Voltage-sensitive Dye Research Articles

Effect of percutaneous pulmonary valve replacement on ventricular electrophysiological remodeling in a porcine model of repaired tetralogy of Fallot

Tetralogy of Fallot is the most common cyanotic congenital heart disease. Late after corrective surgery, patients are at risk of developing right ventricular (RV) failure due to pulmonary valve regurgitation and arrhythmias related to surgical scars and ventricular electrophysiological remodeling. Pulmonary valve replacement has been identified as an interesting strategy to reduce RV dilatation but its impact on the arrhythmic risk remains uncertain. We sought to determine whether percutaneous pulmonary valve implantation (PPVI) at an early pathological stage leads to benefical effects on right and left ventricular electrophysiological remodeling in a porcine model of repaired tetralogy of Fallot (rTOF). Piglets (<10 kg) underwent pulmonary artery banding, pulmonary valve disruption to induce RV hypertrophy and dilatation and a PTFE patch was sewn across the pulmonary annulus. Two-months later, PPVI was performed in a sub-group of rTOF pigs. At 3 months post-surgery, cardiac function was assessed by MRI in rTOF, rTOF-PPVI and Sham-operated pigs. Animals were euthanized and optical mapping of isolated left and right ventricles electrical activity was performed using the voltage-sensitive dye Di-4-ANEPPS. Expression of selected genes was assessed by quantitative RT-PCR. At 3 months post-surgery, pulmonary artery regurgitation, RV end-diastolic and end-systolic volumes normalized to body surface area were significantly increased in rTOF pigs compared to Sham animals. In rTOF-PPVI animals these parameters were partially reversed and not statistically different from Sham pigs. An action potential prolongation was observed in both the left ventricle and the RV epicardium but not in the endocardium of rTOF pigs. Within the RV epicardium, this prolongation was mainly confined to the anterior side. Interestingly, rTOF-PPVI pigs action potential durations were similar to Sham animals. In line with functional investigations, a large increase in BNP expression was found in rTOF RV compared to Sham pigs (fold change = 34) which was not as pronounced in rTOF-PPVi pigs (fold change = 3) while a lower expression of KCND3 and KCNJ2 was found in rTOF pigs. In our rTOF porcine model, PPVI at an early pathological stage reversed RV dilatation. It also successfully reversed action potential prolongation in the left ventricle and the anterior RV epicardium, and may thus have beneficial effects on the arrhythmic risk.

A case study: High resolution optical mapping of a 19 year old male victim of sudden cardiac death during a marathon

Fifty thousand athletes suffer sudden cardiac death every year due to ventricular fibrillation (VF). Most sudden cardiac deaths of young patients have unexplained origin due to the absence of structural or electrical disease. Purkinje network is essential for a normal heart function, however, its role during electrical pathologies remains unclear. A case study to investigate the electrophysiological properties and the Purkinje network role in a 19 years old male after suffering a fatal sudden cardiac arrest during a marathon event. A coronary-perfused left ventricular preparation was imaged using high resolution optical mapping of epicardial and endocardial surfaces using a voltage-sensitive dye, Di-4-ANEPPS. Activation time, repolarization time and action potential duration were assessed during paced activity through the His bundle and endocardial and epicardial surfaces. Conduction system activation origins were identified as distant endocardial origins during His bundle pacing. Short-coupled S1S2S3S4 pacing was used to determine effective refractory period (ERP). Pacing protocols were repeated in the presence of isoprenaline (ISO) at 100 nM, 500 nM and 1 μM to mimic stress conditions. No changes in total activation time are observed during myocardial pacing cycle lengths of 667ms in control (63ms) and under ISO (up to 64ms). In contrary, His-ventricle delay during conduction system pacing was shorted from 61ms in control to 42ms, 44ms and 16ms under 100 nM, 500 nM and 1 μM of ISO respectively. Under His pacing, local APD gradients are observed near Purkinje-muscle junctions under ISO (from 3ms/mm and 3 ms/mm to 40ms/mm and 41ms/mm). ERP under His pacing was 55ms shorter than endocardial pacing. 82% of the ectopy origins observed have less than 4 mm distance from the PMJs. Repeated focal activations were observed during VF and 76% of them had less than 4 mm distance from PMJs. No structural abnormalities were identified under 9.4 T MRI. Conduction system electrical abnormalities play a role in arrhythmogenicity and maintenance of VF in this patient during sudden cardiac death.

