Btg1 and Btg2 regulate neonatal cardiomyocyte cell cycle arrest.

Rodent cardiomyocytes undergo mitotic arrest in the first postnatal week. Here, we investigate the role of transcriptional co-regulator Btg2 (B-cell translocation gene 2) and functionally-similar homolog Btg1 in postnatal cardiomyocyte cell cycling and maturation. Btg1 and Btg2 (Btg1/2) are expressed in neonatal C57BL/6 mouse left ventricles coincident with cardiomyocyte cell cycle arrest. Btg1/2 constitutive double knockout (DKO) mouse hearts exhibit increased pHH3+ mitotic cardiomyocytes compared to Wildtype at postnatal day (P)7, but not at P30. Similarly, neonatal AAV9-mediated Btg1/2 double knockdown (DKD) mouse hearts exhibit increased EdU+ mitotic cardiomyocytes compared to Scramble AAV9-shRNA controls at P7, but not at P14. In neonatal rat ventricular myocyte (NRVM) cultures, siRNA-mediated Btg1/2 single and double knockdown cohorts showed increased EdU+ cardiomyocytes compared to Scramble siRNA controls, without increase in binucleation or nuclear DNA content. RNAseq analyses of Btg1/2-depleted NRVMs support a role for Btg1/2 in inhibiting cell proliferation, and in modulating reactive oxygen species response pathways, implicated in neonatal cardiomyocyte cell cycle arrest. Together, these data identify Btg1 and Btg2 as novel contributing factors in mammalian cardiomyocyte cell cycle arrest after birth.

Transcriptional Regulation of Postnatal Cardiomyocyte Maturation and Regeneration.

During the postnatal period, mammalian cardiomyocytes undergo numerous maturational changes associated with increased cardiac function and output, including hypertrophic growth, cell cycle exit, sarcomeric protein isoform switching, and mitochondrial maturation. These changes come at the expense of loss of regenerative capacity of the heart, contributing to heart failure after cardiac injury in adults. While most studies focus on the transcriptional regulation of embryonic or adult cardiomyocytes, the transcriptional changes that occur during the postnatal period are relatively unknown. In this review, we focus on the transcriptional regulators responsible for these aspects of cardiomyocyte maturation during the postnatal period in mammals. By specifically highlighting this transitional period, we draw attention to critical processes in cardiomyocyte maturation with potential therapeutic implications in cardiovascular disease.

Lens fiber cell differentiation occurs independently of fibroblast growth factor receptor signaling in the absence of Pten.

Fibroblast growth factor receptor (FGFR) signaling patterns multiple tissues in both vertebrates and invertebrates, largely through the activation of intracellular kinases. Recent studies have demonstrated that the phosphatase, PTEN negatively regulates FGFR signaling, such that the loss of PTEN can compensate for reduced FGFR signaling to rescue aspects of normal development. In the developing mouse lens, FGFR signaling promotes cell survival and fiber cell differentiation, and the loss of Pten largely compensates for the loss of Fgfr2 during lens development. To explore this regulatory relationship further, we focused on the phenotypic consequences of Pten loss on lens development and fiber cell differentiation in the absence of all FGFR signaling, both in vivo and in lens epithelial explants. Pten deletion partially rescues primary fiber cell elongation and γ-crystallin accumulation in FGFR-deficient lenses in vivo but fails to rescue cell survival or proliferation. However, in lens epithelial explants, where cells survive without FGFR signaling, Pten deletion rescues vitreous humor-induced lens fiber cell differentiation in the combined absence of Fgfr1, Fgfr2 and Fgfr3. This represents the first evidence that vitreous-initiated signaling cascades, independent of FGFR signaling, can drive mammalian lens fiber cell differentiation, when freed from repression by PTEN.

High-throughput transcriptome analysis reveals that the loss of Pten activates a novel NKX6-1/RASGRP1 regulatory module to rescue microphthalmia caused by Fgfr2-deficient lenses.

FGFR signaling is critical to development and disease pathogenesis, initiating phosphorylation-driven signaling cascades, notably the RAS-RAF-MEK-ERK and PI3 K-AKT cascades. PTEN antagonizes FGFR signaling by reducing AKT and ERK activation. Mouse lenses lacking FGFR2 exhibit microphakia and reduced ERK and AKT phosphorylation, widespread apoptosis, and defective lens fiber cell differentiation. In contrast, simultaneous deletion of both Fgfr2 and Pten restores ERK and AKT activation levels as well as lens size, cell survival and aspects of fiber cell differentiation; however, the molecular basis of this "rescue" remains undefined. We performed transcriptomic analysis by RNA sequencing of mouse lenses with conditional deletion of Fgfr2, Pten or both Fgfr2 and Pten, which reveal new molecular mechanisms that uncover how FGFR2 and PTEN signaling interact during development. The FGFR2-deficient lens transcriptome demonstrates overall loss of fiber cell identity with deregulated expression of 1448 genes. We find that ~ 60% of deregulated genes return to normal expression levels in lenses lacking both Fgfr2 and Pten. Further, application of customized filtering parameters to these RNA-seq data sets identified 68 high-priority candidate genes. Bioinformatics analyses showed that the cis-binding motif of a high-priority homeodomain transcription factor, NKX6-1, was present in the putative promoters of ~ 78% of these candidates. Finally, biochemical reporter assays demonstrate that NKX6-1 activated the expression of the high-priority candidate Rasgrp1, a RAS-activating protein. Together, these data define a novel regulatory module in which NKX6-1 directly activates Rasgrp1 expression to restore the balance of ERK and AKT activation, thus providing new insights into alternate regulation of FGFR downstream events.