hiPSC-CM electrophysiology: impact of temporal changes and study parameters on experimental reproducibility.

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.

Electroanatomical adaptations in the guinea pig heart from neonatal to adulthood.

AIMS: Electroanatomical adaptations during the neonatal to adult phase have not been comprehensively studied in preclinical animal models. To explore the impact of age as a biological variable on cardiac electrophysiology, we employed neonatal and adult guinea pigs, which are a recognized animal model for developmental research. METHODS AND RESULTS: Electrocardiogram recordings were collected in vivo from anaesthetized animals. A Langendorff-perfusion system was employed for the optical assessment of action potentials and calcium transients. Optical data sets were analysed using Kairosight 3.0 software. The allometric relationship between heart weight and body weight diminishes with age, it is strongest at the neonatal stage (R2 = 0.84) and abolished in older adults (R2 = 1E-06). Neonatal hearts exhibit circular activation, while adults show prototypical elliptical shapes. Neonatal conduction velocity (40.6 ± 4.0 cm/s) is slower than adults (younger: 61.6 ± 9.3 cm/s; older: 53.6 ± 9.2 cm/s). Neonatal hearts have a longer action potential duration (APD) and exhibit regional heterogeneity (left apex; APD30: 68.6 ± 5.6 ms, left basal; APD30: 62.8 ± 3.6), which was absent in adults. With dynamic pacing, neonatal hearts exhibit a flatter APD restitution slope (APD70: 0.29 ± 0.04) compared with older adults (0.49 ± 0.04). Similar restitution characteristics are observed with extrasystolic pacing, with a flatter slope in neonates (APD70: 0.54 ± 0.1) compared with adults (younger: 0.85 ± 0.4; older: 0.95 ± 0.7). Neonatal hearts display unidirectional excitation-contraction coupling, while adults exhibit bidirectionality. CONCLUSION: Postnatal development is characterized by transient changes in electroanatomical properties. Age-specific patterns can influence cardiac physiology, pathology, and therapies for cardiovascular diseases. Understanding heart development is crucial to evaluating therapeutic eligibility, safety, and efficacy.

Adapting to a new environment: postnatal maturation of the human cardiomyocyte.

The postnatal mammalian heart undergoes remarkable developmental changes, which are stimulated by the transition from the intrauterine to extrauterine environment. With birth, increased oxygen levels promote metabolic, structural and biophysical maturation of cardiomyocytes, resulting in mature muscle with increased efficiency, contractility and electrical conduction. In this Topical Review article, we highlight key studies that inform our current understanding of human cardiomyocyte maturation. Collectively, these studies suggest that human atrial and ventricular myocytes evolve quickly within the first year but might not reach a fully mature adult phenotype until nearly the first decade of life. However, it is important to note that fetal, neonatal and paediatric cardiac physiology studies are hindered by a number of limitations, including the scarcity of human tissue, small sample size and a heavy reliance on diseased tissue samples, often without age-matched healthy controls. Future developmental studies are warranted to expand our understanding of normal cardiac physiology/pathophysiology and inform age-appropriate treatment strategies for cardiac disease.

Datamining approaches for examining the low prevalence of N-acetylglutamate synthase deficiency and understanding transcriptional regulation of urea cycle genes.

Ammonia, which is toxic to the brain, is converted into non-toxic urea, through a pathway of six enzymatically catalyzed steps known as the urea cycle. In this pathway, N-acetylglutamate synthase (NAGS, EC 2.3.1.1) catalyzes the formation of N-acetylglutamate (NAG) from glutamate and acetyl coenzyme A. NAGS deficiency (NAGSD) is the rarest of the urea cycle disorders, yet is unique in that ureagenesis can be restored with the drug N-carbamylglutamate (NCG). We investigated whether the rarity of NAGSD could be due to low sequence variation in the NAGS genomic region, high NAGS tolerance for amino acid replacements, and alternative sources of NAG and NCG in the body. We also evaluated whether the small genomic footprint of the NAGS catalytic domain might play a role. The small number of patients diagnosed with NAGSD could result from the absence of specific disease biomarkers and/or short NAGS catalytic domain. We screened for sequence variants in NAGS regulatory regions in patients suspected of having NAGSD and found a novel NAGS regulatory element in the first intron of the NAGS gene. We applied the same datamining approach to identify regulatory elements in the remaining urea cycle genes. In addition to the known promoters and enhancers of each gene, we identified several novel regulatory elements in their upstream regions and first introns. The identification of cis-regulatory elements of urea cycle genes and their associated transcription factors holds promise for uncovering shared mechanisms governing urea cycle gene expression and potentially leading to new treatments for urea cycle disorders.

