Machine learning optimization of candidate antibody yields highly diverse sub-nanomolar affinity antibody libraries.

Therapeutic antibodies are an important and rapidly growing drug modality. However, the design and discovery of early-stage antibody therapeutics remain a time and cost-intensive endeavor. Here we present an end-to-end Bayesian, language model-based method for designing large and diverse libraries of high-affinity single-chain variable fragments (scFvs) that are then empirically measured. In a head-to-head comparison with a directed evolution approach, we show that the best scFv generated from our method represents a 28.7-fold improvement in binding over the best scFv from the directed evolution. Additionally, 99% of designed scFvs in our most successful library are improvements over the initial candidate scFv. By comparing a library's predicted success to actual measurements, we demonstrate our method's ability to explore tradeoffs between library success and diversity. Results of our work highlight the significant impact machine learning models can have on scFv development. We expect our method to be broadly applicable and provide value to other protein engineering tasks.

Detection of COVID-19 using multimodal data from a wearable device: results from the first TemPredict Study.

Early detection of diseases such as COVID-19 could be a critical tool in reducing disease transmission by helping individuals recognize when they should self-isolate, seek testing, and obtain early medical intervention. Consumer wearable devices that continuously measure physiological metrics hold promise as tools for early illness detection. We gathered daily questionnaire data and physiological data using a consumer wearable (Oura Ring) from 63,153 participants, of whom 704 self-reported possible COVID-19 disease. We selected 73 of these 704 participants with reliable confirmation of COVID-19 by PCR testing and high-quality physiological data for algorithm training to identify onset of COVID-19 using machine learning classification. The algorithm identified COVID-19 an average of 2.75 days before participants sought diagnostic testing with a sensitivity of 82% and specificity of 63%. The receiving operating characteristic (ROC) area under the curve (AUC) was 0.819 (95% CI [0.809, 0.830]). Including continuous temperature yielded an AUC 4.9% higher than without this feature. For further validation, we obtained SARS CoV-2 antibody in a subset of participants and identified 10 additional participants who self-reported COVID-19 disease with antibody confirmation. The algorithm had an overall ROC AUC of 0.819 (95% CI [0.809, 0.830]), with a sensitivity of 90% and specificity of 80% in these additional participants. Finally, we observed substantial variation in accuracy based on age and biological sex. Findings highlight the importance of including temperature assessment, using continuous physiological features for alignment, and including diverse populations in algorithm development to optimize accuracy in COVID-19 detection from wearables.

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.

Effectiveness of neutral electrolyzed water and copper oxychloride on fungi spores isolated from tropical fruits.

The aim of this study was to assess the antifungal effectiveness of neutral electrolyzed water (NEW) to inhibit the spore germination of post-harvest fungi common in fruits, determine the required available chlorine concentration (ACC) of NEW and to compare it with copper oxychloride (CO) and sterile distilled water (SDW) in vitro. This study evaluated the biological effectiveness of NEW to inactivate pure cultures of 11 different fungi obtained from post-harvest tropical fruits with anthracnose, rottenness or necrosis symptoms. A conidial solution of 1 × 104 spores/mL per culture was prepared and treated with a low, medium and high ACC of NEW (pH 7.0 ± 0.05, 12, 33 and 53 mg/L of ACC and ORP of 850 mV), CO at 0.3 g/L, or sterile distilled water as a control, for 3-, 5- and 10-min contact time. Spore germination of Alternaria alternata, Botrytis cinerea, Cladosporium australiense, Colletotrichum gloeosporioides and C. siamense, Fusarium solani and F. oxysporum, and Lasiodiplodia theobromae was inhibited in 100% by NEW at 12, 33 and 53 ppm ACC; 3,5 and 10 min contact time. Aspergillus niger and A. tamarii required 53 mg/L ACC to inhibit 100% of spore germination. NEW at 33 and 12 mg/L inhibited around 50% and <25% of A. niger spore germination, respectively. NEW at 53 mg/L ACC was the most efficient treatment against Rhizopus stolonifer but only inhibited spore germination in ∼25%. CO inhibited spore germination by 100% of A. alternata, B. cinerea, C. australiense, C. gloeosporioides, C. siamense and L. theobromae. However, CO inhibited <25% of spore germination of F. solani, F. oxysporum, A. niger, A. tamarii and R. stolonifer. NEW and CO had a significant effect on every fungus compared to a SDW treatment. SDW was the least effective treatment, followed by CO. NEW at 12 mg/L and 33 mg/L ACC were equally effective in eliminating the fungi, and more effective than CO. NEW at a concentration of 53 mg/L ACC was the most effective treatment. Results obtained in this study show that NEW has effectively inhibited spore germination of these species, and this treatment could be used as a substitute an ecological novel alternative to CO to avoid spore growth in the above-mentioned fruits.

