Attenuated Mitral Leaflet Enlargement Contributes to Functional Mitral Regurgitation After Myocardial Infarction

JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY

VOL. 75, NO. 4, 2020

ª 2020 BY THE AMERICAN COLLEGE OF CARDIOLOGY FOUNDATION PUBLISHED BY ELSEVIER

Attenuated Mitral Leaflet Enlargement Contributes to Functional Mitral Regurgitation After Myocardial Infarction Ons 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 regurgitation 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; b

Center for Excellence in Vascular Biology, Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital,

Listen to this manuscript’s

Harvard Medical School, Boston, Massachusetts; cDivision of Cardiology, Asan Medical Center, College of Medicine, University of

audio summary by

Ulsan, Seoul, Korea; dCardiac Ultrasound Lab, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts;

Editor-in-Chief

and the eVascular Biology Program and Department of Surgery, Boston Children’s Hospital and Department of Surgery, Harvard

Dr. Valentin Fuster on

Medical School, Boston, Massachusetts. This work has been funded by the Heart and Stroke Foundation of Canada (GIA G-15-

JACC.org.

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.

ISSN 0735-1097/$36.00

https://doi.org/10.1016/j.jacc.2019.11.039

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MI Impairs Valve Adaptation

A

ABBREVIATIONS AND ACRONYMS 3D = 3 dimensional

lthough functional mitral regurgita-

the tricuspid valves (TVs) were also harvested for

tion (FMR) is primarily caused by

analyses.

left ventricular (LV) dilatation and

dysfunction (1–8), intrinsic valvular changes

AR = aortic regurgitation

METHODS

in response to LV disease are also present.

CT = computed tomography FMR = functional mitral regurgitation

LV = left ventricle MI = myocardial infarction MMP = matrix metalloproteinase

MRI = magnetic resonance imaging

MV = mitral valve PCR = polymerase chain

Three-dimensional

(3D)

imaging

studies

MODELS. Sixteen

adult

Dorset

sheep

(weight: 45 kg; 50% male/50% female) were divided

with mitral valve (MV) enlargement, suggest-

equally in to AR and AR þ MI groups. The animals

ing a potential mechanism for preventing

were loaded for 3 days with amiodarone (200 mg/day)

FMR (9–13). However, this compensatory

before the intervention. All procedures and imaging

enlargement is variable, and FMR is predom-

studies were performed under general anesthesia. AR

inantly seen in patients with smaller MVs

was achieved by adapting a method previously used

(10,13,14).

valve

in small animals (21). After carotid cannulation, a

enlargement are not known. Clinical and

bioptome device was introduced to reach the aortic

post-mortem

valve.

The

factors studies

influencing have

suggested

maximal MV enlargement in patients with

reaction

ANIMAL

have shown that LV dilatation is associated

Under

transesophageal

echocardiography

guidance, the valve was grasped and perforated until

(15),

severe AR was obtained (Figure 1) (22). The AR þ MI

factor

whereas relatively smaller valve areas were

group had the same procedure followed by a limited

TV = tricuspid valve

found in patients with ischemic heart dis-

left thoracotomy to access and ligate the distal left

ease, despite comparable LV volumes (13).

anterior descending coronary artery to produce a

Valve growth in AR has been studied experimentally,

small apical MI (Figure 1) (17). Animals were observed

showing the activation of growth pathways in the

for 90 days and killed. Three additional control sheep

valve, similar to what was found in models of

had no intervention and were kept for the same pro-

stretched MV (11,16).

tocol duration to provide normal valve tissue. MVs

TGF = transforming growth

chronic

aortic

regurgitation

(AR)

and TVs were dissected and divided for histopathol-

SEE PAGE 406

ogy (frozen in optimal cutting temperature comConversely, other data suggest that adverse leaflet

pound and stored at –80 C), Western blots and

remodeling can contribute to FMR. Fibrotic leaflet

quantitative polymerase chain reaction (PCR). This

changes induced by myocardial ischemia have been

protocol was approved by the Laval University Ani-

suggested

post-

mal Protection Committee according to the recom-

myocardial infarction (MI) FMR (17,18), and abnor-

mendations of the Canadian Council on Laboratory

mally stiff leaflets have been found in patients with

Animal Care.

