P53 Activation Promotes Maturational Characteristics of Pluripotent Stem Cell-Derived Cardiomyocytes in 3-Dimensional Suspension Culture Via FOXO-FOXM1 Regulation.

BACKGROUND: Current protocols generate highly pure human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro that recapitulate characteristics of mature in vivo cardiomyocytes. Yet, a risk of arrhythmias exists when hiPSC-CMs are injected into large animal models. Thus, understanding hiPSC-CM maturational mechanisms is crucial for clinical translation. Forkhead box (FOX) transcription factors regulate postnatal cardiomyocyte maturation through a balance between FOXO and FOXM1. We also previously demonstrated that p53 activation enhances hiPSC-CM maturation. Here, we investigate whether p53 activation modulates the FOXO/FOXM1 balance to promote hiPSC-CM maturation in 3-dimensional suspension culture. METHODS AND RESULTS: Three-dimensional cultures of hiPSC-CMs were treated with Nutlin-3a (p53 activator, 10 µM), LOM612 (FOXO relocator, 5 µM), AS1842856 (FOXO inhibitor, 1 µM), or RCM-1 (FOXM1 inhibitor, 1 µM), starting 2 days after onset of beating, with dimethyl sulfoxide (0.2% vehicle) as control. P53 activation promoted hiPSC-CM metabolic and electrophysiological maturation alongside FOXO upregulation and FOXM1 downregulation, in n=3 to 6 per group for all assays. FOXO inhibition significantly decreased expression of cardiac-specific markers such as TNNT2. In contrast, FOXO activation or FOXM1 inhibition promoted maturational characteristics such as increased contractility, oxygen consumption, and voltage peak maximum upstroke velocity, in n=3 to 6 per group for all assays. Further, by single-cell RNA sequencing of n=2 LOM612-treated cells compared with dimethyl sulfoxide, LOM612-mediated FOXO activation promoted expression of cardiac maturational pathways. CONCLUSIONS: We show that p53 activation promotes FOXO and suppresses FOXM1 during 3-dimensional hiPSC-CM maturation. These results expand our understanding of hiPSC-CM maturational mechanisms in a clinically-relevant 3-dimensional culture system.

Tissue-embedded stretchable nanoelectronics reveal endothelial cell-mediated electrical maturation of human 3D cardiac microtissues.

Clinical translation of stem cell therapies for heart disease requires electrical integration of transplanted cardiomyocytes. Generation of electrically matured human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is critical for electrical integration. Here, we found that hiPSC-derived endothelial cells (hiPSC-ECs) promoted the expression of selected maturation markers in hiPSC-CMs. Using tissue-embedded stretchable mesh nanoelectronics, we achieved a long-term stable map of human three-dimensional (3D) cardiac microtissue electrical activity. The results revealed that hiPSC-ECs accelerated the electrical maturation of hiPSC-CMs in 3D cardiac microtissues. Machine learning-based pseudotime trajectory inference of cardiomyocyte electrical signals further revealed the electrical phenotypic transition path during development. Guided by the electrical recording data, single-cell RNA sequencing identified that hiPSC-ECs promoted cardiomyocyte subpopulations with a more mature phenotype, and multiple ligand-receptor interactions were up-regulated between hiPSC-ECs and hiPSC-CMs, revealing a coordinated multifactorial mechanism of hiPSC-CM electrical maturation. Collectively, these findings show that hiPSC-ECs drive hiPSC-CM electrical maturation via multiple intercellular pathways.

Localization of leptin receptor mRNA and the long form splice variant (Ob-Rb) in mouse hypothalamus and adjacent brain regions by in situ hybridization.

