Combinatorial Genetic Replenishments in Myocardial and Outflow Tract Tissues Restore Heart Function in Mutant Zebrafish

Cardiac muscle troponin T (Tnnt2) mediates muscle contraction in response to calcium ion dynamics, and 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. 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 and inducible system to restore expression in myocardial tissue in 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 -negative smooth muscle cells on the outflow tract (OFT), was indispensably responsible for the normal mechanical dynamics of OFT. Lastly, expression induced by OFT cells in addition to the myocardial cells successfully rescued heart function and circulation in 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 platform for studying cardiomyopathy and the relevant therapeutic strategies.