nature publishing group Articles Translational Investigation Intrinsic cardiomyopathy in Marfan syndrome: results from in-vivo and ex-vivo studies of the Fbn1C1039G/+ model and longitudinal findings in humans Laurence Campens1, Marjolijn Renard1, Bram Trachet2, Patrick Segers2, Laura Muino Mosquera1, Johan De Sutter3, Lynn Sakai4, Anne De Paepe1 and Julie De Backer1 BACKGROUND: Mild intrinsic cardiomyopathy in patients Cardiovascular involvement in MFS is characterized by pro- with Marfan syndrome (MFS) has consistently been evi- gressive dilatation of the ascending aorta at the level of the denced by independent research groups. So far, little is known sinuses of Valsalva, ensuing a risk for type A aortic dissection about the long-term evolution and pathophysiology of or -rupture and aortic valve regurgitation (2). Other estab- this finding. lished cardiovascular manifestations of MFS include mitral METHODS: To gain more insights into the pathophysiology of valve prolapse and pulmonary artery dilatation. More recently, MFS-related cardiomyopathy, we performed in-vivo and ex-vivo several independent studies demonstrated the presence of— studies of 11 Fbn1C1039G/+ mice and 9 wild-type (WT) littermates. mostly subclinical—intrinsic cardiomyopathy (CMP) with Serial ultrasound findings obtained in mice were correlated to both left and right ventricular (LV and RV) systolic and dia- the human phenotype. We therefore reassessed left ventricular stolic dysfunction in MFS patients (3,4). The current know­ (LV) function parameters over a 6-y follow-up period in 19 pre- ledge of MFS-related CMP is based on cross-sectional studies, viously reported MFS patients, in whom we documented mild but little is known about the long-term evolution and patho- LV dysfunction. physiology of this finding. RESULTS: Fbn1C1039G/+ mice demonstrated LV contractile dys- To unravel the underlying pathophysiology of MFS-related function. Subsequent ex-vivo studies of the myocardium of CMP, the knowledge obtained from the study of other MFS adult mutant mice revealed upregulation of TGFβ-related manifestations may at least be partly translated to the cardiac pathways and consistent abnormalities of the microfibrillar phenotype. Upregulation of TGFβ signaling, with activation of network, implicating a role for microfibrils in the mechanical the canonical (SMAD2/3) and noncanonical (predominantly properties of the myocardium. Echocardiographic parameters ERK1/2) pathways, is implicated in aortic dilatation, mitral did not indicate clinical significant deterioration of LV function valve prolapse, pulmonary emphysema, and skeletal myopa- during follow-up in our patient cohort. thy (reviewed in ref. (5)). Interestingly, TGFβ upregulation CONCLUSION: In analogy with what is observed in the major- has been evidenced in hereditary CMP (dilated, hypertro- ity of MFS patients, the Fbn1C1039G/+ mouse model demonstrates phic, and arrhythmogenic right ventricular CMP), where it is mild intrinsic LV dysfunction. Both extracellular matrix and linked to cardiac interstitial fibrosis (6–10). In the Fbn1mgR/mgR molecular alterations are implicated in MFS-related cardiomy- model, which is a severe murine model for MFS demonstrat- opathy. This model may now enable us to study therapeutic ing dilated CMP from 2 mo of age, selective upregulation of interventions on the myocardium in MFS. the noncanonical signaling pathway was evidenced (11). In addition to its role as a regulator of TGFβ bioavailability, fibrillin-1 has an important structural role, providing stability arfan syndrome (MFS, GenBank accession nos. L13923) and elasticity to tissues. Structural dysfunction is implicated Mis an autosomal dominant inherited connective tissue in several MFS manifestations including lens luxation and disorder caused by mutations in the fibrillin-1 gene (FBN1) aortic dilatation (12). Additionally, loss of mechanical prop- encoding the extracellular matrix protein fibrillin-1. The clini- erties of the microfibrillar network in the myocardium may cal diagnosis of MFS relies on the identification of characteristic evoke ventricular dysfunction (11–13). Another mechanism clinical manifestations, mainly occurring in the cardiovascu- through which aberrant microfibrils could lead to contractile lar, musculoskeletal, and ocular system, as summarized in the dysfunction of the myocardium, is through loss of binding to ‘‘Revised Ghent Nosology’’, and may be supplemented by the integrin receptors, which play a key role in cell-matrix interac- identification of a causal FBN1 mutation (1). tions (extracellular matrix (ECM)) and mechanotransduction 1Center for Medical Genetics Ghent, Ghent University Hospital, Ghent, Belgium; 2IBiTech-bioMMeda, Ghent University, Ghent, Belgium; 3Internal Medicine, Ghent University, Ghent, Belgium; 4Shriners Hospital for Children, Oregon Health & Science University, Portland, Oregon. Correspondence: Laurence Campens ([email protected]) Received 16 November 2014; accepted 