Development of Tg(UAS:SEC-Hsa.ANXA5-YFP,myl7:RFP); Casper(roy-/-,nacre-/-) Transparent Transgenic In Vivo Zebrafish Model to Study the Cardiomyocyte Function.

Surendra K Rajpurohit,Nikhil G Patel,Aaron Gopal,May Ye Mon,Vishal Arora

doi:10.3390/cells10081963

Abstract

The zebrafish provided an excellent platform to study the genetic and molecular approach of cellular phenotype-based cardiac research. We designed a novel protocol to develop the transparent transgenic zebrafish model to study annexin-5 activity in the cardiovascular function by generating homozygous transparent skin Casper(roy−/−,nacre−/−); myl7:RFP; annexin-5:YFP transgenic zebrafish. The skin pigmentation background of any vertebrate model organism is a major obstruction for in vivo confocal imaging to study the transgenic cellular phenotype-based study. By developing Casper(roy−/−,nacre−/−); myl7; annexin-5 transparent transgenic zebrafish strain, we established time-lapse in vivo confocal microscopy to study cellular phenotype/pathologies of cardiomyocytes over time to quantify changes in cardiomyocyte morphology and function over time, comparing control and cardiac injury and cardio-oncology. Casper contributes to the study by integrating a transparent characteristic in adult zebrafish that allows for simpler transparent visualization and observation. The Casper(roy−/−,nacre−/−) transgenic progenies developed through cross-breeding with the transgenic strain of Tg(UAS:SEC-Hsa.ANXA5-YFP,myl7:RFP). Confocal and fluorescent microscopy were being used to obtain accurate, precise imaging and to determine fluorescent protein being activated. This study protocol was conducted under two sections; 1.1: Generation of homozygous Tg(UAS:SEC-Hsa.ANXA5-YFP,myl7:RFP); Casper(roy−/−,nacre−/−) zebrafish (generation F01-F06) and 1.2: Screening and sorting the transparent transgenic progeny and in vivo imaging to validate cardiac morphology through in vivo confocal imaging. We coined the newly developed strain as Tg(UAS:SEC-Hsa.ANXA5-YFP,myl7:RFP); Casper(roy−/−,nacre−/−)gmc1. Thus, the newly developed strain maintains transparency of the skin throughout the entire life of zebrafish and is capable of application of a non-invasive in vivo imaging process. These novel results provide an in vivo whole organism-based platform to design high-throughput screening and establish a new horizon for drug discovery in cardiac cell death and cardio-oncology therapeutics and treatment.

Highlights

The heart is the first organ to form and function in the embryo, and its formation is followed by a series of vital events in the life of the organism
Annexin-5 activities contribute to cardiomyocyte dedifferentiation, proliferation, and epicardial injury responses which lead to cardiac cell death by apoptosis and narcosis pathways
We developed transgenic quantify changes cardiomyocyte morphology and function comparing control progenies throughincross breeding with the transgenic strain ofovertime, Tg

Summary

Introduction

The heart is the first organ to form and function in the embryo, and its formation is followed by a series of vital events in the life of the organism Cardiac malformation at this stage is the most common form of human birth defect, and abnormalities of the adult heart threaten to be the most prevalent cause of morbidity and mortality in the modern era. The early 21st century has marked a transition from a physiological and functional approach towards the heart to a deeper understanding of cardiac function at the genetic and molecular levels. These discoveries have provided new therapeutic dimensions for palliation and prevention of heart pathophysiology, as well as having raised new questions, challenges, and pathways for the generation of cardiac research. Casper(roy−/− ,nacre−/− ) Transparent Skin Pattern Normal WT Skin Pigmentation

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