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.

Stop the beat to see the rhythm: excitation-contraction uncoupling in cardiac research.

Optical mapping is an imaging technique that is extensively used in cardiovascular research, wherein parameter-sensitive fluorescent indicators are used to study the electrophysiology and excitation-contraction coupling of cardiac tissues. Despite many benefits of optical mapping, eliminating motion artifacts within the optical signals is a major challenge, as myocardial contraction interferes with the faithful acquisition of action potentials and intracellular calcium transients. As such, excitation-contraction uncoupling agents are frequently used to reduce signal distortion by suppressing contraction. When compared with other uncoupling agents, blebbistatin is the most frequently used, as it offers increased potency with minimal direct effects on cardiac electrophysiology. Nevertheless, blebbistatin may exert secondary effects on electrical activity, metabolism, and coronary flow, and the incorrect administration of blebbistatin to cardiac tissue can prove detrimental, resulting in erroneous interpretation of optical mapping results. In this "Getting It Right" perspective, we briefly review the literature regarding the use of blebbistatin in cardiac optical mapping experiments, highlight potential secondary effects of blebbistatin on cardiac electrical activity and metabolic demand, and conclude with the consensus of the authors on best practices for effectively using blebbistatin in optical mapping studies of cardiac tissue.

Chronic perinatal hypoxia delays cardiac maturation in a mouse model for cyanotic congenital heart disease.

Compared with acyanotic congenital heart disease (CHD), cyanotic CHD has an increased risk of lifelong mortality and morbidity. These adverse outcomes may be attributed to delayed cardiomyocyte maturation, since the transition from a hypoxic fetal milieu to oxygen-rich postnatal environment is disrupted. We established a rodent model to replicate hypoxic myocardial conditions spanning perinatal development, and tested the hypothesis that chronic hypoxia impairs cardiac development. Pregnant mice were housed in hypoxia beginning at embryonic day 16. Pups stayed in hypoxia until postnatal day (P)8 when cardiac development is nearly complete. Global gene expression was quantified at P8 and at P30, after recovering in normoxia. Phenotypic testing included electrocardiogram, echocardiogram, and ex vivo electrophysiology study. Hypoxic P8 animals were 47% smaller than controls with preserved heart size. Gene expression was grossly altered by hypoxia at P8 (1,427 genes affected), but normalized after recovery (P30). Electrocardiograms revealed bradycardia and slowed conduction velocity in hypoxic animals at P8, with noticeable resolution after recovery (P30). Notable differences that persisted after recovery (P30) included a 65% prolongation in ventricular effective refractory period, sinus node dysfunction, 23% reduction in ejection fraction, and 16% reduction in fractional shortening in animals exposed to hypoxia. We investigated the impact of chronic hypoxia on the developing heart. Perinatal hypoxia was associated with changes in gene expression and cardiac function. Persistent changes to the electrophysiological substrate and contractile function warrant further investigation and may contribute to adverse outcomes observed in the cyanotic CHD population.NEW & NOTEWORTHY We utilized a new mouse model of chronic perinatal hypoxia to simulate the hypoxic myocardial conditions present in cyanotic congenital heart disease. Hypoxia caused numerous abnormalities in cardiomyocyte gene expression, the electrophysiologic substrate of the heart, and contractile function. Taken together, alterations observed in the neonatal period suggest delayed cardiac development immediately following hypoxia.

Age-dependent changes in electrophysiology and calcium handling: implications for pediatric cardiac research.

