PUBLICATION

Combinatorial genetic replenishments in myocardial and outflow tract tissues restore heart function in tnnt2 mutant zebrafish

Authors: Liu, L., Fei, F., Zhang, R., Wu, F., Yang, Q., Wang, F., Sun, S., Zhao, H., Li, Q., Wang, L., Wang, Y., Gui, Y., Wang, X.
ID: ZDB-PUB-191206-5
Date: 2019
Source: Biology Open 8(12): (Journal)
Registered Authors: Li, Qiang, Wang, Xu, Wang, Youhua
Keywords: Cardiac troponin T (TNNT2), Conditional transgene, Dilated cardiomyopathy (DCM), Genetic replenishment, Zebrafish
MeSH Terms: none
PubMed: 31796423 Full text @ Biol. Open

Abstract

Cardiac muscle troponin T (Tnnt2) mediates muscle contraction in response to calcium ion dynamics, and Tnnt2 mutations are associated with multiple types of cardiomyopathy. Here, we employed a zebrafish model to investigate the genetic replenishment strategies of using conditional and inducible promoters to rescue the deficiencies in the heart. tnnt2a mutations were induced in zebrafish via the CRISPR/Cas9 technique, and the mutants displayed heart arrest and dilated cardiomyopathy (DCM)-like phenotypes. We first utilized the classic myocardial promoter of the myl7 and TetOn inducible system to restore tnnt2a expression in myocardial tissue in tnnt2a mutant zebrafish. However, this attempt failed to recover normal heart function and circulation, although heart pumping was partially restored. Further analyses via both RNA-seq and immunofluorescence indicated that Tnnt2a, which was also expressed in a novel group of myl7-negative smooth muscle cells on the outflow tract (OFT), was indispensably responsible for the normal mechanical dynamics of OFT. Lastly, tnnt2 expression induced by OFT cells in addition to the myocardial cells successfully rescued heart function and circulation in tnnt2a mutant zebrafish. Together, our results reveal the significance of OFT expression of Tnnt2 for cardiac function and demonstrate zebrafish larva as a powerful and convenient in vivo platform for studying cardiomyopathy and the relevant therapeutic strategies.

Genes / Markers

Figures

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Expression

Phenotype

Fish	Conditions	Stage	Phenotype	Figure
fdu106Tg	chemical treatment by environment: doxycycline	Protruding-mouth	heart tnnt2a expression increased amount, abnormal	Fig. 2.
tnnt2a^{fdu108/fdu108}; fdu106Tg	standard conditions	Protruding-mouth	atrium distended, abnormal	Fig. 2.
tnnt2a^{fdu108/fdu108}; fdu106Tg	standard conditions	Protruding-mouth	blood circulation decreased rate, abnormal	Fig. 2.
tnnt2a^{fdu108/fdu108}; fdu106Tg	standard conditions	Protruding-mouth	cardiac ventricle distended, abnormal	Fig. 2.
tnnt2a^{fdu108/fdu108}; fdu106Tg	standard conditions	Protruding-mouth	heart actc1a expression increased amount, abnormal	Fig. 3.
tnnt2a^{fdu108/fdu108}; fdu106Tg	standard conditions	Protruding-mouth	heart bmp2b expression decreased amount, abnormal	Fig. 3.
tnnt2a^{fdu108/fdu108}; fdu106Tg	standard conditions	Protruding-mouth	heart gata4 expression increased amount, abnormal	Fig. 3.
tnnt2a^{fdu108/fdu108}; fdu106Tg	standard conditions	Protruding-mouth	heart gata5 expression increased amount, abnormal	Fig. 3.
tnnt2a^{fdu108/fdu108}; fdu106Tg	standard conditions	Protruding-mouth	heart myl7 expression increased amount, abnormal	Fig. 3.
tnnt2a^{fdu108/fdu108}; fdu106Tg	standard conditions	Protruding-mouth	heart tbx2b expression decreased amount, abnormal	Fig. 3.

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
fdu106Tg	Tg(myl7:TET3G,TETRE:tnnt2a-2A-mKate2,myl7:EGFP)	Transgenic Insertion
fdu107Tg	Tg(actb2:GCaMP6s)	Transgenic Insertion
fdu108		Small Deletion	tnnt2a
twu34Tg	Tg(myl7:EGFP)	Transgenic Insertion

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Human Disease / Model

Human Disease	Fish	Conditions	Evidence
cardiomyopathy	tnnt2a^{fdu108/fdu108}	standard conditions	TAS

Sequence Targeting Reagents

Target	Reagent	Reagent Type
tnnt2a	CRISPR6-tnnt2a	CRISPR

Fish

Fish
fdu106Tg
fdu107Tg
tnnt2a^{fdu108/fdu108}
tnnt2a^{fdu108/fdu108}; fdu106Tg
tnnt2a^{fdu108/fdu108}; fdu107Tg
tnnt2a^{fdu108/fdu108}; twu34Tg
tnnt2a^fdu108/+; fdu106Tg/+
twu34Tg
WT

Antibodies

Orthology

Engineered Foreign Genes

Marker	Marker Type	Name
EGFP	EFG	EGFP
GCaMP	EFG	GCaMP
mKate2	EFG	mKate2

Mapping

Liu et al., 2019 ZDB-PUB-191206-5 PMID:31796423

Combinatorial genetic replenishments in myocardial and outflow tract tissues restore heart function in tnnt2 mutant zebrafish

Liu et al., 2019

ZDB-PUB-191206-5
PMID:31796423