Bilateral Functional Electrical Stimulation for the Treatment of Presbyphonia in a Sheep Model.

OBJECTIVES: The aim of the study was to increase muscle volume and improve phonation characteristics of the aged ovine larynx by functional electrical stimulation (FES) using a minimally invasive surgical procedure. METHODS: Stimulation electrodes were placed bilaterally near the terminal adduction branch of the recurrent laryngeal nerves (RLN). The electrodes were connected to battery powered pulse generators implanted subcutaneously at the neck region. Training patterns were programmed by an external programmer using a bidirectional radio frequency link. Training sessions were repeated automatically by the implant every other day for 1 week followed by every day for 8 weeks in the awake animal. Another group of animals were used as sham, with electrodes positioned but not connected to an implant. Outcome parameters included gene expression analysis, histological assessment of muscle fiber size, functional analysis, and volumetric measurements based on three-dimensional reconstructions of the entire thyroarytenoid muscle (TAM). RESULTS: Increase in minimal muscle fiber diameter and an improvement in vocal efficiency were observed following FES, compared with sham animals. CONCLUSION: This is the first study to demonstrate beneficial effects in the TAM of FES at molecular, histological, and functional levels. FES of the terminal branches of the RLN reversed the effects of age-related changes and improved vocal efficiency. LEVEL OF EVIDENCE: NA Laryngoscope, 134:848-854, 2024.

Conserved and species-specific transcriptional responses to daily programmed resistance exercise in rat and mouse.

Mice are often used in gain or loss of function studies to understand how genes regulate metabolism and adaptation to exercise in skeletal muscle. Once-daily resistance training with electrical nerve stimulation produces hypertrophy of the dorsiflexors in rat, but not in mouse. Using implantable pulse generators, we assessed the acute transcriptional response (1-h post-exercise) after 2, 10, and 20 days of training in free-living mice and rats using identical nerve stimulation paradigms. RNA sequencing revealed strong concordance in the timecourse of many transcriptional responses in the tibialis anterior muscles of both species including responses related to "stress responses/immediate-early genes, and "collagen homeostasis," "ribosomal subunits," "autophagy," and "focal adhesion." However, pathways associated with energy metabolism including "carbon metabolism," "oxidative phosphorylation," "mitochondrial translation," "propanoate metabolism," and "valine, leucine, and isoleucine degradation" were oppositely regulated between species. These pathways were suppressed in the rat but upregulated in the mouse. Our transcriptional analysis suggests that although many pathways associated with growth show remarkable similarities between species, the absence of an actual growth response in the mouse may be because the mouse prioritizes energy metabolism, specifically the replenishment of fuel stores and intermediate metabolites.

Adaptation of the transcriptional response to resistance exercise over 4 weeks of daily training.

We present the time course of change in the muscle transcriptome 1 h after the last exercise bout of a daily resistance training program lasting 2, 10, 20, or 30 days. Daily exercise in rat tibialis anterior muscles (5 sets of 10 repetitions over 20 min) induced progressive muscle growth that approached a new stable state after 30 days. The acute transcriptional response changed along with progressive adaptation of the muscle phenotype. For example, expression of type 2B myosin was silenced. Time courses recently synthesized from human exercise studies do not demonstrate so clearly the interplay between the acute exercise response and the longer-term consequences of repeated exercise. We highlight classes of transcripts and transcription factors whose expression increases during the growth phase and declines again as the muscle adapts to a new daily pattern of activity and reduces its rate of growth. Myc appears to play a central role.

PCM1 labeling reveals myonuclear and nuclear dynamics in skeletal muscle across species.

