Recovery Time Constant Research Articles

Blockade of Voltage-Dependent K+ Channels by Benztropine, a Muscarinic Acetylcholine Receptor Inhibitor, in Coronary Arterial Smooth Muscle Cells.

We investigated the effect of the acetylcholine muscarinic receptor inhibitor benztropine on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells. Benztropine inhibited Kv currents in a concentration-dependent manner, with an apparent IC50 value of 6.11 ± 0.80 μM and Hill coefficient of 0.62 ± 0.03. Benztropine shifted the steady-state activation curves toward a more positive potential, and the steady-state inactivation curves toward a more negative potential, suggesting that benztropine inhibited Kv channels by affecting the channel voltage sensor. Train pulse (1 or 2 Hz)-induced Kv currents were effectively reduced by the benztropine treatment. Furthermore, recovery time constants of Kv current inactivation increased significantly in response to benztropine. These results suggest that benztropine inhibited vascular Kv channels in a use (state)-dependent manner. The inhibitory effect of benztropine was canceled by pretreatment with the Kv 1.5 inhibitor, but there was no obvious change after pretreatment with Kv 2.1 or Kv7 inhibitors. In conclusion, benztropine inhibited the Kv current in a concentration- and use (state)-dependent manner. Inhibition of the Kv channels by benztropine primarily involved the Kv1.5 subtype. Restrictions are required when using benztropine to patients with vascular disease.

Frequency-dependent depression of the NTS synapse affects the temporal response of the antihypertensive effect of auricular vagus nerve stimulation (aVNS)

Objective. Auricular vagus nerve stimulation (aVNS) has recently emerged as a promising neuromodulation modality for blood pressure (BP) reduction due to its ease of use although its efficacy is still limited compared to direct baroreflex stimulation. Previous studies have also indicated that synaptic depression of nucleus tractus solitarius (NTS) in the baroreflex pathway depends on stimulus frequency. However, the nature of this frequency dependence phenomenon on antihypertensive effect has been unknown for aVNS. We aimed to investigate the antihypertensive effect of aVNS considering frequency-dependent depression characteristic in the NTS synapse. We explored NTS activation and BP reduction induced by aVNS and by direct secondary neuron stimulation (DS). Approach. Both protocols were performed with recording of NTS activation and BP response with stimulation for each frequency parameter (2, 4, 20, 50, and 80 Hz). Main results. The BP recovery time constant was significantly dependent on the frequency of DS and aVNS (DS—2 Hz: 8.17 ± 4.98; 4 Hz: 9.73 ± 6.3; 20 Hz: 6.61 ± 3.28; 50 Hz: 4.93 ± 1.65; 80 Hz: 4.00 ± 1.43, p < 0.001, Kruskal–Wallis (KW) H-test/aVNS—2 Hz: 4.02 ± 2.55; 4 Hz: 8.13 ± 4.05; 20 Hz: 6.40 ± 3.16; 50 Hz: 5.18 ± 2.37; 80 Hz: 3.13 ± 1.29, p < 0.05, KW H-test) despite no significant BP reduction at 2 Hz compared to sham groups (p > 0.05, Mann–Whitney U-test). Significance. Our observations suggest that the antihypertensive effect of aVNS is influenced by the characteristics of frequency-dependent synaptic depression in the NTS neuron in terms of the BP recovery time. These findings suggest that the antihypertensive effect of aVNS can be improved with further understanding of the neurological properties of the baroreflex associated with aVNS, which is critical to push this new modality for clinical interpretation.

Chemical Exchange Saturation Transfer Magnetic Resonance Imaging Identifies Abnormal Calf Muscle-Specific Energetics in Peripheral Artery Disease.

