Correlation between alveolar ventilation and electrical properties of lung parenchyma.

One key problem in modern medical imaging is linking measured data and actual physiological quantities. In this article we derive such a link between the electrical bioimpedance of lung parenchyma, which can be measured by electrical impedance tomography (EIT), and the magnitude of regional ventilation, a key to understanding lung mechanics and developing novel protective ventilation strategies. Two rat-derived three-dimensional alveolar microstructures obtained from synchrotron-based x-ray tomography are each exposed to a constant potential difference for different states of ventilation in a finite element simulation. While the alveolar wall volume remains constant during stretch, the enclosed air volume varies, similar to the lung volume during ventilation. The enclosed air, serving as insulator in the alveolar ensemble, determines the resulting current and accordingly local tissue bioimpedance. From this we can derive a relationship between lung tissue bioimpedance and regional alveolar ventilation. The derived relationship shows a linear dependence between air content and tissue impedance and matches clinical data determined from a ventilated patient at the bedside.

Influence of V5/6-His Tag on the Properties of Gap Junction Channels Composed of Connexin43, Connexin40 or Connexin45

HeLa cells expressing wild-type connexin43, connexin40 or connexin45 and connexins fused with a V5/6-His tag to the carboxyl terminus (CT) domain (Cx43-tag, Cx40-tag, Cx45-tag) were used to study connexin expression and the electrical properties of gap junction channels. Immunoblots and immunolabeling indicated that tagged connexins are synthesized and targeted to gap junctions in a similar manner to their wild-type counterparts. Voltage-clamp experiments on cell pairs revealed that tagged connexins form functional channels. Comparison of multichannel and single-channel conductances indicates that tagging reduces the number of operational channels, implying interference with hemichannel trafficking, docking and/or channel opening. Tagging provoked connexin-specific effects on multichannel and single-channel properties. The Cx43-tag was most affected and the Cx45-tag, least. The modifications included (1) V j-sensitive gating of I j (V j, gap junction voltage; I j, gap junction current), (2) contribution and (3) kinetics of I j deactivation and (4) single-channel conductance. The first three reflect alterations of fast V j gating. Hence, they may be caused by structural and/or electrical changes on the CT that interact with domains of the amino terminus and cytoplasmic loop. The fourth reflects alterations of the ion-conducting pathway. Conceivably, mutations at sites remote from the channel pore, e.g., 6-His-tagged CT, affect protein conformation and thus modify channel properties indirectly. Hence, V5/6-His tagging of connexins is a useful tool for expression studies in vivo. However, it should not be ignored that it introduces connexin-dependent changes in both expression level and electrophysiological properties.

Regulation of Cx45 hemichannels mediated by extracellular and intracellular calcium

Connexin45 (Cx45) hemichannels (HCs) open in the absence of Ca(2+) and close in its presence. To elucidate the underlying mechanisms, we examined the role of extra- and intracellular Ca(2+) on the electrical properties of HCs. Experiments were performed on HeLa cells expressing Cx45 using electrical (voltage clamp) and optical (Ca(2+) imaging) methods. HCs exhibit a time- and voltage-dependent current (I(hc)), activating with depolarization and inactivating with hyperpolarization. Elevation of [Ca(2+)](o) from 20 nM to 2 μM reversibly decreases I(hc), decelerates its rate of activation, and accelerates its deactivation. Our data suggest that [Ca(2+)](o) modifies the channel properties by adhering to anionic sites in the channel lumen and/or its outer vestibule. In this way, it blocks the channel pore and reversibly lowers I(hc) and modifies its kinetics. Rapid lowering of [Ca(2+)](o) from 2 mM to 20 nM, achieved early during a depolarizing pulse, led to an outward I(hc) that developed with virtually no delay and grew exponentially in time paralleled by unaffected [Ca(2+)](i). A step increase of [Ca(2+)](i) evoked by photorelease of Ca(2+) early during a depolarizing pulse led to a transient decrease of I(hc) superimposed on a growing outward I(hc); a step decrease of [Ca(2+)](i) elicited by photoactivation of a Ca(2+) scavenger provoked a transient increase in I(hc). Hence, it is tempting to assume that Ca(2+) exerts a direct effect on Cx45 hemichannels.