Myonuclei transcriptionally regulate muscle fibers during homeostasis and adaptation to exercise. Their subcellular location and quantity are important when characterizing phenotypes of myopathies, the effect of treatments, and understanding the roles of satellite cells in muscle adaptation and muscle "memory." Difficulties arise in identifying myonuclei due to their proximity to the sarcolemma and closely residing interstitial cell neighbors. We aimed to determine to what extent (pericentriolar material-1) PCM1 is a specific marker of myonuclei in vitro and in vivo. Single isolated myofibers and cross sections from mice and humans were studied from several models including wild-type and Lamin A/C mutant mice after functional overload and damage and recovery in humans following forced eccentric contractions. Fibers were immunolabeled for PCM1, Pax7, and DNA. C2C12 myoblasts were also studied to investigate changes in PCM1 localization during myogenesis. PCM1 was detected at not only the nuclear envelope of myonuclei in mature myofibers and in newly formed myotubes but also centrosomes in proliferating myogenic precursors, which may or may not fuse to join the myofiber syncytium. PCM1 was also detected in nonmyogenic nuclei near the sarcolemma, especially in regenerating areas of the Lmna+/ΔK32 mouse and damaged human muscle. Although PCM1 is not completely specific to myonuclei, the impact that PCM1+ macrophages and interstitial cells have on myonuclei counts would be small in healthy muscle. PCM1 may prove useful as a marker of satellite cell dynamics due to the distinct change in localization during differentiation, revealing satellite cells in their quiescent (PCM1-), proliferating (PCM1+ centrosome), and prefusion states (PCM1+ nuclear envelope).

Metabolomic studies in the inborn error of metabolism alkaptonuria reveal new biotransformations in tyrosine metabolism.

Alkaptonuria (AKU) is an inherited disorder of tyrosine metabolism caused by lack of active enzyme homogentisate 1,2-dioxygenase (HGD). The primary consequence of HGD deficiency is increased circulating homogentisic acid (HGA), the main agent in the pathology of AKU disease. Here we report the first metabolomic analysis of AKU homozygous Hgd knockout (Hgd -/-) mice to model the wider metabolic effects of Hgd deletion and the implication for AKU in humans. Untargeted metabolic profiling was performed on urine from Hgd -/- AKU (n = 15) and Hgd +/- non-AKU control (n = 14) mice by liquid chromatography high-resolution time-of-flight mass spectrometry (Experiment 1). The metabolites showing alteration in Hgd -/- were further investigated in AKU mice (n = 18) and patients from the UK National AKU Centre (n = 25) at baseline and after treatment with the HGA-lowering agent nitisinone (Experiment 2). A metabolic flux experiment was carried out after administration of 13C-labelled HGA to Hgd -/-(n = 4) and Hgd +/-(n = 4) mice (Experiment 3) to confirm direct association with HGA. Hgd -/- mice showed the expected increase in HGA, together with unexpected alterations in tyrosine, purine and TCA-cycle pathways. Metabolites with the greatest abundance increases in Hgd -/- were HGA and previously unreported sulfate and glucuronide HGA conjugates, these were decreased in mice and patients on nitisinone and shown to be products from HGA by the 13C-labelled HGA tracer. Our findings reveal that increased HGA in AKU undergoes further metabolism by mainly phase II biotransformations. The data advance our understanding of overall tyrosine metabolism, demonstrating how specific metabolic conditions can elucidate hitherto undiscovered pathways in biochemistry and metabolism.

Impact of Nitisinone on the Cerebrospinal Fluid Metabolome of a Murine Model of Alkaptonuria.

Background: Nitisinone-induced hypertyrosinaemia is well documented in Alkaptonuria (AKU), and there is uncertainty over whether it may contribute to a decline in cognitive function and/or mood by altering neurotransmitter metabolism. The aim of this work was to evaluate the impact of nitisinone on the cerebrospinal fluid (CSF) metabolome in a murine model of AKU, with a view to providing additional insight into metabolic changes that occur following treatment with nitisinone. Methods: 17 CSF samples were collected from BALB/c Hgd−/− mice (n = 8, treated with nitisinone­4 mg/L and n = 9, no treatment). Samples were diluted 1:1 with deionised water and analysed using a 1290 Infinity II liquid chromatography system coupled to a 6550 quadrupole time-of-flight mass spectrometry (Agilent, Cheadle, UK). Raw data were processed using a targeted feature extraction algorithm and an established in-house accurate mass retention time database. Matched entities (±10 ppm theoretical accurate mass and ±0.3 min retention time window) were filtered based on their frequency and variability. Experimental groups were compared using a moderated t-test with Benjamini−Hochberg false-discovery rate adjustment. Results: L-Tyrosine, N-acetyl-L-tyrosine, γ-glutamyl-L-tyrosine, p-hydroxyphenylacetic acid, and 3-(4-hydroxyphenyl)lactic acid were shown to increase in abundance (log2 fold change 2.6−6.9, 3/5 were significant p < 0.05) in the mice that received nitisinone. Several other metabolites of interest were matched, but no significant differences were observed, including the aromatic amino acids phenylalanine and tryptophan, and monoamine metabolites adrenaline, 3-methoxy-4-hydroxyphenylglycol, and octopamine. Conclusions: Evaluation of the CSF metabolome of a murine model of AKU revealed a significant increase in the abundance of a limited number of metabolites following treatment with nitisinone. Further work is required to understand the significance of these findings and the mechanisms by which the altered metabolite abundances occur.

