Comparing Cardiac Dynamics between Neonatal and Adult Rats

Background: Cardiovascular physiology studies have been largely limited to adult models; yet, significant developmental differences exist between the immature and adult heart. The field of pediatric research has largely been limited to immortalized cardiomyocyte cell lines, which lack physiologically relevant action potentials, and primary neonatal myocytes that have a limited life span and lack physiologically relevant automaticity. As a result, our understanding of developmental changes in ion channel expression, t-tubule development, and excitation-contraction coupling have been deduced from 2D simplified cell models. To fully understand cardiac development from neonate to adult, a physiologically-relevant 3D whole heart model is needed to monitor dynamic changes in electrical activity and excitation-contraction coupling. Objective: This study aimed to establish a pediatric research model to monitor developmental changes in electrical activity and excitation-contraction coupling, using both imaging tools and electrocardiograms. Methods/Design: Sprague-Dawley rat hearts (3 days – adult) were excised, the aorta was cannulated, and then the heart was transferred to a temperature-controlled constant pressure Langendorff-perfusion system. The perfusate was supplemented with 10 mM blebbistatin to reduce motion artifacts by mechanically uncoupling electrical and mechanical activity. Calcium (50 mg Rhod2-AM) and voltage (62 mg RH237) sensitive dyes were used to stain the heart, signals were acquired using a sCMOS camera (Andor, Zyla 4.2 Plus; \u3e500fps). Electrocardiograms were monitored continuously (lead II configuration) and analyzed using ecgAUTO. Results/Discussion: Initial results showed that compared to adult cohorts, neonatal rats displayed a longer action potential duration (APD80: adult= 85.9ms, neonatal=95.5ms, p=0.026), and a steeper Tau Fall (adults: 33.8ms, neonatal 69.9ms, p=.012) which are likely associated with delayed Ito expression. Calcium handling was also slower in the neonatal hearts (Cad80: Adults: 128.9ms, neonatal=138.8, p=.004), likely due to immature calcium handling and less robust calcium-induced calcium release. The developing excitation-contraction coupling machinery will be further probed using pharmacological tools to elucidate underlying mechanisms; and the newly developed pediatric model will be used for toxicological screening. Acknowledgements: The authors gratefully acknowledge Daniel McInerney for technical assistance. This work was supported by the National Institutes of Health (R00ES023477, R01HL139472), Children’s Research Institute and Children’s National Heart Institute

Plastics and cardiovascular disease

Plastics are synonymous with modern life. Nevertheless, the ubiquity of plastics has resulted in their continuous exposure to humans, which can be harmful. The available literature suggests that this daily exposure might be contributing to cardiovascular disease

The adverse cardiac effects of di(2-ethylhexyl)phthalate and bisphenol A

The ubiquitous nature of plastics has raised concerns pertaining to continuous exposure to plastic polymers and human health risks. Of particular concern is the use of endocrine-disrupting chemicals (EDCs) in plastic production, including Di(2-ethylhexyl) phthalate (DEHP) and Bisphenol A (BPA). Widespread and continuous exposure to DEHP and BPA occurs through dietary intake, inhalation, dermal and intravenous exposure via consumer products and medical devices. This article reviews the literature examining the relationship between DEHP and BPA exposure and cardiac toxicity. In vitro and in vivo experimental reports are outlined, as well as epidemiological studies which examine the association between these chemicals and cardiovascular outcomes. Gaps in our current knowledge are also discussed, along with future investigative endeavors that may help resolve whether DEHP and/or BPA exposure has a negative impact on cardiovascular physiology

Optogenetic Release of Norepinephrine from Cardiac Sympathetic Neurons Alters Mechanical and Electrical Function

© 2014 The Author. Aims Release of norepinephrine (NE) from sympathetic neurons enhances heart rate (HR) and developed force through activation of β-adrenergic receptors, and this sympathoexcitation is a key risk for the generation of cardiac arrhythmias. Studies of β-adrenergic modulation of cardiac function typically involve the administration of exogenous β-adrenergic receptor agonists to directly elicit global β-adrenergic receptor activation by bypassing the involvement of sympathetic nerve terminals. In this work, we use a novel method to activate sympathetic fibres within the myocardium of Langendorff-perfused hearts while measuring changes in electrical and mechanical function. Methods and results The light-activated optogenetic protein channelrhodopsin-2 (ChR2) was expressed in murine catecholaminergic sympathetic neurons. Sympathetic fibres were then photoactivated to examine changes in contractile force, HR, and cardiac electrical activity. Incidence of arrhythmia was measured with and without exposure to photoactivation of sympathetic fibres, and hearts were optically mapped to detect changes in action potential durations and conduction velocities. Results demonstrate facilitation of both developed force and HR after photostimulated release of NE, with increases in contractile force and HR of 34.5 ± 5.5 and 25.0 ± 9.3%, respectively. Photostimulation of sympathetic fibres also made hearts more susceptible to arrhythmia, with greater incidence and severity. In addition, optically mapped action potentials displayed a small but significant shortening of the plateau phase (-5.5 ± 1.0 ms) after photostimulation. Conclusion This study characterizes a powerful and clinically relevant new model for studies of cardiac arrhythmias generated by increasing the activity of sympathetic nerve terminals and the resulting activation of myocyte β-adrenergic receptors

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

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