Carl Tucker

Systolic and diastolic ventricular function in zebrafish embryos: Influence of norepenephrine, MS-222 and temperature

BackgroundZebrafish are increasingly used to study the influences of gene mutation and manipulation on cardiac development, structure and function. In this study, a video edge detection system was used to characterise, continuously, cardiac ventricle function in 2–5 days old zebrafish embryos embedded in 0.6% agar and examined under light microscopy at room temperature (22°C). Using video edge detection software (IonOptix Inc), the motion of a small region of the cardiac ventricle wall was converted to a continuous chart trace allowing analysis of wall motion amplitude (WMA) and myocardial wall velocity during systole (MWVs) and diastole (MWVd).ResultsCardiac wall motion characteristics changed progressively from day 2 to 5 (WMA, 2-days, 17.6 ± 4.4 μm vs 5-days, 24.6 ± 4.7 μm, p < 0.01). MWVd was more rapid than MWVs at all developmental time points. Embryonic hearts were also assessed after increasing concentrations of norepenephrine (NE) and the anaesthetic agent MS222 (tricaine) were added to the bathing water. In response to NE, WMA increased significantly more in 4 day embryos compared with 2 day embryos (change in WMA,13.6 ± 8.2 μm vs 4.0 ± 8.8 μm, p = 0.01, respectively) while the decrease in WMA in response to MS222 was similar in both 2 and 4-day embryos. Heart rate, MWVs and MWVd were significantly higher at 28°C compared with 22°C. No differences in cardiac function were observed between AB and Golden strains.ConclusionVideo edge detection appears sufficiently sensitive to detect subtle changes in diastolic and systolic cardiac function during development and changes resulting from pharmacological and environmental interventions. Such measurements could be valuable in assessment of altered cardiac function after genetic manipulation.

Zebrafish as model organisms for studying drug induced liver injury

Drug‐induced liver injury (DILI) is a major challenge in clinical medicine and drug development. New models are needed for predicting which potential therapeutic compounds will cause DILI in humans, and new markers and mediators of DILI still need to be identified. This review highlights the strengths and weaknesses of using zebrafish as a high‐throughput in vivo model for studying DILI. Although the zebrafish liver architecture is different from that of the mammalian liver, the main physiological processes remain similar. Zebrafish metabolize drugs using similar pathways to those in humans; they possess a wide range of cytochrome P450 enzymes that enable metabolic reactions including hydroxylation, conjugation, oxidation, demethylation and de‐ethylation. Following exposure to a range of hepatotoxic drugs, the zebrafish liver develops histological patterns of injury comparable to those of mammalian liver, and biomarkers for liver injury can be quantified in the zebrafish circulation. The zebrafish immune system is similar to that of mammals, but the zebrafish inflammatory response to DILI is not yet defined. In order to quantify DILI in zebrafish, a wide variety of methods can be used, including visual assessment, quantification of serum enzymes and experimental serum biomarkers and scoring of histopathology. With further development, the zebrafish may be a model that complements rodents and may have value for the discovery of new disease pathways and translational biomarkers.

Flavones induce neutrophil apoptosis by down-regulation of Mcl-1 via a proteasomal-dependent pathway

Neutrophil apoptosis and subsequent nonphlogistic clearance by surrounding phagocytes are key to the successful resolution of neutrophilic inflammation, with dysregulated apoptosis reported in multiple human inflammatory diseases. Enhancing neutrophil apoptosis has proresolution and anti‐inflammatory effects in preclinical models of inflammation. Here we investigate the ability of the flavones apigenin, luteolin, and wogonin to induce neutrophil apoptosis in vitro and resolve neutrophilic inflammation in vivo. Human neutrophil apoptosis was assessed morphologically and by flow cytometry following incubation with apigenin, luteolin, and wogonin. All three flavones induced time‐ and concentration‐dependent neutrophil apoptosis (apigenin, EC50=12.2 μM; luteolin, EC50=14.6 μM; and wogonin, EC50=28.9 μM). Induction of apoptosis was caspase dependent, as it was blocked by the broad‐spectrum caspase inhibitor Q‐VD‐OPh and was associated with both caspase‐3 and caspase‐9 activation. Flavone‐induced apoptosis was preceded by down‐regulation of the prosurvival protein Mcl‐1, with proteasomal inhibition preventing flavone‐induced Mcl‐1 down‐regulation and apoptosis. The flavones abrogated the survival effects of mediators that prolong neutrophil life span, including lipoteichoic acid, peptidoglycan, dexamethasone, and granulocyte‐macrophage colony stimulating factor, by driving apoptosis. Furthermore, wogonin enhanced resolution of established neutrophilic inflammation in a zebrafish model of sterile tissue injury. Wogonin‐induced resolution was dependent on apoptosis in vivo as it was blocked by caspase inhibition. Our data show that the flavones induce neutrophil apoptosis and have potential as neutrophil apoptosis‐inducing anti‐inflammatory, proresolution agents.—Lucas, C. D., Allen, K. C., Dorward, D. A., Hoodless, L. J., Melrose, L. A., Marwick, J. A., Tucker, C. S., Haslett, C., Duffin, R., Rossi, A. G. Flavones induce neutrophil apoptosis by down‐regulation of Mcl‐1 via a proteasomal‐dependent pathway. FASEB J. 27, 1084–1094 (2013). www.fasebj.org

