Precision Therapeutics for Pediatric Dilated Cardiomyopathy (DCM) 
(2nd Year)

Kory Lavine, M.D. Ph.D.


Final Report
 
Abstract
Pediatric dilated cardiomyopathy (DCM) is an important cause of mortality and is the most common indication for heart transplantation in children. Unfortunately, outcomes for patients with pediatric DCM remain poor and clinical trials have revealed that children with heart failure do not respond to medications used in adults. In fact, despite modern therapies, there has been no improvement in outcomes for children with heart failure since the implementation of medical regimens established in the 1970s. These observations support the concept that pediatric and adult heart failure represent distinct entities and highlight the clinically unmet need to identify underlying mechanisms that contribute to the progression of pediatric cardiomyopathy, strategies to predict outcomes, and new treatments. These initiatives represent core missions of the Longer Life Foundation.
In the prior award period (LLF 2016-004), we tested the hypothesis that the pathogenesis of pediatric and adult DCMs are distinct in that adverse remodeling (the target of adult heart failure therapies) occurs specifically in adult patients. Using molecular pathology techniques and deep RNA sequencing we demonstrated that adverse remodeling does not occur in children with DCM, thus providing a mechanistic rationale for why adult heart failure medications do not work in children. These observations are paradigm shifting in that they suggest that prognostic and therapeutic approaches routinely employed in adult heart failure patients may not be effective in pediatric populations.
An alternative approach to this challenging disease is to define and target the mechanisms by which specific pediatric DCM mutations result in cardiomyopathy. To accomplish this, we have developed models of pediatric DCM in Zebrafish using Cas9/CRISPR based gene editing. While the Zebrafish heart may be viewed as primitive, previous studies have demonstrated that Zebrafish serve as effective models of human heart failure10. In addition, Zebrafish provide numerous advantages over traditional cell based and mouse models as they are optically translucent and thus ideal for advanced imaging applications, genetically tractable, and amenable to small molecule screens. The goals of this proposal are to generate Zebrafish models of the 6 most prevalent pediatric DCM mutations, perform detailed phenotypic characterization using high content imaging to define mechanistic elements that are unique to and shared by each of these mutations, and to build a platform to execute chemical and genetic screens to identify precision therapies for pediatric DCM.

Project aims 
• Generate Zebrafish models of pediatric DCM and define proximate disease mechanisms
• Design a high-throughput screening strategy to identify precision therapeutics for pediatric DCM 

Introduction
Idiopathic and familial DCMs represent the primary etiology of heart failure in children, adolescents, and young adults. Little is understood regarding pathogenic mechanisms that initiate and drive disease progression in these age groups and no effective strategies exist to predict outcomes or adequately treat these individuals. In this proposal, we aim to develop Zebrafish as a model system to define proximate disease mechanisms and as a tool to identify precision therapeutics for pediatric DCM. These aims fall into the scope and mission of the LLF as they seek to identify strategies to halt disease progression and prognosticators of treatment response. 
Dilated cardiomyopathy (DCM) is a prevalent cause of heart failure in adult patients and represents the most common diagnosis leading to heart transplant in children greater than 1 year of age1-3. Despite advancements in care, pediatric DCM remains a challenging disease with an estimated 50% 5 year transplant-free survival1. Within both pediatric and adult populations, DCM is associated with mutations in a shared subset of saromeric and cytoskeletal genes including, Troponin T, Troponin C, Laminin A, Myosin Binding Protein C, Tropomyosin, and Titin11. Within a single family, patients with a common mutation may present with heart failure over a wide range of ages spanning from early infancy to adulthood. While the genetic basis for pediatric DCM is increasingly recognized12, outside of cardiac transplantation, few options exist for children suffering from this devastating disease exemplifying the clinically unmet need to better understand the pathogenesis of pediatric DCM and develop effective therapies.  

