Nonmyocyte ERK1/2 signaling contributes to load-induced cardiomyopathy in Marfan mice

Abstract

Among children with the most severe presentation of Marfan syndrome (MFS), an inherited disorder of connective tissue caused by a deficiency of extracellular fibrillin-1, heart failure is the leading cause of death. Here, we show that, while MFS mice (Fbn1C1039G/+ mice) typically have normal cardiac function, pressure overload (PO) induces an acute and severe dilated cardiomyopathy in association with fibrosis and myocyte enlargement. Failing MFS hearts show high expression of TGF-β ligands, with increased TGF-β signaling in both nonmyocytes and myocytes; pathologic ERK activation is restricted to the nonmyocyte compartment. Informatively, TGF-β, angiotensin II type 1 receptor (AT1R), or ERK antagonism (with neutralizing antibody, losartan, or MEK inhibitor, respectively) prevents load-induced cardiac decompensation in MFS mice, despite persistent PO. In situ analyses revealed an unanticipated axis of activation in nonmyocytes, with AT1R-dependent ERK activation driving TGF-β ligand expression that culminates in both autocrine and paracrine overdrive of TGF-β signaling. The full compensation seen in wild-type mice exposed to mild PO correlates with enhanced deposition of extracellular fibrillin-1. Taken together, these data suggest that fibrillin-1 contributes to cardiac reserve in the face of hemodynamic stress, critically implicate nonmyocytes in disease pathogenesis, and validate ERK as a therapeutic target in MFS-related cardiac decompensation.

Keywords: Cardiology; Genetics.

Figure 1. Fbn1^C1039G/+ mice develop marked heart failure acutely from pressure overload.

(A) Weekly temporal changes in cardiac dimensions and function after TAC. EDD, end-diastolic diameter; ESD, end-systolic diameter; FS, fractional shortening; LV mass, left ventricular mass; BL, baseline. Comparison pairs shown in brackets on right side of panels. n = 4–8 per group. (B) Representative M-mode echocardiogram 4 weeks after TAC. White arrow, end-diastolic diameter; yellow arrow, end-systolic diameter. (C) Representative whole hearts and summary results for heart weight normalized to tibia length (HW/TL) in Fbn1^+/+ and Fbn1^C1039G/+ mice subjected to 4 weeks of TAC. Wet lung weight (LW) was normalized to TL. n ≥ 5 per group. *P < 0.05, **P < 0.01, ***P < 0.001; ^##P < 0.01 vs. Fbn1^+/+:sham; ††P < 0.01 vs. Fbn1^C1039G/+:sham, 2-way ANOVA, Bonferroni’s correction. (D and E) Representative hematoxylin and eosin and Masson’s trichrome staining of formalin-fixed heart sections from Fbn1^+/+ and Fbn1^C1039G/+ mice subjected to 4 weeks of TAC. Scale bars: 75 μm (D); 150 μm (E). White dotted line (in D) outlines myocyte circumference. Blue stain (in E) indicates fibrosis.

Figure 2. Fibrillin-1 protein fails to deposit in myocardium in load-induced heart failure in Fbn1^C1039G/+ mice.

(A) mRNA expression of Fbn1 normalized to Gapdh and then to Fbn1^+/+:sham data, assessed by real-time RT-PCR. n ≥ 4 per group. (B) Representative images of immunofluorescent fibrillin-1 staining (red) of heart sections from Fbn1^+/+ and Fbn1^C1039G/+ mice subjected to 4 weeks of TAC. Red, fibrillin-1 (interstitial space); blue, DAPI (nuclei); green, lipofuscin (myocytes). Scale bar: 20 μm. (C) Quantitative representation of fibrillin-1 normalized to unit area. n = 4–7 per group. (D and E) Representative Western blot and summary quantification for phosphorylated/total (p/t) Smad2 and ERK1/2 using left ventricular tissue lysates. Lanes were run on the same gel but were noncontiguous. n ≥ 4 per group. (F) mRNA expression of TGF-β targets, normalized to 18S and then to Fbn1^+/+ data, assessed by real-time RT-PCR from left ventricular tissue. Col1a1, collagen type 1 a1; Fn1, fibronectin; Serpine1, plasminogen activator inhibitor, type 1; Postn, periostin; Ctgf, connective tissue growth factor; Spp1, osteopontin. n = 3–6 per group. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA, Tukey’s correction. In box-and-whisker plots, the lower and upper margins of each box define the 25th and 75th percentiles, respectively; the internal line defines the median, and the whiskers define the range. Values outside 1.5 times the interquartile distance are shown.

Figure 3. Smad2 and ERK1/2 activation are differentially increased in the myocyte and nonmyocyte compartments of failing Fbn1^C1039G/+ hearts.