Comparative cardiotoxicity assessment of bisphenol chemicals and estradiol using human induced pluripotent stem cell-derived cardiomyocytes.

Bisphenol A (BPA) is commonly used to manufacture consumer and medical-grade plastics. Due to health concerns, BPA substitutes are being incorporated-including bisphenol S (BPS) and bisphenol F (BPF)-without a comprehensive understanding of their toxicological profile. Previous studies suggest that bisphenol chemicals perturb cardiac electrophysiology in a manner that is similar to 17ß-estradiol (E2). We aimed to compare the effects of E2 with BPA, BPF, and BPS using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Cardiac parameters were evaluated using microelectrode array (MEA) technology and live-cell fluorescent imaging. Cardiac metrics remained relatively stable after exposure to nanomolar concentrations (1-1000 nM) of E2, BPA, BPF, or BPS. At higher micromolar concentrations, chemical exposures decreased the depolarization spike amplitude, and shortened the field potential, action potential duration, and calcium transient duration (E2 ≥ BPA ≥ BPF ≫ BPS). Cardiomyocyte physiology was largely undisturbed by BPS. BPA-induced effects were exaggerated when coadministered with an L-type calcium channel (LTCC) antagonist or E2, and reduced when coadministered with an LTCC agonist or an estrogen receptor alpha antagonist. E2-induced effects were not exaggerated by coadministration with an LTCC antagonist. Although the observed cardiac effects of E2 and BPA were similar, a few distinct differences suggest that these chemicals may act (in part) through different mechanisms. hiPSC-CM are a useful model for screening cardiotoxic chemicals, nevertheless, the described findings should be validated using a more complex ex vivo and/or in vivo model.

Electroanatomical Adaptations in the Guinea Pig Heart from Neonatal to Adulthood.

Background: Electroanatomical adaptations during the neonatal to adult phase have not been comprehensively studied in preclinical animal models. To explore the impact of age as a biological variable on cardiac electrophysiology, we employed neonatal and adult guinea pigs, which are a recognized animal model for developmental research. Methods: Healthy guinea pigs were categorized into three age groups (neonates, n=10; younger adults, n=13; and older adults, n=26). Electrocardiogram (ECG) recordings were collected in vivo from anesthetized animals (2-3% isoflurane). A Langendorff-perfusion system was employed for optical assessment of epicardial action potentials and calcium transients, using intact excised heart preparations. Optical data sets were analyzed and metric maps were constructed using Kairosight 3.0. Results: The allometric relationship between heart weight and body weight diminishes with age, as it is strongest at the neonatal stage (R 2 = 0.84) and completely abolished in older adults (R 2 = 1E-06). Neonatal hearts exhibit circular activation waveforms, while adults show prototypical elliptical shapes. Neonatal conduction velocity (40.6±4.0 cm/s) is slower than adults (younger adults: 61.6±9.3 cm/s; older adults: 53.6±9.2 cm/s). Neonatal hearts have a longer action potential duration (APD) and exhibit regional heterogeneity (left apex; APD30: 68.6±5.6 ms, left basal; APD30: 62.8±3.6), which was absent in adult epicardium. With dynamic pacing, neonatal hearts exhibit a flatter APD restitution slope (APD70: 0.29±0.04) compared to older adults (0.49±0.04). Similar restitution characteristics are observed with extrasystolic pacing, with a flatter slope in neonatal hearts (APD70: 0.54±0.1) compared to adults (Younger adults: 0.85±0.4; Older adults: 0.95±0.7). Finally, neonatal hearts display unidirectional excitation-contraction coupling, while adults exhibit bidirectionality. Conclusion: The transition from neonatal to adulthood in guinea pig hearts is characterized by transient changes in electroanatomic properties. Age-specific patterns can influence cardiac physiology, pathology, and therapies for cardiovascular diseases. Understanding postnatal heart development is crucial to evaluating therapeutic eligibility, safety, and efficacy. What is Known: Age-specific cardiac electroanatomical characteristics have been documented in humans and some preclinical animal models. These age-specific patterns can influence cardiac physiology, pathology, and therapies for cardiovascular diseases. What the Study Adds: Cardiac electroanatomical characteristics are age-specific in guinea pigs, a well-known preclinical model for developmental studies. Age-dependent adaptations in cardiac electrophysiology are readily observed in the electrocardiogram recordings and via optical mapping of epicardial action potentials and calcium transients. Our findings reveal unique activation and repolarization characteristics between neonatal and adult animals.