Expression of the leptin receptor gene has been examined in mouse hypothalamus and other brain regions by in situ hybridization. With a probe recognizing all the known splice variants, receptor mRNA was evident in several brain regions (cortex, hippocampus, thalamus), with strong expression in the hypothalamus (arcuate, ventromedial, paraventricular and ventral premammillary nuclei), choroid plexus and leptomeninges. A probe specific to the long splice variant of the leptin receptor (Ob-Rb), containing the putative intracellular signaling domain, again revealed strong expression in the hypothalamus; there was, however, minimal hybridization to choroid plexus and leptomeninges. These results indicate that the hypothalamus is a key site of leptin action, although other brain regions are also targeted.

Melatonin receptors in the human fetal kidney: 2-[125I]iodomelatonin binding sites correlated with expression of Mel1a and Mel1b receptor genes.

Melatonin receptors in the human fetal kidney were identified and characterized by quantitative in vitro autoradiography using the melatonin agonist, 2-[125I]iodomelatonin. Specific binding was localized to cells in the nephrogenic region at the outer perimeter of the developing kidney and was time-dependent, saturable and inhibited in the presence of guanosine 5'-0-(3-thiotriphosphate) indicative of a G protein-coupled receptor. Expression of the Mel1a and Mel1b melatonin receptors in human fetal kidney was determined using RT-PCR. In situ hybridization confirmed the localization of the Mel1a mRNA transcripts. A role for melatonin in development of the human fetal kidney is postulated.

Localization of [125I]IGF-I binding on the ovine pars tuberalis.

In the sheep, it has been shown that the pars tuberalis of the pituitary may mediate the photoperiodic control of seasonal changes in prolactin secretion. High concentrations of melatonin receptors are present on the ovine pars tuberalis and melatonin is known to inhibit forskolin-stimulated cyclic AMP production in this tissue. Other hormonal inputs to the ovine pars tuberalis have not yet been identified. In the rat mRNA for the IGF-I receptor has been identified in the pars tuberalis using in situ hybridization. In order to define whether IGF-I may influence the function of the ovine pars tuberalis the presence of receptors for IGF-I has been investigated. Using in vitro autoradiography specific [125I]IGF-I binding was found in high concentrations over the ovine pars tuberalis particularly associated with certain of the capillaries. Homogenate receptor assays showed saturable specific binding of [125I]IGF-I with a mean dissociation constant (Kd) of 0.5 +/- 0.1 nM (n = 4). Competition studies revealed a rank order of potency of IGF-I > IGF-II > > > insulin, in displacing [125I]IGF-I binding, indicative of a mixed population of IGF-I and IGF-II/mannose-6-phosphate receptors and insulin-like growth factor binding proteins (IGFBPs). Cross-linking of [125I]IGF-I to pars tuberalis membrane homogenates and analysis by SDS-PAGE under reducing conditions confirmed the presence of both IGF-I receptors and binding proteins. Autophosphorylation of a 97 kDa substrate, compatible with the beta-sub-unit of the IGF-I receptor, was increased in the presence of IGF-I, indicating the existence of functional IGF-I receptors on the ovine pars tuberalis. In contrast in the rat [125I]IGF-I binding was restricted to the median eminence region of the brain and was not detectable over the pars tuberalis.

Coexpression of leptin receptor and preproneuropeptide Y mRNA in arcuate nucleus of mouse hypothalamus.

Leptin, the protein product of the adipose tissue-specific ob (obese) gene (1), reduces the body weight, adiposity and food intake of obese ob/ob mice on peripheral or central injection (2, 3, 4). [125I]leptin binding has been detected in mouse choroid plexus (5), from which a leptin receptor gene was expression cloned (5). The gene has at least 6 splice variants (6, 7). Leptin receptor mRNA was localized in the hypothalamus by in situ hybridization being particularly abundantly expressed in the arcuate nucleus (8). There is evidence linking the physiological effects of injected leptin with hypothalamic neuropeptide Y (9, 10) (NPY), which has potent central effects on food intake and energy balance (11), and is also expressed in the arcuate nucleus. Here we report dual in situ hybridization studies for leptin receptor and NPY gene expression in the mouse arcuate nucleus, where the majority of cells examined expressed both genes. This provides the first direct evidence that leptin acts on cells that express NPY mRNA.

