Sun, Xiaojing

Rationale: Although a cardioprotective function of target of rapamycin (TOR) signaling inhibition has been suggested by pharmacological studies using rapamycin, genetic evidences are still lacking. We explored adult zebrafish as a novel vertebrate model for dissecting signaling pathways in cardiomyopathy. Objective: We generated the second adult zebrafish cardiomyopathy model induced by doxorubicin. By genetically analyzing both the doxorubicin and our previous established anemia-induced cardiomyopathy models, we decipher the functions of TOR signaling in cardiomyopathies of different etiology. Methods and Results: Along the progression of both cardiomyopathy models, we detected dynamic TOR activity at different stages of pathogenesis as well as distinct effects of TOR signaling inhibition. Nevertheless, cardiac enlargement in both models can be effectively attenuated by inhibition of TOR signaling through short-term rapamycin treatment. To assess the long-term effects of TOR reduction, we used a zebrafish target of rapamycin ( ztor ) mutant identified from an insertional mutagenesis screen. We show that TOR haploinsufficiency in the ztor heterozygous fish improved cardiac function, prevented pathological remodeling events, and ultimately reduced mortality in both adult fish models of cardiomyopathy. Mechanistically, these cardioprotective effects are conveyed by the antihypertrophy, antiapoptosis, and proautophagy function of TOR signaling inhibition. Conclusions: Our results prove adult zebrafish as a conserved novel vertebrate model for human cardiomyopathies. Moreover, we provide the first genetic evidence to demonstrate a long-term cardioprotective effect of TOR signaling inhibition on at least 2 cardiomyopathies of distinct etiology, despite dynamic TOR activities during their pathogenesis.

The cardiac neural crest is a subpopulation of neural crest cells (NCCs) that originates from the dorsal neural tube and later migrates to the heart and pharyngeal arch (PA), where they contribute to distinct aspects of cardiogenesis and PA patterning. Wnt family members have been implicated in general neural crest induction; however, the molecular mechanisms underlying the specification of distinct cell types such as cardiac NCCs remain unclear. Here, we have identified cysteine-rich intestinal protein 2 ( crip2 ), a gene encoding for a LIM domain–containing protein, as a novel marker for cardiac NCCs in zebrafish. We observed a dynamic expression pattern for crip2 , with expression being detected in the premigratory NCCs in rhombomere 6 (r6), migrating NCCs, ventricular cardiomyocytes, and aortic vessels in PAs 3 to 6. Interestingly, expression of wnt3a was detected in regions of the dorsal neural tube encompassing r6, and reduction of wnt3a led to disrupted expression of crip2 in r6, abnormal migration of NCCs, and defective cardiac function, as well as PA development. Furthermore, these phenotypes could be recapitulated by reduction of crip2 and rescued by overexpression of crip2 . In summary, our data suggest a specific role for Wnt3a in the development of cardiac NCCs. We propose that this function of wnt3a in r6 is partially mediated by crip2 expression in the premigratory cardiac NCCs, which subsequently affects cardiac function and PA patterning.