A deleterious gene-by-environment interaction imposed by calcium channel blockers in Marfan syndrome

Abstract

Calcium channel blockers (CCBs) are prescribed to patients with Marfan syndrome for prophylaxis against aortic aneurysm progression, despite limited evidence for their efficacy and safety in the disorder. Unexpectedly, Marfan mice treated with CCBs show accelerated aneurysm expansion, rupture, and premature lethality. This effect is both extracellular signal-regulated kinase (ERK1/2) dependent and angiotensin-II type 1 receptor (AT1R) dependent. We have identified protein kinase C beta (PKCβ) as a critical mediator of this pathway and demonstrate that the PKCβ inhibitor enzastaurin, and the clinically available anti-hypertensive agent hydralazine, both normalize aortic growth in Marfan mice, in association with reduced PKCβ and ERK1/2 activation. Furthermore, patients with Marfan syndrome and other forms of inherited thoracic aortic aneurysm taking CCBs display increased risk of aortic dissection and need for aortic surgery, compared to patients on other antihypertensive agents.

Keywords: Amlodipine; ERK; Hydralazine; Marfan syndrome; TGF-β signaling; Verapamil; aortic aneurysm; calcium channel blocker; chromosomes; genes; human; human biology; medicine; mouse; protein kinase C.

Figure 1.. Effect of amlodipine in wild-type (WT) and Marfan mice.

(A) Echocardiography data showing mean (±2 SEM) growth in the aortic root and ascending aorta from 2 to 4 months of age. Number of mice per group (male/female) = WT placebo 11 (5/6), WT amlodipine 10 (6/4), Marfan placebo 14 (7/7), Marfan amlodipine 11 (6/5). Mean (±2 SEM) weight per group (in grams) at 4 months = 27.4 ± 2.4 g (WT placebo), 27.6 ± 2.8 g (WT amlodipine), 28.1 ± 3.0 g (Marfan placebo), 27.9 ± 2.8 g (Marfan amlodipine). (B) Survival curve from 2 to 5 months of age. Number of mice per group (male/female) = WT placebo 11 (5/6), WT amlodipine 10 (6/4), Marfan placebo 14 (7/7), Marfan amlodipine 19 (9/10). (C) Representative VVG staining (upper panel) and Masson's trichrome staining (lower panel) of the proximal ascending aorta in 5-month-old male mice. Scale bar: 40 µm. (D) Mean (±2 SEM) aortic wall architecture score of the proximal ascending aorta in 5-month-old mice. Number of mice per group = 4 (2 male, 2 female). Scale: 1 (normal) to 5 (extensive damage). Plac, placebo; Aml, amlodipine. DOI: http://dx.doi.org/10.7554/eLife.08648.003

Figure 1—figure supplement 1.. Effect of amlodipine in wild-type (WT) and Marfan mice.

(S1) Mean (±2 SEM) systolic and diastolic blood pressure, and heart rate, in 3-month-old mice. Number of mice per group = 8 (4 male; 4 female). (S2) Representative latex-injected images showing ascending aortic size (distance between arrowheads) in 5-month-old male mice. Scale bar: 2 mm. (S3) Representative VVG staining (upper panel) and Masson's trichrome staining (lower panel) of the descending thoracic aorta in 5-month-old male mice. Scale bar: 40 µm. (S4) Mean (±2 SEM) aortic wall architecture score of the descending thoracic aorta in 5-month-old mice. Number of mice per group = 4 (2 male, 2 female). Scale: 1 (normal) to 5 (extensive damage). (S5) Mean (±2 SEM) aortic root and ascending aortic growth from 2 to 4 months of age in mice treated with 3 mg/kg/day amlodipine. Number of mice per group (male/female) = WT placebo 5 (3/2), WT amlodipine 5 (2/3), Marfan placebo 10 (6/4), Marfan amlodipine 8 (4/4). Plac, placebo; Los, losartan; Aml, amlodipine. DOI: http://dx.doi.org/10.7554/eLife.08648.004

Figure 2.. Effect of verapamil in wild-type (WT) and Marfan mice.

