Ion channels are membrane-bound enzymes whose catalytic sites are ion-conducting pores that open and close (gate) in response to specific environmental stimuli. Ion channels are important ...contributors to cell signaling and homeostasis. Our current understanding of gating is the product of 60 plus years of voltage-clamp recording augmented by intervention in the form of environmental, chemical, and mutational perturbations. The need for good phenomenological models of gating has evolved in parallel with the sophistication of experimental technique. The goal of modeling is to develop realistic schemes that not only describe data, but also accurately reflect mechanisms of action. This review covers three areas that have contributed to the understanding of ion channels: traditional Eyring kinetic theory, molecular dynamics analysis, and statistical thermodynamics. Although the primary emphasis is on voltage-dependent channels, the methods discussed here are easily generalized to other stimuli and could be applied to any ion channel and indeed any macromolecule.
Objective: The goal of our study was to determine the importance of electric field orientation in an anisotropic muscle tissue for the extent of irreversible electroporation damage by means of an ...experimentally validated mathematical model. Methods: Electrical pulses were delivered to porcine skeletal muscle in vivo by inserting needle electrodes so that the electric field was applied in direction either parallel or perpendicular to the direction of the muscle fibres. Triphenyl tetrazolium chloride staining was used to determine the shape of the lesions. Next, we used a single cell model to determine the cell-level conductivity during electroporation, and then generalised the calculated conductivity changes to the bulk tissue. Finally, we compared the experimental lesions with the calculated field strength distributions using the Sørensen-Dice similarity coefficient to find the contours of the electric field strength threshold beyond which irreversible damage is thought to occur. Results: Lesions in the parallel group were consistently smaller and narrower than lesions in the perpendicular group. The determined irreversible threshold of electroporation for the selected pulse protocol was 193.4 V/cm with a standard deviation of 42.1 V/cm, and was not dependent on field orientation. Conclusion: Muscle anisotropy is of significant importance when considering electric field distribution in electroporation applications. Significance: The paper presents an important advancement in building up from the current understanding of single cell electroporation to an in silico multiscale model of bulk muscle tissue. The model accounts for anisotropic electrical conductivity and has been validated through experiments in vivo .
Introduction
Contact force has been used to titrate lesion formation for radiofrequency ablation. Pulsed field ablation (PFA) is a field‐based ablation technology for which limited evidence on the ...impact of contact force on lesion size is available.
Methods
Porcine hearts (n = 6) were perfused using a modified Langendorff set‐up. A prototype focal PFA catheter attached to a force gauge was held perpendicular to the epicardium and lowered until contact was made. Contact force was recorded during each PFA delivery. Matured lesions were cross‐sectioned, stained, and the lesion dimensions measured.
Results
A total of 82 lesions were evaluated with contact forces between 1.3 and 48.6 g. Mean lesion depth was 4.8 ± 0.9 mm (standard deviation), mean lesion width was 9.1 ± 1.3 mm, and mean lesion volume was 217.0 ± 96.6 mm3. Linear regression curves showed an increase of only 0.01 mm in depth (depth = 0.01 × contact force + 4.41, R2 = 0.05), 0.03 mm in width (width = 0.03 × contact force + 8.26, R2 = 0.13) for each additional gram of contact force, and 2.20 mm3 in volume (volume = 2.20 × contact force + 162, R2 = 0.10).
Conclusion
Increasing contact force using a bipolar, biphasic focal PFA system has minimal effects on acute lesion dimensions in an isolated porcine heart model and achieving tissue contact is more important than the force with which that contact is made.
Using an isolated swine heart model, a focal ablation catheter was used to apply pulsed electrical fields with varying contact forces as well as with a 2 mm offset (catheter 2 mm away from tissue, not touching). Increasing contact force using a bipolar, biphasic focal PFA system has minimal effects on acute lesion dimensions in an isolated porcine heart model and achieving tissue contact is more important than the force with which that contact is made.
Pulsed field ablation (PFA) is a novel energy modality for treatment of cardiac arrhythmias. The impact of electrode-tissue proximity on lesion formation by PFA has not been conclusively assessed. ...The objective of this investigation was to evaluate the effects of electrode-tissue proximity on cardiac lesion formation with a biphasic, bipolar PFA system.
