Proteins secreted by embryonic stem cells activate cardiomyocytes through ligand binding pathways.

Human embryonic stem cells (hESC) underlie embryogenesis but paracrine signals associated with the process are unknown. This study was designed to 1) profile native proteins secreted by undifferentiated hESC and 2) determine their biological effects on primary neonatal cardiomyocytes. We utilized multi-analyte, immunochemical assays to characterize media conditioned by undifferentiated hESC versus unconditioned media. Expression profiling was performed on cardiomyocytes subjected to these different media conditions and altered transcripts were mapped to critical pathways. Thirty-two of 109 proteins were significantly elevated in conditioned media ranging in concentration from thrombospondin (57.2+/-5.0 ng/ml) to nerve growth factor (7.4+/-1.2pg/ml) and comprising chemokines, cytokines, growth factors, and proteins involved in cell adhesion and extracellular matrix remodeling. Conditioned media induced karyokinesis, cytokinesis and proliferation in mono- and binucleate cardiomyocytes. Pathway analysis revealed comprehensive activation of the ROCK 1 and 2 G-protein coupled receptor (GPCR) pathway associated with cytokinesis, and the RAS/RAF/MEK/ERK receptor tyrosine kinase (RTK) and JAK/STAT-cytokine pathway involved in cell cycle progression. These results provide a partial database of proteins secreted by pluripotent hESC that potentiate cell division in cardiomyocytes via a paracrine mechanism suggesting a potential role for these stem cell factors in cardiogenesis and cardiac repair.

Cardiac fibroblasts influence cardiomyocyte phenotype in vitro.

Cardiac fibroblasts impact myocardial development and remodeling through intercellular contact with cardiomyocytes, but less is known about noncontact, profibrotic signals whereby fibroblasts alter cardiomyocyte behavior. Fibroblasts and cardiomyocytes were harvested from newborn rat ventricles and separated by serial digestion and gradient centrifugation. Cardiomyocytes were cultured in 1) standard medium, 2) standard medium diluted 1:1 with PBS, or 3) standard medium diluted 1:1 with medium conditioned > or =72 h by cardiac fibroblasts. Serum concentrations were held constant under all media conditions, and complete medium exchanges were performed daily. Cardiomyocytes began contracting within 24 h at clonal or mass densities with <5% of cells expressing vimentin. Immunocytochemical analysis revealed progressive expression of alpha-smooth muscle actin in cardiomyocytes after 24 h in all conditions. Only cardiomyocytes in fibroblast-conditioned medium stopped contracting by 72 h. There was a significant, sustained increase in vimentin expression specific to these cultures (means +/- SD: conditioned 46.3 +/- 6.0 vs. control 5.3 +/- 2.9%, P < 0.00025) typically with cardiac myosin heavy chain coexpression. Proteomics assays revealed 10 cytokines (VEGF, GRO/KC, monocyte chemoattractant protein-1, leptin, macrophage inflammatory protein-1alpha, IL-6, IL-10, IL-12p70, IL-17, and tumor necrosis factor-alpha) at or below detection levels in unconditioned medium that were significantly elevated in fibroblast-conditioned medium. Latent transforming growth factor-beta and RANTES were present in unconditioned medium but rose to higher levels in conditioned medium. Only granulocyte-macrophage colony-stimulating factor was present above threshold levels in standard medium but decreased with fibroblast conditioning. These data indicated that under the influence of fibroblast-conditioned medium, cardiomyocytes exhibited marked hypertrophy, diminished contractile capacity, and phenotype plasticity distinct from the dedifferentiation program present under standard culture conditions.

Molecular dynamics of the compensatory response to myocardial infarct.