Over the past two decades, landmark clinical trials have led to the establishment of effective medical therapies for adults with heart failure13. Mechanistically, adult heart failure therapeutics target a process termed adverse remodeling, a common pathway by which the adult heart responds to injury. Pathologically, adverse remodeling is defined by cardiomyocyte hypertrophy, fibrosis, and capillary loss14. While initially beneficial through augmentation of cardiac output, over time adverse remodeling is detrimental as it drives the pathologic progression of heart failure in adults and contributes to poor clinical outcomes14. While it is reasonable to postulate that adult heart failure therapies may be efficacious in children, clinical trials have not supported this idea. For example, despite marked reductions in mortality associated with beta blocker therapy in adult studies, the Pediatric Carvedilol Study failed to demonstrate improvements in clinical outcomes for children with symptomatic heart failure6, 7. Consistent with these findings, registry data has revealed that adult heart failure therapeutics have provided no survival benefit in children over digoxin and diuretic based regimens that were established in the 1970s8. Together, these observations support the concept that pediatric and adult heart failure may represent distinct entities and highlight the clinically unmet need to identify novel therapies for patients with DCM.

One potential explanation for why adult heart failure therapeutics do not improve outcomes in the pediatric population is that adverse remodeling does not govern disease progression in children. In the previous award period, we tested established that adverse remodeling specifically occurs in adult patients and is not present in children with DCM. Our findings have been accepted for publication (Patel et al. JCI Insight 2017) and clearly demonstrate that adverse remodeling does not occur on the pathological or molecular level in children with DCM.  Collectively, these observations show that adverse remodeling does not govern disease progression in pediatric DCM and support the concept that pediatric and adult DCMs represent distinct disease entities. 
Our observations explain several clinical distinctions between pediatric and adult heart failure. For example, the absence of myocardial fibrosis offers a rationale for why children with DCM display substantially higher rates of cardiac recovery and lower rates of sudden cardiac death or ICD placement compared to adults15-18. The absence of adverse remodeling in pediatric DCM may also explain why clinical outcomes for children newly diagnosed with DCM are uniformly determined within 2 years from their incident diagnosis. Children will either experience complete recovery of cardiac function, disease stabilization, or progressive heart failure resulting in death or need for cardiac transplantation19. 

In this context, early delineation of clinical outcomes strongly implies that heart failure progression in children is dictated by proximal events such as the initial cause of heart failure. Therefore, we postulate that an improved understanding of proximate disease mechanisms in children is required to develop appropriate prognostic tools and rationally designed therapies. We have chosen to take 2 approaches to advance prognostic and therapeutic strategies for pediatric DCM. The latter approach will serve as the focus of this application. 
• To define pediatric DCM biomarkers that are predictive of clinical outcomes, we have developed a collaboration with the PCMR investigator group. This large scale biomarker discovery and validation study will utilize the newly available SOMAscan biomarker discovery platform20.
• To define proximate disease mechanisms and precision therapies, we have developed a model system in Zebrafish to introduce DCM variants, define disease mechanisms, and aim to utilize this tool as a platform for drug discovery (FOCUS of this GRANT).  

Methods and Results
The goals of this project are to generate Zebrafish models of the 6 most prevalent pediatric DCM mutations, perform detailed phenotypic characterization including advanced imaging to define mechanistic elements that are unique to and shared by each of these mutations, and to optimize a strategy to execute chemical and genetic screens to identify precision therapies for pediatric DCM (Fig. 1). Zebrafish provide numerous advantages over traditional cell based and mouse models as they are optically translucent and thus ideal for advanced imaging applications, genetically tractable, and amenable to small molecule screens.


Figure 1. Schematic depicting our experimental approach to modeling pediatric DCM in Zebrafish, defining proximate disease mechanisms, and strategy to identify mutation specific precision therapies. Left, bright field image of a Titin N-terminal truncation mutant showing pericardial edema (red arrow). Right, high resolution image of a Titin N-terminal truncation mutant harboring the cmlc::GFP transgene showing normal atrial, ventricular, and outflow tract structure.

Figure 2. Cardiac imaging in Zebrafish. A-B, Diastolic and systolic images of control and Titin mutant Zebrafish at 96 hours post fertilization. cmlc::gfp reporter. Zebrafish expressing Titin truncations display reduced cardiac size and increased wall thickness. C, Quantification of ejection fraction and wall thickening as indices of ventricular function. D, Quantification of cell strain reveals reduced cardiomyocyte longitudinal and axial strain in Titin mutant fish.

