Screening drugs for myocardial disease in vivo with zebrafish: an expert update.

INTRODUCTION: Our understanding of the complexity of cardiovascular disease pathophysiology remains very incomplete and has hampered cardiovascular drug development over recent decades. The prevalence of cardiovascular diseases and their increasing global burden call for novel strategies to address disease biology and drug discovery. Areas covered: This review describes the recent history of cardiovascular drug discovery using in vivo phenotype-based screening in zebrafish. The rationale for the use of this model is highlighted and the initial efforts in the fields of disease modeling and high-throughput screening are illustrated. Finally, the advantages and limitations of in vivo zebrafish screening are discussed, highlighting newer approaches, such as genome editing technologies, to accelerate our understanding of disease biology and the development of precise disease models. Expert opinion: Full understanding and faithful modeling of specific cardiovascular disease is a rate-limiting step for cardiovascular drug discovery. The resurgence of in vivo phenotype screening together with the advancement of systems biology approaches allows for the identification of lead compounds which show efficacy on integrative disease biology in the absence of validated targets. This strategy bypasses current gaps in knowledge of disease biology and paves the way for successful drug discovery and downstream molecular target identification.

Zebrafish as tools for drug discovery.

The zebrafish has become a prominent vertebrate model for disease and has already contributed to several examples of successful phenotype-based drug discovery. For the zebrafish to become useful in drug development more broadly, key hurdles must be overcome, including a more comprehensive elucidation of the similarities and differences between human and zebrafish biology. Recent studies have begun to establish the capabilities and limitations of zebrafish for disease modelling, drug screening, target identification, pharmacology, and toxicology. As our understanding increases and as the technologies for manipulating zebrafish improve, it is hoped that the zebrafish will have a key role in accelerating the emergence of precision medicine.

Future translational applications from the contemporary genomics era: a scientific statement from the American Heart Association.

The field of genetics and genomics has advanced considerably with the achievement of recent milestones encompassing the identification of many loci for cardiovascular disease and variable drug responses. Despite this achievement, a gap exists in the understanding and advancement to meaningful translation that directly affects disease prevention and clinical care. The purpose of this scientific statement is to address the gap between genetic discoveries and their practical application to cardiovascular clinical care. In brief, this scientific statement assesses the current timeline for effective translation of basic discoveries to clinical advances, highlighting past successes. Current discoveries in the area of genetics and genomics are covered next, followed by future expectations, tools, and competencies for achieving the goal of improving clinical care.

Chemical and metabolomic screens identify novel biomarkers and antidotes for cyanide exposure.

Exposure to cyanide causes a spectrum of cardiac, neurological, and metabolic dysfunctions that can be fatal. Improved cyanide antidotes are needed, but the ideal biological pathways to target are not known. To understand better the metabolic effects of cyanide and to discover novel cyanide antidotes, we developed a zebrafish model of cyanide exposure and scaled it for high-throughput chemical screening. In a screen of 3120 small molecules, we discovered 4 novel antidotes that block cyanide toxicity. The most potent antidote was riboflavin. Metabolomic profiling of cyanide-treated zebrafish revealed changes in bile acid and purine metabolism, most notably by an increase in inosine levels. Riboflavin normalizes many of the cyanide-induced neurological and metabolic perturbations in zebrafish. The metabolic effects of cyanide observed in zebrafish were conserved in a rabbit model of cyanide toxicity. Further, humans treated with nitroprusside, a drug that releases nitric oxide and cyanide ions, display increased circulating bile acids and inosine. In summary, riboflavin may be a novel treatment for cyanide toxicity and prophylactic measure during nitroprusside treatment, inosine may serve as a biomarker of cyanide exposure, and metabolites in the bile acid and purine metabolism pathways may shed light on the pathways critical to reversing cyanide toxicity.

An in vivo zebrafish screen identifies organophosphate antidotes with diverse mechanisms of action.

Organophosphates are a class of highly toxic chemicals that includes many pesticides and chemical weapons. Exposure to organophosphates, either through accidents or acts of terrorism, poses a significant risk to human health and safety. Existing antidotes, in use for over 50 years, have modest efficacy and undesirable toxicities. Therefore, discovering new organophosphate antidotes is a high priority. Early life stage zebrafish exposed to organophosphates exhibit several phenotypes that parallel the human response to organophosphates, including behavioral deficits, paralysis, and eventual death. Here, we have developed a high-throughput zebrafish screen in a 96-well plate format to find new antidotes that counteract organophosphate-induced lethality. In a pilot screen of 1200 known drugs, we identified 16 compounds that suppress organophosphate toxicity in zebrafish. Several in vitro assays coupled with liquid chromatography/tandem mass spectrometry-based metabolite profiling enabled determination of mechanisms of action for several of the antidotes, including reversible acetylcholinesterase inhibition, cholinergic receptor antagonism, and inhibition of bioactivation. Therefore, the in vivo screen is capable of discovering organophosphate antidotes that intervene in distinct pathways. These findings suggest that zebrafish screens might be a broadly applicable approach for discovering compounds that counteract the toxic effects of accidental or malicious poisonous exposures.

