Single Component Organic Photosensitizer with NIR-I Emission Realizing Type-I Photodynamic and GSH-Depletion Caused Ferroptosis Synergistic Theranostics.

Phototheranostic agents have thrived as prominent tools for tumor luminescence imaging and therapies. Herein, a series of organic photosensitizers (PSs) with donor-acceptors (D-A) are elaborately designed and synthesized. In particular, PPR-2CN exhibits stable near infrared-I (NIR-I) emission, excellent free radicals generation and phototoxicity. Experimental analysis and calculations imply that a small singlet-triplet energy gap (ΔES1-T1 ) and large spin-orbit coupling (SOC) constant boost the intersystem crossing (ISC), leading to type-I photodynamic therapy (PDT). Additionally, the specific glutamate (Glu) and glutathione (GSH) consumption abilities of PPR-2CN inhibit the intracellular biosynthesis of GSH, resulting in redox dyshomeostasis and GSH-depletion causing ferroptosis. This work first realizes that single component organic PS could be simultaneously used as a type-I photodynamic agent and metal-free ferroptosis inducer for NIR-I imaging-guided multimodal synergistic therapy.

A Bimetallic Metal-Organic-Framework-Based Biomimetic Nanoplatform Enhances Anti-Leukemia Immunity via Synchronizing DNA Demethylation and RNA Hypermethylation.

Epigenetic-alterations-mediated antigenicity reducing in leukemic blasts (LBs) is one of the critical mechanisms of immune escape and resistance to T-cell-based immunotherapy. Herein, a bimetallic metal-organic framework (MOF)-based biomimetic nanoplatform (termed as AFMMB) that consists of a DNA hypomethylating agent, a leukemia stem cell (LSC) membrane, and pro-autophagic peptide is fabricated. These AFMMB particles selectively target not only LBs but also LSCs due to the homing effect and immune compatibility of the LSC membrane, and induce autophagy by binding to the Golgi-apparatus-associated protein. The autophagy-triggered dissolution of AFMMB releases active components, resulting in the restoration of the stimulator of interferon genes pathway by inhibiting DNA methylation, upregulation of major histocompatibility complex class-I molecules, and induction of RNA-methylation-mediated decay of programmed cell death protein ligand transcripts. These dual epigenetic changes eventually enhance T-cell-mediated immune response due to increased antigenicity of leukemic cells. AFMMB also can suppress growth and metastases of solid tumor, which was suggestive of a pan-cancer effect. These findings demonstrate that AFMMB may serve as a promising new nanoplatform for dual epigenetic therapy against cancer and warrants clinical validation.

Cyclodextrin-Functionalized Gold Nanorods Loaded with Meclofenamic Acid for Improving N6-Methyladenosine-Mediated Second Near-Infrared Photothermal Immunotherapy.

Cancer immunotherapy has achieved considerable clinical progress in recent years on account of its potential to treat metastatic tumors and inhibit recurrence. However, low patient response rates and dose-limiting toxicity are the major limitations of immunotherapy. Nanoparticle-based photothermal immunotherapy can amplify antitumor immune responses, although poor tumor penetration depth of near-infrared radiation (NIR) and the immunosuppressive tumor microenvironment significantly dampen its effects. We designed a nanoplatform based on gold nanorods for NIR-II-mediated photothermal therapy (PTT) combined with N6-methyladenosine (m6A) demethylase inhibition to achieve enhanced photothermal immunotherapy against prostate cancer. The GNRs were assembled layer by layer with polystyrenesulfonate as the interconnecting layer and then coated with a cationic polymer of γ-cyclodextrin (CD)-cross-linked low-molecular-weight polyethylenimine that was conjugated to an 8-mer peptide targeting the prostate tumor-specific gastrin-releasing peptide receptor. The m6A RNA demethylase inhibitor meclofenamic acid (MA) was then loaded into the CD cavity through hydrophobic interactions. GNR-CDP8MA specifically targeted the prostate tumor cells and selectively accumulated at the tumor site in vivo. In addition, GNR-CDP8MA almost completely ablated prostate cancer cell-derived tumors upon 1208 nm laser irradiation. Mechanistically, NIR-II triggered the release of MA from GNR-CDP8MA, which increased global mRNA m6A methylation and decreased the stability of PDL1 transcripts. Furthermore, GNR-CDP8MA-mediated PTT-induced immunogenic cell death in the primary tumor and consequently enhanced antitumor immunity by activating the antigen-presenting dendritic cells and tumor-specific effector T cells in the metastatic tumors. This study offers insights into synergistic m6A RNA methylation and PTT as an effective strategy for cancer immunotherapy.