 

To generate Zebrafish models of pediatric DCM, we utilized CRISPR/Cas9 mediated gene editing to insert the following mutations into their corresponding endogenous loci within the Zebrafish genome: Titin N-terminal truncations, Tpm1Lys15Asn, Tnnt2DeltaK210, and LmnaArg399Cys. These variants are strongly associated with pediatric and adult DCM9, 21 and are present in conserved regions of their respective Zebrafish orthologues. 
By generating wild type, heterozygous, and homozygous progeny from F3 founder lines, we demonstrated that Zebrafish harboring homozygous Titin N-terminal truncations, Tnnt2DeltaK210, and LmnaArg399Cys missense mutations develop a heart failure phenotype. Zebrafish homozygous for the Tpm1Lys15Asn mutation were phenotypically normal without a discernable cardiac phenotype. Titin, Tnnt2, and LMNA mutants displayed distinct heart failure phenotypes. Genetic interaction studies showed that each of these mutations represented loss of function alleles. Titin mutants displayed severe heart failure beginning 72 hours post-fertilization (Fig. 2), Tnnt2 mutants developed heart failure at 120 hours post-fertilization, and LMNA mutants showed a heart failure phenotype beginning 3-4 weeks post-fertilization. Moreover, each of these mutants displayed distinct cardiac morphologies (histology and electron microscopy) and no genetic interactions were evident in compound homozygous fish. RNA sequencing on isolated wild type and DCM mutant hearts is ongoing to compare our DCM models at the molecular level.
  
To identify small molecule compounds that suppress or reverse heart failure phenotypes in our Zebrafish DCM models, we designed a high-throughput screening assay. While measurement of cardiac function and contractility using direct microscopy is precise, this method is not well adapted to chemical screens as it is time and labor intensive. Instead, we will designed our screening assay using brain natriuretic peptide (Nppb/BNP) expression as a surrogate marker of heart failure23, 24. We have obtained transgenic Zebrafish harboring the heart failure reporter (nppb::luciferase), which emits light when activated and provided the appropriate substrate (luciferin). This reporter has been demonstrated to be active in Zebrafish expressing human cardiomyopathy mutations and was successfully utilized to conduct a chemical screen10. 
To optimize assay conditions, we utilized our LmnaArg399Cys mutants to determine the impact of adjusting input sample size, luciferin substrate, and duration of signal detection on assay sensitivity and reproducibility. Assay sensitivity was defined by assessing the relationship between reporter activity (relative light units) and ejection fraction. Assay reproducibility was measured by calculating reporter activity variance across 5 independent replicates.  Our initial experiments revealed that at inclusion of 5-7 individual Zebrafish maximized assay precision and Steady-Glo (moderate intensity-5 hour half-life) and Vivo-Glo (endotoxin resistant) provided the best assay sensitivity. 

Discussion: implications and future plans/grants
Our findings establish the feasibility of using Zebrafish DCM. In addition, our observations clearly indicate that distinct DCM mutations give rise to unique heart failure pathologies and highlight the potential impact of pursuing a precision medicine strategy in the treatment of dilated cardiomyopathy. By designing therapeutics targeted based on genetic mutation and mechanism of disease, it may be feasible to develop approaches that correct the molecular underpinnings of a patient’s disease. Future experiments will focus on developing additional Zebrafish models using gene editing and transgenic approaches. In addition, we will conduct chemical screens using small molecule libraries (ICCB bioactives, n=480 and DNA-encoded libraries). These experiments will be the focus of NIH R01 and UG3 applications.