IL-10 and TGF-β Increase Connexin-43 Expression and Membrane Potential of HL-1 Cardiomyocytes Coupled with RAW 264.7 Macrophages.

Cardiac resident macrophages facilitate electrical conduction by interacting with cardiomyocytes via connexin-43 (Cx43) hemichannels. Cx43 is critical for impulse propagation and coordination between muscle contractions. Cardiomyocyte electrophysiology can be altered when coupled with noncardiomyocyte cell types such as M2c tissue-resident macrophages. Using cocultures of murine HL-1 cardiomyocytes and RAW 264.7 macrophages, we examined the hypothesis that cytokine signals, TGF-β1 and IL-10, upregulate Cx43 expression at points of contact between the two cell types. These cytokine signals maintain the macrophages in an M2c anti-inflammatory phenotype, mimicking cardiac resident macrophages. The electrophysiology of cardiomyocytes was examined using di-8-ANEPPS potentiometric dye, which reflects a change in membrane potential. Greater fluorescence intensity of di-8-ANEPPS occurred in areas where macrophages interacted with cardiomyocytes. Suppressor of cytokine signaling 3 (SOCS3) peptide mimetic downregulated fluorescence of this membrane potentiometric stain. Cx43 expression in cocultures was confirmed by fluorescence microscopy and flow cytometry. Confocal images of these interactions demonstrate the Cx43 hemichannel linkages between the cardiomyocytes and macrophages. These results suggest that TGF-β1 and IL-10 upregulate Cx43 hemichannels, thus enhancing macrophage-cardiomyocyte coupling, raising the cellular resting membrane potential and leading to a more excitatory cardiomyocyte.

Electrically controlling and optically observing the membrane potential of supported lipid bilayers

Supported lipid bilayers are a well-developed model system for the study of membranes and their associated proteins, such as membrane channels, enzymes, and receptors. These versatile model membranes can be made from various components, ranging from simple synthetic phospholipids to complex mixtures of constituents, mimicking the cell membrane with its relevant physiochemical and molecular phenomena. In addition, the high stability of supported lipid bilayers allows for their study via a wide array of experimental probes. In this work, we describe a platform for supported lipid bilayers that is accessible both electrically and optically, and demonstrate direct optical observation of the transmembrane potential of supported lipid bilayers. We show that the polarization of the supported membrane can be electrically controlled and optically probed using voltage-sensitive dyes. Membrane polarization dynamics is understood through electrochemical impedance spectroscopy and the analysis of an equivalent electrical circuit model. In addition, we describe the effect of the conducting electrode layer on the fluorescence of the optical probe through metal-induced energy transfer, and show that while this energy transfer has an adverse effect on the voltage sensitivity of the fluorescent probe, its strong distance dependency allows for axial localization of fluorescent emitters with ultrahigh accuracy. We conclude with a discussion on possible applications of this platform for the study of voltage-dependent membrane proteins and other processes in membrane biology and surface science.

Determining the role of uremic toxins on cardiac electrophysiology and pro-arrhythmia during chronic kidney disease