Glutathione-Bioimprinted Nanoparticles Targeting of N6-methyladenosine FTO Demethylase as a Strategy against Leukemic Stem Cells.

The N6-methyladenosine (m6 A) demethylase FTO plays an oncogenic role in acute myeloid leukemia (AML). Despite the promising recent progress for developing some small-molecule FTO inhibitors, the clinical potential remains limited due to mild biological function, toxic side effects and low sensitivity and/or specificity to leukemic stem cells (LSCs). Herein, FTO inhibitor-loaded GSH-bioimprinted nanocomposites (GNPIPP12MA) are developed that achieves targeting of the FTO/m6 A pathway synergized GSH depletion for enhancing anti-leukemogenesis. GNPIPP12MA can selectively target leukemia blasts, especially LSCs, and induce ferroptosis by disrupting intracellular redox status. In addition, GNPIPP12MA increases global m6 A RNA modification and decreases the transcript levels in LSCs. GNPIPP12MA augments the efficacy of the PD-L1 blockade by increasing the infiltration of cytotoxic T cells for enhanced anti-leukemia immunity. This study offers insights for a GSH-bioimprinted nanoplatform targeting m6 A RNA methylation as a synergistic treatment strategy against cancer stem cells that may translate to clinical applications.

Gold Nanorods Exhibit Intrinsic Therapeutic Activity via Controlling N6-Methyladenosine-Based Epitranscriptomics in Acute Myeloid Leukemia.

Reprograming the N6-methyladenosine (m6A) landscape is a promising therapeutic strategy against recalcitrant leukemia. In this study, we synthesized gold nanorods (GNRs) of different aspect ratios using a binary surfactant mixture of hexadecyltrimethylammonium bromide and sodium oleate. Following surface functionalization with chitosan and a 12-mer peptide, GNRa-CSP12 measuring 130 × 21 nm2 was selectively taken up by leukemia cells via targeted endocytosis. Low doses of GNRa-CSP12 inhibited the growth of leukemia cells by disrupting the redox balance and inducing ferroptosis. Mechanistically, GNRa-CSP12 abrogated endogenous Fe2+-dependent m6A demethylase activity, which led to global m6A hypomethylation and post-transcriptional regulation of downstream genes that are involved in glycolysis, hypoxia, and immune checkpoint pathways. In addition, combination treatment with GNRa-CSP12 and tyrosine kinases inhibitors (TKIs) synergistically obviated the m6A-mediated TKI resistance phenotype. Finally, GNRa-CSP12 as a potential immunotherapeutic agent could enhance immunotherapy outcome in leukemia. Our preclinical findings provide the proof-of-concept for targeting m6A-methylation-based epitranscriptomics using nanoparticle as an "epigenetic drug" for cancer therapy.

Expression of the BAD pathway is a marker of triple-negative status and poor outcome.

Triple-negative breast cancer (TNBC) has few therapeutic targets, making nonspecific chemotherapy the main treatment. Therapies enhancing cancer cell sensitivity to cytotoxic agents could significantly improve patient outcomes. A BCL2-associated agonist of cell death (BAD) pathway gene expression signature (BPGES) was derived using principal component analysis (PCA) and evaluated for associations with the TNBC phenotype and clinical outcomes. Immunohistochemistry was used to determine the relative expression levels of phospho-BAD isoforms in tumour samples. Cell survival assays evaluated the effects of BAD pathway inhibition on chemo-sensitivity. BPGES score was associated with TNBC status and overall survival (OS) in breast cancer samples of the Moffitt Total Cancer Care dataset and The Cancer Genome Atlas (TCGA). TNBC tumours were enriched for the expression of phospho-BAD isoforms. Further, the BPGES was associated with TNBC status in breast cancer cell lines of the Cancer Cell Line Encyclopedia (CCLE). Targeted inhibition of kinases known to phosphorylate BAD protein resulted in increased sensitivity to platinum agents in TNBC cell lines compared to non-TNBC cell lines. The BAD pathway is associated with triple-negative status and OS. TNBC tumours were enriched for the expression of phosphorylated BAD protein compared to non-TNBC tumours. These findings suggest that the BAD pathway it is an important determinant of TNBC clinical outcomes.

