Tetanic Electrical Stimulation Research Articles

Validation of a functional human AD model with four AD therapeutics utilizing patterned ipsc-derived cortical neurons integrated with microelectrode arrays

Preclinical methods are needed for screening potential Alzheimer’s disease (AD) therapeutics that recapitulate phenotypes found in the Mild Cognitive Impairment (MCI) stage or even before this stage of the disease. This would require a phenotypic system that reproduces cognitive deficits without significant neuronal cell death to mimic the clinical manifestations of AD during these stages. Long-term potentiation (LTP), which is a correlate of learning and memory, was induced in mature human iPSC-derived cortical neurons cultured on microelectrode arrays utilizing circuit patterns connecting two adjacent electrodes. We demonstrated an LTP system that modeled the MCI and pre-MCI stages of Alzheimer’s and validated this functional system utilizing four AD therapeutics, which was also verified utilizing patch-clamp electrophysiology. LTP was induced by tetanic electrical stimulation, and LTP maintenance was significantly reduced in the presence of Amyloid-Beta 42 (Aβ42) oligomers compared to the controls, however, co-treatment with AD therapeutics (Donepezil, Memantine, Rolipram and Saracatinib) corrected Aβ42-induced LTP impairment. The results illustrate the utility of the system as a validated platform to model MCI AD pathology, and potentially for the pre-MCI phase before significant neuronal death. This system also has the potential to become an ideal platform for high-content therapeutic screening for other neurodegenerative diseases.

Belt electrode tetanus muscle stimulation reduces denervation-induced atrophy of rat multiple skeletal muscle groups

Belt electrode-skeletal muscle electrical stimulation (B-SES) involves the use of belt-shaped electrodes to contract multiple muscle groups simultaneously. Twitch contractions have been demonstrated to protect against denervation-induced muscle atrophy in rats, possibly through mitochondrial biosynthesis. This study examined whether inducing tetanus contractions with B-SES suppresses muscle atrophy and identified the underlying molecular mechanisms. We evaluated the effects of acute (60 Hz, 5 min) and chronic (60 Hz, 5 min, every alternate day for one week) B-SES on the tibialis anterior (TA) and gastrocnemius (GAS) muscles in Sprague–Dawley rats using belt electrodes attached to both ankle joints. After acute stimulation, a significant decrease in the glycogen content was observed in the left and right TA and GAS, suggesting that B-SES causes simultaneous contractions in multiple muscle groups. B-SES enhanced p70S6K phosphorylation, an indicator of the mechanistic target of rapamycin complex 1 activity. During chronic stimulations, rats were divided into control (CONT), denervation-induced atrophy (DEN), and DEN + electrically stimulated with B-SES (DEN + ES) groups. After seven days of treatment, the wet weight (n = 8–11 for each group) and muscle fiber cross-sectional area (CSA, n = 6 for each group) of the TA and GAS muscles were reduced in the DEN and DEN + ES groups compared with that in the CON group. The DEN + ES group showed significantly higher muscle weight and CSA than those in the DEN group. Although RNA-seq and pathway analysis suggested that mitochondrial biogenesis is a critical event in this phenomenon, mitochondrial content showed no difference. In contrast, ribosomal RNA 28S and 18S (n = 6) levels in the DEN + ES group were higher than those in the DEN group, even though RNA-seq showed that the ribosome biogenesis pathway was reduced by electrical stimulation. The mRNA levels of the muscle proteolytic molecules atrogin-1 and MuRF1 were significantly higher in DEN than those in CONT. However, they were more suppressed in DEN + ES than those in DEN. In conclusion, tetanic electrical stimulation of both ankles using belt electrodes effectively reduced denervation-induced atrophy in multiple muscle groups. Furthermore, ribosomal biosynthesis plays a vital role in this phenomenon.