PFA was delivered on the ventricular epicardial surface in an isolated porcine heart model (n=8) via a 4-electrode prototype catheter. An offset tool was designed to control the distance between electrodes and target tissue; deliveries were placed 0 mm (0 mm offset), 2 mm (2 mm offset), and 4 mm away from the tissue (4 mm offset). Lesions were assessed using tetrazolium chloride staining. Numerical models for the experimental setup with and without the offset tool validated and supported results.
Cardiac lesion dimensions decreased proportional to the distance between epicardial surface and electrodes. Lesion depth averaged 4.3±0.4 mm, 2.7±0.4 mm, and 1.3±0.4 mm for the 0, 2, and 4 mm and lesion width averaged 9.4±1.1 mm, 7.5±0.8 mm and 5.8±1.4 mm for the 0, 2, and 4 mm offset distances, respectively. Numerical modeling matched ex vivo results well and predicted lesion creation with and without the offset tool.
Using a biphasic, bipolar PFA system resulted in cardiac lesions even in the 0 mm offset distance case. The relationship between lesion depth and offset distance was linear, and the deepest lesions were created with 0 mm offset distance, that is, with electrodes in contact with tissue. Therefore, close electrode-tissue proximity increases the likelihood of achieving transmural lesions by maximizing the electric field penetration into the target tissue.
Phrenic nerve palsy is a well-known complication of cardiac ablation, resulting from the application of direct thermal energy. Emerging pulsed field ablation (PFA) may reduce the risk of phrenic ...nerve injury but has not been well characterized.
Accelerometers and continuous pacing were used during PFA deliveries in a porcine model. Acute dose response was established in a first experimental phase with ascending PFA intensity delivered to the phrenic nerve (n=12). In a second phase, nerves were targeted with a single ablation level to observe the effect of repetitive ablations on nerve function (n=4). A third chronic phase characterized assessed histopathology of nerves adjacent to ablated cardiac tissue (n=6).
Acutely, we observed a dose-dependent response in phrenic nerve function including reversible stunning (R
=0.965,
<0.001). Furthermore, acute results demonstrated that phrenic nerve function responded to varying levels of PFA and catheter proximity placements, resulting in either: no effect, effect, or stunning. In the chronic study phase, successful isolation of superior vena cava at a dose not predicted to cause phrenic nerve dysfunction was associated with normal phrenic nerve function and normal phrenic nerve histopathology at 4 weeks.
Proximity of the catheter to the phrenic nerve and the PFA dose level were critical for phrenic nerve response. Gross and histopathologic evaluation of phrenic nerves and diaphragms at a chronic time point yielded no injury. These results provide a basis for understanding the susceptibility and recovery of phrenic nerves in response to PFA and a need for appropriate caution in moving beyond animal models.
Excitation-evoked Ca²⁺ influx is the fastest and most ubiquitous chemical trigger for cellular processes, including neurotransmitter release, musde contraction, and gene expression. The voltage ...dependence and timing of Ca²⁺ entry are thought to be functions of voltage-gated calcium (Cav) channels composed of a central pore regulated by four nonidentical voltage-sensing domains (VSDs I-IV). Currently, the individual voltage dependence and the contribution to pore opening of each VSD remain largely unknown. Using an optical approach (voltage-clamp fluorometry) to track the movement of the individual voltage sensors, we discovered that the four VSDs of Cav1.2 channels undergo voltage-evoked conformational rearrangements, each exhibiting distinct voltage-and time-dependent properties over a wide range of potentials and kinetics. The voltage dependence and fast kinetic components in the activation of VSDs II and III were compatible with the ionic current properties, suggesting that these voltage sensors are involved in Cav1.2 activation. This view is supported by an obligatory model, in which activation of VSDs II and III is necessary to open the pore. When these data were interpreted in view of an allosteric model, where pore opening is intrinsically independent but biased by VSD activation, VSDs II and III were each found to supply ~50 meV (~2 kT), amounting to ~85% of the total energy, toward stabilizing the open state, with a smaller contribution from VSD I (~16 meV). VSD IV did not appear to participate in channel opening.
Although we know much about the molecular makeup of the sinus node (SN) in small mammals, little is known about it in humans. The aims of the present study were to investigate the expression of ion ...channels in the human SN and to use the data to predict electrical activity.
