Thrombopoietin receptor agonists protect human cardiac myocytes from injury by activation of cell survival pathways.

Thrombopoietin confers immediate protection against injury caused by ischemia/reperfusion in the rat heart. Eltrombopag is a small molecule agonist of the thrombopoietin receptor, the physiologic target of thrombopoietin. However, the ability of eltrombopag and thrombopoietin to protect human cardiac myocytes against injury and the mechanisms underlying myocyte protection are not known. Human cardiac myocytes (n = 6-10/group) were treated with eltrombopag (0.1-30.0 µM) or thrombopoietin (0.1-30.0 ng/ml) and then subjected to 5 hours of hypoxia (95% N2/5% CO2) and 16 hours of reoxygenation to determine their ability to confer resistance to myocardial injury. The thrombopoietin receptor c-Mpl was detected in unstimulated human cardiac myocytes by Western blotting. Eltrombopag and thrombopoietin confer immediate protection to human cardiac myocytes against injury from hypoxia/reoxygenation by decreasing necrotic and apoptotic cell death in a concentration-dependent manner, with an optimal concentration of 3 µM for eltrombopag and 1.0 ng/ml for thrombopoietin. The extent of protection conferred with eltrombopag is equivalent to that of thrombopoietin. Eltrombopag and thrombopoietin activate multiple prosurvival pathways; inhibition of Janus kinase-2, proto-oncogene tyrosine-protein kinase, protein kinase B/phosphatidylinositol-3 kinase, p44/42 mitogen-activated protein kinase (MAPK), and p38 MAPK abolished cardiac myocyte protection by eltrombopag and thrombopoietin. Eltrombopag and thrombopoietin may represent important and potent agents for immediately and substantially increasing protection of human cardiac myocytes, and may offer a long-lasting benefit through activation of prosurvival pathways during ischemia.

Cardiac injury after 10 gy total body irradiation: indirect role of effects on abdominal organs.

The objective of this study was to determine whether radiation-induced injury to the heart after 10 Gy total body irradiation (TBI) is direct or indirect. Young male WAG/RijCmcr rats received a 10 Gy single dose using TBI, upper hemi-body (UHB) irradiation, lower hemi-body (LHB) irradiation, TBI with the kidneys shielded or LHB irradiation with the intestines shielded. Age-matched, sham-irradiated rats served as controls. The lipid profile, kidney injury, heart and liver morphology and cardiac function were determined up to 120 days after irradiation. LHB, but not UHB irradiation, increased the risk factors for cardiac disease as well as the occurrence of cardiac and kidney injury in a way that was quantitatively and qualitatively similar to that observed after TBI. Shielding of the kidneys prevented the increases in risk factors for cardiac disease. Shielding of the intestines did not prevent the increases in risk factors for cardiac disease. There was no histological evidence of liver injury 120 days after irradiation. Injury to the heart from irradiation appears to be indirect, supporting the notion that injury to abdominal organs, principally the kidneys, is responsible for the increased risk factors for and the occurrence of cardiac disease after TBI and LHB irradiation.

Intestinal microbiota determine severity of myocardial infarction in rats.

Signals from the intestinal microbiota are important for normal host physiology; alteration of the microbiota (dysbiosis) is associated with multiple disease states. We determined the effect of antibiotic-induced intestinal dysbiosis on circulating cytokine levels and severity of ischemia/reperfusion injury in the heart. Treatment of Dahl S rats with a minimally absorbed antibiotic vancomycin, in the drinking water, decreased circulating leptin levels by 38%, resulted in smaller myocardial infarcts (27% reduction), and improved recovery of postischemic mechanical function (35%) as compared with untreated controls. Vancomycin altered the abundance of intestinal bacteria and fungi, measured by 16S and 18S ribosomal DNA quantity. Pretreatment with leptin (0.12 µg/kg i.v.) 24 h before ischemia/reperfusion abolished cardioprotection produced by vancomycin treatment. Dahl S rats fed the commercially available probiotic product Goodbelly, which contains the leptin-suppressing bacteria Lactobacillus plantarum 299v, also resulted in decreased circulating leptin levels by 41%, smaller myocardial infarcts (29% reduction), and greater recovery of postischemic mechanical function (23%). Pretreatment with leptin (0.12 µg/kg i.v.) abolished cardioprotection produced by Goodbelly. This proof-of-concept study is the first to identify a mechanistic link between changes in intestinal microbiota and myocardial infarction and demonstrates that a probiotic supplement can reduce myocardial infarct size.

Making stem cells infarct avid.

A major factor limiting the engraftment of transplanted stem cells after myocardial infarction is the low rate of retention in the infarcted site. Our long-term objective is to improve engraftment by enabling stem cells to recognize and bind infarcted tissue. To this end, we proposed to modify the surface of embryonic stem cells (ESCs) with the C2A domain of synaptotagmin I; this allows the engineered stem cells to bind to dead and dying cardiac cells by recognizing phosphatidylserine (PS). The latter is a molecular marker for apoptotic and necrotic cells. The C2A domain of synaptotagmin I, which binds PS with high affinity and specificity, was attached to the surface of mouse ESCs using the biotin-avidin coupling mechanism. Binding of C2A-ESCs to dead and dying cardiomyocytes was tested in vitro. After the surface modification, cellular physiology was examined for viability, pluripotency, and differentiation potential. C2A covalently attached to the ESC surface at an average of about 1 million C2A molecules per cell under mild conjugation reaction conditions. C2A-ESCs avidly bound to dying, but not viable, cardiomyocytes in culture. The normal physiology of C2A-modified ESCs was maintained. The binding of C2A-ESCs to moribund cardiomyocytes demonstrates that the retention of transplanted cells may be improved by conferring these cells with the ability to bind infarcted tissue. Once established, this novel approach may be applicable to other types of transplanted therapeutic cells.