Rodent models are frequently employed in cardiovascular research, yet our understanding of pediatric cardiac physiology has largely been deduced from more simplified two-dimensional cell studies. Previous studies have shown that postnatal development includes an alteration in the expression of genes and proteins involved in cell coupling, ion channels, and intracellular calcium handling. Accordingly, we hypothesized that postnatal cell maturation is likely to lead to dynamic alterations in whole heart electrophysiology and calcium handling. To test this hypothesis, we employed multiparametric imaging and electrophysiological techniques to quantify developmental changes from neonate to adult. In vivo electrocardiograms were collected to assess changes in heart rate, variability, and atrioventricular conduction (Sprague-Dawley rats). Intact, whole hearts were transferred to a Langendorff-perfusion system for multiparametric imaging (voltage, calcium). Optical mapping was performed in conjunction with an electrophysiology study to assess cardiac dynamics throughout development. Postnatal age was associated with an increase in the heart rate (181 ± 34 vs. 429 ± 13 beats/min), faster atrioventricular conduction (94 ± 13 vs. 46 ± 3 ms), shortened action potentials (APD80: 113 ± 18 vs. 60 ± 17 ms), and decreased ventricular refractoriness (VERP: 157 ± 45 vs. 57 ± 14 ms; neonatal vs. adults, means ± SD, P < 0.05). Calcium handling matured with development, resulting in shortened calcium transient durations (168 ± 18 vs. 117 ± 14 ms) and decreased propensity for calcium transient alternans (160 ± 18- vs. 99 ± 11-ms cycle length threshold; neonatal vs. adults, mean ± SD, P < 0.05). Results of this study can serve as a comprehensive baseline for future studies focused on pediatric disease modeling and/or preclinical testing.NEW & NOTEWORTHY This is the first study to assess cardiac electrophysiology and calcium handling throughout postnatal development, using both in vivo and whole heart models.

Functional response of the isolated, perfused normoxic heart to pyruvate dehydrogenase activation by dichloroacetate and pyruvate.

Dichloroacetate (DCA) and pyruvate activate pyruvate dehydrogenase (PDH), a key enzyme that modulates glucose oxidation and mitochondrial NADH production. Both compounds improve recovery after ischemia in isolated hearts. However, the action of DCA and pyruvate in normoxic myocardium is incompletely understood. We measured the effect of DCA and pyruvate on contraction, mitochondrial redox state, and intracellular calcium cycling in isolated rat hearts during normoxic perfusion. Normalized epicardial NADH fluorescence (nNADH) and left ventricular developed pressure (LVDP) were measured before and after administering DCA (5 mM) or pyruvate (5 mM). Optical mapping of Rhod-2AM was used to measure cytosolic calcium kinetics. DCA maximally activated PDH, increasing the ratio of active to total PDH from 0.48 ± 0.03 to 1.03 ± 0.03. Pyruvate sub-maximally activated PDH to a ratio of 0.75 ± 0.02. DCA and pyruvate increased LVDP. When glucose was the only exogenous fuel, pyruvate increased nNADH by 21.4 ± 2.9 % while DCA reduced nNADH by 21.4 ± 6.1 % and elevated the incidence of premature ventricular contractions (PVCs). When lactate, pyruvate, and glucose were provided together as exogenous fuels, nNADH increased with DCA, indicating that PDH activation with glucose as the only exogenous fuel depletes PDH substrate. Calcium transient time-to-peak was shortened by DCA and pyruvate and SR calcium re-uptake was 30 % longer. DCA and pyruvate increased SR calcium load in myocyte monolayers. Overall, during normoxia when glucose is the only exogenous fuel, DCA elevates SR calcium, increases LVDP and contractility, and diminishes mitochondrial NADH. Administering DCA with plasma levels of lactate and pyruvate mitigates the drop in mitochondrial NADH and prevents PVCs.

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.

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.

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.

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.

Properties of blebbistatin for cardiac optical mapping and other imaging applications.

