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Implementation of optogenetic voltage sensor VSFP2.3 to visualize cardiac excitation
Citation Link: https://doi.org/10.15480/882.1205
Publikationstyp
Doctoral Thesis
Date Issued
2014
Sprache
English
Author(s)
Advisor
Title Granting Institution
Technische Universität Hamburg
Place of Title Granting Institution
Hamburg
Examination Date
2014-04-08
Institut
TORE-DOI
The initiation and propagation of electrical signals play a pivotal role in normal cardiac function. These are carried out by cardiomyocytes, the building blocks of the heart, which generate and conduct electrical signal from one cell to another, throughout the whole heart. The techniques used for investigating electrical activities in cardiomyocytes and hearts, such as applying electrodes and optical mapping, possess different advantages and disadvantages. Microelectrode recordings, for example, provide direct and faithful insight into changes in the current and membrane potential from single cardiomyocytes. The experimental procedure, however, is laborious and invasive, which prevents repeated recordings on the same cells at different time points. Optical mapping using flourescent dyes offers a non-invasive technique with higher spatial-temporal resolution. However, the fluorescent dyes used are usually toxic, and the distribution of the dyes could be inhomogeneous due to the complex tissue structure. Moreover, repeated and simultaneous recordings at different time points in living cell/tissue are most challenging. Thus, in vivo and long-term applications are limited in the abovementioned set up.
In this study, the hypothesis that a voltage sensitive fluorescent protein (VSFP2.3) is applicable for the visualization of cardiac excitability was tested. Optogenetic labeling with VSFP2.3 may overcome some of the aforementioned shortcomings. VSFP2.3 can be stably expressed in defined cell types by using cell type specific promoter elements, making it functional throughout lifetime. Combined with high speed and highly sensitive cameras, chronic studies in a noninvasive manner are feasible.
Firstly, a transgenic mouse model stably expressing VSFP2.3 under the control of the cardiac specific alpha myosin heavy chain promoter (αMHC) was established. The transgene did not impair myocardial structure and cardiac function. Adult cardiomyocytes isolated from these transgenic mice showed clear membrane labeling of VSFP2.3. The electrical activities from single VSFP2.3 cardiomyocytes and whole hearts were optically recorded with high sensitive cameras and photomultipliers and validated the use of this approach.
Secondly, double transgenic induced pluripotent stem cell (iPSC) lines carrying both neomycin resistant gene (neoR) and VSFP2.3 under the control of αMHC promoter were generated. NeoRVSFP iPSCs were differentiated into spontaneously beating cardiomyocytes. Changes in fluorescent signals were recorded from beating cardiomyocytes indicating the function of the protein. Engineered heart muscles (EHMs) generated from neoRVSFP iPSC-derived cardiomyocytes contracted spontaneously and responded to increasing extracellular calcium concentrations with an increase in force development. Fluorescent signals within EHMs were acquired successfully.
Collectively, this study demonstrated for the first time that a genetically encoded voltage sensor expressed in the mammalian heart can serve as a means to precisely assess cardiomyocyte excitability.
In this study, the hypothesis that a voltage sensitive fluorescent protein (VSFP2.3) is applicable for the visualization of cardiac excitability was tested. Optogenetic labeling with VSFP2.3 may overcome some of the aforementioned shortcomings. VSFP2.3 can be stably expressed in defined cell types by using cell type specific promoter elements, making it functional throughout lifetime. Combined with high speed and highly sensitive cameras, chronic studies in a noninvasive manner are feasible.
Firstly, a transgenic mouse model stably expressing VSFP2.3 under the control of the cardiac specific alpha myosin heavy chain promoter (αMHC) was established. The transgene did not impair myocardial structure and cardiac function. Adult cardiomyocytes isolated from these transgenic mice showed clear membrane labeling of VSFP2.3. The electrical activities from single VSFP2.3 cardiomyocytes and whole hearts were optically recorded with high sensitive cameras and photomultipliers and validated the use of this approach.
Secondly, double transgenic induced pluripotent stem cell (iPSC) lines carrying both neomycin resistant gene (neoR) and VSFP2.3 under the control of αMHC promoter were generated. NeoRVSFP iPSCs were differentiated into spontaneously beating cardiomyocytes. Changes in fluorescent signals were recorded from beating cardiomyocytes indicating the function of the protein. Engineered heart muscles (EHMs) generated from neoRVSFP iPSC-derived cardiomyocytes contracted spontaneously and responded to increasing extracellular calcium concentrations with an increase in force development. Fluorescent signals within EHMs were acquired successfully.
Collectively, this study demonstrated for the first time that a genetically encoded voltage sensor expressed in the mammalian heart can serve as a means to precisely assess cardiomyocyte excitability.
Subjects
Herz
genetisch kodierte Spannungsanzeige
optische Bildgebung
Arrhythmie
pluripotenten Stammzellen
heart
genetically encoded voltage indicator
optical imaging
arrhythmia
pluripotent stem cells
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