heart failure with FMR (19,20). These changes include

IMAGING

leaflet thickening, extracellular matrix remodeling

esophageal echocardiography (Philips EPIC 7C ultra-

with the presence of activated myofibroblasts, trans-

sound system, X8-2t transducer; Philips Healthcare,

forming growth factor (TGF)-b , and formation of

Andover, Massachusetts) was used to guide the pro-

microvessels (18). Current knowledge, therefore,

cedure and document AR severity and LV wall motion

suggests a complex role for leaflet remodeling in

abnormality for the AR þ MI group. Contrast-

FMR: compensatory enlargement seems beneficial,

enhanced cardiac computed tomography (CT) (Phi-

whereas adverse fibrotic thickening is potentially

lips iCT 256 slices) and magnetic resonance imaging

harmful. The role of the underlying LV disease on

(MRI) (Philips Achieva 3T) under general anesthesia

mitral biology is still poorly explored. In particular,

were performed at baseline and repeated 90 days

the link between the observed histologic/molecular

later. CT acquisitions were retrospectively gated with

MV changes after MI and the resulting lack of

intravenous beta-blockers as needed to achieve a

compensatory enlargement and occurrence of FMR

target heart rate of <80 beats/min. CT and MRI

have not been clearly demonstrated.

datasets were anonymized for blinded analyses.

as

a

potential

contributor

to

PROTOCOLS

AND

ANALYSES. Trans-

In this study, we hypothesized that fibrotic mitral

CT images were processed in dedicated software

changes associated with MI would negatively affect

(Omni4D, MD Handschumacher, Boston, Massachu-

the compensatory leaflet growth seen in AR. Stan-

setts) as described previously (14) by using zoomed

dardized large-animal models of AR and AR þ MI have

views on the mitral apparatus (excluding the apex).

been created to assess leaflet enlargement and relate

MV area was measured in diastole (Figure 1), and the

it to the occurrence of FMR and subsequent micro-

closure area (3D surface produced by the closed

scopic changes in the valve. To support the idea of

leaflets) was traced in midsystole. The ratio of total

post-MI systemic factors influencing valvular biology,

leaflet area/closure area was calculated to assess the

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MI Impairs Valve Adaptation

F I G U R E 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.

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MI Impairs Valve Adaptation

1A4, catalog no. A5228, Sigma, St. Louis, Missouri).

T A B L E 1 Ventricular and Regurgitant Volumes (MRI) at Baseline and 90 Days

AR þ MI

AR

Other primary antibodies, including matrix metal-

p Intergroups

loproteinase (MMP)-2 (catalog no. IM51, Millipore,

Baseline

Follow-Up

Baseline

Follow-Up

Baseline

Follow-Up

Ontario, Canada) and TGF- b 1 (catalog no. orb214661,

LVEDV, ml

87  7

186  54

84  5

178  53

0.555

0.809

Biorbyt, St. Louis, Missouri), were used to document

LVESV, ml

39  2

110  44

36  8

101  43

0.414

0.692

55  3

42  7

57  10

45  9

extracellular remodeling, and anti-ki67 antibody

LVEF, %

0.700

0.559

RVEDV, ml

72  15

88  10

71  3

82  7

0.742

0.290

(catalog no. ab15580, Abcam, Cambridge, United Kingdom) was used to determine cell proliferation.

RVESV, ml

23  9

32  10

25  8

28  7

0.347

0.434

RVEF, %

69  6

63  10

65  12

65  9

0.132

0.710

Two blinded observers independently measured

MR fraction, %

45

47

12

19  10

0.419

0.019

mitral and tricuspid leaflet thickness using image

AR fraction, %

—

27  8

—

26  10

—

0.812

processing software (Image J, version 1.49, National Institutes of Health, Bethesda, Maryland). Leaflet

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 endsystolic volume.

thickness was determined by measuring the 10 thickest areas across the leaflets on the Masson trichrome coloration. Microvessel count was obtained on the central section of the anterior leaflet. WESTERN