Automated cross-sectional analysis of trained, severely atrophied, and recovering rat skeletal muscles using MyoVision 2.0.

The number of myonuclei within a muscle fiber is an important factor in muscle growth, but its regulation during muscle adaptation is not well understood. We aimed to elucidate the time course of myonuclear dynamics during endurance training, loaded and concentric resistance training, and nerve silencing-induced disuse atrophy with subsequent recovery. We modified tibialis anterior muscle activity in free-living rats with electrical stimulation from implantable pulse generators, or with implantable osmotic pumps delivering tetrodotoxin (TTX) to silence the motor nerve without transection. We used the updated, automated software MyoVision to measure fiber-type-specific responses in whole tibialis anterior cross sections (∼8,000 fibers each). Seven days of continuous low-frequency stimulation (CLFS) reduced muscle mass (-12%), increased slower myosin isoforms and reduced IIX/IIB fibers (-32%), and substantially increased myonuclei especially in IIX/IIB fibers (55.5%). High-load resistance training (spillover) produced greater hypertrophy (∼16%) in muscle mass and fiber cross-sectional area (CSA) than low-load resistance training (concentric, ∼6%) and was associated with myonuclear addition in all fiber types (35%-46%). TTX-induced nerve silencing resulted in progressive loss in muscle mass, fiber CSA, and myonuclei per fiber cross section (-50.7%, -53.7%, and -40.7%, respectively, at 14 days). Myonuclear loss occurred in a fiber-type-independent manner, but subsequent recovery during voluntary habitual activity suggested that type IIX/IIB fibers contained more new myonuclei during recovery from severe atrophy. This study demonstrates the power and accuracy provided by the updated MyoVision software and introduces new models for studying myonuclear dynamics in training, detraining, retraining, repeated disuse, and recovery.NEW & NOTEWORTHY We introduce new models for studying fiber-type-specific myonuclear dynamics in muscle training, detraining, retraining, disuse, and recovery. We show that the various fiber types do not respond identically and that myonuclear number changes during adaptation. We also critically assess an updated version of MyoVision automated image analysis software to quantify whole muscle immunofluorescent microscopical images in a faster and less computer intensive manner. MyoVision remains open source and freely available with more user-controlled features.

Remodeling of the Purkinje Network in Congestive Heart Failure in the Rabbit.