Identification and characterisation of 2-[125I]iodomelatonin binding and Mel1a melatonin receptor expression in the human fetal leptomeninges.

Melatonin binding sites were identified over the leptomeninges surrounding the human fetal brain using quantitative in vitro autoradiography and the melatonin agonist, 2-[125I]iodomelatonin. Binding was found to be saturable and of high affinity (dissociation constant (Kd) = 54 pM and maximal theoretical binding (Bmax) = 13 fmol/mg protein), and inhibited by guanosine-5'-o-(3-thiotriphosphate) (GTPgammaS) suggesting that these binding sites represent G protein-coupled melatonin receptors. RT-PCR performed on mRNA isolated from the human fetal leptomeninges detected expression of the G protein-coupled melatonin receptor Mel1a, but not Mel1b. In situ hybridisation confirmed the localisation of Mel1a mRNA transcripts over the leptomeninges of the fetal brain. The identification of 2-[125I]iodomelatonin and Mel1a melatonin receptor expression in the fetal leptomeninges implies that melatonin may play a role in the early growth and development of the human brain.

Effects of maternal undernutrition during early pregnancy on apoptosis regulators in the ovine fetal ovary.

This study aimed to determine whether reduced fetal ovary folliculogenesis in ewes undernourished during early/midpregnancy is associated with altered ovarian cell proliferation and/or the expression of apoptosis-regulating genes. Groups of ewes (n = 11-19) were fed either 100% (high; H) or 50% (low; L) of metabolisable energy requirements for live-weight maintenance during selected windows of gestation. All animals were killed at days 50, 65 or 110 of gestation. Between mating and slaughter, control animals were fed the H ration, while animals of other subgroups were fed the L ration from (a) mating to slaughter at 50, 65 or 110 days; (b) 0 to 30 days; (c) 31 to 50 or 65 days; or (d), in the day 110 slaughter group only, from 66 to 110 days. Bouin's-fixed fetal ovaries were examined for (a) Ki67 immunoexpression (proliferation) and (b) Bax and Mcl-1 (apoptosis-regulating genes) expression by in situ hybridisation (day 110) and immunohistochemistry (days 50, 65 and 110). At day 50, maternal nutrition had no effect on Ki67, predominant in germ cells, or Bax and Mcl-1, predominant in the oocytes. Restricted maternal food intake from 0 to 30 days significantly reduced staining for Ki67 in germ cells at day 65 (P < 0.05) but increased staining in granulosa cells at day 110 (P < 0.05). In animals fed the L ration for 110 days, primordial follicle Bax and Mcl-1 were significantly increased (Bax: P < 0.01; Mcl-1: P < 0.05). Granulosa cell Bax was also increased (P < 0.05). When the L ration was fed from 66 to 110 days, granulosa cell Bax (P < 0.05) and primordial follicle Mcl-1 (P < 0.01) were also significantly increased. In the fetal ovarian vasculature, animals underfed for 0-110 days had significantly elevated perivascular Mcl-1 (P < 0.001) and endothelial Bax expression (P < 0.05). Moreover, at day 110, endothelial Mcl-1 was increased by underfeeding from 0 to 30 days (P < 0.05). These data indicate that maternal undernutrition alters proliferation and the expression of apoptosis-regulating genes in the developing fetal ovary. The precise mechanism depends on the window of maternal food restriction.

Melatonin receptors in neonatal pig brain and pituitary gland.