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

BLOT

ANALYSIS. Immunoblotting

was

performed as described (Online Appendix). All primary antibodies were used at a 1:1,000 dilution and were purchased from Cell Signaling Technology (Danvers, Massachusetts) or Millipore.

parameters, such as tenting area, tenting volume, and

REAL-TIME

tethering distances (distance between each papillary

messenger RNA levels by quantitative real-time PCR

muscle and contralateral annulus), were measured.

is described in the Online Appendix. IDT (Coralville,

The MRI protocol included T1- and T2-weighted

Iowa) primer assays (pre-optimized specific primer

sequences, balanced steady-state free precession

pairs)

(SSFP) sequences in standard planes (complete short

Supermix (Bio-Rad, Hercules, California) were used.

axis stack, long axis views) for global morphology and

STATISTICS. Imaging data are expressed by mean 

function, phase contrast sequence in the proximal

SD to summarize the characteristics of the animals.

aorta, and late gadolinium enhancement for MI size

Continuous measurements obtained at baseline and

and location. The MI size was quantified on the late

follow-up were analyzed with a linear mixed model to

gadolinium enhancement short axis images by using

compare sheep models of AR and AR þ MI (fixed

and

PCR. The

analysis

SsoAdvanced

of

Universal

MV

and

SYBR

TV

Green

a threshold for enhancement of 5 standard deviations

factor group) measured at 2 periods (fixed factor time)

compared with a normal reference zone (23). Left and

with an interaction term between fixed factors. The

right ventricle volume and function were derived

dependence between measurements was modeled

from the short axis SSFP sequences. AR severity was

with an unstructured covariance matrix of correla-

assessed by using the regurgitant flow from the phase

tion. Because data are correlated, a transformation

contrast sequence in the proximal aorta (Figure 1).

(Cholesky factorization) was performed on the error

The mitral regurgitation mechanism was assessed

distribution from the statistical model to verify the

visually by echocardiography, and regurgitant vol-

normality assumption with the Shapiro-Wilk tests.

ume was computed from the MRI sequences as the

The Brown and Forsythe variation of Levene’s test

difference between LV stroke volume and proximal

statistic was used to verify the homogeneity of vari-

aorta systolic flow (24). MRI images were processed

ances. Some variables (LV end diastolic volume and

with CVI42 (Circle Cardiovascular Imaging Inc., Cal-

LV end systolic volume) were log-transformed to

gary, Canada).

fulfill the normality and variance assumptions from

EXCISED

VALVE

ANALYSES. A

portion of each

the statistical model. Explanted valve analyses

excised MV and TV leaflet was frozen in optimal

(microscopic thickness, microvessels count, Western

cutting temperature compound. Serial transversal

blot, and real-time PCR results) were compared

sections of MV and TV leaflets were obtained for

in both experimental groups and with the control

Masson

immunohisto-

animals using analysis of variance and Student’s

chemistry. Activated valvular interstitial cell pheno-

t-test versus Kruskal-Wallis and Mann-Whitney U

type was achieved with a recognized myofibroblast

analyses according to data distribution. The results

marker anti– a -smooth muscle actin antibody (clone

were considered significant with p values <0.05.

trichrome

coloration

and

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MI Impairs Valve Adaptation

F I G U R E 2 Left Ventricle and Mitral Valve Metrics by Cardiac MRI and CT

300

p = 0.80

200

100

0

p = 0.69

200 150 100 50 0

Baseline Follow-Up Baseline Follow-Up AR AR AR+MI AR+MI

C

Baseline Follow-Up Baseline Follow-Up AR AR AR+MI AR+MI

D 20

p < 0.01

40

p = 0.03

30 15 % MR

Mitral Valve Leaflet Area (cm2)

B LV End-Systolic Volume (ml)

LV End-Diastolic Volume (ml)

A

10

20 10 0

5

–10

Baseline Follow-Up Baseline Follow-Up AR AR AR+MI AR+MI

E

AR

F Leaflet Area/Closure Area Ratio and Infarct Size p = 0.02 1.4 1.2 1.0 0.8

Baseline Follow-Up Baseline Follow-Up AR AR AR+MI AR+MI

Leaflet Area/Closure Area

1.6 Leaflet Area/Closure Area

AR+MI

1.6 r = –0.939 p = 0.002

1.4 1.2 1.0 0.8

0

5

10 15 Infarct Size (g) CTL

20

AR+MI

(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.

