Abstract
Cardiac cells develop within an elaborate electro-mechanical syncytium that continuously generates and reacts to biophysical force. The complexity of the cellular interactions, hemodynamic stresses, and electrical circuitry within the forming heart present significant challenges for mechanistic research into the cellular dynamics of cardiomyocyte maturation. Simply stated, it is prohibitively difficult to replicate the native electro-mechanical cardiac microenvironment in tissue culture systems favorable to high-resolution cellular/subcellular analysis, and current transgenic models of higher vertebrate heart development are limited in their ability to manipulate and assay the behavior of individual cells. As such, cardiac research currently lacks a simple experimental platform for real-time evaluation of cellular function under conditions that replicate native development. Here we report the design and validation of a rapid, low-cost system for stable in vivo somatic transgenesis that allows for individual cells to be genetically manipulated, tracked, and examined at subcellular resolution within the forming four-chambered heart. This experimental platform has several advantages over current technologies, chief among these being that mosaic cellular perturbations can be conducted without globally altering cardiac function. Consequently, direct analysis of cellular behavior can be interrogated in the absence of the organ level adaptions that often confound data interpretation in germline transgenic model organisms.
Highlights
The developing heart presents unique challenges for biomedical investigation
We present a simple, cationic lipid-based transfection system and a toolkit of integrating DNA plasmids that can be used to rapidly create genetically mosaic hearts ideal for high resolution imaging and single cell analysis. This system has several advantages over current technologies including: 1) cellular perturbations can be conducted without globally altering cardiac function, meaning downstream effects can be analyzed under normal hemodynamic conditions; 2) genetically manipulated cells can be compared with control cells within the same heart eliminating many sources of experimental variability; 3) multiple genetic manipulations can be performed in the same cell in vivo; 4) large numbers of manipulated cells can be isolated from a single heart, 5) genetically encoded biosensors can be employed for real-time/longitudinal studies of physiological maturation; and 6) multiple fluorescent molecules can be targeted to subcellular locales in tandem for live-imaging of cytoarchitectural development
Given its relative ease, low cost, and flexibility, we focused on chemical transfection as a potential approach to develop a protocol for in vivo cardiac somatic transgenesis
Summary
The developing heart presents unique challenges for biomedical investigation. During cardiac morphogenesis, juvenile cardiomyocytes undergo cellular diversification, cytoarchitectural specialization, and functional integration as the heart loops, septates, and coalesces into a highly coordinated muscular pump. We present a simple, cationic lipid-based transfection system and a toolkit of integrating DNA plasmids that can be used to rapidly create genetically mosaic hearts ideal for high resolution imaging and single cell analysis This system has several advantages over current technologies including: 1) cellular perturbations can be conducted without globally altering cardiac function, meaning downstream effects can be analyzed under normal hemodynamic conditions; 2) genetically manipulated cells can be compared with control cells within the same heart eliminating many sources of experimental variability (stage, sex, strain, etc.); 3) multiple genetic manipulations can be performed in the same cell in vivo; 4) large numbers of manipulated cells can be isolated from a single heart, 5) genetically encoded biosensors can be employed for real-time/longitudinal studies of physiological maturation; and 6) multiple fluorescent molecules can be targeted to subcellular locales in tandem for live-imaging of cytoarchitectural development. We have identified a simple but powerful platform for examining cardiac development that combines physiological relevance of transgenic models with the flexibility of cell culture techniques
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