI groups (p ¼ 0.25).

REAL-TIME QUANTITATIVE PCR STUDIES. TGF-b1 and TGF-b2 gene expression as well as collagen 1, collagen 3, and MMP-2 were elevated in both experi- mental groups versus controls (all p < 0.05), with maximal expression seen in the AR þ MI group. The same pattern was observed in TVs (Figure 6).

Representative Western blot analysis showing levels of COL1A1 and COL3A1 for 3 control, 4 AR, and 5 AR þ MI mitral valves.Quantification of Western blot. Values are mean ± standard error of the mean. *p < 0.05 between the AR and AR þ MI groups. COL ¼ collagen; ERK ¼ extracellular signal–regulated kinase; other abbreviations as in .

AR+MIARCTL0

AR+MIARCTL0.0

2

1.0

0.5

41.5

6

*2.0

8 p < 0.012.5 p < 0.01B

ERK1/2

COL3A1

COL1A1

AR+MIARCTLA

FIGURE 5 Western Blot for Collagen in Mitral Leaflets

DISCUSSION

In this animal study, we show that: 1) the MV can expand rapidly to match a severe LV dilatation induced by AR, without detectable FMR in the first months of the disease; 2) this valve enlargement is blunted in the presence of a small apical MI, with resulting FMR; and 3) different molecular changes are seen in both MVs and TVs after MI, suggesting a systemic valvular response associated with MI (Central Illustration). Although AR induced compen- satory leaflet enlargement and post-MI fibrotic mitral changes have both been described before, the relation between MI and the lack of mitral compensatory growth (and resulting FMR), as well as the presence of tricuspid changes, are shown, to our knowledge, for the first time. This work further supports the idea that 2 conditions are necessary to induce FMR: LV and/or mitral annulus anomaly (dilatation, dysfunction, or distortion) and the lack of compensatory leaflet enlargement.

COL1

A1 /

Erk

1/2

CO

L3A

1 / E

rk

1/2

Evaluation by quantitative RT-PCR of the (A) mitral and (B) tricuspid valve messenger RNA levels of genes encoding for markers of fibrosis. The results are reported in arbitrary units as the mean ± standard error of the mean. The messenger RNA levels of the CTL group were normalized to 1. Blue columns represent the CTL group, red columns represent the AR group, and black columns represent the AR þ MI group. *p < 0.05 between the AR and AR þ MI groups; ¶ p < 0.05 between the CTL and AR þ MI groups. RT-PCR ¼ real-time polymerase chain reaction; other abbreviations as in .

AR+MIAR

TGF1TGF2Col1a1Col3a1MMP-20

MMP-2CTL

TGF1TGF2Col1a1Col3a10

2

2**

4

¶*¶¶¶

4¶

*¶6

**¶8

6¶¶

10

Tricuspid ValveB Mitral ValveA

FIGURE 6 RT-PCR Results (Mitral and Tricuspid Valves)

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Expr

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on

Rela

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Expr

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The first model (AR alone) did cause LV enlarge- ment and dysfunction, but adequate leaflet enlarge- ment was present, and no FMR was observed by echocardiography or MRI. Our data are consistent with previous clinical (pathology and imaging) and experimental data (13,15,16). Although FMR can be seen in patients with AR (25), it is typically not observed in the compensated stage of the disease, despite significant LV enlargement. Explanted valve analyses did show mild leaflet remodeling; thick- ening, rare microvessels and extracellular matrix remodeling were observed. Overall, this is consistent with previous animal work exploring the effects of mechanical stretch on mitral biology (11).Previous experimental models of apical MI with or without mechanical stretch (3,10) showed extensive cellular changes in the leaflets after an ischemic event but did not cause FMR in the absence of significant LV size/function abnormality. Our second experimental model (AR þ MI) therefore included 2 conditions that, taken alone, did not cause FMR. LV remodeling in this group was nearly identical to that in the AR-alone group: similar LV size and function by MRI as well as identical annulus size and contraction and teth- ering distances by 3D CT reconstructions. The apical MI was small and did not affect the LV variables commonly associated with FMR. This group, howev- er, had mitral leaflet changes at the cellular level, associated with smaller leaflets. This lack of leaflet enlargement combined with LV dilatation was asso- ciated with significant FMR. This experimental work is consistent with a previous clinical 3D echocardi- ography study, in which larger mitral leaflets were found in patients with AR versus those with FMR (mostly ischemic) despite comparable LV size, sug- gesting a potential influence of the underlying LV disease on valve remodeling.

