Human ARF Tumor Suppressor Suppresses Zebrafish Cardiac Regeneration
Solomon Lee, MD, Stanley Tamaki, PhD, Jason Pomerantz, MD.
University of California, San Francisco, San Francisco, CA, USA.
Purpose: This study explores how the Alternative Reading Frame (ARF) tumor suppressor, while preventing oncogenesis, may simultaneously inhibit mammalian epimorphic regeneration. ARF is a tumor suppressor encoded by the Cdkn2a gene in mammals but not lower regenerative vertebrates, previously implicated as a context-sensitive suppressor of regeneration in murine skeletal muscle and humanized ARF-expressing zebrafish fins. We aim to characterize the impact and target of ARF during the more complex and clinically translatable processes of heart regeneration after massive myocardial infarction.
Methods: Transgenic zebrafish lines expressing ARF under control of the heat shock promoter (hs:ARF) and natural human ARF promoter (ARF:ARF) were used. Heart cryoinjury with a liquid nitrogen probe was performed on anesthetized transgenic fish and wild type (WT) controls. Hearts were collected at various days post-injury (dpi) for analysis. Regenerative progress was analyzed using histology, immunofluorescence, and qPCR of tissue-specific regenerative markers.
Results: ARF expression was upregulated during the cardiac regenerative process and slowed the rate of morphological recovery. In hs:ARF fish, AFOG and troponin staining revealed a 48.7% (p<0.01) reduction in myocardial recovery compared to WT fish. In ARF:ARF fish, myocardial recovery was reduced by 2.3% (p=0.96), 20.4% (p=0.47), 41.3% (p<0.01), 36.1% (p=0.05), and 24.3% (p<0.01) at 1, 4, 7, 15, and 30 dpi respectively. A cardiomyocyte proliferation index generated by MEF2/PCNA staining confirmed cardiomyocyte-specific suppression in ARF:ARF heart regeneration by 46.6% (p=0.01) at 11 dpi. Tissue-specific regenerative gene expression was tracked by qPCR in ARF:ARF and WT fish. Fgf17b, vegfaa, and Twist1b were reduced by 42% (p<0.01), 43% (p<0.01), and 55% in ARF:ARF hearts at 11 dpi, reflective of decreases in myocardial regeneration, vascular regeneration, and epithelial-to-mesenchymal (EMT) transition respectively. There was no significant difference in fgfr2c expression (p=0.44), a marker of epicardial regeneration.
Conclusions: Understanding how ARF suppresses cardiac regeneration is important for promoting recovery after heart injury in humans. The timeline of recovery in ARF:ARF fish suggests that ARF does not affect the acute processes of scarring, but rather suppresses cardiomyocyte proliferation. ARF's selective impact on myocardial regeneration, vascular regeneration, and EMT, while not affecting epicardial regeneration, elucidates that in the context of regeneration, ARF is not indiscriminately expressed in all proliferating cells, but is rather localized to cells undergoing dedifferentiation or transdifferentiation. Our findings show that ARF will require alteration in conjunction with other genes to permit regeneration.
Figure 1. Human ARF in ARF:ARF fish suppresses cardiac regeneration. (A) AFOG and troponin staining of zebrafish hearts. (B) Myocardial recovery by troponin infiltration into injury site.
Figure 2. Fgf17b, fgfr2c, vegfaa, and twist1b mRNA expression at 11 dpi reflects myocardial regeneration, epicardial regeneration, vascular regeneration, and epithelial-to-mesenchymal (EMT) transition respectively.
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