RESUMEN
Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are frequently used for preclinical cardiotoxicity testing and remain an important tool for confirming model-based predictions of drug effects in accordance with the comprehensive in vitro proarrhythmia assay (CiPA). Despite the considerable benefits hiPSC-CMs provide, concerns surrounding experimental reproducibility have emerged. We investigated the effects of temporal changes and experimental parameters on hiPSC-CM electrophysiology. iCell cardiomyocytes2 were cultured and biosignals were acquired using a microelectrode array (MEA) system (2-14 days). Continuous recordings revealed a 22.6% increase in the beating rate and 7.7% decrease in the field potential duration (FPD) during a 20-min equilibration period. Location-specific differences across a multiwell plate were also observed, with iCell cardiomyocytes2 in the outer rows beating 8.8 beats/min faster than the inner rows. Cardiac endpoints were also impacted by cell culture duration; from 2 to 14 days, the beating rate decreased (-12.7 beats/min), FPD lengthened (+257 ms), and spike amplitude increased (+3.3 mV). Cell culture duration (4-10 days) also impacted cardiomyocyte drug responsiveness (E-4031, nifedipine, isoproterenol). qRT-PCR results suggest that daily variations in cardiac metrics may be linked to the continued maturation of hiPSC-CMs in culture (2-30 days). Daily experiments were also repeated using a second cell line (Cor.4U). Collectively, our study highlights multiple sources of variability to consider and address when performing hiPSC-CM MEA studies. To improve reproducibility and data interpretation, MEA-based studies should establish a standardized protocol and report key experimental conditions (e.g., cell line, culture time, equilibration time, electrical stimulation settings, and raw data values).NEW & NOTEWORTHY We demonstrate that iCell cardiomyocytes2 electrophysiology measurements are impacted by deviations in experimental techniques including electrical stimulation protocols, equilibration time, well-to-well variability, and length of hiPSC-CM culture. Furthermore, our results indicate that hiPSC-CM drug responsiveness changes within the first 2 wk following defrost.
Asunto(s)
Células Madre Pluripotentes Inducidas , Miocitos Cardíacos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Reproducibilidad de los Resultados , Factores de Tiempo , Potenciales de Acción/efectos de los fármacos , Células Cultivadas , Isoproterenol/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Microelectrodos , Línea Celular , CardiotoxicidadRESUMEN
BACKGROUND: Phthalate chemicals are used to manufacture plastic medical products, including many components of cardiopulmonary bypass (CPB) circuits. We aimed to quantify iatrogenic phthalate exposure in pediatric patients undergoing cardiac surgery and examine the link between phthalate exposure and postoperative outcomes. STUDY DESIGN AND METHODS: The study included pediatric patients undergoing (n=122) unique cardiac surgeries at Children's National Hospital. For each patient, a single plasma sample was collected preoperatively and two additional samples were collected postoperatively upon return from the operating room and the morning after surgery. Concentrations of di(2-ethylhexyl) phthalate (DEHP) and its metabolites were quantified using ultra high-pressure liquid chromatography coupled to mass spectrometry. RESULTS: Patients were subdivided into three groups, according to surgical procedure: (1) cardiac surgery not requiring CPB support, (2) cardiac surgery requiring CPB with a crystalloid prime, and (3) cardiac surgery requiring CPB with red blood cells (RBCs) to prime the circuit. Phthalate metabolites were detected in all patients, and postoperative phthalate levels were highest in patients undergoing CPB with an RBC-based prime. Age-matched (<1 year) CPB patients with elevated phthalate exposure were more likely to experience postoperative complications. RBC washing was an effective strategy to reduce phthalate levels in CPB prime. DISCUSSION: Pediatric cardiac surgery patients are exposed to phthalate chemicals from plastic medical products, and the degree of exposure increases in the context of CPB with an RBC-based prime. Additional studies are warranted to measure the direct effect of phthalates on patient health outcomes and investigate mitigation strategies to reduce exposure.
Asunto(s)
Puente Cardiopulmonar , Humanos , Puente Cardiopulmonar/efectos adversos , Femenino , Masculino , Preescolar , Lactante , Niño , Dietilhexil Ftalato/sangre , Prevalencia , Plásticos , Ácidos Ftálicos/sangre , Procedimientos Quirúrgicos Cardíacos/efectos adversos , Adolescente , Recién NacidoRESUMEN
Background: Nearly 1% or 1.3 million babies are born with congenital heart disease (CHD) globally each year - many of whom will require palliative or corrective heart surgery within the first few years of life. A detailed understanding of cardiac maturation can help to expand our knowledge on cardiac diseases that develop during gestation, identify age-appropriate cardiovascular drug therapies, and inform clinical care decisions related to surgical repair, myocardial preservation, or postoperative management. Yet, to date, our knowledge of the temporal changes that cardiomyocytes undergo during postnatal development is largely limited to animal models. Methods: Right atrial tissue samples were collected from n=117 neonatal, infant, and pediatric patients undergoing correct surgery due to (acyanotic) CHD. Patients were stratified into five age groups: neonate (0-30 days), infant (31-364 days), toddler to preschool (1-5 years), school age (6-11 years), and adolescent to young adults (12-32 years). We measured age-dependent adaptations in cardiac gene expression, and used computational modeling to simulate action potential and calcium transients. Results: Enrichment of differentially expressed genes (DEG) was explored, revealing age-dependent changes in several key biological processes (cell cycle, cell division, mitosis), cardiac ion channels, and calcium handling genes. Gene-associated changes in ionic currents exhibited both linear trends and sudden shifts across developmental stages, with changes in calcium handling ( I NCX ) and repolarization ( I K1 ) most strongly associated with an age-dependent decrease in the action potential plateau potential and increase in triangulation, respectively. We also note a shift in repolarization reserve, with lower I Kr expression in younger patients, a finding likely tied to the increased amplitude of I Ks triggered by elevated sympathetic activation in pediatric patients. Conclusion: This study provides valuable insights into age-dependent changes in human cardiac gene expression and electrophysiology among patients with CHD, shedding light on molecular mechanisms underlying cardiac development and function across different developmental stages.