An important additional observation of the present study is the presence of cellular changes in the TV leaflets in the AR þ MI group. Those changes did not result in detectable tricuspid regurgitation (which is expected, given the normal right ventricle size and function) but suggest systemic valve remodeling after MI. This is consistent with previously reported whole-heart changes after MI, including remote (noninfarcted) myocardial remodeling (26). The precise mechanisms of these changes remain to be elucidated. After MI, numerous neurohumoral and inflammatory processes are activated; some but not all of them are also present in chronic AR. Precise mechanisms underlying post-MI leaflet remodeling will require additional investigation because it could represent a key target for promoting valve adaptation and preventing FMR.

CENTRAL ILLUSTRATION Left Ventricular and Mitral Valve Changes in Aortic Regurgitation With and Without Myocardial Infarction

Marsit, O. et al. J Am Coll Cardiol. 2020;75(4):395–405.

(A) Adequate leaflet enlargement has the potential to match a severe left ventricular dilatation induced by aortic regurgitation and prevent mitral regurgitation. (B) Myocardial infarction is associated with mitral valve thickening, expression of extracellular matrix genes, and insufficient leaflet enlargement. These modifications are associated with the development of functional mitral regurgitation.

STUDY LIMITATIONS. This is an animal study exploring a single time point of dynamic and evolving processes. The induced AR resulted in severe LV dilatation and depressed ejection fraction, repre- senting an advanced stage of AR but not the entire continuum of the disease. Although aortic regurgita- tion fraction appeared to be lower in our series than what is observed in the clinical setting, there was a clear increase in LV volumes, strongly suggesting that the amount of AR we created was physiologically relevant. Exploration of mitral biology in both earlier (before LV dysfunction) and later (as heart failure progresses and eventual FMR can appear) stages will be interesting in future studies. Although the relation found between infarct size and leaflet adaptation is suggestive, this study was not primarily designed to assess this element. The sample size was small, and p values were not corrected for multiple testing. The sample size is comparable, however, to previously published large-animal data and with consistent re- sults. The small sample size is counterbalanced by animal model standardization and advanced multi- modality imaging techniques, giving precise and reproducible geometric and physiological metrics.

CONCLUSIONS

As opposed to isolated AR, the presence of a small apical MI produces MV changes associated with altered MV growth and the development of FMR. Tricuspid leaflets are also affected by those changes, suggesting a systemic process. Understanding these mechanisms could lead to new therapeutic opportu- nities to prevent FMR.

ACKNOWLEDGMENTS The authors thank the animal facility staff (Sébastien Poulin, Vincent Tellier, and Justin Robillard), Serge Simard for the biostatistical support, and Ahmed Benhamadi for graphic design.

ADDRESS FOR CORRESPONDENCE: Dr. Jonathan Beaudoin, Institut Universitaire de Cardiologie et de Pneumologie de Québec, 2725 Chemin Sainte-Foy, Québec City, Québec G1V4G5, Canada. E-mail: jonathan.beaudoin@criucpq.ulaval.ca.Twitter: @universitelaval.

PERSPECTIVES

COMPETENCY IN MEDICAL KNOWLEDGE: Impaired mo-lecular remodeling in mitral and tricuspid leaflets attenuates leaflet expansion in response to ventricular dilatation after myocardial infarction and contributes to the development of functional mitral regurgitation.

TRANSLATIONAL OUTLOOK: Further studies are needed toelucidate the mechanisms by which myocardial ischemia influ- ences valve biology and growth

RE FE RE NCE S

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KEY WORDS aortic regurgitation, LV remodeling, mitral regurgitation, mitral valve, myocardial infarction