Prevalence and clinical implications of heightened plastic chemical exposure in pediatric patients undergoing cardiopulmonary bypass.

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.

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.

The Impact of Chronic Phthalate Exposure on Rodent Anxiety and Cognition.

Background: There is a growing importance for environmental contributions to psychiatric disorders and understanding the impact of the exposome (i.e., pollutants and toxins). For example, increased biomonitoring and epidemiological studies suggest that daily phthalate chemical exposure contributes to neurological and behavioral abnormalities; however, these mechanisms remain poorly understood. Therefore, the current study was aimed at examining the effects of chronic phthalate exposure on rodent anxiety behaviors and cognition and the impact on hypothalamic-pituitary-adrenal axis function. Methods: Adult male mice (C57BL6/J) were administered MEHP via drinking water (1 mg/mL), and anxiety-like behavior and cognition combined with hypothalamic-pituitary-adrenal axis and inflammatory assays were assessed after 3 weeks of MEHP exposure. Results: MEHP-treated mice exhibited enhanced generalized anxiety-like behaviors, as demonstrated by reduced time spent in the open-arm of the elevated plus maze and center exploration in the open field. Tests of spatial memory and cognition were unchanged. Following MEHP administration, circulating levels of corticosterone and proinflammatory cytokines were significantly increased, while at the tissue level, there were MEHP-dependent reductions in glucocorticoid metabolism genes Hsd11b1 and Hsd11b2. Conclusions: These data suggest that chronic MEHP exposure leads to enhanced generalized anxiety behaviors independent of rodent measures of cognition and memory, which may be driven by MEHP-dependent effects on hypothalamic-pituitary-adrenal axis and peripheral glucocorticoid metabolism function.

Changes in attachment and metabolic activity of rat neonatal cardiomyocytes and nonmyocytes caused by Macrovipera lebetina obtusa venom.

The Caucasian viper Macrovipera lebetina obtusa (MLO) is one of the most prevalent and venomous snakes in the Caucasus and the surrounding regions, yet the effects of MLO venom on cardiac function remain largely unknown. We examined the influence of MLO venom (crude and with inhibited metalloproteinases and phospholipase A2) on attachment and metabolic activity of rat neonatal cardiomyocytes (CM) and nonmyocytes (nCM), assessed at 1 and 24 h. After exposing both CM and nCM to varying concentrations of MLO venom, we observed immediate cytotoxic effects at a concentration of 100 µg/ml, causing detachment from the culture substrate. At lower MLO venom concentrations both cell types detached in a dose-dependent manner. Inhibition of MLO venom metalloproteinases significantly improved CM and nCM attachment after 1-hour exposure. At 24-hour exposure to metalloproteinases inhibited venom statistically significant enhancement was observed only in nCM attachment. However, metabolic activity of CM and nCM did not decrease upon exposure to the lower dose of the venom. Moreover, we demonstrated that metalloproteinases and phospholipases A2 are not the components of the MLO venom that change metabolic activity of both CM and nCM. These results provide a valuable platform to study the impact of MLO venom on prey cardiac function. They also call for further exploration of individual venom components for pharmaceutical purposes.

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) initiative. Despite the considerable benefits hiPSC-CMs provide, concerns surrounding experimental reproducibility have emerged. Our study aimed to investigate the effects of temporal changes and experimental parameters on hiPSC-CM electrophysiology. hiPSC-CMs (iCell cardiomyocyte 2 ) were cultured for 14 days and biosignals were acquired using a microelectrode array (MEA) system. Continuous recordings revealed a 22.6% increase in the beating rate and 7.7% decrease in the field potential duration (FPD) during a 20-minute equilibration period. Location specific differences across a multiwell plate were also observed, with hiPSC-CMs in the outer rows beating 8.8 beats per minute (BPM) faster than the inner rows. Cardiac endpoints were also impacted by cell culture duration; from 2-14 days the beating rate decreased (-12.7 BPM), FPD lengthened (+257 ms), and spike amplitude increased (+3.3 mV). Cell culture duration (4-10 days) also impacted hiPSC-CM drug responsiveness (E-4031, nifedipine, isoproterenol). Our study highlights multiple sources of variability that should be considered and addressed 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., culture time, equilibration time, electrical stimulation settings, report raw data values). New & Noteworthy: We demonstrate that hiPSC-CM electrophysiology measurements are significantly impacted by slight 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 two weeks following defrost.

The Impact of Chronic Phthalate Exposure on Rodent Anxiety and Cognition.