Peripheral artery disease (PAD) results in exercise-induced ischemia in leg muscles. 31Phosphorus (P) magnetic resonance spectroscopy demonstrates prolonged phosphocreatine recovery time constant after exercise in PAD but has low signal to noise, low spatial resolution, and requires multinuclear hardware. Chemical exchange saturation transfer (CEST) is a quantitative magnetic resonance imaging method for imaging substrate (CEST asymmetry [CESTasym]) concentration by muscle group. We hypothesized that kinetics measured by CEST could distinguish between patients with PAD and controls. Patients with PAD and age-matched normal subjects were imaged at 3T with a transmit-receive coil around the calf. Four CEST mages were acquired over 24-second intervals. The subjects then performed plantar flexion exercise on a magnetic resonance imaging-compatible ergometer until calf exhaustion. Twenty-five CEST images were obtained at end exercise. Regions of interest were drawn around individual muscle groups, and (CESTasym) decay times were fitted by exponential curve to CEST values. In 10 patients and 11 controls, 31P spectra were obtained 20 minutes later after repeat exercise. Five patients and 5 controls returned at a mean of 1±1 days later for repeat CEST studies. Thirty-five patients with PAD (31 male, age 66±8 years) and 29 controls (11 male, age 63±8 years) were imaged with CEST. The CESTasym decay times for the whole calf (341±332 versus 153±72 seconds; P<0.03) as well as for the gastrocnemius and posterior tibialis were longer in patients with PAD. Agreement between CESTasym decay and phosphocreatine recovery time constant was good. CEST is a magnetic resonance imaging method that can distinguish energetics in patients with PAD from age-matched normal subjects on a per muscle group basis. CEST agrees reasonably well with the gold standard 31P magnetic resonance spectroscopy. Moreover, CEST has higher spatial resolution, creates an image, and does not require multinuclear hardware and thus may be more suitable for clinical studies in PAD.

Inhibitory effects of the atypical antipsychotic, clozapine, on voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells.

To investigate the adverse effects of clozapine on cardiovascular ion channels, we examined the inhibitory effect of clozapine on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells. Clozapine-induced inhibition of Kv channels occurred in a concentration-dependent manner with an half-inhibitory concentration value of 7.84 ± 4.86 µM and a Hill coefficient of 0.47 ± 0.06. Clozapine did not shift the steady-state activation or inactivation curves, suggesting that it inhibited Kv channels regardless of gating properties. Application of train pulses (1 and 2 Hz) progressively augmented the clozapine-induced inhibition of Kv channels in the presence of the drug. Furthermore, the recovery time constant from inactivation was increased in the presence of clozapine, suggesting that clozapine-induced inhibition of Kv channels is use (state)-dependent. Pretreatment of a Kv1.5 subtype inhibitor decreased the Kv current amplitudes, but additional application of clozapine did not further inhibit the Kv current. Pretreatment with Kv2.1 or Kv7 subtype inhibitors partially blocked the inhibitory effect of clozapine. Based on these results, we conclude that clozapine inhibits arterial Kv channels in a concentration- and use (state)-dependent manner. Kv1.5 is the major subtype involved in clozapine-induced inhibition of Kv channels, and Kv2.1 and Kv7 subtypes are partially involved.

Promising Novel Barium Carbonate One-Dimensional Nanostructures and Their Gas Sensing Application: Preparation and Characterization

Recently, barium carbonate-based nanomaterials have been used for sensor and catalysis applications. The sensing performance can be improved with a suitable one-dimensional nanostructure. In this regard, novel nanosized BaCO3 materials were fabricated by a one-pot designed thermal evaporation system. Ten milligrams of Ba as raw material were used to deposit BaCO3 nanostructures at a pressure of 0.85 torr and a temperature of 850 °C in a partial oxygen atmosphere of the ambient. This simple method for fabricating novel BaCO3 nanostructures is presented here. X-ray diffraction was indexed on the orthorhombic polycrystalline structure of the prepared BaCO3. The nanostructures deposited here could be described as Datura-like structures linked with nanowires of 20–50 nm in diameter and 5 µm in length. The BaCO3 nanostructure prepared by the current method exhibited a semiconductor-like behavior with an activation energy of 0.68 eV. This behavior was ascribed to the nature of the morphology, which may possess large defective points. Thus, this nanostructure was subjected to gas sensing measurements, showing high activity toward NO2 gas. The proposed sensor also underwent deep investigation toward NO2 at various gas concentrations and working. The response and recovery time constants were recorded in the ranges of 6–20 s and 30–150 s, respectively. The sensor showed its reversibility toward NO2 when the sensor signal was repeated at various cycles of various concentrations. The sensor was exposed to different levels of humidity, showing high performance toward NO2 gas at 250 °C. The sensor exhibited fast response and recovery toward NO2 gas.

Method to Distinguish Between Buffer and Surface Trapping in Stressed Normally-ON GaN GITs Using the Gate-Voltage Dependence of Recovery Time Constants

Drain current recovery transients are analyzed in normally-ON (NON) gate injection transistors (GITs) subjected to different kinds of stress. The recovery is measured as a function of forward gate bias, <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">${V}_{\text {G}}$ </tex-math></inline-formula> , which controls the number of holes injected from the gate and speeds up the recovery. Unlike conventional normally-OFF GITs, the newly designed NON GIT test structures allow simple characterization of buffer trapping during back-gating. By comparing the <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">${V}_{\text {G}}$ </tex-math></inline-formula> -dependence of recovery time constants for OFF-state and semi- ON state stress with those obtained from back-gating experiments we are able to distinguish between buffer and surface trapping. Our approach is simple and does not require time-consuming temperature-dependent measurements. It is found that OFF-state stress causes negative charge accumulation in the buffer, while semi- ON state stress leads to accumulation in both, buffer and surface. The use of NON GITs enables to measure the recovery time constants related to the buffer over a wide span of six orders of magnitude ( <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$10^{-{4}}$ </tex-math></inline-formula> –100 s).

