Human Genetics of Hypoplastic Left Heart Syndrome.

Hypoplastic left heart syndrome (HLHS) is a severe congenital cardiovascular malformation characterized by hypoplasia of the left ventricle, aorta, and other structures on the left side of the heart. The pathologic definition includes atresia or stenosis of both the aortic and mitral valves. Despite considerable progress in clinical and surgical management of HLHS, mortality and morbidity remain concerns. One barrier to progress in HLHS management is poor understanding of its cause. Several lines of evidence point to genetic origins of HLHS. First, some HLHS cases have been associated with cytogenetic abnormalities (e.g., Turner syndrome). Second, studies of family clustering of HLHS and related cardiovascular malformations have determined HLHS is heritable. Third, genomic regions that encode genes influencing the inheritance of HLHS have been identified. Taken together, these diverse studies provide strong evidence for genetic origins of HLHS and related cardiac phenotypes. However, using simple Mendelian inheritance models, identification of single genetic variants that "cause" HLHS has remained elusive, and in most cases, the genetic cause remains unknown. These results suggest that HLHS inheritance is complex rather than simple. The implication of this conclusion is that researchers must move beyond the expectation that a single disease-causing variant can be found. Utilization of complex models to analyze high-throughput genetic data requires careful consideration of study design.

New Insights into the Education of Children with Congenital Heart Disease with and without Trisomy 21.

Background and Objectives: Patients with congenital heart disease (CHD), especially as a concomitant syndromal disease of trisomy 21 (T21), are at risk for impaired neurodevelopment. This can also affect these patients' education. However, there continues to be a research gap in the educational development of CHD patients and T21 CHD patients. Materials and Methods: In total, data from 2873 patients from the German National Register for Congenital Heart Defects were analyzed. The data are based on two online education surveys conducted among patients registered in the National Register for Congenital Heart Defects (2017, 2020). Results: Of 2873 patients included (mean age: 14.1 ± 4.7 years, 50.5% female), 109 (3.8%) were identified with T21 (mean age: 12.9 ± 4.4 years, 49.5% female). T21 CHD participants had a high demand for early specific interventions (overall cohort 49.1%; T21 cohort 100%). T21 CHD children more frequently attended special schools and, compared to non-trisomy 21 (nT21) CHD patients, the probability of attending a grammar school was reduced. In total, 87.1% of nT21 CHD patients but 11% of T21 CHD patients were enrolled in a regular elementary school, and 12.8% of T21 CHD patients could transfer to a secondary school in contrast to 35.5% of nT21 CHD patients. Most of the T21 CHD patients were diagnosed with psychiatric disorders, e.g., learning, emotional, or behavioral disorders (T21 CHD patients: 82.6%; nT21 CHD patients: 31.4%; p < 0.001). Conclusions: CHD patients are at risk for impaired academic development, and the presence of T21 is an aggravating factor. Routine follow-up examinations should be established to identify developmental deficits and to provide targeted interventions.

Cooling and Sterile Inflammation in an Oxygen-Glucose-Deprivation/Reperfusion Injury Model in BV-2 Microglia.