Transcriptome analysis revealed a novel nine-gene prognostic risk score of clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) continues to pose a significant global health concern, with rising incidence and high mortality rate. Accordingly, identifying molecular alternations associated with ccRCC is crucial to boost our understanding of its onset, persistence, and progression as well as developing prognostic biomarkers and novel therapies. Bulk RNA sequencing data and its associated clinicopathological variables of ccRCC were obtained from The Cancer Genome Atlas Program. Atypical differential gene expression analysis of advanced disease states using the extreme categories of staging and grading components was performed. Upregulated differentially expressed genes shared across the aforementioned components were selected. The risk-score construction pipeline started with univariate Cox logistic regression analysis, least absolute shrinkage and selection operator, and multivariate Cox logistic regression analysis in sequence. The generated risk score classified patients into low- vs high-risk groups. The predictive power of the constructed risk score was assessed using Kaplan-Meier curves analysis, multivariate Cox logistic regression analysis, and receiver operator curve of the overall survival. External validation of the risk score was performed using the E-MTAB-1980 cohort. The analysis work scheme established a novel nine-gene prognostic risk score composed of the following genes: ZIC2, TNNT1, SAA1, OTX1, C20orf141, CDHR4, HOXB13, IGFL2, and IGFN1. The high-risk group was associated with shortened overall survival and possessed an independent predictive power (hazard ratio: 1.942, 95% CI: 1.367-2.758, P < .0001, area under the curve = 0.719). In addition, the high-risk score was associated with advance clinicopathological parameters. The same pattern was observed within the external validation dataset (E-MTAB-1980 cohort), in which the high-risk score held a poor prognostic signature as well as independent predictive potential (hazard ratio: 5.121, 95% CI: 1.412-18.568, P = .013, area under the curve = 0.787). In the present work, a novel nine-gene prognostic risk score was constructed and validated. The risk score correlated with tumor immune microenvironment, somatic mutation patterns, and altered molecular pathways involved in tumorigenesis. Further experimental data are warranted to expand the work.

Deep skin fibroblast-mediated macrophage recruitment supports acute wound healing.

Epithelial and immune cells have long been appreciated for their contribution to the early immune response after injury; however, much less is known about the role of mesenchymal cells. Using single nuclei RNA-sequencing, we defined changes in gene expression associated with inflammation at 1-day post-wounding (dpw) in mouse skin. Compared to keratinocytes and myeloid cells, we detected enriched expression of pro-inflammatory genes in fibroblasts associated with deeper layers of the skin. In particular, SCA1+ fibroblasts were enriched for numerous chemokines, including CCL2, CCL7, and IL33 compared to SCA1- fibroblasts. Genetic deletion of Ccl2 in fibroblasts resulted in fewer wound bed macrophages and monocytes during injury-induced inflammation with reduced revascularization and re-epithelialization during the proliferation phase of healing. These findings highlight the important contribution of deep skin fibroblast-derived factors to injury-induced inflammation and the impact of immune cell dysregulation on subsequent tissue repair.