3 March 2015; advance online publication 1 July 2015. doi:10.1038/pr.2015.110 256 Pediatric ReseArcH Volume 78 | Number 3 | September 2015 Copyright © 2015 International Pediatric Research Foundation, Inc. Marfan-related cardiomyopathy Articles across the cell surface through the cytoskeleton (14–16). This developed significant aortic valve regurgitation with holodia- hypothesis is supported by the observation that disruption stolic flow reversal in the descending aorta; in one of them this of the integrin pathway leads to the development of CMP was already apparent on the scan at 6 mo. None of the WT in both mice and humans (17–19). Interestingly, the down- mice presented significant aortic valve dysfunction. After 12 stream integrin pathway is downregulated in the myocardium mo, two additional MFS mice died, one of dissection at 60 wk, of the Fbn1mgR/mgR model (11). the other died shortly after the last scan and demonstrated The objective of the current study is to gain more insight into no evidence for aortic dissection or internal hemorrhage on the underlying pathophysiology of MFS-related CMP. To this necropsy. aim, we first performed longitudinal ultrasound studies using LV fractional shortening was lower in MFS mice at all time a representative and well-characterized murine model for points with significantly lower values at 6 mo (30.9% ± 7.9) MFS, the Fbn1C1039G/+ model, and compared our findings to the vs. 41.2% ± 3.0; P = 0.001) (Figure 1). From 6 mo onward, human cardiac phenotype. Subsequently, we explored struc- fractional shortening remained stable (31.6% ± 5.5 at 12 mo, tural and molecular alterations in the mouse myocardium. P = 0.71). Heart rate, weight, sex, sinus diameter, and aortic regurgitation were not identified as confounding factors of LV RESULTS fractional shortening with univariate regression analysis (P = Serial Ultrasound Data in the Fbn1C1039G/+ Mouse Model 0.61, P = 0.99, P = 0.53, P = 0.68, and P = 0.81, respectively). Although there was a nonsignificant trend toward larger aortic We found no differences in diastolic function between MFS diameters at 1 and 3 mo, aortic diameters in the Fbn1C1039G/+ and WT mice (Table 1). mice were only significantly increased from month 6 onward when compared to wild-type (WT) mice. Aortic dilatation Clinical Studies extended from the sinuses of Valsalva to the ascending and Impaired ventricular function has been established in humans transverse aorta (Table 1)—which is different from the human (3,4), but so far, no data on evolutionary changes have been aortic phenotype being limited to the level of the aortic sinus reported. In view of our results obtained in mice, we aimed to in most cases. Main pulmonary artery diameters were not sig- provide longitudinal human data for which we analyzed echo- nificantly different between both groups. During follow-up, cardiographic studies at different time-points available from two MFS mice died of aortic dissection at 47 and 48 wk of age; our MFS patient population. An initial detailed study of left on their scan at 6 mo they exhibited severe aortic dilatation ventricular function in this population indicated mild intrinsic associated with significant aortic valve regurgitation. At the LV systolic and diastolic dysfunction (20). Data obtained from end of the follow-up period, three additional MFS mice had the subsequent preliminary retrospective analysis demonstrate stable LV dimensions and no clinically significant decline of Table 1. Echocardiographic parameters of Fbn1C1039G/+ mice at 12 mo LV systolic or diastolic function (Table 2). Two patients devel- of age oped moderate valvular dysfunction upon follow-up (one WT MFS patient with aortic and one with mitral valve dysfunction) N = 9 N = 9 P value Females 4 (44%) 6 (66%) 0.64 50 Weight (kg) 27.7 ± 3.4 26.2 ± 4.7 0.34 ** HR (bpm) 423.8 ± 19.1 425.8 ± 27.0 0.95 40 Aortic diameters Sinus (mm) 1.9 ± 0.2 2.6 ± 0.5 0.003 30 Ascending aorta (mm) 1.7 ± 0.2 2.3 ± 0.7 0.014 Arch (mm) 1.4 ± 0.1 1.8 ± 0.2 0.006 20 Descending aorta (mm) 1.2 ± 0.1 1.3 ± 0.2 0.014 Main pulmonary artery (mm) 1.4 ± 0.2 1.6 ± 0.3 0.38 Fractional shortening (FS; %) 10 LVEDD (mm) 3.7 ± 0.4 3.7 ± 0.7 1.0 LVESD (mm) 2.1 ± 0.2 2.5 ± 0.5 0.063 0 FS (%) 42.0 ± 6.3 31.6 ± 5.5 0.011 13612 E/A 1.4 ± 0.4 1.1 ± 0.2 0.14 Age (mo) DT (ms) 14.9 ± 3.7 17.7 ± 7.1 0.74 Figure 1. Bar chart with error bars (representing 1 standard error) of left C1039G/+ Em (mm/s) 21.4 ± 8.2 23.9 ± 11.1 0.73 ventricular fractional shortening of 11 Fbn1 and 9 wild-type (WT) mice at 1, 3, and 6 mo and in 9 Fbn1C1039G/+ and 9 WT mice at 12 mo of age. E/E 37.1 ± 23.7 23.4 ± 5.8 0.20 m Fbn1C1039G/+ mice demonstrate significant lower fractional shortening from DT, deceleration time; FS, fractional shortening; HR, heart rate; LVEDD, left ventricular 6 mo of age on in comparison to WT mice. White bars represent WT mice end diastolic diameter; LVESD, left ventricular end systolic diameter; MFS, Marfan and black bars Fbn1C1039G/+ mice.