OBJECTIVE: Cold-inducible RNA-binding protein (CIRBP) has been shown to be involved not only in cooling-induced cellular protection but also as a mediator of sterile inflammation, a critical mechanism of the innate immune response in ischemia/reperfusion (I/R) injury. The role of microglia and its activation in cerebral I/R injury warrants further investigation as both detrimental and regenerative properties have been described. Therefore, we investigated the effects of cooling, specifically viability, activation, and release of damage associated molecular patterns (DAMPs) on oxygen glucose deprivation/reperfusion- (OGD/R-) induced injury in murine BV-2 microglial cells. METHODS: Murine BV-2 microglial cells were exposed to 2 to 6 h OGD (0.2% O2 in glucose- and serum-free medium) followed by up to 19 h of reperfusion, simulated by restoration of oxygen (21% O2) and nutrients. Cells were maintained at either normothermia (37°C) or cooled to 33.5°C, 1 h after experimental start. Cultured supernatants were harvested after exposure to OGD for analysis of DAMP secretions, including high-mobility group box 1 (HMGB1), heat shock protein 70 (HSP70), and CIRBP, and cytotoxicity was assessed by lactate dehydrogenase releases after exposure to OGD and reperfusion. Intracellular cold-shock proteins CIRBP and RNA-binding motif 3 (RBM3) as well as caspases 9, 8, and 3 were also analyzed via Western blot analysis. Furthermore, inducible nitric oxide synthase (iNOS), ionized calcium-binding adaptor molecule 1 (Iba1), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1ß (IL-1ß), interleukin-1α (IL-1α), monocyte chemotactic protein 1 (MCP-1), transforming growth factor ß (TGFß), CIRBP, and RBM3 gene expressions were assessed via reverse transcription polymerase chain reaction, and TNF-α, IL-6, and IL-1ß releases into the cultured supernatants were assessed via enzyme-linked immunosorbent assays (ELISA). RESULTS: Prolonged exposure to OGD resulted in increased BV-2 necrotic cell death, which was attenuated by cooling. Cooling also significantly induced cold-shock proteins CIRBP and RBM3 gene expressions, with CIRBP expression more rapidly regulated than RBM3 and translatable to significantly increased protein expression. DAMPs including HMGB-1, HSP70, and CIRBP could be detected in cultured supernatants after 6 h of OGD with CIRBP release being significantly attenuated by cooling. Exposure to OGD suppressed cytokine gene expressions of IL-1ß, TNF-α, MCP-1, and TGFß independently of temperature management, whereas cooling led to a significant increase in IL-1α gene expression after 6 h of OGD. In the reperfusion phase, TNF-α and MCP-1 gene expressions were increased, and cooling was associated with significantly lower TGFß gene expression. Interestingly, cooled Normoxia groups had significant upregulations of microglial activation marker, Iba1, IL-1ß, and TNF-α gene expressions. CONCLUSION: BV-2 microglial cells undergo necrotic cell death resulting in DAMP release due to OGD/R-induced injury. Cooling conveyed neuroprotection in OGD/R-injury as observable in increased cell viability as well as induced gene expressions of cold shock proteins. As cooling alone resulted in both upregulation of microglial activation, expression of proinflammatory cytokines, and cold shock protein transcript and protein expression, temperature management might have ambiguous effects in sterile inflammation. However, cooling resulted in a significant decrease of extracellular CIRBP, which has recently been characterized as a novel DAMP and a potent initiator and mediator of inflammation.

Survival and Mid-Term Neurologic Outcome After Extracorporeal Cardiopulmonary Resuscitation in Children.

OBJECTIVES: Extracorporeal cardiopulmonary resuscitation in children with refractory cardiac arrest has been shown to improve survival, however, risk factors associated with mortality and neurologic impairments are not well defined. We analyzed our recent institutional experience with pediatric extracorporeal cardiopulmonary resuscitation to identify variables associated with survival and neurocognitive outcome. DESIGN: Retrospective observational study. SETTING: Pediatric cardiology and congenital heart surgery departments of a tertiary referral heart center. PATIENTS: Seventy-two consecutive children (median age, 0.3 yr [0.0-1.9 yr]) who underwent extracorporeal cardiopulmonary resuscitation at our institution during the study period from 2005 to 2016. INTERVENTIONS: Not applicable. MEASUREMENTS AND MAIN RESULTS: Median duration of resuscitation was 60 minutes (42-80 min) and median extracorporeal support duration was 5.4 days (2.2-7.9 d). Forty-three (59.7%) extracorporeal cardiopulmonary resuscitation events occurred during off-hours, however, neither duration of resuscitation (65 min [49-89 min] vs 51 min [35-80 min]; p = 0.16) nor survival (34.9% vs 37.9%; p = 0.81) differed significantly compared to working hours. Congenital heart disease was present in 84.7% of the patients. Survival to hospital discharge was 36.1%; younger age, higher lactate levels after resuscitation, acute kidney injury, renal replacement therapy, hepatic injury, and complexity of prior cardiothoracic surgical procedures were significantly associated with mortality. At mid-term follow-up (median, 4.1 yr [3.7-6.1 yr]), 22 patients (84.6% of discharge survivors) were still alive with 77.3% having a favorable neurologic outcome. High lactate levels, arrest location other than ICU, and requirement for renal replacement therapy were associated with unfavorable neurologic outcome. Interestingly, longer duration of resuscitation did not negatively impact survival or neurologic outcome. CONCLUSIONS: Extracorporeal cardiopulmonary resuscitation is a valuable tool for the treatment of children with refractory cardiac arrest and a favorable neurologic outcome can be achieved in the majority of survivors even after prolonged resuscitation. Mortality after extracorporeal cardiopulmonary resuscitation in postcardiac surgery children is associated with procedural complexity.