Acute alcohol exposure during mouse gastrulation alters lipid metabolism in placental and heart development: Folate prevention.

BACKGROUND: Embryonic acute exposure to ethanol (EtOH), lithium, and homocysteine (HCy) induces cardiac defects at the time of exposure; folic acid (FA) supplementation protects normal cardiogenesis (Han et al., , ; Serrano et al., ). Our hypothesis is that EtOH exposure and FA protection relate to lipid and FA metabolism during mouse cardiogenesis and placentation. METHODS: On the morning of conception, pregnant C57BL/6J mice were placed on either of two FA-containing diets: a 3.3 mg health maintenance diet or a high FA diet of 10.5 mg/kg. Mice were injected a binge level of EtOH, HCy, or saline on embryonic day (E) 6.75, targeting gastrulation. On E15.5, cardiac and umbilical blood flow were examined by ultrasound. Embryonic cardiac tissues were processed for gene expression of lipid and FA metabolism; the placenta and heart tissues for neutral lipid droplets, or for medium chain acyl-dehydrogenase (MCAD) protein. RESULTS: EtOH exposure altered lipid-related gene expression on E7.5 in comparison to control or FA-supplemented groups and remained altered on E15.5 similarly to changes with HCy, signifying FA deficiency. In comparison to control tissues, the lipid-related acyl CoA dehydrogenase medium length chain gene and its protein MCAD were altered with EtOH exposure, as were neutral lipid droplet localization in the heart and placenta. CONCLUSION: EtOH altered gene expression associated with lipid and folate metabolism, as well as neutral lipids, in the E15.5 abnormally functioning heart and placenta. In comparison to controls, the high FA diet protected the embryo and placenta from these effects allowing normal development. Birth Defects Research (Part A) 106:749-760, 2016. © 2016 The Authors Birth Defects Research Part A: Clinical and Molecular Teratology Published by Wiley Periodicals, Inc.

Embryonic exposures of lithium and homocysteine and folate protection affect lipid metabolism during mouse cardiogenesis and placentation.

Embryonic exposures can increase the risk of congenital cardiac birth defects and adult disease. The present study identifies the predominant pathways modulated by an acute embryonic mouse exposure during gastrulation to lithium or homocysteine that induces cardiac defects. High dose periconceptional folate supplementation normalized development. Microarray bioinformatic analysis of gene expression demonstrated that primarily lipid metabolism is altered after the acute exposures. The lipid-related modulation demonstrated a gender bias with male embryos showing greater number of lipid-related Gene Ontology biological processes altered than in female embryos. RT-PCR analysis demonstrated significant change of the fatty acid oxidation gene Acadm with homocysteine exposure primarily in male embryos than in female. The perturbations resulting from the exposures resulted in growth-restricted placentas with disorganized cellular lipid droplet distribution indicating lipids have a critical role in cardiac-placental abnormal development. High folate supplementation protected normal heart-placental function, gene expression and lipid localization.

Changes in vitelline and utero-placental hemodynamics: implications for cardiovascular development.

