an-intraperitoneal-injection-technique-adult-zebrafish-that-minimizes

An Intraperitoneal Injection Technique in Adult Zebrafish that Minimizes Body Damage and Associated Mortality

The adult zebrafish (Danio rerio), which is genetically accessible, is being employed as a valuable vertebrate model to study human disorders such as ...

Mayo Clinic

Central South University

Myosin heavy chain 7 (MYH7) is a major causative gene for hypertrophic cardiomyopathy, but the affected signaling pathways and therapeutics remain elusive. In this research, we identified ventricle myosin heavy chain like (vmhcl) as a zebrafish homolog of human MYH7, and we generated vmhcl frameshift mutants. We noted vmhcl-based embryonic cardiac dysfunction (VEC) in the vmhcl homozygous mutants and vmhcl-based adult cardiomyopathy (VAC) phenotypes in the vmhcl heterozygous mutants. Using the VEC model, we assessed 7 known cardiomyopathy signaling pathways pharmacologically and 11 candidate genes genetically via CRISPR/Cas9 genome editing technology based on microhomology-mediated end joining (MMEJ). Both studies converged on therapeutic benefits of mTOR or mitogen-activated protein kinase (MAPK) inhibition of VEC. While mTOR inhibition rescued the enlarged nuclear size of cardiomyocytes, MAPK inhibition restored the prolonged cell shape in the VEC model. The therapeutic effects of mTOR and MAPK inhibition were later validated in the VAC model. Together, vmhcl/myh7 loss of function is sufficient to induce cardiomyopathy in zebrafish. The VEC and VAC models in zebrafish are amenable to both efficient genetic and chemical genetic tools, offering a rapid in vivo platform for discovering candidate signaling pathways of MYH7 cardiomyopathy.

Inhibition of mTOR or MAPK ameliorates vmhcl/myh7 cardiomyopathy in zebrafish.

Modifier genes contribute significantly to our understanding of pathophysiology in human diseases; however, effective approaches to identify modifier genes are still lacking. Here, we aim to develop a rapid F0-based genetic assay in adult zebrafish using the bag3 gene knockout (bag3e2/e2) cardiomyopathy model as a paradigm. First, by utilizing a classic genetic breeding approach, we identified dnajb6b as a deleterious modifier gene for bag3 cardiomyopathy. Next, we established an F0-based genetic assay in adult zebrafish through injection of predicted microhomology-mediated end joining (MMEJ)-inducing single guide RNA/Cas9 protein complex. We showed that effective gene knockdown is maintained in F0 adult fish, enabling recapitulation of both salutary modifying effects of the mtor haploinsufficiency and deleterious modifying effects of the dnajb6b gene on bag3 cardiomyopathy. We finally deployed the F0-based genetic assay to screen differentially expressed genes in the bag3 cardiomyopathy model. As a result, myh9b was identified as a novel modifier gene for bag3 cardiomyopathy. Together, these data prove the feasibility of an F0 adult zebrafish-based genetic assay that can be effectively used to discover modifier genes for inherited cardiomyopathy.

Application of an F0-based genetic assay in adult zebrafish to identify modifier genes of an inherited cardiomyopathy.

Sprinkled snow melting salt (SMS) exerts a snow melting effect and also has a negative impact on the asphalt pavement and the environment. Salt storage pavement technology can alleviate these two problems. However, non-alkaline SMSs may have the risk of affecting asphalt mastic properties and further affecting the mechanical properties of asphalt pavements. Therefore, the general properties and rheological properties of two styrene-butadiene-styrene-modified asphalts with and without high elastic polymer were studied after adding SMS. The asphalt mastic without a high elastic agent is defined as the SBS group, and the other group is the HEA group. Our results show that the HEA group shows a lower penetration and a higher softening point, ductility, and viscosity than the SBS group. The more the SMS, the more the reduction effect of the general performance. The elastic recovery of asphalt mastic decreases with the content of SMS. SMS has no obvious effect on the ratio of the viscous and elastic composition of asphalt mastic. The creep of asphalt mastic increases with the content of SMS. The high elastic polymer can significantly reduce the creep, and even the strain of HEA100 is smaller than that of SBS00. SMS increases the creep stiffness and reduces the creep rate at low temperature. Although SMS increases the potential of asphalt pavement to melt ice and snow, it also reduces the high-temperature rutting resistance and low-temperature crack resistance of asphalt mastic. Salt storage pavement materials can be used in combination with high elastic polymers to reduce the negative effects brought by SMSs.