High‐Density Lipoprotein is Independently Associated with Muscle Mitochondrial Function in Healthy Humans

High‐density lipoprotein cholesterol (HDL‐C) is well known to play an important anti‐atherogenic role via reverse cholesterol transport. Increasing number of studies in mice have also suggested a protective role of HDL in preserving muscle mitochondrial function via ApoA1‐induced enhancement of cellular respiration of glucose. However, data in humans are lacking. We therefore hypothesize that HDL levels and/or function are correlated with muscle mitochondrial function in humans. Accordingly, we conducted a cross‐sectional study to determine the relationship between levels and function of HDL and skeletal muscle mitochondrial function in 31 healthy adults without diabetes mellitus or cardiovascular disease. To estimate muscle mitochondrial function, we measured the oxygen recovery time constant (Tau) during supra‐systolic cuff‐occlusions following 2 minutes of rhythmic handgrip exercise at 30% maximal voluntary contraction in the forearm muscle, using near infrared spectroscopy (NIRS). To assess cholesterol efflux capacity (CEC), we used J774 macrophages, radiolabeled cholesterol, and ApoB‐depleted plasma to calculate cholesterol efflux normalized to a pooled sample. Of the 31 subjects, 13 (42%) were female, mean age was 40 +/‐ 16, mean BMI was 23.9 +/‐ 3.4 kg/m^2, and the mean total serum cholesterol was 198.4 +/‐ 43.1 mg/dL. We found a significant inverse correlation between HDL‐C levels and Tau, with a correlation coefficient (r) of ‐0.51 (p &lt; 0.01, Figure. 1a). As expected, a positive correlation is observed between BMI and Tau (r= 0.50, p &lt; 0.01, Figure 1c). In contrast, no significant correlation between fasting triglyceride, plasma glucose, insulin levels or HDL efflux function with Tau were found (all p‐values &gt; 0.05). In conclusion, our study identifies a novel association between circulating HDL levels with muscle mitochondrial function. However, the CEC of our sample was not significantly correlated with Tau, therefore future investigations with larger studies or more measures of HDL function and composition may elucidate these findings. The association of HDL‐C and muscle mitochondrial function may explain increased prevalence of physical inactivity among populations with low HDL‐C, such as those with metabolic syndrome. Additionally, future studies are needed to determine if strategies to improve HDL‐C levels will result in improved muscle mitochondrial function and exercise capacity.

Effects of JZTX-V on the wild type Kv4.3 Expressed in HEK293T and Molecular Determinants in the Voltage-sensing Domains of Kv4.3 Interacting with JZTX-V

ABSTRACT JZTX-V is a toxin isolated from the venom of the Chinese spider Chilobrachys jingzhao. Previous studies had shown that JZTX-V could inhibit the transient outward potassium current of Kv4.2 and Kv4.3 expressed in Xenopus oocytes but had no effects on Kv1.2–1.4. However, the underlying action mechanism of JZTX-V on Kv4.3 remains unclear. In our study, JZTX-V could inhibit not only transient outward potassium currents evoked in small-sized DRG neurons but also Kv4.3-encoded currents expressed in HEK293T cells in the concentration and voltage dependence. The half maximal inhibitory concentration of JZTX-V on Kv4.3 was 9.6 ± 1.2 nM. In addition, the time course for JZTX-V inhibition and release of inhibition after washout were 15.8 ± 1.54 s and 58.8 ± 4.35 s. Electrophysiological assays indicated that 25 nM JZTX-V could shift significantly the voltage dependence of steady-state activation and steady-state inactivation to depolarization. Meanwhile, 25 nM JZTX-V decreased markedly the time constant of activation and inactivation but had no effect on the time constant of recovery from inactivation. To study the molecular determinants of Kv4.3, we performed alanine scanning on a conserved motif of Kv4.3 and assayed the affinity between mutants and JZTX-V. The results not only showed that I273, L275, V283, and F287 were molecular determinants in the conserved motif of Kv4.3 for interacting with JZTX-V but also speculated the underlying action mechanism that the hydrophobic interaction and steric effects played key roles in the binding of JZTX-V with Kv4.3. In summary, our studies have laid a scientific theoretical foundation for further research on the interaction mechanism between JZTX-V and Kv4.3.

