Blockade to pathological remodeling of infarcted heart tissue using a porcupine antagonist

The secreted Wnt signaling molecules are essential to the coordination of cell-fate decision making in multicellular organisms. In adult animals, the secreted Wnt proteins are critical for tissue regeneration and frequently contribute to cancer. Small molecules that disable the Wnt acyltransferase Porcupine (Porcn) are candidate anticancer agents in clinical testing. Here we have systematically assessed the effects of the Porcn inhibitor (WNT-974) on the regeneration of several tissue types to identify potentially unwanted chemical effects that could limit the therapeutic utility of such agents. An unanticipated observation from these studies is proregenerative responses in heart muscle induced by systemic chemical suppression of Wnt signaling. Using in vitro cultures of several cell types found in the heart, we delineate the Wnt signaling apparatus supporting an antiregenerative transcriptional program that includes a subunit of the nonfibrillar collagen VI. Similar to observations seen in animals exposed to WNT-974, deletion of the collagen VI subunit, COL6A1, has been shown to decrease aberrant remodeling and fibrosis in infarcted heart tissue. We demonstrate that WNT-974 can improve the recovery of heart function after left anterior descending coronary artery ligation by mitigating adverse remodeling of infarcted tissue. Injured heart tissue exposed to WNT-974 exhibits decreased scarring and reduced Col6 production. Our findings support the development of Porcn inhibitors as antifibrotic agents that could be exploited to promote heart repair following injury

A calcineurin–Hoxb13 axis regulates growth mode of mammalian cardiomyocytes

10.1038/s41586-020-2228-6Nature5827811271-27

Derivation and characterization of zebrafish embryonic germ cell cultures and the effects of kit ligand a

A stem cell-mediated gene targeting approach is currently not available for zebrafish. To address this problem our lab has developed methods for the culture of pluripotent stem cells derived from early-stage zebrafish embryos. Embryonic stem (ES) cell cultures were derived from blastula-stage zebrafish embryos and primary cultures of pluripotent embryonic germ (EG) cells were initiated from primordial germ cells (PGCs). To be useful for gene targeting studies, optimization of the culture conditions is required to maintain the stem cells in a pluripotent condition for multiple passages. Also methods must be established to introduce targeting plasmids into the cells by homologous recombination and selection strategies are needed to isolate colonies of cells that have undergone the targeting event. In this study, I have developed methods to initiate multiple-passage EG cell cultures derived from zebrafish PGCs. A transgenic approach involving drug selection was used to isolate homogeneous populations of PGCs for cell culture. Optimization of the PGC culture conditions using recombinant zebrafish growth factors demonstrated that Kit ligand a and b (Kitlga and Kitlgb) and stromal cell-derived factor 1a and 1b (Sdf-1a and Sdf-1b) promoted the in vitro growth of the PGCs. Although PGCs do not express kit receptor in vivo, in culture the cells expressed the receptor and responded to recombinant zebrafish Kitlga added to the medium. After several days in the presence of these factors, the PGC cultures began to exhibit in vitro characteristics of pluripotent EG cells. Kitlga was especially important for promoting the expression of pluripotency markers including nanos, alkaline phosphatase and SSEA-1. The EG cells also began to express endogenous kitlga and inhibition of its expression or function with antisensense morpholinos or MAP kinase inhibitors demonstrated that the factor was important for maintaining pluripotency. In a separate project, gene targeting methods were established for zebrafish ES cell cultures. The results of these studies demonstrated that the ES cells are able to incorporate plasmid in a targeted fashion by homologous recombination. Two selection strategies were used to isolate ES cell colonies that contained targeted plasmid insertions in either the no tail or myostatin I gene

Zebrafish Primordial Germ Cell Cultures Derived from vasa::RFP Transgenic Embryos

Although embryonic germ (EG) cell-mediated gene transfer has been successful in the mouse for more than a decade, this approach is limited in other species due to the difficulty of isolating the small numbers of progenitors of germ cell lineage (PGCs) from early-stage embryos and the lack of information on the in vitro culture requirements of the cells. In this study, methods were established for the culture of PGCs obtained from zebrafish embryos. Transgenic embryos that express the red fluorescent protein (RFP) under the control of the PGC-specific vasa promoter were used, making it possible to isolate pure populations of PGCs by fluorescence-activated cell sorting (FACS) and to optimize the culture conditions by counting the number of fluorescent PGC colonies produced in different media. Cultures initiated from 26-somite-stage embryos contained the highest percentage of PGCs that proliferated in vitro to generate colonies. The effect of growth factors, including Kit ligand a and b (Kitlga and Kitlgb) and stromal cell-derived factor 1a and 1b (Sdf-1a and Sdf-1b), on PGC proliferation was studied. Optimal in vitro growth and survival of the zebrafish PGCs was achieved when recombinant Kitlga and Sdf-1b were added to the culture medium through transfected feeder cells, resulting in a doubling of the number of PGC colonies. Results from RT-PCR and in situ hybridization analysis demonstrated that PGCs maintained in culture expressed the kita receptor, even though receptor expression was not detected in PGCs isolated by FACS directly from dissociated embryos. In optimal growth conditions, the PGCs continued to proliferate for at least 4 months in culture. The capacity to establish long-term PGC cultures from zebrafish will make it possible to conduct in vitro studies of germ cell differentiation and EG cell pluripotency in this model species and may be valuable for the development of a cell-mediated gene transfer approach

The Oxygen-Rich Postnatal Environment Induces Cardiomyocyte Cell-Cycle Arrest through DNA Damage Response

The mammalian heart has a remarkable regenerative capacity for a short period of time after birth, after which the majority of cardiomyocytes permanently exit cell cycle. We sought to determine the primary postnatal event that results in cardiomyocyte cell-cycle arrest. We hypothesized that transition to the oxygen-rich postnatal environment is the upstream signal that results in cell-cycle arrest of cardiomyocytes. Here, we show that reactive oxygen species (ROS), oxidative DNA damage, and DNA damage response (DDR) markers significantly increase in the heart during the first postnatal week. Intriguingly, postnatal hypoxemia, ROS scavenging, or inhibition of DDR all prolong the postnatal proliferative window of cardiomyocytes, whereas hyperoxemia and ROS generators shorten it. These findings uncover a protective mechanism that mediates cardiomyocyte cell-cycle arrest in exchange for utilization of oxygen-dependent aerobic metabolism. Reduction of mitochondrial-dependent oxidative stress should be an important component of cardiomyocyte proliferation-based therapeutic approaches