Author + information
- Received August 5, 2014
- Revision received October 9, 2014
- Accepted October 28, 2014
- Published online February 17, 2015.
- Leah Cannon, PhD∗,
- Ze-Yan Yu, MBBS, PhD†,
- Tadeusz Marciniec, PhD∗,
- Ashley J. Waardenberg, PhD‡,
- Siiri E. Iismaa, PhD∗,§,
- Vesna Nikolova-Krstevski, PhD∗,§,
- Elysia Neist, B Biotech (Hons)†,
- Monique Ohanian, BMedSci (Hons)∗,
- Min Ru Qiu, MBBS, PhD‖,
- Stephen Rainer, MBBS‖,
- Richard P. Harvey, PhD‡,§,¶,
- Michael P. Feneley, MD†,§,#,
- Robert M. Graham, MD∗,§,¶,#∗ ( and )
- Diane Fatkin, MD∗,§,#∗ ()
- ∗Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, Australia
- †Cardiac Physiology and Transplantation Division, Victor Chang Cardiac Research Institute, Darlinghurst, Australia
- ‡Cardiac Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, Australia
- §Faculty of Medicine, University of New South Wales, Kensington, Australia
- ¶School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, Australia
- ‖Anatomical Pathology Department, St. Vincent’s Hospital, Darlinghurst, Australia
- #Cardiology Department, St. Vincent’s Hospital, Darlinghurst, Australia
- ↵∗Reprint requests and correspondence:
Dr. Diane Fatkin or Dr. Robert M. Graham, Victor Chang Cardiac Research Institute, Lowy Packer Building, 405 Liverpool Street, P.O. Box 699, Darlinghurst NSW 2010, Australia.
Background Hypertrophic cardiomyopathy (HCM) is caused by mutations in sarcomere protein genes, and left ventricular hypertrophy (LVH) develops as an adaptive response to sarcomere dysfunction. It remains unclear whether persistent expression of the mutant gene is required for LVH or whether early gene expression acts as an immutable inductive trigger.
Objectives The aim of this study was to use a regulatable murine model of HCM to study the reversibility of pathological LVH.
Methods The authors generated a double-transgenic mouse model, tTA x αMHCR403Q, in which expression of the HCM-causing Arg403Gln mutation in the α-myosin heavy chain (MHC) gene is inhibited by doxycycline administration. Cardiac structure and function were evaluated in groups of mice that received doxycycline for varying periods from 0 to 40 weeks of age.
Results Untreated tTA x αMHCR403Q mice showed increased left ventricular (LV) mass, contractile dysfunction, myofibrillar disarray, and fibrosis. In contrast, mice treated with doxycycline from conception to 6 weeks had markedly less LVH and fibrosis at 40 weeks. Transgene inhibition from 6 weeks reduced fibrosis but did not prevent LVH or functional changes. There were no differences in LV parameters at 40 weeks between mice with transgene inhibition from 20 weeks and mice with continuous transgene expression.
Conclusions These findings highlight the critical role of the early postnatal period in HCM pathogenesis and suggest that mutant sarcomeres manifest irreversible cardiomyocyte defects that induce LVH. In HCM, mutation-silencing therapies are likely to be ineffective for hypertrophy regression and would have to be administered very early in life to prevent hypertrophy development.
Hypertrophic cardiomyopathy (HCM) is a heritable myocardial disorder associated with an increased risk of congestive heart failure, myocardial ischemia, cardiac arrhythmias, and sudden death, particularly in young individuals (1,2). Given the substantial morbidity and mortality of HCM, discovering strategies that induce regression or prevent left ventricular hypertrophy (LVH) would have substantial clinical impact.
The contemporary mechanistic paradigm for HCM is that primary perturbations of sarcomere function resulting from disease gene mutations trigger a cascade of cardiomyocyte responses including changes in calcium homeostasis, energy use, and gene expression that culminate in the hallmark pathological features of myocardial hypertrophy, disarray, and fibrosis (1,2). Although numerous interventions that modify downstream hypertrophic pathways have been evaluated, the effects of therapies that primarily inactivate or interfere with expression of mutant HCM genes have not been fully explored.
