Voltage-sensitive Fluorescent Dye Research Articles

Voltage-Sensitive Fluorescence of Indocyanine Green in the Heart

So far, the optical mapping of cardiac electrical signals using voltage-sensitive fluorescent dyes has only been performed in experimental studies because these dyes are not yet approved for clinical use. It was recently reported that the well-known and widely used fluorescent dye indocyanine green (ICG), which has FDA approval, exhibits voltage sensitivity in various tissues, thus raising hopes that electrical activity could be optically mapped in the clinic. The aim of this study was to explore the possibility of using ICG to monitor cardiac electrical activity. Optical mapping experiments were performed on Langendorff rabbit hearts stained with ICG and perfused with electromechanical uncouplers. The residual contraction force and electrical action potentials were recorded simultaneously. Our research confirms that ICG is a voltage-sensitive dye with a dual-component (fast and slow) response to membrane potential changes. The fast component of the optical signal (OS) can have opposite polarities in different parts of the fluorescence spectrum. In contrast, the polarity of the slow component remains the same throughout the entire spectrum. Separating the OS into these components revealed two different voltage-sensitivity mechanisms for ICG. The fast component of the OS appears to be electrochromic in nature, whereas the slow component may arise from the redistribution of the dye molecules within or around the membrane. Both components quite accurately track the time of electrical signal propagation, but only the fast component is suitable for estimating the shape and duration of action potentials. Because ICG has voltage-sensitive properties in the entire heart, we suggest that it can be used to monitor cardiac electrical behavior in the clinic.

Blockade of Calcium/Calmodulin-Dependent Kinase II (CaMKII) Induces Heterogeneous Depression of Conduction and Promotes Ischemic Ventricular Fibrillation (VF)

CaMKII regulates amongst many targets the sodium channel (INa), the primary determinant of cardiac excitability and conduction. Cellular studies diverge on the downstream effect of CaMKII on INa. Successful pharmacological therapy targeting CaMKII requires understanding how it regulates excitability and conduction in intact tissue, especially during myocardial ischemia when reduced sodium channel availability causes unsafe conduction. We recorded fluorescence of voltage-sensitive dye Di-4-ANEPPS in the left (LV) and right (RV) ventricles of Langendorff-perfused rabbit hearts (n=11) before and during 30 minutes of global ischemia, with and without perfusion of CaMKII blocker KN-93 (2.75 microM). We computed the local conduction delay (LCD, measured in ms/mm) and compared the 10-90% range of LCD values in the RV and LV at baseline and 4 minutes of ischemia. KN-93 caused heterogeneous slowing of conduction at baseline, which was further exacerbated by ischemia. Without major effect on the 10% level, KN-93 extended the 90% level of the LCD distribution (at baseline: 63% increase in LV; 211% increase in RV. At 4 minutes of ischemia: 230% increase in LV; 344% increase in RV; all p<0.05). During ischemia, KN-93 prompted the initiation (at 5.6±0.47 min of ischemia vs. 17.4±1.89 min in control, p<0.05) of VF through recurrent conduction block at a basal RV site of marked conduction depression. The “chemical phosphatase” butanedione monoxime (BDM, 20 mM) reduced almost threefold the KN-93 concentration required for depression of conduction and early ischemic VF. Conclusions: Constitutive activity of CaMKII is essential to maintain robust and uniform conduction in normal and acutely ischemic heart. CaMKII block is pro-arrhythmic during ischemia. The increased sensitivity to KN-93 in the presence of BDM suggests a delicate balance between kinase and phosphatase activity in regulation of cardiac conduction.