Blebbistatin is a recently discovered myosin II inhibitor. It is rapidly becoming a compound of choice to reduce motion artifacts during cardiac optical mapping, as well as to study cell motility and cell invasion. Although blebbistatin has a number of advantages over other electromechanical uncouplers, many of its properties have yet to be addressed. Here we describe several methodological issues associated with the use of blebbistatin, including its spectral properties, reversibility, and its effect on tissue metabolic state. We show that if precautions are not taken, perfusion with blebbistatin may result in blebbistatin precipitate that accumulates in the vasculature. Although such precipitate is fluorescent, it is not detectable within wavelength bands that are typically used for transmembrane voltage fluorescence imaging (i.e., emission wavelengths >600 nm). Therefore, blockage of the microcirculation by blebbistatin may cause data misinterpretation in studies that use voltage-sensitive dyes. Blebbistatin may also impact imaging of green fluorophores due to the spectral shift it causes in endogenous tissue fluorescence. 3D excitation-emission matrices of blebbistatin in precipitate form and in various solutions (DMSO, water, and 1 % aqueous albumin) revealed significant changes in the fluorescence of this molecule in different environments. Finally, we examined the reversibility of blebbistatin's uncoupling effect on cardiac contraction. Our findings provide important new information about the properties of this myosin II inhibitor, which will aid in the proper design and interpretation of studies that use this compound.

Use of endogenous NADH fluorescence for real-time in situ visualization of epicardial radiofrequency ablation lesions and gaps.

Radiofrequency ablation (RFA) aims to produce lesions that interrupt reentrant circuits or block the spread of electrical activation from sites of abnormal activity. Today, there are limited means for real-time visualization of cardiac muscle tissue injury during RFA procedures. We hypothesized that the fluorescence of endogenous NADH could be used as a marker of cardiac muscle injury during epicardial RFA procedures. Studies were conducted in blood-free and blood-perfused hearts from healthy adult Sprague-Dawley rats and New Zealand rabbits. Radiofrequency was applied to the epicardial surface of the heart using a 4-mm standard blazer ablation catheter. A dual camera optical mapping system was used to monitor NADH fluorescence upon ultraviolet illumination of the epicardial surface and to record optical action potentials using the voltage-sensitive probe RH237. Epicardial lesions were seen as areas of low NADH fluorescence. The lesions appeared immediately after ablation and remained stable for several hours. Real-time monitoring of NADH fluorescence allowed visualization of viable tissue between the RFA lesions. Dual recordings of NADH and epicardial electrical activity linked the gaps between lesions to postablation reentries. We found that the fluorescence of endogenous NADH aids the visualization of injured epicardial tissue caused by RFA. This was true for both blood-free and blood-perfused preparations. Gaps between NADH-negative regions revealed unablated tissue, which may promote postablation reentry or provide pathways for the conduction of abnormal electrical activity.

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.

KairoSight: Open-Source Software for the Analysis of Cardiac Optical Data Collected From Multiple Species.

Cardiac optical mapping, also known as optocardiography, employs parameter-sensitive fluorescence dye(s) to image cardiac tissue and resolve the electrical and calcium oscillations that underly cardiac function. This technique is increasingly being used in conjunction with, or even as a replacement for, traditional electrocardiography. Over the last several decades, optical mapping has matured into a "gold standard" for cardiac research applications, yet the analysis of optical signals can be challenging. Despite the refinement of software tools and algorithms, significant programming expertise is often required to analyze large optical data sets, and data analysis can be laborious and time-consuming. To address this challenge, we developed an accessible, open-source software script that is untethered from any subscription-based programming language. The described software, written in python, is aptly named "KairoSight" in reference to the Greek word for "opportune time" (Kairos) and the ability to "see" voltage and calcium signals acquired from cardiac tissue. To demonstrate analysis features and highlight species differences, we employed experimental datasets collected from mammalian hearts (Langendorff-perfused rat, guinea pig, and swine) dyed with RH237 (transmembrane voltage) and Rhod-2, AM (intracellular calcium), as well as human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) dyed with FluoVolt (membrane potential), and Fluo-4, AM (calcium indicator). We also demonstrate cardiac responsiveness to ryanodine (ryanodine receptor modulator) and isoproterenol (beta-adrenergic agonist) and highlight regional differences after an ablation injury. KairoSight can be employed by both basic and clinical scientists to analyze complex cardiac optical mapping datasets without requiring dedicated computer science expertise or proprietary software.

Bisphenol S and Bisphenol F Are Less Disruptive to Cardiac Electrophysiology, as Compared With Bisphenol A.

