Increasing Stimulation Frequency Research Articles

Perceptibility and Pain Thresholds in Low- and High-Frequency Alternating Current Stimulation: Implications for tACS and tTIS

AbstractTranscranial electrical stimulation (tES) has emerged as a promising tool for neuromodulation, but its application is often limited by the discomfort associated with higher stimulation intensities. Newer variants like transcranial temporal interference stimulation (tTIS) utilize high-frequency alternating currents (≥ 500 Hz) to penetrate deeper brain regions while mitigating perceptual discomfort. This study sought to examine sensation and pain thresholds across various stimulation frequencies of alternating currents, aiming to explore the boundaries of comfortable intensities. Additionally, we sought to evaluate the efficacy of an anesthetizing topical cream in increasing participant comfort and potentially extending the range of tolerable stimulation levels. We recruited 37 participants and applied alternating current stimulation to the head at various frequencies (10 Hz, 20 Hz, 500 Hz, 1000 Hz, and 2000 Hz) to determine intensity-dependent perception and pain thresholds. Additionally, thresholds were determined under the influence of a topical anesthetic. Our findings confirm that as stimulation frequency increases, perceptibility decreases, with higher frequencies allowing a manyfold increase in stimulation intensity before becoming perceptible or causing pain. Additionally, the anesthetizing cream was efficacious in further reducing perceptibility and pain sensations across all frequencies. This study lays the groundwork for future research by establishing comfortable limits for stimulation intensities, particularly in the context of high-frequency stimulation. The reduced perceptibility of high-frequency stimulation, coupled with the effectiveness of anesthetizing creams, enables the administration of higher stimulation intensities for more potent neuromodulatory interventions without causing discomfort.

Biomechanical response of ultrathin slices of hypertrophic cardiomyopathy tissue to myosin modulator mavacamten

Hypertrophic cardiomyopathy (HCM) is the most common inherited myocardial disorder of the heart, but effective treatment options remain limited. Mavacamten, a direct myosin modulator, has been presented as novel pharmacological therapy for HCM. The aim of this study was to analyze the biomechanical response of HCM tissue to Mavacamten using living myocardial slices (LMS). LMS (n = 58) from patients with HCM (n = 10) were cultured under electromechanical stimulation, and Verapamil and Mavacamten were administered on consecutive days to evaluate their effects on cardiac biomechanics. Mavacamten and Verapamil reduced contractile force and dF/dt and increased time-to-relaxation in a similar manner. Yet, the time-to-peak of the cardiac contraction was prolonged after administration of Mavacamten (221.0 ms (208.8 – 236.3) vs. 237.7 (221.0 – 254.7), p = 0.004). In addition, Mavacamten prolonged the functional refractory period (FRP) (330 ms (304 − 351) vs. 355 ms (313 − 370), p = 0.023) and better preserved twitch force with increasing stimulation frequencies, compared to Verapamil. As such, Mavacamten reduced (hyper-)contractility and prolonged contraction duration of HCM LMS, suggesting a reduction in cardiac wall stress. Also, Mavacamten might protect against the development of ventricular tachyarrhythmias due to prolongation of the FRP, and improve toleration of tachycardia due to better preservation of twitch force at tachycardiac stimulation frequencies.

Disease severity, arrhythmogenesis, and fibrosis are related to longer action potentials in tetralogy of Fallot.

Arrhythmias may originate from surgically unaffected right ventricular (RV) regions in patients with tetralogy of Fallot (TOF). We aimed to investigate action potential (AP) remodelling and arrhythmia susceptibility in RV myocardium of patientswith repaired and with unrepaired TOF, identify possible correlations with clinical phenotype and myocardial fibrosis, and compare findings with data from patients with atrial septal defect (ASD), a less severe congenital heart disease. Intracellular AP were recorded ex vivo in RV outflow tract samples from 22 TOF and three ASD patients. Arrhythmias were provoked by superfusion with solutions containing reduced potassium and barium chloride, or isoprenaline. Myocardial fibrosis was quantified histologically and associations between clinical phenotype, AP shape, tissue arrhythmia propensity, and fibrosis were examined. Electrophysiological abnormalities (arrhythmias, AP duration [APD] alternans, impaired APD shortening at increased stimulation frequencies) were generally present in TOF tissue, even from infants, but rare or absent in ASD samples. More severely diseased and acyanotic patients, pronounced tissue susceptibility to arrhythmogenesis, and greater fibrosis extent were associated with longer APD. In contrast, APD was shorter in tissue from patients with pre-operative cyanosis. Increased fibrosis and repaired-TOF status were linked to tissue arrhythmia inducibility. Functional and structural tissue remodelling may explain arrhythmic activity in TOF patients, even at a very young age. Surprisingly, clinical acyanosis appears to be associated with more severe arrhythmogenic remodelling. Further research into the clinical drivers of structural and electrical myocardial alterations, and the relation between them, is needed to identify predictive factors for patients at risk.