458 Reproducibility of Maximum ATP Production Following Isotonic versus Isometric Exercise

OBJECTIVES/GOALS: This project aimed to investigate differences between isometric and isotonic systems for low and high frequency contractions. Intracellular changes in pH caused by the rate of exercise differ in their effect on PCr. The project assessed the reproducibility between these exercise methods. METHODS/STUDY POPULATION: Four subjects were studied (2F/2M, avg. age 27 years). 31P-MRS spectra were measured in the vastus lateralis (VL) using a 3T MRI scanner using a 31P/1H surface coil. In the isometric studies, the (supine) subjects legs were supported by a wedge pad and restrained by straps over the hips and knee with a resistance strap over one ankle. In the isometric studies, the (prone) subjects legs were fit into machine designed to induce isometric exercise. There were 4 exercise bouts that differed by exercise type and frequency and, consequently, duration. 31P-MR spectra were acquired from a 25mm slab of leg positioned parasagittal along the VL. Analyses were done using jMRUI and R. RESULTS/ANTICIPATED RESULTS: With an increase in sample size, we expect differences between recovery time constants, tau, of PCr between the different exercise frequency groups alluded to by other studies investigating recovery rates in bicep skeletal muscle. From the preliminary data, we obtained large limits of agreement of tau = 0.053 s-1 &amp; tau =-0.074 s-1 from using Bland-Altman analysis. As we increase our sample size, we anticipate that we will see differences of PCr recovery rates between the exercise types as well. DISCUSSION/SIGNIFICANCE: Current studies that investigate the differences in PCr recovery rates between different metabolic states differ in the exercise methods employed on subjects. This study aims to standardize PCr recovery assessment in the VL for more interpretable and applicable assessments of metabolic function.

Quantitative Analysis of the Cytoskeleton's Role in Inward Rectifier K IR 2.1 Forward and Backward Trafficking.

Alteration of the inward rectifier current IK1, carried by KIR2.1 channels, affects action potential duration, impacts resting membrane stability and associates with cardiac arrhythmias. Congenital and acquired KIR2.1 malfunction frequently associates with aberrant ion channel trafficking. Cellular processes underlying trafficking are intertwined with cytoskeletal function. The extent to which the cytoskeleton is involved in KIR2.1 trafficking processes is unknown. We aimed to quantify the dependence of KIR2.1 trafficking on cytoskeleton function. GFP or photoconvertible Dendra2 tagged KIR2.1 constructs were transfected in HEK293 or HeLa cells. Photoconversion of the Dendra2 probe at the plasma membrane and subsequent live imaging of trafficking processes was performed by confocal laser-scanning microscopy. Time constant of green fluorescent recovery (τg,s) represented recruitment of new KIR2.1 at the plasma membrane. Red fluorescent decay (τr,s) represented internalization of photoconverted KIR2.1. Patch clamp electrophysiology was used to quantify IKIR2.1. Biochemical methods were used for cytoskeleton isolation and detection of KIR2.1-cytoskeleton interactions. Cytochalasin B (20 μM), Nocodazole (30 μM) and Dyngo-4a (10 nM) were used to modify the cytoskeleton. Chloroquine (10 μM, 24 h) was used to impair KIR2.1 breakdown. Cytochalasin B and Nocodazole, inhibitors of actin and tubulin filament formation respectively, strongly inhibited the recovery of green fluorescence at the plasma membrane suggestive for inhibition of KIR2.1 forward trafficking [τg,s 13 ± 2 vs. 131 ± 31* and 160 ± 40* min, for control, Cytochalasin B and Nocodazole, respectively (*p < 0.05 vs. control)]. Dyngo-4a, an inhibitor of dynamin motor proteins, strongly slowed the rate of photoconverted channel internalization, whereas Nocodazole and Cytochalasin B had less effect [τr,s 20 ± 2 vs. 87 ± 14*, 60 ± 16 and 64 ± 20 min (*p < 0.05 vs. control)]. Cytochalasin B treatment (20 μM, 24 h) inhibited IKIR2.1. Chloroquine treatment (10 μM, 24 h) induced intracellular aggregation of KIR2.1 channels and enhanced interaction with the actin/intermediate filament system (103 ± 90 fold; p < 0.05 vs. control). Functional actin and tubulin cytoskeleton systems are essential for forward trafficking of KIR2.1 channels, whereas initial backward trafficking relies on a functional dynamin system. Chronic disturbance of the actin system inhibits KIR2.1 currents. Internalized KIR2.1 channels become recruited to the cytoskeleton, presumably in lysosomes.