The first mutation discovered to have an association with HCM was an Arg403Gln substitution in the β-myosin heavy chain (MHC) gene (3), and this mutation has been studied in murine and rabbit models (4–10). The 2 isoforms of cardiac MHC, α and β, vary in expression among species and developmental stages (11). In humans and rabbits, β is the major isoform in the normal adult ventricle. In mice, there is a postnatal isoform switch from β to α, and thus human β mutations are typically modeled in the murine α gene. The first Arg403Gln α-MHC model was generated using a “hit-and-run” approach (4). Heterozygous mice developed HCM during adult life, whereas their homozygous littermates showed fulminant dilated cardiomyopathy (DCM) and died during the first postnatal week (4–6). In a seminal study, Jiang et al. (12) reported that ribonucleic acid interference (RNAi)–induced silencing of the mutant allele in heterozygous mice suppressed the development of HCM only when silencing was induced in mice at 1 day of age, with no effect on mice with overt HCM.
Here we report a novel regulatable mouse model of HCM in which mice carrying an Arg403Gln α-MHC transgene under the control of a tetracycline operon were crossed to mice that carried a tetracycline transactivator (tTA) transgene under the control of the α-MHC promoter (13). In the double-transgenic mice, tTA x α-MHCR403Q, Arg403Gln is constitutively expressed but can be inhibited by adding the tetracycline analogue, doxycycline, to drinking water. We used this model to determine whether transgene inhibition at varying times in adult mice would enable HCM to be reversed or prevented. Our data support the use of mutation-silencing strategies for primary prevention of HCM but suggest that there is a limited time window in which such therapies are likely to be effective.
A construct containing murine Myh6 complementary deoxyribonucleic acid (cDNA) with the Arg403Gln mutation and a Kozak consensus sequence (accAUGg) was cloned into a pTet splice plasmid vector downstream of 7 copies of the tetracycline operator and a single copy of the human cytomegalovirus (CMV) minimal promoter (Figure 1A). DNA was injected into pronuclei of zygotes from FVB/N mice, and transgenic mice were generated and maintained on an FVB/N background. Heterozygous carriers of the Arg403Gln α-MHC (αMHCR403Q) transgene were then crossed to mice carrying a single tTA transgene under the control of the α-MHC promoter on an FVB/N background (Jackson Laboratory, Bar Harbor, Maine) (13) to produce double-transgenic offspring (referred to as tTA x αMHCR403Q). Expression of the αMHCR403Q transgene was turned off by administering 1 mg/ml doxycycline mixed with 25 mg/ml sucrose in mouse drinking water. Wild-type (WT) controls were littermates of tTA x αMHCR403Q mice that did not carry either the αMHCR403Q or tTA transgenes. Mice were genotyped by polymerase chain reaction (PCR) amplification of tail genomic DNA and were maintained and evaluated according to protocols approved by the Garvan-St Vincent’s Hospital Animal Ethics Committee (Darlinghurst, Australia).
Mice were sedated with 2.5% tribromoethanol (Avertin), 0.005 to 0.01 ml/g) by intraperitoneal injection, and transthoracic echocardiography was performed using a Sonos 5500 ultrasonograph (Philips Medical Systems, Andover, Massachusetts) and a 12- to 15-mHz probe. Heart rate was monitored by continuous electrocardiography.
Hearts were dissected, washed in 0.9% sodium chloride, fixed in 4% paraformaldehyde overnight, dehydrated in 70% ethanol, and embedded in paraffin. Tissue blocks were cut into 3-μm sections, placed on glass slides, and stained with hematoxylin and eosin or picrosirius red. Sections were reviewed by an independent cardiac histopathologist for the presence of myofibrillar disarray. Interstitial fibrosis was quantitatively estimated by collagen volume fraction (CVF), defined as the number of picrosirius red–positive pixels to the total number of pixels in each field (ImageJ software, National Institutes of Health, Bethesda, Maryland). Ten random fields were evaluated for each mouse. For electron microscopy, hearts were fixed in 2.5% glutaraldehyde, 0.1 mol/l sodium cacodylate buffer, washed, post-fixed with 2% osmium tetroxide and 2% uranyl acetate solution, dehydrated in ethanol, and embedded in epoxy resin. Sections were viewed using a JEM-1400 transmission electron microscope (Jeol, Peabody, Massachusetts) at ×3,500 magnification. Z-disc alignment was assessed using ImageJ software by measuring the longitudinal distance from a perpendicular line drawn across neighboring myofibrils (14). A minimum of 25 measurements from 10 fields in each mouse was made.