Multifrequency Analysis of Masseter Vestibular Evoked Myogenic Potentials in Young Adults.

The purpose of this study was to explore the dynamics of multifrequency tone bursts on the masseter vestibular evoked myogenic potential (mVEMP) parameters. Furthermore, it sought to determine the optimal frequency tuning of mVEMP responses. Twenty young adults with normal hearing sensitivity participated in the study. Bilateral tone burst evoked mVEMPs were obtained using the zygomatic montage at 250-, 500-, 750-, 1000-, 1500-, and 2000-Hz stimulation frequencies. Self-monitoring biofeedback was given during the procedure to confirm the tension of the masseter muscle between 49.9 and 150.6 rms. Furthermore, the electromyography (EMG) scaling was done to avoid any muscle-related irregularities. Tone burst evoked mVEMPs were found to be 100% present at the stimulation frequencies of 250, 500, 750, and 1000 Hz. There were no ear and gender effects seen for any of the frequencies. Significant shortening of the P1 and N1 latencies with increasing stimulation frequencies was observed. The peak-to-peak amplitude was the highest at 500 Hz and lowest at 2000 Hz tone bursts under both EMG scaled and unscaled conditions. The present revealed higher response rates and larger amplitudes study of mVEMP at lower frequencies, and, therefore, the frequency tuning was seen for the stimulation frequency at 500 Hz.

Abstract P3134: Impaired Calcium Homeostasis Underlies Lamin A/C Mutation R331Q Associated Atrial Fibrillation

Background: Atrial fibrillation (AF) is one of the first cardiac manifestations in patients harboring mutations in nucleoskeletal protein lamin A/C (LMNA). However, the underlying arrhythmogenic mechanisms are unclear. Objective: We hypothesize that impaired intracellular Ca 2+ -homeostasis contribute to LMNA mutation-associated atrial arrhythmogenesis. Methods: LMNA mutation R331Q was introduced in a healthy control induced pluripotent stem cell (iPSC) line using CRISPR/Cas9 and cells were differentiated into atrial iPSC-derived cardiomyocytes (iPSC-CMs). L-type Ca 2+ current and intracellular Ca 2+ concentration were measured using patch-clamp technique and epifluorescence microscopy (Fluo-3), respectively. Results: R331Q iPSC-CMs demonstrated increased L-type Ca 2+ current density followed by increased amplitude of systolic Ca 2+ transient (CaT) compared to isogenic control (Fig. A). Sarcoplasmic reticulum (SR) Ca 2+ content was evaluated from caffeine-induced CaT amplitude and integrated Na + -Ca 2+ exchange current, and found to be greater in R331Q iPSC-CMs (Fig. B). Confocal line scan analysis revealed an increased Ca 2+ spark frequency in R331Q iPSC-CMs (Fig. C). In addition, by applying a stepwise increase in stimulation frequency (1-5 Hz), R331Q iPSC-CMs developed Ca 2+ alternans at lower threshold frequency (Fig. D). Conclusions: LMNA mutation R331Q is associated with disturbed intracellular Ca 2+ cycling, promoting occurrence of diastolic Ca 2+ sparks and arrhythmogenic Ca 2+ alternans. Collectively, our findings suggest that impaired Ca 2+ homeostasis may contribute to atrial arrhythmogenesis in patients harboring LMNA mutations.

Species-Specific Adaptation for Ongoing High-Frequency Action Potential Generation in MNTB Neurons.