Abstract Funding Acknowledgements Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Young Talent Programme, CVON Introduction Chronic kidney disease (CKD) is represented by a diminished filtration capacity of the kidneys. End stage renal disease patients need dialysis treatment to remove waste and toxins from the circulation. However, endogenously produced uremic toxins (UTs) cannot always be filtered during dialysis. UTs are one of the CKD-related factors already linked to maladaptive and pathophysiological remodeling of the heart. Importantly, 50% of the deaths in dialysis patients is cardiovascular related, with sudden cardiac death predominating. However, the mechanisms responsible still remain poorly understood. Purpose To assess the potentially increased pro-arrhythmic risk of pre-identified UTs at clinically relevant concentrations: 1) acutely in isolated adult dog ventricular cardiomyocytes (ADCMs) and human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs), 2) chronically in iPSC-CMs and 3) both conditions in human embryonic kidney (HEK) cells transfected with constructs that generate different ion channels, using optical and manual electrophysiological in vitro approaches. Methods and results Using the voltage sensitive membrane dye FluoVolt and manual patch clamp, action potential duration (APD) prolongation, considered as a pro-arrhythmic parameter, was assessed in ADCMs and iPSC-CMs. Acute exposure of indoxyl sulfate (IS), kynurenine (KYN) or kynurenic acid (KYNA) induced no alterations in APD in ADCMs and iPSC-CMs. iPSC-CMs, although having rather immature electrophysiological characteristics, showed significant APD prolongation when exposed chronically (48h) to those UTs. Manual patch clamp on HEK293 cells was performed to investigate individual ion currents to reveal underlying mechanisms. The repolarizing current IKr, often most sensitive and responsible for APD alterations, was not acutely affected by IS, KYN, or KYNA. However, chronic exposure (48h) to IS or KYNA significantly decreased IKr at clinically relevant concentrations. Finally, the protein level of the ion channel conducting IKr was determined in HEK293 cells, to further unveil the mechanism behind the effects of UTs on channels conducting IKr. Chronic exposure (48-72h) of the three UTs decreased total ion channel expression at pathological concentrations, which could be responsible for the decreased IKr and prolonged APD. Conclusion The results show the proof of concept to identify potentially pro-arrhythmogenic UTs and their mode of action, allowing the provision of a clinically relevant overview of (patho)physiological concentrations of UTs in both acute and chronic exposure.

Dynamic voltage attenuation identifies atrial fibrosis in a rabbit model: simultaneous assessment with optical mapping and contact electrogram mapping

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): British Heart Foundation. Academy of Medical Sciences. Background Bipolar voltage amplitude is a widely-used clinical parameter in atrial electrophysiology procedures. However, voltage amplitude is variable, and it has been shown that increasing activation rate decreases bipolar voltage amplitude in patients with atrial fibrillation. It is not known whether such voltage attenuation is a marker of the presence of atrial fibrosis which could therefore be used to improve intra-procedural assessment of atrial cardiomyopathy. Purpose We sought to investigate the effect of increasing activation rate on bipolar voltage amplitude in both healthy and fibrotic left atrial tissue. Methods 10 New Zealand Rabbits were fed a high cholesterol diet (0.75%) for a period of 12 weeks to create an atrial fibrosis model. 10 Animals were fed normal chow. After terminal anaesthesia the heart was excised, and optical and voltage mapping of the excised left atrial tissue was performed. Blebbistatin was used to maintain cardiac stasis and the voltage sensitive dye RH237 was used for optical mapping. Voltage and optical recordings were made during pacing was from 3 different directions at rates from 2-6Hz and at 3 sites across the atrial tissue. Voltage amplitude was recorded as the mean amplitude over 10 beats during steady-state pacing. Optical recordings were used to measure conduction velocity and action potential characteristics. Only pacing runs showing 1:1 conduction were included in analysis. Atrial fibrosis was assessed using Masson’s Trichrome staining. Results The degree of atrial fibrosis was significantly greater in the atrial fibrosis model compared to healthy controls (15±3.24% vs. 9.74±4.98%, p=0.0069). Median voltage at base rate pacing of 2Hz was not significantly different between control and fibrotic atria (11.63mV, IQR 6.35mV vs. 10.3mV, IQR 6.81mV, p=0.71, respectively). Median voltage was significantly lower at 6Hz than at 2Hz in the control group (9.84mV, IQR 6.87mV, p=0.046). The degree of voltage attenuation between study groups was not significantly different between when pacing at 3hz or 4hz, whereas pacing at 5Hz and 6Hz showed significantly greater attenuation in fibrotic atria. At 5Hz the median reduction in amplitude from baseline in control vs fibrotic atria was 0.88mV, IQR 2.36mV vs 1.92mV, IQR 1.63mV (p=0.031). At 6 Hz the median reduction was 0.94mV, IQR 1.69mV vs 2.68mV, IQR 1.11mV, p=0.013 in control and fibrotic groups respectively. Discussion High cholesterol diet increased atrial fibrosis in a rabbit model. Bipolar voltage amplitude attenuation occurred in both control and fibrotic atria however the degree of voltage attenuation was significantly greater in fibrotic atria. These findings support the further evaluation of dynamic voltage attenuation for intraprocedural identification of atrial fibrosis.