Ventricular Myocytes Electrically Couple with Nonmyocytes in the Infarcted Mouse Heart

In vitro and in silico studies have claimed that electrically non-excitable cells, such as myofibroblasts populating the chronically infarcted heart, couple by gap junctions to adjacent cardiomyocytes, evoking arrhythmogenic electrotonic interactions. However, direct evidence for the existence of such connections in the infarcted, fibrotically remodeled myocardium is lacking. Here, we used two-photon laser scanning microscopy (TPLSM)-based voltage imaging to determine whether nonmyocytes electrically couple to myocytes in the infarct border zone (BZ). Seven to ten days after permanent coronary artery ligation, isolated perfused mouse hearts expressing a fluorescent reporter protein exclusively in cardiomyocytes were loaded with the voltage-sensitive fluorescent dye ANNINE-6plus and subjected to TPLSM in the presence of the excitation-contraction uncouplers (±)blebbistatin (40 microM) and ryanodine (1 microM). BZ nonmyocytes were found to exhibit depolarizing transients at a 1:1 coupling ratio with the action potentials recorded simultaneously from adjacent ventricular myocytes. The electrotonic responses in nonmyocytes exhibited markedly lower peak (F-F0)/F0 amplitudes (where F is fluorescence intensity and F0 is F at baseline) compared to the action potential waveform of juxtaposed myocytes (0.039 ± 0.003 [mean ± SEM] versus 0.112 ± 0.005; n=38 heterocellular pairs from 13 hearts; P<0.001), as well as slower on- and off-kinetics (10%-90% rise time: 19.5 ± 2.2 ms versus 3.4 ± 0.3 ms, P<0.001; 70% recovery time: 68.5 ± 5.7 ms versus 29.8 ± 3.0 ms, P<0.001). Immunohistochemistry revealed low expression levels of the gap junction proteins connexin43 or connexin45 at the abutment of BZ myocytes and alpha-smooth muscle actin-expressing cells, i.e., myofibroblasts. In conclusion, these results demonstrate that cardiomyocytes electrically couple with juxtaposed myofibroblasts in the BZ of chronic infarctions, possibly via gap junctions, expanding the cellular network of the infarct border zone to include direct electrotonic interactions between nonmyocytes and myocytes.

Optical mapping design for murine atrial electrophysiology

Optical mapping is an important tool for assessment of cardiac electrophysiology. We demonstrate a system for quantification and automatic measurement of electrophysiological parameters in isolated cardiac tissue. The system makes use of voltage sensitive fluorescent dyes. These shift in wavelength in response to millivolt changes in potential across cell membranes located on the left atrium. Automated analysis of the pixel-wise measurements yields information on action potential durations and isochronal maps allowing for high throughput of data analysis. The algorithms that we propose reliably describe activation sequences and allow for quantification of conduction velocities.

2015 4thTERMIS World CongressBoston, MassachusettsSeptember 8–11, 2015

2015 4^thTERMIS World CongressBoston, MassachusettsSeptember 8–11, 2015

A fluorescent approach for identifying P2X1 ligands

There are no commercially available, small, receptor-specific P2X1 ligands. There are several synthetic derivatives of the natural agonist ATP and some structurally-complex antagonists including compounds such as PPADS, NTP-ATP, suramin and its derivatives (e.g. NF279, NF449). NF449 is the most potent and selective ligand, but potencies of many others are not particularly high and they can also act at other P2X, P2Y and non-purinergic receptors. While there is clearly scope for further work on P2X1 receptor pharmacology, screening can be difficult owing to rapid receptor desensitisation. To reduce desensitisation substitutions can be made within the N-terminus of the P2X1 receptor, but these could also affect ligand properties. An alternative is the use of fluorescent voltage-sensitive dyes that respond to membrane potential changes resulting from channel opening. Here we utilised this approach in conjunction with fragment-based drug-discovery. Using a single concentration (300 μM) we identified 46 novel leads from a library of 1443 fragments (hit rate = 3.2%). These hits were independently validated by measuring concentration-dependence with the same voltage-sensitive dye, and by visualising the competition of hits with an Alexa-647-ATP fluorophore using confocal microscopy; confocal yielded kon (1.142 × 106 M−1 s−1) and koff (0.136 s−1) for Alexa-647-ATP (Kd = 119 nM). The identified hit fragments had promising structural diversity. In summary, the measurement of functional responses using voltage-sensitive dyes was flexible and cost-effective because labelled competitors were not needed, effects were independent of a specific binding site, and both agonist and antagonist actions were probed in a single assay. The method is widely applicable and could be applied to all P2X family members, as well as other voltage-gated and ligand-gated ion channels.This article is part of the Special Issue entitled ‘Fluorescent Tools in Neuropharmacology’.