The value of pupillary diameter in evaluating pain perception after awakening in patients undergoing general anesthesia during orthopedic surgery

BackgroundThe pupillary response to tetanic electrical stimulation reflects the balance between nociceptive stimulation and analgesia. Although pupillary pain index (PPI) was utilized to predict postoperative pain, it depended on tetanic stimulation and was complex. We aim to describe the potential relationship between PD in the presence of surgical stimulation and pain levels after awakening.MethodsAccording to the Verbal Rating Scale (VRS) score after extubation, the patients were divided into painless group (VRS = 0) and pain group (VRS ≥ 1). Pupillary diameter (PD) and pupillary light reflex velocity (PLRV) were compared between two groups when patients entered the operating room (T1), before incision (T2), 10 s after incision (T3), 30 s after incision (T4), 1 h after incision (T5), at the end of surgery (T6), shortly after extubation (T7), and when patients expressed pain clearly (T8). The magnitude of PD change (ΔPD) compared to the baseline value after anesthesia induction (T2) was calculated. The correlations between pupillary parameters and pain after awakening were calculated.ResultsPatients with VRS ≥ 1 had greater PD than painless patients at T3-7 (P = 0.04, 0.04, 0.003, <0.001, <0.001), and it was positively correlated with VRS score after awakening at T4-7 (r = 0.188, 0.217, 0.684, 0.721). The ability of T6ΔPD to predict VRS ≥ 1 was strong [threshold: 20.53%, area under the curve (AUC): 0.93, 95% confidence interval (CI): 0.89–0.97 ].ConclusionOur study indicates that PD is a useful index to direct the individualized analgesics used during operation, to better avoid the occurrence of pain during the postoperative emergence period.Trial registrationThis study was registered with the Chinese Clinical Trial Registry (registration number: ChiCTR2000040908, registration date: 15/12/2020).

Structural and functional connectivity from the dorsomedial hypothalamus to the ventral medulla as a chronological amplifier of sympathetic outflow

Psychological stress activates the hypothalamus, augments the sympathetic nervous output, and elevates blood pressure via excitation of the ventral medullary cardiovascular regions. However, anatomical and functional connectivity from the hypothalamus to the ventral medullary cardiovascular regions has not been fully elucidated. We investigated this issue by tract-tracing and functional imaging in rats. Retrograde tracing revealed the rostral ventrolateral medulla was innervated by neurons in the ipsilateral dorsomedial hypothalamus (DMH). Anterograde tracing showed DMH neurons projected to the ventral medullary cardiovascular regions with axon terminals in contiguity with tyrosine hydroxylase-immunoreactive neurons. By voltage-sensitive dye imaging, dynamics of ventral medullary activation evoked by electrical stimulation of the DMH were analyzed in the diencephalon-lower brainstem-spinal cord preparation of rats. Although the activation of the ventral medulla induced by single pulse stimulation of the DMH was brief, tetanic stimulation caused activation of the DMH sustained into the post-stimulus phase, resulting in delayed recovery. We suggest that prolonged excitation of the DMH, which is triggered by tetanic electrical stimulation and could also be triggered by psychological stress in a real life, induces further prolonged excitation of the medullary cardiovascular networks, and could contribute to the pathological elevation of blood pressure. The connectivity from the DMH to the medullary cardiovascular networks serves as a chronological amplifier of stress-induced sympathetic excitation. This notion will be the anatomical and pathophysiological basis to understand the mechanisms of stress-induced sustained augmentation of sympathetic activity.