Post-TTM Rebound Pyrexia after Ischemia-Reperfusion Injury Results in Sterile Inflammation and Apoptosis in Cardiomyocytes.

INTRODUCTION: Fever is frequently observed after acute ischemic events and is associated with poor outcome and higher mortality. Targeted temperature management (TTM) is recommended for neuroprotection in comatose cardiac arrest survivors, but pyrexia after rewarming is proven to be detrimental in clinical trials. However, the cellular mechanisms and kinetics of post-TTM rebound pyrexia remain to be elucidated. Therefore, we investigated the effects of cooling and post-TTM pyrexia on the inflammatory response and apoptosis in a cardiomyocyte ischemia-reperfusion (IR) injury model. METHODS: HL-1 cardiomyocytes were divided into the following groups to investigate the effect of oxygen-glucose deprivation/reperfusion (OGD/R), hypothermia (33.5°C), and pyrexia (40°C): normoxia controls maintained at 37°C and warmed to 40°C, OGD/R groups maintained at 37°C and cooled to 33.5°C for 24 h with rewarming to 37°C, and OGD/R pyrexia groups further warmed from 37 to 40°C. Caspase-3 and RBM3 were assessed by Western blot and TNF-α, IL-6, IL-1ß, SOCS3, iNOS, and RBM3 transcriptions by RT-qPCR. RESULTS: OGD-induced oxidative stress (iNOS) in cardiomyocytes was attenuated post-TTM by cooling. Cytokine transcriptions were suppressed by OGD, while reperfusion induced significant TNF-α transcription that was exacerbated by cooling. Significant inductions of TNF-α, IL-6, IL-1ß, and SOCS3 were observed in noncooled, but not in cooled and rewarmed, OGD/R-injured cardiomyocytes. Further warming to pyrexia induced a sterile inflammatory response in OGD/R-injured groups that was attenuated by previous cooling, but no inflammation was observed in pyrexic normoxia groups. Moreover, cytoprotective RBM3 expression was induced by cooling but suppressed by pyrexia, correlating with apoptotic caspase-3 activation. CONCLUSION: Our findings show that maintaining a period of post-TTM "therapeutic normothermia" is effective in preventing secondary apoptosis-driven myocardial cell death, thus minimizing the infarct area and further release of mediators of the innate sterile inflammatory response after acute IR injury.

First steps to a clinical research unit for developmental research in paediatric cardiology: conception and progress of the LEADER project (Long Term Early Development Research) in CHD.

OBJECTIVE: We developed the Long-term Early Development Research (LEADER) project to investigate the development of children with CHD and/or after cardiopulmonary resuscitation. Both populations are at risk for delays in motor, cognitive, and language development. However, few studies to date have investigated the longitudinal development in these children. METHODS: To establish a clinical research unit, we planned three studies: a cross-sectional study in children after cardiopulmonary resuscitation (LEADER-REA Pilot Study), a longitudinal study in children after cardiopulmonary resuscitation, with a focus on evaluating various biomarkers as predictors for developmental outcome (LEADER-CPR study), and a longitudinal study in children with ventricular septal defect, tetralogy of Fallot, or transposition of the great arteries after cardiac surgery (LEADER-CHD study). RESULTS: Implementation of all three LEADER studies was successful and study protocols were conducted as planned. Findings from the LEADER-REA Pilot study have been recently published and data collection for both prospective trials is ongoing. Descriptive analysis of the first 20 assessments of the LEADER-CHD study showed no severe deficits in overall cognitive, motor, and language developments in the children. CONCLUSIONS: Children with CHD and/or after cardiopulmonary resuscitation are at risk for developmental delay. Therefore, a detailed developmental assessment is necessary as a pre-requisite for individual developmental support. Our LEADER project has been shown to be feasible in a clinical setting and is the first step towards the establishment of a clinical research unit in our clinic with a focus on longitudinal research.