Oxidative Crosstalk with Venular Capillary Mitochondria Mediates Barrier Failure in Acute Lung Injury

OBJECTIVE. To elucidate mechanisms underlying alveolar‐capillary crosstalk in the context of lipopolysaccharide (LPS)‐induced Acute Lung Injury (ALI), which causes The Acute Respiratory Distress Syndrome (ARDS).HYPOTHESISOxidative crosstalk between alveolar epithelium and adjacent endothelium causes mitochondrial injury and barrier failure in Acute Lung InjuryMETHODS. We instilled LPS (5mg/kg) by the intranasal (i.n.) route in anesthetized mice. We evaluated microvascular endothelia of isolated blood‐perfused mouse lungs by confocal microscopy, and ALI by bronchoalveolar lavage (BAL) 48h later. For microscopy, we gave the potentiometric mitochondrial dye MTDR, and the reactive oxygen species (ROS) indicator DCF by intravascular infusion. We determined the role of endothelial uncoupling protein 2 (UCP2) by (1) UCP2 knockdown in endothelia by vascular siRNA injection, and (2) endothelial‐specific knockout of UCP2. We transfected alveolar epithelium by i.n. instillation of catalase‐expressing plasmid, or depleted neutrophils by intraperitoneal injection of anti‐Gr1 antibody, 24 hours before i.n. LPS. We quantified barrier permeability through BAL protein content.RESULTS. Confocal imaging indicated that baseline fluorescence intensity of both endothelial MTDR and DCF was steady, indicating that endothelial mitochondrial potential and endothelial ROS were stable. However, 48 hours after LPS instillation, MTDR fluorescence decreased in venular capillaries more than in alveolar capillaries (n=4, p&lt;0.05), indicating endothelial mitochondrial depolarization. Simultaneously, DCF fluorescence increased in venular capillaries more than in alveolar capillaries (n=4, p&lt;0.05), indicating an increase in endothelial ROS. LPS instillation increased BAL protein (n=4, p&lt;0.05). Knockdown of endothelial UCP2 expression, or expression of transfected catalase in the alveolar epithelium blocked LPS‐induced endothelial mitochondrial depolarization and BAL protein increase (p&lt;0.05). Endothelial‐specific knockout of UCP2, but not neutrophil depletion, blocked LPS‐induced BAL protein increase (p&lt;0.05).CONCLUSIONS. We show here for the first time that the hyper‐permeable responses of LPS‐induced ALI result from mitochondrial depolarization in venular capillaries. Transfer of H2O2 from alveoli, not neutrophils activated UCP2, causing proton influx into the mitochondrial matrix. Our findings reveal a novel mechanism of LPS‐induced ALI, implicating mitochondria‐oxidant coupling in barrier failure. Thus, UCP2 presents a potential therapeutic target for ARDS.

Patient-specific, re-engineered cardiomyocyte model confirms the circumstance-dependent arrhythmia risk associated with the African-specific common SCN5A polymorphism p.S1103Y: Implications for the increased sudden deaths observed in black individuals during the COVID-19 pandemic.

