Voltage-sensitive dye mapping in Langendorff-perfused rat hearts
ABSTRACT An imaging system suitable for recordings from Langendorff-perfused rat hearts using the voltage-sensitive dye 4-[beta-[2-(di-n-butylamino)-6-naphthyl]vinyl]pyridinium (di-4-ANEPPS) has been developed. Conduction velocity was measured under hyper- and hypokalemic conditions, as well as at physiological and reduced temperature. Elevation of extracellular [K(+)] to 9 mM from 5.9 mM caused a slowing of conduction velocity from 0.66 +/- 0.08 to 0.43 +/- 0.07 mm/ms (35%), and reduction of the temperature to 32 degrees C from 37 degrees C caused a slowing from 0.64 +/- 0.07 to 0.46 +/- 0.05 mm/ms (28%). Ventricular activation patterns in sinus rhythm showed areas of early activation (breakthrough) in both the right and left ventricle, with breakthrough at a site near the apex of the right ventricle usually occurring first. The effects of mechanically immobilizing the preparation to reduce motion artifact were also characterized. Activation patterns in epicardially paced rhythm were insensitive to this procedure over the range of applied force tested. In sinus rhythm, however, a relatively large immobilizing force caused prolonged PQ intervals as well as altered ventricular activation patterns. The time-dependent effects of the dye on the rat heart were characterized and include 1) a transient vasodilation at the onset of dye perfusion and 2) a long-lasting prolongation of the PQ interval of the electrocardiogram, frequently resulting in brief episodes of atrioventricular block.
- SourceAvailable from: Marieke W Veldkamp
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- "CV of freshly isolated tissue was relatively low (18.2±1.0 cm/s) compared to reported values for adult rat ventricle (66±8 cm/s) . Only few papers have reported CV in neonatal rat heart –, and showed values ranging from 21 cm/s in 2-day old rats to 31 cm/s in 10-day old rats. "
ABSTRACT: Testing cardiac gene and cell therapies in vitro requires a tissue substrate that survives for several days in culture while maintaining its physiological properties. The purpose of this study was to test whether culture of intact cardiac tissue of neonatal rat ventricles (organ explant culture) may be used as a model to study gene and cell therapy. We compared (immuno) histology and electrophysiology of organ explant cultures to both freshly isolated neonatal rat ventricular tissue and monolayers. (Immuno) histologic studies showed that organ explant cultures retained their fiber orientation, and that expression patterns of α-actinin, connexin-43, and α-smooth muscle actin did not change during culture. Intracellular voltage recordings showed that spontaneous beating was rare in organ explant cultures (20%) and freshly isolated tissue (17%), but common (82%) in monolayers. Accordingly, resting membrane potential was -83.9±4.4 mV in organ explant cultures, -80.5±3.5 mV in freshly isolated tissue, and -60.9±4.3 mV in monolayers. Conduction velocity, measured by optical mapping, was 18.2±1.0 cm/s in organ explant cultures, 18.0±1.2 cm/s in freshly isolated tissue, and 24.3±0.7 cm/s in monolayers. We found no differences in action potential duration (APD) between organ explant cultures and freshly isolated tissue, while APD of monolayers was prolonged (APD at 70% repolarization 88.8±7.8, 79.1±2.9, and 134.0±4.5 ms, respectively). Organ explant cultures and freshly isolated tissue could be paced up to frequencies within the normal range for neonatal rat (CL 150 ms), while monolayers could not. Successful lentiviral (LV) transduction was shown via Egfp gene transfer. Co-culture of organ explant cultures with spontaneously beating cardiomyocytes increased the occurrence of spontaneous beating activity of organ explant cultures to 86%. We conclude that organ explant cultures of neonatal rat ventricle are structurally and electrophysiologically similar to freshly isolated tissue and a suitable new model to study the effects of gene and cell therapy.PLoS ONE 03/2013; 8(3):e59290. DOI:10.1371/journal.pone.0059290 · 3.23 Impact Factor
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- "To bypass the difficulties of using intracellular electrophysiological approaches to monitor electrical activity from contracting muscle cells, many laboratories now use voltage-or calcium-sensitive dyes (Herron et al., 2012). Although very effective for monitoring the beating rhythms, spread of APs and assessing the effects of drugs on these parameters, the loading of indicators may affect the electrophysiological properties of the cells (Nygren et al., 2003). Furthermore, imaging of the free intracellular calcium concentration kinetics reflects complex processes of calcium influx through voltage gated calcium channels, the release of calcium from intracellular stores, and the removal of calcium by a large number of mechanisms. "