Quantitative polymerase chain reaction, in situ hybridization, and immunofluorescence were used to analyze 6 human tissue samples. Messenger RNA (mRNA) for 120 ion channels (and some related proteins) was measured in the SN, a novel paranodal area, and the right atrium (RA). The results showed, for example, that in the SN compared with the RA, there was a lower expression of Na(v)1.5, K(v)4.3, K(v)1.5, ERG, K(ir)2.1, K(ir)6.2, RyR2, SERCA2a, Cx40, and Cx43 mRNAs but a higher expression of Ca(v)1.3, Ca(v)3.1, HCN1, and HCN4 mRNAs. The expression pattern of many ion channels in the paranodal area was intermediate between that of the SN and RA; however, compared with the SN and RA, the paranodal area showed greater expression of K(v)4.2, K(ir)6.1, TASK1, SK2, and MiRP2. Expression of ion channel proteins was in agreement with expression of the corresponding mRNAs. The levels of mRNA in the SN, as a percentage of those in the RA, were used to estimate conductances of key ionic currents as a percentage of those in a mathematical model of human atrial action potential. The resulting SN model successfully produced pacemaking.
Ion channels show a complex and heterogeneous pattern of expression in the SN, paranodal area, and RA in humans, and the expression pattern is appropriate to explain pacemaking.
Irreversible electroporation is an energy form utilizing high-voltage pulsed electric field, leading to cellular homeostasis disruption and cell death. Recently, irreversible electroporation has ...shown promising results for the treatment of cardiac arrhythmias. However, reversible and irreversible effects of pulsed electric field on cardiac myocytes remain poorly understood. Here, we evaluated the influence of a monophasic single electric pulse (EP) on the contractility, Ca
homeostasis and recovery of cardiac myocytes.
Isolated rat left ventricular myocytes were electroporated using single monophasic EP of different durations and voltages. Sarcomere length and intracellular Ca
were simultaneously monitored for up to 20 minutes after EP application in Fura-2 loaded left ventricular myocytes. Lethal voltage thresholds were determined using 100 µs and 10 ms pulses and by discriminating cell orientation with respect to the electric field.
Electroporation led to an immediate increase in intracellular Ca
which was dependent upon the voltage delivered to the cell. Intermediate-voltage EP (140 V, 100 µs) increased sarcomere shortening, Ca
transient amplitude, and diastolic Ca
level measured 1 minute post-EP. Although sarcomere shortening returned to pre-EP level within 5 minutes, Ca
transient amplitude decreased further below pre-EP level and diastolic Ca
level remained elevated within 20 minutes post-EP. Spontaneous contractions were observed after sublethal EP application but their frequency decreased progressively within 20 minutes. Lethal EP voltage threshold was lower in myocytes oriented perpendicular than parallel to the electric field using 100 µs pulses while an opposite effect was found using 10 ms pulses.
Sublethal EP affected rat left ventricular myocytes contractility and disrupted Ca
homeostasis as a function of the EP voltage. Moreover, EP-induced lethality was preceded by a large increase in intracellular Ca
and was dependent upon the EP duration, amplitude and left ventricular myocytes orientation with respect to the electric field. These findings provide new insights into the effect of pulsed electric field on cardiac myocytes.
Potassium-selective inward rectifier (Kir) channels are a class of membrane proteins necessary for maintaining stable resting membrane potentials, controlling excitability, and shaping the final ...repolarization of action potentials in excitable cells. In addition to the strong inward rectification of the ionic current caused by intracellular blockers, Kir2.1 channels possess "weak" inward rectification observed in inside-out patches after prolonged washout of intracellular blockers. The mechanisms underlying strong inward rectification have been attributed to voltage-dependent block by intracellular Mg
and polyamines; however, the mechanism responsible for weak rectification remains elusive. Hypotheses include weak voltage-dependent block and intrinsic voltage-dependent gating. Here, we performed a conductance Hill analysis of currents recorded with a double-ramp protocol to evaluate different mechanisms proposed for weak inward rectification of Kir2.1 channels. Linkage analysis in the form of a Hill plot revealed that the ramp currents could be best explained by allosteric coupling between a mildly voltage-dependent pore gate (gating charge ∼0.18 e
) and a voltage sensor (gating charge ∼1.7 e
). The proposed voltage sensor stabilized the closing of the pore gate (coupling factor ∼31). We anticipate that the use of linkage analysis will broaden understanding of functional coupling in ion channels and proteins in general.
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