Todd Heallen

Hippo Pathway Inhibits Wnt Signaling to Restrain Cardiomyocyte Proliferation and Heart Size

Heart size is controlled through an antagonistic interaction between Hippo and Wnt signaling pathways. Genetic regulation of mammalian heart size is poorly understood. Hippo signaling represents an organ-size control pathway in Drosophila, where it also inhibits cell proliferation and promotes apoptosis in imaginal discs. To determine whether Hippo signaling controls mammalian heart size, we inactivated Hippo pathway components in the developing mouse heart. Hippo-deficient embryos had overgrown hearts with elevated cardiomyocyte proliferation. Gene expression profiling and chromatin immunoprecipitation revealed that Hippo signaling negatively regulates a subset of Wnt target genes. Genetic interaction studies indicated that β-catenin heterozygosity suppressed the Hippo cardiomyocyte overgrowth phenotype. Furthermore, the Hippo effector Yap interacts with β-catenin on Sox2 and Snai2 genes. These data uncover a nuclear interaction between Hippo and Wnt signaling that restricts cardiomyocyte proliferation and controls heart size.

Hippo signaling impedes adult heart regeneration

Heart failure due to cardiomyocyte loss after ischemic heart disease is the leading cause of death in the United States in large part because heart muscle regenerates poorly. The endogenous mechanisms preventing mammalian cardiomyocyte regeneration are poorly understood. Hippo signaling, an ancient organ size control pathway, is a kinase cascade that inhibits developing cardiomyocyte proliferation but it has not been studied postnatally or in fully mature adult cardiomyocytes. Here, we investigated Hippo signaling in adult cardiomyocyte renewal and regeneration. We found that unstressed Hippo-deficient adult mouse cardiomyocytes re-enter the cell cycle and undergo cytokinesis. Moreover, Hippo deficiency enhances cardiomyocyte regeneration with functional recovery after adult myocardial infarction as well as after postnatal day eight (P8) cardiac apex resection and P8 myocardial infarction. In damaged hearts, Hippo mutant cardiomyocytes also have elevated proliferation. Our findings reveal that Hippo signaling is an endogenous repressor of adult cardiomyocyte renewal and regeneration. Targeting the Hippo pathway in human disease might be beneficial for the treatment of heart disease.

Actin cytoskeletal remodeling with protrusion formation is essential for heart regeneration in Hippo-deficient mice.

Inhibiting the Hippo pathway could promote heart regeneration by encouraging heart cells to move into injured areas. Helping cardiomyocytes fill in a broken heart Activation of the Hippo pathway prevents the overgrowth of organs, such as the heart, by inhibiting the activity of the transcriptional coactivator Yap. This function of the Hippo pathway is important during development, but limits the ability of some organs to regenerate after injury. Morikawa et al. found that Yap target genes not only included cell cycle genes but also genes encoding cytoskeletal remodeling proteins or proteins that link the cytoskeleton to the extracellular matrix. Unlike cultured cardiomyocytes from control mice, those from Hippo signaling–deficient mice migrated through collagen, a key component of the extracellular matrix in the heart. Cardiomyocytes in these mice formed cellular protrusions typical of migrating cells and more readily moved toward scar sites after cardiac injury. Thus, inhibiting the Hippo pathway could help with heart regeneration. The mammalian heart regenerates poorly, and damage commonly leads to heart failure. Hippo signaling is an evolutionarily conserved kinase cascade that regulates organ size during development and prevents adult mammalian cardiomyocyte regeneration by inhibiting the transcriptional coactivator Yap, which also responds to mechanical signaling in cultured cells to promote cell proliferation. To identify Yap target genes that are activated during cardiomyocyte renewal and regeneration, we performed Yap chromatin immunoprecipitation sequencing (ChIP-Seq) and mRNA expression profiling in Hippo signaling–deficient mouse hearts. We found that Yap directly regulated genes encoding cell cycle progression proteins, as well as genes encoding proteins that promote F-actin polymerization and that link the actin cytoskeleton to the extracellular matrix. Included in the latter group were components of the dystrophin glycoprotein complex, a large molecular complex that, when defective, results in muscular dystrophy in humans. Cardiomyocytes near the scar tissue of injured Hippo signaling–deficient mouse hearts showed cellular protrusions suggestive of cytoskeletal remodeling. The hearts of mdx mutant mice, which lack functional dystrophin and are a model for muscular dystrophy, showed impaired regeneration and cytoskeleton remodeling, but normal cardiomyocyte proliferation, after injury. Our data showed that, in addition to genes encoding cell cycle progression proteins, Yap regulated genes that enhance cytoskeletal remodeling. Thus, blocking the Hippo pathway input to Yap may tip the balance so that Yap responds to mechanical changes associated with heart injury to promote repair.

