Abstract
The alpha regulator subunit B'' of protein phosphatase 2 (PPP2R3A), a regulatory subunit of protein phosphatase 2A (PP2A), was reported to present a special subcellular localization in cardiomyocytes and elevate in non-ischemia failing hearts. PPP2R3A has two transcriptions PR72 and PR130. PR72 acts as a negative regulator of the Wnt signaling cascade, while the Wnt signaling cascade plays a pivotal role in cardiac development. And PR130 was found to be involved in cardiac development of zebrafish in our previous study. Thus, to investigate the function of PR72 in heart, two stable pr72 knockout (KO) zebrafish lines were generated using Transcription Activator-Like Effector Nuclease (TALEN) technology. Homozygous pr72 KO fish struggled to survive to adulthood and exhibited cardiac developmental defects, including enlarged ventricular chambers, reduced cardiomyocytes and decreased cardiac function. And the defective sarcomere ultrastructure that affected mitochondria, I bands, Z lines, and intercalated disks was also observed. Furthermore, the abnormal heart looping was detected in mutants which could be rescued by injection with wild type pr72 mRNA. Additionally, it was found that Wnt effectors were elevated in mutants. Those indicated that deletion of pr72 in zebrafish interrupted cardiac development, probably through activation of the Wnt pathway.
Highlights
Dysregulation of protein phosphorylation is an important molecular mechanism for numerous cardiac diseases, including heart failure and cardiomyopathy [1, 2]
The wild type (Wt) pr72 cDNA of zebrafish was transcribed from the extracted zebrafish mRNA by reverse transcription PCR (RT-PCR), and subcloned into the pSP64 plasmid expression vector (Primer sequences were presented in S1 Table)
Two stable deletion mutations were produced using Transcription Activator-Like Effector Nuclease (TALEN), and were detected by PCR-RFLP and were validated by DNA sequencing on the fragment containing TALEN target site (Figs 1 and 2), and the western blotting result which showing no Pr72 protein in the mutants (Fig 1)
Summary
Dysregulation of protein phosphorylation is an important molecular mechanism for numerous cardiac diseases, including heart failure and cardiomyopathy [1, 2]. There are at least four families of PP2A regulatory B subunits, and each family has more than one familiar members: identified as B. The activity, specificity and subcellular localization of the PP2A heterotrimeric holoenzyme complex, are highly regulated through the interaction of regulatory B subunit family members with the substrates [6]. Deletion of the regulatory B56γ subunit, results in heart development defects, including reduced cardiomyocytes (CMs) and a ventricular septal defect [12]. The role and regulation of this critical enzyme family especially subunit B, was still largely elusive in cardiac diseases
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