Analyses of cardiovascular development have shown an important interplay between heart function, blood flow, and morphogenesis of heart structure during the formation of a four-chambered heart. It is known that changes in vitelline and placental blood flow seemingly contribute substantially to early cardiac hemodynamics. This suggests that in order to understand mammalian cardiac structure-hemodynamic functional relationships, blood flow from the extra-embryonic circulation needs to be taken into account and its possible impact on cardiogenesis defined. Previously published Doppler ultrasound analyses and data of utero-placental blood flow from human studies and those using the mouse model are compared to changes observed with environmental exposures that lead to cardiovascular anomalies. Use of current concepts and models related to mechanotransduction of blood flow and fluid forces may help in the future to better define the characteristics of normal and abnormal utero-placental blood flow and the changes in the biophysical parameters that may contribute to congenital heart defects. Evidence from multiple studies is discussed to provide a framework for future modeling of the impact of experimental changes in blood flow on the mouse heart during normal and abnormal cardiogenesis.

Expression of myotilin during chicken development.

Several missense mutations in the Z-band protein, myotilin, have been implicated in human muscle diseases such as myofibrillar myopathy, spheroid body myopathy, and distal myopathy. Recently, we have reported the cloning of chicken myotilin cDNA. In this study, we have investigated the expression of myotilin in cross-striated muscles from developing chicken by qRT-PCR and in situ hybridizations. In situ hybridization of embryonic stages shows myotilin gene expression in heart, somites, neural tissue, eyes and otocysts. RT-PCR and qRT-PCR data, together with in situ hybridization results point to a biphasic transcriptional pattern for MYOT gene during early heart development with maximum expression level in the adult. In skeletal muscle, the expression level starts decreasing after embryonic day 20 and declines in the adult skeletal muscles. Western blot assays of myotilin in adult skeletal muscle reveal a decrease in myotilin protein compared with levels in embryonic skeletal muscle. Our results suggest that MYOT gene may undergo transcriptional activation and repression that varies between tissues in developing chicken. We believe this is the first report of the developmental regulation on myotilin expression in non-mammalian species.

Effects of alcohol, lithium, and homocysteine on nonmuscle myosin-II in the mouse placenta and human trophoblasts.

OBJECTIVE: Mouse embryonic exposure to alcohol, lithium, and homocysteine results in intrauterine growth restriction (IUGR) and cardiac defects. Our present study focused on the placental effects. We analyzed the hypothesis that expression of nonmuscle myosin (NMM)-II isoforms involved in cell motility, mechanosensing, and extracellular matrix assembly are altered by the 3 factors in human trophoblast (HTR8/SVneo) cells in vitro and in the mouse placenta in vivo. STUDY DESIGN: After exposure during gastrulation to alcohol, homocysteine, or lithium, ultrasonography defined embryos exhibiting abnormal placental blood flow. RESULTS: NMM-IIA/NMM-IIB are differentially expressed in trophoblasts and in mouse placental vascular endothelial cells under pathological conditions. Misexpression of NMM-IIA/NMM-IIB in the affected placentas continued stably to midgestation but can be prevented by folate and myoinositol supplementation. CONCLUSION: It is concluded that folate and myoinositol initiated early in mouse pregnancy can restore NMM-II expression, permit normal placentation/embryogenesis, and prevent IUGR induced by alcohol, lithium, and homocysteine.

Blood flow dynamics of one cardiac cycle and relationship to mechanotransduction and trabeculation during heart looping.

Analyses of form-function relationships during heart looping are directly related to technological advances. Recent advances in four-dimensional optical coherence tomography (OCT) permit observations of cardiac dynamics at high-speed acquisition rates and high resolution. Real-time observation of the avian stage 13 looping heart reveals that interactions between the endocardial and myocardial compartments are more complex than previously depicted. Here we applied four-dimensional OCT to elucidate the relationships of the endocardium, myocardium, and cardiac jelly compartments in a single cardiac cycle during looping. Six cardiac levels along the longitudinal heart tube were each analyzed at 15 time points from diastole to systole. Using image analyses, the organization of mechanotransducing molecules, fibronectin, tenascin C, α-tubulin, and nonmuscle myosin II was correlated with specific cardiac regions defined by OCT data. Optical coherence microscopy helped to visualize details of cardiac architectural development in the embryonic mouse heart. Throughout the cardiac cycle, the endocardium was consistently oriented between the midline of the ventral floor of the foregut and the outer curvature of the myocardial wall, with multiple endocardial folds allowing high-volume capacities during filling. The cardiac area fractional shortening is much higher than previously published. The in vivo profile captured by OCT revealed an interaction of the looping heart with the extra-embryonic splanchnopleural membrane providing outside-in information. In summary, the combined dynamic and imaging data show the developing structural capacity to accommodate increasing flow and the mechanotransducing networks that organize to effectively facilitate formation of the trabeculated four-chambered heart.

