Zebrafish as a tractable model of human cardiovascular disease

Freire C., Fish R.J., Vilar R., Di Sanza C., Grzegorski S.J., Richter C.E., Shavit J.A., Neerman-Arbez M.

Abstract Plasma fibrinogen molecules comprise 2 copies of Aα, Bβ, and γ chains folded into a hexameric protein. A minor fibrinogen isoform with an extended Aα chain (AαE) is more abundant in newborn human blood than in adults. Larval zebrafish produce predominantly AαE-containing fibrinogen, but its functional significance is unclear. In 3-day-old zebrafish, when hemostasis is reliant on fibrinogen and erythrocyte-rich clotting but is largely thrombocyte-independent, we measured the time to occlusion (TTO) in a laser-induced venous thrombosis assay in 3 zebrafish strains (AB, TU, and AB × TL hybrids). AB larvae showed delayed TTO compared with the TU and AB × TL strains. Mating AB with TU or TL produced larvae with a TU-like TTO. In contrast to TU, AB larvae failed to produce fibrinogen AαE, due to a mutation in the AαE-specific coding region of fibrinogen α-chain gene (fga). We investigated whether the lack of AαE explained the delayed AB TTO. Transgenic expression of AαE, but not Aα, shortened the AB TTO to that of TU. AαE rescued venous occlusion in fibrinogen mutants or larvae with morpholino-targeted fibrinogen α-chain messenger RNA, but Aα was less effective. In 5-day-old larvae, circulating thrombocytes contribute to hemostasis, as visualized in Tg(itga2b:EGFP) transgenics. Laser-induced venous thrombocyte adhesion and aggregation is reduced in fibrinogen mutants, but transgenic expression of Aα or AαE restored similar thrombocyte accumulation at the injury site. Our data demonstrate a genetic modifier of venous thrombosis and a role for fibrinogen AαE in early developmental blood coagulation, and suggest a link between differentially expressed fibrinogen isoforms and the cell types available for clotting.

Michel J.

The evolution of the circulatory system from invertebrates to mammals has involved the passage from an open system to a closed in-parallel system via a closed in-series system, accompanying the increasing complexity and efficiency of life’s biological functions. The archaic heart enables pulsatile motion waves of hemolymph in invertebrates, and the in-series circulation in fish occurs with only an endothelium, whereas mural smooth muscle cells appear later. The present review focuses on evolution of the circulatory system. In particular, we address how and why this evolution took place from a closed, flowing, longitudinal conductance at low pressure to a flowing, highly pressurized and bifurcating arterial compartment. However, although arterial pressure was the latest acquired hemodynamic variable, the general teleonomy of the evolution of species is the differentiation of individual organ function, supported by specific fueling allowing and favoring partial metabolic autonomy. This was achieved via the establishment of an active contractile tone in resistance arteries, which permitted the regulation of blood supply to specific organ activities via its localized function-dependent inhibition (active vasodilation). The global resistance to viscous blood flow is the peripheral increase in frictional forces caused by the tonic change in arterial and arteriolar radius, which backscatter as systemic arterial blood pressure. Consequently, the arterial pressure gradient from circulating blood to the adventitial interstitium generates the unidirectional outward radial advective conductance of plasma solutes across the wall of conductance arteries. This hemodynamic evolution was accompanied by important changes in arterial wall structure, supported by smooth muscle cell functional plasticity, including contractility, matrix synthesis and proliferation, endocytosis and phagocytosis, etc. These adaptive phenotypic shifts are due to epigenetic regulation, mainly related to mechanotransduction. These paradigms actively participate in cardio-arterial pathologies such as atheroma, valve disease, heart failure, aneurysms, hypertension, and physiological aging.

Salgado-Almario J., Vicente M., Vincent P., Domingo B., Llopis J.

Zebrafish embryos have been proposed as a cost-effective vertebrate model to study heart function. Many fluorescent genetically encoded Ca2+ indicators (GECIs) have been developed, but those with ratiometric readout seem more appropriate to image a moving organ such as the heart. Four ratiometric GECIs based on troponin C, TN-XXL, Twitch-1, Twitch-2B, and Twitch-4 were expressed transiently in the heart of zebrafish embryos. Their emission ratio reported the Ca2+ levels in both the atrium and the ventricle. We measured several kinetic parameters of the Ca2+ transients: systolic and diastolic ratio, the amplitude of the systolic Ca2+ rise, the heart rate, as well as the rise and decay times and slopes. The systolic ratio change decreased in cells expressing high biosensor concentration, possibly caused by Ca2+ buffering. The GECIs were able to report the effect of nifedipine and propranolol on the heart, which resulted in changes in heart rate, diastolic and systolic Ca2+ levels, and Ca2+ kinetics. As a result, Twitch-1 and Twitch-4 (Kd 0.25 and 2.8 µM, respectively) seem the most promising GECIs for generating transgenic zebrafish lines, which could be used for modeling heart disorders, for drug screening, and for cardiotoxicity assessment during drug development.

von der Heyde B., Emmanouilidou A., Mazzaferro E., Vicenzi S., Höijer I., Klingström T., Jumaa S., Dethlefsen O., Snieder H., de Geus E., Ameur A., Ingelsson E., Allalou A., Brooke H.L., den Hoed M.

A meta-analysis of genome-wide association studies (GWAS) identified eight loci that are associated with heart rate variability (HRV), but candidate genes in these loci remain uncharacterized. We developed an image- and CRISPR/Cas9-based pipeline to systematically characterize candidate genes for HRV in live zebrafish embryos. Nine zebrafish orthologues of six human candidate genes were targeted simultaneously in eggs from fish that transgenically express GFP on smooth muscle cells (Tg[acta2:GFP]), to visualize the beating heart. An automated analysis of repeated 30 s recordings of beating atria in 381 live, intact zebrafish embryos at 2 and 5 days post-fertilization highlighted genes that influence HRV (hcn4 and si:dkey-65j6.2 [KIAA1755]); heart rate (rgs6 and hcn4); and the risk of sinoatrial pauses and arrests (hcn4). Exposure to 10 or 25 µM ivabradine—an open channel blocker of HCNs—for 24 h resulted in a dose-dependent higher HRV and lower heart rate at 5 days post-fertilization. Hence, our screen confirmed the role of established genes for heart rate and rhythm (RGS6 and HCN4); showed that ivabradine reduces heart rate and increases HRV in zebrafish embryos, as it does in humans; and highlighted a novel gene that plays a role in HRV (KIAA1755).

Bise T., Sallin P., Pfefferli C., Jaźwińska A.

Zebrafish can regenerate their damaged hearts throughout their lifespan. It is, however, unknown, whether regeneration remains effective when challenged with successive cycles of cardiac damage in the same animals. Here, we assessed ventricular restoration after two, three and six cryoinjuries interspaced by recovery periods. Using transgenic cell-lineage tracing analysis, we demonstrated that the second cryoinjury damages the regenerated area from the preceding injury, validating the experimental approach. We identified that after multiple cryoinjuries, all hearts regrow a thickened myocardium, similarly to hearts after one cryoinjury. However, the efficiency of scar resorption decreased with the number of repeated cryoinjuries. After six cryoinjuries, all examined hearts failed to completely resolve the fibrotic tissue, demonstrating reduced myocardial restoration. This phenotype was associated with enhanced recruitment of neutrophils and decreased cardiomyocyte proliferation and dedifferentiation at the early regenerative phase. Furthermore, we found that each repeated cryoinjury increased the accumulation of collagen at the injury site. Our analysis demonstrates that the cardiac regenerative program can be successfully activated many times, despite a persisting scar in the wounded area. This finding provides a new perspective for regenerative therapies, aiming in stimulation of organ regeneration in the presence of fibrotic tissue in mammalian models and humans.

Withers S.E., Parry-Jones A.R., Allan S.M., Kasher P.R.

Apart from acute and chronic blood pressure lowering, we have no specific medications to prevent intracerebral haemorrhage (ICH) or improve outcomes once bleeding has occurred. One reason for this may be related to particular limitations associated with the current pre-clinical models of ICH, leading to a failure to translate into the clinic. It would seem that a breakdown in the ‘drug development pipeline’ currently exists for translational ICH research which needs to be urgently addressed. Here, we review the most commonly used pre-clinical models of ICH and discuss their advantages and disadvantages in the context of translational studies. We propose that to increase our chances of successfully identifying new therapeutics for ICH, a bi-directional, 2- or 3-pronged approach using more than one model species/system could be useful for confirming key pre-clinical observations. Furthermore, we highlight that post-mortem/ex-vivo ICH patient material is a precious and underused resource which could play an essential role in the verification of experimental results prior to consideration for further clinical investigation. Embracing multidisciplinary collaboration between pre-clinical and clinical ICH research groups will be essential to ensure the success of this type of approach in the future.

Fang Y., Sun Y., Luo C., Gu J., Shi Z., Lu G., Silvestre J., Chen Z.

Recently, the zebrafish has gained attention as an innovative experimental model to decipher molecular and cellular mechanisms involved in cardiovascular development and diseases. Nevertheless, the use of zebrafish models has been challenged because the transparency of these fish, which allows for accurate cardiac evaluation, disappears in adulthood. In this study, the epicardial outline method was performed to investigate the feasibility of echocardiography in assessing cardiac function in pathological adult zebrafish.We attempted to estimate heart failure in adult zebrafish treated with three distinct regulators of cardiac function: phenylhydrazine hydrochloride (PHZ), doxorubicin (DOX), and ethanol. B-mode and Doppler images were evaluated at frequencies of up to 50 MHz and 40 MHz, respectively. The correlation between alterations in cardiac function, haemoglobin concentration, and myocardial histopathology were assessed.Cardiac output (CO) in PHZ-treated zebrafish was significantly higher than that in control zebrafish (151 ± 67 vs. 84 ± 37 μl/min, P = 0.004), whereas ejection fraction (EF) was lower (36.3 ± 10.9 vs. 50.9 ± 8.7%, P < 0.001), indicating typical high output heart failure derived from anaemia. Additionally, ventricular dysfunction in DOX-treated zebrafish was characterised by low CO (57 ± 38 μl/min) and EF (28.8 ± 10.4%), accompanied by an enlarged ventricle in diastole and systole, representing low output heart failure. For ethanol-treated zebrafish, EF was markedly reduced (39.6 ± 7.2%) indicating a dilated heart, while CO remained unchanged (90 ± 40 μl/min).The epicardial outline method is an effective way of using echocardiography to assess cardiac dysfunction in pathological adult zebrafish, unlocking a major bottleneck in this research field with limited cardiac functional assays.

Baehr A., Umansky K.B., Bassat E., Jurisch V., Klett K., Bozoglu T., Hornaschewitz N., Solyanik O., Kain D., Ferraro B., Cohen-Rabi R., Krane M., Cyran C., Soehnlein O., Laugwitz K.L., et. al.

Background: Ischemic heart diseases are leading causes of death and reduced life quality worldwide. Although revascularization strategies significantly reduce mortality after acute myocardial infarction (MI), a large number of patients with MI develop chronic heart failure over time. We previously reported that a fragment of the extracellular matrix protein agrin promotes cardiac regeneration after MI in adult mice. Methods: To test the therapeutic potential of agrin in a preclinical porcine model, we performed ischemia–reperfusion injuries using balloon occlusion for 60 minutes followed by a 3-, 7-, or 28-day reperfusion period. Results: We demonstrated that local (antegrade) delivery of recombinant human agrin to the infarcted pig heart can target the affected regions in an efficient and clinically relevant manner. A single dose of recombinant human agrin improved heart function, infarct size, fibrosis, and adverse remodeling parameters 28 days after MI. Short-term MI experiments along with complementary murine studies revealed myocardial protection, improved angiogenesis, inflammatory suppression, and cell cycle reentry as agrin’s mechanisms of action. Conclusions: A single dose of agrin is capable of reducing ischemia–reperfusion injury and improving heart function, demonstrating that agrin could serve as a therapy for patients with acute MI and potentially heart failure.

Ren F., Ji C., Huang Y., Aniagu S., Jiang Y., Chen T.

Recent studies have shown an association between maternal exposure to ambient fine particle matter (PM2.5) and congenital heart defects in the offspring, but the underlying molecular mechanisms are yet to be elucidated. Previously, we demonstrated that extractable organic matter (EOM) from PM2.5 induced heart defects in zebrafish embryos by activating the aromatic hydrocarbon receptor (AHR). Hence, we hypothesized that AHR mediates excessive reactive oxygen species (ROS) production, leading to the cardiac developmental toxicity of PM2.5. To test our hypothesis, we examined AHR activity and ROS levels in the heart of zebrafish embryos under a fluorescence microscope. mRNA expression levels were then quantified using qPCR whereas DNA damage and apoptosis were detected by immunofluorescence. Our results showed that the AHR inhibitor, CH223191 (CH) as well as the ROS scavenger, N-Acetyl-L-cysteine (NAC), significantly mitigated the PM2.5-induced cardiac malformations in zebrafish embryos. Furthermore, both CH and NAC diminished the EOM-elevated ROS generation, DNA damage and apoptosis in the test system. Incidentally, both CH and NAC attenuated the EOM-induced changes in the mRNA expression of genes involved in cardiac development (nkx2.5, sox9b), oxidative stress (nrf2a, nrf2b, gstp1, gstp2, sod2, ho1, cat) and apoptosis (p53, bax). We further confirmed that AHR activity is a necessary condition for EOM-induced ROS generation, DNA damage and apoptosis, through AHR knockdown. However, the ROS scavenger NAC did not counteract the EOM-induced AHR activity. In conclusion, our findings suggest that AHR mediates EOM-induced oxidative stress, resulting in DNA damage and apoptosis, thereby contributing to the cardiac developmental toxicity of PM2.5.

Bahrami N., Childs S.J.

ABSTRACT The thin endothelial wall of a newly formed vessel is under enormous stress at the onset of blood flow, rapidly acquiring support from mural cells (pericytes and vascular smooth muscle cells; vSMCs) during development. Mural cells then develop vasoactivity (contraction and relaxation) but we have little information as to when this first develops or the extent to which pericytes and vSMCs contribute. For the first time, we determine the dynamic developmental acquisition of vasoactivity in vivo in the cerebral vasculature of zebrafish. We show that pericyte-covered vessels constrict in response to α1-adrenergic receptor agonists and dilate in response to nitric oxide donors at 4 days postfertilization (dpf) but have heterogeneous responses later, at 6 dpf. In contrast, vSMC-covered vessels constrict at 6 dpf, and dilate at both stages. Using genetic ablation, we demonstrate that vascular constriction and dilation is an active response. Our data suggest that both pericyte- and vSMC-covered vessels regulate their diameter in early development, and that their relative contributions change over developmental time.

Yan J., Li H., Bu H., Jiao K., Zhang A.X., Le T., Cao H., Li Y., Ding Y., Xu X.

Because of its powerful genetics, the adult zebrafish has been increasingly used for studying cardiovascular diseases. Considering its heart rate of ~100 beats per minute at ambient temperature, which is very close to human, we assessed the use of this vertebrate animal for modeling heart rhythm disorders such as sinus arrest (SA) and sick sinus syndrome (SSS). We firstly optimized a protocol to measure electrocardiogram in adult zebrafish. We determined the location of the probes, implemented an open-chest microsurgery procedure, measured the effects of temperature, and determined appropriate anesthesia dose and time. We then proposed an PP interval of more than 1.5 seconds as an arbitrary criterion to define an SA episode in an adult fish at ambient temperature, based on comparison between the current definition of an SA episode in humans and our studies of candidate SA episodes in aged wild-type fish and Tg(SCN5A-D1275N) fish (a fish model for inherited SSS). With this criterion, a subpopulation of about 5% wild-type fish can be considered to have SA episodes, and this percentage significantly increases to about 25% in 3-year-old fish. In response to atropine, this subpopulation has both common SSS phenotypic traits that are shared with the Tg(SCN5A-D1275N) model, such as bradycardia; and unique SSS phenotypic traits, such as increased QRS/P ratio and chronotropic incompetence. In summary, this study defined baseline SA and SSS in adult zebrafish and underscored use of the zebrafish as an alternative model to study aging-associated SSS.

Lam P., Kutchukian P., Anand R., Imbriglio J., Andrews C., Padilla H., Vohra A., Lane S., Parker D.L., Cornella Taracido I., Johns D.G., Beerens M., MacRae C.A., Caldwell J.P., Sorota S., et. al.