There is a growing importance for environmental contributions to psychiatric disorders and understanding the impact of the exposome (i.e., pollutants and toxins). Increased biomonitoring and epidemiological studies, for example, suggest that daily phthalate chemical exposure contribute to neurological and behavioral abnormalities, however these mechanisms remain poorly understood. The current study therefore aimed to examine the effects of chronic phthalate exposure on rodent anxiety behaviors, cognition, and the impact on hypothalamic-pituitary- adrenal (HPA)-axis function. Adult male mice (C57BL6/J) were administered mono-2-ethylhexyl phthalate (MEHP) via drinking water (1 mg/ml), and anxiety-like behavior, cognition combined with HPA- axis and inflammatory assays were assessed after 3 weeks of MEHP exposure. MEHP-treated mice exhibited enhanced generalized anxiety-like behaviors, as demonstrated by reduced time spent in the open-arm of the elevated plus maze (EPM) and center exploration in the open field (OF). Tests of spatial, cognition and memory function were unchanged. Following MEHP administration, circulating levels of corticosterone and pro- inflammatory cytokines were significantly increased, while at the tissue level, MEHP-dependent reductions in glucocorticoid metabolism genes 11ß-hydroxysteroid dehydrogenase (11ß-HSD) 1 and 2. These data suggest that chronic MEHP exposure leads to enhanced generalized-anxiety behaviors independent of rodent measures of cognition and memory, which maybe driven by MEHP-dependent effects on HPA-axis and peripheral glucocorticoid metabolism function.

KairoSight-3.0: A validated optical mapping software to characterize cardiac electrophysiology, excitation-contraction coupling, and alternans.

Background: Cardiac optical mapping is an imaging technique that measures fluorescent signals across a cardiac preparation. Dual optical imaging of voltage-sensitive and calcium-sensitive probes allows for simultaneous recordings of cardiac action potentials and intracellular calcium transients with high spatiotemporal resolution. The analysis of these complex optical datasets is both time intensive and technically challenging; as such, we have developed a software package for semi-automated image processing and analysis. Herein, we report an updated version of our software package (KairoSight-3.0) with features to enhance the characterization of cardiac parameters using optical signals. Methods: To test software validity and applicability, we used Langendorff-perfused heart preparations to record transmembrane voltage and intracellular calcium signals from the epicardial surface. Isolated hearts from guinea pigs and rats were loaded with a potentiometric dye (RH237) and/or calcium indicator dye (Rhod-2AM) and fluorescent signals were acquired. We used Python 3.8.5 programming language to develop the KairoSight-3.0 software. Cardiac maps were validated with a user-specified manual mapping approach. Results: Manual maps of action potential duration (30 or 80 % repolarization), calcium transient duration (30 or 80 % reuptake), action potential and calcium transient alternans were constituted to validate the accuracy of software-generated maps. Manual and software maps had high accuracy, with >97 % of manual and software values falling within 10 ms of each other and >75 % within 5 ms for action potential duration and calcium transient duration measurements (n = 1000-2000 pixels). Further, our software package includes additional measurement tools to analyze signal-to-noise ratio, conduction velocity, action potential and calcium transient alternans, and action potential-calcium transient coupling time to produce physiologically meaningful optical maps. Conclusions: KairoSight-3.0 has enhanced capabilities to perform measurements of cardiac electrophysiology, calcium handling, alternans, and the excitation-contraction coupling with satisfactory accuracy.

Evidence for the cardiodepressive effects of the plasticizer di-2-ethylhexyl phthalate.

Di-2-ethylhexyl phthalate (DEHP) is commonly used in the manufacturing of plastic materials, including intravenous bags, blood storage bags, and medical-grade tubing. DEHP can leach from plastic medical products, which can result in inadvertent patient exposure. DEHP concentrations were measured in red blood cell units stored between 7 and 42 days (17-119 µg/ml). Using these concentrations as a guide, Langendorff-perfused rat heart preparations were acutely exposed to DEHP. Sinus activity remained stable with lower doses of DEHP (25-50 µg/ml), but sinus rate declined by 43% and sinus node recovery time (SNRT) prolonged by 56.5% following 30-min exposure to 100 µg/ml DEHP. DEHP exposure also exerted a negative dromotropic response, as indicated by a 69.4% longer PR interval, 108.5% longer Wenckebach cycle length (WBCL), and increased incidence of atrioventricular (AV) uncoupling (60-min exposure). Pretreatment with doxycycline partially rescued the effects of DEHP on sinus activity, but did not ameliorate the effects on AV conduction. DEHP exposure also prolonged the ventricular action potential and effective refractory period, but had no measurable effect on intracellular calcium transient duration. Follow-up studies using human-induced pluripotent stem cell-derived cardiomyocytes confirmed that DEHP slows electrical conduction in a time (15 min-3 h) and dose-dependent manner (10-100 µg/ml). Previous studies have suggested that phthalate toxicity is specifically attributed to metabolites of DEHP, including mono-2-ethylhexylphthalate. This study demonstrates that DEHP exposure also contributes to cardiac dysfunction in a dose- and time-dependent manner. Future work is warranted to investigate the impact of DEHP (and its metabolites) on human health, with special consideration for clinical procedures that employ plastic materials.