BACKGROUND: Purkinje fibers (PFs) control timing of ventricular conduction and play a key role in arrhythmogenesis in heart failure (HF) patients. We investigated the effects of HF on PFs. METHODS: Echocardiography, electrocardiography, micro-computed tomography, quantitative polymerase chain reaction, immunohistochemistry, volume electron microscopy, and sharp microelectrode electrophysiology were used. RESULTS: Congestive HF was induced in rabbits by left ventricular volume- and pressure-overload producing left ventricular hypertrophy, diminished fractional shortening and ejection fraction, and increased left ventricular dimensions. HF baseline QRS and corrected QT interval were prolonged by 17% and 21% (mean±SEMs: 303±6 ms HF, 249±11 ms control; n=8/7; P=0.0002), suggesting PF dysfunction and impaired ventricular repolarization. Micro-computed tomography imaging showed increased free-running left PF network volume and length in HF. mRNA levels for 40 ion channels, Ca2+-handling proteins, connexins, and proinflammatory and fibrosis markers were assessed: 50% and 35% were dysregulated in left and right PFs respectively, whereas only 12.5% and 7.5% changed in left and right ventricular muscle. Funny channels, Ca2+-channels, and K+-channels were significantly reduced in left PFs. Microelectrode recordings from left PFs revealed more negative resting membrane potential, reduced action potential upstroke velocity, prolonged duration (action potential duration at 90% repolarization: 378±24 ms HF, 249±5 ms control; n=23/38; P<0.0001), and arrhythmic events in HF. Similar electrical remodeling was seen at the left PF-ventricular junction. In the failing left ventricle, upstroke velocity and amplitude were increased, but action potential duration at 90% repolarization was unaffected. CONCLUSIONS: Severe volume- followed by pressure-overload causes rapidly progressing HF with extensive remodeling of PFs. The PF network is central to both arrhythmogenesis and contractile dysfunction and the pathological remodeling may increase the risk of fatal arrhythmias in HF patients.

Pulse Shaping Strategies for Electroceuticals: A Comprehensive Survey of the Use of Interphase Gaps in Miniature Stimulation Systems.

OBJECTIVE: Interphase gaps (IPGs) are among the most commonly suggested pulse shape variations to try to enhance neural stimulation efficiency by reducing the action potential (AP) suppressing effect of an early anodic hyperpolarization. The majority of published literature on the effect of IPGs is based on investigations of monopolar stimulation configurations. However, many contemporary neuromodulation applications including the emerging field of electroceutical devices operate in a bipolar electrode configuration. METHODS: We investigated the effect of IPGs and asymmetric biphasic current controlled pulses with reduced anodic amplitude on neural activation in both principal electrode configurations in a rodent in-vivo nerve muscle preparation. RESULTS: In the monopolar electrode configuration, our findings of 10.9 ± 1.5% decreased stimulation amplitude with 200 µs IPGs in biphasic pulses of 40 µs phase width are in agreement with published literature in this configuration. Surprisingly, using the bipolar configuration, opposite effects of IPGs were observed and neural activation required up to 18.6 ± 3.1% (phase width 100 µs, IPG = 1000 µs) higher amplitudes. Electroneurogram recordings of the stimulated nerve revealed temporal differences in AP generation between the monopolar and bipolar configuration. In the bipolar configuration excitation first occurred in response to the middle field transition of biphasic pulses. CONCLUSION: This is the first study to report consistently increased amplitude requirements with IPGs in bipolar stimulation configurations. SIGNIFICANCE: Our findings must be taken into consideration when designing stimulation waveforms for neuromodulation devices that operate in a bipolar mode to avoid increased amplitude requirements that result in increased energy consumption.

Mechanical loading of bioengineered skeletal muscle in vitro recapitulates gene expression signatures of resistance exercise in vivo.

Understanding the role of mechanical loading and exercise in skeletal muscle (SkM) is paramount for delineating the molecular mechanisms that govern changes in muscle mass. However, it is unknown whether loading of bioengineered SkM in vitro adequately recapitulates the molecular responses observed after resistance exercise (RE) in vivo. To address this, the transcriptional and epigenetic (DNA methylation) responses were compared after mechanical loading in bioengineered SkM in vitro and after RE in vivo. Specifically, genes known to be upregulated/hypomethylated after RE in humans were analyzed. Ninety-three percent of these genes demonstrated similar changes in gene expression post-loading in the bioengineered muscle when compared to acute RE in humans. Furthermore, similar differences in gene expression were observed between loaded bioengineered SkM and after programmed RT in rat SkM tissue. Hypomethylation occurred for only one of the genes analysed (GRIK2) post-loading in bioengineered SkM. To further validate these findings, DNA methylation and mRNA expression of known hypomethylated and upregulated genes post-acute RE in humans were also analyzed at 0.5, 3, and 24 h post-loading in bioengineered muscle. The largest changes in gene expression occurred at 3 h, whereby 82% and 91% of genes responded similarly when compared to human and rodent SkM respectively. DNA methylation of only a small proportion of genes analyzed (TRAF1, MSN, and CTTN) significantly increased post-loading in bioengineered SkM alone. Overall, mechanical loading of bioengineered SkM in vitro recapitulates the gene expression profile of human and rodent SkM after RE in vivo. Although some genes demonstrated differential DNA methylation post-loading in bioengineered SkM, such changes across the majority of genes analyzed did not closely mimic the epigenetic response to acute-RE in humans.

