Regulation of Notch signaling in the heart by epigenetic modifications

Synopsis: Understanding the molecular mechanisms regulating cardiac cell proliferation during the embryonic, fetal and adult life is of paramount importance in view of developing innovative strategies aimed at inducing myocardial regeneration after cardiac damage. The Notch pathway plays a key role in the regulation of cardiomyocyte proliferation during mammalian embryonic life. Moreover, it is essentially involved in the cardiac regeneration process after injury in Zebrafish. Therefore, we assessed the efficacy of Notch pathway activation to sustain cardiac regeneration in a model of myocardial infarction in mice. During early postnatal life, cardiomyocytes exit the cell cycle. We demonstrated that this event is paralleled by a decrease of Notch signaling and by the establishment of a repressive chromatin environment at Notch target genes, characterized by Polycomb Group protein 2-mediated silencing. The stimulation of the Notch pathway through Adeno-associated virus-mediated gene transfer of activated Notch1 or of the soluble form of the ligand Jagged1 prolonged the capacity of cardiomyocytes to replicate, which correlated with an increased rate of Notch target gene expression and the maintenance of an open chromatin conformation at Notch target gene promoters. However, the same vectors were ineffective in stimulating cardiac repair in a model of myocardial infarction in adult mice, despite efficient transgene expression. We identified the molecular cause of the lack of action of Notch signaling stimulation in adults in the increased DNA methylation at Notch target gene promoters, which correlated with permanent switch off of the Notch pathway. Our results confirm that the Notch pathway is an important regulator of neonata adults, due to the permanent epigenetic modifications at the DNA level at Notch responsive genes l

Reversible Notch1 acetylation tunes proliferative signalling in cardiomyocytes

Aims: The Notch signalling pathway regulates the balance between proliferation and differentiation in several tissues, including the heart. Our previous work has demonstrated that the proliferative potential of neonatal cardiomyocytes relies on Notch1 activity. A deep investigation on the biochemical regulation of the Notch signalling in cardiomyocytes is the focus of the current research. Methods and results: We show that the Notch1 intracellular domain is acetylated in proliferating neonatal rat cardiomyocytes and that acetylation tightly controls the amplitude and duration of Notch signalling. We found that acetylation extends the half-life of the protein, and enhanced its transcriptional activity, therefore counteracting apoptosis and sustaining cardiomyocyte proliferation. Sirt1 acted as a negative modulator of Notch1 signalling; its overexpression in cardiomyocytes reverted Notch acetylation and dampened its stability. A constitutively acetylated fusion protein between Notch1 and the acetyltransferase domain of p300 promoted cardiomyocyte proliferation, which was remarkably sustained over time. Viral vector-mediated expression of this protein enhanced heart regeneration after apical resection in neonatal mice. Conclusion: These results identify the reversible acetylation of Notch1 as a novel mechanism to modulate its signalling in the heart and tune the proliferative potential of cardiomyocytes

Epigenetic Modification at Notch Responsive Promoters Blunts Efficacy of Inducing Notch Pathway Reactivation After Myocardial Infarction

Rationale: The Notch pathway plays a key role in stimulating mammalian cardiomyocyte proliferation during development and in the early postnatal life; in adult zebrafish, reactivation of this pathway is also essential to drive cardiac regeneration after injury. Objective: We wanted to assess efficacy of Notch pathway stimulation in neonatal and adult hearts as a means to induce cardiac regeneration after myocardial infarction in mice. Methods and Results: In early postnatal life, cardiomyocyte exit from the cell cycle was paralleled by decreased Notch signaling and the establishment of a repressive chromatin environment at Notch-responsive genes, characterized by recruitment of the polycomb group enhancer of zeste homolog 2 methyltransferase and the acquisition of the histone 3 Lysine 27 trimethylation histone mark, as detected by chromatin immunoprecipitation. Forced Notch pathway activation by adenoassociated virus gene transfer of activated Notch1 or its ligand Jagged1 expanded the proliferative capacity of neonatal cardiomyocytes; this correlated with increased transcription of Notch target genes and maintenance of an open chromatin conformation at their promoters. The same adenoassociated virus vectors, however, were largely ineffective in stimulating cardiac repair after myocardial infarction in adult mice, despite optimal and long-lasting transgene expression. Analysis of Notch-responsive promoters in adult cardiomyocytes showed marks of repressed chromatin and irreversible CpG DNA methylation. Induction of adult cardiomyocyte re-entry into the cell cycle with microRNAs was independent from Notch pathway reactivation. Conclusions: Notch pathway activation is crucial in regulating cardiomyocyte proliferation during the early postnatal life, but it is largely ineffective in driving cardiac regeneration in adults, because of permanent epigenetic modification at Notch-responsive promoters.</p