Age related changes in surface myoelectric signals

The initial values and the time course of muscle fiber conduction velocity and of surface myoelectric signal spectral variables were studied during voluntary or electrically elicited contractions of the tibialis anterior muscle of 15 healthy elderly human subjects. Age ranged from 65 to 84 years. Isometric voluntary contractions were performed at 20% MVC (Maximal Voluntary Contraction) and 80% MVC for 20 s. Tetanic electrical stimulation was then applied to the main muscle motor point for 20 s with surface electrodes. Two stimulation rates (20 Hz and 40 Hz) and two stimulation amplitudes were used to induce different degrees of fatigue. One stimulation amplitude was supramaximal, the second was adjusted to induce a response (M-wave) about 30% of the maximal. Results were compared with those reported in previous work on healthy adults (age range 18 to 43 years). The main findings of this work are: (a) when voluntary contraction level is increased from 20% MVC to 80% MVC conduction velocity and spectral variables increase; this increase is significantly smaller in elderly subjects, (b) during sustained contractions at 80% MVC the decrease of conduction velocity and spectral variables is significantly smaller in elderly subjects, (c) during contractions induced by supramaximal stimulation at 40 Hz the decrease of conduction velocity and spectral variables is not significantly different in the two age groups. It is concluded that points a and b reflect the age related decrease of number and size of fast twitch fibers indicated by histological data. Point c is discussed and possible explanations are suggested.

Effects of varying remifentanil concentrations on Analgesia Nociception Index® under propofol: an observational study.

Analgesia Nociception Index monitor provides a measurement of the nociception and anti-nociception balance based on heart-rate variability. The aim was to assess the ability of Analgesia Nociception Index (ANI) to detect standard noxious stimulation during anesthesia at different opioid concentrations in comparison to hemodynamic and Bispectral Index parameters. Sixteen patients undergoing general anesthesia with propofol and remifentanil. Standardized tetanic electrical stimulation was applied without any other concurrent stimuli, while different effect site concentrations of remifentanil were increased step-by-step (0.5, 1.5, 3.0, 5 and 7ng/ml). For each tetanic stimulus, values of the different variables 60s before and 120s following the stimulus were analyzed. ANI values decreased significantly 120s after the stimulus when compared to the mean ANI 60s before the stimulus (P < 0.01). At lower remifentanil concentrations (0.5ng/ml), all variables except electromyogram from the Bispectral Index significantly changed after the stimulus but in the higher concentrations (5.0 and 7.0ng/ml) there was not a significant change. The other variables did not change significantly after the noxious stimulation. ANI was the only variable that positively correlated with the different remifentanil concentrations (R = 0.959, P = 0.01). Our study showed that there was a significant decrease in Analgesia Nociception Index after a tetanic stimulation while hemodynamic and BIS parameters did not change, suggesting that the Analgesia Nociception Index may perform better than traditional hemodynamic parameters at reflecting noxious stimulation. Analgesia Nociception Index significantly changed at lower, but not at higher, remifentanil concentrations.

Effects of Neuromuscular Blockages on Entropy Monitoring During Sevoflurane Anesthesia.

BackgroundThere are no data available on the effects of different degrees of neuromuscular blockade on spectral entropy during sevoflurane anesthesia. This study aimed to observe the effects of different degrees of neuromuscular blockade on state and response entropy during sevoflurane anesthesia.Material/MethodsEighty-one female patients were randomized to 9 groups (n=9 per group) according to the concentration of sevoflurane and degree of neuromuscular blockade. Response and state entropy were monitored. The endpoints were: 1) impact of neuromuscular blockade on state entropy and response entropy, and the difference between response entropy and state entropy; and 2) the response of entropy after cutaneous tetanic electrical noxious stimulation to the ulnar nerve under different degrees of neuromuscular blockade and concentrations of sevoflurane.ResultsThese were no significant differences in response entropy or state entropy, or differences between response entropy and state entropy among the groups in the awake state (P>0.05). Without noxious stimulation, sevoflurane concentrations and neuromuscular blockade had no significant effects on response entropy or state entropy, or on the difference between response entropy and state entropy (all P>0.05), but sevoflurane concentrations showed a significant effect on state entropy (P<0.05). After noxious stimulation, sevoflurane concentrations and neuromuscular blockade had significant effects on response entropy and state entropy, and on the difference between response entropy and state entropy.ConclusionsResponse entropy and state entropy decreased with increasing sevoflurane concentration. Neuromuscular blockade did not affect entropy without noxious stimulation. With stimulation, muscle relaxants significantly reduced the changes in entropy, and there were significant effects of neuromuscular blockade and sevoflurane on entropy.