The inhibitory effects of pimozide, an antipsychotic drug, on voltage-gated K+ channels in rabbit coronary arterial smooth muscle cells

Pimozide is an antipsychotic drug used to treat chronic psychosis, such as Tourette’s syndrome. Despite its widespread clinical use, pimozide can cause unexpected adverse effects, including arrhythmias. However, the adverse effects of pimozide on vascular K+ channels have not yet been determined. Therefore, we investigated the effects of pimozide on voltage-gated K+ (Kv) channels in rabbit coronary arterial smooth muscle cells. Pimozide concentration-dependently inhibited the Kv currents with an IC50 value of 1.78 ± 0.17 μM and a Hill coefficient of 0.90 ± 0.05. The inhibitory effect on the Kv current by pimozide was highly voltage-dependent in the voltage range of Kv channel activation, and additive inhibition of the Kv current by pimozide was observed in the full activation voltage range. The decay rate of inactivation was significantly accelerated by pimozide. Pimozide shifted the inactivation curve to a more negative potential. The recovery time constant from inactivation increased in the presence of pimozide. Furthermore, pimozide-induced inhibition of the Kv current was augmented by applying train pulses. Although pretreatment with the Kv2.1 subtype inhibitor guangxitoxin and the Kv7 subtype inhibitor linopirdine did not alter the degree of pimozide-induced inhibition of the Kv currents, pretreatment with the Kv1.5 channel inhibitor DPO-1 reduced the inhibitory effects of pimozide on Kv currents. Pimozide induced membrane depolarization. We conclude that pimozide inhibits Kv currents in voltage-, time-, and use (state)-dependent manners. Furthermore, the major Kv channel target of pimozide is the Kv1.5 channel.

Kinetics and Connectivity Properties of Parvalbumin- and Somatostatin-Positive Inhibition in Layer 2/3 Medial Entorhinal Cortex.

Parvalbumin-positive (Pvalb+) and somatostatin-positive (Sst+) cells are the two largest subgroups of inhibitory interneurons. Studies in visual cortex indicate that synaptic connections between Pvalb+ cells are common while connections between Sst+ interneurons have not been observed. The inhibitory connectivity and kinetics of these two interneuron subpopulations, however, have not been characterized in medial entorhinal cortex (mEC). Using fluorescence-guided paired recordings in mouse brain slices from interneurons and excitatory cells in layer 2/3 mEC, we found that, unlike neocortical measures, Sst+ cells inhibit each other, albeit with a lower probability than Pvalb+ cells (18% vs 36% for unidirectional connections). Gap junction connections were also more frequent between Pvalb+ cells than between Sst+ cells. Pvalb+ cells inhibited each other with larger conductances, smaller decay time constants, and shorter delays. Similarly, synaptic connections between Pvalb+ and excitatory cells were more likely and expressed faster decay times and shorter delays than those between Sst+ and excitatory cells. Inhibitory cells exhibited smaller synaptic decay time constants between interneurons than on their excitatory targets. Inhibition between interneurons also depressed faster, and to a greater extent. Finally, inhibition onto layer 2 pyramidal and stellate cells originating from Pvalb+ interneurons were very similar, with no significant differences in connection likelihood, inhibitory amplitude, and decay time. A model of short-term depression fitted to the data indicates that recovery time constants for refilling the available pool are in the range of 50–150 ms and that the fraction of the available pool released on each spike is in the range 0.2–0.5.

Time limit at maximal aerobic power, heart rate kinetics and performance in time-trial cycling test of 3 km

The performance of cyclists in short terms workouts can be associate with several factors, including the maximal aerobic power (MAP), heart rate (HR) and its kinetics parameters, and the capacity to tolerate maximal efforts to exhaustion (TLimMAP). Thereby, the main of this study was analyze the presumable relation among TLimMAP and performance in a time-trial cycling test of 3 km. (TT3km). Seven cyclists were involved in this study, performing the following tests with a minimum interval of 48h: (1) initial familiarization and anthropometric evaluation, (2) maximal progressive test to exhaustion, (3) TLimMAP and (4) TT3km test. There was a tendency of subjects with higher values to TLimMAP performed the TT3km faster (r = -0,71; p = 0,07). It showed positive correlation among TLimMAP and the first time constant of the heart rate at the beginning of exercise (r = 0,95 e p &lt; 0,01), and negative to TLimMAP and the first time constant of the heart rate recovery (r = -0,67 e p = 0,04). The tendency to association among the TLimMAP and performance at the TT3km indicate that the TLimMAP could be utilized in the assessment of cyclists – although with caveats – since there was not significant correlation. Additional investigations to enlighten the relations among TLimMAP, HR kinetics and performance would characterize a proficuous field of research.