Regional Oxidative Stress Disrupts the Normal Propagation of Voltage Waves and Promotes Reentry in Monolayers of Cardiac Myocytes

It has been suggested that the oxidative stress due to ischemic injury plays an important role in arrhythmogenesis. Reactive oxygen species can activate the mitochondrial inner membrane anion channel causing mitochondrial depolarization, which can lead to arrhythmias as we previously showed using experimental and computational models. In particular, our previous experiments showed that coverslip-induced ischemia leads to oxidation of GSH/GSSG couple. Here, we study the effects of regional depletion of GSH pool on mitochondrial function and propagation of the voltage wave in monolayers of cardiac myocytes. Neonatal rat ventricular myocytes were isolated and cultured on fibronectin-coated plastic coverslips (D = 2.1 cm) for 5-7 days. Mitochondrial inner membrane potential was observed using potentiometric fluorescent dye, TMRM, in the dequench mode (2 μM). Optical mapping was used to record the changes in sarcolemmal membrane potential using voltage-sensitive fluorescent dye, di-4-ANEPPS (5 μM), as the voltage waves elicited by a stimulation pulse train, propagated through the monolayer. A thiol oxidizing agent, diamide (1 mM) was locally perfused in the center part (D = 0.5 cm) of the monolayer, while the rest of the coverslip received normal Tyrode's solution. The resulting GSH oxidation led to mitochondrial depolarization (left figure). Action potential amplitude decrease was followed by complete inexcitability in 20 minutes and reentry occurred in 2 out of 3 monolayers (right figure). There was no incidence of reentry in control. The results emphasize the role of oxidative stress in mitochondrial dysfunction and the following post-ischemic arrhythmias. This work was supported by NIH's R01HL105216 and R37HL54598 (B.O'R.) and AHA's 14POST20000018 (S.S.).

Cardiac response to low-energy field pacing challenges the standard theory of defibrillation.

The electric response of myocardial tissue to periodic field stimuli has attracted significant attention as the basis for low-energy antifibrillation pacing, potentially more effective than traditional single high-energy shocks. In conventional models, an electric field produces a highly nonuniform response of the myocardial wall, with discrete excitations, or hot spots (HS), occurring at cathodal tissue surfaces or large coronary vessels. We test this prediction using novel 3-dimensional tomographic optical imaging. Experiments were performed in isolated coronary perfused pig ventricular wall preparations stained with near-infrared voltage-sensitive fluorescent dye DI-4-ANBDQBS. The 3-dimensional coordinates of HS were determined using alternating transillumination. To relate HS formation with myocardial structures, we used ultradeep confocal imaging (interrogation depths, >4 mm). The peak HS distribution is located deep inside the heart wall, and the depth is not significantly affected by field polarity. We did not observe the strong colocalization of HS with major coronary vessels anticipated from theory. Yet, we observed considerable lateral displacement of HS with field polarity reversal. Models that de-emphasized lateral intracellular coupling and accounted for resistive heterogeneity in the extracellular space showed similar HS distributions to the experimental observations. The HS distributions within the myocardial wall and the significant lateral displacements with field polarity reversal are inconsistent with standard theories of defibrillation. Extended theories based on enhanced descriptions of cellular scale electric mechanisms may be necessary. The considerable lateral displacement of HS with field polarity reversal supports the hypothesis of biphasic stimuli in low-energy antifibrillation pacing being advantageous.

Two-Photon Excitation of Fluorescent Voltage-Sensitive Dyes: Monitoring Membrane Potential in the Infrared.

Functional imaging microscopy based on voltage-sensitive dyes (VSDs) has proven effective for revealing spatio-temporal patterns of activity in vivo and in vitro. Microscopy based on two-photon excitation of fluorescent VSDs offers the possibility of recording sub-millisecond membrane potential changes on micron length scales in cells that lie upwards of one millimeter below the brain's surface. Here we describe progress in monitoring membrane voltage using two-photon excitation (TPE) of VSD fluorescence, and detail an application of this emerging technology in which action potentials were recorded in single trials from individual mammalian nerve terminals in situ. Prospects for, and limitations of this method are reviewed.