Developmental Outcome in Infants with Cardiovascular Disease After Cardiopulmonary Resuscitation: A Pilot Study.

Unfavorable neurological outcome in children after cardiopulmonary resuscitation in infancy is frequent. However, few studies have investigated the development of these patients using comprehensive developmental tests and the feasibility of the Bayley Scales of Infant Development, 3rd Edition (BSID-III) has not been reported for this population. In this cross-sectional pilot study, we assessed the cognitive, language, and motor development in infants after cardiopulmonary resuscitation of ≥ 5 min with the BSID-III at the age of 12 or 24 months, depending on recruitment age. For analysis, 11 patients with in-hospital (n = 8) and out-of-hospital (n = 3) cardiac arrest were included. BSID-III results could not be quantified in three patients because of visual/hearing and/or motor impairment. In patients with quantifiable scores, 50.0% scored average in composite BSID-III scores, while the other 50.0% showed developmental delays, scoring distinctly below average. We conclude that the BSID-III is feasible for developmental assessment in the majority of the study population, but the use of instruments suitable for hearing/visually impaired and/or severely disabled infants is crucial to avoid biased results. Accurate characterization of developmental deficits is important to facilitate early identification and therapy of deficits.

Educational level and employment status in adults with congenital heart disease.

Purpose Through this study we aimed to assess the educational level and employment status of adults with CHD in Germany. METHODS: Data were acquired from an online survey carried out in 2015 by the German National Register for Congenital Heart Defects. A total of 1458 adults with CHD participated in the survey (response rate: 37.6%). For 1198 participants, detailed medical information, such as main cardiac diagnosis and information from medical reports, was available. RESULTS: Of the participants surveyed (n=1198), 54.5% (n=653) were female, and the mean age was 30 years. The majority of respondents (59.4%) stated that they had high education levels and that they were currently employed (51.1%). Patients with simple CHD had significantly higher levels of education (p<0.001) and were more likely to be employed (p=0.01) than were patients with complex CHD. CONCLUSIONS: More than half of the participants had high education levels and the majority were employed. The association between CHD and its severity and individuals' educational attainment should be investigated more closely in future studies.

Cardiac MRI in patients with complex CHD following primary or secondary implantation of MRI-conditional pacemaker system.

OBJECTIVES: In patients with CHD, cardiac MRI is often indicated for functional and anatomical assessment. With the recent introduction of MRI-conditional pacemaker systems, cardiac MRI has become accessible for patients with pacemakers. The present clinical study aims to evaluate safety, susceptibility artefacts, and image reading of cardiac MRI in patients with CHD and MRI-conditional pacemaker systems. Material and methods CHD patients with MRI-conditional pacemaker systems and a clinical need for cardiac MRI were examined with a 1.5-T MRI system. Lead function was tested before and after MRI. Artefacts and image readings were evaluated using a four-point grading scale. RESULTS: A total of nine patients with CHD (mean age 34.0 years, range 19.5-53.6 years) received a total of 11 cardiac MRI examinations. Owing to clinical indications, seven patients had previously been converted from conventional to MRI-conditional pacemaker systems. All MRI examinations were completed without adverse effects. Device testing immediately after MRI and at follow-up showed no alteration of pacemaker device and lead function. Clinical questions could be addressed and answered in all patients. CONCLUSION: Cardiac MRI can be performed safely with high certainty of diagnosis in CHD patients with MRI-conditional pacemaker systems. In case of clinically indicated lead and box changing, CHD patients with non-MRI-conditional pacemaker systems should be considered for complete conversion to MRI-conditional systems.

Effects of incremental beta-blocker dosing on myocardial mechanics of the human left ventricle: MRI 3D-tagging insight into pharmacodynamics supports theory of inner antagonism.