Systematic approaches to toxicology in the zebrafish.

As the current paradigms of drug discovery evolve, it has become clear that a more comprehensive understanding of the interactions between small molecules and organismal biology will be vital. The zebrafish is emerging as a complement to existing in vitro technologies and established preclinical in vivo models that can be scaled for high-throughput. In this review, we highlight the current status of zebrafish toxicology studies, identify potential future niches for the model in the drug development pipeline, and define the hurdles that must be overcome as zebrafish technologies are refined for systematic toxicology.

Zebrafish genetic models for arrhythmia.

Over the last decade the zebrafish has emerged as a major genetic model organism. While stimulated originally by the utility of its transparent embryos for the study of vertebrate organogenesis, the success of the zebrafish was consolidated through multiple genetic screens, sequencing of the fish genome by the Sanger Center, and the advent of extensive genomic resources. In the last few years the potential of the zebrafish for in vivo cell biology, physiology, disease modeling and drug discovery has begun to be realized. This review will highlight work on cardiac electrophysiology, emphasizing the arenas in which the zebrafish complements other in vivo and in vitro models; developmental physiology, large-scale screens, high-throughput disease modeling and drug discovery. Much of this work is at an early stage, and so the focus will be on the general principles, the specific advantages of the zebrafish and on future potential.

Adenosine monophosphate-activated protein kinase disease mimicks hypertrophic cardiomyopathy and Wolff-Parkinson-White syndrome: natural history.

OBJECTIVES: The aim of this study was to investigate the clinical expression of adenosine monophosphate-activated protein kinase (AMPK) gene mutations (PRKAG2) in adenosine monophosphate (AMP) kinase disease based on 12 years follow-up of known mutation carriers and to define the prevalence of PRKAG2 mutations in hypertrophic cardiomyopathy (HCM). BACKGROUND: Adenosine monophosphate-activated protein kinase gene mutations cause HCM with Wolff-Parkinson-White syndrome and conduction disease. METHODS: Clinical evaluation of 44 patients with known AMP kinase disease was analyzed. Mutation analysis of PRKAG2 was performed by fluorescent single-strand confirmation polymorphism analysis and direct sequencing of abnormal conformers in 200 patients with HCM. RESULTS: Only one additional mutation was identified. The mean age at clinical diagnosis in the 45 gene carriers was 24 years (median 20 years, range 9 to 55 years). Symptoms of palpitation, dypspnea, chest pain, or syncope were present in 31 (69%) gene carriers; 7 (15%) complained of myalgia and had clinical evidence of proximal myopathy. Skeletal muscle biopsy showed excess mitochondria and ragged red fibers with minimal glycogen accumulation. Disease penetrance defined by typical electrocardiogram abnormalities was 100% by age 18 years. Thirty-two of 41 adults (78%) had left ventricular hypertrophy (LVH) on echocardiography, and progressive LVH was documented during follow-up. Survival was 91% at a mean follow-up of 12.2 years. Progressive conduction disease required pacemaker implantation in 17 of 45 (38%) at a mean age of 38 years. CONCLUSIONS: The AMP kinase disease is uncommon in HCM and is characterized by progressive conduction disease and cardiac hypertrophy and includes extracardiac manifestations such as a skeletal myopathy, consistent with a systemic metabolic storage disease. Defects in adenosine triphosphate utilization or in specific cellular substrates, rather than mere passive deposition of amylopectin, may account for these clinical features.

High-throughput assay for small molecules that modulate zebrafish embryonic heart rate.

To increase the facility and throughput of scoring phenotypic traits in embryonic zebrafish, we developed an automated micro-well assay for heart rate using automated fluorescence microscopy of transgenic embryos expressing green fluorescent protein in myocardium. The assay measures heart rates efficiently and accurately over a large linear dynamic range, and it rapidly characterizes dose dependence and kinetics of small molecule-induced changes in heart rate. This is the first high-throughput micro-well assay for organ function in an intact vertebrate.