A near-infrared fluorescent voltage-sensitive dye allows for moderate-throughput electrophysiological analyses of human induced pluripotent stem cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM)-based assays are emerging as a promising tool for the in vitro preclinical screening of QT interval-prolonging side effects of drugs in development. A major impediment to the widespread use of human iPSC-CM assays is the low throughput of the currently available electrophysiological tools. To test the precision and applicability of the near-infrared fluorescent voltage-sensitive dye 1-(4-sulfanatobutyl)-4-{β[2-(di-n-butylamino)-6-naphthyl]butadienyl}quinolinium betaine (di-4-ANBDQBS) for moderate-throughput electrophysiological analyses, we compared simultaneous transmembrane voltage and optical action potential (AP) recordings in human iPSC-CM loaded with di-4-ANBDQBS. Optical AP recordings tracked transmembrane voltage with high precision, generating nearly identical values for AP duration (AP durations at 10%, 50%, and 90% repolarization). Human iPSC-CMs tolerated repeated laser exposure, with stable optical AP parameters recorded over a 30-min study period. Optical AP recordings appropriately tracked changes in repolarization induced by pharmacological manipulation. Finally, di-4-ANBDQBS allowed for moderate-throughput analyses, increasing throughput >10-fold over the traditional patch-clamp technique. We conclude that the voltage-sensitive dye di-4-ANBDQBS allows for high-precision optical AP measurements that markedly increase the throughput for electrophysiological characterization of human iPSC-CMs.

Flow cytometric-membrane potential detection of sodium channel active marine toxins: application to ciguatoxins in fish muscle and feasibility of automating saxitoxin detection.

Ciguatoxins are potent neurotoxins with a significant public health impact. Cytotoxicity assays have allowed the most sensitive means of detection of ciguatoxin-like activity without reliance on mouse bioassays and have been invaluable in studying outbreaks. An improvement of these cell-based assays is presented here in which rapid flow cytometric detection of ciguatoxins and saxitoxins is demonstrated using fluorescent voltage sensitive dyes. A depolarization response can be detected directly due to ciguatoxin alone; however, an approximate 1000-fold increase in sensitivity is observed in the presence of veratridine. These results demonstrate that flow cytometric assessment of ciguatoxins is possible at levels approaching the trace detection limits of our earlier cytotoxicity assays, however, with a significant reduction in analysis time. Preliminary results are also presented for detection of brevetoxins and for automation and throughput improvements to a previously described method for detecting saxitoxins in shellfish extracts.

Indocyanine Green is a Voltage-Sensitive Fluorescent Dye

Indocyanine green (ICG) is an infrared fluorescent dye with widespread use in clinical applications, including hepatic function tests, cardiac output monitoring, and ophthalmic angiography. Using Xenopus laevis oocytes under voltage clamp, we have found that ICG is voltage-sensitive and capable of responding to changes in cellular membrane potential with a roughly linear voltage dependency. ICG's voltage response does not display the sub-millisecond kinetics characteristic of electrochromic dyes; however, it is faster than many so-called slow-response voltage probes. Tests of oocytes performed under current clamp-like conditions show that ICG can clearly follow action potentials produced at approximately 70 Hz. ICG has low expected cytotoxicity; an infrared wavelength amenable to deep tissue imaging; and the ability to label excitable cells, including cardiac myocytes and neurons. Consequently, the voltage-sensitivity of ICG presented here may have significant implications for novel applications in research and the clinic. Support: NIH GM030376.