Comparative analysis of evolutionarily conserved neuronal circuits between phylogenetically distant mammals highlights the relevant mechanisms and specific adaptations to information processing. The medial nucleus of the trapezoid body (MNTB) is a conserved mammalian auditory brainstem nucleus relevant for temporal processing. While MNTB neurons have been extensively investigated, a comparative analysis of phylogenetically distant mammals and the spike generation is missing. To understand the suprathreshold precision and firing rate, we examined the membrane, voltage-gated ion channel and synaptic properties in Phyllostomus discolor (bat) and in Meriones unguiculatus (rodent) of either sex. Between the two species, the membrane properties of MNTB neurons were similar at rest with only minor differences, while larger dendrotoxin (DTX)-sensitive potassium currents were found in gerbils. Calyx of Held-mediated EPSCs were smaller and frequency dependence of short-term plasticity (STP) less pronounced in bats. Simulating synaptic train stimulations in dynamic clamp revealed that MNTB neurons fired with decreasing success rate near conductance threshold and at increasing stimulation frequency. Driven by STP-dependent conductance decrease, the latency of evoked action potentials increased during train stimulations. The spike generator showed a temporal adaptation at the beginning of train stimulations that can be explained by sodium current inactivation. Compared with gerbils, the spike generator of bats sustained higher frequency input-output functions and upheld the same temporal precision. Our data mechanistically support that MNTB input-output functions in bats are suited to sustain precise high-frequency rates, while for gerbils, temporal precision appears more relevant and an adaptation to high output-rates can be spared.SIGNIFICANCE STATEMENT Neurons in the mammalian medial nucleus of the trapezoid body (MNTB) convey precise, faithful inhibition vital for binaural hearing and gap detection. The MNTB's structure and function appear evolutionarily well conserved. We compared the cellular physiology of MNTB neurons in bat and gerbil. Because of their adaptations to echolocation or low frequency hearing both species are model systems for hearing research, yet with largely overlapping hearing ranges. We find that bat neurons sustain information transfer with higher ongoing rates and precision based on synaptic and biophysical differences in comparison to gerbils. Thus, even in evolutionarily conserved circuits species-specific adaptations prevail, highlighting the importance for comparative research to differentiate general circuit functions and their specific adaptations.

Neuromodulation with transcutaneous spinal stimulation reveals different groups of motor profiles during robot-guided stepping in humans with incomplete spinal cord injury.

Neuromodulation via spinal stimulation has been investigated for improving motor function and reducing spasticity after spinal cord injury (SCI) in humans. Despite the reported heterogeneity of outcomes, few investigations have attempted to discern commonalities among individual responses to neuromodulation, especially the impact of stimulation frequencies. Here, we examined how exposure to continuous lumbosacral transcutaneous spinal stimulation (TSS) across a range of frequencies affects robotic torques and EMG patterns during stepping in a robotic gait orthosis on a motorized treadmill. We studied nine chronic motor-incomplete SCI individuals (8/1 AIS-C/D, 8 men) during robot-guided stepping with body-weight support without and with TSS applied at random frequencies between 1 and up to 100Hz at a constant, individually selected stimulation intensity below the common motor threshold for posterior root reflexes. The hip and knee robotic torques needed to maintain the predefined stepping trajectory and EMG in eight bilateral leg muscles were recorded. We calculated the standardized mean difference between the stimulation conditions grouped into frequency bins and the no stimulation condition to determine changes in the normalized torques and the average EMG envelopes. We found heterogeneous changes in robotic torques across individuals. Agglomerative clustering of robotic torques identified four groups wherein the patterns of changes differed in magnitude and direction depending mainly on the stimulation frequency and stance/swing phase. On one end of the spectrum, the changes in robotic torques were greater with increasing stimulation frequencies (four participants), which coincided with a decrease in EMG, mainly due to the reduction of clonogenic motor output in the lower leg muscles. On the other end, we found an inverted u-shape change in torque over the mid-frequency range along with an increase in EMG, reflecting the augmentation of gait-related physiological (two participants) or pathophysiological (one participant) output. We conclude that TSS during robot-guided stepping reveals different frequency-dependent motor profiles among individuals with chronic motor incomplete SCI. This suggests the need for a better understanding and characterization of motor control profiles in SCI when applying TSS as a therapeutic intervention for improving gait.

Green-in-green biohybrids as transient biotriboelectric nanogenerators

Green self-powered devices based on biodegradable materials have attracted widespread attention. Here, we propose the construction of the transient biotriboelectric nanogenerator (TENG) using green-in-green bionanocompoites. The green-in-green nanocomposites, cellulose nanocrystal (CNC)/polyhydroxybutyrate (PHB), are prepared with a high-pressure molding method. The CNC promotes the degradation and enhances the dielectric constant of CNC/PHB. It further allows for the significant improvement of the triboelectric output of CNC/PHB-based TENG. The voltage output and current output of CNC/PHB-based TENG are 5.7 and 12.5 times higher than those of pristine PHB-based TENG, respectively. Also, the bio-TENG exhibits admirable signal stability in over 20000 cycles. Despite the high hardness of CNC/PHB, a soft but simple-structured arch sensor is successfully assembled using CNC/PHB-based TENG. It can attain the precise real-time monitoring of various human motions. This study may provide new insights into the design/fabrication of green functional materials, and initiate the next wave of innovations in eco-friendly self-powered devices.