RNA preparation and quantitative PCR
Total RNA was extracted from left ventricular (LV) tissue, and cDNA was synthesized using the SuperScript III First Strand Synthesis SuperMix Kit (Invitrogen, Carlsbad, California) for real-time quantitative PCR, which was performed on a LightCycler480 thermal cycler (Roche, Basel, Switzerland). Gene expression levels were normalized to reference genes Hprt1 and Ppia.
To identify Q403 mutant myosin, a custom polyclonal α-MHC antibody was synthesized (Peptide Specialty Laboratories, Heidelberg, Germany). WT and mutant peptides were generated that comprised 15 amino acids in the vicinity of residue 403 in murine α-MHC. The WT peptide contained an R at residue 403 (LKGLXHPRVKVGNEY), and the mutant peptide had a Q at this site (LKGLXHPQVKVGNEY).
Total protein was extracted from LV tissue, separated on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel, and incubated with purified polyclonal Q403 α-MHC antibody (1:1,000 dilution) or specific antibodies against R403 α-MHC (1:2,500 dilution; Santa Cruz Biotechnology, Inc., Dallas, Texas) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2,000 dilution; Abcam, Cambridge, United Kingdom), with antimouse, antigoat, and antirabbit horseradish peroxidase–conjugated secondary antibodies, respectively (Abcam). Hybridization signals were quantified and normalized to GAPDH.
Differences among groups were assessed using analysis of variance and Student t test. Data are expressed as mean ± SD; p values <0.05 were considered statistically significant, with no adjustments made for multiple comparisons.
Generation of double-transgenic mice
Heterozygous mice bearing the αMHCR403Q or tTA transgenes were crossed to produce double-transgenic (tTA x αMHCR403Q) offspring (Figures 1A and 1B). The tTA x αMHCR403Q mice were viable and phenotypically normal at birth. Real-time PCR showed cardiac-specific expression of the αMHCR403Q transgene (Figure 1C) in LV samples from tTA x αMHCR403Q mice, and significant reductions of mutant α-MHC transcript were induced by doxycycline (Figure 1D). Presence of Q403 α-MHC protein in tTA x αMHCR403Q mice was demonstrated by Western blotting using a mutation-specific antibody (Figure 1E, top panel; weak cross-reaction of this antibody occurred with the WT R403 protein; see WT lanes). In contrast to the levels of R403 protein, which were similar in WT and tTA x αMHCR403Q mice and unchanged after doxycycline treatment, levels of Q403 α-MHC were markedly reduced by doxycycline (Figures 1E and 1F).
Cardiac phenotype analysis
Cardiac morphology and function were evaluated in 6-, 12-, 20-, and 40-week-old tTA x αMHCR403Q mice and in WT littermates. Both groups had similar low rates of mortality (5%). Male tTA x αMHCR403Q mice showed increased heart weight/body weight ratio and echocardiographically derived LV mass (Figure 2A to 2C), as well as increased mean LV end-diastolic diameter (LVDD) and left atrial diameter (LAD) and reductions in LV fractional shortening (LVFS) (Figure 2D to 2F, Online Table 1). Female tTA x αMHCR403Q mice showed similar but less severe morphological and functional changes (Online Figures 1A to 1H, Online Table 2).
Light microscopy of LV sections stained with hematoxylin and eosin showed myofibrillar disarray in all 4 male tTA x αMHCR403Q mice and in 2 of 3 female tTA x αMHCR403Q mice evaluated at 40 weeks, but in none of 6 WT mice (3 male, 3 female) (Figure 2G). Assessment of LV fibrosis by collagen-specific picrosirius red staining showed that 40-week-old male tTA x αMHCR403Q mice had a significantly higher CVF than WT mice (WT, n = 6, 1.6 ± 1.3%, versus tTA x αMHCR403Q, n = 6, 3.3 ± 2.4%; p < 0.001; Figure 2H). There were no differences in CVF between female WT and tTA x αMHCR403Q mice (WT, n = 3, 1.3 ± 0.6 % versus tTA x αMHCR403Q, n = 3, 1.5 ± 0.6%; p = 0.35). On electron microscopy, tTA x αMHCR403Q mice showed misalignment of Z-discs in adjacent myofibrils (mean displacement, 200 ± 204 nm) when compared with WT mice (119 ± 82 nm; p < 0.001; Online Figure 2).