Abstract 12812: Creatine Chemical Exchange Transfer MRI Demonstrates Abnormal Calf Muscle Energetics in Peripheral Arterial Disease

Introduction: Creatine chemical exchange saturation transfer (CrCEST) is a novel MRI technique utilizing radiofrequency pulses to monitor creatine concentrations at high spatial resolution. The study utilized CrCEST kinetics to compare post-exercise creatine decay in peripheral arterial disease (PAD) patients to normal subjects and to validate against phosphocreatine (PCr) recovery by 31 phosphorus (P) MR spectroscopy (MRS). Methods: 23 subjects with PAD (claudication and ankle-brachial index&lt;0.9) and 28 healthy subjects were enrolled. All underwent 3T MRI (Siemens Prisma) using a knee coil around the calf. Pre- and post-exercise images were obtained around plantar flexion ergometry (Ergospect) until exhaustion. Regions of interests were drawn around major muscle groups. Creatine decay times were obtained by fitting an exponential curve to the CrCEST values. 31 P spectra were obtained 20 minutes later at peak exercise using a surface-coil localized, free induction decay acquisition with 25 signal averages at a repetition time of 550s with calculation of PCr recovery time constant. Results: 23 PAD subjects (mean ABI 0.66 ± 0.11, age 64 ± 8, 17% females) had prolonged median overall calf muscle creatine decay time of 286 seconds (s) (Interquartile Range (IQR) 134 to 370) versus 152 s (IQR 114 to 193) in controls (age 63 ± 9, 70% females), p = 0.009. The gastrocnemius muscle demonstrated a creatine decay time of 316 s in PAD (IQR 189 to 510) versus 181 s in controls (IQR 102 to 236), p = 0.003 and the posterior tibialis muscle, 186 s (IQR 119 to 653) versus 125 s (IQR 72 to 221), p = 0.013. 8 PAD patients and 6 controls underwent CrCEST and 31 P MRS which were compared over the entire calf. Bland-Altman analysis (Figure 1B) yielded a mean difference of 71s (SD 109). Conclusions: Imaging with CrCEST demonstrates prolonged creatine kinetics in PAD patients compared to normal subjects with high spatial resolution and good agreement with 31 P MRS, although PCr recovery appears faster.

Study and modulation of cardiac electroporation with a novel model utilizing human induced pluripotent stem cells

Abstract Background Cardiac electroporation is a promising novel non-thermal ablation method, gaining significant interest with recent first-in-man data suggesting effective cardiac lesion generation with no collateral damage. Nevertheless, significant knowledge gaps exist regarding its electrophysiological consequences in cardiomyocytes, including; cell specificity, protocol optimization, irreversibility threshold, recovery time-constants, and the mechanistic nature of its cytolytic and anti-arrhythmic properties. Purpose Establishing an innovative in-vitro model for the study of cardiac electroporation-ablation, utilizing human induced pluripotent stem cells (hiPSCs). Methods and results Healthy-control hiPSC-derived cardiomyocytes were enzymatically dissociated and seeded as circular cell sheets (hiPSC-CCSs). Electroporation-ablation experiments were performed using a custom designed high-frequency electroporation (HF-EP) generator. Two needle-shaped electrodes were used for HF-EP delivery (Figure 1). Subsequently, detailed voltage- and Ca2+-mapping studies of the hiPSC-CCSs were conducted (Figure 2). HF-EP application resulted in the generation of electrically isolated lesions within the hiPSC-CCSs (Figure 3). Further characterization of the temporal changes and electrophysiological properties following electroporation revealed that; (1) lesions persisted over prolonged periods of time (days), indicating irreversible electroporation, (2) a temporal decrease in lesion dimensions was noted, consistent with a significant reversible electroporation component (Figures 3–5), (3) most tissue recovery had occurred within the first 15 minutes following electroporation, with little recovery beyond that time-frame, (4) increasing pulse-number augmented lesion area as well as the proportion of irreversible damage, and (5) electroporation sensitization was achieved by increasing extracellular Ca2+, indicating its crucial role in electroporation cytolysis, potentially via direct cellular toxicity and apoptosis facilitation (Figures 5–6). Finally, evaluating for HF-EP anti-arrhythmic properties, we targeted multiple rotors or focal triggered-activity generated in the hiPSC-CCSs. HF-EP application generated sustained line-blocks, isolating arrhythmogenic substrates within the hiPSC-CCSs while blocking the propagation of arrhythmic wavefronts (Figure 7). Conclusion Our results demonstrate the ability to study cardiac electroporation utilizing hiPSC-derived cardiomyocytes, provide novel insights into its temporal and electrophysiological characteristics, facilitate electroporation protocol optimization, screen for potential electroporation sensitizers, and to study its mechanistic nature and anti-arrhythmic properties. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Division of Cardiology, and Tamman Cardiovascular Research Institute, Leviev Heart Center, Sheba Medical Center - Tel Hashomer, Ramat-Gan, Israel Figures 1–4Figures 5–7

