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.

A calcineurin-Hoxb13 axis regulates growth mode of mammalian cardiomyocytes.

A major factor in the progression to heart failure in humans is the inability of the adult heart to repair itself after injury. We recently demonstrated that the early postnatal mammalian heart is capable of regeneration following injury through proliferation of preexisting cardiomyocytes1,2 and that Meis1, a three amino acid loop extension (TALE) family homeodomain transcription factor, translocates to cardiomyocyte nuclei shortly after birth and mediates postnatal cell cycle arrest3. Here we report that Hoxb13 acts as a cofactor of Meis1 in postnatal cardiomyocytes. Cardiomyocyte-specific deletion of Hoxb13 can extend the postnatal window of cardiomyocyte proliferation and reactivate the cardiomyocyte cell cycle in the adult heart. Moreover, adult Meis1-Hoxb13 double-knockout hearts display widespread cardiomyocyte mitosis, sarcomere disassembly and improved left ventricular systolic function following myocardial infarction, as demonstrated by echocardiography and magnetic resonance imaging. Chromatin immunoprecipitation with sequencing demonstrates that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and cell cycle. Finally, we show that the calcium-activated protein phosphatase calcineurin dephosphorylates Hoxb13 at serine-204, resulting in its nuclear localization and cell cycle arrest. These results demonstrate that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and proliferation and provide mechanistic insights into the link between hyperplastic and hypertrophic growth of cardiomyocytes.

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.

Diverse chemical scaffolds support direct inhibition of the membrane-bound O-acyltransferase porcupine.

Secreted Wnt proteins constitute one of the largest families of intercellular signaling molecules in vertebrates with essential roles in embryonic development and adult tissue homeostasis. The functional redundancy of Wnt genes and the many forms of cellular responses they elicit, including some utilizing the transcriptional co-activator ß-catenin, has limited the ability of classical genetic strategies to uncover their roles in vivo. We had previously identified a chemical compound class termed Inhibitor of Wnt Production (or IWP) that targets Porcupine (Porcn), an acyltransferase catalyzing the addition of fatty acid adducts onto Wnt proteins. Here we demonstrate that diverse chemical structures are able to inhibit Porcn by targeting its putative active site. When deployed in concert with small molecules that modulate the activity of Tankyrase enzymes and glycogen synthase kinase 3 ß (GSK3ß), additional transducers of Wnt/ß-catenin signaling, the IWP compounds reveal an essential role for Wnt protein fatty acylation in eliciting ß-catenin-dependent and -independent forms of Wnt signaling during zebrafish development. This collection of small molecules facilitates rapid dissection of Wnt gene function in vivo by limiting the influence of redundant Wnt gene functions on phenotypic outcomes and enables temporal manipulation of Wnt-mediated signaling in vertebrates.

Longitudinal assessment of B-RAF V595E levels in the peripheral cell-free tumor DNA of a 10-year-old spayed female Korean Jindo dog with unresectable metastatic urethral transitional cell carcinoma for monitoring the treatment response to a RAF inhibitor (sorafenib).

Transitional cell carcinoma (TCC) is the most common malignant tumor of the canine urinary tract. In this case study, a dog with metastatic urethral TCC was treated with sorafenib. The tumor expression levels of receptor tyrosine kinase genes, including VEGFR-1, VEGFR-2, PDGFR-α, PDGFR-ß, ALK, EGFR, ErbB2, and B-RAF, were analyzed. VEGFR was overexpressed in tumor tissues compared to the normal tissues. Considering the high frequency of B-RAF mutation in canine urological tumors, the B-RAF gene was examined, and the B-RAF V595E mutation was detected in the tumor tissue. Therefore, the antitumor effect of sorafenib, a multi-tyrosine kinase inhibitor, on unresectable metastatic urethral TCC characterized by B-RAF V595E was evaluated and circulating cell-free tumor DNA (ctDNA) was assessed for monitoring the treatment response. After the initiation of oral sorafenib therapy (4 mg/kg/day escalated to 10 mg/kg/day), the dysuria was alleviated gradually, and the patient remained stable for 3 months. During that treatment period, the patient showed various levels of changes associated with B-RAF V595E mutation in ctDNA as evident from longitudinal plasma samples after initiation of sorafenib therapy. The findings of this study suggest that ctDNA may serve as a useful non-invasive tool for monitoring the treatment response to anticancer drugs.