Multiparametric One-Color Assays for Functional Assessment of Cardiomyocytes

Heart-related health problems remain a leading cause of mortality in developed countries. To efficiently fight these problems, cardiology needs an information-rich screening for new drug candidates. Recent achievements in stem cell research provide a long-awaited solution by producing human stem-cell derived cardiomyocytes and, thus, providing various disease-in-a-dish models of cardiac disorders.Two biologically distinct parameters are required to efficiently characterize the cardiomyocytes function: changes in cell membrane potential and distribution of intracellular calcium ions. Unfortunately, due to experimental limitations and lack of appropriate fluorescent indicators, the traditional approach to perform such an assessment is to record one signal at a time.Here we present novel approach to perform multi-parametric assessment of cardiomyocytes while using two functionally different fluorescent indicators excited by the same wavelength of light. Specifically, using the IC200 HCS screening system (Vala Sciences), we performed a functional assessment of stem cell-derived cardiomyocytes by parallel optical recording of two physiologically distinct signals in real time: dynamic changes in cell membrane voltage as well as redistribution of calcium ions in cells. To do that, in addition to a calcium indicator Fluo4, we used a novel highly efficient voltage-sensitive fluorescent dye (VF 2.1) to detect voltage changes in cardiomyocytes.To perform data analysis, we have developed a dedicated software program that by creating spatially distinct subsets of masks is separating calcium and voltage signals. Using our multiparametric assay, we have performed several screening campaigns using benchmark compounds such as modulators of ion channels involved in generation of cardiac action potential (hERG channels, sodium and calcium voltage-gated ion channels), as well as drugs that affect the intracellular calcium handling. In summary, we have developed a multi-parametric approach for comprehensive screening of a human stem cell-derived cardiomyocytes for drug discovery and cardiotoxicity purposes.

Selective inhibition of late sodium current suppresses ventricular tachycardia and fibrillation in intact rat hearts

Enhanced late inward Na current (INa-L) modulates action potential duration (APD) and plays a key role in the genesis of early afterdepolarizations (EADs) and delayed afterdepolarizations (DADs) and triggered activity. The purpose of this study was to define the influence of selective block of INa-L on EAD- and DAD-mediated triggered ventricular tachycardia (VT) and ventricular fibrillation (VF) in intact hearts using (GS967), a selective and potent (IC50 = 0.13 ± 0.01 μM) blocker of INa-L. VT/VF were induced either by local aconitine injection (50 μg) in the left ventricular muscle of adult (3-4 months) male rats (N = 21) or by arterial perfusion of 0.1 mM hydrogen peroxide (H2O2) in aged male rats (24-26 months, N = 16). The left ventricular epicardial surface of the isolated-perfused hearts was optically mapped using fluorescent voltage-sensitive dye, and microelectrode recordings of action potentials were made adjacent to the aconitine injection site. The suppressive and preventive effects of GS967 (1 μM) against EAD/DAD-mediated VT/VF were then determined. Aconitine induced VT in all 13 hearts studied. Activation map (N = 6) showed that the VT was initiated by a focal activity arising from the aconitine injection site (cycle length [CL] 84 ± 12) that degenerated to VF (CL 52 ± 8 ms) within a few seconds. VF was maintained by multifocal activity with occasional incomplete reentrant wavefronts. Administration of GS967 suppressed the VT/VF in 10 of 13 hearts (P < .001). Preexposure to GS967 for 15 minutes before aconitine injection prevented initiation of VT/VF in 5 of 8 additional hearts (P < .02). VF reoccurred within 10 minutes on washout of GS967. Microelectrode recordings (N = 7) showed that VT/VF was initiated by EAD- and DAD-mediated triggered activity at CL of 86 ± 14 ms (NS from VT CL) that preceded the VF. GS967 shortened APD, flattened the slope of the dynamic APD restitution curve, and reduced APD dispersion from 42 ± 12 ms to 8 ± 3 ms (P < .01). H2O2 perfusion in eight fibrotic aged hearts promoted EAD-mediated focal VT/VF, which was suppressed by GS967 in five hearts (P < .02). The selective INa-L blocker GS967 effectively suppresses and prevents aconitine and oxidative stress-induced EADs, DADs, and focal VT/VF. Suppression of EADs, DADs, and reduction of APD dispersion make GS967 a potentially useful antiarrhythmic drug in conditions of enhanced INa-L.

