Abstract
ABSTRACTCardiac 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-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.
Highlights
Generation and phenotype analyses of tnnt2 mutant zebrafish Tnnt2a mutant zebrafish were successfully generated via the CRISPR/Cas9 technique (Liu et al, 2017; Wang et al, 2018)
To determine whether the cardiomyocytes in mutant zebrafish maintained normal electrocardiac rhythm, we introduced the Tg(bactin2:GCamp6s) transgenic background that expresses a slower version of fluorescent calcium sensor ubiquitously (Chen et al, 2013)
Our results reveal the importance of outflow tract (OFT) expression of Tnnt2 for cardiac function and indicate the complexity of assessing therapeutic strategies for cardiomyopathy caused by sarcomerebased mutations (Fig. 7)
Summary
Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) are two common cardiomyopathies with an estimated prevalence of 1:250 and 1:200 in adults Mutations in a single sarcomere gene like cardiac troponin T (TNNT2) are sufficient to cause cardiomyopathy, and TNNT2 mutations are the most common ‘drivers’ of thin filament deficiency in both DCM and HCM (Hershberger et al, 2013; Veselka et al, 2017). Most human TNNT2 mutations are located in central and C-terminal domains of cardiac troponin T and are responsible for both familial cardiomyopathy and sporadic cardiomyopathy (Forissier et al, 1996; Van Driest, 2003; McConnell et al, 2017)
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