Peripheral impairments of oxidative metabolism after a 10-day bed rest are upstream of mitochondrial respiration.

In order to identify peripheral biomarkers of impaired oxidative metabolism during exercise following a 10‐day bed rest, 10 males performed an incremental exercise (to determine peak pulmonary V̇O2 (V̇O2 p)) and moderate‐intensity exercises, before (PRE) and after (POST) bed rest. Blood flow response was evaluated in the common femoral artery by Eco‐Doppler during 1 min of passive leg movements (PLM). The intramuscular matching between O2 delivery and O2 utilization was evaluated by near‐infrared spectroscopy (NIRS). Mitochondrial respiration was evaluated ex vivo by high‐resolution respirometry in isolated muscle fibres, and in vivo by NIRS by the evaluation of skeletal muscle V̇O2 (V̇O2 m) recovery kinetics. Resting V̇O2 m was estimated by NIRS. Peak V̇O2 p was lower in POST vs. PRE. The area under the blood flow vs. time curve during PLM was smaller (P = 0.03) in POST (274 ± 233 mL) vs. PRE (427 ± 291). An increased (P = 0.03) overshoot of muscle deoxygenation during a metabolic transition was identified in POST. Skeletal muscle citrate synthase activity was not different (P = 0.11) in POST (131 ± 16 nmol min–1 mg–1) vs. PRE (138 ± 19). Maximal ADP‐stimulated mitochondrial respiration (66 ± 18 pmol s–1 mg–1 (POST) vs. 72 ± 14 (PRE), P = 0.41) was not affected by bed rest. Apparent K m for ADP sensitivity of mitochondrial respiration was reduced in POST vs. PRE (P = 0.04). The V̇O2 m recovery time constant was not different (P = 0.79) in POST (22 ± 6 s) vs. PRE (22 ± 6). Resting V̇O2 m was reduced by 25% in POST vs. PRE (P = 0.006). Microvascular‐endothelial function was impaired following a 10‐day bed rest, whereas mitochondrial mass and function (both in vivo and ex vivo) were unaffected or slightly enhanced.Key points Ten days of horizontal bed rest impaired in vivo oxidative function during exercise.Microvascular impairments were identified by different methods.Mitochondrial mass and mitochondrial function (evaluated both in vivo and ex vivo) were unchanged or even improved (i.e. enhanced mitochondrial sensitivity to submaximal [ADP]).Resting muscle oxygen uptake was significantly lower following bed rest, suggesting that muscle catabolic processes induced by bed rest/inactivity are less energy‐consuming than anabolic ones.

Growth of Defect-Induced Carbon Nanotubes for Low-Temperature Fruit Monitoring Sensor

Herein, a carbon nanotubes-based sensor has been grown for the purpose of ethylene detection. The prepared CNTs had a crystalline structure with a smooth surface of 11.0 nm in diameter and 10.0 µm in length. The low-intensity graphite peak (G-band) as compared to the peak of the defect (D-band) characterizes the defects in the CNTs. An MWNTs-gas sensor was fabricated for monitoring the ethylene gas. The highest response was recorded at a low operating temperature of 30 °C. The sensor was also examined at 300 ppb up to 10 ppm and it showed a response of 2% up to 28%. The sensor response and recovery time constants were varied from 60 to 300 s, depending on the gas concentration. The results that were obtained for the synthetic ethylene gas were also compared with the real measurements for banana ripening. The results confirmed that the sensor is appropriate for the monitoring of fruit ripening.