The Role of Ca2+ Sparks in Force Frequency Relationships in Guinea Pig Ventricular Myocytes.

Calcium sparks are the elementary Ca2+ release events in excitation-contraction coupling that underlie the Ca2+ transient. The frequency-dependent contractile force generated by cardiac myocytes depends upon the characteristics of the Ca2+ transients. A stochastic computational local control model of a guinea pig ventricular cardiomyocyte was developed, to gain insight into mechanisms of force-frequency relationship (FFR). This required the creation of a new three-state RyR2 model that reproduced the adaptive behavior of RyR2, in which the RyR2 channels transition into a different state when exposed to prolonged elevated subspace [Ca2+]. The model simulations agree with previous experimental and modeling studies on interval-force relations. Unlike previous common pool models, this local control model displayed stable action potential trains at 7 Hz. The duration and the amplitude of the [Ca2+]myo transients increase in pacing rates consistent with the experiments. The [Ca2+]myo transient reaches its peak value at 4 Hz and decreases afterward, consistent with experimental force-frequency curves. The model predicts, in agreement with previous modeling studies of Jafri and co-workers, diastolic sarcoplasmic reticulum, [Ca2+]sr, and RyR2 adaptation increase with the increased stimulation frequency, producing rising, rather than falling, amplitude of the myoplasmic [Ca2+] transients. However, the local control model also suggests that the reduction of the L-type Ca2+ current, with an increase in pacing frequency due to Ca2+-dependent inactivation, also plays a role in the negative slope of the FFR. In the simulations, the peak Ca2+ transient in the FFR correlated with the highest numbers of SR Ca2+ sparks: the larger average amplitudes of those sparks, and the longer duration of the Ca2+ sparks.

Regeneration of Functional Bladder Using Cell-seeded Amnion and P(LA/CL) Scaffolds.

Long-term bladder regeneration has not been successful instead of augmentation with gastrointestinal segments, as is commonly performed for bladder reconstruction. To evaluate whether or not cell-seeded bioabsorbable materials regenerate half-resected bladder in a rabbit model. Female Japanese white rabbits were divided two groups: cell-seeded material (CSM) group and Control (n = 6 each). Control rabbits underwent resection of half the bladder. CSM rabbits were sutured with cell-seeded amniotic membrane and P(LA/CL) material after bladder resection. After 6, 12, and 18 months, rabbits underwent X-ray and cystometry, and bladder tissues after 18 months were subjected to functional and histological analyses. X-ray confirmed the peristaltic movements of the reconstructed bladders in the CSM group. On cystometry, the mean maximum bladder volume, maximum bladder pressure, and 25 mL bladder volume compliance in the CSM group were significantly greater than in the Control group at 6, 12, and 18 months. In addition, organ bath studies showed good contraction under electrical stimulation with increasing stimulation frequency in the CSM group, while, the Control group showed weak contraction on both tests in the central marginal zone. Furthermore, the rates of neovascularization, urothelial and smooth muscle formation, and neurofilamentation in the CSM group were significantly greater than in the Control group. Oral mucosal cell-seeded amniotic membrane and stomach smooth muscle cell-seeded P(LA/CL) scaffold with omentum after abdominal implantation regenerated functional bladder with satisfactory epithelium and smooth muscle without scarring more than 1 year. Impact Statement Regeneration of functional bladder without using gastrointestinal segments has been a huge challenge to urological reconstruction. Various materials, such as nonbioabsorbable materials and biomaterials have been attempted to reconstruct bladder in animal models. However, the long-term results more than a year failed due to the low biocompatibility, high risks, and difficulty creating the materials. In this study, we revealed long-term bladder regeneration using cell-seeded amniotic membrane and P(LA/CL) material in a rabbit model. The new method of bladder reconstruction seems able to regenerate functional bladder with satisfactory bladder epithelium and bladder smooth muscle function without scarring for more than 1 year successfully.

Functional remodelling of perinuclear mitochondria alters nucleoplasmic Ca2+ signalling in heart failure.

