Smooth muscle LDL receptor-related protein-1 deletion induces aortic insufficiency and promotes vascular cardiomyopathy in mice

Abstract

Valvular disease is common in patients with Marfan syndrome and can lead to cardiomyopathy. However, some patients develop cardiomyopathy in the absence of hemodynamically significant valve dysfunction, suggesting alternative mechanisms of disease progression. Disruption of LDL receptor-related protein-1 (Lrp1) in smooth muscle cells has been shown to cause vascular pathologies similar to Marfan syndrome, with activation of smooth muscle cells, vascular dysfunction and aortic aneurysms. This study used echocardiography and blood pressure monitoring in mouse models to determine whether inactivation of Lrp1 in vascular smooth muscle leads to cardiomyopathy, and if so, whether the mechanism is a consequence of valvular disease. Hemodynamic changes during treatment with captopril were also assessed. Dilation of aortic roots was observed in young Lrp1-knockout mice and progressed as they aged, whereas no significant aortic dilation was detected in wild type littermates. Diastolic blood pressure was lower and pulse pressure higher in Lrp1-knockout mice, which was normalized by treatment with captopril. Aortic dilation was followed by development of aortic insufficiency and subsequent dilated cardiomyopathy due to valvular disease. Thus, smooth muscle cell Lrp1 deficiency results in aortic dilation and insufficiency that causes secondary cardiomyopathy that can be improved by captopril. These findings provide novel insights into mechanisms of cardiomyopathy associated with vascular activation and offer a new model of valvular cardiomyopathy.

Figure 1. Lrp1 disruption in cardiovascular tissues and isolated cardiac myocyte function.

A, Immunoblots of Lrp1 in tissue and cell extracts from heart, aorta, liver and cardiomyocytes demonstrated disruption of Lrp1 protein in cardiovascular tissues of sm22-cre Lrp1^flox/flox mice. Cardiomyoctye mechanics were demonstrated in (B) representative cell shortening tracings of smLrp1^+/+ and smLrp1^-/- cardiomycytes, as well as (C) fractional shortening, (D) rates of relaxation, -dL/dt, and (E) rates of contraction, +dL/dt. n = 3 mice per group for Lrp1 immunoblots and n = 27 cells from 3 smLrp1^-/- hearts; n = 20 cells from 2 smLrp1^+/+ hearts. Data are mean ± SEM.

Figure 2. Aortic root diameter and aortic insufficiency.

A, Changes in aortic root diameter during the 22 week study period were measured with echocardiograms at indicated intervals. *P<0.01, versus aortic root diameter of eight week old smLrp1 ^-/- mice; †P<0.05, pair wise comparison among age-matched smLrp1 ^+/+ mice. B, Kaplan Meier curve analysis of aortic insufficiency events in smLrp1 ^-/- and smLrp1 ^+/+ littermates (P=0.05). n = 6 mice per genotype. C, Representative 2D image of aortic root with M-mode imaging at 16 weeks of smLrp1 ^-/- (bottom) and wildtype littermate (top). D, Hematoxylin-eosin-stained, cross-section of ascending aorta at ~3mm from aortic valve from 40-week old smLrp1 ^+/+ (left) and smLrp1 ^-/- (right) Scale bar, 500μm. E, Detection of AI jet (blue) in 2D long axis image with color Doppler on a 24 week old smLrp1 ^-/- mouse with the same AI jet projected along pulsed Doppler M-mode.

Figure 3. Cardiac output and left ventricular dilation.

A, Fractional Shortening (FS), Ejection Fraction (EF) and (B) Left ventricular diameter in diastole (LVVD) measurements collected from 36 week old smLrp1 ^+/+ and smLrp1 ^-/- mice. * P<0.05 C, Left ventricular volumes during diastole were determined during the 22 week study period with echocardiograms at indicated intervals. *P<0.01, versus aortic root diameter of eight week old smLrp1 ^-/- mice; †P<0.05, pair wise comparison among age-matched smLrp1 ^+/+ mice.

Figure 4. Heart defects in smLrp1-deficient mice.

A, Ventral view of intact heart from 40 week old mice, formalin-fixed dissected heart and parasternal long axis view echocardiogram, scale bar=5 mm. B, Heart mass (M_h) to body mass (M_b) ratios among 24 smLrp1 ^+/+ (solid line) and 42 smLrp1 ^-/- (dashed line) mice across the experimental age spectrum from 29 smLrp1 ^+/+ mice and 42 smLrp1 ^-/- mice. (Spearman Correlation: r_s -0.489 and P 0.0074 for smLrp1 ^+/+ and r_s -0.402 and P 0.0086 for smLrp1 ^-/-; Regression Analysis: Slope -0.933 for smLrp1^+/+ and -0.461 for smLrp1 ^-/-). C, Mason’s trichrome-stained short axis sections from 40 week old mice, left panels (bar = 5mm); hematoxylin and eosin and Mason’s trichrome staining of parenchymal area of left ventricle (LV), center and right panels, respectively (bar = 30 μm). D, Longitudinal cell surface area of LV cardiomyocytes. E-I, Perivascular fibrosis and interstitial fibrosis (Mason’s trichrome, blue area) observed in intramural coronary arteries of LV free wall; arrows indicate arteries (E, F), papillary muscle (G, H) and right ventricle parenchyma and coronary arteries of hearts from smLrp1 ^-/- (I) and smLrp1 ^+/+ mice (J) (bar = 30 μm). * P < 0.05.

Figure 5. Aorta and coronary artery fibrosis and vascular CTGF accumulation.

A, Mason’s Trichrome staining of ascending aortas of 15 and 40 week old mice and immunostaining with anti-CTGF antibodies (brown staining at arrows) or isotype control antibody and hematoxylin counterstained nuclei. Solid arrows indicate regions with abundant CTGF-positive signal, asterisk denotes vessel lumen (bar = 30 μm). B, Immunoblots of tissue CTGF full length and hydrolytic fragment content in aortas sampled from 30 week old mice. Bar graphs represent densitometric means ± SEM of CTGF (full length and fragment) normalized to GAPDH from three smLrp1 ^+/+ and three smLrp1 ^-/- tissue preparations. * P<0.05 between genotypes C, Mason’s trichrome staining of LV coronary arteries and surrounding myocytes (fibrosis, blue area; asterisk denotes vessel lumen) with inset showing internal (IEL) and external (EEL) elastic laminae; Vanhoeff-Van Geison (VVG) staining of internal and external elastic laminae (black colored lines) (bar = 30 μm). D, Tissue expression of CTGF in hearts collected from 30 week old mice.

Figure 6. ERK and SMAD2 signaling in thoracic aortas.

A, Immunoblots of tissue (p)SMAD2 and total SMAD2 detected in thoracic aortas sampled from 30 week old mice; 3 mice per genotype. Bar graphs represent densitometric means ± SEM following normalization to GAPDH. B, Tissue expression of (p)ERK1/2 and ERK2 in thoracic aortas from 30 week old mice. * P<0.05 between genotypes.