Physiological roles of glucocorticoids during early embryonic development of the zebrafish (Danio rerio)

•  Glucocorticoids are known to be present in the developing zebrafish embryo but little is known about their physiological role at this early stage. •  The zebrafish embryo demonstrates a functional glucocorticoid system from around 48 h post fertilisation. •  This system and the stress response is amenable to pharmacological and genetic manipulation in a manner predicted by mammalian physiology. •  Glucocorticoids play a key developmental role in hatching, swimming and stress response. •  The zebrafish embryo is a relevant model for the study of glucocorticoid physiology.

Class III antiarrhythmic methanesulfonanilides inhibit leukocyte recruitment in zebrafish

Understanding fundamental molecular mechanisms that govern the transmigration and interstitial migration of leukocytes to sites of tissue damage and infection is of potential significance in identifying novel therapeutic targets for the management of chronic inflammatory disorders. CD31 is a mammalian cell adhesion molecule that regulates the recruitment of leukocytes from the circulation. Our recent unpublished work has suggested that homophilic ligation of CD31 can negatively regulate the ether‐à‐go‐go‐related gene (ERG) current within leukocytes to regulate cell‐cell adhesion. To validate and probe the functional significance of ERG in leukocytes, we developed an infected wound model of inflammation in zebrafish and assessed the efficacy of two ERG‐specific inhibitors, dofetilide and E4031, as well as an ERG‐specific antisense RNA morpholino on neutrophil recruitment. Our data confirm a hitherto undescribed role for ERG in leukocytes, where inhibition or translational knockdown of ERG resulted in significant attenuation of the inflammatory response to an infectious stimulus. Inhibition of ERG was verified independently by a decrease in the ventricular heart rate, where ERG also functions in the repolarization of the cardiac action potential. Our results suggest that ERG‐specific Class III antiarrhythmic drugs can modulate inflammatory responses to infection.

Skin exposure to micro- and nano-particles can cause haemostasis in zebrafish larvae

Low mass ambient exposure to airborne particles is associated with atherothrombotic events that may be a consequence of the combustion-derived nanoparticle content. There is concern also over the potential cardiovascular impact of manufactured nanoparticles. To better understand the mechanism by which toxic airborne particles can affect cardiovascular function we utilised zebrafish as a genetically tractable model. Using light and confocal fluorescence video-microscopy, we measured heart-rate and blood flow in the dorsal aorta and caudal artery of zebrafish larvae that had been exposed to a number of toxic and non-toxic microparticles and nanoparticles. Diesel exhaust particles (DEP), carboxy-charged Latex beads (carboxy-beads) and toxic alumina (Taimicron TM300), but not non-toxic alumina (Baikalox A125), were found to promote both skin and gut cell damage, increased leukocyte invasion into the epidermis, tail muscle ischaemia and haemostasis within the caudal artery of free swimming zebrafish larvae. The presence of sodium sulfite, a reducing agent, or warfarin, an anticoagulant, within the system water abrogated the effects of both toxic alumina and carboxy-beads but not DEP. Genetic manipulation of skin barrier function augmented skin damage and haemostasis, even for the non-toxic alumina. The toxic effects of carboxy-beads were still apparent after leukocyte numbers were depleted with anti-Pu.1 morpholino. We conclude that particle uptake across skin epithelium and gut mucosal barriers, or the presence of leukocytes, is not required for particle-induced haemostasis while a compromised skin barrier function accentuated tissue injury and haemostasis.

Laser-targeted ablation of the zebrafish embryonic ventricle : a novel model of cardiac injury and repair.

