Pingzhu Zhou

Adult mouse epicardium modulates myocardial injury by secreting paracrine factors

The epicardium makes essential cellular and paracrine contributions to the growth of the fetal myocardium and the formation of the coronary vasculature. However, whether the epicardium has similar roles postnatally in the normal and injured heart remains enigmatic. Here, we have investigated this question using genetic fate-mapping approaches in mice. In uninjured postnatal heart, epicardial cells were quiescent. Myocardial infarction increased epicardial cell proliferation and stimulated formation of epicardium-derived cells (EPDCs), which remained in a thickened layer on the surface of the heart. EPDCs did not adopt cardiomyocyte or coronary EC fates, but rather differentiated into mesenchymal cells expressing fibroblast and smooth muscle cell markers. In vitro and in vivo assays demonstrated that EPDCs secreted paracrine factors that strongly promoted angiogenesis. In a myocardial infarction model, EPDC-conditioned medium reduced infarct size and improved heart function. Our findings indicate that epicardium modulates the cardiac injury response by conditioning the subepicardial environment, potentially offering a new therapeutic strategy for cardiac protection.

PRC2 directly methylates GATA4 and represses its transcriptional activity

Polycomb-repressive complex 2 (PRC2) promotes tissue-specific differentiation by depositing trimethylated histone H3 Lys 27 (H3K27me3) epigenetic marks to silence ectopic gene expression programs. Here, we show that EZH2, the catalytic subunit of PRC2, is required for cardiac morphogenesis. Both in vitro and in fetal hearts, EZH2 interacted with cardiac transcription factor GATA4 and directly methylated it at Lys 299. PRC2 methylation of GATA4 attenuated its transcriptional activity by reducing its interaction with and acetylation by p300. Our results reveal a new mechanism of PRC2-mediated transcriptional repression in which PRC2 methylates a transcription factor to inhibit its transcriptional activity.

Polycomb Repressive Complex 2 Regulates Normal Development of the Mouse Heart

Rationale: Epigenetic marks are crucial for organogenesis, but their role in heart development is poorly understood. Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27, which establishes H3K27me3 repressive epigenetic marks that promote tissue-specific differentiation by silencing ectopic gene programs. Objective: We studied the function of PRC2 in murine heart development using a tissue-restricted conditional inactivation strategy. Methods and Results: Inactivation of the PRC2 subunit Ezh2 by Nkx2–5Cre (Ezh2NK) caused lethal congenital heart malformations, namely, compact myocardial hypoplasia, hypertrabeculation, and ventricular septal defect. Candidate and genome-wide RNA expression profiling and chromatin immunoprecipitation analyses of Ezh2NK heart identified genes directly repressed by EZH2. Among these were the potent cell cycle inhibitors Ink4a/b (inhibitors of cyclin-dependent kinase 4 A and B), the upregulation of which was associated with decreased cardiomyocyte proliferation in Ezh2NK. EZH2-repressed genes were enriched for transcriptional regulators of noncardiomyocyte expression programs such as Pax6, Isl1, and Six1. EZH2 was also required for proper spatiotemporal regulation of cardiac gene expression, because Hcn4, Mlc2a, and Bmp10 were inappropriately upregulated in ventricular RNA. PRC2 was also required later in heart development, as indicated by cardiomyocyte-restricted TNT-Cre inactivation of the PRC2 subunit Eed. However, Ezh2 inactivation by TNT-Cre did not cause an overt phenotype, likely because of functional redundancy with Ezh1. Thus, early Ezh2 inactivation by Nk2–5Cre caused later disruption of cardiomyocyte gene expression and heart development. Conclusions: Our study reveals a previously undescribed role of EZH2 in regulating heart formation and shows that perturbation of the epigenetic landscape early in cardiogenesis has sustained disruptive effects at later developmental stages.

