1. Date of download: 6/3/2016 Copyright © The American College of Cardiology. All rights reserved. From: Irreversible Triggers for Hypertrophic Cardiomyopathy Are Established in the Early Postnatal Period J Am Coll Cardiol. 2015;65(6):560-569. doi:10.1016/j.jacc.2014.10.069 Generation of Regulatable tTA x αMHC R403Q Mice (A) Construct design used to generate double-transgenic tetracycline transactivator (tTA) x α-myosin heavy chain (MHC) R403Q mice. (B) Polymerase chain reaction (PCR) of left ventricular (LV) genomic deoxyribonucleic acid demonstrating presence of the transgenes, αMHC R403Q (1095 bp product) and tTA (300 bp product) in single- and double-transgenic mice. (C) Real-time PCR (RT- PCR) showing expression of αMHC R403Q transcript in the heart (H), but not in gastrocnemius skeletal muscle (Sk) or bladder smooth muscle (Sm). (D) RT-PCR showing expression of the αMHC R403Q transgene in LV tissue from 12-week-old male tTA x αMHC R403Q mice treated with vehicle or doxycycline (dox) post-weaning (n = 3 each group). (E) Western blot showing levels of Q403 α-MHC and R403 α-MHC protein in 12-week-old male wild-type (WT) mice, untreated tTA x αMHC R403Q mice, and dox-treated tTA x αMHC R403Q mice. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (F) Mean levels of Q403 α-MHC (left panel) and R403 α-MHC (right panel) normalized to GAPDH in WT mice (n = 4, blue bars), untreated tTA x αMHC R403Q mice (n = 6, salmon bars), and dox-treated tTA x αMHC R403Q mice (n = 6, grey bars). *p < 0.05, ***p < 0.001; analysis of variance (ANOVA) and Student t test. CMVmin = CMV minimal promoter; (tetO)7, 7 copies of the tetracycline operator; Poly-A, polyadenylated tail. Figure Legend:

2. Date of download: 6/3/2016 Copyright © The American College of Cardiology. All rights reserved. From: Irreversible Triggers for Hypertrophic Cardiomyopathy Are Established in the Early Postnatal Period J Am Coll Cardiol. 2015;65(6):560-569. doi:10.1016/j.jacc.2014.10.069 Cardiac Phenotype of Male tTA x αMHC R403Q Mice (A) Longitudinal sections of hearts from WT and tTA x αMHC R403Q mice at 40 weeks of age. Scale bar = 2.5 mm. (B) Changes in the ratio of heart weight (HW) to body weight (BW) in WT (blue bars) and tTA x αMHC R403Q (salmon bars) mice from 6 to 40 weeks of age. (C) to (F) Echocardiographic data in WT (squares) and tTA x αMHC R403Q (circles) mice aged 6 weeks (n = 7), 12 weeks (n = 22), 20 weeks (n = 21), and 40 weeks (n = 18) showing changes in LV mass (LVM), LV end-diastolic diameter (LVDD), left atrial diameter (LAD), and LV fractional shortening (LVFS). *p < 0.05 versus WT; ANOVA and Student t test. LV sections from 40-week-old mice stained with (G) hematoxylin and eosin or (H) picrosirius red. Scale bar = 100 μm. Abbreviations as in Figure 1. Figure Legend:

3. Date of download: 6/3/2016 Copyright © The American College of Cardiology. All rights reserved. From: Irreversible Triggers for Hypertrophic Cardiomyopathy Are Established in the Early Postnatal Period J Am Coll Cardiol. 2015;65(6):560-569. doi:10.1016/j.jacc.2014.10.069 Effects of Transgene Inhibition in Male tTA x αMHC R403Q Mice (A) to (F) tTA x αMHC R403Q mice were treated with vehicle (V) or dox from conception to 6 weeks (D0-6), 6 weeks to 40 weeks (D6-40), and 20 weeks to 40 weeks (D20-40). Pooled data are shown for WT mice (see Online Tables 3 to 5 for expanded WT results). Endpoints evaluated at 40 weeks were: (A) HW/BW ratio; (B) LVM; (C) LVDD; (D) LAD, (E) LVFS; (F) collagen volume fraction (CVF); n = 3 to 11 each group. *p < 0.05; ANOVA and Student t test. LV sections from 40-week-old mice stained with (G) hematoxylin and eosin or (H) picrosirius red. Scale bar = 100 μm. Abbreviations as in Figures 1 and 2. Figure Legend:

4. Date of download: 6/3/2016 Copyright © The American College of Cardiology. All rights reserved. From: Irreversible Triggers for Hypertrophic Cardiomyopathy Are Established in the Early Postnatal Period J Am Coll Cardiol. 2015;65(6):560-569. doi:10.1016/j.jacc.2014.10.069 LVH Reversibility Through Mutation-Silencing Therapy In a double-transgenic mouse model, doxycycline administration was evaluated for its ability to inhibit expression of the hypertrophic cardiomyopathy–causing Arg403Gln mutation in the α-myosin heavy chain gene. Doxycycline was given at 3 time points: 0 to 6 weeks, 6 to 40 weeks, and 20 to 40 weeks (the latter 2 groups representing early and established disease). Although transgene inhibition at 6 weeks reduced fibrosis, it did not prevent left ventricular hypertrophy (LVH) or functional changes; no effects were seen in the 20- to 40-week group. Disease suppression was experienced only in the 0- to 6-week group treated from conception. Figure Legend: