An Efficient and Reproducible Method for the Isolation and Culture of Primary Cardiomyocytes from Adult Zebrafish.

Zebrafish is a popular animal model in regeneration studies due to their ability to regenerate the heart. Primary cardiomyocytes could be an alternative tool for studying the intrinsic mechanisms of cardiovascular disease in vitro. Thus, our objective is to develop an efficient protocol to isolate primary cardiomyocytes from zebrafish hearts. Low concentration of digestive enzyme (0.5 mg/mL collagenase type II) was utilized in our protocol to obtain single-cell suspension. The ventricles were fragmented, mechanically pipetted, and constantly shaken to ensure adequate contact between the tissues and the enzyme. Preplating the cell suspension onto culture plates for 2 h helped remove cardiac fibroblasts. The purity of isolated cells was validated by flow cytometry analysis of transgenic zebrafish with cardiomyocyte-specific expression of enhanced green fluorescent protein (EGFP) or endothelial cell-specific expression of mCherry. Quantitative real-time PCR analysis revealed a high level of the purity, with cardiac fibroblasts, endothelial cells, and epicardial cell markers scarcely detected in the purified cells. Altogether, this study established a reproducible protocol for isolating primary cardiomyocytes with high purity and activity from adult zebrafish hearts that can be cultured in vitro for up to 4 weeks. This protocol provides a valuable tool for studying the intrinsic mechanisms of cardiovascular disease in vitro using primary cardiomyocytes.

Isolation and analyses of lamina propria lymphocytes from mouse intestines.

Investigating intestinal immune responses is critical to understanding local and systemic immunity. However, obtaining resident intestinal immune cells with high cell viability can be challenging. Here, we provide an optimized protocol to isolate lamina propria lymphocytes from the small and large intestines, including lymphocyte activation for cytokine expression analysis and techniques for surface and intracellular antibody staining and flow cytometry. This protocol can be used for isolating and analyzing tissue-resident immune cells from other tissues with specified modifications. For complete details on the use and execution of this protocol, please refer to Kim et al. (2022).

Splenic Marginal Zone B Lymphocytes Regulate Cardiac Remodeling After Acute Myocardial Infarction in Mice.

BACKGROUND: Mature B lymphocytes alter the recovery of cardiac function after acute myocardial infarction (MI) in mice. Follicular B cells and marginal zone B (MZB) cells are spatially distinct mature B-cell populations in the spleen, and they exert specific functional properties. microRNA-21 (miR21)/hypoxia-inducible factor-α (HIF-α)-related pathways have been shown to govern B-cell functions. OBJECTIVES: The goal of this study was to unravel the distinct role of MZB cells and that of endogenous activation of miR21/HIF-α signaling in MZB cells during post-ischemic injury. METHODS: Acute MI was induced in mice by permanent ligation of the left anterior descending coronary artery. Cardiac function and remodeling were assessed by using echocardiography and immunohistochemistry. To determine the specific role of MZB cells, the study used mice with B-cell lineage-specific conditional deletion of Notch signaling, which leads to selection deficiency of MZB cells. To evaluate the role of the HIF-1α isoform, mice were generated with MZB-cell lineage-specific conditional deletion of Hif1a. RESULTS: Acute MI prompted an miR21-dependent increase in HIF-1α, particularly in splenic MZB cells. MZB cell deficiency and MZB cell-specific deletion of miR21 or Hif1a improved cardiac function after acute MI. miR21/HIF-1α signaling in MZB cells was required for Toll-like receptor dependent expression of the monocyte chemoattractant protein CCL7, leading to increased mobilization of inflammatory monocytes to the ischemic myocardium and to adverse post-ischemic cardiac remodeling. CONCLUSIONS: This work reveals a novel function for the miR21/HIF-1α pathway in splenic MZB cells with potential major implications for the modulation of cardiac function after acute MI.

Innate Lymphoid Cells Promote Recovery of Ventricular Function After Myocardial Infarction.

BACKGROUND: Innate lymphoid cells type 2 (ILC2s) play critical homeostatic functions in peripheral tissues. ILC2s reside in perivascular niches and limit atherosclerosis development. OBJECTIVES: ILC2s also reside in the pericardium but their role in postischemic injury is unknown. METHODS: We examined the role of ILC2 in a mouse model of myocardial infarction (MI), and compared mice with or without genetic deletion of ILC2. We determined infarct size using histology and heart function using echocardiography. We assessed cardiac ILC2 using flow cytometry and RNA sequencing. Based on these data, we devised a therapeutic strategy to activate ILC2 in mice with acute MI, using exogenous interleukin (IL)-2. We also assessed the ability of low-dose IL-2 to activate ILC2 in a double-blind randomized clinical trial of patients with acute coronary syndromes (ACS). RESULTS: We found that ILC2 levels were increased in pericardial adipose tissue after experimental MI, and genetic ablation of ILC2 impeded the recovery of heart function. RNA sequencing revealed distinct transcript signatures in ILC2, and pointed to IL-2 axis as a major upstream regulator. Treatment of T-cell-deficient mice with IL-2 (to activate ILC2) significantly improved the recovery of heart function post-MI. Administration of low-dose IL-2 to patients with ACS led to activation of circulating ILC2, with significant increase in circulating IL-5, a prototypic ILC2-derived cytokine. CONCLUSIONS: ILC2s promote cardiac healing and improve the recovery of heart function after MI in mice. Activation of ILC2 using low-dose IL-2 could be a novel therapeutic strategy to promote a reparative response after MI.

Endothelial Cell Indoleamine 2, 3-Dioxygenase 1 Alters Cardiac Function After Myocardial Infarction Through Kynurenine.

BACKGROUND: Ischemic cardiovascular diseases, particularly acute myocardial infarction (MI), is one of the leading causes of mortality worldwide. Indoleamine 2, 3-dioxygenase 1 (IDO) catalyzes 1 rate-limiting step of L-tryptophan metabolism, and emerges as an important regulator of many pathological conditions. We hypothesized that IDO could play a key role to locally regulate cardiac homeostasis after MI. METHODS: Cardiac repair was analyzed in mice harboring specific endothelial or smooth muscle cells or cardiomyocyte or myeloid cell deficiency of IDO and challenged with acute myocardial infarction. RESULTS: We show that kynurenine generation through IDO is markedly induced after MI in mice. Total genetic deletion or pharmacological inhibition of IDO limits cardiac injury and cardiac dysfunction after MI. Distinct loss of function of IDO in smooth muscle cells, inflammatory cells, or cardiomyocytes does not affect cardiac function and remodeling in infarcted mice. In sharp contrast, mice harboring endothelial cell-specific deletion of IDO show an improvement of cardiac function as well as cardiomyocyte contractility and reduction in adverse ventricular remodeling. In vivo kynurenine supplementation in IDO-deficient mice abrogates the protective effects of IDO deletion. Kynurenine precipitates cardiomyocyte apoptosis through reactive oxygen species production in an aryl hydrocarbon receptor-dependent mechanism. CONCLUSIONS: These data suggest that IDO could constitute a new therapeutic target during acute MI.

Evaluation of cardiac dysfunction in adult zebrafish using high frequency echocardiography.

AIMS: 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. MATERIALS AND METHODS: 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. KEY FINDINGS: 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). SIGNIFICANCE: 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.

Histone Deacetylase Inhibitors in Cancer Therapy.

Histone deacetylases (HDACs), as epigenetic modifiers, are essential for gene transcriptional activities. The alternation of HDACs expression, mutation and/or inappropriate recruitments has been discovered in a broad range of tumors contributing to the tumorigenesis through a serial of biological pathways. HDACs, therefore, are characterized as promising cancer therapeutic targets, and their inhibitors are under rapid development. Here, we discuss HDAC inhibitors in terms of their functional mechanism establishing the anti-tumor effects and potential clinical applications including the synergistic effects in combinational treatment.

Activation of the Nkx2.5-Calr-p53 signaling pathway by hyperglycemia induces cardiac remodeling and dysfunction in adult zebrafish.

Hyperglycemia is an independent risk factor for diabetic cardiomyopathy in humans; however, the underlying mechanisms have not been thoroughly elucidated. Zebrafish (Danio rerio) was used in this study as a novel vertebrate model to explore the signaling pathways of human adult cardiomyopathy. Hyperglycemia was induced by alternately immersing adult zebrafish in a glucose solution or water. The hyperglycemic fish gradually exhibited some hallmarks of cardiomyopathy such as myocardial hypertrophy and apoptosis, myofibril loss, fetal gene reactivation, and severe arrhythmia. Echocardiography of the glucose-treated fish demonstrated diastolic dysfunction at an early stage and systolic dysfunction at a later stage, consistent with what is observed in diabetic patients. Enlarged hearts with decreased myocardial density, accompanied by decompensated cardiac function, indicated that apoptosis was critical in the pathological process. Significant upregulation of the expression of Nkx2.5 and its downstream targets calreticulin (Calr) and p53 was noted in the glucose-treated fish. High-glucose stimulation in vitro evoked marked apoptosis of primary cardiomyocytes, which was rescued by the p53 inhibitor pifithrin-µ. In vitro experiments were performed using compound treatment and genetically via cell infection. Genetically, knockout of Nkx2.5 induced decreased expression of Nkx2.5, Calr and p53 Upregulation of Calr resulted in increased p53 expression, whereas the level of Nkx2.5 remained unchanged. An adult zebrafish model of hyperglycemia-induced cardiomyopathy was successfully established. Hyperglycemia-induced myocardial apoptosis was mediated, at least in part, by activation of the Nkx2.5-Calr-p53 pathway in vivo, resulting in cardiac dysfunction and hyperglycemia-induced cardiomyopathy.

Evidence of an Association between Age-Related Functional Modifications and Pathophysiological Changes in Zebrafish Heart.

BACKGROUND: Zebrafish have become a valuable model for the study of developmental biology and human disease, such as cardiovascular disease. It is difficult to discriminate between disease-related and age-related alterations. OBJECTIVE: This study was aimed to investigate the effects and potential mechanisms of age-related cardiac modifications in an older zebrafish population. METHODS: In this study, we calculated the survival rate and measured the spinal curvature through the aging process. A swimming challenge test was performed and showed that swimming capacity and endurance dramatically dropped in older fish groups. RESULTS: To find out the effect of stress on zebrafish during the aging process, we recorded electrocardiograms on zebrafish and showed that during stress, aging not only led to a significant reduction in heart rate, but also caused other age-related impairments, such as arrhythmias and ST-T depression. Echocardiography showed a marked increase in end-diastolic ventricular dimensions and in isovolumic relaxation time and a notably slower mean and peak velocity of the bulboventricular valve in older zebrafish, but stroke volume and cardiac output were not different in young and old zebrafish. Both nppa and nppb (cardiac fetal genes for natriuretic factor) expression detected by real-time polymerase chain reaction analysis increased in older fish compared to the younger group. Histological staining revealed fibrosis within cardiomyocytes and an increase in ventricular myocardial density and a decrease in epicardial vessel dimensions in older fish hearts that may correlate with a deterioration of cardiac function and exercise capacity. CONCLUSION: These data suggest that cardiac functional modifications in zebrafish are comparable to those in humans and may partly be due to changes in the cardiovascular system including cardiac fetal gene reprogramming, myocardial density, and epicardial vessel dimensions.

Unusual superconducting state at 49 K in electron-doped CaFe2As2 at ambient pressure.

We report the detection of unusual superconductivity up to 49 K in single crystalline CaFe(2)As(2) via electron-doping by partial replacement of Ca by rare-earth. The superconducting transition observed suggests the possible existence of two phases: one starting at 49 K, which has a low critical field < 4 Oe, and the other at 21 K, with a much higher critical field > 5 T. Our observations are in strong contrast to previous reports of doping or pressurizing layered compounds AeFe(2)As(2) (or Ae122), where Ae = Ca, Sr, or Ba. In Ae122, hole-doping has been previously observed to generate superconductivity with a transition temperature (T(c)) only up to 38 K and pressurization has been reported to produce superconductivity with a T(c) up to 30 K. The unusual 49 K phase detected will be discussed.

[Voltammetric behaviors of diethylstilbestrol and its determination at multi-wall carbon nanotubes modified glassy carbon electrode].

AIM: To fabricate multi-wall carbon nanotube (MWNT) modified electrode and study the electrochemical behaviors of diethylstilbestrol at the MWNT-modified electrode. METHODS: Cyclic voltammetry and linear sweep voltammetry. RESULTS: The oxidation peak current of diethylstilbestrol increased remarkably and the peak potential shifted negatively at the MWNT-dihexadecyl hydrogen phosphate (DHP) modified glassy carbon electrode (GCE), in contrast to that at the bare GC electrode and DHP-modified GC electrode. The oxidation peak current is linear with the concentration of diethylstilbestrol over the range from 1 x 10(-8) to 2 x 10(-6) mol.L-1. The detection limit was 2.5 x 10(-9) mol.L-1. The relative standard deviation (n = 10) was 2.9% for 1 x 10(-6) mol.L-1 diethylstilbestrol. CONCLUSION: The MWNT-DHP modified GCE exhibits catalytic activity to the oxidation of diethylstilbestrol.

Simultaneous electrochemical determination of xanthine and uric acid at a nanoparticle film electrode.

A sensitive electrochemical method was developed for simultaneous determination of uric acid (UA) and xanthine (XA) at a glassy carbon electrode modified with multi-wall carbon nanotubes (MWNTs) film. The oxidation peak currents of UA and XA were increased at the MWNTs film electrode significantly. The experimental parameters, which influence the peak currents of UA and XA, such as the amount of MWNTs on the glassy carbon electrode, the pH of the solution, accumulation time, and scan rate, were optimized. Under optimum conditions, the peak currents were linear to the concentration of UA over the wide range from 1 x 10(-7) mol L(-1) to 1 x 10(-4) mol L(-1) and to that of XA over the wide range from 2 x 10(-8) mol L(-1) to 2 x 10(-5) mol L(-1). The interferences studies showed that the MWNTs-modified electrode exhibited excellent selectivity in the presence of ascorbic acid, dopamine, and hypoxanthine. The proposed procedure was successfully applied to detect UA and XA in human serum without any preliminary treatment.