Summer infertility remains a major problem in domestic pigs. It has been proposed that sows which display this trait are inherent seasonal breeders. The influence of photoperiod on domestic pigs has been difficult to ascertain as significant diurnal fluctuations in blood levels of the pineal hormone, melatonin, which provide a direct neuroendocrine transduction of the ambient photoperiod in other species, remain questionable in adult pigs. To investigate whether the pig is potentially receptive to melatonin, central sites of action for this hormone were localized and characterized within the brain and pituitary of the neonatal pig by in vitro autoradiography using 2-((125)I)iodomelatonin. Specific binding was distributed over a number of discrete regions of the brain including the cerebral cortex, hypothalamus, thalamus, brainstem, and cerebellum. The choroid plexus, and the pars tuberalis and pars distalis of the pituitary were also specifically labeled. Specific binding was completely abolished in the presence of 10(-7) M melatonin, and inhibited in the presence of 10(-4) M GTPgammaS (guanosine-5-0-(3-thiotriphosphate)), a non-hydrolysable analogue of GTP, in all regions examined, indicating that binding is representative of a G-protein coupled receptor. Characterization studies showed that 2-((125)I)iodomelatonin binding was saturable with a dissociation constant (Kd) in the low picomolar range (approximately 30 pM). Competition studies with iodomelatonin, melatonin, N-acetylserotonin and serotonin (5-HT) gave IC50 values similar to those previously characterized for the melatonin receptor in the ovine pars tuberalis.

Changes in regional protein synthesis in rat brain and pituitary after systemic interleukin-1 beta administration.

A convenient and sensitive method has been developed for measuring changes in protein synthesis in discrete areas of the brain and pituitary of conscious freely moving rats. A single injection of high-concentration low-specific activity L-[35S]methionine is given to flood amino acid precursor pools, thereby equalizing the specific activity of the L-[35S]methionine throughout the tissue. Unincorporated L-[35S]methionine is removed from cryostat sections by treatment with perchloric acid (2%) before quantitative autoradiography. The sensitivity of this technique is demonstrated by the detection of changes in protein synthesis in regions of the brain and pituitary after systemic administration of interleukin-1 beta, a cytokine that has centrally mediated effects but which is not thought to cross the blood-brain barrier. Areas of the brain found to exhibit significant increases in protein synthesis were the subfornical organ, the choroid plexus, the medial habenular, the dentate gyrus, and the anterior and posterior lobes of the pituitary. In the brain, the cingulate cortex and the pineal gland showed significant decreases in the rate of protein synthesis.

Placental leptin in normal, diabetic and fetal growth-retarded pregnancies.

Leptin expression in third trimester placenta (p) and leptin concentrations in umbilical cord blood (cb) were investigated in normal pregnancies [n = 10 (p), 31 (cb)] and abnormal pregnancies complicated with (i) maternal insulin-dependent diabetes [IDDM: n = 3 (p), 13 (cb)], (ii) gestational diabetes [GD: n = 2 (p), 10 (cb)] and (iii) fetal growth retardation [FGR: n = 5 (p), 5 (cb)]. By in-situ hybridization and immunohistochemistry, placental leptin mRNA and protein were co-localized to the syncytiotrophoblast and villous vascular endothelial cells. Leptin receptor was immunolocalized to the syncytiotrophoblast. Relative to controls, the FGR group was characterized by low concentrations of placental and cord blood leptin. In a twin pregnancy, the normal-sized infant exhibited more placental and cord blood leptin than its growth-retarded twin. In contrast, both diabetic groups exhibited high concentrations of placental leptin mRNA and protein. The IDDM group exhibited the highest concentrations of leptin in cord blood. No change was observed in the expression of the leptin receptor in either the growth-retarded or diabetic pregnancies. In conclusion, the localization of placental leptin suggests that it may be released into both maternal and fetal blood. Furthermore, in fetal growth-retarded and diabetic pregnancies, the changes in leptin expression in the placenta and in leptin concentrations in umbilical cord blood appear to be related.

Melatonin receptors in red deer fetuses (Cervus elaphus).

