1. Chromosomes and Gene Expression
  2. Human Biology and Medicine
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A deleterious gene-by-environment interaction imposed by calcium channel blockers in Marfan syndrome

  1. Jefferson J Doyle  Is a corresponding author
  2. Alexander J Doyle
  3. Nicole K Wilson
  4. Jennifer P Habashi
  5. Djahida Bedja
  6. Ryan E Whitworth
  7. Mark E Lindsay
  8. Florian Schoenhoff
  9. Loretha Myers
  10. Nick Huso
  11. Suha Bachir
  12. Oliver Squires
  13. Benjamin Rusholme
  14. Hamid Ehsan
  15. David Huso
  16. Craig J Thomas
  17. Mark J Caulfield
  18. Jennifer E Van Eyk
  19. Daniel P Judge
  20. Harry C Dietz  Is a corresponding author
  21. GenTAC Registry Consortium
  22. MIBAVA Leducq Consortium
  1. Howard Hughes Medical Institute and Institute of Genetic Medicine, Johns Hopkins University School of Medicine, United States
  2. Johns Hopkins University School of Medicine, United States
  3. Queen Mary University of London, United Kingdom
  4. Macquarie University, Australia
  5. Research Triangle Institute International, United States
  6. Harvard Medical School, United States
  7. Inselspital, Switzerland
  8. National Center for Advancing Translational Sciences, United States
Research Article
Cite this article as: eLife 2015;4:e08648 doi: 10.7554/eLife.08648
7 figures and 1 table

Figures

Figure 1 with 1 supplement
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.

https://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.

https://doi.org/10.7554/eLife.08648.004
Figure 2 with 1 supplement
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.

https://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.

https://doi.org/10.7554/eLife.08648.006
Figure 3 with 1 supplement
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.

https://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.

https://doi.org/10.7554/eLife.08648.008
Figure 4 with 1 supplement
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.

https://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.

https://doi.org/10.7554/eLife.08648.010
Figure 5 with 1 supplement
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.

https://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.

https://doi.org/10.7554/eLife.08648.012

Tables

Table 1

GenTAC human data

https://doi.org/10.7554/eLife.08648.013
Aortic dissectionAortic surgery
MarfanOtherMarfanOther
n = 531n = 1819n = 531n = 1819
Odds in CCB5.1%0.57%28.1%10.70%
Odds in non-CCB0.41%0.12%5.1%4.4%
Odds ratio12.54.75.52.4
 p-value0.032NS<0.001<0.01
Odds ratio (BP)*12.75.65.42.2
 p-value0.06NS<0.0010.016
Odds ratio (Aortic Size)*11.24.15.02.2
 p-value0.08NS<0.010.017
Odds ratio (β-blocker)*15.93.75.72.0
 p-value0.045NS<0.010.026
  1. Odds of ‘aortic dissection’ and ‘aortic surgery’ in patients with Marfan syndrome (‘Marfan’) and other forms of inherited thoracic aortic aneurysm (‘Other’). Odds (written as %) = number of people who incurred an event (i.e., dissection or surgery) divided by the number who did not. Odds of aortic dissection or surgery were calculated separately for patients who had used CCBs (‘Odds in CCB’) compared to those who had not (i.e., ‘Odds in non-CCB’). Odds Ratio = Odds of aortic dissection or surgery in patients who had taken CCBs divided by the odds in patients who had not taken CCBs.

  2. *

    The Odds Ratio was then adjusted for blood pressure (‘BP’), aortic size (‘Aortic Size’), and β-blocker use (‘β-blocker’) at enrollment, with corresponding p-values.

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