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.

Evidence for the cardiodepressive effects of the plasticizer di-2-ethylhexylphthalate (DEHP).

Di-2-ethylhexylphthalate (DEHP) is commonly used in the manufacturing of plastic materials, including intravenous bags, blood storage bags, and medical-grade tubing. DEHP can leach from plastic medical products, which can result in inadvertent patient exposure. DEHP concentrations were measured in red blood cell (RBC) units stored between 7-42 days (23-119 µg/mL). Using these concentrations as a guide, Langendorff-perfused rat heart preparations were acutely exposed to DEHP. Sinus activity remained stable with lower doses of DEHP (25-50 µg/mL), but sinus rate declined by 43% and sinus node recovery time prolonged by 56.5% following 30-minute exposure to 100 µg/ml DEHP. DEHP exposure also exerted a negative dromotropic response, as indicated by a 69.4% longer PR interval, 108.5% longer Wenckebach cycle length, and increased incidence of atrioventricular uncoupling. Pretreatment with doxycycline partially rescued the effects of DEHP on sinus activity, but did not ameliorate the effects on atrioventricular conduction. DEHP exposure also prolonged the ventricular action potential and effective refractory period, but had no measurable effect on intracellular calcium transient duration. Follow-up studies using hiPSC-CM confirmed that DEHP slows electrical conduction in a time (15 min - 3 hours) and dose-dependent manner (10-100 µg/mL). Previous studies have suggested that phthalate toxicity is specifically attributed to metabolites of DEHP, including mono-2-ethylhexyl phthalate (MEHP). This study demonstrates that DEHP exposure also contributes to cardiac dysfunction in a dose- and time-dependent manner. Future work is warranted to investigate the impact of DEHP (and its metabolites) on human health, with special consideration for clinical procedures that employ plastic materials.

KairoSight-3.0 : A Validated Optical Mapping Software to Characterize Cardiac Electrophysiology, Excitation-Contraction Coupling, and Alternans.

Background: Cardiac optical mapping is an imaging technique that measures fluorescent signals across a cardiac preparation. Dual optical mapping of voltage-sensitive and calcium-sensitive probes allow for simultaneous recordings of cardiac action potentials and intracellular calcium transients with high spatiotemporal resolution. The analysis of these complex optical datasets is both time intensive and technically challenging; as such, we have developed a software package for semi-automated image processing and analysis. Herein, we report an updated version of our software package ( KairoSight-3 . 0 ) with features to enhance characterization of cardiac parameters using optical signals. Methods: To test software validity and applicability, we used Langendorff-perfused heart preparations to record transmembrane voltage and intracellular calcium signals from the epicardial surface. Isolated hearts from guinea pigs and rats were loaded with a potentiometric dye (RH237) and/or calcium indicator dye (Rhod-2AM) and fluorescent signals were acquired. We used Python 3.8.5 programming language to develop the KairoSight-3 . 0 software. Cardiac maps were validated with a user-specified manual mapping approach. Results: Manual maps of action potential duration (30 or 80% repolarization), calcium transient duration (30 or 80% reuptake), action potential and calcium transient alternans were constituted to validate the accuracy of software-generated maps. Manual and software maps had high accuracy, with >97% of manual and software values falling within 10 ms of each other and >75% within 5 ms for action potential duration and calcium transient duration measurements (n=1000-2000 pixels). Further, our software package includes additional cardiac metric measurement tools to analyze signal-to-noise ratio, conduction velocity, action potential and calcium transient alternans, and action potential-calcium transient coupling time to produce physiologically meaningful optical maps. Conclusions: KairoSight-3 . 0 has enhanced capabilities to perform measurements of cardiac electrophysiology, calcium handling, and the excitation-contraction coupling with satisfactory accuracy. Graphical Abstract Demonstrating Experimental and Data Analysis Workflow: Created with Biorender.com.