Doxorubicin is a highly effective chemotherapy agent used to treat many common malignancies. However, its use is limited by cardiotoxicity, and cumulative doses exponentially increase the risk of heart failure. To identify novel heart failure treatment targets, we previously established a zebrafish model of doxorubicin-induced cardiomyopathy for small molecule screening. Using this model, we previously identified several small molecules that prevent doxorubicin-induced cardiotoxicity both in zebrafish as well as in mouse models. In this study, we have expanded our exploration of doxorubicin cardiotoxicity by screening 2,271 small molecules from a proprietary, target-annotated tool compound collection. We found 120 small molecules that can prevent doxorubicin-induced cardiotoxicity, including seven highly-effective compounds. Of these, all seven exhibited inhibitory activity towards Cytochrome P450 family 1 (CYP1). These results are consistent with our previous findings in which visnagin, a CYP1 inhibitor, also prevented doxorubicin-induced cardiotoxicity. Importantly, genetic mutation of cyp1a protected zebrafish against doxorubicin-induced cardiotoxicity phenotypes. Together, these results provide strong evidence that CYP1 is an important contributor to doxorubicin-induced cardiotoxicity and highlight the CYP1 pathway as a candidate therapeutic target for clinical cardioprotection.

Gierten J., Pylatiuk C., Hammouda O.T., Schock C., Stegmaier J., Wittbrodt J., Gehrig J., Loosli F.

Accurate quantification of heartbeats in fish models is an important readout to study cardiovascular biology, disease states and pharmacology. However, dependence on anaesthesia, laborious sample orientation or requirement for fluorescent reporters have hampered the use of high-throughput heartbeat analysis. To overcome these limitations, we established an efficient screening assay employing automated label-free heart rate determination of randomly oriented, non-anesthetized medaka (Oryzias latipes) and zebrafish (Danio rerio) embryos in microtiter plates. Automatically acquired bright-field data feeds into an easy-to-use HeartBeat software with graphical user interface for automated quantification of heart rate and rhythm. Sensitivity of the assay was demonstrated by profiling heart rates during entire embryonic development. Our analysis revealed rapid adaption of heart rates to temperature changes, which has implications for standardization of experimental layout. The assay allows scoring of multiple embryos per well enabling a throughput of >500 embryos per 96-well plate. In a proof of principle screen for compound testing, we captured concentration-dependent effects of nifedipine and terfenadine over time. Our novel assay permits large-scale applications ranging from phenotypic screening, interrogation of gene functions to cardiovascular drug development.

Simões F.C., Cahill T.J., Kenyon A., Gavriouchkina D., Vieira J.M., Sun X., Pezzolla D., Ravaud C., Masmanian E., Weinberger M., Mayes S., Lemieux M.E., Barnette D.N., Gunadasa-Rohling M., Williams R.M., et. al.

Canonical roles for macrophages in mediating the fibrotic response after a heart attack include extracellular matrix turnover and activation of cardiac fibroblasts to initiate collagen deposition. Here we reveal that macrophages directly contribute collagen to the forming post-injury scar. Unbiased transcriptomics shows an upregulation of collagens in both zebrafish and mouse macrophages following heart injury. Adoptive transfer of macrophages, from either collagen-tagged zebrafish or adult mouse GFPtpz-collagen donors, enhances scar formation via cell autonomous production of collagen. In zebrafish, the majority of tagged collagen localises proximal to the injury, within the overlying epicardial region, suggesting a possible distinction between macrophage-deposited collagen and that predominantly laid-down by myofibroblasts. Macrophage-specific targeting of col4a3bpa and cognate col4a1 in zebrafish significantly reduces scarring in cryoinjured hosts. Our findings contrast with the current model of scarring, whereby collagen deposition is exclusively attributed to myofibroblasts, and implicate macrophages as direct contributors to fibrosis during heart repair. Macrophages mediate the fibrotic response after a heart attack by extracellular matrix turnover and cardiac fibroblasts activation. Here the authors identify an evolutionarily-conserved function of macrophages that contributes directly to the forming post-injury scar through cell-autonomous deposition of collagen.

Kioka H., Kato H., Fujita T., Asano Y., Shintani Y., Yamazaki S., Tsukamoto O., Imamura H., Kogo M., Kitakaze M., Sakata Y., Takashima S.