During the early stages of the coronavirus disease 2019 (COVID-19) pandemic, a marked increase in sudden cardiac death (SCD) was observed. The p.S1103Y-SCN5A common variant, which is present in ∼8% of individuals of African descent, may be a circumstance-dependent, SCD-predisposing, proarrhythmic polymorphism in the setting of hypoxia-induced acidosis or QT-prolonging drug use. The purpose of this study was to ascertain the effects of acidosis and hydroxychloroquine (HCQ) on the action potential duration (APD) in a patient-specific induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model of p.S1103Y-SCN5A. iPSC-CMs were generated from a 14-year-old p.S1103Y-SCN5A-positive African American male. The patient's variant-corrected iPSC-CMs (isogenic control [IC]) were generated using CRISPR/Cas9 technology. FluoVolt voltage-sensitive dye was used to assess APD90 values in p.S1103Y-SCN5A iPSC-CMs compared to IC before and after an acidotic state (pH 6.9) or 24 hours of treatment with 10 μM HCQ. Under baseline conditions (pH 7.4), there was no difference in APD90 values of p.S1103Y-SCN5A vs IC iPSC-CMs (P = NS). In the setting of acidosis (pH 6.9), there was a significant increase in fold-change of APD90 in p.S1103Y-SCN5A iPSC-CMs compared to IC iPSC-CMs (P <.0001). Similarly, with 24-hour 10 μM HCQ treatment, the fold-change of APD90 was significantly higher in p.S1103Y-SCN5A iPSC-CMs compared to IC iPSC-CMs (P <.0001). Although the African-specific p.S1103Y-SCN5A common variant had no effect on APD90 under baseline conditions, the physiological stress of either acidosis or HCQ treatment significantly prolonged APD90 in patient-specific, re-engineered heart cells.

Ultra-parallel label-free optophysiology of neural activity

SummaryThe electrical activity of neurons has a spatiotemporal footprint that spans three orders of magnitude. Traditional electrophysiology lacks the spatial throughput to image the activity of an entire neural network; besides, labeled optical imaging using voltage-sensitive dyes and tracking Ca2+ ion dynamics lack the versatility and speed to capture fast-spiking activity, respectively. We present a label-free optical imaging technique to image the changes to the optical path length and the local birefringence caused by neural activity, at 4,000 Hz, across a 200 × 200 μm2 region, and with micron-scale spatial resolution and 300-pm displacement sensitivity using Superfast Polarization-sensitive Off-axis Full-field Optical Coherence Microscopy (SPoOF OCM). The undulations in the optical responses from mammalian neuronal activity were matched with field-potential electrophysiology measurements and validated with channel blockers. By directly tracking the widefield neural activity at millisecond timescales and micrometer resolution, SPoOF OCM provides a framework to progress from low-throughput electrophysiology to high-throughput ultra-parallel label-free optophysiology.

Altered cortical processing of sensory input in Huntington disease mouse models

Huntington disease (HD), a hereditary neurodegenerative disorder, manifests as progressively impaired movement and cognition. Although early abnormalities of neuronal activity in striatum are well established in HD models, there are fewer in vivo studies of the cortex. Here, we record local field potentials (LFPs) in YAC128 HD model mice versus wild-type mice. In multiple cortical areas, limb sensory stimulation evokes a greater change in LFP power in YAC128 mice. Mesoscopic imaging using voltage-sensitive dyes reveals more extensive spread of evoked sensory signals across the cortical surface in YAC128 mice. YAC128 layer 2/3 sensory cortical neurons ex vivo show increased excitatory events, which could contribute to enhanced sensory responses in vivo. Cortical LFP responses to limb stimulation, visual and auditory input are also significantly increased in zQ175 HD mice. Results presented here extend knowledge of HD beyond ex vivo studies of individual neurons to the intact cortical network.

Metabotropic 5-HT receptor-mediated effects in the human submucous plexus.

Serotonin (5-HT) is an important mediator in the gastrointestinal tract, acting on different neuronal 5-HT receptors. The ionotropic 5-HT3 receptor mediates immediate but transient spike discharge in human enteric neurons. We studied the role of the metabotropic 5-HT1P , 5-HT4 , and 5-HT7 receptors to activate human submucous neurons. Neuroimaging using the voltage sensitive dye Di-8-ANEPPS was performed in submucous plexus preparations from human surgical specimens of the small and large intestine. We synthesized a new, stable 5-HT1P agonist, 5-benzyloxyhydrazonoindalpine (5-BOHIP). 5-HT evoked a fast and late-onset spike discharge in enteric neurons. The fast component was blocked by the 5-HT3 receptor antagonist cilansetron, while the remaining sustained response was significantly reduced by the 5-HT1P receptor antagonist 5-hydroxytryptophanyl-5-hydroxytryptophan amide (5-HTP-DP). The newly synthesized 5-HT1P agonist 5-BOHIP induced a slowly developing, long-lasting activation of submucous neurons, which was blocked by 5-HTP-DP. We could not demonstrate any 5-HT7 receptor-induced spike discharge based on the lack of response to 5-carboxamidotryptamine. Similarly, the 5-HT4 agonists 5-methoxytryptamine and prucalopride evoked no immediate or late-onset spike discharge. Our work demonstrated for the first time the presence of functional 5-HT1P receptors on human submucous neurons. Furthermore, we found no evidence for a role of 5-HT4 or 5-HT7 receptors in the postsynaptic activation of human submucous neurons by 5-HT.