Standardised noxious stimulation-guided individual adjustment of remifentanil target-controlled infusion to prevent haemodynamic responses to laryngoscopy and surgical incision: A randomised controlled trial.

The surgical plethysmographic index (SPI) is one of the available indexes of the nociception-antinociception (NAN) balance. Individually adjusting the NAN balance to prevent somatic responses to noxious stimulation remains a challenge. To assess whether guiding remifentanil administration according to the SPI response to a calibrated noxious stimulus (NANCAL) can blunt the haemodynamic response to tracheal intubation and surgical incision. Randomised multicentre study. Two Belgian university hospitals from January 2014 to April 2015. After ethic review board approval and informed consent, 48 American Society of Anesthesiologists I or II adult patients scheduled for surgery under general anaesthesia were enrolled. Patients were randomly assigned to a SPI group, where remifentanil effect-site concentration was adjusted according to NANCAL, or a control group, where it was fixed at 4 ng ml. Propofol concentration was always adjusted to maintain the bispectral index close to 40. NANCAL consisted of a 100 Hz, 60 mA electrical tetanic stimulation during 30 s at the wrist before tracheal intubation and before surgical incision. The primary endpoint was the efficacy of the NANCAL-guided remifentanil administration to prevent the haemodynamic response to tracheal intubation and surgical incision. The secondary aim was to compare the ability of SPI, analgesia nociception index, pupil diameter and mean arterial pressure response to NANCAL to predict the haemodynamic response to tracheal intubation and surgical incision. Our SPI response to NANCAL-based correcting scheme for remifentanil administration was not superior to a fixed remifentanil concentration at blunting the haemodynamic response to tracheal intubation or surgical incision. Among all tested NAN balance indices, only mean arterial pressure had significant predictive ability with regard to the haemodynamic response to surgical incision. Further research is needed to define the best NANCAL stimulus and the best remifentanil correcting scheme to help individualised tailoring of antinociception for each specific subpopulation of surgical patients. Clinicaltrials.gov NCT: 02884310; https://clinicaltrials.gov/ct2/show/NCT02884310.

Statistical Mechanics of the Human Placenta: A Stationary State of a Near-Equilibrium System in a Linear Regime.

All near-equilibrium systems under linear regime evolve to stationary states in which there is constant entropy production rate. In an open chemical system that exchanges matter and energy with the exterior, we can identify both the energy and entropy flows associated with the exchange of matter and energy. This can be achieved by applying statistical mechanics (SM), which links the microscopic properties of a system to its bulk properties. In the case of contractile tissues such as human placenta, Huxley’s equations offer a phenomenological formalism for applying SM. SM was investigated in human placental stem villi (PSV) (n = 40). PSV were stimulated by means of KCl exposure (n = 20) and tetanic electrical stimulation (n = 20). This made it possible to determine statistical entropy (S), internal energy (E), affinity (A), thermodynamic force (A / T) (T: temperature), thermodynamic flow (v) and entropy production rate (A / T x v). We found that PSV operated near equilibrium, i.e., A ≺≺ 2500 J/mol and in a stationary linear regime, i.e., (A / T) varied linearly with v. As v was dramatically low, entropy production rate which quantified irreversibility of chemical processes appeared to be the lowest ever observed in any contractile system.