Mitochondrial dysfunction in cardiomyocytes is a hallmark of heart failure development. Although initial studies recognized the importance of different mitochondrial subpopulations, there is a striking lack of direct comparison of intrafibrillar (IF) versus perinuclear (PN) mitochondria during the development of HF. Here, we use multiple approaches to examine the morphology and functional properties of IF versus PN mitochondria in pressure overload-induced cardiac remodelling in mice, and in non-failing and failing human cardiomyocytes. We demonstrate that PN mitochondria from failing cardiomyocytes are more susceptible to depolarization of mitochondrial membrane potential, reactive oxygen species generation and impairment in Ca2+ uptake compared with IF mitochondria at baseline and under physiological stress protocol. We also demonstrate, for the first time to our knowledge, that under normal conditions PN mitochondrial Ca2+ uptake shapes nucleoplasmic Ca2+ transients (CaTs) and limits nucleoplasmic Ca2+ loading. The loss of PN mitochondrial Ca2+ buffering capacity translates into increased nucleoplasmic CaTs and may explain disproportionate rise in nucleoplasmic [Ca2+] in failing cardiomyocytes at increased stimulation frequencies. Therefore, a previously unidentified benefit of restoring the mitochondrial Ca2+ uptake may be normalization of nuclear Ca2+ signalling and alleviation of altered excitation–transcription, which could be an important therapeutic approach to prevent adverse cardiac remodelling.This article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’.

Pain-Related Vertex Evoked Potentials. Comparison of Surface Electrical to Heat Stimulation.

Demonstration of nociceptive fiber abnormality is important for diagnosing neuropathic pain and small fiber neuropathies. This is usually assessed by brief heat pulses using lasers, contact heat, or special electrodes. We hypothesized that pain-related evoked potentials to conventional surface electrical stimulation (PREPse) can index Aδ afferences despite tactile Aß fibers coactivation. PREPse may be more readily used clinically than contact heat evoked potentials (CHEPS). Twenty-eight healthy subjects. Vertex (Cz-A1/A2) recordings. Electrical stimulation of middle finger and second toe with conventional ring, and forearm/leg skin with cup, electrodes. Contact heat stimulation to forearm and leg. Compression ischemic nerve blockade. PREPse peripheral velocities were within the midrange of Aδ fibers. N1-P1 amplitude increased with pain numerical rating scale graded (0-10) electrical stimulation (n = 25) and decreased with increasing stimulation frequency. Amplitudes were unchanged by different presentation orders of four stimulation intensities. PREPse N1 (∼130 milliseconds) and N2 (∼345 milliseconds) peaks were approximately 40 milliseconds earlier than that with CHEPS. PREPse and CHEPS N1-N2 interpeak latency (∼207 milliseconds) were similar. PREPse became unrecordable with nerve blockade of Aδ fibers. PREPse earlier N1 and N2 peaks, and similar interpeak N1-N2 latencies and central conduction velocities, or synaptic delays, to CHEPS are consistent with direct stimulation of Aδ fibers. The relation of vertex PREPse amplitude and pain, or the differential effects of frequency stimulation, is similar to pain-related evoked potential to laser, special electrodes, or contact heat stimulation. The relationship to Aδ was validated by conduction velocity and nerve block. Clinical utility of PREPse compared with CHEPS needs validation in somatosensory pathways lesions.

Exploring phase–amplitude coupling from primary motor cortex-basal ganglia–thalamus network model

The purpose of this study is to develop a primary motor cortex (M1)-basal ganglia–thalamus model capable of reproducing the physiological phenomenon of exaggerated phase–amplitude coupling (PAC) in Parkinson’s disease and exploring the potential sources of PAC anomalies in M1. The subthalamic nucleus (STN) phase-STN amplitude coupling, STN phase–M1 amplitude coupling, and M1 phase–M1 amplitude coupling are reproduced, where the phase frequencies are distributed in the beta band and the amplitude frequencies are distributed in the broad gamma band. We mainly study the impacts of thalamus →M1 connections and STN↔M1 bidirectional synaptic connections. Abnormal beta oscillations generated within the basal ganglia are found to be transmitted to M1 through the STN or thalamus and could be one of the potential sources of PAC-related beta oscillations in M1, thereby interfering with high-frequency signals in the motor cortex. Furthermore, the weakening of M1→STN leads to a shift of the oscillations of the STN from the high beta band to the low beta band, which is more consistent with pathological experiments, thus supporting the experimental results that the hyper-direct path from M1 to STN drives the beta oscillations of STN. Finally, the suppression effect of STN deep brain stimulation on PAC is investigated. As the stimulation frequency increases, the PAC modulation index within different regions gradually decreases, in general agreement with the trend of synchronization level and beta oscillation energy, indirectly indicating that PAC can be used as a feedback indicator of parkinsonian state.

Effect of stimulation frequency on force, power and fatigue of isolated mouse extensor digitorum longus muscle.