Fetal Calcium Regulates Branching Morphogenesis in the Developing Human and Mouse Lung: Involvement of Voltage-Gated Calcium Channels

Airway branching morphogenesis in utero is essential for optimal postnatal lung function. In the fetus, branching morphogenesis occurs during the pseudoglandular stage (weeks 9–17 of human gestation, embryonic days (E)11.5–16.5 in mouse) in a hypercalcaemic environment (∼1.7 in the fetus vs. ∼1.1–1.3 mM for an adult). Previously we have shown that fetal hypercalcemia exerts an inhibitory brake on branching morphogenesis via the calcium-sensing receptor. In addition, earlier studies have shown that nifedipine, a selective blocker of L-type voltage-gated Ca2+ channels (VGCC), inhibits fetal lung growth, suggesting a role for VGCC in lung development. The aim of this work was to investigate the expression of VGCC in the pseudoglandular human and mouse lung, and their role in branching morphogenesis. Expression of L-type (CaV1.2 and CaV1.3), P/Q type (CaV2.1), N-type (CaV2.2), R-type (CaV2.3), and T-type (CaV3.2 and CaV3.3) VGCC was investigated in paraffin sections from week 9 human fetal lungs and E12.5 mouse embryos. Here we show, for the first time, that Cav1.2 and Cav1.3 are expressed in both the smooth muscle and epithelium of the developing human and mouse lung. Additionally, Cav2.3 was expressed in the lung epithelium of both species. Incubating E12.5 mouse lung rudiments in the presence of nifedipine doubled the amount of branching, an effect which was partly mimicked by the Cav2.3 inhibitor, SNX-482. Direct measurements of changes in epithelial cell membrane potential, using the voltage-sensitive fluorescent dye DiSBAC2(3), demonstrated that cyclic depolarisations occur within the developing epithelium and coincide with rhythmic occlusions of the lumen, driven by the naturally occurring airway peristalsis. We conclude that VGCC are expressed and functional in the fetal human and mouse lung, where they play a role in branching morphogenesis. Furthermore, rhythmic epithelial depolarisations evoked by airway peristalsis would allow for branching to match growth and distension within the developing lung.

Functional brain fluorescence plurimetry in rat by implantable concatenated CMOS imaging system

Measurement of brain activity in multiple areas simultaneously by minimally invasive methods contributes to the study of neuroscience and development of brain machine interfaces. However, this requires compact wearable instruments that do not inhibit natural movements. Application of optical potentiometry with voltage-sensitive fluorescent dye using an implantable image sensor is also useful. However, the increasing number of leads required for the multiple wired sensors to measure larger domains inhibits natural behavior. For imaging broad areas by numerous sensors without excessive wiring, a web-like sensor that can wrap the brain was developed. Kaleidoscopic potentiometry is possible using the imaging system with concatenated sensors by changing the alignment of the sensors. This paper describes organization of the system, evaluation of the system by a fluorescence imaging, and finally, functional brain fluorescence plurimetry by the sensor. The recorded data in rat somatosensory cortex using the developed multiple-area imaging system compared well with electrophysiology results.

Motion Artefact in Voltage-Sensitive Fluorescent Dye Emission During Repeated Ischemia of Isolated Heart

Motion artefact (MA) in voltage-sensitive fluorescent signals causes significant debasement of action potential. During ischemia and reperfusion in isolated rabbit heart, this artefact develops in a manner which may be described by the time of its onset, level, and shape. The MA during ischemia: (a) may become substantial with approximately two minutes delay after establishing global ischemia; (b) may be almost twice as high as the physiological action potential and decreases both with time and repetition of ischemia; (c) the MA shape is unpredictable and depends on individual rabbit.

Abstract 341: Estrogen Prevents Sudden Death and Spontaneous Ventricular Fibrillation Associated with Pulmonary Hypertension via Estrogen Receptor Beta Mediated Mechanism