Enhancing a sedation score to include truly noxious stimulation: the Extended Observer's Assessment of Alertness and Sedation (EOAA/S)

Although the Modified Observer's Assessment of Alertness and Sedation (MOAA/S) is frequently used in sedation-related drug and device studies, a major shortcoming is that it does not differentiate between lighter and deeper levels of general anaesthesia because the only noxious stimulus of the MOAA/S is a trapezius squeeze. The primary aim of this investigation was to expand the MOAA/S score to include truly noxious stimulation, thereby extending the dynamic range of the assessment to include sedation states consistent with deeper levels of general anaesthesia. Twenty healthy volunteers received target controlled infusions of fentanyl (target=0.8 ng ml(-1)) and propofol (starting at 0.5 µg ml(-1) and gradually increasing to 5 µg ml(-1)). At each propofol concentration, a MOAA/S score was obtained before and after tetanic electrical stimulation. The tetanic electrical stimulation current was gradually increased until the subject responded or until 50 mA was delivered without a response. A pharmacodynamic model was constructed to characterize the concentration-effect relationship between propofol and the MOAA/S scores. All subjects required a significantly higher propofol concentration to produce unresponsiveness to tetanic electrical stimulation at 50 mA compared with a standardized trapezius squeeze. The pharmacodynamic model adequately characterized the concentration-effect relationship. The Extended Observer's Assessment of Alertness and Sedation (or EOAA/S) extends the range of the widely used MOAA/S score to include truly noxious stimulation, thereby enabling the identification of drug-induced central nervous system depression representative of surgical anaesthesia.

Depression of corticomotor excitability after muscle fatigue induced by electrical stimulation and voluntary contraction.

In this study, we examined the effect of muscle fatigue induced by tetanic electrical stimulation (ES) and submaximal isometric contraction on corticomotor excitability. Experiments were performed in a cross-over design. Motor-evoked potentials (MEPs) were elicited by transcranial magnetic stimulation (TMS). Corticomotor excitability was recorded before and after thumb opposition muscle fatigue tasks, in which 10% of the maximal tension intensity was induced by tetanic ES or voluntary contraction (VC). The participants were 10 healthy individuals who performed each task for 10 min. Surface electrodes placed over the abductor pollicis brevis (APB) muscle recorded MEPs. F- and M-waves were elicited from APB by supramaximal ES of the median nerve. After the tetanic ES- and VC tasks, MEP amplitudes were significantly lower than before the task. However, F- and M-wave amplitudes remained unchanged. These findings suggest that corticospinal excitability is reduced by muscle fatigue as a result of intracortical inhibitory mechanisms. Our results also suggest that corticomotor excitability is reduced by muscle fatigue caused by both VC and tetanic ES.

Influence of joint angular velocity on electrically evoked concentric force potentiation induced by stretch-shortening cycle in young adults.

BackgroundDuring a stretch- shortening cycle (SSC), muscle force attained during concentric contractions (shortening phase) is potentiated by the preceding eccentric contractions (lengthening phase). The purpose of this study was to examine the influence of joint angular velocity on force potentiation induced by SSC (SSC effect).FindingsTwelve healthy men (age, 24.2 ± 3.2 years; height, 1.73 ± 0.05 m; body mass, 68.1 ± 11.0 kg) participated in this study. Ankle joint angle was passively moved by a dynamometer, with range of motion from dorsiflexion (DF) 15° to plantarflexion (PF) 15°. Muscle contractions were evoked by tetanic electrical stimulation. Joint angular velocity of concentric contraction was set at 30°/s and 150°/s. Magnitude of SSC effect was calculated as the ratio of joint torque obtained by concentric contraction with preliminary eccentric contraction trial relative to that obtained by concentric contraction without preliminary eccentric contraction trial. As a result, magnitude of SSC effect calculated at three joint angles was significantly larger in the 150°/s condition than in the 30°/s condition (p < 0.05).ConclusionsThese results indicate that the magnitude of SSC effect is affected by joint angular velocity, which is larger when joint angular velocity is larger. This phenomenon would be caused by insufficient duration to increase activation level in the large joint angular velocity condition. When the duration to increase activation level is insufficient due to short contraction duration, preactivation (one of the factors of SSC effect) leads to a significant increase in joint torque.