Homozygous disruption of the Tip60 gene causes early embryonic lethality.

Tat-interactive protein 60 (Tip60) is a member of the MYST family, proteins of which are related by an atypical histone acetyltransferase (HAT) domain. Although Tip60 has been implicated in cellular activities including DNA repair, apoptosis, and transcriptional regulation, its function during embryonic development is unknown. We ablated the Tip60 gene (Htatip) from the mouse by replacing exons 1-9 with a neomycin resistance cassette. Development and reproduction of wild-type and heterozygous animals were normal. However, homozygous ablation of the Tip60 gene caused embryolethality near the blastocyst stage of development, as evidenced by inability of cells in Tip60-null blastocysts to hatch and survive in culture. Monitoring cell proliferation and death by detecting EdU-substituted DNA and TUNEL labeling revealed suppression of cell proliferation concomitant with increased cell death as Tip60-null cells attempted to hatch from blastocysts. These findings indicate that Tip60 is essential for cellular survival during the blastocyst-gastrula transition of embryogenesis.

Cardiogenic effects of trichloroethylene and trichloroacetic acid following exposure during heart specification of avian development.

Trichloroethylene (TCE) and its metabolite trichloroacetic acid (TCA) are common drinking water contaminants in the United States. Both chemicals have been implicated in causing congenital heart defects (CHD) in human epidemiological and animal model studies. However, the latter studies have primarily focused on assessment of cardiac morphology at late embryonic stages. Here, we tested whether treating avian embryos with TCE or TCA during an exposure window encompassing cardiac specification (Hamburger-Hamilton [HH] 3+) until the onset of chambering (HH 17) informs the etiology of CHD at later stages of development. Embryos were exposed to TCE or TCA via direct injection into the yolk, over a range of doses that included each compound's maximum contaminant level as established by the U.S. Environmental Protection Agency. A modified TUNEL (Terminal deoxynucleotide transferase mediated dUTP-biotin Nick-End Labeling) assay indicated that neither compound induced apoptotic cell death in ventricular myocytes or endocardiocytes at HH 18. However, mid-range dosages of TCE increased myocyte and endocardiocyte proliferation by this time, as determined by monitoring BrdU incorporation; in contrast, an intermediate dose of TCA inhibited proliferation in endocardiocytes. These cellular changes had no apparent functional consequences because all measured hemodynamic parameters were normal for TCE- and TCA-exposed embryos at HH 18, HH 21, and HH 23. In summary, TCE or TCA exposure during the cardiac specification window has only minimal effects on the developing avian heart. These results sharply contrast with our previously reported observations following administration of equivalent doses during a window of valvuloseptal morphogenesis. Taken together, these findings indicate that, as for other teratogens, sensitivity is dictated by the embryo's stage of development.

Trichloroethylene exposure during cardiac valvuloseptal morphogenesis alters cushion formation and cardiac hemodynamics in the avian embryo.

It is controversial whether trichloroethylene (TCE) is a cardiac teratogen. We exposed chick embryos to 0, 0.4, 8, or 400 ppb TCE/egg during the period of cardiac valvuloseptal morphogenesis (2-3.3 days' incubation) . Embryo survival, valvuloseptal cellularity, and cardiac hemodynamics were evaluated at times thereafter. TCE at 8 and 400 ppb/egg reduced embryo survival to day 6.25 incubation by 40-50%. At day 4.25, increased proliferation and hypercellularity were observed within the atrioventricular and outflow tract primordia after 8 and 400 ppb TCE. Doppler ultrasound revealed that the dorsal aortic and atrioventricular blood flows were reduced by 23% and 30%, respectively, after exposure to 8 ppb TCE. Equimolar trichloroacetic acid (TCA) was more potent than TCE with respect to increasing mortality and causing valvuloseptal hypercellularity. These results independently confirm that TCE disrupts cardiac development of the chick embryo and identifies valvuloseptal development as a period of sensitivity. The hypercellular valvuloseptal profile is consistent with valvuloseptal heart defects associated with TCE exposure. This is the first report that TCA is a cardioteratogen for the chick and the first report that TCE exposure depresses cardiac function. Valvuloseptal hypercellularity may narrow the cardiac orifices, which reduces blood flow through the heart, thereby compromising cardiac output and contributing to increased mortality. The altered valvuloseptal formation and reduced hemodynamics seen here are consistent with such an outcome. Notably, these effects were observed at a TCE exposure (8 ppb) that is only slightly higher than the U.S. Environmental Protection Agency maximum containment level for drinking water (5 ppb) .

Gastrulating chick embryo as a model for evaluating teratogenicity: a comparison of three approaches.

BACKGROUND: Teratology studies must be carefully designed to minimize potential secondary effects of vehicle and delivery routes. A systematic method to evaluate chick models of early embryogenesis is lacking. METHODS: We investigated 3 experimental approaches that are popular for studies of early avian development, in terms of their utility for teratogen assessment starting at gastrulation. These included in vitro embryo culture, egg windowing followed by direct application of a carrier vehicle to the embryo, and injection of a carrier vehicle into the egg yolk. We also developed a morphologically based scoring system to assess development of the early embryo. RESULTS: The in vitro culture and egg windowing approaches both caused an unacceptably high incidence of central nervous system and cardiac abnormalities in vehicle-treated embryos, which made it difficult to identify teratogen-specific defects. In contrast, exposing chick embryos to vehicle via direct egg yolk injection did not induce developmental anomalies. CONCLUSIONS: Optimization of the exposure route of potential toxicants to the embryo is critical because control treatments can cause developmental anomalies. In ovo yolk injection minimizes perturbation of young embryos and may be appropriate for teratogen delivery.