Hippo Coactivator YAP1 Upregulates SOX9 and Endows Esophageal Cancer Cells with Stem-like Properties

Cancer stem cells (CSC) are purported to initiate and maintain tumor growth. Deregulation of normal stem cell signaling may lead to the generation of CSCs; however, the molecular determinants of this process remain poorly understood. Here we show that the transcriptional coactivator YAP1 is a major determinant of CSC properties in nontransformed cells and in esophageal cancer cells by direct upregulation of SOX9. YAP1 regulates the transcription of SOX9 through a conserved TEAD binding site in the SOX9 promoter. Expression of exogenous YAP1 in vitro or inhibition of its upstream negative regulators in vivo results in elevated SOX9 expression accompanied by the acquisition of CSC properties. Conversely, shRNA-mediated knockdown of YAP1 or SOX9 in transformed cells attenuates CSC phenotypes in vitro and tumorigenicity in vivo. The small-molecule inhibitor of YAP1, verteporfin, significantly blocks CSC properties in cells with high YAP1 and a high proportion of ALDH1(+). Our findings identify YAP1-driven SOX9 expression as a critical event in the acquisition of CSC properties, suggesting that YAP1 inhibition may offer an effective means of therapeutically targeting the CSC population.

Pitx2 promotes heart repair by activating the antioxidant response after cardiac injury

Summary Myocardial infarction results in compromised myocardial function with heart failure due to insufficient cardiomyocyte self-renewal1. Unlike lower vertebrates, mammalian hearts only have a transient neonatal renewal capacity2. Reactivating primitive reparative ability in the mature heart requires knowledge of the mechanisms promoting early heart repair. By testing an established Hippo-deficient heart regeneration model for renewal promoting factors, we found that Pitx2 expression was induced in injured, Hippo-deficient ventricles. Pitx2-deficient neonatal hearts failed to repair after apex resection while Pitx2-gain-of-function in adult cardiomyocytes conferred reparative ability after myocardial infarction. Genomic analyses indicated that Pitx2 activated genes encoding electron transport chain components and reactive oxygen species scavengers. A subset of Pitx2 target genes was cooperatively regulated with the Hippo effector, Yap. Furthermore, Nrf2, a regulator of antioxidant response3, directly regulated Pitx2 expression and subcellular localization. Pitx2 mutant myocardium had elevated reactive oxygen species levels while antioxidant supplementation suppressed the Pitx2-loss-of-function phenotype. These findings reveal a genetic pathway, activated by tissue damage that is essential for cardiac repair.

Dystrophin–glycoprotein complex sequesters Yap to inhibit cardiomyocyte proliferation

The regenerative capacity of the adult mammalian heart is limited, because of the reduced ability of cardiomyocytes to progress through mitosis. Endogenous cardiomyocytes have regenerative capacity at birth but this capacity is lost postnatally, with subsequent organ growth occurring through cardiomyocyte hypertrophy. The Hippo pathway, a conserved kinase cascade, inhibits cardiomyocyte proliferation in the developing heart to control heart size and prevents regeneration in the adult heart. The dystrophin–glycoprotein complex (DGC), a multicomponent transmembrane complex linking the actin cytoskeleton to extracellular matrix, is essential for cardiomyocyte homeostasis. DGC deficiency in humans results in muscular dystrophy, including the lethal Duchenne muscular dystrophy. Here we show that the DGC component dystroglycan 1 (Dag1) directly binds to the Hippo pathway effector Yap to inhibit cardiomyocyte proliferation in mice. The Yap–Dag1 interaction was enhanced by Hippo-induced Yap phosphorylation, revealing a connection between Hippo pathway function and the DGC. After injury, Hippo-deficient postnatal mouse hearts maintained organ size control by repairing the defect with correct dimensions, whereas postnatal hearts deficient in both Hippo and the DGC showed cardiomyocyte overproliferation at the injury site. In the hearts of mature Mdx mice (which have a point mutation in Dmd)—a model of Duchenne muscular dystrophy—Hippo deficiency protected against overload-induced heart failure.

Hippo pathway deficiency reverses systolic heart failure after infarction

Mammalian organs vary widely in regenerative capacity. Poorly regenerative organs, such as the heart are particularly vulnerable to organ failure. Once established, heart failure commonly results in mortality. The Hippo pathway, a kinase cascade that prevents adult cardiomyocyte proliferation and regeneration, is upregulated in human heart failure. Here we show that deletion of the Hippo pathway component Salvador (Salv) in mouse hearts with established ischaemic heart failure after myocardial infarction induces a reparative genetic program with increased scar border vascularity, reduced fibrosis, and recovery of pumping function compared with controls. Using translating ribosomal affinity purification, we isolate cardiomyocyte-specific translating messenger RNA. Hippo-deficient cardiomyocytes have increased expression of proliferative genes and stress response genes, such as the mitochondrial quality control gene, Park2. Genetic studies indicate that Park2 is essential for heart repair, suggesting a requirement for mitochondrial quality control in regenerating myocardium. Gene therapy with a virus encoding Salv short hairpin RNA improves heart function when delivered at the time of infarct or after ischaemic heart failure following myocardial infarction was established. Our findings indicate that the failing heart has a previously unrecognized reparative capacity involving more than cardiomyocyte renewal.