Peripheral NMDA Receptors Mediate Antidromic Nerve Stimulation-Induced Tactile Hypersensitivity in the Rat.

We investigated the role of peripheral NMDA receptors (NMDARs) in antidromic nerve stimulation-induced tactile hypersensitivity outside the skin area innervated by stimulated nerve. Tetanic electrical stimulation (ES) of the decentralized L5 spinal nerve, which induced enlargement of plasma extravasation, resulted in tactile hypersensitivity in the L4 plantar dermatome of the hind-paw. When intraplantar (i.pl.) injection was administered into the L4 dermatome before ES, NMDAR and group-I metabotropic Glu receptor (mGluR) antagonists and group-II mGluR agonist but not AMPA/kainate receptor antagonist prevented ES-induced hypersensitivity. I.pl. injection of PKA or PKC inhibitors also prevented ES-induced hypersensitivity. When the same injections were administered after establishment of ES-induced hypersensitivity, hypersensitivity was partially reduced by NMDAR antagonist only. In naïve animals, i.pl. Glu injection into the L4 dermatome induced tactile hypersensitivity, which was blocked by NMDAR antagonist and PKA and PKC inhibitors. These results suggest that the peripheral release of Glu, induced by antidromic nerve stimulation, leads to the expansion of tactile hypersensitive skin probably via nociceptor sensitization spread due to the diffusion of Glu into the skin near the release site. In addition, intracellular PKA- and PKC-dependent mechanisms mediated mainly by NMDAR activation are involved in Glu-induced nociceptor sensitization and subsequent hypersensitivity.

Ultraslow myosin molecular motors of placental contractile stem villi in humans.

Human placental stem villi (PSV) present contractile properties. In vitro mechanics were investigated in 40 human PSV. Contraction of PSV was induced by both KCl exposure (n = 20) and electrical tetanic stimulation (n = 20). Isotonic contractions were registered at several load levels ranging from zero-load up to isometric load. The tension-velocity relationship was found to be hyperbolic. This made it possible to apply the A. Huxley formalism for determining the rate constants for myosin cross-bridge (CB) attachment and detachment, CB single force, catalytic constant, myosin content, and maximum myosin ATPase activity. These molecular characteristics of myosin CBs did not differ under either KCl exposure or tetanus. A comparative approach was established from studies previously published in the literature and driven by mean of a similar method. As compared to that described in mammalian striated muscles, we showed that in human PSV, myosin CB rate constants for attachment and detachment were about 103 times lower whereas myosin ATPase activity was 105 times lower. Up to now, CB kinetics of contractile cells arranged along the long axis of the placental sheath appeared to be the slowest ever observed in any mammalian contractile tissue.

Rapid recovery of serratus anterior muscle function after microneurolysis of long thoracic nerve injury

BackgroundInjury to the long thoracic nerve is a common cause of winging scapula. When the serratus anterior muscle is unable to function, patients often lose the ability to raise their arm overhead on the affected side.MethodsSerratus anterior function was restored through decompression, neurolysis, and tetanic electrical stimulation of the long thoracic nerve. This included partial release of constricting middle scalene fibers and microneurolysis of epineurium and perineurium of the long thoracic nerve under magnification. Abduction angle was measured on the day before and the day following surgery.ResultsIn this retrospective study of 13 neurolysis procedures of the long thoracic nerve, abduction is improved by 10% or greater within one day of surgery. The average improvement was 59° (p < 0.00005). Patients had been suffering from winging scapula for 2 months to 12 years. The improvement in abduction is maintained at last follow-up, and winging is also reduced.ConclusionIn a notable number of cases, decompression and neurolysis of the long thoracic nerve leads to rapid improvements in winging scapula and the associated limitations on shoulder movement. The duration of the injury and the speed of improvement lead us to conclude that axonal channel defects can potentially exist that do not lead to Wallerian degeneration and yet cause a clear decrease in function.