Effect of neuromuscular electrical stimulation on the recovery of people with COVID-19 admitted to the intensive care unit: A narrative review.

The rehabilitation of patients with COVID-19 after prolonged treatment in the intensive care unit is often complex and challenging. Patients may develop a myriad of long-term multiorgan impairments, affecting the respiratory, cardiac, neurological, digestive and musculoskeletal systems. Skeletal muscle dysfunction of respiratory and limb muscles, commonly referred to as intensive care unit acquired weakness, occurs in approximately 40% of all patients admitted to intensive care. The impact on mobility and return to activities of daily living is severe. Furthermore, many patients experience ongoing symptoms of fatigue, weakness and shortness of breath, in what is being described as "long COVID". Neuromuscular electrical stimulation is a technique in which small electrical impulses are applied to skeletal muscle to cause contractions when voluntary muscle contraction is difficult or impossible. Neuromuscular electrical stimulation can prevent muscle atrophy, improve muscle strength and function, maintain blood flow and reduce oedema. This review examines the evidence, current guidelines, and proposed benefits of using neuromuscular electrical stimulation with patients admitted to the intensive care unit. Practical recommendations for using electrical muscle stimulation in patients with COVID-19 are provided, and suggestions for further research are proposed. Evidence suggests NMES may play a role in the weaning of patients from ventilators and can be continued in the post-acute and longer-term phases of recovery. As such, NMES may be a suitable treatment modality to implement within rehabilitation pathways for COVID-19, with consideration of the practical and safety issues highlighted within this review.

Knockdown of the E3 ubiquitin ligase UBR5 and its role in skeletal muscle anabolism.

UBR5 is an E3 ubiquitin ligase positively associated with anabolism, hypertrophy, and recovery from atrophy in skeletal muscle. The precise mechanisms underpinning UBR5's role in the regulation of skeletal muscle mass remain unknown. The present study aimed to elucidate these mechanisms by silencing the UBR5 gene in vivo. To achieve this aim, we electroporated a UBR5-RNAi plasmid into mouse tibialis anterior muscle to investigate the impact of reduced UBR5 on anabolic signaling MEK/ERK/p90RSK and Akt/GSK3ß/p70S6K/4E-BP1/rpS6 pathways. Seven days after UBR5 RNAi electroporation, although reductions in overall muscle mass were not detected, the mean cross-sectional area (CSA) of green fluorescent protein (GFP)-positive fibers were reduced (-9.5%) and the number of large fibers were lower versus the control. Importantly, UBR5-RNAi significantly reduced total RNA, muscle protein synthesis, ERK1/2, Akt, and GSK3ß activity. Although p90RSK phosphorylation significantly increased, total p90RSK protein levels demonstrated a 45% reduction with UBR5-RNAi. Finally, these early events after 7 days of UBR5 knockdown culminated in significant reductions in muscle mass (-4.6%) and larger reductions in fiber CSA (-18.5%) after 30 days. This was associated with increased levels of phosphatase PP2Ac and inappropriate chronic elevation of p70S6K and rpS6 between 7 and 30 days, as well as corresponding reductions in eIF4e. This study demonstrates that UBR5 plays an important role in anabolism/hypertrophy, whereby knockdown of UBR5 culminates in skeletal muscle atrophy.

Anatomical Distribution of Ochronotic Pigment in Alkaptonuric Mice is Associated with Calcified Cartilage Chondrocytes at Osteochondral Interfaces.

