Real-Time Monitoring and Analysis of Zebrafish Electrocardiogram with Anomaly Detection.

Heart disease is the leading cause of mortality in the U.S. with approximately 610,000 people dying every year. Effective therapies for many cardiac diseases are lacking, largely due to an incomplete understanding of their genetic basis and underlying molecular mechanisms. Zebrafish (Danio rerio) are an excellent model system for studying heart disease as they enable a forward genetic approach to tackle this unmet medical need. In recent years, our team has been employing electrocardiogram (ECG) as an efficient tool to study the zebrafish heart along with conventional approaches, such as immunohistochemistry, DNA and protein analyses. We have overcome various challenges in the small size and aquatic environment of zebrafish in order to obtain ECG signals with favorable signal-to-noise ratio (SNR), and high spatial and temporal resolution. In this paper, we highlight our recent efforts in zebrafish ECG acquisition with a cost-effective simplified microelectrode array (MEA) membrane providing multi-channel recording, a novel multi-chamber apparatus for simultaneous screening, and a LabVIEW program to facilitate recording and processing. We also demonstrate the use of machine learning-based programs to recognize specific ECG patterns, yielding promising results with our current limited amount of zebrafish data. Our solutions hold promise to carry out numerous studies of heart diseases, drug screening, stem cell-based therapy validation, and regenerative medicine

Epicardial cells derived from human embryonic stem cells augment cardiomyocyte-driven heart regeneration.

The epicardium and its derivatives provide trophic and structural support for the developing and adult heart. Here we tested the ability of human embryonic stem cell (hESC)-derived epicardium to augment the structure and function of engineered heart tissue in vitro and to improve efficacy of hESC-cardiomyocyte grafts in infarcted athymic rat hearts. Epicardial cells markedly enhanced the contractility, myofibril structure and calcium handling of human engineered heart tissues, while reducing passive stiffness compared with mesenchymal stromal cells. Transplanted epicardial cells formed persistent fibroblast grafts in infarcted hearts. Cotransplantation of hESC-derived epicardial cells and cardiomyocytes doubled graft cardiomyocyte proliferation rates in vivo, resulting in 2.6-fold greater cardiac graft size and simultaneously augmenting graft and host vascularization. Notably, cotransplantation improved systolic function compared with hearts receiving either cardiomyocytes alone, epicardial cells alone or vehicle. The ability of epicardial cells to enhance cardiac graft size and function makes them a promising adjuvant therapeutic for cardiac repair.: This work was supported by the British Heart Foundation (BHF; Grants NH/11/1/28922, G1000847, FS/13/29/30024 and FS/18/46/33663), Oxford-Cambridge Centre for Regenerative Medicine (RM/13/3/30159), the UK Medical Research Council (MRC) and the Cambridge Hospitals National Institute for Health Research Biomedical Research Centre funding (SS), as well as National Institutes of Health Grants P01HL094374, P01GM081619, R01HL12836 and a grant from the Fondation Leducq Transatlantic Network of Excellence (CEM). J.B. was supported by a Cambridge National Institute for Health Research Biomedical Research Centre Cardiovascular Clinical Research Fellowship and subsequently, by a BHF Studentship (Grant FS/13/65/30441). DI received a University of Cambridge Commonwealth Scholarship. LG is supported by BHF Award RM/l3/3/30159 and LPO is funded by a Wellcome Trust Fellowship (203568/Z/16/Z). NF was supported by BHF grants RG/13/14/30314. NL was supported by the Biotechnology and Biological Sciences Research Council (Institute Strategic Programmes BBS/E/B/000C0419 and BBS/E/B/000C0434). SS and MB were supported by the British Heart Foundation Centre for Cardiovascular Research Excellence. Core support was provided by the Wellcome-MRC Cambridge Stem Cell Institute (203151/Z/16/Z), The authors thank Osiris for provision of the primary mesenchymal stem cells (59

TFPa/HADHA is required for fatty acid beta-oxidation and cardiolipin re-modeling in human cardiomyocytes