Comparative evaluation of canine cadaver embalming methods for veterinary anatomy education.

Formalin-embalmed cadavers have been extensively used to teach anatomy. Although they ensure the preservation of anatomical structures without microbial contamination, they are considerably rigid and cannot be used to study the joint and muscle movements. Moreover, formalin irritates the eyes and airways and is carcinogenic on chronic exposure. To overcome the disadvantages of formalin-fixed cadavers, we investigated the usefulness of alternative embalming methods using saturated salt solution (SS) and Thiel's solution (TS). We compared the three solutions based on the following parameters: cost of the embalming solution; preservation of anatomical structure, color, flexibility, and texture; and microbial contamination. Convenience of anatomical structure identification and preferences in anatomical laboratory practice were evaluated using questionnaires answered by veterinary undergraduate students. Cost of the embalming solution was the lowest for formalin solution (FS) and most expensive for TS. All cadavers were successfully preserved without significant putrefaction and were useful for teaching veterinary anatomy. Cadavers embalmed with SS or TS were superior in facilitating joint and muscle movement. Compared to FS, the color and texture of muscles and internal organs were similar to those of living animals and there was no irritating and offensive smell in SS and TS cadavers. Students preferred the SS and TS cadavers for their usefulness in identification of anatomical structures, highlighting their usefulness in veterinary anatomy education.

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.

Interaction between -786TC polymorphism in the endothelial nitric oxide synthase gene and smoking for myocardial infarction in Korean population.

BACKGROUND: Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) mediates endothelium-dependent vasodilation and antithrombotic action. Controversial results regarding the association of eNOS gene polymorphisms with myocardial infarction (MI) have been reported. METHODS: A total of 932 individuals living in Seoul and the suburb, Korea, were randomly selected. Genomic DNA was prepared from blood leukocytes. A GT missense mutation in exon 7 (894GT) was screened using PCR-RFLP analysis. The genotypes of 3 mutations (-786TC, -922AG, and -1468TA) in the 5'-flanking region were determined by a minisequencing protocol (SNaPshot), respectively. RESULTS: Pair-wise linkage analysis revealed that 3 mutations of -786TC, -922AG, and -1468TA were completely linked with each other (mid R:D'mid R:=1, r(2)=0.96-1.0). Furthermore, each of these mutant alleles (-786C, -922G, or -1468A), but not 894T allele, was associated with the risk of MI. Multiple logistic regression analysis revealed that each of these mutant alleles was a predictive independent risk factor for the risk of MI (odds ratio, 1.69 for dominant effects, P<0.05) after age and sex adjustments. Smoking further increased the odds ratio by 2.04 for the risk of MI when it was combined with the mutant alleles. CONCLUSION: Each of 3 mutations (-786TC, -922AG, or -1468TA) in the 5'-flanking region of eNOS gene may play a role in the pathogenesis of MI in Korean population, and also provides an evidence for a significant interaction between these mutations and smoking.

Biochemical Analysis of Tankyrase Activity in Zebrafish In Vitro and In Vivo.

The activity of tankyrase (Tnks) enzymes modulates the activity of the ß-catenin destruction complex in the Wnt/ß-catenin signaling pathway. Here, we describe a method for determining the accessibility of various zebrafish tissues in vivo and in vitro to small molecule inhibitors of Tankyrase enzymes. This biochemical assay will facilitate chemically based studies focused on understanding the role of Tankyrase in cell fate reprogramming and tissue homeostasis and provide insights into the potential role of Wnt/ß-catenin signaling in these processes.