Alkaptonuria (AKU) is characterised by increased circulating homogentisic acid and deposition of ochronotic pigment in collagen-rich connective tissues (ochronosis), stiffening the tissue. This process over many years leads to a painful and severe osteoarthropathy, particularly affecting the cartilage of the spine and large weight bearing joints. Evidence in human AKU tissue suggests that pigment binds to collagen. The exposed collagen hypothesis suggests that collagen is initially protected from ochronosis, and that ageing and mechanical loading causes loss of protective molecules, allowing pigment binding. Schmorl's staining has previously demonstrated knee joint ochronosis in AKU mice. This study documents more comprehensively the anatomical distribution of ochronosis in two AKU mouse models (BALB/c Hgd-/-, Hgd tm1a-/-), using Schmorl's staining. Progression of knee joint pigmentation with age in the two AKU mouse models was comparable. Within the knee, hip, shoulder, elbow and wrist joints, pigmentation was associated with chondrons of calcified cartilage. Pigmented chondrons were identified in calcified endplates of intervertebral discs and the calcified knee joint meniscus, suggesting that calcified tissues are more susceptible to pigmentation. There were significantly more pigmented chondrons in lumbar versus tail intervertebral disc endplates (p = 0.002) and clusters of pigmented chondrons were observed at the insertions of ligaments and tendons. These observations suggest that loading/strain may be associated with increased pigmentation but needs further experimental investigation. The calcified cartilage may be the first joint tissue to acquire matrix damage, most likely to collagen, through normal ageing and physiological loading, as it is the first to become susceptible to pigmentation.

MiniVStimA: A miniaturized easy to use implantable electrical stimulator for small laboratory animals.

According to PubMed, roughly 10% of the annually added publications are describing findings from the small animal model (mice and rats), including investigations in the field of muscle physiology and training. A subset of this research requires neural stimulation with flexible adjustments of stimulation parameters, highlighting the need for reliable implantable electrical stimulators, small enough (~1 cm3), that even mice can tolerate them without impairing their movement. The MiniVStimA is a battery-powered implant for nerve stimulation with an outer diameter of 15 mm and an encapsulated volume of 1.2 cm3 in its smallest variation. It can be pre-programmed according to the experimental protocol and controlled after implantation with a magnet. It delivers constant current charge-balanced monophasic rectangular pulses up to 2 mA and 1 ms phase width (1 kΩ load). The circuitry is optimized for small volume and energy efficiency. Due to the variation of the internal oscillator (31 kHz ± 10%), calibration measures must be implemented during the manufacturing process, which can reduce the deviation of the frequency related parameters down to ± 1%. The expected lifetime of the smaller (larger) version is 100 (480) days for stimulation with 7 Hz all day and 10 (48) days for stimulation with 100 Hz. Devices with complex stimulation patterns for nerve stimulation have been successfully used in two in-vivo studies, lasting up to nine weeks. The implant worked fully self-contained while the animal stayed in its familiar environment. External components are not required during the entire time.

Efficacy and safety of once-daily nitisinone for patients with alkaptonuria (SONIA 2): an international, multicentre, open-label, randomised controlled trial.