Connecting Transcriptomics with Computational Modeling to Reveal Developmental Adaptations in the Human Pediatric Myocardium.

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.

Comparative cardiotoxicity assessment of bisphenol chemicals and estradiol using human induced pluripotent stem cell-derived cardiomyocytes.

Background: Bisphenol A (BPA) is commonly used to manufacture consumer and medical-grade plastics. Due to health concerns, BPA substitutes are being incorporated - including bisphenol S (BPS) and bisphenol F (BPF) - without a comprehensive understanding of their toxicological profile. Objective: Previous studies suggest that bisphenol chemicals perturb cardiac electrophysiology in a manner that is similar to 17ß-estradiol (E2). We aimed to compare the effects of E2 with BPA, BPF, and BPS using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Methods: Cardiac parameters were evaluated using microelectrode array (MEA) technology and live-cell fluorescent imaging at baseline and in response to chemical exposure (0.001-100 µM). Results: Cardiac metrics remained relatively stable after exposure to nanomolar concentrations (1-1,000 nM) of E2, BPA, BPF, or BPS. At higher micromolar concentrations, chemical exposures resulted in a decrease in the depolarizing spike amplitude, shorter field potential and action potential duration, shorter calcium transient duration, and decrease in hiPSC-CM contractility (E2 > BPA > BPF >> BPS). Cardiomyocyte physiology was largely undisturbed by BPS exposure. BPA-induced effects were exaggerated when co-administered with an L-type calcium channel antagonist (verapamil) or E2 - and reduced when co-administered with an L-type calcium channel agonist (Bay K8644) or an estrogen receptor alpha antagonist (MPP). E2-induced effects generally mirrored those of BPA, but were not exaggerated by co-administration with an L-type calcium channel antagonist. Discussion: Collectively across multiple cardiac endpoints, E2 was the most potent and BPS was the least potent disruptor of hiPSC-CM function. Although the observed cardiac effects of E2 and BPA were similar, a few distinct differences suggest that these chemicals may act (in part) through different mechanisms. hiPSC-CM are a useful model for screening cardiotoxic chemicals, nevertheless, the described in vitro findings should be validated using a more complex ex vivo and/or in vivo model.

Use of the Arabic version of Oral Health Impact Profile-14 to evaluate the impact of periodontal disease on oral health-related quality of life among Jordanian adults.

We used the short-form of the Oral Health Impact Profile (OHIP-14) to assess the impact of periodontal diseases on the quality of life of Jordanian adults. A systematic random sample of 400 individuals was selected from patients referred to the periodontics clinic at the Dental Teaching Center in Irbid, Jordan. Those willing to participate were examined by specifically trained dentists and requested to complete the Arabic short-form version of the OHIP-14 questionnaire. Multivariate analysis of differences in OHIP-14 subscales among the periodontal disease groups was conducted using the general linear model multivariate procedure. This study included 400 adults (164 men and 236 women) aged between 18 and 60 years, with a mean (SD) of 36.7 (11.9) years. Of the 400 participants, 41.8% had chronic gingivitis, 19.8% had mild periodontitis, 23.3% had moderate periodontitis, and 15.3% had severe periodontitis. "Fairly often" or "very often" was reported for one or more items of the OHIP-14 by fewer than one-third of patients with gingivitis (32.9%) or mild periodontitis (31.6%), by about one-half of patients with moderate periodontitis (53.8%), and by about two-thirds of those with severe periodontitis (63.9%). There was a statistically significant association between the severity of periodontal disease and OHIP-14 scores (P < 0.05). Severe chronic periodontitis had a significantly greater impact on quality of life, specifically with regard to physical pain and physical disability (P < 0.05). Physical pain and physical disability were the dimensions most affected, and all OHIP-14 scores were significantly associated with severity of periodontal disease after adjusting for common confounders.