Myocardial infarct via occlusion of the left anterior descending coronary in rats caused overriding depression in transcription, signal transduction, inflammation and extracellular matrix pathways in the infarct zone within 24 h. In contrast, remote zone gene expression was reciprocally activated during the immediate post-infarct period. Infarct zone signal transduction occurred primarily through TGFbeta1 induction while the remote zone exhibited elevated WNT, NOTCH, GPCR and transmembrane signaling. A minimal day 1 acute phase, inflammatory response was detected in the infarct zone while interleukins (IL1alpha, IL1beta, IL6, IL12alpha, IL18) and the TNFalpha superfamily were activated in the remote zone. Different cytochrome subsets were activated in each left ventricular region on day 1 while anti-oxidant genes were elevated only in the remote zone. The infarct zone exhibited mixed early transcription factor activation across all binding domains with a balance favoring constitutive gene activation and differentiation pathways as opposed to cell proliferation. In contrast, the remote zone exhibited activation of extensive developmental transcription factors involved in specification of cell phenotype, tissue-specific interactions and position-specific cell proliferation on day 1. The day 28 infarct zone response mirrored the day 1 remote zone response including activation of genes associated with matrix remodeling (metallothionein and metalloproteinase 9, 12, 23), as well as genes associated with cell proliferation and phenotype specification (MYC, EGR2, ATF3, HOXA1) recapitulating developmental histogenesis programs.

Effect of muscle origin and phenotype on satellite cell muscle-specific gene expression.

Satellite cells from adult mouse tongue, diaphragm, vastus lateralis, rectus femoris, tibialis anterior, soleus, and extensor digitorum longus muscles were isolated, expanded, and differentiated under identical culture conditions. Proliferating myoblasts and differentiated myofiber cultures were analyzed via SDS-PAGE, immunochemical, and PCR methods for expression of myosin heavy chains (MyHC) and muscle creatine kinase (MCK) as indices of muscle fiber type. Contralateral muscles were harvested for simultaneous, parallel analysis utilizing these assays. The MyHC profile of differentiated primary satellite cells was equivalent across all cultures with MyHC(2A) and MyHC(1/slow) co-expressed in all myotube and myofiber structures. Trace amounts of MyHC(2B) and MyHC(neo) were detected in a few myofibers. MCK was expressed at a uniform, similar level among these cultures. In contrast, contralateral muscles expressed each muscle-specific indicator at levels correlated with the fiber-type distribution within each muscle. MM14 and C2C12 cells, mouse satellite cell lines, were expanded and compared to primary cell cultures. MM14 cells had a high differentiation index (>95%) and co-expressed MyHC(2A) and MyHC(1/slow) along with trace amounts of MyHC(neo) throughout myotube cultures. Myofibers obtained from C2C12 cells exhibited less differentiation (~75%) with MyHC(2A) as the dominant isoform. These data indicate that primary satellite cells from adult muscle form a uniform differentiated cell type regardless of the fiber-type, anatomic location, and embryonic origin of the donor muscles. MM14 cells expressed an adult MyHC isoform profile similar to primary satellite cells. The results suggest that satellite cells provide a uniform cell source for use in autologous transplantation studies and do not acquire a heritable fiber-type-specific phenotype from their host muscle.

Muscle type-specific myosin isoforms in crustacean muscles.

Differential expression of multiple myosin heavy chain (MyHC) genes largely determines the diversity of critical physiological, histochemical, and enzymatic properties characteristic of skeletal muscle. Hypotheses to explain myofiber diversity range from intrinsic control of expression based on myoblast lineage to extrinsic control by innervation, hormones, and usage. The unique innervation and specialized function of crayfish (Procambarus clarkii) appendicular and abdominal musculature provide a model to test these hypotheses. The leg opener and superficial abdominal extensor muscles are innervated by tonic excitatory motoneurons. High resolution SDS-PAGE revealed that these two muscles express the same MyHC profile. In contrast, the deep abdominal extensor muscles, innervated by phasic motoneurons, express MyHC profiles different from the tonic profiles. The claw closer muscles are dually innervated by tonic and phasic motoneurons and a mixed phenotype was observed, albeit biased toward the phasic profile seen in the closer muscle. These results indicate that multiple MyHC isoforms are present in the crayfish and that differential expression is associated with diversity of muscle type and function.