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T A B L E 2 Mitral Geometry (CT) at Baseline and 90 Days

AR þ MI

AR

p Intergroups

Baseline

Follow-Up

Baseline

Follow-Up

Baseline

Follow-Up

MV leaflet area, cm2

9.7  0.6

15.1  1.6

9.4  1.2

12.8  1.3

0.760

0.030

Closure area, cm2

7.8  0.5

12.0  1.4

7.5  0.9

12.0  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

procedure, and AR could not be induced in another

adjusted for multiplicity, and therefore inferences

animal. In the AR þ MI group, AR could not be

drawn from these p values may not be reproducible.

induced in 2 animals. Therefore, the final analysis

All analyses were conducted with the statistical

includes 5 AR and 6 AR þ MI animals. All animals

packages R, version 3.0.2 (R Foundation for Statistical

surviving the procedure remained alive until the end

Computing, Vienna, Austria) and SAS, version 9.4

of protocol.

(SAS Institute, Cary, North Carolina).

MYOCARDIAL FUNCTION AND VALVE REGURGITATION.

RESULTS

Baseline values of LV volume and function were normal and comparable between groups (Table 1).

Eight animals in each group underwent the planned

There was no significant valve regurgitation before the

procedures. The first 2 AR animals died during the

procedure. At 90 days, both groups had similar AR

F I G U R E 3 Microscopic Morphologic Analyses

A

Control

AR

AR+MI

Microvessels

C 4

p < 0.01

*

3 2 1 0

Control AR

AR+MI

Mitral Leaflet Thickness (mm)

B # of Microvessels / Leaflet (Anterior)

400

1.5

p < 0.01

*

1.0

0.5

0.0

Control AR

AR+MI

(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 Figure 2.

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MI Impairs Valve Adaptation

F I G U R E 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

(12.8  1.3 cm 2 vs. 15.1  1.6 cm 2; p ¼ 0.03) (Figure 2).

regurgitant volume (20  10 ml vs. 17  10 ml; p ¼ 0.56),

At 90 days, the leaflet size/closure area ratio was

severe LV dilatation (LV volume was 2.2 times larger

stable in the AR group but was significantly reduced

in both groups vs. baseline; p ¼ 0.80 between

in the AR þ MI group (p ¼ 0.023) (Table 2, Figure 2). In

groups) (Figure 2), and mild systolic dysfunction

the AR þ MI group, there was a negative correlation

(LVEF: 42%  7% vs. 45%  9%; p ¼ 0.55). Similar

(R 2 ¼ 0.88) between infarct size and this ra-

results for volumes and LVEF were obtained by using

tio (Figure 2).

CT datasets (Online Figure 1). MRI-derived infarct

EXPLANTED VALVE ANALYSES. MV leaflets in the

size was 7.9  4.0 g in the AR þ MI group. Right

AR þ MI group were 1.4-fold thicker versus the AR

ventricular function remained normal and similar in

group and 2.1-fold thicker versus the control group

both groups. By MRI, mitral regurgitant fraction at

(1.12  0.16 mm vs. 0.81  0.08 mm vs. 0.53 

90 days was 4%  7% in the AR group versus 19% 

0.05 mm in the AR þ MI, AR, and control groups,

10% in the AR þ MI group (p ¼ 0.01) (Figure 2).