Chemical Targeting of Rhodol Voltage-Sensitive Dyes to Dopaminergic Neurons.

Optical imaging of changes in the membrane potential of living cells can be achieved by means of fluorescent voltage-sensitive dyes (VSDs). A particularly challenging task is to efficiently deliver these highly lipophilic probes to specific neuronal subpopulations in brain tissue. We have tackled this task by designing a solubilizing, hydrophilic polymer platform that carries a high-affinity ligand for a membrane protein marker of interest and a fluorescent VSD. Here, we disclose an improved design of polymer-supported probes for chemical, nongenetic targeting of voltage sensors to axons natively expressing the dopamine transporter in ex vivo mouse brain tissue. We first show that for negatively charged rhodol VSDs functioning on the photoinduced electron transfer principle, poly(ethylene glycol) as a carrier enables targeting with higher selectivity than the polysaccharide dextran in HEK cell culture. In the same experimental setting, we also demonstrate that incorporation of an azetidine ring into the rhodol chromophore substantially increases the brightness and voltage sensitivity of the respective VSD. We show that the superior properties of the optimized sensor are transferable to recording of electrically evoked activity from dopaminergic axons in mouse striatal slices after averaging of multiple trials. Finally, we suggest the next milestones for the field to achieve single-scan recordings with nongenetically targeted VSDs in native brain tissue.

Label-Free Imaging of Membrane Potentials by Intramembrane Field Modulation, Assessed by Second Harmonic Generation Microscopy.

Optical interrogation of cellular electrical activity has proven itself essential for understanding cellular function and communication in complex networks. Voltage-sensitive dyes are important tools for assessing excitability but these highly lipophilic sensors may affect cellular function. Label-free techniques offer a major advantage as they eliminate the need for these external probes. In this work, it is shown that endogenous second-harmonic generation (SHG) from live cells is highly sensitive to changes in transmembrane potential (TMP). Simultaneous electrophysiological control of a living human embryonic kidney (HEK293T) cell, through a whole-cell voltage-clamp reveals a linear relation between the SHG intensity and membrane voltage. The results suggest that due to the high ionic strengths and fast optical response of biofluids, membrane hydration is not the main contributor to the observed field sensitivity. A conceptual framework is further provided that indicates that the SHG voltage sensitivity reflects the electric field within the biological asymmetric lipid bilayer owing to a nonzero tensor. Changing the TMP without surface modifications such as electrolyte screening offers high optical sensitivity to membrane voltage (≈40% per 100mV), indicating the power of SHG for label-free read-out. These results hold promise for the design of a non-invasive label-free read-out tool for electrogenic cells.

Spatiotemporal patterns of population response in the visual cortex under isoflurane: from wakefulness to loss of consciousness.

Anesthetic drugs are widely used in medicine and research to mediate loss of consciousness (LOC). Isoflurane is a commonly used anesthetic drug; however, its effects on cortical sensory processing, in particular around LOC, are not well understood. Using voltage-sensitive dye imaging, we measured visually evoked neuronal population response from the visual cortex in awake and anesthetized mice at 3 increasing concentrations of isoflurane, thus controlling the level of anesthesia from wakefulness to deep anesthesia. At low concentration of isoflurane, the effects on neuronal measures were minor relative to the awake condition. These effects augmented with increasing isoflurane concentration, while around LOC point, they showed abrupt and nonlinear changes. At the network level, we found that isoflurane decreased the stimulus-evoked intra-areal spatial spread of local neural activation, previously reported to be mediated by horizontal connections, and also reduced intra-areal synchronization of neuronal population. The synchronization between different visual areas decreased with higher isoflurane levels. Isoflurane reduced the population response amplitude and prolonged their latencies while higher visual areas showed increased vulnerability to isoflurane concentration. Our results uncover the changes in neural activity and synchronization at isoflurane concentrations leading to LOC and suggest reverse hierarchical shutdown of cortical areas.