Background While the adult zebrafish (Danio rerio) heart demonstrates a remarkable capacity for self-renewal following apical resection little is known about the response to injury in the embryonic heart. Methods Injury to the beating zebrafish embryo heart was induced by laser using a transgenic zebrafish expressing cardiomyocyte specific green fluorescent protein. Changes in ejection fraction (EF), heart rate (HR), and caudal vein blood flow (CVBF) assessed by video capture techniques were assessed at 2, 24 and 48 h post-laser. Change in total and mitotic ventricular cardiomyocyte number following laser injury was also assessed by counting respectively DAPI (VCt) and Phospho-histone H3 (VCm) positive nuclei in isolated hearts using confocal microscopy. Results Laser injury to the ventricle resulted in bradycardia and mild bleeding into the pericardium. At 2 h post-laser injury, there was a significant reduction in cardiac performance in lasered-hearts compared with controls (HR 117 ± 11 vs 167 ± 9 bpm, p ≤ 0.001; EF 14.1 ± 1.8 vs 20.1 ± 1.3%, p ≤ 0.001; CVBF 103 ± 15 vs 316 ± 13μms− 1, p ≤ 0.001, respectively). Isolated hearts showed a significant reduction in VCt at 2 h post-laser compared to controls (195 ± 15 vs 238 ± 15, p ≤ 0.05). Histology showed necrosis and apoptosis (TUNEL assay) at the site of laser injury. At 24 h post-laser cardiac performance and VCt had recovered fully to control levels. Pretreatment with the cell-cycle inhibitor, aphidicolin, significantly inhibited functional recovery of the ventricle accompanied by a significant inhibition of cardiomyocyte proliferation. Conclusions Laser-targeted injury of the zebrafish embryonic heart is a novel and reproducible model of cardiac injury and repair suitable for pharmacological and molecular studies.

Cardiomyocyte proliferation in zebrafish and mammals: lessons for human disease

Cardiomyocytes proliferate profusely during early development and for a brief period after birth in mammals. Within a month after birth, this proliferative capability is dramatically reduced in mammals unlike lower vertebrates where it persists into adult life. The zebrafish, for example, retains the ability to regenerate the apex of the heart following resection by a mechanism predominantly driven by cardiomyocyte proliferation. Differences in proliferative capacity of cardiomyocytes in adulthood between mammals and lower vertebrates are closely liked to ontogenetic or phylogenetic factors. Elucidation of these factors has the potential to provide enormous benefits if they lead to the development of therapeutic strategies that facilitate cardiomyocyte proliferation. In this review, we highlight the differences between Mammalian and Zebrafish cardiomyocytes, which could explain at least in part the different proliferative capacities in these two species. We discuss the advantages of the zebrafish as a model of cardiomyocyte proliferation, particularly at the embryonic stage. We also identify a number of key molecular pathways with potential to reveal key steps in switching cardiomyocytes from a quiescent to a proliferative phenotype.

Genetic and pharmacological inhibition of CDK9 drives neutrophil apoptosis to resolve inflammation in zebrafish in vivo

Neutrophilic inflammation is tightly regulated and subsequently resolves to limit tissue damage and promote repair. When the timely resolution of inflammation is dysregulated, tissue damage and disease results. One key control mechanism is neutrophil apoptosis, followed by apoptotic cell clearance by phagocytes such as macrophages. Cyclin-dependent kinase (CDK) inhibitor drugs induce neutrophil apoptosis in vitro and promote resolution of inflammation in rodent models. Here we present the first in vivo evidence, using pharmacological and genetic approaches, that CDK9 is involved in the resolution of neutrophil-dependent inflammation. Using live cell imaging in zebrafish with labelled neutrophils and macrophages, we show that pharmacological inhibition, morpholino-mediated knockdown and CRISPR/cas9-mediated knockout of CDK9 enhances inflammation resolution by reducing neutrophil numbers via induction of apoptosis after tailfin injury. Importantly, knockdown of the negative regulator La-related protein 7 (LaRP7) increased neutrophilic inflammation. Our data show that CDK9 is a possible target for controlling resolution of inflammation.

CDK9 and its repressor LARP7 modulate cardiomyocyte proliferation and response to injury in the zebrafish heart

ABSTRACT Cyclin dependent kinase (Cdk)9 acts through the positive transcription elongation factor-b (P-TEFb) complex to activate and expand transcription through RNA polymerase II. It has also been shown to regulate cardiomyocyte hypertrophy, with recent evidence linking it to cardiomyocyte proliferation. We hypothesised that modification of CDK9 activity could both impair and enhance the cardiac response to injury by modifying cardiomyocyte proliferation. Cdk9 expression and activity were inhibited in the zebrafish (Danio rerio) embryo. We show that dephosphorylation of residue Ser2 on the C-terminal domain of RNA polymerase II is associated with impaired cardiac structure and function, and cardiomyocyte proliferation and also results in impaired functional recovery following cardiac laser injury. In contrast, de-repression of Cdk9 activity, through knockdown of La-related protein (Larp7) increases phosphorylation of Ser2 in RNA polymerase II and increases cardiomyocyte proliferation. Larp7 knockdown rescued the structural and functional phenotype associated with knockdown of Cdk9. The balance of Cdk9 and Larp7 plays a key role in cardiomyocyte proliferation and response to injury. Larp7 represents a potentially novel therapeutic target to promote cardiomyocyte proliferation and recovery from injury. Summary: The balance of CDK9 and LARP7 plays a key role in cardiomyocyte proliferation and response to injury. LARP7 represents a potentially novel therapeutic target in promoting recovery from injury.