Prevalence and Clinical Implications of Heightened Plastic Chemical Exposure in Pediatric Patients Undergoing Cardiopulmonary Bypass.

Importance: Phthalate chemicals are used to manufacture disposable plastic medical products, including blood storage bags and components of cardiopulmonary bypass (CPB) circuits. During cardiac surgery, patients can be inadvertently exposed to phthalate chemicals that are released from these plastic products. Objective: To quantify iatrogenic phthalate chemical exposure in pediatric patients undergoing cardiac surgery, and examine the link between phthalate exposure and post-operative outcomes. Design Setting and Participants: The study cohort included 122 pediatric patients undergoing cardiac surgery at Children's National Hospital. For each patient, a single plasma sample was collected pre-operatively and two additional samples were collected post-operatively upon return from the operating room (post-operative day 0) and the morning after surgery (post-operative day 1). Exposures: Concentrations of di(2-ethylhexyl)phthalate (DEHP) and its metabolites were quantified using ultra high-pressure liquid chromatography coupled to mass spectrometry. Main Outcomes and Measures: Plasma concentrations of phthalates, post-operative blood gas measurements, and post-operative complications. Results: Study subjects were subdivided into three groups, according to surgical procedure: 1) cardiac surgery not requiring CPB support, 2) cardiac surgery requiring CPB with 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 RBC-based prime. Age-matched (<1 year) CPB patients with elevated phthalate exposure were more likely to experience post-operative complications, including arrhythmias, low cardiac output syndrome, and additional post-operative interventions. RBC washing was an effective strategy to reduce DEHP levels in CPB prime. Conclusions and Relevance: 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 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. Key Points: Question: Is cardiac surgery with cardiopulmonary bypass a significant source of phthalate chemical exposure in pediatric patients?Findings: In this study of 122 pediatric cardiac surgery patients, phthalate metabolites were quantified from blood samples before and after surgery. Phthalate concentrations were highest in patients undergoing cardiopulmonary bypass with red blood cell-based prime. Heightened phthalate exposure was associated with post-operative complications.Meaning: Cardiopulmonary bypass is a significant source of phthalate chemical exposure, and patients with heightened exposure may be at greater risk for postoperative cardiovascular complications.

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.

Diversity of cells and signals in the cardiovascular system.

This white paper is the outcome of the seventh UC Davis Cardiovascular Research Symposium on Systems Approach to Understanding Cardiovascular Disease and Arrhythmia. This biannual meeting aims to bring together leading experts in subfields of cardiovascular biomedicine to focus on topics of importance to the field. The theme of the 2022 Symposium was 'Cell Diversity in the Cardiovascular System, cell-autonomous and cell-cell signalling'. Experts in the field contributed their experimental and mathematical modelling perspectives and discussed emerging questions, controversies, and challenges in examining cell and signal diversity, co-ordination and interrelationships involved in cardiovascular function. This paper originates from the topics of formal presentations and informal discussions from the Symposium, which aimed to develop a holistic view of how the multiple cell types in the cardiovascular system integrate to influence cardiovascular function, disease progression and therapeutic strategies. The first section describes the major cell types (e.g. cardiomyocytes, vascular smooth muscle and endothelial cells, fibroblasts, neurons, immune cells, etc.) and the signals involved in cardiovascular function. The second section emphasizes the complexity at the subcellular, cellular and system levels in the context of cardiovascular development, ageing and disease. Finally, the third section surveys the technological innovations that allow the interrogation of this diversity and advancing our understanding of the integrated cardiovascular function and dysfunction.

The effect of sex and age on ex vivo cardiac electrophysiology: insight from a guinea pig model.