ATP signaling in skeletal muscle: from fiber plasticity to regulation of metabolism.

Tetanic electrical stimulation releases adenosine triphosphate (ATP) from muscle fibers through pannexin-1 channels in a frequency-dependent manner; extracellular ATP activates signals that ultimately regulate gene expression and is able to increase glucose transport through activation of P2Y receptors, phosphatidylinositol 3-kinase, Akt, and AS160. We hypothesize that this mechanism is an important link between exercise and the regulation of muscle fiber plasticity and metabolism.

Role of both actin–myosin cross bridges and NO-cGMP pathway modulators in the contraction and relaxation of human placental stem villi

IntroductionHuman placental stem villi (PSV) present contractile properties. We studied the role of actin–myosin cross bridges (CBs) and the effects of NO-cGMP pathway modulators in the PSV contraction and relaxation. MethodsIn vitro contractile properties were investigated in 71 PSV from term human placentas studied according to their long axis. Contraction was induced by both KCl and electrical tetanic stimulation. Relaxation was induced by inhibiting the CB cycle with either 2,3-butanedione monoxime (BDM) or blebbistatin (BLE) and by activating the NO-cGMP pathway with isosorbide dinitrate (ISDN), sildenafil (SIL) or ISDN + SIL. ResultsPSV tension slowly increased by 140% of the basal tone after KCl exposure and by 85% after tetanus. The addition of BDM, BLE, ISDN, SIL and ISDN + SIL induced a relaxation of PSV, the overall time course of relaxation (in s) was respectively (means ± SD) 3412 ± 1904, 14,250 ± 3095*, 3813 ± 1383, 2883 ± 1188 and 2440 ± 477; significantly longer in BLE compared with BDM, ISDN, SIL and ISDN + SIL:*p < 0.001). the overall time course of relaxation (in s) was respectively (means ± SD) 3412 ± 1904, 14,250 ± 3095*, 3813 ± 1383, 2883 ± 1188 and 2440 ± 477; significantly longer in BLE compared with BDM, ISDN, SIL and ISDN + SIL:*p < 0.001). These relaxation kinetics were particularly slow. Other relaxation parametres, i.e., maximum lengthening, -peak dT/dt, and resting tension, did not differ between these 5 subgroups. Discussion and conclusionIsolated human PSV were able to contract after both KCl exposure and tetanus. This increase in contractility was reversed by inhibiting the CB cycle with BDM or BLE and by stimulating the NO-cGMP pathway with ISDN or SIL. The association ISDN + SIL did not potentiate the relaxing processes

ATP Sensitivity and IP 3-Dependent Calcium Transients Which Regulate Gene Expression in Adult Muscle Fibers are Altered in Mdx Mice

ATP has been shown to be released from muscles during exercise and after tetanic electrical stimulation (ES) and induces IP3 dependent slow calcium transients involved in gene expression in myotubes. The aim of this study was to characterize these signals in adult fibers and to establish differences between normal and mdx mouse. Experiments were performed in cultured isolated muscle fibers obtained from normal and mdx adult mice. The release of ATP induced by ES was measured using luciferin-luciferase and for calcium release studies the fibers were loaded with fluo-3. The location and expression of the nucleotide receptors were determined by immunofluorescence and western blot. Slow, post tetanic calcium signals were partly inhibited by pharmacological agents targeting pannexin-1 (a pathway for ATP release), purinergic receptors and extracellular ATP. These signals had slower decay constants in mdx fibers. ES induced significant ATP release in normal fibers but this release was not detected in mdx fibers. The cellular location of ATP receptors was similar in both fiber types but their level of expression was higher in mdx fibers. Pannexin-1 expression was significantly augmented in mdx fibers. External ATP induced Ca2+ signals in both, normal and mdx muscle fibers, but the sensitivity to ATP was much higher in mdx fibers. This study demonstrates that ATP signaling is profoundly altered in mdx fibers and could be implicated in the calcium disturbance described in DMD; a compensatory mechanism may be involved in the reduced ATP extrusion apparent in dystrophic cells. FONDAP 15010006, AFM 14562, FONDECYT 1080120, CONICYT PhD fellowship (DV)