Bisphenol A (BPA) is a high-production volume chemical used to manufacture consumer and medical-grade plastic products. Due to its ubiquity, the general population can incur daily environmental exposure to BPA, whereas heightened exposure has been reported in intensive care patients and industrial workers. Due to health concerns, structural analogs are being explored as replacements for BPA. This study aimed to examine the direct effects of BPA on cardiac electrophysiology compared with recently developed alternatives, including BPS (bisphenol S) and BPF (bisphenol F). Whole-cell voltage-clamp recordings were performed on cell lines transfected to express the voltage-gated sodium channel (Nav1.5), L-type voltage-gated calcium channel (Cav1.2), or the rapidly activating delayed rectifier potassium channel (hERG). Cardiac electrophysiology parameters were measured using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and intact, whole rat heart preparations. BPA was the most potent inhibitor of fast/peak (INa-P) and late (INa-L) sodium channel (IC50 = 55.3, 23.6 µM, respectively), L-type calcium channel (IC50 = 30.8 µM), and hERG channel current (IC50 = 127 µM). Inhibitory effects on L-type calcium channels were supported by microelectrode array recordings, which revealed a shortening of the extracellular field potential (akin to QT interval). BPA and BPF exposures slowed atrioventricular (AV) conduction and increased AV node refractoriness in isolated rat heart preparations, in a dose-dependent manner (BPA: +9.2% 0.001 µM, +95.7% 100 µM; BPF: +20.7% 100 µM). BPS did not alter any of the cardiac electrophysiology parameters tested. Results of this study demonstrate that BPA and BPF exert an immediate inhibitory effect on cardiac ion channels, whereas BPS is markedly less potent. Additional studies are necessary to fully elucidate the safety profile of bisphenol analogs on the heart.

Clinically relevant concentrations of di (2-ethylhexyl) phthalate (DEHP) uncouple cardiac syncytium.

Di(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer found in a variety of polyvinyl chloride (PVC) medical products. The results of studies in experimental animals suggest that DEHP leached from flexible PVC tubing may cause health problems in some patient populations. While the cancerogenic and reproductive effects of DEHP are well recognized, little is known about the potential adverse impact of phthalates on the heart. This study examined the effects of clinically relevant concentrations of DEHP on neonatal rat cardiomyocytes. It was found that application of DEHP to a confluent, synchronously beating cardiac cell network, leads to a marked, concentration-dependent decrease in conduction velocity and asynchronous cell beating. The mechanism behind these changes was a loss of gap junctional connexin-43, documented using Western blot analysis, dye-transfer assay and immunofluorescence. In addition to its effect on electrical coupling, DEHP treatment also affected the mechanical movement of myocyte layers. The latter was linked to the decreased stiffness of the underlying fibroblasts, as the amount of triton-insoluble vimentin was significantly decreased in DEHP-treated samples. The data indicate that DEHP, in clinically relevant concentrations, can impair the electrical and mechanical behavior of a cardiac cell network. Applicability of these findings to human patients remains to be established.

Lights, camera, path splitter: a new approach for truly simultaneous dual optical mapping of the heart with a single camera.