Endothelial nitric oxide synthase gene is associated with vessel stenosis in Korean population.

BACKGROUND: Nitric oxide (NO) produced by endothelial NO synthase (eNOS) mediates endothelium-dependent vasodilation and antithrombotic action. Controversial results regarding the association of eNOS gene polymorphisms with vessel stenosis have been reported. METHODS: Age- and sex-matched 932 individuals (656 subjects having 1-, 2-, and 3-stenosed vessels and 276 controls without stenosis) living in Seoul and surrounding suburbs were selected. A GT missense mutation in exon 7 (894GT) was screened using PCR-restriction fragment length polymorphism analysis. The genotypes of a 27-bp insertion/deletion in intron 4 (eNOS4b/a) and a TC mutation in promoter region of -786 (-786TC) were determined by the banding pattern on gel electrophoresis and a commercially available minisequencing protocol (SNaPshot), respectively. RESULTS: The eNOS4a allele was highly linked to the -786C allele (r=0.93, P<0.0001) while there was no linkage between eNOS4a allele and 894T allele or between 894T allele and -786C allele. Furthermore, 894T allele, but not eNOS4a (-786C) allele, was associated with the presence, but not the number, of stenosed vessels (odds ratio=1.57 for dominant effect of the T allele, P<0.05, and 1.49 for additive effect, P<0.05). Multiple logistic regression analysis revealed that 894T allele and hypertension were predictive independent risk factors for the presence of vessel stenosis. CONCLUSION: Our data suggest that eNOS gene polymorphisms may play an important role in the pathogenesis of vessel stenosis in Korean population.

Lack of evidence for contribution of Glu298Asp (G894T) polymorphism of endothelial nitric oxide synthase gene to plasma nitric oxide levels.

INTRODUCTION: Both positive and negative associations between a rare allele of 27-bp repeat polymorphism in intron 4 of endothelial nitric oxide synthase and plasma nitric oxide (NO) levels were previously reported, and further, these conflicting results were suggested to be partly accounted for smoking status of subjects. However, the genetic contribution of Glu298Asp (G894T) polymorphism to plasma NO levels with respect to smoking status has not been published. METHODS: In a group of 411 healthy Korean subjects aged 19-81 years, the end product of NO (NO(x): nitrite plus nitrate) as an index of plasma NO levels was measured by the Griess method. The genotypes of G894T polymorphism were determined by the banding patterns on gel electrophoresis after restriction enzyme digestion. RESULTS: Comparison of plasma NO(x) levels revealed no significant differences across the genotypes and alleles of G894T polymorphism, which is independently of smoking status. However, significant differences in plasma NO(x) levels between nonsmokers and smokers were observed (P = 0.0040). Furthermore, only the common G allele was found to be responsible for these differences. Multiple regression analysis showed that the most independent contributing factor for plasma NO(x) levels was smoking (P = 0.0119) and followed by triglycerides (P = 0.0384). CONCLUSIONS: Our results indicate no substantial effect of G894T polymorphism on the variance of plasma NO(x) levels in healthy Korean population.

Smoking status-dependent association of the 27-bp repeat polymorphism in intron 4 of endothelial nitric oxide synthase gene with plasma nitric oxide concentrations.