Defining and measuring quality of diabetes care.

The literature on diabetes mellitus has increasingly focused on the quality of diabetes care and its measurement. Serious and widespread quality problems exist throughout American medicine. Current efforts to improve will not succeed unless we undertake a major, systematic effort to overhaul how we deliver health care services, educate and train clinicians, and assess and improve quality. This article defines the components of quality of diabetes care provision and discusses approaches to their measurement individually and globally.

Computer classification of sleep in preterm and full-term neonates at similar postconceptional term ages.

A classification strategy of neonatal sleep is being developed by comparing visually scored minutes of 21 channels of electroencephalographic (EEG)/polygraphic recordings with the corresponding values for each physiological signal derived from either visual or computer analyses. Continuous 3-hour sleep studies on 54 preterm and full-term neonates at similar postconceptional term ages were acquired under environmentally controlled conditions using a computerized monitoring system. An on-line event marker program recorded behavioral observations. One of three EEG sleep states was assigned to each of 8,995 minutes by traditional visual analysis criteria. EEG spectral values, spectral and nonspectral cardiorespiratory calculations and behaviorally observed movements, arousals and rapid eye movement counts were submitted for discriminant analysis. Based on the total minutes known for each of three states (i.e. active, quiet and awake), linear combinations of all specified digitized parameters were formed into an arithmetic algorithm by use of discriminant analysis, which served as the basis of a state assignment for each minute. Fifty percent of the data were arbitrarily used as the training set to derive the state classification model. The remaining fifty percent of the data were used as the cross-validation "test sample" to determine the accuracy of the classification when compared to the visually analyzed score for each corresponding minute. Thirteen out of 32 physiological measures best predicted state of both preterm and full-term neonatal groups. For both groups, the correct classification for active sleep was 90.3%, quiet sleep was 97.4%, awake was 97% and the overall accuracy was 93.3%. However, the order of significance for specific variables differed between these two neonatal groups. Differences in the order of variables that predict sleep states between preterm and full-term infants may reflect adaptation of brain function of the preterm infant to prematurity and/or prolonged extrauterine experience.

Effect of pulse dexamethasone therapy on the incidence and severity of chronic lung disease in the very low birth weight infant.

We conducted a prospective, randomized, double-blind trial to assess the efficacy and safety of pulse doses of dexamethasone on survival without supplemental oxygen in very low birth weight infants at high risk of having chronic lung disease. Seventy-eight infants with birth weights < or = 1500 gm who were ventilator dependent at 7 days of postnatal age were randomly assigned to receive pulse doses of dexamethasone, 0.5 mg/kg per day, divided twice daily (n = 39), or an equivalent volume of saline solution placebo (n = 39), for 3 days at 10-day intervals until they no longer required supplemental oxygen or assisted ventilation, or reached 36 weeks of postmenstrual age. At study entry, the groups did not differ by birth weight, gestational age, or severity of lung disease. At 36 weeks of postmenstrual age, there was both a significant increase in survival rates without oxygen supplementation (p = 0.03) and a significant decrease in the incidence of chronic lung disease (p = 0.047) in the group that received pulse therapy. Supplemental oxygen requirements were less throughout the study period in the group that received repeated pulse doses of dexamethasone (p = 0.013). The total numbers of deaths and the durations of supplemental oxygen, ventilator support, and hospital stay did not differ between groups. Recorded side effects in the pulse therapy group were minimal and included an increase in the use of insulin therapy for hyperglycemia (p < 0.05). We conclude that in this population of very low birth weight infants, treatment with pulse doses of dexamethasone resulted in improvement in pulmonary outcome without clinically significant side effects.

Maturational trends of EEG-sleep measures in the healthy preterm neonate.

