Rafael Jaimes

Bisphenol A exposure and cardiac electrical conduction in excised rat hearts.

Background: Bisphenol A (BPA) is used to produce polycarbonate plastics and epoxy resins that are widely used in everyday products, such as food and beverage containers, toys, and medical devices. Human biomonitoring studies have suggested that a large proportion of the population may be exposed to BPA. Recent epidemiological studies have reported correlations between increased urinary BPA concentrations and cardiovascular disease, yet the direct effects of BPA on the heart are unknown. Objectives: The goal of our study was to measure the effect of BPA (0.1–100 μM) on cardiac impulse propagation ex vivo using excised whole hearts from adult female rats. Methods: We measured atrial and ventricular activation times during sinus and paced rhythms using epicardial electrodes and optical mapping of transmembrane potential in excised rat hearts exposed to BPA via perfusate media. Atrioventricular activation intervals and epicardial conduction velocities were computed using recorded activation times. Results: Cardiac BPA exposure resulted in prolonged PR segment and decreased epicardial conduction velocity (0.1–100 μM BPA), prolonged action potential duration (1–100 μM BPA), and delayed atrioventricular conduction (10–100 μM BPA). These effects were observed after acute exposure (≤ 15 min), underscoring the potential detrimental effects of continuous BPA exposure. The highest BPA concentration used (100 μM) resulted in prolonged QRS intervals and dropped ventricular beats, and eventually resulted in complete heart block. Conclusions: Our results show that acute BPA exposure slowed electrical conduction in excised hearts from female rats. These findings emphasize the importance of examining BPA’s effect on heart electrophysiology and determining whether chronic in vivo exposure can cause or exacerbate conduction abnormalities in patients with preexisting heart conditions and in other high-risk populations. Citation: Posnack NG, Jaimes R III, Asfour H, Swift LM, Wengrowski AM, Sarvazyan N, Kay MW. 2014. Bisphenol A exposure and cardiac electrical conduction in excised rat hearts. Environ Health Perspect 122:384–390; http://dx.doi.org/10.1289/ehp.1206157

Oxygen demand of perfused heart preparations: how electromechanical function and inadequate oxygenation affect physiology and optical measurements.

What is the topic of this review? This review discusses how the function and electrophysiology of isolated perfused hearts are affected by oxygenation and energy utilization. The impact of oxygenation on fluorescence measurements in perfused hearts is also discussed. What advances does it highlight? Recent studies have illuminated the inherent differences in electromechanical function, energy utilization rate and oxygen requirements between the primary types of excised heart preparations. A summary and analysis of how these variables affect experimental results are necessary to elevate the physiological relevance of these approaches in order to advance the field of whole‐heart research.

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures3. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model7, 8. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode8. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours8. When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration12 and provides a measure of mitochondrial redox state13. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions14, 15 and to monitor the progression of hypoxic conditions over time10. The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution16 at 37 °C. Aortic, LV, pulmonary artery, and left & right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350±25 nm) and used to illuminate the epicardium. Emitted light was filtered (460±20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.

Exposure to phthalates affects calcium handling and intercellular connectivity of human stem cell-derived cardiomyocytes.

Background The pervasive nature of plastics has raised concerns about the impact of continuous exposure to plastic additives on human health. Of particular concern is the use of phthalates in the production of flexible polyvinyl chloride (PVC) products. Di-2-ethylhexyl-phthalate (DEHP) is a commonly used phthalate ester plasticizer that imparts flexibility and elasticity to PVC products. Recent epidemiological studies have reported correlations between urinary phthalate concentrations and cardiovascular disease, including an increased risk of high blood pressure and coronary risk. Yet, there is little direct evidence linking phthalate exposure to adverse effects in human cells, including cardiomyocytes. Methods and Results The effect of DEHP on calcium handling was examined using monolayers of gCAMP3 human embryonic stem cell-derived cardiomyocytes, which contain an endogenous calcium sensor. Cardiomyocytes were exposed to DEHP (5 – 50 μg/mL), and calcium transients were recorded using a Zeiss confocal imaging system. DEHP exposure (24 – 72 hr) had a negative chronotropic and inotropic effect on cardiomyocytes, increased the minimum threshold voltage required for external pacing, and modified connexin-43 expression. Application of Wy-14,643 (100 μM), an agonist for the peroxisome proliferator-activated receptor alpha, did not replicate DEHP’s effects on calcium transient morphology or spontaneous beating rate. Conclusions Phthalates can affect the normal physiology of human cardiomyocytes, including DEHP elicited perturbations in cardiac calcium handling and intercellular connectivity. Our findings call for additional studies to clarify the extent by which phthalate exposure can alter cardiac function, particularly in vulnerable patient populations who are at risk for high phthalate exposure.

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.

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 Tyrodes 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.

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.

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.

Visualization of Epicardial Cryoablation Lesions Using Endogenous Tissue Fluorescence

Background—Percutaneous cryoballoon ablation is a commonly used procedure to treat atrial fibrillation. One of the major limitations of the procedure is the inability to directly visualize tissue damage and functional gaps between the lesions. We seek to develop an approach that will enable real-time visualization of tissue necrosis during cryo- or radiofrequency ablation procedures. Methods and Results—Cryoablation of either blood-perfused or saline-perfused hearts was associated with a marked decrease in nicotinamide adenine dinucleotide (NADH) fluorescence, leading to a 60% to 70% loss of signal intensity at the lesion site. The total lesion area observed on the NADH channel exhibited a strong correlation with the area identified by triphenyl tetrazolium staining (r=0.89, P<0.001). At physiological temperatures, loss of NADH became visually apparent within 26±8 s after detachment of the cryoprobe from the epicardial surface and plateaued within minutes after which the boundaries of the lesions remained stable for several hours. The loss of electrical activity within the cryoablation site exhibited a close spatial correlation with the loss of NADH (r=0.84±0.06, P<0.001). Cryoablation led to a decrease in diffuse reflectance across the entire visible spectrum, which was in stark contrast to radiofrequency ablation that markedly increased the intensity of reflected light at the lesion sites. Conclusions—We confirmed the feasibility of using endogenous NADH fluorescence for the real-time visualization of cryoablation lesions in blood-perfused cardiac muscle preparations and revealed similarities and differences between imaging cryo- and radiofrequency ablation lesions when using ultraviolet and visible light illumination.

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.