Abnormal muscle mechanosignaling triggers cardiomyopathy in mice with Marfan syndrome

Abstract

Patients with Marfan syndrome (MFS), a multisystem disorder caused by mutations in the gene encoding the extracellular matrix (ECM) protein fibrillin 1, are unusually vulnerable to stress-induced cardiac dysfunction. The prevailing view is that MFS-associated cardiac dysfunction is the result of aortic and/or valvular disease. Here, we determined that dilated cardiomyopathy (DCM) in fibrillin 1-deficient mice is a primary manifestation resulting from ECM-induced abnormal mechanosignaling by cardiomyocytes. MFS mice displayed spontaneous emergence of an enlarged and dysfunctional heart, altered physical properties of myocardial tissue, and biochemical evidence of chronic mechanical stress, including increased angiotensin II type I receptor (AT1R) signaling and abated focal adhesion kinase (FAK) activity. Partial fibrillin 1 gene inactivation in cardiomyocytes was sufficient to precipitate DCM in otherwise phenotypically normal mice. Consistent with abnormal mechanosignaling, normal cardiac size and function were restored in MFS mice treated with an AT1R antagonist and in MFS mice lacking AT1R or β-arrestin 2, but not in MFS mice treated with an angiotensin-converting enzyme inhibitor or lacking angiotensinogen. Conversely, DCM associated with abnormal AT1R and FAK signaling was the sole abnormality in mice that were haploinsufficient for both fibrillin 1 and β1 integrin. Collectively, these findings implicate fibrillin 1 in the physiological adaptation of cardiac muscle to elevated workload.

Figures

Figure 1. DCM detection in MFS mice.

(A) Top: representative echo M-mode images taken at the level of the mid-papillary muscle from 3-month-old Fbn1+/+ and Fbn1mgR/mgR mice; red and green double-headed arrows highlight LV internal diameter in diastole (LVIDd) and systole (LVIDs), respectively. Percentage of fractional shortening (FS) was calculated as (LVIDd – LVIDs) / LVIDd × 100. Bottom: Bar graphs summarize the LVIDd and percentage of FS data in Fbn1+/+ and Fbn1mgR/mgR mice (n >15 per genotype). (B) Representative in vivo PV loop tracings in Fbn1mgR/mgR (blue) compared with Fbn1+/+ (green) mice. PV loop quantification of cardiac function including EDV, ESV, ESPVR, PRSW, and dP/dT maximum and minimum values (n = 5 per genotype). *P < 0.05; bars indicate the mean ± SD.

Figure 2. DCM characterization in MFS mice.

(A) Representative Fbn1+/+ and Fbn1mgR/mgR hearts collected at the indicated stages of postnatal life with the respective ventricular HW/BW estimates shown by the bar graphs above them (n > 5 per genotype and time point). Scale bar: 5 mm. H&E staining of representative heart sections from 3-month-old Fbn1+/+ and Fbn1mgR/mgR mice are shown below. Scale bar: 1 mm. (B) Representative WGA-stained cross sections of Fbn1+/+ and Fbn1mgR/mgR hearts (white tracings highlight myocyte cross-sectional area [CSA]), with overall estimates of cardiac hypertrophy expressed as CSA. Levels of hypertrophy-related transcripts β-myosin heavy chain (Mhy7) and natriuretic peptide A (Nppa) are shown on the right. Scale bar: 100 μm. (C) Representative trichrome-stained cross sections of Fbn1+/+ and Fbn1mgR/mgR hearts (left) shown together with quantitative PCR estimates of fibrogenic transcripts periostin (Pstn) and collagen I (Col1) (right), and Smad3 immunoblots of total protein extracts (bottom) from 3-month-old Fbn1+/+ and Fbn1mgR/mgR myocardium. Scale bar: 100 μm. (D) Mean opening angle (degree) of the LV free wall (left) and elastic modulus (kPa) measurement (right) in the myocardium of 2-month-old Fbn1+/+ and Fbn1mgR/mgR hearts (n = 5 per genotype and assay). *P < 0.05; bars indicate the mean ± SD.