respectively; p ¼ 0.002) (Figures 3 and 4). There was

MITRAL

MORPHOLOGY. Midsystolic

no significant difference in TV leaflet thickness (0.71

mitral closure area and tethering distances increased,

APPARATUS

 0.13 mm vs. 0.71  0.21 mm vs. 0.70  0.15 mm in

whereas annulus contraction decreased at 90 days

the AR þ MI, AR, and control groups, respectively)

versus baseline. Those changes were similar in both

(p ¼ 0.99). Immunohistochemistry showed qualita-

groups (Table 2). Mitral leaflet area increased versus

tively increased a -smooth muscle actin, TGF- b , Ki67,

baseline in both groups, but enlargement was smaller

and MMP-2 staining in the AR þ MI group versus AR

in the AR þ MI group compared with the AR group

and control valves. Finally, the AR þ MI group

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MI Impairs Valve Adaptation

F I G U R E 5 Western Blot for Collagen in Mitral Leaflets

A

CTL

AR

AR+MI

COL1A1

COL3A1

ERK1/2 2.5

8

p < 0.01

2.0

COL3A1 / Erk 1/2

B

COL1A1 / Erk 1/2

402

1.5 1.0 0.5 0.0

CTL

AR

AR+MI

p < 0.01

*

6 4 2 0

CTL

AR

AR+MI

(A) Representative Western blot analysis showing levels of COL1A1 and COL3A1 for 3 control, 4 AR, and 5 AR þ MI mitral valves. (B) 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 Figure 2.

showed numerous microvessels in the MV, unlike the

DISCUSSION

other groups. Western blot in mitral leaflets showed an increased protein level of collagen in the AR þ MI

In this animal study, we show that: 1) the MV can

group compared with the AR group (p < 0.01)

expand rapidly to match a severe LV dilatation

(Figure 5). A similar trend was found in the TVs but

induced by AR, without detectable FMR in the first

without a statistically significant difference between

months of the disease; 2) this valve enlargement is

the AR and AR þ MI groups (p ¼ 0.25).

blunted in the presence of a small apical MI, with

REAL-TIME QUANTITATIVE PCR STUDIES. TGF- b 1

resulting FMR; and 3) different molecular changes are

and TGF-b2 gene expression as well as collagen 1,

seen in both MVs and TVs after MI, suggesting a

collagen 3, and MMP-2 were elevated in both experi-

systemic

mental groups versus controls (all p < 0.05), with

(Central Illustration). Although AR induced compen-

maximal expression seen in the AR þ MI group. The

satory leaflet enlargement and post-MI fibrotic mitral

same pattern was observed in TVs (Figure 6).

changes have both been described before, the relation

valvular

response

associated

with

MI

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MI Impairs Valve Adaptation

F I G U R E 6 RT-PCR Results (Mitral and Tricuspid Valves)

A

¶

8 6

* *

¶

¶

*

4

*

2 0

TGFβ β1

TGFβ β2

Col1a1

Tricuspid Valve 6

¶

¶

Relative Expression

10 Relative Expression

B

Mitral Valve

Col3a1

CTL

AR

¶

4

¶

¶

*

¶

2

0

MMP-2

*

TGFβ β1

TGFβ β2

Col1a1

Col3a1

MMP-2

AR+MI

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 Figures 2 to 4.

between MI and the lack of mitral compensatory

group was nearly identical to that in the AR-alone

growth (and resulting FMR), as well as the presence of

group: similar LV size and function by MRI as well

tricuspid changes, are shown, to our knowledge, for

as identical annulus size and contraction and teth-

the first time. This work further supports the idea that

ering distances by 3D CT reconstructions. The apical

2 conditions are necessary to induce FMR: LV and/or

MI was small and did not affect the LV variables

mitral annulus anomaly (dilatation, dysfunction, or

commonly associated with FMR. This group, howev-

distortion) and the lack of compensatory leaflet

er, had mitral leaflet changes at the cellular level,

enlargement.

associated with smaller leaflets. This lack of leaflet

The first model (AR alone) did cause LV enlarge-

enlargement combined with LV dilatation was asso-

ment and dysfunction, but adequate leaflet enlarge-

ciated with significant FMR. This experimental work

ment was present, and no FMR was observed by

is consistent with a previous clinical 3D echocardi-

echocardiography or MRI. Our data are consistent

ography study, in which larger mitral leaflets were

with previous clinical (pathology and imaging) and

found in patients with AR versus those with FMR

experimental data (13,15,16). Although FMR can be

(mostly ischemic) despite comparable LV size, sug-

seen in patients with AR (25), it is typically not

gesting a potential influence of the underlying LV

observed in the compensated stage of the disease,

disease on valve remodeling.