Five physiologic groupings of 45 EEG-sleep measures were acquired from serial 24-channel EEG-sleep recordings (i.e., sleep architecture, continuity, EEG spectral, phasic, and autonomic measures), utilizing 129 studies on 56 healthy preterm infants from 28 to 43 weeks postconceptional age (PCA) who were neurodevelopmentally normal on follow-up. Regression analyses chose the least number of measures that best reflected maturation. Four of 45 variables (i.e., spectral alpha energy during quiet sleep, total spectral EEG energy, arousal number during active sleep, and percentage of EEG discontinuity) most significantly explained brain maturation in neonates < 36 weeks PCA. Three of 45 variables (i.e., spectral theta and beta energies during active sleep and spectral alpha energy during quiet sleep) were most representative after 36 weeks PCA. Spectral EEG energies were the strongest indicators of maturation compared with other measures, particularly in near-term neonates.

Grating acuity and visual field development in infants following perinatal asphyxia.

Grating acuity and visual fields were assessed in 66 children who had had perinatal asphyxia (ASPH). Also tested were 41 healthy preterm children (H-PT). Subjects were tested at birth to one month, and four, nine, 12, 17, 24, 30 and 36 months corrected age. The mean acuity scores of the ASPH group were lower than those of the H-PT group at most test ages, and significantly so at 30 and 36 months. The mean visual field size of the infants in the ASPH group was significantly smaller than that of the H-PT group at nine, 12, 30 and 36 months. No significant effects were found for preterm vs term birth, gestational age at birth, degree of asphyxia and presence of strabismus. However, central nervous system abnormalities (intraventricular haemorrhage and periventricular leukomalacia) were related to deficits in acuity and visual field size.

Maturation of phasic and continuity measures during sleep in preterm neonates.

Different physiologic measures during EEG sleep periods in preterm neonates are postulated to change with maturation and reflect functional brain development. Forty-three healthy preterm neonates received 3-h EEG sleep studies in an environmentally controlled setting. Postconceptional ages of neonates at each recording session ranged from 28 to 35 wk. Minute-by-minute analyses of EEG discontinuity, motility, arousals, and REM were performed. Eight phasic events and continuity measures of sleep were tabulated. Data were analyzed using Spearman rank order correlation coefficients. Increases in arousal numbers (p < 0.001) and durations (p < 0.001) were noted with age only during continuous periods of EEG activity (i.e. active sleep). REM also increased with corrected age during indeterminate or transitional sleep (p < 0.002) and decreased during quiet sleep (p < 0.01). Decreases in small body movements per minute (p = 0.02) and large body movements per minute (p < 0.001) occurred only during discontinuous periods of EEG activity (i.e. quiet sleep). Sleep efficiency (p < 0.001), maintenance (p < 0.001), and latency (p = 0.01) also decreased with increasing postconceptional age. Cycle length between two segments of continuous EEG with an intervening period of EEG discontinuity also lengthened with maturation (p < 0.001). These findings are discussed in the context of previously reported differences in phasic and continuity measures noted between preterm and full-term infants at matched full-term postconceptional ages. Changes in phasic and continuity measures with increasing postconceptional ages reflect maturation of specific neuronal processes of the CNS within a rudimentary sleep cycle of the preterm neonate.

Cardiorespiratory behavior during sleep in full-term and preterm neonates at comparable postconceptional term ages.

Cardiorespiratory behavior during sleep has been investigated by comparing visually analyzed minutes of EEG sleep with the digitized values of these two physiologic variables for each corresponding minute. Continuous 3-h nighttime sleep studies on 37 full-term and preterm neonates at comparable postconceptional term ages were acquired under controlled conditions, using a 24-channel computerized monitoring system and an automated event-marker program. Five thousand, two hundred ninety-four minutes were assigned an EEG state by traditional criteria. Eighteen preterm infants were compared with 19 full-term infants with respect to six cardiac and six respiratory measures: two nonspectral calculations (i.e. average per minute and variance of the means) and four spectral calculations of the cardiorespiratory signal (i.e. bandwidth, spectral edge, mean frequency, and ratio of harmonics). The relative capabilities of these measures to predict a sleep state change were investigated using discriminant analysis. A stepwise selection algorithm in discriminant analysis was used to identify the order of significance for the remaining variables. Eight cardiorespiratory measures were then submitted to multivariate analysis of variance to assess sleep state or preterm-full-term differences: mean frequency, bandwidth, average per minute, and ratio of harmonics for cardiac signals; and spectral edge, mean frequency, logarithm of variance, and ratio of harmonics for respiratory signals. Differences among the sleep states and between neonatal groups were highly significant (p < 0.0001). Interaction between sleep state and neonatal group was also significant (p < 0.034). Two variables differentiated preterm from full-term respiratory behavior: ratio (p < or = 0.001) and mean frequency (p < or = 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of conventional and matched filtering techniques for rapid eye movement detection of the newborn.