To determine whether the presence of the tTA transgene could have independent phenotype-modifying effects, male tTA mice were evaluated (Online Figures 3A to 3G). There were modest differences in heart weight/body weight ratio and LV mass between tTA mice and male WT mice at 40 weeks. When male tTA x αMHCR403Q mice and tTA mice were compared, however, there were substantially greater differences in heart weight/body weight ratio, LV mass, LVDD, LVFS, and LAD, similar to comparisons between WT and tTA x αMHCR403Q mice. LV sections from tTA mouse hearts showed no myocardial disarray, and CVF was similar to that in WT mice.
αMHCR403Q transgene suppression at varying ages
To determine whether inhibition of αMHCR403Q transgene expression would prevent or induce regression of LVH, male WT and tTA x αMHCR403Q mice were given oral doxycycline or vehicle (25 mg/ml sucrose) from 6 weeks (D6-40, early disease) or 20 weeks (D20-40, established disease) and were evaluated at 40 weeks of age. In the early disease group, there were no differences in indices of LVH or LV function between doxycycline-treated and vehicle-treated tTA x αMHCR403Q mice (Figures 3A to 3H, Online Table 3). Transgene inhibition from 6 weeks did not prevent myofibrillar disarray, but it did reduce the CVF. Transgene inhibition from 20 weeks in the established disease group produced no effects on any LV functional or histopathological parameters (Figure 3, Online Table 4). The lack of LVH prevention or regression was not the result of any pro-hypertrophic actions of doxycycline because there were no significant effects of drug treatment in WT mice or in tTA mice (Figure 3, Online Figure 4).
These observations suggest that the pathological processes that promote LVH in tTA x αMHCR403Q mice are set in place very early in life, and removal of the genetic trigger after 6 weeks cannot prevent or reverse these processes. To investigate the effects of transgene exposure during cardiac development further, a third group of male WT and tTA x αMHCR403Q mice was treated with doxycycline from conception to 6 weeks of age (D0-6) and then evaluated at 40 weeks of age. When compared with the previous groups of tTA x αMHCR403Q mice studied, the D0-6 treated mice had lower LV mass (Figure 3B, Online Table 5). Although this finding was modestly higher than LV mass in treated WT mice, there were no differences between D0-6 treated mutant mice and untreated tTA mice (p = 0.29). Heart weight/body weight ratio in D0-6 treated tTA x αMHCR403Q mice was similar to that in D0-6 treated WT mice (Online Table 5) and untreated tTA mice (p = 0.20). Myocardial histopathological features were also better preserved in D0-6 treated tTA x αMHCR403Q mice than in vehicle-treated tTA x αMHCR403Q mice, with less myofibrillar disarray, less Z-disc misalignment, and lower CVF (Figures 3F to 3H, Online Figure 2).
We found that inhibiting expression of the mutant αMHCR403Q transgene in adult tTA x αMHCR403Q mice from 6 weeks (early disease) or 20 weeks (established disease) had no effect on LVH remodeling, but transgene inhibition from conception to 6 weeks had protective effects. These observations suggest that cardiomyocyte defects established very early in life act as an inductive trigger for sustained LVH and have important implications for the timing and efficacy of targeted mutation-silencing therapies.
To undertake these studies of LVH prevention and reversal, we generated a novel mouse model in which expression of an Arg403Gln α-MHC transgene could be temporally regulated by doxycycline administration. The “gene on” tTA x αMHCR403Q mice showed progressive LVH, disarray, and fibrosis. These changes replicate those seen in humans with HCM (1,2) and in 2 other mouse models of Arg403Gln α-MHC generated by knock-in and transgenic approaches, respectively (4,7), as well as a rabbit Arg403Gln β-MHC model (9).
Although all Arg403Gln MHC animal models displayed hallmark histopathological features of HCM, there were some differences in LV dimensions and functional parameters. LV systolic function in patients with HCM is generally normal or hyperdynamic but may become progressively impaired in patients with end-stage disease (15). Age-related progression to DCM has been noted in male single-transgenic Arg403Gln α-MHC mice (8) and in Arg403Gln β-MHC rabbits (10), whereas homozygous knock-in Arg403Gln α-MHC mice have severe neonatal-onset DCM (6). Fulminant postnatal DCM rather than HCM is also seen in heterozygous knock-in Arg403Gln α-MHC mice when they are crossed to transgenic mice with a troponin I mutation (16). In tTA-regulated murine models, the presence of the tTA transgene was reported to cause mild cardiomyopathy in some studies but not others (17,18). We found no differences in LVDD or LVFS between 40-week-old male tTA and WT mice, a finding indicating that the tTA transgene did not determine the changes seen in these parameters in tTA x αMHCR403Q mice. Similar to other Arg403Gln MHC murine models (4,7), sex differences in LV functional parameters were present in tTA x αMHCR403Q mice, with male mice exhibiting relatively earlier and more severe changes. Taken together, these observations suggest that several factors, including mutant MHC “dose,” age, sex, and background genetic context, can modify Arg403Gln MHC LV phenotype.