This study examined the effect of stimulation frequency (140, 200, 230 and 260 Hz) on isometric force, work loop (WL) power and the fatigue resistance of extensor digitorum longus (EDL) muscle (n=32), isolated from 8- to 10-week-old CD-1 female mice. Stimulation frequency had significant effects on isometric properties of isolated mouse EDL, whereby increasing stimulation frequency evoked increased isometric force, quicker activation and prolonged relaxation (P<0.047) up to 230 Hz and above; thereafter, force and activation did not differ (P>0.137). Increasing stimulation frequency increased maximal WL power output (P<0.001; 140 Hz, 71.3±3.5; 200 Hz, 105.4±4.1; 230 Hz, 115.5±4.1; 260 Hz, 121.1±4.1 W kg-1), but resulted in significantly quicker rates of fatigue during consecutive WLs (P<0.004). WL shapes indicate impaired muscle relaxation at the end of shortening and subsequent increased negative work appeared to contribute to fatigue at 230 and 260 Hz, but not at lower stimulation frequencies. Cumulative work was unaffected by stimulation frequency, except at the start of the fatigue protocol, where 230 and 260 Hz produced more work than 140 Hz (P<0.039). We demonstrate that stimulation frequency affects force, power and fatigue, but these effects are not uniform between different assessments of contractile performance. Therefore, future work examining the contractile properties of isolated skeletal muscle should consider increasing the stimulation frequency beyond that needed for maximal force when examining maximal power but should utilise a sub-maximal stimulation frequency for fatigue assessments to avoid a high degree of negative work atypical of in vivo function.

BS-526-04 IMPAIRED REDOX RESPONSE TO INCREASED WORKLOAD IN ATRIAL MITOCHONDRIA FROM PATIENTS WITH ATRIAL FIBRILLATION

Calcium handling abnormalities contribute to the pathophysiology of atrial fibrillation (AF). In ventricular myocytes, Ca2+ has been shown to play an important role in mitochondrial response to increased workload by activating key dehydrogenases in the Krebs cycle, necessary for NAD(P)H regeneration and, ultimately, neutralisation. We hypothesise that workload response of mitochondria is impaired in atrial cardiomyocytes from patients with AF. Membrane currents (patch clamp), cytosolic Ca2+ (Fluo-3) and NAD(P)H/FAD autofluorescence were recorded in right atrial myocytes from sinus rhythm (CTL) or AF patients. Immunofluorescence labeling together with STED microscopy and EM microscopy were employed to characterize interaction between mitochondria and sarcoplasmic reticulum (SR). Diastolic [Ca2+]i of voltage-clamped myocytes was comparable, whereas amplitudes of L-type Ca2+ current and triggered of Ca2+ transients (CaTs) were decreased by 49% and 43%, respectively. Basal level of NAD(P)H at 0.5 Hz stimulation frequency was comparable in CTL and AF (73.08±2.62%, n=14/7 vs. 81.05±5.39%, n=4/3 respectively), suggesting a similar redox index. In all CTL cells, upon increasing stimulation frequency to 3 Hz and subsequent β-adrenergic stimulation, the NAD(P)H level initially decreased but recovered to a level comparable to basal state. In contrast, 35% of AF myocytes lost this capacity to recover. Electron and STED microscopy images (Figure) show a disturbance in mitochondrial organisation and their interaction with the SR. Impaired cytosolic Ca2+ handling and disturbed interaction between mitochondrial and Ca2+ release sites of the SR may contribute to the impaired redox response of mitochondria to increased workload which we observed in AF patients. This likely results in impaired ATP production and ROS neutralisation, which may finally contribute to atrial arrhythmogenesis in AF patients.

Characterization of bilateral, pudendal nerve-evoked, urethral rhabdosphincter contractions in anesthetized cats

Pudendal (PUD) efferent activity contracts urethral rhabdosphincters to resist urine leakage. This study evaluates the potential for PUD nerve stimulation to provide benefit for urinary incontinence. Acute responses of the urethral rhabdosphincter to modulation of current amplitude, pulse duration (PD), and frequency of stimulation were examined. Urethral pressure was measured using a microtip catheter in chloralose-anesthetized, male cats with or without acute spinalization at T10. Stimulation was applied from an external pulse generator coupled to cuff electrodes placed around left and right PUD nerves in the ischiorectal fossa, central to its branching. Bladder pressure clamp techniques were used for testing effects of stimulation on time and rate of leakage during PUD stimulation. Single pulses (0.1 ms) produced single transient increases in urethral pressure that peaked at 50 ms and diminished to 50% of peak at 90 ms. Threshold current was 88 ± 30μA with maximal responses occurring with 50% increases in current. Peak pressures increased with increasing PDs between 40–180μs. Summation, tetany, peak pressures, and fatigue of responses to increasing stimulation frequencies during 2 min periods of stimulation are described. Stimulation under pressures that are typical during bladder filling or during bladder contractions (30–40 cm H2O) prevented leakage up to an hour in some animals, increased the median time to leak from 0.3 min to 56.0 min, and decreased the median rate of leakage from 1.44 ml/min to 0.04 ml/min when leakage did occur in less than hour. During rapid elevations in bladder pressure to simulate a Valsalva-like bladder pressure (120 cm H2O), PUD stimulation increased the median time to leak from 9 s to 93 s and decreased the median rate of leakage from 5.5 ml/min to 3.4 ml/min. These results support future studies of bilateral PUD stimulation in a clinically-relevant, animal model of urinary incontinence.