Impact of Membrane Voltage on Formation and Stability of Human Renal Proximal Tubules in Vitro.

More than 15% of adults in the United States suffer from some form of chronic kidney disease (CKD). Current strategies for CKD consist of dialysis or kidney transplant, which, however, can take several years. In this light, tissue engineering and regenerative medicine approaches are the key to improving people's living conditions by advancing previous tissue engineering approaches and seeking new targets as intervention methods for kidney repair or replacement. The membrane voltage (Vm) dynamics of a cell have been associated with cell migration, cell cycle progression, differentiation, and pattern formation. Furthermore, bioelectrical stimuli have been used as a means in the treatment of diseases and wound healing. Here, we investigated the role of Vm as a novel target to guide and manipulate in vitro renal tissue models. Human-immortalized renal proximal tubule epithelial cells (RPTECs-TERT1) were cultured on Matrigel to support the formation of 3D proximal tubular-like structures with the incorporation of a voltage-sensitive dye indicator─bis-(1,3-dibutylbarbituric acid)timethine oxonol (DiBAC). The results demonstrated a correlation between the depolarization and the reorganization of human renal proximal tubule cells, indicating Vm as a candidate variable to control these events. Accordingly, Vm was pharmacologically manipulated using glibenclamide and pinacidil, KATP channel modulators, and proximal tubule formation and tubule stability over 21 days were assessed. Chronic manipulation of KATP channels induced changes in the tubular network topology without affecting lumen formation. Thus, a relationship was found between the preluminal tubulogenesis phase and KATP channels. This relationship may provide future options as a control point during kidney tissue development, treatment, and regeneration goals.

Ratiometry for improving the signal-to-noise with the red-shifted voltage dye di-4ANBDQBS

Voltage-sensitive fluorescent dyes allow non-invasive recording of action potentials in single cells. These experiments often have low signal-to-noise ratios (SNR). To improve SNR, ratiometric fluorescence methods have been utilized. In these studies, we used di-4-ANBDQBS (Cytovolt1), a far-red-shifted voltage-sensitive dye with a stable signal that works through the Stark effect. HEK cells were loaded with CytoVolt1 that was excited at 528 nm using our optical system. Fluorescence emission was measured at 720 nm with a long pass filter and 655 nm with a bandpass filter, corresponding to opposite slope regions of the emission spectrum.

Electrical activity in exercise trained infarcted rat hearts

Myocardial infarction (MI) is a major cause of cardiac arrhythmias. The aim of the study was to determine if exercise training changes the electrical conduction into, within and emerging from an infarcted area of a rat heart. Rats were divided into three groups: sedentary sham, sedentary MI, and exercise training MI. The impact of MI and exercise training on susceptibility to ventricular tachycardias (VTs) was evaluated by in vivo intra-cardiac pacing. Rats with sustained VTs were converted before sacrifice and electrophysiological experiments and optical imaging with voltage-sensitive dye were performed on perfused hearts.

Bacterial resting membrane potential: a case study with Bacillus subtilis

Bacterial membrane potential changes (Δψ) play an important role in bacterial metabolism and cellular processes as well as cell to cell communications in a biofilm. However, existing tools for reading Δψ quantitatively in individual bacterial cells as well as in bacterial communities are limited. A fluorescence lifetime imaging microscopy (FLIM) technique in combination with photo-induced electron transfer (PeT) based small molecule voltage sensitive dyes have been employed to quantify absolute resting membrane potential for individual Bacillus subtilis cells.

Electrophysiological heterogeneity in large populations of rabbit ventricular cardiomyocytes.