Reference List
(1) Towbin JA, Lowe AM, Colan SD et al. Incidence, causes, and outcomes of dilated cardiomyopathy in children. JAMA 2006;296(15):1867-1876.
(2) Taylor DO, Stehlik J, Edwards LB et al. Registry of the International Society for Heart and Lung Transplantation: Twenty-sixth Official Adult Heart Transplant Report-2009. J Heart Lung Transplant 2009;28(10):1007-1022.
(3) Go AS, Mozaffarian D, Roger VL et al. Heart Disease and Stroke Statistics--2013 Update A Report From the American Heart Association. Circulation 2012.
(4) Hartupee J, Mann DL. Neurohormonal activation in heart failure with reduced ejection fraction. Nat Rev Cardiol 2017;14(1):30-38.
(5) Mann DL. Basic mechanisms of left ventricular remodeling: the contribution of wall stress. J Card Fail 2004;10(6 Suppl):S202-S206.
(6) Shaddy RE, Boucek MM, Hsu DT et al. Carvedilol for children and adolescents with heart failure: a randomized controlled trial. JAMA 2007;298(10):1171-1179.
(7) Bristow MR, Gilbert EM, Abraham WT et al. Carvedilol produces dose-related improvements in left ventricular function and survival in subjects with chronic heart failure. MOCHA Investigators. Circulation 1996;94(11):2807-2816.
(8) Kantor PF, Abraham JR, Dipchand AI, Benson LN, Redington AN. The impact of changing medical therapy on transplantation-free survival in pediatric dilated cardiomyopathy. J Am Coll Cardiol 2010;55(13):1377-1384.
(9) McNally EM, Golbus JR, Puckelwartz MJ. Genetic mutations and mechanisms in dilated cardiomyopathy. J Clin Invest 2013;123(1):19-26.
(10) Asimaki A, Kapoor S, Plovie E et al. Identification of a new modulator of the intercalated disc in a zebrafish model of arrhythmogenic cardiomyopathy. Sci Transl Med 2014;6(240):240ra74.
(11) McNally EM, Barefield DY, Puckelwartz MJ. The genetic landscape of cardiomyopathy and its role in heart failure. Cell Metab 2015;21(2):174-182.
(12) Rampersaud E, Siegfried JD, Norton N, Li D, Martin E, Hershberger RE. Rare variant mutations identified in pediatric patients with dilated cardiomyopathy. Prog Pediatr Cardiol 2011;31(1):39-47.
(13) Goldberg LR. Heart failure. Ann Intern Med 2010;152(11):ITC61-15.
(14) Burchfield JS, Xie M, Hill JA. Pathological ventricular remodeling: mechanisms: part 1 of 2. Circulation 2013;128(4):388-400.
(15) Pahl E, Sleeper LA, Canter CE et al. Incidence of and risk factors for sudden cardiac death in children with dilated cardiomyopathy: a report from the Pediatric Cardiomyopathy Registry. J Am Coll Cardiol 2012;59(6):607-615.
(16) Everitt MD, Sleeper LA, Lu M et al. Recovery of Echocardiographic Function in Children with Idiopathic Dilated Cardiomyopathy: Results from the Pediatric Cardiomyopathy Registry. J Am Coll Cardiol 2014.
(17) Mann DL, Barger PM, Burkhoff D. Myocardial recovery and the failing heart: myth, magic, or molecular target? J Am Coll Cardiol 2012;60(24):2465-2472.
(18) Mann DL, Bogaev R, Buckberg GD. Cardiac remodelling and myocardial recovery: lost in translation? Eur J Heart Fail 2010;12(8):789-796.
(19) Alvarez JA, Orav EJ, Wilkinson JD et al. Competing risks for death and cardiac transplantation in children with dilated cardiomyopathy: results from the pediatric cardiomyopathy registry. Circulation 2011;124(7):814-823.
(20) Gold L, Ayers D, Bertino J et al. Aptamer-based multiplexed proteomic technology for biomarker discovery. PLoS One 2010;5(12):e15004.
(21) Rampersaud E, Siegfried JD, Norton N, Li D, Martin E, Hershberger RE. Rare variant mutations identified in pediatric patients with dilated cardiomyopathy. Prog Pediatr Cardiol 2011;31(1):39-47.
(22) Chi NC, Shaw RM, Jungblut B et al. Genetic and physiologic dissection of the vertebrate cardiac conduction system. PLoS Biol 2008;6(5):e109.
(23) Motiwala SR, Gaggin HK. Biomarkers to Predict Reverse Remodeling and Myocardial Recovery in Heart Failure. Curr Heart Fail Rep 2016;13(5):207-218.
(24) Price JF, Thomas AK, Grenier M et al. B-type natriuretic peptide predicts adverse cardiovascular events in pediatric outpatients with chronic left ventricular systolic dysfunction. Circulation 2006;114(10):1063-1069.


 
Project Overview:
Dilated cardiomyopathy (DCM) is the most common indication for heart transplantation in children. While it is recognized that mutations in sarcomeric and cytoskeletal genes underlie pediatric DCM, outcomes remain poor and few therapeutics exist. The goal of this proposal is to generate Zebrafish models of the six most prevalent pediatric DCM mutations, perform detailed phenotyping to define mechanistic elements that are unique to and shared by each of these mutations, and optimize experimental approaches to identify mutation specific compounds that either suppress or reverse heart failure. We envision these studies would provide the requisite information to design small molecule and genetic screens to identify novel therapies for pediatric DCM, a core mission of the LLF.