(A) Mean (±2 SEM) growth in the aortic root and ascending aorta from 2 to 6 months of age. Number of mice per group (male/female) = WT placebo 8 (4/4), WT verapamil 10 (4/6), Marfan placebo 9 (5/4), Marfan verapamil 11 (6/5). Mean (±2 SEM) weight per group (in grams) at 6 months = 30.3 ± 2.6 g (WT placebo), 30.6 ± 2.2 g (WT verapamil), 31.3 ± 3.3 g (Marfan placebo), 31.5 ± 3.2 g (Marfan verapamil). (B) Representative VVG staining (upper panel) and Masson's trichrome staining (lower panel) of the proximal ascending aorta in 6-month-old male mice. Scale bar: 40 µm. (C) Mean (±2 SEM) aortic wall architecture score of the proximal ascending aorta in 6-month-old mice. Number of mice per group = 4 (2 male, 2 female). Scale: 1 (normal) to 5 (extensive damage). Plac, placebo; Ver, verapamil. DOI: http://dx.doi.org/10.7554/eLife.08648.005

Figure 2—figure supplement 1.. Effect of verapamil in wild-type (WT) and Marfan mice.

(S1) Representative latex-injected images showing aortic size (distance between arrowheads) in 6-month-old male mice. Scale bar: 2 mm. (S2) Representative VVG staining (upper panel) and Masson's trichrome staining (lower panel) of the descending thoracic aorta in 6-month-old male mice. Scale bar: 40 µm. (S3) Mean (±2 SEM) aortic wall architecture score of the descending thoracic aorta in 6-month-old mice. Number of mice per group = 4 (2 male, 2 female). Scale: 1 (normal) to 5 (extensive damage). Plac, placebo; Ver, verapamil. DOI: http://dx.doi.org/10.7554/eLife.08648.006

Figure 3.. CCB effect is ERK1/2- and AT1R-dependent in wild-type (WT) and Marfan mice.

(A) Western blot analysis of the aortic root and ascending aorta in 5-month-old mice. Number of mice per group = 4 (2 male, 2 female). (B) Mean (±2 SEM) ascending aortic growth from 2 to 4 months of age. Number of mice per group (male/female) = WT placebo 9 (5/4), WT amlodipine 8 (4/4), WT amlodipine + RDEA119 7 (3/4), Marfan placebo 9 (4/5), Marfan amlodipine 10 (6/4), Marfan amlodipine + RDEA119 11 (6/5). (C) Survival curve from 2 to 4 months of age. (D) Western blot analysis of the aortic root and ascending aorta in 4-month-old mice. Number of mice per group = 4 (2 male, 2 female). (E) Mean (±2 SEM) ascending aortic growth from 2 to 4 months of age. Number of mice per group (male/female) = WT placebo 11 (6/5), WT amlodipine 11 (6/5), WT amlodipine + losartan 8 (4/4), Marfan placebo 7 (4/3), Marfan amlodipine 6 (3/3), Marfan amlodipine + losartan 9 (4/5). (F) Western blot analysis of the aortic root and ascending aorta in 4-month-old mice. Number of mice per group = 3 (2 male, 1 female; or 1 male, 2 female). Plac, placebo; Aml, amlodipine; RDEA, RDEA119; Los, losartan; Geno, genotype; Treat, treatment; I/A, interaction. DOI: http://dx.doi.org/10.7554/eLife.08648.007

Figure 3—figure supplement 1.. CCB effect is ERK1/2- and AT1R-dependent in wild-type (WT) and Marfan mice.

(S1) Representative latex images, VVG and trichrome staining in 4-month-old male mice. Latex scale bar: 2 mm. VVG and trichrome scale bar: 40 μm. (S2) Mean (±2 SEM) aortic architecture score in 4-month-old mice. Number of mice per group = 4 (2 male, 2 female). Plac, placebo; Aml, amlodipine; RDEA, RDEA119. DOI: http://dx.doi.org/10.7554/eLife.08648.008

Figure 4.. PKC activation in placebo- and CCB-treated wild-type (WT) and Marfan mice.