BACKGROUND: Optical mapping of transmembrane voltage and intracellular calcium is a powerful tool for investigating cardiac physiology and pathophysiology. However, simultaneous dual mapping of two fluorescent probes remains technically challenging. We introduce a novel, easy-to-use approach that requires a path splitter, single camera and excitation light to simultaneously acquire voltage and calcium signals from whole heart preparations, which can be applied to other physiological models - including neurons and isolated cardiomyocytes. RESULTS: Complementary probes were selected that could be excited with a single wavelength light source. Langendorff-perfused hearts (rat, swine) were stained and imaged using a sCMOS camera outfitted with an optical path splitter to simultaneously acquire two emission fields at high spatial and temporal resolution. Voltage (RH237) and calcium (Rhod2) signals were acquired concurrently on a single sensor, resulting in two 384 × 256 images at 814 frames per second. At this frame rate, the signal-to-noise ratio was 47 (RH237) and 85 (Rhod2). Imaging experiments were performed on small rodent hearts, as well as larger pig hearts with sufficient optical signals. In separate experiments, each dye was used independently to assess crosstalk and demonstrate signal specificity. Additionally, the effect of ryanodine on myocardial calcium transients was validated - with no measurable effect on the amplitude of optical action potentials. To demonstrate spatial resolution, ventricular tachycardia was induced - resulting in the novel finding that spatially discordant calcium alternans can be present in different regions of the heart, even when electrical alternans remain concordant. The described system excels in providing a wide field of view and high spatiotemporal resolution for a variety of cardiac preparations. CONCLUSIONS: We report the first multiparametric mapping system that simultaneously acquires calcium and voltage signals from cardiac preparations, using a path splitter, single camera and excitation light. This approach eliminates the need for multiple cameras, excitation light patterning or frame interleaving. These features can aid in the adoption of dual mapping technology by the broader cardiovascular research community, and decrease the barrier of entry into panoramic heart imaging, as it reduces the number of required cameras.

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

Plasticizer Interaction With the Heart: Chemicals Used in Plastic Medical Devices Can Interfere With Cardiac Electrophysiology.

BACKGROUND: Phthalates are used as plasticizers in the manufacturing of flexible, plastic medical products. Patients can be subjected to high phthalate exposure through contact with plastic medical devices. We aimed to investigate the cardiac safety and biocompatibility of mono-2-ethylhexyl phthalate (MEHP), a phthalate with documented exposure in intensive care patients. METHODS: Optical mapping of transmembrane voltage and pacing studies were performed on isolated, Langendorff-perfused rat hearts to assess cardiac electrophysiology after MEHP exposure compared with controls. MEHP dose was chosen based on reported blood concentrations after an exchange transfusion procedure. RESULTS: Thirty-minute exposure to MEHP increased the atrioventricular node (147 versus 107 ms) and ventricular (117 versus 77.5 ms) effective refractory periods, compared with controls. Optical mapping revealed prolonged action potential duration at slower pacing cycle lengths, akin to reverse use dependence. The plateau phase of the action potential duration restitution curve steepened and became monophasic in MEHP-exposed hearts (0.18 versus 0.06 slope). Action potential duration lengthening occurred during late-phase repolarization resulting in triangulation (70.3 versus 56.6 ms). MEHP exposure also slowed epicardial conduction velocity (35 versus 60 cm/s), which may be partly explained by inhibition of Nav1.5 (874 and 231 µmol/L half-maximal inhibitory concentration, fast and late sodium current). CONCLUSIONS: This study highlights the impact of acute MEHP exposure, using a clinically relevant dose, on cardiac electrophysiology in the intact heart. Heightened clinical exposure to plasticized medical products may have cardiac safety implications-given that action potential triangulation and electrical restitution modifications are a risk factor for early after depolarizations and cardiac arrhythmias.

Controlled regional hypoperfusion in Langendorff heart preparations.

We describe a new approach that combines several techniques to allow abnormal electrical and calcium activity to be visualized within hypoperfused myocardial tissue. A flexible microcannula was inserted into the left anterior descending artery of Langendorff perfused rat hearts, an air-tight seal between the coronary artery and the cannula was created, and an HPLC pump was used to deliver a specified flowrate through the microcannula. High resolution optical mapping of NADH/calcium, NADH/voltage or calcium/voltage was then conducted using a dual camera system. The ECG was acquired using surface electrodes. This perfusion technique is superior to occluding a vessel by either a tie or a clamp because it allows precise control of the composition and amount of flow to a defined ischemic bed. Another advantage is that flow can be stopped and resumed remotely, without touching the heart. This allows ectopic beats, or other arrhythmogenic activity, such as alternans, to be recorded immediately after changes in flow are imposed. Altogether, the described method provides a powerful new tool to assess how coronary flow rate affects the degree of local ischemia by the ability to record abnormal patterns of electrical activity and associated intracellular calcium transients with high spatiotemporal resolution from epicardial areas as small as 100 x 100 microm.