Luther M. Swift

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.

Phthalate exposure changes the metabolic profile of cardiac muscle cells.

Background: Phthalates are common plasticizers present in medical-grade plastics and other everyday products. They can also act as endocrine-disrupting chemicals and have been linked to the rise in metabolic disorders. However, the effect of phthalates on cardiac metabolism remains largely unknown. Objectives: We examined the effect of di(2-ethylhexyl)phthalate (DEHP) on the metabolic profile of cardiomyocytes because alterations in metabolic processes can lead to cell dysfunction. Methods: Neonatal rat cardiomyocytes were treated with DEHP at a concentration and duration comparable to clinical exposure (50–100 μg/mL, 72 hr). We assessed the effect of DEHP on gene expression using microarray analysis. Physiological responses were examined via fatty acid utilization, oxygen consumption, mitochondrial mass, and Western blot analysis. Results: Exposure to DEHP led to up-regulation of genes associated with fatty acid transport, esterification, mitochondrial import, and β-oxidation. The functional outcome was an increase in myocyte fatty acid–substrate utilization, oxygen consumption, mitochondrial mass, PPARα (peroxisome proliferator-activated receptor α) protein expression, and extracellular acidosis. Treatment with a PPARα agonist (Wy-14643) only partially mimicked the effects observed in DEHP-treated cells. Conclusions: Data suggest that DEHP exposure results in metabolic remodeling of cardiomyocytes, whereby cardiac cells increase their dependence on fatty acids for energy production. This fuel switch may be regulated at both the gene expression and posttranscription levels. Our findings have important clinical implications because chronic dependence on fatty acids is associated with an accumulation in lipid intermediates, lactate, protons, and reactive oxygen species. This dependence can sensitize the heart to ischemic injury and ventricular dysfunction.

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

Locations of ectopic beats coincide with spatial gradients of NADH in a regional model of low-flow reperfusion

We studied the origins of ectopic beats during low-flow reperfusion after acute regional ischemia in excised rat hearts. The left anterior descending coronary artery was cannulated. Perfusate was delivered to the cannula using an high-performance liquid chromatography pump. This provided not only precise control of flow rate but also avoided mechanical artifacts associated with vessel occlusion and deocclusion. Optical mapping of epicardial transmembrane potential served to identify activation wavefronts. Imaging of NADH fluorescence was used to quantify local ischemia. Our experiments suggest that low-flow reperfusion of ischemic myocardium leads to a highly heterogeneous ischemic substrate and that the degree of ischemia between adjacent patches of tissue changes in time. In contrast to transient ectopic activity observed during full-flow reperfusion, persistent ectopic arrhythmias were observed during low-flow reperfusion. The origins of ectopic beats were traceable to areas of high spatial gradients of changes in NADH fluorescence caused by low-flow reperfusion.

Signal Decomposition of Transmembrane Voltage-Sensitive Dye Fluorescence Using a Multiresolution Wavelet Analysis

Fluorescence imaging of transmembrane voltage-sensitive dyes is used to study electrical activation in cardiac tissue. However, the fluorescence signals, typically, have low SNRs and may be contaminated with motion artifact. In this report, we introduce a new processing approach for fluoresced transmembrane potentials (fTmps) that is based upon a discrete wavelet transform. We show how fTmp signals can be decomposed and reconstructed to form three subsignals that contain signal noise (noise signal), the early depolarization phase of the action potential (rTmp signal), and motion artifact (rMA signal). A coiflet4 wavelet is used for fTmp decomposition and reconstruction of these subsignals. Results using fTmp signals that are contaminated with motion artifact indicate that the approach is a useful processing step to remove baseline drift, reduce noise, and reveal wavefronts. It streamlines the preprocessing of fTmps for the subsequent measurement of activation times and conduction velocities. It is a promising approach for studying wavefronts without aggressive mechanical tissue constraint or electromechanical uncoupling agents and is, useful for single-camera systems that do not provide for ratiometric imaging.

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.

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.

Anthracycline-induced phospholipase A2 inhibition

The purpose of this essay is to overview our findings that membrane-associated calcium-independent phospholipase A2 is markedly inhibited by low, clinically relevant concentrations of anthracyclines. Our studies suggest that due to the essential role of this enzyme in membrane homeostasis, its inhibition can be one of the early culprits leading to anthracycline-induced cardiac dysfunction. The clinical importance and potential pharmaceutical use of this new phenomenon await further studies.

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.

Oxidant-induced inhibition of myocardial calcium-independent phospholipase A2

We discovered the acute inhibition of myocardial phospholipase A2 activity by micromolar concentrations of tert-butyl hydroperoxide and hydrogen peroxide. Specifically, freshly isolated adult rat cardiomyocytes were treated with the oxidants for 30 min, and phospholipase A2 activity was assessed in cell subcellular fractions using (16∶0, [3H]18∶1) plasmenylcholine and phosphatidylcholine substrates in the absence or presence of calcium. Calcium-independent phospholipase A2 activity was inhibited by approx 50% in both the cytosolic and membrane fractions by the oxidants. The inhibition of the phospholipase A2 activity was concentration dependent and preceded detectable changes in cell viability as assessed by lactate dehydrogenase release and rod-shaped morphology. Taking into account that oxidized sn-2 fatty acyl groups must first be hydrolyzed by phospholipase A2 to be repaired by glutathione peroxidase, we suggest that the observed inhibition of phospholipase A2 activity by oxidants compromises the myocytes ability to deal with lipid peroxidation. This conclusion was further validated by the experiments in which pretreatment with the calcium-independent phospholipase A2 inhibitor bromoenol lactone exacerbated cardiotoxicity of tert-butyl hydroperoxide in myocyte cultures.