Association of Dnmt3a and thymine DNA glycosylase links DNA methylation with base-excision repair

While methylcytosines serve as the fifth base encoding epigenetic information, they are also a dangerous endogenous mutagen due to their intrinsic instability. Methylcytosine undergoes spontaneous deamination, at a rate much higher than cytosine, to generate thymine. In mammals, two repair enzymes, thymine DNA glycosylase (TDG) and methyl-CpG binding domain 4 (MBD4), have evolved to counteract the mutagenic effect of methylcytosines. Both recognize G/T mismatches arising from methylcytosine deamination and initiate base-excision repair that corrects them to G/C pairs. However, the mechanism by which the methylation status of the repaired cytosines is restored has remained unknown. We show here that the DNA methyltransferase Dnmt3a interacts with TDG. Both the PWWP domain and the catalytic domain of Dnmt3a are able to mediate the interaction with TDG at its N-terminus. The interaction affects the enzymatic activity of both proteins: Dnmt3a positively regulates the glycosylase activity of TDG, while TDG inhibits the methylation activity of Dnmt3a in vitro. These data suggest a mechanistic link between DNA repair and remethylation at sites affected by methylcytosine deamination.

Cardiac-Specific YAP Activation Improves Cardiac Function and Survival in an Experimental Murine MI Model

Rationale: Yes-associated protein (YAP), the terminal effector of the Hippo signaling pathway, is crucial for regulating embryonic cardiomyocyte proliferation. Objective: We hypothesized that YAP activation after myocardial infarction (MI) would preserve cardiac function and improve survival. Methods and Results: We used a cardiac-specific, inducible expression system to activate YAP in adult mouse heart. Activation of YAP in adult heart promoted cardiomyocyte proliferation and did not deleteriously affect heart function. Furthermore, YAP activation after MI preserved heart function and reduced infarct size. Using adeno-associated virus subtype 9 (AAV9) as a delivery vector, we expressed human YAP (hYAP) in the adult murine myocardium immediately after MI. We found that AAV9:hYAP significantly improved cardiac function and mouse survival. AAV9:hYAP did not exert its salutary effects by reducing cardiomyocyte apoptosis. Rather, AAV9:hYAP stimulated adult cardiomyocyte proliferation. Gene expression profiling indicated that AAV9:hYAP stimulated expression of cell cycle genes and promoted a less mature cardiac gene expression signature. Conclusions: Cardiac-specific YAP activation after MI mitigated myocardial injury, improved cardiac function, and enhanced survival. These findings suggest that therapeutic activation of YAP or its downstream targets, potentially through AAV-mediated gene therapy, may be a strategy to improve outcome after MI.

Cardiac-Specific YAP Activation Improves Cardiac Function and Survival in an Experimental Murine Myocardial Infarction Model

Rationale: Yes-associated protein (YAP), the terminal effector of the Hippo signaling pathway, is crucial for regulating embryonic cardiomyocyte proliferation. Objective: We hypothesized that YAP activation after myocardial infarction (MI) would preserve cardiac function and improve survival. Methods and Results: We used a cardiac-specific, inducible expression system to activate YAP in adult mouse heart. Activation of YAP in adult heart promoted cardiomyocyte proliferation and did not deleteriously affect heart function. Furthermore, YAP activation after MI preserved heart function and reduced infarct size. Using adeno-associated virus subtype 9 (AAV9) as a delivery vector, we expressed human YAP (hYAP) in the adult murine myocardium immediately after MI. We found that AAV9:hYAP significantly improved cardiac function and mouse survival. AAV9:hYAP did not exert its salutary effects by reducing cardiomyocyte apoptosis. Rather, AAV9:hYAP stimulated adult cardiomyocyte proliferation. Gene expression profiling indicated that AAV9:hYAP stimulated expression of cell cycle genes and promoted a less mature cardiac gene expression signature. Conclusions: Cardiac-specific YAP activation after MI mitigated myocardial injury, improved cardiac function, and enhanced survival. These findings suggest that therapeutic activation of YAP or its downstream targets, potentially through AAV-mediated gene therapy, may be a strategy to improve outcome after MI.

Pi3kcb Links Hippo-YAP and PI3K-AKT Signaling Pathways to Promote Cardiomyocyte Proliferation and Survival