Atrial dilation, present in male and female tTA x αMHCR403Q mice from 12 weeks of age, has been observed in human subjects with Arg403Gln β-MHC (19) and in some animal models (4,10) but not others (7), and it has numerous potential causes. Abnormalities of diastolic relaxation commonly manifest in HCM and precede hypertrophy development in humans and mice (5,20), whereas elevated filling pressures can result from severe systolic dysfunction or LV outflow tract obstruction. Interestingly, Arg403Gln MHC animal models have a concentric pattern of LVH, and LV outflow tract obstruction has not been described. Because α-MHC is expressed in the fetal and adult atrium in mice (11), a contribution of primary atrial myopathic changes to atrial dilatation cannot be excluded.
Arg403 is situated in the myosin head at the base of a loop that interacts with actin, and the Gln403 substitution at this site increases actin-myosin sliding velocities, adenosine triphosphate hydrolysis, and force generation (21). These changes are associated with increased end-diastolic and end-systolic stiffness and slowed relaxation dynamics of individual cardiomyocytes, and they manifest as increased end-diastolic and end-systolic chamber elastance and diastolic dysfunction at the whole-heart level (22). The primary mutation-induced defects trigger a cascade of downstream effects including alterations in intracellular calcium homeostasis, mitochondrial function, myocardial energetics, and cell signaling (1,2). Several interventions that modify components of these hypertrophic responses have beneficial effects in Arg403Gln murine and rabbit models, including diltiazem (L-type calcium channel inhibitor) (23), simvastatin and atorvastatin (3-hydroxy-3-methylglutaryl–coenzyme A [HMG-CoA] reductase inhibitors) (24,25), N-acetylcysteine (antioxidant) (26), and exercise training (27).
Researchers have proposed that small molecules designed to bind specifically to mutant myosin protein may directly target the sarcomeric defects underpinning HCM and prevent disease onset (28). Jiang et al. (12) provided proof of concept for mutation-inhibitory therapies by demonstrating that allele-specific RNAi-mediated silencing of Arg403Gln α-MHC in 1-day-old mice prevented later development of LVH, disarray, and fibrosis. Notably, RNAi was ineffective in mice with established disease. Using a tTA-regulated gene expression system, we found that LVH was unable to be prevented or reversed by inhibiting an αMHCR403Q transgene from 6 weeks (early disease) or 20 weeks (established disease), respectively. The inability to induce disease regression at these time points could not be explained by effects of the tTA transgene or doxycycline’s failure to suppress expression of the αMHCR403Q transgene adequately. We do not believe that persistent expression of the mutant protein can explain our findings, given that the reported turnover time (half-life) of myosin is 1 to 12 days (29,30).
What is the inductive trigger for sustained LVH?
In the embryonic mouse, the tubular heart contains high levels of both α-MHC and β-MHC. As chamber formation progresses, β-MHC becomes restricted to the ventricles, where levels remain high until birth, when there is a rapid fall (within 1 day). In the atria, α-MHC is expressed at high levels throughout development and after birth. In the ventricles, α-MHC is expressed at relatively lower levels (up to 8-fold) than β-MHC from embryonic day 10.5 onward. However, immediately after birth, α-MHC levels increase dramatically, and an α/β ratio of 16:1 is maintained throughout adult life (11,31). Coinciding with this isoform switch, the ventricular effects of the αMHCR403Q transgene would be predominantly manifest from the first postnatal week onward, but they could affect heart development.
One plausible hypothesis is that the combination of WT and hypercontractile mutant MHC in cardiac sarcomeres may result in heterogeneous force production and profound changes in the mechanical stresses within cardiomyocytes during critical periods of maturation and growth. This could affect not only the structural integrity of individual sarcomeres and Z-discs but also myofibril alignment, mitochondrial surface tension and function, intercalated disc function, nuclear shape and function, and epigenetic state. The “dose” of mutant protein is a further consideration, and we are unable to exclude the possibility that transient high levels of the αMHCR403Q transgene in mice could have deleterious effects not seen in human heterozygous mutation carriers. Undoubtedly, multiple factors are involved, and elucidation of the full spectrum of developmental defects that stimulate lifelong hypertrophic remodeling in HCM remains an important avenue for future research.