Red deer (Cervus elaphus) exhibit highly seasonal rhythms in physiology and behaviour. The influence of photoperiod on the timing of these changes begins in utero where the fetus receives photoperiodic information via the diurnal pattern of maternal melatonin secretion. The potential sensitivity of deer fetuses to melatonin was ascertained by mapping specific receptors and characterizing them using 2-[125I]iodomelatonin and quantitative autoradiography in vitro. Specific binding occurred from day 31 of gestation onwards (term = 233 days) over the spinal nerves and respiratory system. At later stages of gestation binding occurred over the brain, particularly the brainstem, the pituitary gland, thyroid gland, gastrointestinal tract including the pancreas, metanephros, cochlea of the ear, spinal cord, and spinal and cranial nerves. Binding was abolished in the presence of 10(-7) mol melatonin l-1 and diminished in the presence of 10(-4) mol GTP gamma S l-1 (guanosine-5-O-(3-thiotriphosphate)), confirming that binding represented functional G-protein-coupled melatonin receptors. Characterization studies, carried out on fetal lung, revealed that binding was time-dependent, reaching equilibrium at about 3 h at room temperature (22 degrees C), and saturable with a dissociation constant (Kd) of 104 pmol l-1. This study demonstrates the presence of G-protein-coupled melatonin receptors over a wide range of tissues in red deer fetuses from early in gestation, indicating that in addition to its role in the communication of photoperiodic information to the fetus in this species, melatonin may be involved in fetal growth and development.

Central melatonin binding sites in rainbow trout (Onchorhynchus mykiss).

A combination of in vitro autoradiography and membrane homogenate receptor assays has been used to localize and characterized 2-[125I]iodomelatonin binding sites in the brain of the rainbow trout (Onchorhynchus mykiss). Specific 2-[125I]iodomelatonin binding, defined as that displaced by 1 microM melatonin, increased linearly with increasing protein concentration in membrane homogenates of whole trout brain. Specific binding was both time and temperature dependent and reversible in the presence of 1 microM melatonin. Binding was saturable at between 100-150 pM 2-[125I]iodomelatonin and Scatchard analysis of saturation isotherms revealed a dissociation constant (Kd) of 15.00 +/- 0.95 pM and a maximum receptor number (Bmax) of 42.35 +/- 2.70 fm/mg protein (n = 16). Addition of 10(-4) M GTP gamma S (an analogue of guanosine triphosphate) to saturation isotherms apparently reduced the Bmax by 75% on average with no apparent change in the affinity of the binding. Scatchard analysis of saturation isotherms generated from whole brain membrane homogenates of trout kept on long days (15 hr light:9 hr dark) and killed either during the midlight or middark phase showed no significant differences in either the Kd or the Bmax of 2-[125I]iodomelatonin binding, although a robust rhythm in melatonin concentration was confirmed in these fish. Displacement of 2-[125I]iodomelatonin binding with increasing concentrations of competing ligands gave an order of potency of 2-iodomelatonin > melatonin >> 5-HT. Localization of specific central 2-[125I]iodomelatonin binding in the rainbow trout showed high levels of binding associated with neuronal areas involved in the processing of visual signals, particularly the optic tectum and nucleus rotundus.(ABSTRACT TRUNCATED AT 250 WORDS)

Central melatonin receptors in red deer (Cervus elaphus).

Red deer display characteristic seasonal changes in appetite, growth, and reproduction which are mediated by the pineal hormone, melatonin, which provides a direct neuroendocrine transduction of the ambient photoperiod. To identify potential central sites of action for this hormone, [2(-125)I]iodomelatonin binding sites were localized and characterized within the cervine brain and pituitary by in vitro autoradiography. Specific binding was distributed over a number of discrete regions of the brain, including the cortex, septum, putamen, hippocampus, cerebellum, and superior colliculus. The choroid plexus, the pars tuberalis, and the pars distalis of the pituitary were also specifically labeled. Specific binding was completely abolished in the presence of 10(-7) M melatonin and inhibited in the presence of 10(-4) M GTP gamma S (guanosine-5-O-(3-thiotriphosphate)), a nonhydrolysable analogue of GTP, in the pars tuberalis, pars distalis, choroid plexus, and all neuronal regions examined apart from the hippocampus and layer III of the cerebral cortex. Inhibition of [2(-125)I]iodomelatonin binding by GTP gamma S is indicative that binding is representative of a G-protein-coupled receptor. Characterization studies showed that [2(-125)I]iodomelatonin binding was time-dependent and saturable with a dissociation constant (Kd) in the low picomolar range (approximately 10 pM). Competition studies with iodomelationin, melatonin, and N-acetylserotonin gave IC50 values similar to those previously characterized for the melatonin receptor in the ovine pars tuberalis.