Beta-blockers contribute to treatment of heart failure. Their mechanism of action, however, is incompletely understood. Gradients in beta-blocker sensitivity of helically aligned cardiomyocytes compared with counteracting transversely intruding cardiomyocytes seem crucial. We hypothesize that selective blockade of transversely intruding cardiomyocytes by low-dose beta-blockade unloads ventricular performance. Cardiac magnetic resonance imaging (MRI) 3D tagging delivers parameters of myocardial performance. We studied 13 healthy volunteers by MRI 3D tagging during escalated intravenous administration of esmolol. The circumferential, longitudinal, and radial myocardial shortening was determined for each dose. The curves were analyzed for peak value, time-to-peak, upslope, and area-under-the-curve. At low doses, from 5 to 25 µg·kg(-1)·min(-1), peak contraction increased while time-to-peak decreased yielding a steeper upslope. Combining the values revealed a left shift of the curves at low doses compared with baseline without esmolol. At doses of 50 to 150 µg·kg(-1)·min(-1), a right shift with flattening occurred. In healthy volunteers we found more pronounced myocardial shortening at low compared with clinical dosage of beta-blockers. In patients with ventricular hypertrophy and higher prevalence of transversely intruding cardiomyocytes selective low-dose beta-blockade could be even more effective. MRI 3D tagging could help to determine optimal individual beta-blocker dosing avoiding undesirable side effects.

CCN1 mutation is associated with atrial septal defect.

The genetic basis of congenital heart disease remains unknown in most of the cases. Recently, a novel mouse model shed new light on the role of CCN1/CYR61, a matricellular regulatory factor, in cardiac morphogenesis. In a candidate gene approach, we analyzed a cohort of 143 patients with atrial septal defects (ASD) by sequencing the coding exons of CCN1. In addition to three frequent polymorphisms, we identified an extremely rare novel heterozygous missense mutation (c.139C > T; p.R47W) in one patient with severe ASD. The mutation leads to an exchange of residues with quite different properties in a highly conserved position of the N-terminal insulin-like growth factor binding protein module. Further bioinformatic analysis, exclusion of known ASD disease genes as well as the exclusion of the mutation in a very high number of ethnically matched controls (more than 1,000 individuals) and in public genetic databases, indicates that the p.R47W variant is a probable disease-associated mutation. The report about ASD in mice in heterozygous Ccn 1 +/- animals strongly supports this notion. Our study is the first to suggest a relationship between a probable CCN1 mutation and ASD. Our purpose here was to draw attention to CCN1, a gene that we believe may be important for genetic analysis in patients with congenital heart disease.

Promising results of a clinical feasibility study: CIRBP as a potential biomarker in pediatric cardiac surgery.

Objective: Cold-inducible RNA binding Protein (CIRBP) has been shown to be a potent inflammatory mediator and could serve as a novel biomarker for inflammation. Systemic inflammatory response syndrome (SIRS) and capillary leak syndrome (CLS) are frequent complications after pediatric cardiac surgery increasing morbidity, therefore early diagnosis and therapy is crucial. As CIRBP serum levels have not been analyzed in a pediatric population, we conducted a clinical feasibility establishing a customized magnetic bead panel analyzing CIRBP in pediatric patients undergoing cardiac surgery. Methods: A prospective hypothesis generating observational clinical study was conducted at the German Heart Center Berlin during a period of 9 months starting in May 2020 (DRKS00020885, https://drks.de/search/de/trial/DRKS00020885). Serum samples were obtained before the cardiac operation, upon arrival at the pediatric intensive care unit, 6 and 24 h after the operation in patients up to 18 years of age with congenital heart disease (CHD). Customized multiplex magnetic bead-based immunoassay panels were developed to analyze CIRBP, Interleukin-1ß (IL-1ß), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-10 (IL-10), Monocyte chemotactic protein 1 (MCP-1), Syndecan-1 (SDC-1), Thrombomodulin (TM), Vascular endothelial growth factor (VEGF-A), Angiopoietin-2 (Ang-2), and Fibroblast growth factor 23 (FGF-23) in 25 µl serum using the Luminex MagPix® system. Results: 19 patients representing a broad range of CHD (10 male patients, median age 2 years, 9 female patients, median age 3 years) were included in the feasibility study. CIRBP was detectable in the whole patient cohort. Relative to individual baseline values, CIRBP concentrations increased 6 h after operation and returned to baseline levels over time. IL-6, IL-8, IL-10, and MCP-1 concentrations were significantly increased after operation and except for MCP-1 concentrations stayed upregulated over time. SDC-1, TM, Ang-2, as well as FGF-23 concentrations were also significantly increased, whereas VEGF-A concentration was significantly decreased after surgery. Discussion: Using customized magnetic bead panels, we were able to detect CIRBP in a minimal serum volume (25 µl) in all enrolled patients. To our knowledge this is the first clinical study to assess CIRBP serum concentrations in a pediatric population.