Implications for targeted mutation-silencing therapies
The failure of αMHCR403Q transgene inhibition to demonstrate preventive effects in 6-week-old tTA x αMHCR403Q mice implies that irreversible inductive stimuli for LVH are already established by this age. Given that the most rapid period of hypertrophic remodeling in patients with HCM is typically seen during the pubertal growth spurt (32), targeted mutation-silencing therapies may need to be administered before adolescence to be effective.
Our data suggest that primary prevention of HCM may be possible only when mutation-silencing therapies are commenced in utero or in the early post-partum period because protective effects were seen only in tTA x αMHCR403Q mice that received doxycycline from conception to 6 weeks of age. Given that β-MHC is the predominant MHC isoform in human fetal and adult ventricles (33), the effects of HCM-causing β-MHC mutations could extend during an equivalent prenatal and postnatal timespan. The demonstrated efficacy of RNAi-induced Arg403Gln β-MHC allelic silencing in 1-day-old mice (12) suggests that postnatal therapy could be sufficient to prevent HCM. Our group recently described a thyroid hormone–mediated proliferative burst in cardiomyocyte numbers at 15 days in pre-adolescent mice (34). This period of accelerated growth falls within the timeframe of successful mutation-silencing in tTA x αMHCR403Q mice and clearly does not mitigate against subsequent hypertrophy prevention. Further studies are clearly required to define the therapeutic window for effective mutation-silencing interventions in humans and mice more closely. Identifying genetic causes of HCM in families and prenatal or early postnatal genetic testing may be necessary to identify individuals likely to benefit from these therapies, but it is hoped that intervention before the pubertal cardiac growth spurt may be sufficient.
Due to genetic and species differences, observations in double-transgenic tTA x αMHCR403Q mice cannot be extrapolated directly to humans with HCM, and further studies in patient cohorts are warranted.
This study generated a regulatable double-transgenic Arg403Gln α-MHC murine model of HCM. Inhibiting expression of the αMHCR403Q transgene in mice with early (6 weeks) or established disease (20 weeks) does not prevent or reverse the extent of LVH present at 40 weeks. In contrast, transgene inhibition from conception up to 6 weeks provides a sustained protective effect (Central Illustration). These data suggest that irreversible mutation-induced perturbations of cardiomyocyte structure and function are established in the postnatal period, and hence mutation-silencing therapies for HCM prevention would need to be administered very early to be effective.
COMPETENCY IN MEDICAL KNOWLEDGE: Mutations in genes encoding sarcomeric proteins play a crucial role in the pathogenesis of inherited forms of HCM.
COMPETENCY IN PATIENT CARE: Identifying the genetic causes of HCM in families facilitates prenatal and early postnatal identification of mutation carriers and provides an opportunity for preventative therapies.
TRANSLATIONAL OUTLOOK: Mutation-silencing interventions in genetically predisposed individuals early in development could prevent HCM development later in life, whereas regression of hypertrophy in patients with established disease may be possible by modifying downstream signaling pathways.
The authors thank David Allen, Karen Brennan, the BioCore staff, Sara Holman, David Humphreys, Scott Kesteven, Christiana Leimena, Bryony Mearns, Thomas Preiss, Molly Vale, Xiao-Hui Xiao, and Li Sze Yeo, for helpful discussions, assistance with laboratory experiments, data analysis, and animal maintenance.
This work was supported by the National Health and Medical Research Council, Canberra, Australian Capital Territory (grant numbers 142009, 354400, 404808, 573731, and 573732). Dr. Cannon was the recipient of an Australian Postgraduate Award (University of New South Wales, Kensington, Australia). All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.
Drs. Graham and Fatkin are joint senior authors.
- Abbreviations and Acronyms
- collagen volume fraction
- dilated cardiomyopathy
- hypertrophic cardiomyopathy
- left ventricular
- left ventricular end-diastolic diameter
- left ventricular fractional shortening
- left ventricular hypertrophy
- myosin heavy chain
- ribonucleic acid interference
- tetracycline transactivator
- Received August 5, 2014.
- Revision received October 9, 2014.
- Accepted October 28, 2014.
- American College of Cardiology Foundation
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