ABSTRACT: Cardiological research greatly rely on the use of cultured primary cardiomyocytes (CMs). The prime methodology to assess CM network electrophysiology is based on the use of extracellular recordings by substrate-integrated planar Micro-Electrode Arrays (MEAs). Whereas this methodology permits simultaneous, long-term monitoring of the CM electrical activity, it limits the information to extracellular field potentials (FPs). The alternative method of intracellular action potentials (APs) recordings by sharp- or patch-microelectrodes is limited to a single cell at a time. Here, we began to merge the advantages of planar MEA and intracellular microelectrodes. To that end we cultured rat CM on micrometer size protruding gold mushroom-shaped microelectrode (gMμEs) arrays. Cultured CMs engulf the gMμE permitting FPs recordings from individual cells. Local electroporation of a CM converts the extracellular recording configuration to attenuated intracellular APs with shape and duration similar to those recorded intracellularly. The procedure enables to simultaneously record APs from an unlimited number of CMs. The electroporated membrane spontaneously recovers. This allows for repeated recordings from the same CM a number of times (>8) for over 10 days. The further development of CM-gMμE configuration opens up new venues for basic and applied biomedical research.Frontiers in Neuroengineering 08/2012; 5:21. DOI:10.3389/fneng.2012.00021
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- "The action potentials on the epicardial surface are recorded in a specific imaging field of view (e.g., from 2 Â 2 mm 2 to 3 Â 3 cm 2 , although panoramic imaging systems recording from the entire anterior or posterior surface of the heart have recently been implemented), after perfusion with voltagesensitive dyes. The isochronal, or isophasis, maps of activation, or depolarization, sequence, conduction direction and velocity are obtained through spatiotemporal analysis  . Due to depth penetration and mechanical artifact limitations, optical imaging is typically performed on the epicardial surface of ex vivo perfusion models and cannot map the cardiac electrical activity in vivo  . "
ABSTRACT: Electromechanical wave imaging is a novel technique for the noninvasive mapping of conduction waves in the left ventricle through the combination of ECG gating, high frame rate ultrasound imaging and radio-frequency (RF)-based displacement estimation techniques. In this paper, we describe this new technique and characterize the origin and velocity of the wave under distinct pacing schemes. First, in vivo imaging (30 MHz) was performed on anesthetized, wild-type mice (n = 12) at high frame rates in order to take advantage of the transient electromechanical coupling occurring in the myocardium. The RF signal acquisition in a long-axis echocardiographic view was gated between consecutive R-wave peaks of the mouse electrocardiogram (ECG) and yielded an ultra-high RF frame rate of 8000 frames/s (fps). The ultrasound RF signals in each frame were digitized at 160 MHz. Axial, frame-to-frame displacements were estimated using 1D cross-correlation (window size of 240 μm, overlap of 90%). Three pacing protocols were sequentially applied in each mouse: (1) sinus rhythm (SR), (2) right-atrial (RA) pacing and (3) right-ventricular (RV) pacing. Pacing was performed using an eight-electrode catheter placed into the right side of the heart with the capability of pacing from any adjacent bipole. During a cardiac cycle, several waves were depicted on the electromechanical wave images that propagated transmurally and/or from base to apex, or apex to base, depending on the type of pacing and the cardiac phase. Through comparison between the ciné-loops and their corresponding ECG obtained at different pacing protocols, we were able to identify and separate the electrically induced, or contraction, waves from the hemodynamic (or, blood-wall coupling) waves. In all cases, the contraction wave was best observed along the posterior wall starting at the S-wave of the ECG, which occurs after Purkinje fiber, and during myocardial, activation. The contraction wave was identified based on the fact that it changed direction only when the pacing origin changed, i.e., it propagated from the apex to the base at SR and RA pacing and from base to apex at RV pacing. This reversal in the wave propagation direction was found to be consistent in all mice scanned and the wave velocity values fell within the previously reported conduction wave range with statistically significant differences between SR/RA pacing (0.85 ± 0.22 m/s and 0.84 ± 0.20 m/s, respectively) and RV pacing (−0.52 ± 0.31 m/s; p < 0.0001). This study thus shows that imaging the electromechanical function of the heart noninvasively is feasible. It may therefore constitute a unique noninvasive method for conduction wave mapping of the entire left ventricle. Such a technology can be extended to 3D mapping and/or used for early detection of dyssynchrony, arrhythmias, left-bundle branch block, or other conduction abnormalities as well as diagnosis and treatment thereof.Ultrasonics 02/2010; 50(2-50):208-215. DOI:10.1016/j.ultras.2009.09.026 · 1.81 Impact Factor