Cardiac electrophysiological heterogeneity includes: (i) regional differences in action potential (AP) waveform, (ii) AP waveform differences in cells isolated from a single region, (iii) variability of the contribution of individual ion currents in cells with similar AP durations (APDs). The aim of this study is to assess intra-regional AP waveform differences, to quantify the contribution of specific ion channels to the APD via drug responses and to generate a population of mathematical models to investigate the mechanisms underlying heterogeneity in rabbit ventricular cells. APD in ∼50 isolated cells from subregions of the LV free wall of rabbit hearts were measured using a voltage-sensitive dye. When stimulated at 2 Hz, average APD90 value in cells from the basal epicardial region was 254 ± 25 ms (mean ± standard deviation) in 17 hearts with a mean interquartile range (IQR) of 53 ± 17 ms. Endo-epicardial and apical-basal APD90 differences accounted for ∼10% of the IQR value. Highly variable changes in APD occurred after IK(r) or ICa(L) block that included a sub-population of cells (HR) with an exaggerated (hyper) response to IK(r) inhibition. A set of 4471 AP models matching the experimental APD90 distribution was generated from a larger population of models created by random variation of the maximum conductances (Gmax) of 8 key ion channels/exchangers/pumps. This set reproduced the pattern of cell-specific responses to ICa(L) and IK(r) block, including the HR sub-population. The models exhibited a wide range of Gmax values with constrained relationships linking ICa(L) with IK(r), ICl, INCX, and INaK. Modelling the measured range of inter-cell APDs required a larger range of key Gmax values indicating that ventricular tissue has considerable inter-cell variation in channel/pump/exchanger activity. AP morphology is retained by relationships linking specific ionic conductances. These interrelationships are necessary for stable repolarization despite large inter-cell variation of individual conductances and this explains the variable sensitivity to ion channel block.

Transient Hypoperfusion to Ischemic/Anoxic Spreading Depolarization is Related to Autoregulatory Failure in the Rat Cerebral Cortex

BackgroundIn ischemic stroke, cerebral autoregulation and neurovascular coupling may become impaired. The cerebral blood flow (CBF) response to spreading depolarization (SD) is governed by neurovascular coupling. SDs recur in the ischemic penumbra and reduce neuronal viability by the insufficiency of the CBF response. Autoregulatory failure and SD may coexist in acute brain injury. Here, we set out to explore the interplay between the impairment of cerebrovascular autoregulation, SD occurrence, and the evolution of the SD-coupled CBF response.MethodsIncomplete global forebrain ischemia was created by bilateral common carotid artery occlusion in isoflurane-anesthetized rats, which induced ischemic SD (iSD). A subsequent SD was initiated 20–40 min later by transient anoxia SD (aSD), achieved by the withdrawal of oxygen from the anesthetic gas mixture for 4–5 min. SD occurrence was confirmed by the recording of direct current potential together with extracellular K+ concentration by intracortical microelectrodes. Changes in local CBF were acquired with laser Doppler flowmetry. Mean arterial blood pressure (MABP) was continuously measured via a catheter inserted into the left femoral artery. CBF and MABP were used to calculate an index of cerebrovascular autoregulation (rCBFx). In a representative imaging experiment, variation in transmembrane potential was visualized with a voltage-sensitive dye in the exposed parietal cortex, and CBF maps were generated with laser speckle contrast analysis.ResultsIschemia induction and anoxia onset gave rise to iSD and aSD, respectively, albeit aSD occurred at a longer latency, and was superimposed on a gradual elevation of K+ concentration. iSD and aSD were accompanied by a transient drop of CBF (down to 11.9 ± 2.9 and 7.4 ± 3.6%, iSD and aSD), but distinctive features set the hypoperfusion transients apart. During iSD, rCBFx indicated intact autoregulation (rCBFx < 0.3). In contrast, aSD was superimposed on autoregulatory failure (rCBFx > 0.3) because CBF followed the decreasing MABP. CBF dropped 15–20 s after iSD, but the onset of hypoperfusion preceded aSD by almost 3 min. Taken together, the CBF response to iSD displayed typical features of spreading ischemia, whereas the transient CBF reduction with aSD appeared to be a passive decrease of CBF following the anoxia-related hypotension, leading to aSD.ConclusionsWe propose that the dysfunction of cerebrovascular autoregulation that occurs simultaneously with hypotension transients poses a substantial risk of SD occurrence and is not a consequence of SD. Under such circumstances, the evolving SD is not accompanied by any recognizable CBF response, which indicates a severely damaged neurovascular coupling.