Figure 3. Origin of MFS-related DCM.

(A) Representative hearts (left) collected from 6-week-old Fbn1+/+, Fbn1Col2–/–, Fbn1Wnt1–/mgR, and Fbn1αMHC–/– mice together with the indicated parameters of cardiac function or HW/BW. *P < 0.05 (n ≥ 5 per genotype) compared with control samples; bars indicate the mean ± SD; ANOVA P < 0.0001. Scale bar: 5 mm. (B) Collagen IV (Col IV, green) and fibrillin 1 (FBN1, red) immunofluorescence staining of neonatal myocardia of the indicated genotypes. Whenever detectable, immunostaining of interstitial and BM-containing pericellular matrices are highlighted by a yellow arrow and white arrowhead, respectively. Scale bar: 30 μm. Far left panels are higher-power images of Fbn1+/+ samples showing fibrillin 1 and collagen IV codistribution in the pericellular matrix. Scale bar: 5 μm. (C) Lower-power images of fibrillin 1 (FBN1, red) and vimentin (Vim, green) immunostaining of neonatal Fbn1+/+ myocardia that illustrate the almost exclusive distribution of cardiac fibroblasts along interstitial cables of fibrillin 1 microfibrils. Scale bar: 30 μm.

Figure 4. Molecular pathogenesis of MFS-related DCM.

(A) ERK1/2 immunoblots of total protein extracts from 3-month-old Fbn1+/+ and Fbn1mgR/mgR myocardia, with the numbers above each lane indicating ratios between phosphorylated and nonphosphorylated proteins. A statistical summary of these data is shown in Table 4. (B) Representative hearts collected from placebo-treated 2- to 3-month-old Fbn1+/+ and Fbn1mgR/mgR mice; Fbn1mgR/mgR mice treated with losartan (LOS), enalapril (ENA), propranolol (PPL), or pan–TGF-β–neutralizing antibody (1D11); or mice genetically deficient for AT1R (At1r–/–), β-arrestin 2 (Arrb2–/–), or angiotensinogen (Agt–/–). Scale bar: 5 mm. Below these images are evaluations of the same animals for the indicated parameters of cardiac function and size. Data relevant to Fbn1mgR/mgR;Agt–/– are shown separately because they are based on the analysis of 1-month-old control and experimental animals. *P < 0.05 (n ≥ 5 per genotype) relative to Fbn1+/+ mice, #P < 0.05 relative to untreated Fbn1mgR/mgR mice, ANOVA P < 0.0001; bars indicate the mean ± SD. (C) FAK immunoblots of total protein extracts from 2-month-old myocardia of Fbn1+/+ and mutant mice of the indicated genotypes. Numbers above each lane indicate ratios between phosphorylated and nonphosphorylated proteins, and bar graphs on the right summarize the data. *P < 0.05; n ≥ 5 per genotype.

Figure 5. Integrin involvement in MFS-related DCM.

(A) Illustrative immunoblots of pFAK and FAK proteins and β1 integrin in myocardial extracts from 3-month-old Fbn1+/+ and Fbn1mgR/mgR mice. Ponceau staining is shown below each immunoblot as an indication of total protein loading. (B) Cardiac function in 3-month-old mice of the indicated genotypes (cHI denotes compound haploinsufficient Fbn1+/–;Itgb1+/– mice), with the representative hearts shown below the assays. *P < 0.05 relative to Fbn1+/+ samples (n = 5 per genotype), and mean ± SD. ANOVA P < 0.05. Scale bar: 5 mm. (C) Illustrative immunoblots of FAK and ERK1/2 proteins in myocardial extracts from 3-month-old Fbn1+/+ and cHI mice (n = 5). In A and C, numbers above each lane indicate ratios between phosphorylated and nonphosphorylated proteins. FAK immunoblot in C includes lanes that were run on the same gel but were not contiguous.