BACKGROUND: Both positive and negative associations between a rare allele of 27-bp repeat polymorphism (eNOS4b/a polymorphism) in intron 4 of endothelial nitric oxide synthase and plasma nitric oxide (NO) concentrations were previously reported. Although these conflicting results were suggested to be partly accounted for smoking status of subjects, no further studies have been accomplished. METHODS: We analyzed eNOS4b/a polymorphism in a group of 393 healthy Korean subjects and measured their plasma nitrite and nitrate (NO(x)) concentrations. NO(x) concentrations were measured by the Griess method and the genotypes of eNOS4b/a polymorphism determined by the banding pattern on gel electrophoresis. RESULTS: The frequency of eNOS4a allele in this study was 11.6%. The plasma NO(x) concentrations (in micromol/l) in subjects with eNOS4a allele was found to be significantly higher relative to those in eNOS4b allele (49.68 +/- 18.62 and 55.25 +/- 20.87, respectively, P < 0.05), which was valid only in smokers. Multiple regression analysis revealed that the most predictive contributing factor for plasma NO(x) concentrations was eNOS4a allele (P < 0.01), followed by smoking (P < 0.05), total cholesterol (P < 0.05), and triglycerides (P < 0.05). CONCLUSION: Our data indicate that there is substantial effect of eNOS4b/a polymorphism on the variance of plasma NO(x) concentrations in Korean population and that this effect is dependent on smoking status.

The development of highly potent inhibitors for porcupine.

Porcupine is a member of the membrane-bound O-acyltransferase family of proteins. It catalyzes the palmitoylation of Wnt proteins, a process required for their secretion and activity. We recently disclosed a class of small molecules (IWPs) as the first reported Porcn inhibitors. We now describe the structure-activity relationship studies and the identification of subnanomolar inhibitors. We also report herein the effects of IWPs on Wnt-dependent developmental processes, including zebrafish posterior axis formation and kidney tubule formation.

Homologous recombination in zebrafish ES cells.

Targeted insertion of a plasmid by homologous recombination was demonstrated in zebrafish ES cell cultures. Two selection strategies were used to isolate ES cell colonies that contained targeted plasmid insertions in either the no tail or myostatin I gene. One selection strategy involved the manual isolation of targeted cell colonies that were identified by the loss of fluorescent protein gene expression. A second strategy used the diphtheria toxin A-chain gene in a positive-negative selection approach. Homologous recombination was confirmed by PCR, sequence and Southern blot analysis and colonies isolated using both selection methods were expanded and maintained for multiple passages. The results demonstrate that zebrafish ES cells have potential for use in a cell-mediated gene targeting approach.

Hypoxia-induced endothelial NO synthase gene transcriptional activation is mediated through the tax-responsive element in endothelial cells.

Although hypoxia is known to induce upregulation of endothelial NO synthase (eNOS) gene expression, the underlying mechanism is largely unclear. In this study, we show that hypoxia increases eNOS gene expression through the binding of phosphorylated cAMP-responsive element binding (CREB) protein (pCREB) to the eNOS gene promoter. Hypoxia (1% O2) increased both eNOS expression and NO production, peaking at 24 hours, in bovine aortic endothelial cells, and these increases were accompanied by increases in pCREB. Treatment with the protein kinase A inhibitor H-89 or transfection with dominant-negative inhibitor of CREB reversed the hypoxia-induced increases in eNOS expression and NO production, with concomitant inhibition of the phosphorylation of CREB induced by hypoxia, suggesting an involvement of protein kinase A/pCREB-mediated pathway. To map the regulatory elements of the eNOS gene responsible for pCREB binding under hypoxia, we constructed an eNOS gene promoter (-1600 to +22 nucleotides) fused with a luciferase reporter gene [pGL2-eNOS(-1600)]. Hypoxia (for 24-hour incubation) increased the promoter activity by 2.36+/-0.18-fold in the bovine aortic endothelial cells transfected with pGL2-eNOS(-1600). However, progressive 5'-deletion from -1600 to -873 completely attenuated the hypoxia-induced increase in promoter activity. Electrophoretic mobility shift, anti-pCREB antibody supershift, and site-specific mutation analyses showed that pCREB is bound to the Tax-responsive element (TRE) site, a cAMP-responsive element-like site, located at -924 to -921 of the eNOS promoter. Our data demonstrate that the interaction between pCREB and the Tax-responsive element site within the eNOS promoter may represent a novel mechanism for the mediation of hypoxia-stimulated eNOS gene expression.