Highlighting the importance of sex as a biological variable, we recently reported sex differences in guinea pig in vivo electrocardiogram (ECG) measurements. However, substantial inconsistencies exist in this animal model, with conflicting reports of sex-specific differences in cardiac electrophysiology observed in vivo and in vitro. Herein, we evaluated whether sexual dimorphism persists in ex vivo preparations, using an isolated intact heart preparation. Pseudo-ECG recordings were collected in conjunction with dual optical mapping of transmembrane voltage and intracellular calcium from Langendorff-perfused hearts. In contrast to our in vivo results, we did not observe sex-specific differences in ECG parameters collected from isolated hearts. Furthermore, we observed significant age-specific differences in action potential duration (APD) and Ca2+ transient duration (CaD) during both normal sinus rhythm (NSR) and in response to dynamic pacing but only a modest sex-specific difference in CaD30. Similarly, the alternans fluctuation coefficient, conduction velocity during sinus rhythm or in response to pacing, and electrophysiology parameters (atrioventricular nodal effective refractory period, Wenckebach cycle length) were comparable between males and females. Results of our study suggest that the observed sex-specific differences in in vivo ECG parameters from guinea pigs are diminished in ex vivo isolated heart preparations, although age-specific patterns are prevalent. To assess sex as a biological variable in cardiac electrophysiology, a comprehensive approach may be necessary using both in vitro measurements from cardiomyocyte or intact heart preparations with secondary follow-up in vivo studies.NEW & NOTEWORTHY We evaluated whether the guinea pig heart has intrinsic sex-specific differences in cardiac electrophysiology. Although we observed sex-specific differences in in vivo ECGs, these differences did not persist ex vivo. Using a whole heart model, we observed similar APD, CaD, conduction velocity, and alternans susceptibility in males and females. We conclude that sex-specific differences in guinea pig cardiac electrophysiology are likely influenced by the in vivo environment and less dependent on the intrinsic electrical properties of the heart.

Guidelines for assessment of cardiac electrophysiology and arrhythmias in small animals.

Cardiac arrhythmias are a major cause of morbidity and mortality worldwide. Although recent advances in cell-based models, including human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM), are contributing to our understanding of electrophysiology and arrhythmia mechanisms, preclinical animal studies of cardiovascular disease remain a mainstay. Over the past several decades, animal models of cardiovascular disease have advanced our understanding of pathological remodeling, arrhythmia mechanisms, and drug effects and have led to major improvements in pacing and defibrillation therapies. There exist a variety of methodological approaches for the assessment of cardiac electrophysiology and a plethora of parameters may be assessed with each approach. This guidelines article will provide an overview of the strengths and limitations of several common techniques used to assess electrophysiology and arrhythmia mechanisms at the whole animal, whole heart, and tissue level with a focus on small animal models. We also define key electrophysiological parameters that should be assessed, along with their physiological underpinnings, and the best methods with which to assess these parameters.

Demographic and Methodological Heterogeneity in Electrocardiogram Signals From Guinea Pigs.

Electrocardiograms (ECG) are universally used to measure the electrical activity of the heart; however, variations in recording techniques and/or subject demographics can affect ECG interpretation. In this study, we investigated variables that are likely to influence ECG metric measurements in cardiovascular research, including recording technique, use of anesthesia, and animal model characteristics. Awake limb lead ECG recordings were collected in vivo from adult guinea pigs using a platform ECG system, while recordings in anesthetized animals were performed using both a platform and needle ECG system. We report significant heterogeneities in ECG metric values that are attributed to methodological differences (e.g., ECG lead configuration, ECG recording platform, presence or absence of anesthesia) that persist even within the same cohort of animals. Further, we report that variability in animal demographics is preserved in vivo ECG recordings-with animal age serving as a significant contributor, while sex-specific influences were less pronounced. Methodological approaches and subject demographics should be fully considered when interpreting ECG values in animal models, comparing datasets between studies, or developing artificial intelligence algorithms that utilize an ECG database.

Characteristics of Bisphenol Cardiotoxicity: Impaired Excitability, Contractility, and Relaxation.

Bisphenol a (BPA) is a high production volume chemical that is frequently used to manufacture epoxy resins and polycarbonate plastics. BPA-containing products are now pervasive, and as a result, biomonitoring studies report widespread exposure in > 90% of adults, adolescents, and children. Both epidemiological and experimental studies have reported associations between BPA exposure and adverse cardiovascular health outcomes. With increasing concerns regarding BPA exposure, a few structurally similar bisphenol chemicals have been introduced as replacements, including bisphenol s (BPS) and bisphenol f (BPF). In accordance with the recently established "Key characteristics of cardiovascular toxicants", we reviewed the literature to highlight the immediate effects of bisphenol chemicals on (1) cardiac excitability, and (2) contractility and relaxation. BPA inhibits key cardiac ion channels, impairs cardiac excitability, and acts as a more potent inhibitor as compared to BPF and BPS. Through the inhibition of calcium current, some studies report that bisphenol chemicals can act as negative inotropic agents. Yet, others suggest that low dose exposures may increase contractility and precipitate triggered arrhythmias via the phosphorylation of key calcium handling proteins. Accordingly, we propose additional considerations for future work to comprehensively address the cardiac safety profile of BPA, as compared to replacement chemicals.