BACKGROUND: Alkaptonuria is a rare, genetic, multisystem disease characterised by the accumulation of homogentisic acid (HGA). No HGA-lowering therapy has been approved to date. The aim of SONIA 2 was to investigate the efficacy and safety of once-daily nitisinone for reducing HGA excretion in patients with alkaptonuria and to evaluate whether nitisinone has a clinical benefit. METHODS: SONIA 2 was a 4-year, open-label, evaluator-blind, randomised, no treatment controlled, parallel-group study done at three sites in the UK, France, and Slovakia. Patients aged 25 years or older with confirmed alkaptonuria and any clinical disease manifestations were randomly assigned (1:1) to receive either oral nitisinone 10 mg daily or no treatment. Patients could not be masked to treatment due to colour changes in the urine, but the study was evaluator-blinded as far as possible. The primary endpoint was daily urinary HGA excretion (u-HGA24) after 12 months. Clinical evaluation Alkaptonuria Severity Score Index (cAKUSSI) score was assessed at 12, 24, 36, and 48 months. Efficacy variables were analysed in all randomly assigned patients with a valid u-HGA24 measurement at baseline. Safety variables were analysed in all randomly assigned patients. The study was registered at ClinicalTrials.gov (NCT01916382). FINDINGS: Between May 7, 2014, and Feb 16, 2015, 139 patients were screened, of whom 138 were included in the study, with 69 patients randomly assigned to each group. 55 patients in the nitisinone group and 53 in the control group completed the study. u-HGA24 at 12 months was significantly decreased by 99·7% in the nitisinone group compared with the control group (adjusted geometric mean ratio of nitisinone/control 0·003 [95% CI 0·003 to 0·004], p<0·0001). At 48 months, the increase in cAKUSSI score from baseline was significantly lower in the nitisinone group compared with the control group (adjusted mean difference -8·6 points [-16·0 to -1·2], p=0·023). 400 adverse events occurred in 59 (86%) patients in the nitisinone group and 284 events occurred in 57 (83%) patients in the control group. No treatment-related deaths occurred. INTERPRETATION: Nitisinone 10 mg daily was well tolerated and effective in reducing urinary excretion of HGA. Nitisinone decreased ochronosis and improved clinical signs, indicating a slower disease progression. FUNDING: European Commission Seventh Framework Programme.

Adaptation of rat fast-twitch muscle to endurance activity is underpinned by changes to protein degradation as well as protein synthesis.

Muscle adaptations to exercise are underpinned by alterations to the abundance of individual proteins, which may occur through a change either to the synthesis or degradation of each protein. We used deuterium oxide (2 H2 O) labeling and chronic low-frequency stimulation (CLFS) in vivo to investigate the synthesis, abundance, and degradation of individual proteins during exercise-induced muscle adaptation. Independent groups of rats received CLFS (10 Hz, 24 h/d) and 2 H2 O for 0, 10, 20, or 30 days. The extensor digitorum longus (EDL) was isolated from stimulated (Stim) and contralateral non-stimulated (Ctrl) legs. Proteomic analysis encompassed 38 myofibrillar and 46 soluble proteins and the rates of change in abundance, synthesis, and degradation were reported in absolute (ng/d) units. Overall, synthesis and degradation made equal contributions to the adaptation of the proteome, including instances where a decrease in protein-specific degradation primarily accounted for the increase in abundance of the protein.

Fractional Synthesis Rates of Individual Proteins in Rat Soleus and Plantaris Muscles.

Differences in the protein composition of fast- and slow-twitch muscle may be maintained by different rates of protein turnover. We investigated protein turnover rates in slow-twitch soleus and fast-twitch plantaris of male Wistar rats (body weight 412 ± 69 g). Animals were assigned to four groups (n = 3, in each), including a control group (0 d) and three groups that received deuterium oxide (D2O) for either 10 days, 20 days or 30 days. D2O administration was initiated by an intraperitoneal injection of 20 µL of 99% D2O-saline per g body weight, and maintained by provision of 4% (v/v) D2O in the drinking water available ad libitum. Soluble proteins from harvested muscles were analysed by liquid chromatography-tandem mass spectrometry and identified against the SwissProt database. The enrichment of D2O and rate constant (k) of protein synthesis was calculated from the abundance of peptide mass isotopomers. The fractional synthesis rate (FSR) of 44 proteins in soleus and 34 proteins in plantaris spanned from 0.58%/day (CO1A1: Collagen alpha-1 chain) to 5.40%/day NDRG2 (N-myc downstream-regulated gene 2 protein). Eight out of 18 proteins identified in both muscles had a different FSR in soleus than in plantaris (p < 0.05).

Reply to "Energy Optimal Stimulation Waveforms, Or Not: Comments on 'An Investigation of Neural Stimulation Efficiency With Gaussian Waveforms'".