(A) Representative sections of pSmad2 (red) immunostaining. Scale bar: 20 μm. White arrowheads point to examples of positively stained myocyte nuclei. (B) Summary quantification of total intensity normalized to Fbn1^+/+:sham and labeling index (percentage positive cells) of pSmad2 in the nonmyocyte and myocyte compartments. (C and D) Representative sections and summary quantification of pERK1/2 (red) immunostaining. Scale bar: 20 μm. n = 6 fields per group. **P < 0.01, ***P < 0.001, 1-way ANOVA, Tukey’s correction.

Figure 4. Treatment with TGF-β NAb, losartan, or MEKi improves load-induced cardiac decompensation in Fbn1^C1039G/+ hearts, despite persistent load.

(A) Temporal changes of cardiac dimensions and function of Fbn1^C1039G/+ hearts after TAC, with treatment arms. Fbn1^+/+, blue circle with solid line; Fbn1^C1039G/+, red square with solid line; Fbn1^C1039G/+ with TGF-β NAb treatment, orange triangle with dotted line; Fbn1^C1039G/+ with losartan (Los) treatment, green diamond with dotted line; Fbn1^C1039G/+ with MEK1/2 inhibitor (MEKi) treatment, purple circle with dotted line. Early time point, 1 week after TAC; mid time point, 2–4 weeks after TAC; end time point, 3–5 weeks after TAC. EDD, end-diastolic diameter; ESD, end-systolic diameter; FS, fractional shortening; LV mass, left ventricular mass; BL, baseline. Comparison pairs shown in brackets on right side of respective panel. n ≥ 5 per group. (B) Representative M-mode echocardiograms at end time point, Fbn1^C1039G/+ sham vs. TAC groups, with and without treatment. (C) Summary quantification of heart weight normalized to tibia length (HW/TL). n ≥ 5 per group. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA, Tukey’s correction.

Figure 5. Functional improvement of mechanically loaded Fbn1^C1039G/+ hearts correlates with reduced fibrosis.

(A) Representative Masson’s trichrome–stained heart sections of Fbn1^C1039G/+ sham vs. TAC groups, with and without treatment, showing fibrosis stained in blue. Scale bars: 750 μm (top); 100 μm (bottom). (B) Quantification of fibrosis, quantified by total number of blue pixels normalized to whole tissue area and then to Fbn1^+/+:sham data. n = 4–7 per group. (C) mRNA expression of TGF-β–responsive fibrogenic genes, normalized to Gapdh and then to Fbn1^+/+:sham data, assessed by real-time RT-PCR. Col1a1, collagen type 1 a1; Spp1, osteopontin. n = 4–6 per group. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA, Tukey’s correction. In box-and-whisker plots, the lower and upper margins of each box define the 25th and 75th percentiles, respectively; the internal line defines the median, and the whiskers define the range. Values outside 1.5 times the interquartile distance are shown.

Figure 6. ERK1/2 activation is upstream of Smad2 activation in Fbn1^C1039G/+ hearts.

(A–D) Representative Western blot and summary quantification for Smad2 and ERK1/2 phosphorylation from left ventricular tissue lysates. n = 4–6 per group. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA, Tukey’s correction. (E) Diagram illustrating the pathogenic sequence of load-induced heart failure in the fibrillin-1–deficient myocardium of Fbn1^C1039G/+ mice. NM, nonmyocytes; M, myocytes. In box-and-whisker plots, the lower and upper margins of each box define the 25th and 75th percentiles, respectively; the internal line defines the median, and the whiskers define the range. Values outside 1.5 times the interquartile distance are shown.

Figure 7. Inhibition of ERK1/2 activation suppresses Smad2 activation in both nonmyocyte and myocyte compartments.

Representative coimmunostaining of (A) pSmad2 (red, row 1) and vimentin (pink, row 2) and of (B) pERK1/2 (red, row 1) and vimentin (pink, row 2) of myocyte-enriched sections of Fbn1^C1039G/+ hearts subjected to sham vs. TAC, with treatment groups. Scale bars: 20 μm; zoom inset: ×2 of nonmyocyte-enriched areas. Blue, DAPI (nuclei); green, lipofuscin (myocytes).

Figure 8. Inhibition of ERK1/2 activation suppresses expression of all three TGF-β isoforms.

mRNA expression of Tgfb1 (TGF-β1), Tgfb2 (TGF-β2), and Tgfb3 (TGF-β3) isoforms, normalized to Gapdh and then to Fbn1^+/+:sham data, assessed by real-time RT-PCR. n = 4–6 per group. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA, Tukey’s correction. In box-and-whisker plots, the lower and upper margins of each box define the 25th and 75th percentiles, respectively; the internal line defines the median, and the whiskers define the range. Values outside 1.5 times the interquartile distance are shown.