C3orf58 , a Novel Paracrine Protein, Stimulates Cardiomyocyte Cell-Cycle Progression Through the PI3K–AKT–CDK7 Pathway

Abstract

Rational The regenerative capacity of the heart is markedly diminished shortly after birth coinciding with overall withdrawal of cardiomyocytes from cell cycle. Consequently, the adult mammalian heart has limited capacity to regenerate after injury. The discovery of factors that can induce cardiomyocyte proliferation is therefore of high interest and has been the focus of extensive investigation over the past years. Objective We have recently identified C3orf58 as a novel Hypoxia and Akt induced Stem cell Factor (HASF) secreted from mesenchymal stem cells that can promote cardiac repair through cytoprotective mechanisms. Here, we tested the hypothesis that HASF can also contribute to cardiac regeneration by stimulating cardiomyocyte division and proliferation. Methods and Results Neonatal ventricular cardiomyocytes were stimulated in culture for seven days with purified recombinant HASF protein. Compared to control untreated cells, HASF-treated neonatal cardiomyocytes exhibited 60% increase in DNA synthesis as measured by BrdU incorporation. These results were confirmed by immunofluorescence confocal microscopy showing a 50–100% increase in the number of cardiomyocytes in the mitotic and cytokinesis phases. Importantly, in vivo cardiac overexpression of HASF in a transgenic mouse model resulted in enhanced level of DNA synthesis and cytokinesis in neonatal and adult cardiomyocytes. These proliferative effects were modulated by a PI3K-AKT-CDK7 pathway as revealed by the use of PI3K pathway specific inhibitors and silencing of the Cdk7 gene. Conclusion Our studies support the hypothesis that HASF induces cardiomyocyte proliferation via a PI3K-AKT-CDK7 pathway. The implications of this finding may be significant for cardiac regeneration biology and therapeutics.

Cloning and sequence analysis of the LOC339524 gene in Sprague-Dawley rats

“…ExPASy ProtParam online analysis showed that the LOC339524 protein instability index was so large that it could be classified as an unstable protein, suggesting that the protein is active and important. Transmembrane prediction software indicated that at residues 47-68, the protein may have a transmembrane helix, which is consistent with the results reported by Ota et al (2004), suggesting that the protein is a single transmembrane protein without a putative signal peptide, just like hypoxia and Akt-induced stem cell factor (HASF) (Beigi et al, 2013). Further analysis showed that the functional domain of the LOC339524 protein is located at residues 138-236, a region that is likely to induce and regulate cytokine production (Boissanet al, 2014;Turnquistet al, 2014), thereby regulating the immune system.…”

Emerging Concepts in Paracrine Mechanisms in Regenerative Cardiovascular Medicine and Biology

“…Importantly, evidence of cytokinesis was observed in a murine model 73 . The proliferative effects of HASF were found to be mediated by PI3K and the cell-cycle regulator cyclin-dependent kinase 7 (CDK7) 73 . We are currently investigating in more detail the molecular pathways by which HASF promotes cardiomyocyte proliferation.…”

“…As noted in a recent editorial HASF has a number of novel features as a cytoprotective factor 136 . Over-expression of PKCε promotes cardiac hypertrophy 137 , whereas that of HASF does not 73 . Moreover, pharmacological inhibition of PKCε did not affect HASF mediated activation of Akt and it is curious feature of HASF that high levels of Akt activity are not important for the cytoprotective effects of this paracrine factor 136 .…”

“…HASF is a much larger molecule than IGF-1, and indeed many of the commonly investigated growth factors, and may activate different pathways. Indeed, whereas IGF-1 promotes cardiac hypertrophy 75 , HASF does not 73 . This finding itself highlights the potential clinical benefits of the HASF protein.…”

Underlying mechanisms and prospects of heart regeneration

“…Thus, we successfully used this model to identify Meis1, one of the key regulators of neonatal cardiac regeneration, and report that Meis1 inhibits cardiomyocyte proliferation through transcriptional activation of CDKIs p15, p16, and p21 . Meis1 knockout 9 (pH3 + CMs, Aurora B + CMs) GSK-3 inhibition (BIO) 5 (pH3 + CMs) (Tseng et al, 2006) Foxo1 dominant negative 2.5 (pH3 + CMs) (Evans-Anderson et al, 2008) miR-133a knockout 2.5 (pH3 + CMs) (Liu et al, 2008) Jumonji knockout 2.2 (pH3 + CMs) (Jung et al, 2005) p27 KIP1 knockout 2-3 (troponin I + CMs) (Poolman et al, 1999) Constitutively active ERBB2 >12 (Ki67 + CMs, pH3 + CMs, Aurora B+ CMs) (D'Uva et al, 2015) Nrg1 (or FGF1, periostin) treatment >4 (BrdU + CMs, Aurora B + CMs, pH3 + CMs,) (Bersell et al, 2009) Activated Yap1 >7 (Ki67 + CMs, pH3 + CMs, Aurora B+ CMs) (von Gise et al, 2012) Salv knockout >4 (pH3 + CMs) (Heallen et al, 2011) IL13 (or IL3, CTGF, Nrg1) treatment >1.5 (3H Thymidine CMs, Ki67 + CMs, BrdU + CMs) Oncostatin M treatment >2 (EdU + CMs) (Kubin et al, 2011) TWEAK treatment 6.2 (BrdU + CMs) (Novoyatleva et al, 2010) C3orf58 treatment >2 (Ki67 + CMs, BrdU + CMs, Aurora B + CMs) (Beigi et al, 2013) Periostin treatment >5 (BrdU + CMs, pH3 + CMs, Aurora B+ CMs) (Kühn et al, 2007) FGF10 treatment 2 (Ki67 + CMs, pH3 + CMs,) (Rochais et al, 2014) Cyclin D2 overexpression >5 (MHC-nLAC + CMs) (Pasumarthi et al, 2005) Cyclin B1-CDC2 or cyclin a2 overexpression >1.4 (CMs) (Bicknell et al, 2004) (Cheng et al, 2007) Activated notch >7 (Ki67 + CMs, BrdU + CMs, Aurora B + CMs) (Campa et al, 2008) c-myc or E1A overexpression 2 (CMs) (Jackson et al, 1990) (Liu and Kitsis, 1996) CM: cardiomyocytes.…”