Microcephaly is associated with impaired educational development in children with congenital heart disease.

Objectives: This study aims to evaluate the school careers of patients with congenital heart disease (CHD) and microcephaly. Methods: An exploratory online survey was conducted on patients from a previous study on somatic development in children with CHD in 2018 (n = 2818). A total of 750 patients participated in the online survey (26.6%). This publication focuses on 91 patients (12.1%) diagnosed with CHD and microcephaly who participated in the new online survey. Results: Microcephaly was significantly associated with CHD severity (p < 0.001). Microcephalic patients suffered from psychiatric comorbidity two times as often (67.0%) as non-microcephalic patients (29.8%). In particular, the percentage of patients with developmental delay, intellectual debility, social disability, learning disorder, or language disorder was significantly increased in microcephalic CHD patients (p < 0.001). A total of 85.7% of microcephalic patients and 47.6% of non-microcephalic patients received early interventions to foster their development. The school enrollment of both groups was similar at approximately six years of age. However, 89.9% of non-microcephalic but only 51.6% of microcephalic patients were enrolled in a regular elementary school. Regarding secondary school, only half as many microcephalic patients (14.3%) went to grammar school, while the proportion of pupils at special schools was eight times higher. Supportive interventions, e.g., for specific learning disabilities, were used by 52.7% of microcephalic patients and 21.6% of non-microcephalic patients. Conclusion: Patients with CHD and microcephaly are at high risk for impaired educational development. Early identification should alert clinicians to provide targeted interventions to optimize the developmental potential.

The German EMPATHIC-30 Questionnaire Showed Reliability and Convergent Validity for Use in an Intermediary/General Pediatric Cardiology Unit: A Psychometric Evaluation.

Background: Family-Centered Care is a useful framework for improving care for hospitalized children with congenital heart disease. The EMpowerment of PArents in THe Intensive Care-30 (EMPATHIC-30) questionnaire is a widely accepted tool to measure parental satisfaction with Family-Centered Care. Psychometric properties of the EMPATHIC-30 have been evaluated in neonatal and pediatric intensive care units, but not in pediatric cardiac care units. Therefore, our aim was to assess the psychometric properties of the German EMPATHIC-30 in an intermediary/general pediatric cardiology unit. Methods: We used data from a quality management survey comprising the German EMPATHIC-30, a sociodemographic questionnaire and four general satisfaction items. Data were collected at the intermediary/general pediatric cardiology unit of a specialized heart center in Germany (n = 366). We split the data randomly into two subsets. In the first subset, we assessed internal consistency reliability with McDonald's omega and Cronbach's alpha, and convergent validity using Spearman's rank correlation. Furthermore, we explored the internal structure with Principal Component Analysis (PCA). In the second subset, we validated the resulting structure using Confirmatory Factor Analysis (CFA). Results: The reliability estimates exceeded 0.70 for all five domain scores and 0.90 for the full-scale score. Convergent validity between EMPATHIC-30 domain scores/ the full-scale score and the four general satisfaction items was adequate (rs = 0.40-0.74). The PCA suggested three components, accounting for 56.8% of the total variance. Cross-validation via CFA showed poor model fit (χ2 = 1545.78, χ2/df = 3.85, CFI = 0.70, TLI = 0.66, RMSEA = 0.13), indicating that the EMPATHIC-30 shows no clear and generalizable factor structure in this sample. Discussion: The German version of the EMPATHIC-30 exhibited reasonable psychometric properties in an intermediary/general pediatric cardiology unit. Follow-up studies should investigate the factor structure of the EMPATHIC-30 in other pediatric inpatient care settings.

A gain-of-function TBX20 mutation causes congenital atrial septal defects, patent foramen ovale and cardiac valve defects.