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.

An Artificial Intelligence Model to Predict the Mortality of COVID-19 Patients at Hospital Admission Time Using Routine Blood Samples: Development and Validation of an Ensemble Model.

BACKGROUND: COVID-19, which is accompanied by acute respiratory distress, multiple organ failure, and death, has spread worldwide much faster than previously thought. However, at present, it has limited treatments. OBJECTIVE: To overcome this issue, we developed an artificial intelligence (AI) model of COVID-19, named EDRnet (ensemble learning model based on deep neural network and random forest models), to predict in-hospital mortality using a routine blood sample at the time of hospital admission. METHODS: We selected 28 blood biomarkers and used the age and gender information of patients as model inputs. To improve the mortality prediction, we adopted an ensemble approach combining deep neural network and random forest models. We trained our model with a database of blood samples from 361 COVID-19 patients in Wuhan, China, and applied it to 106 COVID-19 patients in three Korean medical institutions. RESULTS: In the testing data sets, EDRnet provided high sensitivity (100%), specificity (91%), and accuracy (92%). To extend the number of patient data points, we developed a web application (BeatCOVID19) where anyone can access the model to predict mortality and can register his or her own blood laboratory results. CONCLUSIONS: Our new AI model, EDRnet, accurately predicts the mortality rate for COVID-19. It is publicly available and aims to help health care providers fight COVID-19 and improve patients' outcomes.

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.

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.

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.

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.

Disruption of neonatal cardiomyocyte physiology following exposure to bisphenol-a.

Bisphenol chemicals are commonly used in the manufacturing of polycarbonate plastics, polyvinyl chloride plastics, resins, and thermal printing applications. Humans are inadvertently exposed to bisphenols through contact with consumer products and/or medical devices. Recent reports have shown a link between bisphenol-a (BPA) exposure and adverse cardiovascular outcomes; although these studies have been limited to adult subjects and models. Since cardiac physiology differs significantly between the developing and adult heart, we aimed to assess the impact of BPA exposure on cardiac function, using a neonatal cardiomyocyte model. Neonatal rat ventricular myocytes were monitored to assess cell viability, spontaneous beating rate, beat rate variability, and calcium-handling parameters in the presence of control or bisphenol-supplemented media. A range of doses were tested to mimic environmental exposure (10-9-10-8M), maximum clinical exposure (10-5M), and supraphysiological exposure levels (10-4M). Acute BPA exposure altered cardiomyocyte functionality, resulting in a slowed spontaneous beating rate and increased beat rate variability. BPA exposure also impaired intracellular calcium handling, resulting in diminished calcium transient amplitudes, prolonged calcium transient upstroke and duration time. Alterations in calcium handling also increased the propensity for alternans and skipped beats. Notably, the effect of BPA-treatment on calcium handling was partially reversible. Our data suggest that acute BPA exposure could precipitate secondary adverse effects on contractile performance and/or electrical alternans, both of which are dependent on intracellular calcium homeostasis.

Cardiac performance is limited by oxygen delivery to the mitochondria in the crystalloid-perfused working heart.

The left ventricular working, crystalloid-perfused heart is used extensively to evaluate basic cardiac function, pathophysiology, and pharmacology. Crystalloid-perfused hearts may be limited by oxygen delivery, as adding oxygen carriers increases myoglobin oxygenation and improves myocardial function. However, whether decreased myoglobin oxygen saturation impacts oxidative phosphorylation (OxPhos) is unresolved, since myoglobin has a much lower affinity for oxygen than cytochrome c oxidase (COX). In the present study, a laboratory-based synthesis of an affordable perfluorocarbon (PFC) emulsion was developed to increase perfusate oxygen carrying capacity without impeding optical absorbance assessments. In left ventricular working hearts, along with conventional measurements of cardiac function and metabolic rate, myoglobin oxygenation and cytochrome redox state were monitored using a novel transmural illumination approach. Hearts were perfused with Krebs-Henseleit (KH) or KH supplemented with PFC, increasing perfusate oxygen carrying capacity by 3.6-fold. In KH-perfused hearts, myoglobin was deoxygenated, consistent with cytoplasmic hypoxia, and the mitochondrial cytochromes, including COX, exhibited a high reduction state, consistent with OxPhos hypoxia. PFC perfusate increased aortic output from 76 ± 6 to 142 ± 4 ml/min and increased oxygen consumption while also increasing myoglobin oxygenation and oxidizing the mitochondrial cytochromes. These results are consistent with limited delivery of oxygen to OxPhos resulting in an adapted lower cardiac performance with KH. Consistent with this, PFCs increased myocardial oxygenation, and cardiac work was higher over a wider range of perfusate Po2. In summary, heart mitochondria are limited by oxygen delivery with KH; supplementation of KH with PFC reverses mitochondrial hypoxia and improves cardiac performance, creating a more physiological tissue oxygen delivery. NEW & NOTEWORTHY Optical absorbance spectroscopy of intrinsic chromophores reveals that the commonly used crystalloid-perfused working heart is oxygen limited for oxidative phosphorylation and associated cardiac work. Oxygen-carrying perfluorocarbons increase myocardial oxygen delivery and improve cardiac function, providing a more physiological mitochondrial redox state and emphasizing cardiac work is modulated by myocardial oxygen delivery.

