miR133b Microinjection during Early Development Targets Transcripts of Cardiomyocyte Ion Channels and Induces Oil-like Cardiotoxicity in Zebrafish (Danio rerio) Embryos.

ABSTRACT

Previous studies have shown that altered expression of a family of small noncoding RNAs (microRNAs, or miRs) regulates the expression of downstream mRNAs and is associated with diseases and developmental disorders. miR133b is highly expressed in mammalian cardiac and skeletal muscle, and aberrant expression is associated with cardiac disorders and electrophysiological changes in cardiomyocytes. Similarly, cardiac dysfunction has been observed in early life-stage mahi-mahi (Coryphaena hippurus) exposed to crude oil, a phenotype that has been associated with an upregulation of miR133b as well as subsequent downregulation of a delayed rectifier potassium channel (IKr) and calcium signaling genes that are important for proper heart development during embryogenesis. To examine the potential role of miR133b in oil-induced early life-stage cardiotoxicity in fish, cleavage-stage zebrafish (Danio rerio) embryos were either (1) microinjected with ∼3 nL of negative control miR (75 µM) or miR133b (75 µM) or (2) exposed to a treatment solution containing 5 µM benzo(a)pyrene (BaP), a model polycyclic aromatic hydrocarbon, as a positive control. At 72 h post fertilization (hpf), miR133b-injected fish exhibited BaP-like cardiovascular malformations, including a significantly increased pericardial area relative to negative control miR-injected embryos, as well as a significantly reduced eye area. qPCR revealed that miR133b microinjection decreased the abundance of cardiac-specific IKr kcnh6 at 5 hpf, which may contribute to action potential elongation in oil-exposed cardiomyocytes. Additionally, ryanodine receptor 2, a crucial calcium receptor in the sarcoplasmic reticulum, was also downregulated by miR133b. These results indicate that an oil-induced increase in miR133b may contribute to cardiac abnormalities in oil-exposed fish by targeting cardiac-specific genes essential for proper heart development.