We thank Professors Grill and Wongsarnpigoon for their detailed responses prompted by our recent publication [1] in this journal. We welcome the opportunity we have had for discussion and thank the Editor for his invitation to make this further contribution. We made our experiments partly in the light of the statements in the abstract of their relevant paper [2] that energy optimal genetic algorithm (GA) waveforms "resembled truncated Gaussian curves" and that "if used in implantable neural stimulators, GA-optimized waveforms could prolong battery life, thereby reducing the frequency of recharge intervals, the volume of implanted pulse generators, and the costs and risks of battery-replacement surgeries." The authors discussed the practical consequences of both a 5% increase and a 60% increase in energy efficiency in the final statement of the paper and acknowledged that to include the cost of generating the GA waveforms might change the predicted optimal shape. Although mentioned only once, a claim of up to 60% relative energy savings is of great interest to those who design and use neural stimulators. Based on our in-vivo investigation of neural stimulation efficiency with biphasic Gaussian waveforms, we reported an upper potential of 17% energy savings over rectangular biphasic pulses. Furthermore, we added the consideration of instantaneous power requirement in our paper to further inform this discussion. This is a crucial parameter, especially to set the practical performance of the sharp-peaked GA waveforms into perspective, given that they deliver the majority of the cathodic charge in a time much shorter than the nominal phase duration.

Nitisinone causes acquired tyrosinosis in alkaptonuria.

For over two decades, nitisinone (NTBC) has been successfully used to manipulate the tyrosine degradation pathway and save the lives of many children with hereditary tyrosinaemia type 1. More recently, NTBC has been used to halt homogentisic acid accumulation in alkaptonuria (AKU) with evidence suggesting its efficacy as a disease modifying agent. NTBC-induced hypertyrosinaemia has been associated with cognitive impairment and potentially sight-threatening keratopathy. In the context of a non-lethal condition (ie, AKU), these serious risks call for an evaluation of the wider impact of NTBC on the tyrosine pathway. We hypothesised that NTBC increases the tyrosine pool size and concentrations in tissues. In AKU mice tyrosine concentrations of tissue homogenates were measured before and after treatment with NTBC. In humans, pulse injection with l-[13 C9 ]tyrosine and l-[d8 ]phenylalanine was used along with compartmental modelling to estimate the size of tyrosine pools before and after treatment with NTBC. We found that NTBC increased tyrosine concentrations in murine tissues by five to nine folds. It also significantly increased the tyrosine pool size in humans (P < .001), suggesting that NTBC increases tyrosine not just in serum but also in tissues (ie, acquired tyrosinosis). This study provides, for the first time, the experimental proof for the magnitude of NTBC-related acquired tyrosinosis which should be overcome to ensure the safe use of NTBC in AKU.

The Effect of Sub-Threshold Pre-Pulses on Neural Activation Depends on Electrode Configuration.

OBJECTIVE: Published research on nerve stimulation with sub-threshold conditioning pre-pulses is contradictory. Like most early research on electrical stimulation (ES), the pioneer work on the use of pre-pulses was modelled and measured only for monopolar electrodes. However, many contemporary ES applications, including miniaturized neuromodulation implants, known as electroceuticals, operate in bipolar mode. METHODS: We compared depolarizing (DPPs) and hyperpolarizing (HPPs) pre-pulses on neural excitability in rat nerve with monopolar and bipolar electrodes. The rat common peroneal nerve was stimulated with biphasic stimuli with and without ramp and square DPPs or HPPs of 1, 5 and 10 ms duration and 10%-20% of the amplitude of the following pulse. RESULTS: The effects were opposite for the monopolar and bipolar configurations. With monopolar electrodes DPPs increased the amplitude required to activate 50% of the motoneuron pool (between 0.7% and 10.3%) and HPPs decreased the threshold (between 1.7% and 4.7%). With bipolar electrodes both pre-pulse types had the opposite effect: DPPs decreased thresholds (between 1.8% and 5.5%) whereas HPPs increased thresholds (between 0.5% and 4.1%). Electroneurograms from the stimulated nerve revealed spatial and temporal differences in action potential generation for monopolar and bipolar electrodes. In bipolar biphasic stimulation, excitation first occurred at the return electrode as a response to the transition between the cathodic and anodic phase. CONCLUSION: These data help to resolve the contradictions in the published data over two decades. SIGNIFICANCE: They also show that fundamental research carried out in monopolar configuration is not directly applicable to contemporary bipolar ES applications.