The Hippo pathway in the heart: pivotal roles in development, disease, and regeneration

The Hippo–YAP (Yes-associated protein) pathway is an evolutionarily and functionally conserved regulator of organ size and growth with crucial roles in cell proliferation, apoptosis, and differentiation. This pathway has great potential for therapeutic manipulation in different disease states and to promote organ regeneration. In this Review, we summarize findings from the past decade revealing the function and regulation of the Hippo–YAP pathway in cardiac development, growth, homeostasis, disease, and regeneration. In particular, we highlight the roles of the Hippo–YAP pathway in endogenous heart muscle renewal, including the pivotal role of the Hippo–YAP pathway in regulating cardiomyocyte proliferation and differentiation, stress response, and mechanical signalling. The human heart lacks the capacity to self-repair; therefore, the loss of cardiomyocytes after injury such as myocardial infarction can result in heart failure and death. Despite substantial advances in the treatment of heart failure, an enormous unmet clinical need exists for alternative treatment options. Targeting the Hippo–YAP pathway has tremendous potential for developing therapeutic strategies for cardiac repair and regeneration for currently intractable cardiovascular diseases such as heart failure. The lessons learned from cardiac repair and regeneration studies will also bring new insights into the regeneration of other tissues with limited regenerative capacity.This Review summarizes the current understanding on the roles of the Hippo–YAP pathway in cardiac development, growth, homeostasis, disease, and regeneration, with a particular focus on the roles of the Hippo–YAP pathway in endogenous cardiac muscle renewal, including the pivotal role of this pathway in regulating cardiomyocyte proliferation, differentiation, stress response, and mechanical signalling.Key pointsThe Hippo–YAP (Yes-associated protein) pathway is an evolutionarily conserved pathway that controls organ size.Hippo signalling restrains cardiomyocyte proliferation during development to control cardiac size.The Hippo–YAP pathway regulates the activity of growth pathways during prenatal and postnatal life and is important for cardiac homeostasis.Hippo signalling inhibits adult cardiac regeneration.The Hippo–YAP pathway regulates various events during cardiac regeneration, including cardiomyocyte proliferation and differentiation, injury resistance, stress response, and mechanical signals.Manipulating the Hippo–YAP pathway is a potential therapeutic tool for treating cardiac injury.

Heart repair via cardiomyocyte-secreted vesicles

The sustained delivery of extracellular vesicles, secreted by induced-pluripotent-stem-cell-derived cardiomyocytes, through a hydrogel patch promotes cardiac recovery after myocardial infarction in rats.

Abstract 3896: The Hippo coactivator YAP1 upregulates SOX9 and endows cancer stem cell properties in non-transformed cells and esophageal cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Cancer stem cells are proposed to initiate and maintain tumor growth. Dysregulation of normal stem cell signaling may lead to the generation of cancer stem cells (CSCs), however, the molecular determinants of this process remain poorly understood. Here we show that the transcriptional co-activator YAP1 through direct regulation of SOX9 promotes the generation of CSCs and that the inhibition of YAP1 and SOX9 attenuates CSC formation. SOX9 transcripts and expression are upregulated upon YAP1 activation and several lines of evidence indicate that SOX9 is a direct target of YAP1. The Chromatin Immunoprecipitation analysis and luciferase assays demonstrate direct binding of YAP1 to the SOX9 promoter through a conserved TEAD binding site. Mutation of this site abrogates transcriptional regulation of SOX9 by YAP1 and Tead2. Functional studies demonstrate that YAP1 regulation of SOX9 is necessary and sufficient to confer CSC properties and tumorigenesis in vitro and in vivo. The small molecule inhibitor of YAP1, Verteporfin (VP) significantly blocks CSC self-renewal properties in cells with high YAP1 and a high proportion of the CSC marker aldehyde dehydrogenase 1 (ALDH1) indicating that VP targets the CSC population. These data identify YAP1 as a driver of esophageal cancer (EC) stem cells, in part, by regulation of SOX9 and suggest that pharmacological inhibition of YAP1 may be an effective means of specifically targeting EC stem cells. Citation Format: Shumei Song, Jaffer A. Ajani, Soichiro Honjo, Dipen M. Maru, Qiongrong Chen, Jiankang Jin, Ailing W. Scott, Todd R. Heallen, Lianchun Xiao, Wayne L. Hofstetter, Brian Weston, Jeffrey H. Lee, Roopma Wadhwa, Kazuki Sudo, James F. Martin, John R. Stroehlein, Mien-Chie Hung, Randy L. Johnson. The Hippo coactivator YAP1 upregulates SOX9 and endows cancer stem cell properties in non-transformed cells and esophageal cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3896. doi:10.1158/1538-7445.AM2014-3896