Introduction: Previously we showed that right ventricular failure (RVF) secondary to pulmonary hypertension (PH) is associated with spontaneous ventricular fibrillation (VF) and sudden death in rats. We also discovered that estrogen (E2) therapy rescues severe PH and RVF. Estrogen receptor b (ERb) has been shown to play a protective role both in heart and lung. Here we hypothesize that E2 prevents sudden death and abolishes spontaneous VF associated with RVF through ERb. Methods: Male rats were treated with monocrotaline (MCT 60 mg/kg, s.c.) or PBS (CTRL, n=9). At day 21, MCT rats were left untreated to develop RVF by day 30 (n=9), or treated with E2 (42.5 ug/kg/day, s.c., n=9) with or without ERb antagonist PHTPP (850 ug/kg/day, s.c., n=8) from day 21 to day 30. RV ejection fraction (RVEF) was serially monitord with echocardiography. Direct RV catheterisation was performed terminally to record RV pressure (RVP). At ~day 30, hearts were studied in isolated-perfused Langendorff setting and microelectrode recordings were made. RV-epicardial activation pattern was optically mapped using fluorescent voltage-sensitive dye (RH-237). Trichrome staining was performed to assess cardiac fibrosis. Values are expressed as Mean±SEM. Results: By day 30, MCT rats developed severe PH (RVP=72±4 vs. 30±2 mmHg in CTRL, p&lt;0.05) and RV failure (RVEF=33±3% vs. 65±1 in CTRL, p&lt;0.05) with moderate RV fibrosis and ∼30% of RVF rats died suddenly. Early after depolarization (EADs) and spontaneous VF were observed in RVF group during normal Tyrode perfusion with wave front dynamics supported by both focal and multiple wavelet patterns. E2 therapy resulted in complete rescue of PH (RVP=38±3 mmHg, p&lt;0.05 vs. RVF; RVEF=60±2%, p&lt;0.05 vs. RVF; no signs of fibrosis) and none of the rats died in E2-groups. No VF was initiated in any of the control or E2 treated rat hearts. In the presence of ERb antagonist PHTPP, E2 could not rescue PH (RVP=60±4 vs. 38±3 in E2; RVEF=42±2 vs. 60±2 % in E2; p&lt;0.05 vs. E2); moderate RV fibrosis was present and spontaneous EADs and VF were observed in all the hearts similar to RVF group. Conclusions: In conclusion, E2-therapy rescues PH-induced RVF and prevents VF by reversing PH-induced RV-dysfunction and fibrosis most likely through ERb-mediated mechanism.

Optical mapping at increased illumination intensities

Voltage-sensitive fluorescent dyes have become a major tool in cardiac and neuro-electrophysiology. Achieving high signal-to-noise ratios requires increased illumination intensities, which may cause photobleaching and phototoxicity. The optimal range of illumination intensities varies for different dyes and must be evaluated individually. We evaluate two dyes: di-4-ANBDQBS (excitation 660 nm) and di-4-ANEPPS (excitation 532 nm) in the guinea pig heart. The light intensity varies from 0.1 to 5 mW/mm2, with the upper limit at 5 to 10 times above values reported in the literature. The duration of illumination was 60 s, which in guinea pigs corresponds to 300 beats at a normal heart rate. Within the identified duration and intensity range, neither dye shows significant photobleaching or detectable phototoxic effects. However, light absorption at higher intensities causes noticeable tissue heating, which affects the electrophysiological parameters. The most pronounced effect is a shortening of the action potential duration, which, in the case of 532-nm excitation, can reach ∼30%. At 660-nm excitation, the effect is ∼10%. These findings may have important implications for the design of optical mapping protocols in biomedical applications.

Electrical Inhomogeneities of Guinea Pig Atrial Tissue in High Resolution Optical Potential Mapping

Inhomogeneities in the tissue are a determining factor for cardiac function and can be either structural or functional. Using the voltage sensitive fluorescent dye di-4-ANEPPS on incubation stained guinea pig preparations allows to map the tissue's response to externally applied electric shocks. Previous work on ventricular muscle preparations revealed a membrane potential morphology that could be relevant during defibrillation [1]. Using a two pulse stimulus protocol we are able to map the excitation response to a short triggering field pulse and to consequently map the membrane potential morphology resulting from a second field pulse, which is applied in the depolarized state and is attributed to the interaction of the electric field with the tissue. This approach is now carried over to atrial preparations. A custom developed object-positioning and image-stitching method allows for wide view analysis of the atrial tissue structure. Objective-magnifications of 10x, 20x, 40x and 63x enable a multi-scale based analysis with resolutions up to 15 micrometers. This is augmented with information from transmitted light microscopy so that it is possible to correlate the membrane potential patterns with the visible structure of the atrial tissue, providing further insight into atrial excitation and conduction.[1] Windisch,H. et al. (2007) Quantification of shock-induced microscopic virtual electrodes assessed by subcellular resolution optical potential mapping in guinea pig papillary muscle. J Cardiovasc Electrophysiol; 18(10):1086-1094