Fetal alcohol syndrome: cardiac birth defects in mice and prevention with folate.

OBJECTIVE: Alcohol (ethanol) consumption during pregnancy is linked to congenital heart defects that are associated with fetal alcohol syndrome. Recent reports have associated ethanol exposure with the Wnt/beta-catenin pathway. Therefore, we defined whether ethanol affects Wnt/beta-catenin signaling during cardiac cell specification. STUDY DESIGN: Pregnant mice on embryonic day 6.75 during gastrulation were exposed by an intraperitoneal injection to a binge-drinking dose of ethanol. Folic acid supplementation of mouse diet was tested for the prevention of ethanol-induced cardiac birth defects. RESULTS: Acute ethanol exposure induced myocardial wall changes and atrioventricular and semilunar valve defects, which was determined by echocardiography on embryonic day 15.5. A high folate diet prevented the ethanol-induced cardiac defects. Ethanol exposure in avian embryos suppressed 2 key Wnt-modulated genes that are involved in cardiac induction; folic acid rescued normal gene expression. CONCLUSION: Folic acid supplementation alone or with myoinositol prevented alcohol potentiation of Wnt/beta-catenin signaling that allowed normal gene activation and cardiogenesis.

Folate rescues lithium-, homocysteine- and Wnt3A-induced vertebrate cardiac anomalies.

Elevated plasma homocysteine (HCy), which results from folate (folic acid, FA) deficiency, and the mood-stabilizing drug lithium (Li) are both linked to the induction of human congenital heart and neural tube defects. We demonstrated previously that acute administration of Li to pregnant mice on embryonic day (E)6.75 induced cardiac valve defects by potentiating Wnt-beta-catenin signaling. We hypothesized that HCy may similarly induce cardiac defects during gastrulation by targeting the Wnt-beta-catenin pathway. Because dietary FA supplementation protects from neural tube defects, we sought to determine whether FA also protects the embryonic heart from Li- or HCy-induced birth defects and whether the protection occurs by impacting Wnt signaling. Maternal elevation of HCy or Li on E6.75 induced defective heart and placental function on E15.5, as identified non-invasively using echocardiography. This functional analysis of HCy-exposed mouse hearts revealed defects in tricuspid and semilunar valves, together with altered myocardial thickness. A smaller embryo and placental size was observed in the treated groups. FA supplementation ameliorates the observed developmental errors in the Li- or HCy-exposed mouse embryos and normalized heart function. Molecular analysis of gene expression within the avian cardiogenic crescent determined that Li, HCy or Wnt3A suppress Wnt-modulated Hex (also known as Hhex) and Islet-1 (also known as Isl1) expression, and that FA protects from the gene misexpression that is induced by all three factors. Furthermore, myoinositol with FA synergistically enhances the protective effect. Although the specific molecular epigenetic control mechanisms remain to be defined, it appears that Li or HCy induction and FA protection of cardiac defects involve intimate control of the canonical Wnt pathway at a crucial time preceding, and during, early heart organogenesis.

Cellular nonmuscle myosins NMHC-IIA and NMHC-IIB and vertebrate heart looping.

Flectin, a protein previously described to be expressed in a left-dominant manner in the embryonic chick heart during looping, is a member of the nonmuscle myosin II (NMHC-II) protein class. During looping, both NMHC-IIA and NMHC-IIB are expressed in the mouse heart on embryonic day 9.5. The patterns of localization of NMHC-IIB, rather than NMHC-IIA in the mouse looping heart and in neural crest cells, are equivalent to what we reported previously for flectin. Expression of full-length human NMHC-IIA and -IIB in 10 T1/2 cells demonstrated that flectin antibody recognizes both isoforms. Electron microscopy revealed that flectin antibody localizes in short cardiomyocyte cell processes extending from the basal layer of the cardiomyocytes into the cardiac jelly. Flectin antibody also recognizes stress fibrils in the cardiac jelly in the mouse and chick heart; while NMHC-IIB antibody does not. Abnormally looping hearts of the Nodal(Delta 600) homozygous mouse embryos show decreased NMHC-IIB expression on both the mRNA and protein levels. These results document the characterization of flectin and extend the importance of NMHC-II and the cytoskeletal actomyosin complex to the mammalian heart and cardiac looping.