BACKGROUND: Ostium secundum atrial septal defects (ASDII) account for approximately 10% of all congenital heart defects (CHD), and mutations in cardiac transcription factors, including TBX20, were identified as an underlying cause for ASDII. However, very little is known about disease penetrance in families and functional consequences of inherited TBX20 mutations. METHODS: The coding region of TBX20 was directly sequenced in 170 ASDII patients. Functional consequences of one novel mutation were investigated by surface plasmon resonance, CD spectropolarymetry, fluorescence spectrophotometry, luciferase assay and chromatin immunoprecipitation. RESULTS: We found a novel mutation in a highly conserved residue in the T-box DNA binding domain (I121M) segregating with CHD in a three generation kindred. Four mutation carriers revealed cardiac phenotypes in terms of cribriform ASDII, large patent foramen ovale or cardiac valve defects. Interestingly, tertiary hydrophobic interactions within the mutant TBX20 T-box were significantly altered leading to a more dynamic structure of the protein. Moreover, Tbx20-I121M resulted in a significantly enhanced transcriptional activity, which was further increased in the presence of co-transcription factors GATA4/5 and NKX2-5. Occupancy of DNA binding sites on target genes was also increased. CONCLUSIONS: We suggest that TBX20-I121M adopts a more fluid tertiary structure leading to enhanced interactions with cofactors and more stable transcriptional complexes on target DNA sequences. Our data, combined with that of others, suggest that human ASDII may be related to loss-of-function as well as gain-of-function TBX20 mutations.

Labeling of primary human hepatocytes with micron-sized iron oxide particles in suspension culture suitable for large-scale preparation.

Labeling of hepatocytes with micron-sized iron oxide particles (MPIOs) enables cell detection using clinical magnetic resonance equipment. For clinical applications, large numbers of cells must be labeled in a simple and rapid manner and have to be applied in suspension. However, all existing protocols are based on adhesion culture labeling with subsequent resuspension, only suitable for small experimental settings. The aim of this study was to investigate the feasibility of preparing MPIO-labeled primary human hepatocytes in a temporary suspension culture. Human hepatocytes were isolated from 16 donors and labeled with MPIOs in suspension, using the Rotary Cell Culture System. Particle incorporation was investigated by light and electron microscopy. Cells were compared with adhesion culture-labeled and subsequently enzymatically resuspended cells. During a period of 5 days, hepatocyte-specific parameters of cell damage (aspartate aminotransferase and alanine aminotransferase) and metabolic activity (urea and albumin) were analyzed (n=7). Suspension cultures showed a higher outcome in cell recovery compared with the conventional labeling method. When incubated with 180 particles/viable cell for 4 h, the mean particle uptake was 28.8 particles/cell at a labeling efficiency of 95.1%. Labeling in suspension had no adverse effects on cell integrity or metabolic activity. We conclude that labeling of human hepatocytes in suspension is feasible and simple and may serve future large-scale processing of cells.

Family-Centered Care at Pediatric Cardiac Intensive Care Units in Germany and the Relationship With Parent and Infant Well-Being: A Study Protocol.

Rationale and Aim: Infants with Congenital Heart Disease (CHD) are at risk for neurodevelopmental delays, emotional, social and behavioral difficulties. Hospitalization early in life and associated stressors may contribute to these challenges. Family-centered Care (FCC) is a health care approach that is respectful of and responsive to the needs and values of a family and has shown to be effective in improving health outcomes of premature infants, as well as the mental well-being of their parents. However, there is limited empirical data available on FCC practices in pediatric cardiology and associations with parent and infant outcomes. Methods and Analysis: In this cross-sectional study, we will explore FCC practices at two pediatric cardiac intensive care units in Germany, assess parent satisfaction with FCC, and investigate associations with parental mental well-being and parenting stress, as well as infant physical and mental well-being. We will collect data of 280 infants with CHD and their families. Data will be analyzed using multivariate statistics and multilevel modeling. Implications and Dissemination: The study protocol was approved by the medical ethics committees of both partner sites and registered with the German registry for clinical trials (NR DRKS00023964). This study serves as a first step to investigate FCC practices in a pediatric cardiology setting, providing insight into the relationship between FCC and parent and infant outcomes in a population of infants with CHD. Results will be disseminated in peer-reviewed journals.

Education of Children With Cyanotic Congenital Heart Disease After Neonatal Cardiac Surgery.