Plastics and cardiovascular health: phthalates may disrupt heart rate variability and cardiovascular reactivity.

Plastics have revolutionized medical device technology, transformed hematological care, and facilitated modern cardiology procedures. Despite these advances, studies have shown that phthalate chemicals migrate out of plastic products and that these chemicals are bioactive. Recent epidemiological and research studies have suggested that phthalate exposure adversely affects cardiovascular function. Our objective was to assess the safety and biocompatibility of phthalate chemicals and resolve the impact on cardiovascular and autonomic physiology. Adult mice were implanted with radiofrequency transmitters to monitor heart rate variability, blood pressure, and autonomic regulation in response to di-2-ethylhexyl-phthalate (DEHP) exposure. DEHP-treated animals displayed a decrease in heart rate variability (-17% SD of normal beat-to-beat intervals and -36% high-frequency power) and an exaggerated mean arterial pressure response to ganglionic blockade (31.5% via chlorisondamine). In response to a conditioned stressor, DEHP-treated animals displayed enhanced cardiovascular reactivity (-56% SD major axis Poincarè plot) and prolonged blood pressure recovery. Alterations in cardiac gene expression of endothelin-1, angiotensin-converting enzyme, and nitric oxide synthase may partly explain these cardiovascular alterations. This is the first study to show an association between phthalate chemicals that are used in medical devices with alterations in autonomic regulation, heart rate variability, and cardiovascular reactivity. Because changes in autonomic balance often precede clinical manifestations of hypertension, atherosclerosis, and conduction abnormalities, future studies are warranted to assess the downstream impact of plastic chemical exposure on end-organ function in sensitive patient populations. This study also highlights the importance of adopting safer biomaterials, chemicals, and/or surface coatings for use in medical devices.NEW & NOTEWORTHY Phthalates are widely used in the manufacturing of consumer and medical products. In the present study, di-2-ethylhexyl-phthalate exposure was associated with alterations in heart rate variability and cardiovascular reactivity. This highlights the importance of investigating the impact of phthalates on health and identifying suitable alternatives for medical device manufacturing.

Enzyme-dependent fluorescence recovery of NADH after photobleaching to assess dehydrogenase activity of isolated perfused hearts.

Reduction of NAD+ by dehydrogenase enzymes to form NADH is a key component of cellular metabolism. In cellular preparations and isolated mitochondria suspensions, enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) of NADH has been shown to be an effective approach for measuring the rate of NADH production to assess dehydrogenase enzyme activity. Our objective was to demonstrate how dehydrogenase activity could be assessed within the myocardium of perfused hearts using NADH ED-FRAP. This was accomplished using a combination of high intensity UV pulses to photobleach epicardial NADH. Replenishment of epicardial NADH fluorescence was then imaged using low intensity UV illumination. NADH ED-FRAP parameters were optimized to deliver 23.8 mJ of photobleaching light energy at a pulse width of 6 msec and a duty cycle of 50%. These parameters provided repeatable measurements of NADH production rate during multiple metabolic perturbations, including changes in perfusate temperature, electromechanical uncoupling, and acute ischemia/reperfusion injury. NADH production rate was significantly higher in every perturbation where the energy demand was either higher or uncompromised. We also found that NADH production rate remained significantly impaired after 10 min of reperfusion after global ischemia. Overall, our results indicate that myocardial NADH ED-FRAP is a useful optical non-destructive approach for assessing dehydrogenase activity.

A technical review of optical mapping of intracellular calcium within myocardial tissue.