Molecular effects of lithium exposure during mouse and chick gastrulation and subsequent valve dysmorphogenesis.

BACKGROUND: Lithium (Li) has been associated with cardiac teratogenicity in the developing fetus. We took advantage of the association of therapeutic administration of Li with an increase in heart defects to gain insight into both normal and pathological heart and valve development with GSK-3 inhibition. The objective of this study was to define whether Li mimicry of canonical Wnt/beta-catenin signaling induces cardiac valve defects. METHODS: Li was administered by a single intraperitoneal injection to the pregnant mouse on embryonic day E6.75, much earlier than heretofore analyzed. On E15.5 developing heart defects were defined by Doppler ultrasound. The embryonic hearts were analyzed for changes in patterning of active canonical Wnt expression and nuclear factor of the activated T cells-c1 (NFATc1), both key regulators of valve development. Li-exposed chick embryos were used to define the early cell populations during gastrulation that are susceptible to GSK-3 inhibition and may relate to valve formation. RESULTS: Li exposure during gastrulation decreased the number of prechordal plate (PP) cells that reached the anterior intestinal portal, a region associated with valve development. Li decreased expression of Hex, an endoderm cardiac inducing molecule, normally also expressed by the PP cells, and of Sox 4 at the anterior intestinal portal and NFAT, critical factors in valvulogenesis. CONCLUSIONS: Cells existing already during gastrulation are associated with valve formation days later. The Wnt/beta-catenin signaling in PP cells is normally repressed by Wnt antagonists and Hex is up-regulated. The antagonism occurring at the receptor level is bypassed by Li exposure by its intracellular inactivation of GSK-3 directly to augment Wnt signaling.

Tropomyosin expression and dynamics in developing avian embryonic muscles.

The expression of striated muscle proteins occurs early in the developing embryo in the somites and forming heart. A major component of the assembling myofibrils is the actin-binding protein tropomyosin. In vertebrates, there are four genes for tropomyosin (TM), each of which can be alternatively spliced. TPM1 can generate at least 10 different isoforms including the striated muscle-specific TPM1alpha and TPM1kappa. We have undertaken a detailed study of the expression of various TM isoforms in 2-day-old (stage HH 10-12; 33 h) heart and somites, the progenitor of future skeletal muscles. Both TPM1alpha and TPM1kappa are expressed transiently in embryonic heart while TPM1alpha is expressed in somites. Both RT-PCR and in situ hybridization data suggest that TPM1kappa is expressed in embryonic heart whereas TPM1alpha is expressed in embryonic heart, and also in the branchial arch region of somites, and in the somites. Photobleaching studies of Yellow Fluorescent Protein-TPM1alpha and -TPM1kappa expressed in cultured avian cardiomyocytes revealed that the dynamics of the two probes was the same in both premyofibrils and in mature myofibrils. This was in sharp contrast to skeletal muscle cells in which the fluorescent proteins were more dynamic in premyofibrils. We speculate that the differences in the two muscles is due to the appearance of nebulin in the skeletal myocytes premyofibrils transform into mature myofibrils.

Early temporal-specific responses and differential sensitivity to lithium and Wnt-3A exposure during heart development.