despite significant LV enlargement. Explanted valve

An important additional observation of the present

analyses did show mild leaflet remodeling; thick-

study is the presence of cellular changes in the TV

ening, rare microvessels and extracellular matrix

leaflets in the AR þ MI group. Those changes did not

remodeling were observed. Overall, this is consistent

result in detectable tricuspid regurgitation (which is

with previous animal work exploring the effects of

expected, given the normal right ventricle size and

mechanical stretch on mitral biology (11).

function) but suggest systemic valve remodeling after

Previous experimental models of apical MI with or

MI. This is consistent with previously reported

without mechanical stretch (3,10) showed extensive

whole-heart changes after MI, including remote

cellular changes in the leaflets after an ischemic event

(noninfarcted) myocardial remodeling (26). The pre-

but did not cause FMR in the absence of significant LV

cise mechanisms of these changes remain to be

size/function abnormality. Our second experimental

elucidated. After MI, numerous neurohumoral and

model (AR þ MI) therefore included 2 conditions that,

inflammatory processes are activated; some but not

taken alone, did not cause FMR. LV remodeling in this

all of them are also present in chronic AR. Precise

403

404

Marsit et al.

JACC VOL. 75, NO. 4, 2020 FEBRUARY 4, 2020:395–405

MI Impairs Valve Adaptation

C E N T R A L IL L U ST R A T I O N 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.

mechanisms underlying post-MI leaflet remodeling

found between infarct size and leaflet adaptation is

will require additional investigation because it could

suggestive, this study was not primarily designed to

represent a key target for promoting valve adaptation

assess this element. The sample size was small, and

and preventing FMR.

p values were not corrected for multiple testing. The

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, representing an advanced stage of AR but not the entire continuum of the disease. Although aortic regurgita-

sample size is comparable, however, to previously published large-animal data and with consistent results. The small sample size is counterbalanced by animal model standardization and advanced multimodality imaging techniques, giving precise and reproducible geometric and physiological metrics.

tion fraction appeared to be lower in our series than what is observed in the clinical setting, there was a

CONCLUSIONS

clear increase in LV volumes, strongly suggesting that the amount of AR we created was physiologically

As opposed to isolated AR, the presence of a small

relevant. Exploration of mitral biology in both earlier

apical MI produces MV changes associated with

(before LV dysfunction) and later (as heart failure

altered MV growth and the development of FMR.

progresses and eventual FMR can appear) stages will

Tricuspid leaflets are also affected by those changes,

be interesting in future studies. Although the relation

suggesting a systemic process. Understanding these

Marsit et al.

JACC VOL. 75, NO. 4, 2020 FEBRUARY 4, 2020:395–405

MI Impairs Valve Adaptation

mechanisms could lead to new therapeutic opportu-

PERSPECTIVES

nities to prevent FMR. ACKNOWLEDGMENTS The authors thank the animal

COMPETENCY IN MEDICAL KNOWLEDGE: Impaired mo-

facility staff (Sébastien Poulin, Vincent Tellier, and

lecular remodeling in mitral and tricuspid leaflets attenuates

Justin Robillard), Serge Simard for the biostatistical

leaflet expansion in response to ventricular dilatation after

support, and Ahmed Benhamadi for graphic design.

myocardial infarction and contributes to the development of functional mitral regurgitation.

ADDRESS FOR CORRESPONDENCE: Dr. Jonathan

Beaudoin, Institut Universitaire de Cardiologie et de

TRANSLATIONAL OUTLOOK: Further studies are needed to

Pneumologie de Québec, 2725 Chemin Sainte-Foy,

elucidate the mechanisms by which myocardial ischemia influ-

Québec

ences valve biology and growth.

City,

Québec

G1V4G5,

Canada.

[email protected].

E-mail: Twitter:

@universitelaval.

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A PPE NDI X For supplemental Methods, a figure, and videos, please see the online version of this paper.

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