Human ARF Specifically Inhibits Epimorphic Regeneration in the Zebrafish Heart.

Solomon Lee,Robert Hesse,Catharine Garland,Stanley Tamaki,Jason H Pomerantz

doi:10.3390/genes11060666

Abstract

The Alternative Reading Frame (ARF) protein is a tumor suppressor encoded by the Cyclin Dependent Kinase Inhibitor 2A gene in mammals but not lower regenerative vertebrates, and has been previously implicated as a context-sensitive suppressor of regeneration in murine skeletal muscle and humanized ARF-expressing zebrafish fins. This study extends our investigation of the role of ARF in the regeneration of other solid tissues, including the zebrafish heart and the mammalian digit. Heart regeneration after cryoinjury was used to mimic massive myocardial infarction. ARF gene expression was upregulated during the cardiac regenerative process and slowed the rate of morphological recovery. ARF specifically impacts cardiomyocytes, neovascularization, and the endothelial-mesenchymal transition, while not affecting epicardial proliferation. This suggests that in the context of regeneration, ARF is specifically expressed in cells undergoing dedifferentiation. To investigate ARF as a suppressor of epimorphic regeneration in mammalian systems, we also tested whether the absence of ARF was permissive for murine digit regeneration, but found that ARF absence alone was insufficient to significantly alter digit restoration. These findings provide additional evidence that ARF suppresses epimorphic regeneration, but suggests that modulation of ARF alone is insufficient to permit regeneration.

Highlights

Humans do not undergo epimorphic regeneration after significant injury, which instead leads to collagen deposition, scar formation, and functional impairment
Alternative Reading Frame (ARF) is a protein encoded by Cyclin dependent kinase inhibitor 2A (Cdkn2a) [12] that responds to inappropriate retinoblastoma (Rb) pathway signaling [13] by sequestering Mouse double minute 2 (MDM2) to regulate Tumor protein 53 (p53) [14,15] and promoting cell cycle arrest or apoptosis to maintain the postmitotic state
Arf deficient digits did contain intramedullary sclerosis and disorganized periosteal osseous proliferation near the amputation margin compared to WT mice, but no mature ossification or cortication was noted by histology or micro-CT (Figure S1b). In this extension of our initial findings that mammalian ARF is a suppressor of fin regeneration, we show this property of ARF to be active in the context of heart regeneration

Summary

Introduction

Humans do not undergo epimorphic regeneration after significant injury, which instead leads to collagen deposition, scar formation, and functional impairment. Zebrafish have served as excellent models to advance our knowledge of epimorphic regeneration and elucidate limitations to human regeneration. Tumor suppressor genes have increasing importance in cancer protection manifested most strongly in mammalian phyla, but may adversely affect mammalian regeneration [9]. Reading Frame (ARF) gene product is an unusual tumor suppressor differentiated from others because it does not have any orthologs represented in highly-regenerative species [10,11], suggesting that the absence of ARF may be permissive for regeneration in those species. ARF is a protein encoded by Cyclin dependent kinase inhibitor 2A (Cdkn2a) [12] that responds to inappropriate retinoblastoma (Rb) pathway signaling [13] by sequestering Mouse double minute 2 (MDM2) to regulate Tumor protein 53 (p53) [14,15] and promoting cell cycle arrest or apoptosis to maintain the postmitotic state

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Results

Conclusion