Members of both Wnt and bone morphogenetic protein (BMP) families of signaling molecules are important in heart development. We previously demonstrated that beta-catenin, a key downstream intermediary of the canonical Wnt signaling pathway, delineates the dorsal boundary of the cardiac compartments in an anteroposterior progression. We hypothesized the progression involves canonical Wnt signaling and reflects development of the primary body axis of the embryo. A similar anteroposterior signaling wave leading to cardiac cell specification involves inductive signaling by BMP-2 synthesized by the underlying endoderm in anterior bilateral regions. Any molecule that disrupts the normal balance of Wnt and BMP concentrations within the heart field may be expected to affect early heart development. The canonical Wnt signaling step mimicked by lithium involves inactivation of glycogen synthase kinase-3beta (GSK-3beta; Klein and Melton [1996] Proc. Natl. Acad. Sci. U. S. A. 93:8455-8459). We show that lithium, Wnt-3A, and an inhibitor of GSK-3beta, SB415286, affect early heart development at the cardiac specification stages. We demonstrate that normal expression patterns of key signaling molecules as Notch-1 and Dkk-1 are altered in the anterior mesoderm within the heart fields by a one-time exposure to lithium, or by noggin inhibition of BMP, at Hamburger and Hamilton (HH) stage 3 during chick embryonic development. The severity of developmental defects is greatest with exposure to lithium or Wnt-3A at HH stage 3 and decreases at HH stage 4. Taken together, our results demonstrate that there are temporal-specific responses and differential sensitivities to lithium/Wnt-3A exposure during early heart development.

MCT-4, A511/Basigin and EF5 expression patterns during early chick cardiomyogenesis indicate cardiac cell differentiation occurs in a hypoxic environment.

We have identified the presence of the hypoxia marker EF5 in the stage 4/5 chick heart fields. This suggests that cardiac cell differentiation occurs in a relatively anaerobic environment. Monocarboxylate transporter (MCT) studies in adult cardiac myocytes have demonstrated that MCTs catalyze proton-linked pyruvate and lactate transport activity. 5A11/Basigin is an ancillary protein that targets MCTs to the plasma membrane for their function. MCT-4 expression is most evident in cells with a high glycolytic rate associated with hypoxic energy production. Subsequent to the immunohistochemical localization of EF5 in the early heart field, we continued in our analysis during stages 5 to 12 for the expression of indicators of cellular glycolytic metabolism in the developing heart, such as MCT-4, MCT-1, and 5A11 (Basigin/CD147). Our observations indicate that MCT-4 and 5A11/Basigin are expressed early, in a differential left-right pattern, in the bi-lateral plate mesoderm, as the cardiac compartment is forming. At stage 11, MCT-4/5A11 continues to be highly expressed in the myocardial wall of the looping heart, but not in the dorsal mesocardium. RT-PCR analyses for MCT-1, -4, and 5A11 indicate that MCT-4 and 5A11 are expressed throughout precardiac, embryonic, and fetal stages in the heart. MCT-1 is first detected in the heart on embryonic day 3 and then remains expressed throughout development to hatching. These results indicate that cardiac precursor cells are equipped for differentiating in a hypoxic environment using anaerobic metabolism for energy production.

Cross talk between cell-cell and cell-matrix adhesion signaling pathways during heart organogenesis: implications for cardiac birth defects.

The anterior-posterior and dorsal-ventral progression of heart organogenesis is well illustrated by the patterning and activity of two members of different families of cell adhesion molecules: the calcium-dependent cadherins, specifically N-cadherin, and the extracellular matrix glycoproteins, fibronectin. N-cadherin by its binding to the intracellular molecule beta-catenin and fibronectin by its binding to integrins at focal adhesion sites, are involved in regulation of gene expression by their association with the cytoskeleton and through signal transduction pathways. The ventral precardiac mesoderm cells epithelialize and become stably committed by the activation of these cell-matrix and intracellular signaling transduction pathways. Cross talk between the adhesion signaling pathways initiates the characteristic phenotypic changes associated with cardiomyocyte differentiation: electrical activity and organization of myofibrils. The development of both organ form and function occurs within a short interval thereafter. Mutations in any of the interacting molecules, or environmental insults affecting either of these signaling pathways, can result in embryonic lethality or fetuses born with severe heart defects. As an example, we have defined that exposure of the embryo temporally to lithium during an early sensitive developmental period affects a canonical Wnt pathway leading to beta-catenin stabilization. Lithium exposure results in an anterior-posterior progression of severe cardiac defects.