This paper compares an extended conventional filter technique for automated detection and analysis of rapid eye movements (REM) in neonates, using amplitude, synchrony, velocity, and coherence threshold criteria, with a matched filtering technique using the morphology of the REM waveform. Analyses of both simulated and real data were carried out. Automated REM tabulations are compared with visual scoring by a trained observer. Both preterm and fullterm neonates were used to test these methods. Both the advantages and disadvantages of these two techniques are discussed as compared with conventional methods which use only amplitude and synchrony threshold criteria. The major advantage of the extended conventional over the conventional method, as well as the matched filtering over the extended conventional technique, is the increased REM detection rate for ten minute intervals of artifact-free sleep. More accurate methods of automated REM detection that can be applied over extended monitoring periods are still needed.

Rectal temperature changes during sleep state transitions in term and preterm neonates at postconceptional term ages.

Mean rectal temperatures in neonates were investigated during sleep state transitions as assessed by visually analyzed electroencephalographic-polygraphic recordings. Continuous 3-hour studies were obtained on 3 term and 5 preterm infants at postconceptional term ages using a 24-channel computerized monitoring system. In the study, 1,461 min were assigned an EEG state by traditional criteria. Mean rectal temperature measurements were tabulated for each minute of sleep. Data were analyzed both as 1,461 consecutive minutes of sleep, and as 28 complete ultradian neonatal sleep cycles. Exploratory analyses were performed using t tests, Mann-Whitney U tests, and one-way analysis of variance. Decreases in mean rectal temperatures followed a transition from active to quiet sleep for only the term group. The preterm group had higher temperatures at sleep onset than the term group and demonstrated no changes during state transitions. Higher temperatures were maintained in the preterm group during both active and quiet sleep (i.e., 36.7 degrees C versus 36.4 degrees C, P = .02) when 28 complete cycles of sleep were compared and during the transition when 658 min of active sleep were compared to 617 min of quiet sleep. These findings are preliminary; however, the phenomenon of state-dependent changes in mean rectal temperature in neonates based on electroencephalographic sleep is unreported. Higher mean rectal temperatures during active sleep and altered temperature responses during transition to quiet sleep in the preterm infant suggest altered brain function because of the preterm infant's adaptation to the extrauterine experience.

Comparisons of EEG spectral and correlation measures between healthy term and preterm infants.

Continuous 12-hour electroencephalography (EEG)-sleep studies were acquired by a computerized monitoring system under environmentally controlled conditions for 2 groups of neonates. Eighteen health preterm infants at a postconceptional term age were matched to 18 term infants. These 2 groups were also matched for gender, race, and socioeconomic class. For the entire 12-hour recording, relative spectral power values (i.e., ratio of specific EEG power in specific frequency band compared to total EEG power) were significantly reduced in the preterm group for theta (P < or = .007), alpha (P < or = .001), and beta (P < or = .018) frequency bands, while delta remained unchanged. Correlations between 91 pairs of EEG channels were also calculated and the preterm infants had significantly higher correlation values in 27 of the 91 pairs of channels (P < .05); 14 interhemispheric, 8 intrahemispheric, and 5 sagittal combinations, while 3 intrahemispheric combinations were higher in the term group. Fewer functional neuronal aggregates generate less oscillatory potential (i.e., lower spectral power) in the theta through beta frequency ranges in the preterm infant, while greater cortical connectivity (i.e., higher correlations) exists in many brain regions by postconceptional term ages in this group. These findings suggest a functional alteration in brain development of the preterm infant as a result of prolonged extrauterine experience and/or prematurity.