Inhibition of voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells by the class Ic antiarrhythmic agent lorcainide

Voltage-dependent K+ (Kv) channels play the role of returning the membrane potential to the resting state, thereby maintaining the vascular tone. Here, we used native smooth-muscle cells from rabbit coronary arteries to investigate the inhibitory effect of lorcainide, a class Ic antiarrhythmic agent, on Kv channels. Lorcainide inhibited Kv channels in a concentration-dependent manner with an IC50 of 4.46 ± 0.15 μM and a Hill coefficient of 0.95 ± 0.01. Although application of lorcainide did not change the activation curve, it shifted the inactivation curve toward a more negative potential, implying that lorcainide inhibits Kv channels by changing the channels’ voltage sensors. The recovery time constant from channel inactivation increased in the presence of lorcainide. Furthermore, application of train steps (of 1 or 2 Hz) in the presence of lorcainide progressively augmented the inhibition of Kv currents, implying that lorcainide-induced inhibition of Kv channels is use (state)-dependent. Pretreatment with Kv1.5 or Kv2.1/2.2 inhibitors effectively reduced the amplitude of the Kv current but did not affect the inhibitory effect of lorcainide. Based on these results, we conclude that lorcainide inhibits vascular Kv channels in a concentration and use (state)-dependent manner by changing their inactivation gating properties. Considering the clinical efficacy of lorcainide, and the pathophysiological significance of vascular Kv channels, our findings should be considered when prescribing lorcainide to patients with arrhythmia and vascular disease.

Effects of Antiarrhythmic Drugs on hERG Gating in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes From a Patient With Short QT Syndrome Type 1.

Aims: The short QT syndrome type 1 (SQT1) is linked to hERG channel mutations (e.g., N588K). Drug effects on hERG channel gating kinetics in SQT1-cells have not been investigated. Methods: This study used hiPSC-CMs of a healthy donor and a SQT1-patient carrying the N588K mutation and patch clamp to examine the drug effects on hERG channel gating kinetics. Results: Ajmaline, amiodarone, ivabradine, flecainide, quinidine, mexiletine and ranolazine inhibited the hERG channel current (IKr) less strongly in hiPSC-CMs from the SQTS1-patient (SQT1-hiPSC-CMs) comparing with cells from the healthy donor (donor-hiPSC-CMs). Quinidine and mexiletine reduced, but ajmaline, amiodarone, ivabradine and ranolazine increased the time to peak of IKr similarly in SQT1-hiPSC-CMs and donor-hiPSC-CMs. Although regarding the shift of activation and inactivation curves, tested drugs showed differential effects in donor- and SQT1-hiPSC-CMs, quinidine, ajmaline, ivabradine and mexiletine but not amiodarone, flecainide and ranolazine reduced the window current in SQT1-hiPSC-CMs. Quinidine, ajmaline, ivabradine and mexiletine differentially changed the time constant of recovery from inactivation, but all of them increased the time constant of deactivation in SQT1-hiPSC-CMs. Conclusion: The window current-reducing and deactivation-slowing effects may be important for the antiarrhythmic effect of ajmaline, ivabradine, quinidine and mexiletine in SQT1-cells. This information may be helpful for selecting drugs for treating SQT1-patients with hERG channel mutation.

Effects of magnetic stimulation at different frequencies on neuronal excitability and voltage-gated potassium channels in vitro brain slices

As a noninvasive neuromodulation technique, transcranial magnetic stimulation (TMS) is widely used in the clinical treatment of neurological and psychiatric diseases, but the mechanism of its action is still unclear. The purpose of this paper is to investigate the effects of different frequencies of magnetic stimulation (MS) on neuronal excitability and voltage-gated potassium channels in the in vitro brain slices from the electrophysiological perspective of neurons. The experiment was divided into stimulus groups and control group, and acute isolated mice brain slices were applied to MS with the same intensity (0.3 T) at different frequencies (20 Hz and 0.5 Hz, 500 pulses) respectively in the stimulus groups. The whole-cell patch clamp technique was used to record the resting membrane potential (RMP), action potential (AP), voltage-gated potassium channels current of hippocampal dentate gyrus (DG) granule cells. The results showed that 20 Hz MS significantly increased the number of APs released and the maximum slope of a single AP, reduced the threshold of AP, half width and time to AP peak amplitude, and improved the excitability of hippocampal neurons. The peak currents of potassium channels were decreased, the inactivation curve of transient outward potassium channels shifted to the left significantly, and the time constant of recovery after inactivation increased significantly. 0.5 Hz MS significantly inhibited neuronal excitability and increased the peak currents of potassium channels, but the dynamic characteristics of potassium channels had little change. The results suggest that the dynamic characteristics of voltage-gated potassium channels and the excitability of hippocampal DG granule neurons may be one of the potential mechanisms of neuromodulation by MS.