BACKGROUND: The aim of the study was to evaluate the educational achievement of patients diagnosed with univentricular heart physiology (UVHP) or transposition of the great arteries (TGA) after neonatal cardiac surgery. METHODS: An exploratory online survey was performed with patients registered with the National Register for Congenital Heart Defects in Germany. For this publication, a subgroup analysis was conducted among patients diagnosed with TGA (n = 173; 36.3%) and UVHP (n = 304; 63.7%). RESULTS: Median age of the sample at school enrollment was 6 years (range, 5-8 years). The majority were enrolled at a standard elementary school (n = 368 of 477; 77.1%), although patients with UVHP were enrolled significantly more often at a special needs school (n = 52 of 304; 17.1%, TGA patients n = 11/ of 173; 6.4%, P < .001). A total of 45.8% (n = 66 of 144) of the patients graduated with a high school diploma. A substantial number of patients had been diagnosed with behavioral or learning disorders (TGA patients n = 63 of 173 [36.4%], UVHP patients n = 148 of 304 [48.7%]) and received early supportive therapy or remedial teaching before (TGA patients n = 89 of 173 [51.4%], UVHP patients n = 209 of 304 [68.8%]) and/or during their school careers (TGA patients n = 54 of 173 [31.2%], UVHP patients n = 120 of 304 [39.5%]). CONCLUSIONS: A large proportion of patients who underwent neonatal cardiac surgery graduated with a high school diploma. These results are of great importance to congenital heart defect patients, affected families, and treating physicians. Nevertheless, study participants, especially patients with UVHP, face some academic challenges. We conclude that long-term follow-up examinations and regular developmental assessments may be beneficial.

Mechanisms of hypothermia-induced cell protection mediated by microglial cells in vitro.

Despite the widespread interest in the clinical applications of hypothermia, the cellular mechanisms of hypothermia-induced neuroprotection have not yet been clearly understood. Therefore, the aim of this study was to elucidate the cellular effects of clinically relevant hypothermia and rewarming on the morphological and functional characteristics of microglia. Microglial cells were exposed to a dynamic cooling and rewarming protocol. For stimulation, microglial cells were treated with 1 microg/mL lipopolysaccharide (LPS). We found that hypothermia led to morphological changes from ramified to ameboid cell shapes. At 2 h after hypothermia and rewarming, microglial cells were again ramified with extended branches. Moreover, we found enhanced cell activation after rewarming, accompanied by increased phagocytosis and adenosine triphosphate consumption. Interestingly, hypothermia and rewarming led to a time-dependent significant up-regulation of the anti-inflammatory cytokines interleukin-10 and interleukin-1 receptor antagonist in stimulated microglial cells. This is in line with the reduced proliferation and time-dependent down-regulation of the pro-inflammatory cytokines tumor necrosis factor-alpha and monocyte chemotactic protein-1 in comparison to normothermic control cells after LPS stimulation. Furthermore, degradation of the inhibitor of the nuclear transcription factor-kappaB (IkappaB-alpha) was diminished and delayed under conditions of cooling and rewarming in LPS-stimulated microglial cells. Thus, our results show that hypothermia and rewarming activate microglial cells, increase phagocytosis and shift the balance of cytokine release in stimulated microglial cells towards the anti-inflammatory cytokines. This could be a new cellular mechanism of hypothermia-induced neuroprotection mediated by activated microglial cells.

Vascular endothelial growth factor and its soluble receptor in infants with congenital cardiac disease.

Patients with cyanotic congenital cardiac disease often develop major aortopulmonary collaterals. Vascular endothelial growth factor is a key promoter of angiogenesis. Its soluble receptor-1 acts as a potent antagonist. We studied 30 infants with cyanotic congenital cardiac disease and 27 infants with acyanotic congenital cardiac disease. Central venous plasma vascular endothelial growth factor and soluble vascular endothelial growth factor receptor-1 levels were measured before, and 24 and 96 hours after surgery. There was no difference between plasma vascular endothelial growth factor levels in infants with cyanotic and those with acyanotic congenital cardiac disease. In cyanotic infants, the soluble vascular endothelial growth factor receptor-1 levels tended to be higher than in the acyanotic infants. In conclusion, there is no significant difference in the plasma levels of vascular endothelial growth factor and its soluble receptor-1 between infants with cyanotic and those with acyanotic congenital cardiac disease.