Mechanisms of Arrhythmogenicity of Hypertrophic Cardiomyopathy-Associated Troponin T (TNNT2) Variant I79N.

Hypertrophic cardiomyopathy (HCM) is the most common heritable cardiovascular disease and often results in cardiac remodeling and an increased incidence of sudden cardiac arrest (SCA) and death, especially in youth and young adults. Among thousands of different variants found in HCM patients, variants of TNNT2 (cardiac troponin T—TNNT2) are linked to increased risk of ventricular arrhythmogenesis and sudden death despite causing little to no cardiac hypertrophy. Therefore, studying the effect of TNNT2 variants on cardiac propensity for arrhythmogenesis can pave the way for characterizing HCM in susceptible patients before sudden cardiac arrest occurs. In this study, a TNNT2 variant, I79N, was generated in human cardiac recombinant/reconstituted thin filaments (hcRTF) to investigate the effect of the mutation on myofilament Ca2+ sensitivity and Ca2+ dissociation rate using steady-state and stopped-flow fluorescence techniques. The results revealed that the I79N variant significantly increases myofilament Ca2+ sensitivity and decreases the Ca2+ off-rate constant (k off). To investigate further, a heterozygous I79N+/− TNNT2 variant was introduced into human-induced pluripotent stem cells using CRISPR/Cas9 and subsequently differentiated into ventricular cardiomyocytes (hiPSC-CMs). To study the arrhythmogenic properties, monolayers of I79N+/− hiPSC-CMs were studied in comparison to their isogenic controls. Arrhythmogenesis was investigated by measuring voltage (V m) and cytosolic Ca2+ transients over a range of stimulation frequencies. An increasing stimulation frequency was applied to the cells, from 55 to 75 bpm. The results of this protocol showed that the TnT-I79N cells had reduced intracellular Ca2+ transients due to the enhanced cytosolic Ca2+ buffering. These changes in Ca2+ handling resulted in beat-to-beat instability and triangulation of the cardiac action potential, which are predictors of arrhythmia risk. While wild-type (WT) hiPSC-CMs were accurately entrained to frequencies of at least 150 bpm, the I79N hiPSC-CMs demonstrated clear patterns of alternans for both V m and Ca2+ transients at frequencies >75 bpm. Lastly, a transcriptomic analysis was conducted on WT vs. I79N+/− TNNT2 hiPSC-CMs using a custom NanoString codeset. The results showed a significant upregulation of NPPA (atrial natriuretic peptide), NPPB (brain natriuretic peptide), Notch signaling pathway components, and other extracellular matrix (ECM) remodeling components in I79N+/− vs. the isogenic control. This significant shift demonstrates that this missense in the TNNT2 transcript likely causes a biophysical trigger, which initiates this significant alteration in the transcriptome. This TnT-I79N hiPSC-CM model not only reproduces key cellular features of HCM-linked mutations but also suggests that this variant causes uncharted pro-arrhythmic changes to the human action potential and gene expression.

Intramuscular pressure of human tibialis anterior muscle detects age-related changes in muscle performance

Intramuscular pressure (IMP) reflects forces produced by a muscle. Age is one of the determinants of skeletal muscle performance. The present study aimed to test whether IMP mirrors known age-related muscular changes. We simultaneously measured the tibialis anterior (TA) IMP, compound muscle action potential (CMAP), and ankle torque in thirteen older adults (60–80 years old) in vivo by applying different stimulation intensities and frequencies. We found significant positive correlations between the stimulation intensity and IMP and CMAP. Increasing stimulation frequency caused ankle torque and IMP to increase. The electromechanical delay (EMD) (36 ms) was longer than the onset of IMP (IMPD) (29 ms). Compared to the previously published data collected from young adults (21–40 years old) in identical conditions, the TA CMAP and IMP of older adults at maximum intensity of stimulation were 23.8% and 39.6% lower, respectively. For different stimulation frequencies, CMAP, IMP, as well as ankle torque of older adults were 20.5%, 24.2%, and 13.2% lower, respectively. Surprisingly, the EMD did not exhibit any difference between young and older adults and the IMPD was consistent with the EMD. Data supporting the hypotheses suggest that IMP measurement is an indicator of muscle performance in older adults.