(A) Western blot analysis of the aortic root and proximal ascending aorta in 4-month-old mice. Number of mice per group = 4 (2 male, 2 female). (B) Western blot analysis of the aortic root and proximal ascending aorta in 4-month-old mice. Number of mice per group = 3 (2 male, 1 female; or 1 male, 2 female). (C) Mean (±2 SEM) ascending aortic growth from 2 to 4 months of age. Number of mice per group (male/female) = WT placebo 8 (4/4), WT amlodipine 9 (4/5), WT amlodipine + enzastaurin 8 (3/5), Marfan placebo 12 (7/5), Marfan amlodipine 8 (5/3), Marfan amlodipine + enzastaurin 8 (5/3). (D) Mean (±2 SEM) aortic root growth from 2 to 4 months of age. Number of mice per group (male/female) = WT placebo 8 (4/4), WT enzastaurin 6 (3/3), Marfan placebo 12 (7/5), Marfan enzastaurin 8 (4/4). (E) Western blot analysis of the aortic root and ascending aorta in 4-month-old mice. Number of mice per group = 4 (2 male, 2 female). Plac, placebo; NAb, neutralizing antibody; Los, losartan; Aml, amlodipine; Enz, enzastaurin; Geno, genotype; Treat, treatment; I/A, interaction. DOI: http://dx.doi.org/10.7554/eLife.08648.009

Figure 4—figure supplement 1.. PKC activation in placebo- and CCB-treated wild-type (WT) and Marfan mice.

(S1) Representative latex-injected images of 4-month-old male mice. Scale bar: 2 mm. (S2) Representative VVG staining (upper panel) and Masson's trichrome staining (lower panel) of the proximal ascending aorta in 4-month-old male mice. Scale bar: 40 µm. (S3) Mean (±2 SEM) aortic architecture score of the aortic root and proximal ascending aorta in 4-month-old mice. Number of mice per group = 4 (2 male, 2 female). Scale: 1 (normal) to 5 (extensive damage). Plac, placebo; Aml, amlodipine; Enz, enzastaurin. DOI: http://dx.doi.org/10.7554/eLife.08648.010

Figure 5.. Effect of hydralazine in wild-type (WT) and Marfan mice.

(A) Mean (±2 SEM) aortic root growth from 2 to 6 months of age. Number of mice per group (male/female) = WT placebo 9 (4/5), WT hydralazine 12 (7/5), Marfan placebo 15 (6/9), Marfan hydralazine 12 (6/6). Mean (±2 SEM) weight per group (in grams) at 6 months = 31.4 ± 2.4 g (WT placebo), 31.2 ± 3.1 g (WT hydralazine), 31.0 ± 2.7 g (Marfan placebo), 30.4 ± 3.5 g (Marfan hydralazine). (B) Western blot analysis of the aortic root in 6-month-old mice. Number of mice per group = 4 (2 male, 2 female). (C) Diagram illustrating key nodal points in Marfan mouse aortic disease pathogenesis. Drugs shown in red ameliorate aneurysm progression, while manipulations shown in blue exacerbate it. Plac, placebo; Hyd, hydralazine; Geno, genotype; Treat, treatment; I/A, interaction. DOI: http://dx.doi.org/10.7554/eLife.08648.011

Figure 5—figure supplement 1.. Effect of hydralazine in wild-type (WT) and Marfan mice.

(S1) Mean (±2 SEM) systolic and diastolic blood pressure, and heart rate, in 3-month-old mice. Number of mice per group = 8 (4 male; 4 female). (S2) Representative parasternal long-axis in vivo echocardiography images of 6-month-old male mice. Scale bar: 1 mm. (S3) Representative VVG staining (upper panel) and Masson's trichrome staining (lower panel) of the aortic root in 6-month-old mice. Scale bar: 40 µm. (S4) Mean (±2 SEM) aortic wall architecture score of the aortic root in 6-month-old mice. Number of mice per group = 4 (2 male, 2 female). Scale: 1 (normal) to 5 (extensive damage). (S5) Western blot analysis of the aortic root in 4-month-old mice. Number of mice per group = 4 (2 male, 2 female). Plac, placebo; Hyd, hydralazine; Aml, amlodipine; RDEA, RDEA119; Geno, genotype; Treat, treatment. DOI: http://dx.doi.org/10.7554/eLife.08648.012

Author response image 1.

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