Subconductance states of Cx30 gap junction channels: data from transfected HeLa cells versus data from a mathematical model

Human HeLa cells expressing mouse connexin30 were used to study the electrical properties of gap junction channel substates. Experiments were performed on cell pairs using a dual voltage-clamp method. Single-channel currents revealed discrete levels attributable to a main state, a residual state, and five substates interposed, suggesting the operation of six subgates provided by the six connexins of a gap junction hemichannel. Substate conductances, gamma(j,substate), were unevenly distributed between the main-state and the residual-state conductance (gamma(j,main state) = 141 pS, gamma(j,residual state) = 21 pS). Activation of the first subgate reduced the channel conductance by approximately 30%, and activation of subsequent subgates resulted in conductance decrements of 10-15% each. Current transitions between the states were fast (<2 ms). Substate events were usually demarcated by transitions from and back to the main state; transitions among substates were rare. Hence, subgates are recruited simultaneously rather than sequentially. The incidence of substate events was larger at larger gradients of V(j). Frequency and duration of substate events increased with increasing number of synchronously activated subgates. Our mathematical model, which describes the operation of gap junction channels, was expanded to include channel substates. Based on the established V(j)-sensitivity of gamma(j,main state) and gamma(j,residual state), the simulation yielded unique functions gamma(j,substate) = f(V(j)) for each substate. Hence, the spacing of subconductance levels between the channel main state and residual state were uneven and characteristic for each V(j).

Calcium indicator loading of neurons using single-cell electroporation

Studies of subcellular Ca(2+) signaling rely on methods for labeling cells with fluorescent Ca(2+) indicator dyes. In this study, we demonstrate the use of single-cell electroporation for Ca(2+) indicator loading of individual neurons and small neuronal networks in rat neocortex in vitro and in vivo. Brief voltage pulses were delivered through glass pipettes positioned close to target cells. This approach resulted in reliable and rapid (within seconds) loading of somata and subsequent complete labeling of dendritic and axonal arborizations. By using simultaneous whole-cell recordings in brain slices, we directly addressed the effect of electroporation on neurons. Cell viability was high (about 85%) with recovery from the membrane permeabilization occurring within a minute. Electrical properties of recovered cells were indistinguishable before and after electroporation. In addition, Ca(2+) transients with normal appearance could be evoked in dendrites, spines, and axonal boutons of electroporated cells. Using negative-stains of somata, targeted single-cell electroporation was equally applicable in vivo. We conclude that electroporation is a simple approach that permits Ca(2+) indicator loading of multiple cells with low background staining within a short amount of time, which makes it especially well suited for functional imaging of subcellular Ca(2+) dynamics in small neuronal networks.

Functional Characterization and Comparison of Intercellular Communication in Stem Cell-Derived Cardiomyocytes

One novel treatment strategy for the diseased heart focuses on the use of pluripotent stem cell-derived cardiomyocytes (SC-CMs) to overcome the heart's innate deficiency for self-repair. However, targeted application of SC-CMs requires in-depth characterization of their true cardiogenic potential in terms of excitability and intercellular coupling at cellular level and in multicellular preparations. In this study, we elucidated the electrical characteristics of single SC-CMs and intercellular coupling quality of cell pairs, and concomitantly compared them with well-characterized murine native neonatal and immortalized HL-1 cardiomyocytes. Firstly, we investigated the electrical properties and Ca2+ signaling mechanisms specific to cardiac contraction in single SC-CMs. Despite heterogeneity of the new cardiac cell population, their electrophysiological activity and Ca2+ handling were similar to native cells. Secondly, we investigated the capability of paired SC-CMs to form an adequate subunit of a functional syncytium and analyzed gap junctions and signal transmission by dye transfer in cell pairs. We discovered significantly diminished coupling in SC-CMs compared with native cells, which could not be enhanced by a coculture approach combining SC-CMs and primary CMs. Moreover, quantitative and structural analysis of gap junctions presented significantly reduced connexin expression levels compared with native CMs. Strong dependence of intercellular coupling on gap junction density was further confirmed by computational simulations. These novel findings demonstrate that despite the cardiogenic electrophysiological profile, SC-CMs present significant limitations in intercellular communication. Inadequate coupling may severely impair functional integration and signal transmission, which needs to be carefully considered for the prospective use of SC-CMs in cardiac repair.

CNT and PDCs: A fruitful association? Study of a polycarbosilane–MWCNT composite

The effect of MWCNT introduction in a polycarbosilane based ceramic on its electrical properties is presented. The electrical conductivity of two MWCNT powders was measured under dynamic compaction up to 20 MPa when it reached 3–5 S/cm. The compaction behavior was also analyzed and modeled. A composite was then realized using allylhydridopolycarbosilane SMP10® and divinylbenzene as matrix. Intact 10 mm MWCNT-SiC ceramic discs samples with 2 wt.% filler load were produced pressure-less via liquid route despite the linear shrinkage of about 30%. Nanotubes microstructure and distribution in the matrix were confirmed after pyrolysis with TEM and SEM analysis. Anyhow similar electrical conductivity values after pyrolysis between the loaded and unloaded samples were measured. The microstructure analysis via XRD and TEM revealed that the percolative carbon network formed through the use of divinylbenzene improves the electric conductivity more than that of MWCNT addition and also simplifies the whole process.