Melatonin receptors in the rat brain and pituitary.

2-(125I)iodomelatonin binding has been mapped and characterized in the brain and pituitary of the male laboratory rat using quantitative in vitro autoradiography. Specific binding was defined as that completely displaced in the presence of 1 microM melatonin. In the brain high levels of binding were localized over the suprachiasmatic nucleus (SCN), the area postrema (AP), and the spinal tract of the trigeminal nerve (Sp5). Lower densities of binding were found over the medial preoptic area (MPA), the septohypothalamic nuclei (SHy), the anterior hypothalamic area (AHA), the nuclei of the lateral olfactory tract (LOT), the paraventricular (PV), anteroventral (AV) and intermediodorsal (IMD) nuclei of the thalamus, the medial region of the lateral habenular (Lhb), the nuclei of the stria medullaris (SM), the basolateral (BL) and medial (ME) amygdaloid nuclei, the ventromedial nuclei (VMH), the arcuate nuclei (Arc), the subiculum of the hippocampus (S) and the lateral mammillary nuclei (LM). High levels of binding were also present over the pars tuberalis of the pituitary (PT) and the anterior and posterior cerebral arteries (CA). In both neuronal and non-neuronal areas, specific binding was time dependent and partially reversible in the presence of 1 microM melatonin. Binding was also saturable and of high affinity with dissociation constants (Kd) in the low picomolar range and was significantly inhibited in the presence of 10(-4)M guanosine 5'-0-(3-thiotriphosphate) (GTP gamma S) and 150 mM NaCl in all regions examined, indicating the presence of high affinity G-protein coupled melatonin receptors.

Oestrogen and progesterone receptor immunoreactivity and c-fos expression in the ovine cervix.

Immunocytochemistry was used to detect the presence of oestrogen and progesterone receptors in the cervices of prepubertal lambs, seasonally anoestrous ewes, cyclic ewes, and pregnant ewes of known gestational stages, to define the roles of gonadal steroids in cervical function. The presence of the immediate early gene product, c-Fos, a marker for cellular activation, was also investigated using immunocytochemistry and in situ hybridization. Oestrogen receptor immunoreactivity was restricted to the endometrium on days 0-3 of the oestrous cycle (day 0 = oestrus). In immature animals, very few scattered nuclei in the endometrium were immunoreactive. Oestrogen receptor immunoreactivity was not apparent in the endometrium during the remainder of the oestrous cycle or in this region in anoestrous animals. In pregnant ewes, oestrogen receptor immunostaining appeared as relatively few isolated nuclei in the connective tissue stroma. Progesterone receptor immunoreactivity was found in the endometrium at days 0-3 of the oestrous cycle and also in the luminal epithelium, the myometrium and the blood vessels. Progesterone receptor immunoreactivity was also found in these regions, with the exception of the endometrium, at all other stages examined. Immunostaining for c-Fos was present in the endometrium at days 0-3 of the oestrous cycle, and some scattered immunopositive nuclei were present in prepubertal animals. c-Fos immunoreactivity was also found in the myometrium and in blood vessels at all other stages examined. Visualization of c-fos gene expression by in situ hybridization showed that it occurred in the luminal epithelium and blood vessels at oestrus, but was restricted to the blood vessels in all other samples examined.