Optical mapping of Ca(2+)-sensitive fluorescence probes has become an extremely useful approach and adopted by many cardiovascular research laboratories to study a spectrum of myocardial physiology and disease conditions. Optical mapping data are often displayed as detailed pseudocolor images, providing unique insight for interpreting mechanisms of ectopic activity, action potential and Ca(2+) transient alternans, tachycardia, and fibrillation. Ca(2+)-sensitive fluorescent probes and optical mapping systems continue to evolve in the ongoing effort to improve therapies that ease the growing worldwide burden of cardiovascular disease. In this technical review we provide an updated overview of conventional approaches for optical mapping of Cai (2+) within intact myocardium. In doing so, a brief history of Cai (2+) probes is provided, and nonratiometric and ratiometric Ca(2+) probes are discussed, including probes for imaging sarcoplasmic reticulum Ca(2+) and probes compatible with potentiometric dyes for dual optical mapping. Typical measurements derived from optical Cai (2+) signals are explained, and the analytics used to compute them are presented. Last, recent studies using Cai (2+) optical mapping to study arrhythmias, heart failure, and metabolic perturbations are summarized.

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.

Feeding the fibrillating heart: Dichloroacetate improves cardiac contractile dysfunction following VF.

Ventricular fibrillation (VF) is an important cause of sudden cardiac arrest following myocardial infarction. Following resuscitation from VF, decreased cardiac contractile function is a common problem. During and following myocardial ischemia, decreased glucose oxidation, increased anaerobic glycolysis for cardiac energy production are harmful and energetically expensive. The objective of the present study is to determine the effects of dichloroacetate (DCA), a glucose oxidation stimulator, on cardiac contractile dysfunction following ischemia-induced VF. Male Sprague-Dawley rat hearts were Langendorff perfused in Tyrode's buffer. Once stabilized, hearts were subjected to 15 min of global ischemia and 5 min of aerobic reperfusion in the presence or absence of DCA. At the 6th min of reperfusion, VF was induced electrically, and terminated. Left ventricular (LV) pressure was measured using a balloon. Pretreatment with DCA significantly improved post-VF left ventricular developed pressure (LVDP) and dp/dtmax. In DCA-pretreated hearts, post-VF lactate production and pyruvate dehydrogenase (PDH) phosphorylation were significantly reduced, indicative of stimulated glucose oxidation, and inhibited anaerobic glycolysis by activation of PDH. Epicardial NADH fluorescence was increased during global ischemia above preischemic levels, but decreased below preischemia levels following VF, with no differences between nontreated controls and DCA-pretreated hearts, whereas DCA pretreatment increased NADH production in nonischemic hearts. With exogenous fatty acids (FA) added to the perfusion solution, DCA pretreatment also resulted in improvements in post-VF LVDP and dp/dtmax, indicating that the presence of exogenous FA did not affect the beneficial actions of DCA. In conclusion, enhancement of PDH activation by DCA mitigates cardiac contractile dysfunction following ischemia-induced VF.

Physiological response of cardiac tissue to bisphenol A: alterations in ventricular pressure and contractility.

Biomonitoring studies have indicated that humans are routinely exposed to bisphenol A (BPA), a chemical that is commonly used in the production of polycarbonate plastics and epoxy resins. Epidemiological studies have shown that BPA exposure in humans is associated with cardiovascular disease; however, the direct effects of BPA on cardiac physiology are largely unknown. Previously, we have shown that BPA exposure slows atrioventricular electrical conduction, decreases epicardial conduction velocity, and prolongs action potential duration in excised rat hearts. In the present study, we tested if BPA exposure also adversely affects cardiac contractile performance. We examined the impact of BPA exposure level, sex, and pacing rate on cardiac contractile function in excised rat hearts. Hearts were retrogradely perfused at constant pressure and exposed to 10(-9)-10(-4) M BPA. Left ventricular developed pressure and contractility were measured during sinus rhythm and during pacing (5, 6.5, and 9 Hz). Ca(2+) transients were imaged from whole hearts and from neonatal rat cardiomyocyte layers. During sinus rhythm in female hearts, BPA exposure decreased left ventricular developed pressure and inotropy in a dose-dependent manner. The reduced contractile performance was exacerbated at higher pacing rates. BPA-induced effects on contractile performance were also observed in male hearts, albeit to a lesser extent. Exposure to BPA altered Ca(2+) handling within whole hearts (reduced diastolic and systolic Ca(2+) transient potentiation) and neonatal cardiomyocytes (reduced Ca(2+) transient amplitude and prolonged Ca(2+) transient release time). In conclusion, BPA exposure significantly impaired cardiac performance in a dose-dependent manner, having a major negative impact upon electrical conduction, intracellular Ca(2+) handing, and ventricular contractility.