Most eukaryotic cells generate adenosine triphosphate (ATP) through the oxidative phosphorylation system (OXPHOS) to support cellular activities. In cultured cell-based experiments, we recently identified the hypoxia-inducible protein G0/G1 switch gene 2 (G0s2) as a positive regulator of OXPHOS, and showed that G0s2 protects cultured cardiomyocytes from hypoxia. In this study, we examined the in vivo protective role of G0s2 against hypoxia by generating both loss-of-function and gain-of-function models of g0s2 in zebrafish. Zebrafish harboring transcription activator-like effector nuclease (TALEN)-mediated knockout of g0s2 lost hypoxic tolerance. Conversely, cardiomyocyte-specific transgenic zebrafish hearts exhibited strong tolerance against hypoxia. To clarify the mechanism by which G0s2 protects cardiac function under hypoxia, we introduced a mitochondrially targeted FRET-based ATP biosensor into zebrafish heart to visualize ATP dynamics in in vivo beating hearts. In addition, we employed a mosaic overexpression model of g0s2 to compare the contraction and ATP dynamics between g0s2-expressing and non-expressing cardiomyocytes, side-by-side within the same heart. These techniques revealed that g0s2-expressing cardiomyocyte populations exhibited preserved contractility coupled with maintained intra-mitochondrial ATP concentrations even under hypoxic condition. Collectively, these results demonstrate that G0s2 provides ischemic tolerance in vivo by maintaining ATP production, and therefore represents a promising therapeutic target for hypoxia-related diseases.

Wang H., Huang Y., Zhou C., Gong F., Wang J., Liu H., Zhao W., Zhao Y., Gao X.

Seomoon K., Park J., Song G., Lim W.

Tian Z., Chi B., Liu W., Gao X., Wang D., Gu Z., Tian L.

Identification, targeted isolation, and evaluation of the in vivo and in vitro protective effects of bioactive compounds from Acanthopanax senticosus against vascular endothelial injury.

Morshead M.L., Tanguay R.L.

Lachowicz-Radulska J., Widelski J., Nowaczyński F., Serefko A., Sobczyński J., Ludwiczuk A., Kasica N., Szopa A.

The aim of this review is to summarize the current knowledge on the use of coumarin-derived compounds in the zebrafish (Danio rerio) model. Coumarins, a class of naturally occurring compounds with diverse biological activities, including compounds such as coumarin, angelicin, and warfarin, have attracted considerable attention in the study of potential therapeutic agents for cancer, central nervous system disorders, and infectious diseases. The capabilities of coumarins as active compounds have led to synthesizing various derivatives with their own properties. While such variety is certainly promising, it is also cumbersome due to the large amount of research needed to find the most optimal compounds. The zebrafish model offers unique advantages for such studies, including high genetic and physiological homology to mammals, optical transparency of the embryos, and rapid developmental processes, facilitating the assessment of compound toxicity and underlying mechanisms of action. This review provides an in-depth analysis of the chemical properties of coumarins, their mechanisms of biological activity, and the results of previous studies evaluating the toxicity and efficacy of these compounds in zebrafish assays. The zebrafish model allows for a holistic assessment of the therapeutic potential of coumarin derivatives, offering valuable insights for advancing drug discovery and development.

Mizoguchi T., Maki A., Nakase Y., Okita M., Minami Y., Fukunaga M., Itoh M.

Feng W., Weng Y., Shi W., Liang S., Liao X., Chu R., Ai Q., Mai K., Wan M.

Liu Y., Lin Y., Lei Y., Xie W., Wei Y., Zhang H., Zhuang X., Cao H., Wang X.

The C.florida. is one of the common medicines used by She population in China, with therapeutic effects of promoting blood circulation and anti-inflammatory. According to the acute toxicity grading standard of chemical substances, this herb is a low-toxicity herb. At present, the safety of C.florida., especially its impact on early embryonic development, is still unclear.

Chang J., Zhang L., Zhao J., Zhang Z., Wang Z., Wang H., Wan B.

Bowley G., Irving S., Hoefer I., Wilkinson R., Pasterkamp G., Darwish H.M., White S., Francis S.E., Chico T., Noel E., Serbanovic-Canic J., Evans P.C.

AbstractLocal haemodynamics control arterial homeostasis and dysfunction by generating wall shear stress (WSS) which regulates endothelial cell (EC) physiology. Here we use a zebrafish model to identify genes that regulate EC proliferation in response to flow. Suppression of blood flow in zebrafish embryos (by targeting cardiac troponin) reduced EC proliferation in the intersegmental vessels (ISVs) compared to controls exposed to flow. The expression of candidate regulators of proliferation was analysed in EC isolated from zebrafish embryos by qRT-PCR. Genes shown to be expressed in EC were analysed for the ability to regulate proliferation in zebrafish vasculature exposed to flow or no-flow conditions using a knockdown approach. wnk1 negatively regulated proliferation in no-flow conditions, whereas fzd5, gsk3β, trpm7 and bmp2a promoted proliferation in EC exposed to flow. Immunofluorescent staining of mammalian arteries revealed that WNK1 is expressed at sites of low WSS in the murine aorta, and in EC overlying human atherosclerotic plaques. We conclude that WNK1 is expressed in EC at sites of low WSS and in diseased arteries and may influence vascular homeostasis by reducing EC proliferation.