Project aims:

            Generate zebrafish models of pediatric DCM and define proximate disease mechanisms

            Design a high-throughput screening strategy to identify precision therapeutics for pediatric DCM

 

Progress Report:

One potential explanation for why adult heart failure therapeutics do not improve outcomes in the pediatric population is that adverse remodeling does not govern disease progression in children. In the previous award period, we tested the hypothesis that adverse remodeling specifically occurs in adult patients and is not present in children with DCM. Our findings have been accepted for publication (Patel et al. JCI Insight 2017).

 

Data from Previous Award Period: To test the hypothesis that adverse remodeling does not occur in children with DCM, we obtained LV myocardial specimens from the apex and lateral wall of non-failing pediatric donor controls (n=11), pediatric DCM patients (n=31), adult non-failing donor controls (n=14), and adult DCM (n=34) patients. To our knowledge, this represents the largest collection of pediatric and adult DCM tissue specimens studied to date. Control tissue was obtained from unused donors with normal cardiac function by echocardiography. DCM specimens were obtained from children and adults with advanced stage idiopathic or familial DCN at the time of cardiac transplantation or implantation of a left ventricular assist device. To evaluate the presence and extent of adverse remodeling at the tissue level, we measured cardiomyocyte and sarcomere size, myocardial fibrosis, and capillary density in pediatric and adult donor control and DCM specimens. In addition, we performed RNA sequencing on pediatric and adult DCM specimens to examine gene expression signatures of adverse remodeling and define pathways that differentiate pediatric from adult DCM. Control and DCM specimens were obtained in collaboration with the Translational Cardiovascular Biobank and Repository at Washington University, University of Colorado, and the Pediatric Cardiomyopathy Registry (PCMR) tissue repository.

 

Using a combination of quantitative histology, immunostaining, electron microscopy, and RNA sequencing, we demonstrated that adverse remodeling does not uniformly occur in children with DCM. Myocardial specimens obtained from patients with pediatric DCM display no evidence of cardiomyocyte hypertrophy or increased sarcomere thickness compared to age matched donor controls. In contrast, myocardial specimens obtained from adults with DCM, displayed robust increases in cardiomyocyte hypertrophy and sarcomere thickness. Examination of myocardial fibrosis similarly revealed no evidence of increased collagen deposition in pediatric DCM specimens compared to age matched donor controls. In contrast, myocardial specimens obtained from adults with DCM, displayed robust increases in myocardial fibrosis compared to age matched donor controls. Importantly, these findings appear to be irrespective of patient age across pediatric and adult subgroups, as both linear reqression and subgroup analyses stratifying pediatric and adult groups by patient age demonstrated that only adult DCM displayed increased cardiomyocyte hypertrophy and myocardial fibrosis across the spectrum of ages examined. Measurement of capillary density revealed coronary expansion only in pediatric DCM specimens.

 

Careful analysis of clinical and demographic information revealed several key differences between our pediatric and adult DCM cohorts including increased length of disease duration and increased frequency of hypertension, diabetes, and chronic kidney disease in the adult DCM group. As these differences may represent potential confounding variables, we examined if disease duration, hypertension, diabetes, and chronic kidney disease were associated with the extent of pathological remodeling (cardiomyocyte hypertrophy or myocardial fibrosis). Linear regression and subgroup analyses failed to demonstrate statistically significant associations between these variables, indicating that differences in adverse remodeling between pediatric and adult DCM were not due to unbalanced comorbidities or disease duration.

 

Consistent with pathology data, RNA sequencing revealed divergent gene expression profiles between pediatric and adult DCM tissue  specimens with adult specimens displaying marked induction of transcripts associated with adverse remodeling and inflammation (Fig. 3). In addition, we noted that compared to children, adult DCM specimens displayed marked increases in transcripts involved in metabolite signaling, fatty acid utilization, and oxidative metabolism. These data confirm the importance of metabolism/substrate utilization in adult heart failure and signify the relevance of innate immune activation in the pathogenesis of adult DCM. Collectively, these data demonstrate that adverse remodeling does not govern disease progression in pediatric DCM and support the innovative concept that pediatric and adult DCMs represent distinct disease entities.