Ip3-Dependent Calcium Transients Differentially Modulate Nf-Kappa B Activity in Wild Type and Mdx Myotubes

In normal skeletal myotubes, tetanic electrical stimulation activates NFκB through intracellular calcium increase released by inositol 1,4,5 triphosphate receptor (IP3R) and by Ryanodine receptors (RyR). In this work, we studied the differences in calcium transients evoked by electrical stimuli and NFκB activation in wild type (C57) and in mdx (animal model of Duchenne muscular dystrophy, DMD) myotubes. Myoblasts were obtained from C57 or mdx neonatal mice (1-3 days). Myotubes were loaded with fluo-3AM and stimulated with 250 pulses of 0.5 ms duration at 20 Hz and the calcium signal was assessed by fluorescence microscopy. NFκB activation was determined by luciferase reporter assay, p65 immunofluorescence and p65-GFP translocation. Fast calcium signal (RyR dependent) was significantly reduced in electrically stimulated mdx compared to wild type C57 myotubes (0.62 vs 1.41 RLU, p<0.01). However, the amplitude and kinetics of (IP3 dependent) slow calcium signals were similar in mdx and C57 myotubes. Electrical stimulation increased the NFκB luciferase activity in normal but not in mdx myotubes. By immunofluorescence, p65-GFP translocation studies and reporter assays we determined that the basal activity of this transcription factor is up-regulated in mdx myotubes and this could be blocked by inhibitors of IP3-related pathways (100 µM suramine, 10 µM BAPTA-AM, 5 µM xestospongin B) but not by ryanodine (30 µM). We propose that NFκB up regulation in mdx myotubes is modulated by intracellular calcium release through IP3R. The differences in NFκB activation between normal and dystrophic muscle cells may help to understand the mechanisms of pro-inflamatory cytokines gene expression in Duchenne muscular dystrophy. FONDAP 15010006, AFM14562. CONICYT graduate student fellowship and doctoral support AT-24100066.

Cav1.1 Acts as a Voltage Sensor for Two Separate Processes in Skeletal Muscle with Different Voltage Dependence

In adult muscle fibers, tetanic electrical stimulation induces two separate calcium signals, a fast one related to contraction and dependent on Cav1.1 (DHPR) and RyRs and a slow signal, dependent on DHPR and IP3Rs. We have recently shown that the amplitude of this slow signal is dependent on frequency of stimulation, having a maximum at 20 Hz. Importantly, this signal participates in the activation of genes related to slow muscle fiber type phenotype. This signal is inhibited by nifedipine, supporting a role for the DHPR in its onset. We have stimulated adult muscle fibers with different concentrations of extracellular K+, and found calcium signals at more negative membrane potential than those described for triggering calcium release associated with RyR. Moreover, in the same conditions, we observe an increase in mRNA of the slow isoform of TroponinI, as seen in fibers stimulated with a 20Hz train of pulses. These signals have an intracellular origin, because they are still present when working in solutions with no extracellular calcium and 0.1 mM EGTA. Interestingly, they are inhibited by incubation of fibers with 20 uM XestosponginB, a specific inhibitor of IP3R. These results strongly suggest that the slow signal fires at membrane potential values more negative (Nernst potential estimated −50 mV) than the fast signal (E-C coupling related), where the fast signal threshold (−30 to −20 mV) has not yet been reached. This opens the possibility for a different part of the DHPR molecule being involved in the signal transmission of depolarization to IP3-production machinery, independently of the process of E-C coupling.FONDECYT 1080120, FONDAP 15010006.