Liu Z., Wang M., Ding X., Tian J., Sun D., Gao X., Jin C., Peng D., Gui S., Wang X.

Krylov V.V., Sizov D.A., Machikhin A.S., Guryleva A.V., Tchougounov V., Burlakov A.B.

Teranikar T.A., Saeed S., LE T.V., Kang Y., Hernandez G., Nguyen P., Ding Y., Chuong C., Lee J.Y., Ko H., Lee J.

ABSTRACTLight Sheet Microscopy (LSM) in conjunction with embryonic zebrafish, is rapidly advancing three-dimensional,in vivocharacterization of myocardial contractility. Preclinical cardiac deformation imaging is predominantly restricted to a low-order dimensionality image space (2i) or suffers from poor reproducibility. In this regard, LSM has enabled high throughput, non-invasive 4i (3d+time) characterization of dynamic organogenesis within the transparent zebrafish model. More importantly, LSM offers cellular resolution across large imaging Field-of-Views at millisecond camera frame rates, enabling single cell localization for global cardiac deformation analysis. However, manual labeling of cells within multilayered tissue is a time-consuming task and requires substantial expertise. In this study, we applied the 3i nnU-Net with Linear Assignment Problem (LAP) framework for automated segmentation and tracking of myocardial cells. Using binarized labels from the neural network, we quantified myocardial deformation of the zebrafish ventricle across 4-6 days post fertilization (dpf). Our study offers tremendous promise for developing highly scalable and disease-specific biomechanical quantification of myocardial microstructures.

Gu M., Xu X., Wang X., Wang Y., Zhao Y., Hu X., Zhu L., Deng Z., Han C.

Ricciotti E., Haines P., Beerens M., Kartoun U., Castro C., Tang S.Y., Ghosh S., Das U., Lahens N., Wang T., Griffin J., Shaw S., Macrae C., FitzGerald G.A.

AbstractHeart failure (HF) is one of the most strongly associated adverse cardiovascular events linked to the use of cyclooxygenase (COX)-2 selective and non-selective nonsteroidal anti-inflammatory drug (NSAID). Nevertheless, it remains uncertain whether NSAID exposure is more likely to lead to heart failure with reduced ejection fraction (HFrEF) or preserved ejection fraction (HFpEF).In adult mice, postnatal genetic deletion or pharmacological inhibition of COX-2 did not affect cardiac function. In contrast, aged female inducible COX-2 (iCOX-2) knockout (KO) mice displayed diastolic dysfunction, cardiac hypertrophy, pulmonary congestion, and elevated levels of plasma N-terminal pro B-type natriuretic peptide (BNP) when compared to age- and sex- matched controls, while their ejection fraction (EF) remained preserved (≥ 50%). No such phenotype was observed in aged male iCox-2 KO mice. Aged female iCox-2 KO mice showed a shift from prostanoid to leukotriene biosynthesis, along with changes in the expression of mitochondrial genes and calcium-handling proteins in the myocardium. The ratio of phospholamban to SERCA2a was increased, indicating an inhibitory effect on SERCA2a activity, which may contribute to impaired myocardial relaxation. In larval zebrafish, COX-2 inhibition by celecoxib caused a modest yet significant reduction in heart rate and diastolic function, while EF was preserved. Additionally, celecoxib increased BNP expression and ventricular calcium transient amplitude. Diabetic patients in the Harvard-Partners electronic medical record exposed to NSAIDs selective for COX-2 inhibition were more strongly associated with an increased risk of HFpEF compared to HFrEF.Collectively, these findings indicate that COX-2 deletion or inhibition does not impair systolic cardiac function but instead leads to an HFpEF phenotype in mice, zebrafish, and humans. An imbalance in calcium handling may mediate the impairment of myocardial relaxation following COX-2 suppression.SummaryGenetic deletion or pharmacological inhibition of COX-2 results in heart failure with preserved ejection fraction across zebrafish, mice, and humans.