Right-ventricular dysfunction in HFpEF is linked to altered cardiomyocyte Ca2+ homeostasis and myofilament sensitivity.

AimsHeart failure with preserved ejection fraction (HFpEF) is frequently (30%) associated with right ventricular (RV) dysfunction, which increases morbidity and mortality in these patients. Yet cellular mechanisms of RV remodelling and RV dysfunction in HFpEF are not well understood. Here, we evaluated RV cardiomyocyte function in a rat model of metabolically induced HFpEF.Methods and resultsHeart failure with preserved ejection fraction‐prone animals (ZSF‐1 obese) and control rats (Wistar Kyoto) were fed a high‐caloric diet for 13 weeks. Haemodynamic characterization by echocardiography and invasive catheterization was performed at 22 and 23 weeks of age, respectively. After sacrifice, organ morphometry, RV histology, isolated RV cardiomyocyte function, and calcium (Ca2+) transients were assessed. ZSF‐1 obese rats showed a HFpEF phenotype with left ventricular (LV) hypertrophy, LV diastolic dysfunction (including increased LV end‐diastolic pressures and E/e′ ratio), and preserved LV ejection fraction. ZSF‐1 obese animals developed RV dilatation (50% increased end‐diastolic area) and mildly impaired RV ejection fraction (42%) with evidence of RV hypertrophy. In isolated RV cardiomyocytes from ZSF‐1 obese rats, cell shortening amplitude was preserved, but cytosolic Ca2+ transient amplitude was reduced. In addition, augmentation of cytosolic Ca2+ release with increased stimulation frequency was lost in ZSF‐1 obese rats. Myofilament sensitivity was increased, while contractile kinetics were largely unaffected in intact isolated RV cardiomyocytes from ZSF‐1 obese rats. Western blot analysis revealed significantly increased phosphorylation of cardiac myosin‐binding protein C (Ser282 cMyBP‐C) but no change in phosphorylation of troponin I (Ser23, 24 TnI) in RV myocardium from ZSF‐1 obese rats.ConclusionsRight ventricular dysfunction in obese ZSF‐1 rats with HFpEF is associated with intrinsic RV cardiomyocyte remodelling including reduced cytosolic Ca2+ amplitudes, loss of frequency‐dependent augmentation of Ca2+ release, and increased myofilament Ca2+ sensitivity.

CA2+ Handling in Non-Failing Hypertrophic Cardiomyocytes Subjected to Inotropic Interventions

Impaired force development during ventricular hypertrophy has been associated with structural and functional alterations of Ca2+ handling proteins. Previous findings in whole hearts and multicellular trabeculae from a rat model of right ventricular (RV) hypertrophy showed spontaneous contractile activity and asynchronous Ca2+ release during inotropic interventions. Our aim was to investigate whether these changes occurred at the single cell level. Male Wistar rats were injected with 60 mg kg−1 of monocrotaline (MCT, n=3) or saline as control (CON, n=4). Four weeks post injection, hearts were isolated and enzymatically digested to yield rod-shaped quiescent cardiomyocytes. Aliquots of RV myocytes were collected and measurements of cell shortening and intracellular Ca2+ (fura-2AM, 340/380 ratio) were made in response to inotropic interventions (increased stimulation frequency and ß-adrenergic stimulation). Application of 20mM caffeine revealed Ca2+ content of the sarcoplasmic reticulum (SR). A separate group of cardiomyocytes were fixed and key proteins were labelled with antibodies for subsequent confocal imaging. MCT myocytes had the largest cross-sectional area (4338 ± 292µm2) versus CON (3170 ± 262µm2, P=0.01), although no difference in cell shortening was observed in response to 1 Hz stimulation. MCT myocytes had larger Ca2+ transients at all stimulation frequencies (P= 0.004) and increased spontaneous activity during ß-adrenergic stimulation. No difference between groups was found in Ca2+ store content, although the time constant of caffeine transient decay was prolonged in MCT (17.3 ± 1.5s) versus CON (13.7 ± 0.6s, P=0.05). Although there was no difference in shortening or SR Ca2+ content, there was evidence of changes in Ca2+ handling in MCT myocytes during inotropic stimulation. Furthermore, hypertrophic MCT myocytes exhibited slower trans-sarcolemmal Ca2+ removal during 20mM caffeine, potentially as a result of disorganized T-tubular arrangement and/or reduced Na+/Ca2+ exchanger abundance.