Mitochondrial trifunctional protein deficiency, due to mutations in hydratase subunit A (HADHA), results in sudden infant death syndrome with no cure. To reveal the disease etiology, we generated stem cell-derived cardiomyocytes from HADHA-deficient hiPSCs and accelerated their maturation via an engineered microRNA maturation cocktail that upregulated the epigenetic regulator, HOPX. Here we report, matured HADHA mutant cardiomyocytes treated with an endogenous mixture of fatty acids manifest the disease phenotype: defective calcium dynamics and repolarization kinetics which results in a pro-arrhythmic state. Single cell RNA-seq reveals a cardiomyocyte developmental intermediate, based on metabolic gene expression. This intermediate gives rise to mature-like cardiomyocytes in control cells but, mutant cells transition to a pathological state with reduced fatty acid beta-oxidation, reduced mitochondrial proton gradient, disrupted cristae structure and defective cardiolipin remodeling. This study reveals that HADHA (tri-functional protein alpha), a monolysocardiolipin acyltransferase-like enzyme, is required for fatty acid beta-oxidation and cardiolipin remodeling, essential for functional mitochondria in human cardiomyocytes.Peer reviewe

Cardiac development in zebrafish and human embryonic stem cells is inhibited by exposure to tobacco cigarettes and ecigarettes

Background Maternal smoking is a risk factor for low birth weight and other adverse developmental outcomes. Objective We sought to determine the impact of standard tobacco cigarettes and e-cigarettes on heart development in vitro and in vivo. Methods Zebrafish (Danio rerio) were used to assess developmental effects in vivo and cardiac differentiation of human embryonic stem cells (hESCs) was used as a model for in vitro cardiac development. Results In zebrafish, exposure to both types of cigarettes results in broad, dose-dependent developmental defects coupled with severe heart malformation, pericardial edema and reduced heart function. Tobacco cigarettes are more toxic than e-cigarettes at comparable nicotine concentrations. During cardiac differentiation of hESCs, tobacco smoke exposure results in a delayed transition through mesoderm. Both types of cigarettes decrease expression of cardiac transcription factors in cardiac progenitor cells, suggesting a persistent delay in differentiation. In definitive human cardiomyocytes, both e-cigarette- and tobacco cigarette-treated samples showed reduced expression of sarcomeric genes such as MLC2v and MYL6. Furthermore, tobacco cigarette-treated samples had delayed onset of beating and showed low levels and aberrant localization of N-cadherin, reduced myofilament content with significantly reduced sarcomere length, and increased expression of the immature cardiac marker smooth muscle alpha-actin. Conclusion These data indicate a negative effect of both tobacco cigarettes and e-cigarettes on heart development in vitro and in vivo. Tobacco cigarettes are more toxic than E-cigarettes and exhibit a broader spectrum of cardiac developmental defects

ALPK2 promotes cardiogenesis in zebrafish and human pluripotent stem cells

Cardiac development requires coordinated biphasic regulation of the WNT/beta-catenin signaling pathway. By intersecting gene expression and loss-of-function siRNA screens we identified Alpha Protein Kinase 2 (ALPK2) as a candidate negative regulator of WNT/beta-catenin signaling in cardiogenesis. In differentiating human embryonic stem cells (hESCs), ALPK2 is highly induced as hESCs transition from mesoderm to cardiac progenitors. Using antisense knockdown and CRISPR/Cas9 mutagenesis in hESCs and zebrafish, we demonstrate that ALPK2 promotes cardiac function and cardiomyocyte differentiation. Quantitative phosphoproteomics, protein expression profiling, and beta-catenin reporter assays demonstrate that loss of ALPK2 led to stabilization of beta-catenin and increased WNT signaling. Furthermore, cardiac defects attributed to ALPK2 depletion can be rescued in a dose-dependent manner by direct inhibition of WNT signaling through the small molecule XAV939. Together, these results demonstrate that ALPK2 regulates beta-catenin-dependent signaling during developmental commitment of cardiomyocytes

Zebrafish embryo-larval survival analysis following escalating doses of nicotine, e-cigarette and tobacco extracts.

<p>Assessment of zebrafish survival over 72 h with increasing concentrations of pure nicotine, e-cigarettes and tobacco cigarettes extracts or vehicle control (0.1% DMSO). n ≥ 3 (independent experiments with each n containing between 24–48 animals per treatment). * P < 0.05, hpe = hours post exposure.</p