Comparisons of EEG sleep state-specific spectral values between healthy full-term and preterm infants at comparable postconceptional ages.

Differences in state-specific electroencephalographic (EEG) spectral values are described between groups of preterm and full-term neonates at comparable postconceptional term ages. Eighteen healthy preterm neonates of < or = 32 weeks gestation were selected from an inborn population of a neonatal intensive care unit. Twenty-four-channel recordings were obtained at a full-term age and compared with studies of 22 healthy full-term neonates. The initial three hours of each 12-hour study were recorded on paper from which EEG sleep state scores per minute were visually assessed. Six mean spectral values (i.e. total EEG, electromyogram, delta, theta, alpha and beta energies) were calculated from each corresponding minute of digitized data, which was also assigned one of six EEG sleep states. Each neonatal group displayed statistically significant differences among sleep-state segments for all spectral values. The alpha- and beta-range spectral values of the preterm group, compared to the full-term control group, were lower during all sleep state segments. Spectral values for the theta band were lower during both quiet sleep segments only, whereas spectral values for delta were lower during all sleep stages, except tracé-alternant quiet sleep. Significant differences in EEG spectral values were noted among states of sleep for both preterm and full-term infants of similar postconceptional term ages. These data also suggest differences in central nervous system maturation between neonatal populations. These findings strengthen our previously stated contention that there is a functional alteration in brain development of the preterm infant as reflected in sleep organization that results from a prolonged extrauterine experience and/or prematurity.

Hypertrophic cardiomyopathy associated with dexamethasone therapy for chronic lung disease in preterm infants.

To assess whether long-term dexamethasone therapy for chronic lung disease (CLD) in infancy is associated with any deleterious cardiac structural effects, we conducted a retrospective review of all preterm infants with CLD born between October 1, 1989, and October 1, 1990, who had serial echocardiographic data available. These infants were divided into three groups based on the length of their exposure to dexamethasone. Group 1 contained nine infants with CLD who did not receive dexamethasone. Group 2 was comprised of six infants who received dexamethasone for less than 8 days. Group 3 contained one infant who received a 26-day course, and 13 infants who received at least one 42-day course of dexamethasone for CLD. Left ventricular hypertrophy was noted in 8 of 14 (57%) infants in group 3; hypertrophy usually was noted near the end of the treatment course. Five of these eight affected infants died; the hypertrophic cardiomyopathy was considered to have contributed to mortality in three of these five infants. Regression of the hypertrophy was noted in the three surviving infants in group 3 after the dexamethasone course was completed. We speculate that prolonged dexamethasone treatment for CLD is associated with hypertrophic cardiomyopathy in a significant portion of preterm infants.

Effect of undernutrition on contractile and fatigue properties of rat diaphragm during development.

The present study was designed to assess the effects of combined pre- and postnatal undernutrition on the in vitro contractile and fatigue properties of the rat diaphragm during development. In vitro direct stimulation of costal diaphragm from control (CTL) and undernourished (UN) rats was done on postnatal days 1, 4, 14, 21, 30, 40, 50, and 60. Combined pre- and postnatal undernutrition resulted in stunted animal growth but did not alter the diaphragm-to-total body weight ratio. Twitch contraction time, half-relaxation time, and force-frequency relationships were not consistently affected by undernutrition. Specific twitch force and specific tetanic force were also unchanged in the UN group. Fatigue resistance was high and comparable in UN and CTL groups at days 1 and 4. At day 14 and thereafter, fatigue resistance declined but was consistently higher in the UN than in the CTL group. We conclude that combined pre- and postnatal undernutrition results in a significant increase in fatigue resistance of the diaphragm compared with CTL, whereas diaphragm muscle contractile properties are not appreciably affected by prolonged undernutrition.