Rationale: Yes-associated protein (YAP), the nuclear effector of Hippo signaling, regulates cellular growth and survival in multiple organs, including the heart, by interacting with TEA (transcriptional enhancer activator)-domain sequence–specific DNA-binding proteins. Recent studies showed that YAP stimulates cardiomyocyte proliferation and survival. However, the direct transcriptional targets through which YAP exerts its effects are poorly defined. Objective: To identify direct YAP targets that mediate its mitogenic and antiapoptotic effects in the heart. Methods and Results: We identified direct YAP targets by combining differential gene expression analysis in YAP gain- and loss-of-function with genome-wide identification of YAP-bound loci using chromatin immunoprecipitation and high throughput sequencing. This screen identified Pik3cb, encoding p110&bgr;, a catalytic subunit of phosphoinositol-3-kinase, as a candidate YAP effector that promotes cardiomyocyte proliferation and survival. YAP and TEA-domain occupied a conserved enhancer within the first intron of Pik3cb, and this enhancer drove YAP-dependent reporter gene expression. Yap gain- and loss-of-function studies indicated that YAP is necessary and sufficient to activate the phosphoinositol-3-kinase-Akt pathway. Like Yap, Pik3cb gain-of-function stimulated cardiomyocyte proliferation, and Pik3cb knockdown dampened YAP mitogenic activity. Reciprocally, impaired heart function in Yap loss-of-function was significantly rescued by adeno-associated virus–mediated Pik3cb expression. Conclusions: Pik3cb is a crucial direct target of YAP, through which the YAP activates phosphoinositol-3-kinase-AKT pathway and regulates cardiomyocyte proliferation and survival.

Regulation of GATA4 transcriptional activity in cardiovascular development and disease.

Transcription factors regulate formation and function of the heart, and perturbation of transcription factor expression and regulation disrupts normal heart structure and function. Multiple mechanisms regulate the level and locus-specific activity of transcription factors, including transcription, translation, subcellular localization, posttranslational modifications, and context-dependent interactions with other transcription factors, chromatin remodeling enzymes, and epigenetic regulators. The zinc finger transcription factor GATA4 is among the best-studied cardiac transcriptional factors. This review focuses on molecular mechanisms that regulate GATA4 transcriptional activity in the cardiovascular system, providing a framework to investigate and understand the molecular regulation of cardiac gene transcription by other transcription factors.

Optimization of Genome Engineering Approaches with the CRISPR/Cas9 System

Designer nucleases such as TALENS and Cas9 have opened new opportunities to scarlessly edit the mammalian genome. Here we explored several parameters that influence Cas9-mediated scarless genome editing efficiency in murine embryonic stem cells. Optimization of transfection conditions and enriching for transfected cells are critical for efficiently recovering modified clones. Paired gRNAs and wild-type Cas9 efficiently create programmed deletions, which facilitate identification of targeted clones, while paired gRNAs and the Cas9D10A nickase generated smaller targeted indels with lower chance of off-target mutagenesis. Genome editing is also useful for programmed introduction of exogenous DNA sequences at a target locus. Increasing the length of the homology arms of the homology-directed repair template strongly enhanced targeting efficiency, while increasing the length of the DNA insert reduced it. Together our data provide guidance on optimal design of scarless gene knockout, modification, or knock-in experiments using Cas9 nuclease.

Interrogating translational efficiency and lineage-specific transcriptomes using ribosome affinity purification

Significance We developed reagents and approaches to pull down ribosome-associated RNAs from Cre-labeled cells. We show that this strategy is useful to probe cell type-specific gene expression and the extent of transcript binding to ribosomes. Transcriptional profiling is a useful strategy to study development and disease. Approaches to isolate RNA from specific cell types, or from specific cellular compartments, would extend the power of this strategy. Previous work has shown that isolation of genetically tagged ribosomes (translating ribosome affinity purification; TRAP) is an effective means to isolate ribosome-bound RNA selectively from transgene-expressing cells. However, widespread application of this technology has been limited by available transgenic mouse lines. Here we characterize a TRAP allele (Rosa26fsTRAP) that makes this approach more widely accessible. We show that endothelium-specific activation of Rosa26fsTRAP identifies endothelial cell-enriched transcripts, and that cardiomyocyte-restricted TRAP is a useful means to identify genes that are differentially expressed in cardiomyocytes in a disease model. Furthermore, we show that TRAP is an effective means for studying translational regulation, and that several nuclear-encoded mitochondrial genes are under strong translational control. Our analysis of ribosome-bound transcripts also shows that a subset of long intergenic noncoding RNAs are weakly ribosome-bound, but that the majority of noncoding RNAs, including most long intergenic noncoding RNAs, are ribosome-bound to the same extent as coding transcripts. Together, these data show that the TRAP strategy and the Rosa26fsTRAP allele will be useful tools to probe cell type-specific transcriptomes, study translational regulation, and probe ribosome binding of noncoding RNAs.