Rheological Properties of Styrene-Butadiene-Styrene Asphalt Mastic Containing High Elastic Polymer and Snow Melting Salt.

The traditional method of removing ice and snow on roads carries the risk of damaging roads and the environment. In this circumstance, the technology of salt-storage asphalt pavement has gradually attracted attention. However, snow-melting salts may also have an impact on asphalt mixture performance. To explore the effect of snow-melting salts on the mechanical and surface properties of salt-storage asphalt mixtures (SSAM), SSAMs were prepared with styrene-butadiene-styrene (SBS)-modified asphalt and high-elastic asphalt (HEA) as binders and snow-melting salts as fillers. The influence of the type of asphalt binder and the content of snow-melting salt on the performance of the SSAM was preliminarily investigated through laboratory tests. The results show that the high-temperature, low-temperature, and moisture resistance performance of the SBS group SSAM decreased by 9.8-15.1%, 1.6-12.3%, and 6.3-19.4%, respectively, compared with SBS00. The higher the amount of snow-melting salt, the greater the performance drop. The three mechanical properties of the HEA group containing high-elastic agent TPS are 11.3-19.7%, 4.2-12.3%, and 4.8-13.3% higher than that of the SBS group. Even when the content of snow-melting salt is 50% or 75%, the mechanical properties of the HEA group are better than that of SBS00 without snow-melting salt. Snow-melting salt has clear advantages in improving the anti-skid performance but decreases the anti-spalling performance. The surface properties of the HEA group were also better than that of the SBS group. Considering the mechanical properties and surface properties, the comprehensive performance of the HEA group is better than that of the SBS group, and HEA50 has the best comprehensive performance. In addition, the construction performance of the SSAM has also been verified, and the production of SSAM according to the hot mix asphalt can meet the specification requirements.

Laboratory Study and Field Validation of the Performance of Salt-Storage Asphalt Mixtures.

Allelic heterogeneity (AH) has been noted in truncational TTN (TTNtv)-associated dilated cardiomyopathy (DCM), i.e., mutations affecting A-band-encoding exons are pathogenic, but those affecting Z-disc-encoding exons are likely benign. The lack of an in vivo animal model that recapitulates AH hinders the deciphering of the underlying mechanism. Here, we explored zebrafish as a candidate vertebrate model by phenotyping a collection of zebrafish ttntv alleles. We noted that cardiac function and sarcomere structure are more severely disrupted in ttntv-A than in ttntv-Z homozygous embryos. Consistently, cardiomyopathy-like phenotypes were presented in ttntv-A but not ttntv-Z adult heterozygous mutants. The phenotypes observed in ttntv-A alleles were recapitulated in null mutants with the entire titin-encoding sequences removed. Defective autophagic flux, largely due to impaired autophagosome-lysosome fusion, was also only noted in ttntv-A but not ttntv-Z models. Moreover, we found that genetic manipulation of ulk1a restored autophagy flux and rescued cardiac dysfunction in ttntv-A animals. Together, our findings presented adult zebrafish as an in vivo animal model for studying AH in TTNtv DCM, demonstrated TTN loss-of-function sufficient to trigger ttntv DCM in zebrafish, and uncovered ulk1a as a potential therapeutic target gene for TTNtv DCM.

Article	Year
Inhibition of mTOR or MAPK ameliorates vmhcl/myh7 cardiomyopathy in zebrafish. JCI insight\| PubMed ID: 34935644	2021
Application of an F0-based genetic assay in adult zebrafish to identify modifier genes of an inherited cardiomyopathy. Disease models & mechanisms\| PubMed ID: 35481478	2023
Rheological Properties of Styrene-Butadiene-Styrene Asphalt Mastic Containing High Elastic Polymer and Snow Melting Salt. Polymers\| PubMed ID: 36080727	2022
Laboratory Study and Field Validation of the Performance of Salt-Storage Asphalt Mixtures. Materials (Basel, Switzerland)\| PubMed ID: 36234058	2022
Allelic heterogeneity of TTNtv cardiomyopathy can be modeled in adult zebrafish. JCI insight\| PubMed ID: 38412038	2024

Jiarong Li

Biography

JoVE Journal Publications

Other Publications