Excitability Of Muscle Cells Research Articles

Sural sparing reversible sensorimotor abnormalities in hypokalemia: A close mimic of GBS

Nerve conduction studies (NCS) in hypokalemic paralysis usually show decreased compound muscle action potential (CMAP) which improves with correction of hypokalemia and normalizes in between the attacks. Inactivity of Na+ K+ ATPase pump due to hypokalemia results in decreased excitability of muscle fibers resulting in reduced CMAP on NCS. Sensory symptoms, as well as abnormalities in sensory NCS is a rare phenomenon. Here we describe a 40-year-old male who presented with acute onset rapidly progressive ascending quadriparesis with type 2 respiratory failure due to hypokalemia following hepatorenal dysfunction. NCS done before hypokalemia correction, suggested sural sparing with generalized reduced CMAPs, nonrecordable median and ulnar nerves sensory nerve action potential (SNAP) and abnormal F waves. After hypokalemia correction there was improvement in CMAPs as well as SNAPs. Thus, hypokalemia can present as an axonal sensorimotor involvement with sural sparing on NCS study similar to Guillain-Barré syndrome subtype.

Evaluation of intravenous magnesium sulphate on haemodynamic responses to laryngoscopy and tracheal intubation in surgeries of ear, nose and throat

Heart rate, arterial blood pressure, and arrhythmias significantly surge during laryngoscopy and intubation. Anesthesiologists constantly look for ways to minimize these unwanted reactions. Several medicinal interventions have been suggested to mitigate these side effects; intravenous magnesium sulphate injection is one such intervention. Magnesium sulphate is a divalent salt widely recognized for its capacity to lower sympathetic nervous system muscle cell excitability, which lowers blood pressure and causes vasodilation. It is approved as a medicine to treat preeclampsia and control blood pressure. Moreover, there is growing recognition of its benefits on hemodynamic parameters in patients suffering from ischemic infective endocarditis and heart disease. Magnesium is an asset for reducing negative cardiovascular reactions occurring at the time of laryngoscopy and intubation because of its capacity to reduce catecholamine synthesis from adrenergic nerve ends and the adrenal medulla. Intravenous magnesium sulphate will be used in this trial to assess its effect on reducing adverse hemodynamic responses in candidates for elective laryngoscopy and intubation for ENT surgeries.

Loss of anoctamin 1 reveals a subtle role for BK channels in lymphatic muscle action potentials.

Ca2+ signalling plays a crucial role in determining lymphatic muscle cell excitability and contractility through its interaction with the Ca2+-activated Cl- channel anoctamin 1 (ANO1). In contrast, the large-conductance (BK) Ca2+-activated K+ channel (KCa) and other KCa channels have prominent vasodilatory actions by hyperpolarizing vascular smooth muscle cells. Here, we assessed the expression and contribution of the KCa family to mouse and rat lymphatic collecting vessel contractile function. The BK channel was the only KCa channel consistently expressed in fluorescence-activated cell sorting-purified mouse lymphatic muscle cell lymphatic muscle cells. We used a pharmacological inhibitor of BK channels, iberiotoxin, and small-conductance Ca2+-activated K+ channels, apamin, to inhibit KCa channels acutely in ex vivo isobaric myography experiments and intracellular membrane potential recordings. In basal conditions, BK channel inhibition had little to no effect on either mouse inguinal-axillary lymphatic vessel (MIALV) or rat mesenteric lymphatic vessel contractions or action potentials (APs). We also tested BK channel inhibition under loss of ANO1 either by genetic ablation (Myh11CreERT2-Ano1 fl/fl, Ano1ismKO) or by pharmacological inhibition with Ani9. In both Ano1ismKO MIALVs and Ani9-pretreated MIALVs, inhibition of BK channels increased contraction amplitude, increased peak AP and broadened the peak of the AP spike. In rat mesenteric lymphatic vessels, BK channel inhibition also abolished the characteristic post-spike notch, which was exaggerated with ANO1 inhibition, and significantly increased the peak potential and broadened the AP spike. We conclude that BK channels are present and functional on mouse and rat lymphatic muscle cells but are otherwise masked by the dominance of ANO1. KEY POINTS: Mouse and rat lymphatic muscle cells express functional BK channels. BK channels make little contribution to either rat or mouse lymphatic collecting vessel contractile function in basal conditions across a physiological pressure range. ANO1 limits the peak membrane potential achieved in the action potential and sets a plateau potential limiting the voltage-dependent activation of BK. BK channels are activated when ANO1 is absent or blocked and slightly impair contractile strength by reducing the peak membrane potential achieved in the action potential spike and accelerating the post-spike repolarization.

From Psychostasis to the Discovery of Cardiac Nerves: The Origins of the Modern Cardiac Neuromodulation Concept.

This review explores the historical development of cardiology knowledge, from ancient Egyptian psychostasis to the modern comprehension of cardiac neuromodulation. In ancient Egyptian religion, psychostasis was the ceremony in which the deceased was judged before gaining access to the afterlife. This ritual was also known as the "weighing of the heart" or "weighing of the soul". The Egyptians believed that the heart, not the brain, was the seat of human wisdom, emotions, and memory. They were the first to recognize the cardiocentric nature of the body, identifying the heart as the center of the circulatory system. Aristotle (fourth century BC) considered the importance of the heart in human physiology in his philosophical analyses. For Galen (third century AD), the heart muscle was the site of the vital spirit, which regulated body temperature. Cardiology knowledge advanced significantly in the 15th century, coinciding with Leonardo da Vinci and Vesalius's pioneering anatomical and physiological studies. It was William Harvey, in the 17th century, who introduced the concept of cardiac circulation. Servet's research and Marcello Malpighi's discovery of arterioles and capillaries provided a more detailed understanding of circulation. Richard Lower emerged as the foremost pioneer of experimental cardiology in the late 17th century. He demonstrated the heart's neural control by tying off the vagus nerve. In 1753, Albrecht von Haller, a professor at Göttingen, was the first to discover the heart's automaticity and the excitation of muscle fibers. Towards the end of the 18th century, Antonio Scarpa challenged the theories of Albrecht von Haller and Johann Bernhard Jacob Behrends, who maintained that the myocardium possessed its own "irritability", on which the heartbeat depended, and was independent of neuronal sensitivity. Instead, Scarpa argued that the heart required innervation to maintain life, refuting Galenic notions. In contemporary times, the study of cardiac innervation has regained prominence, particularly in understanding the post-acute sequelae of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) infection (PASC), which frequently involves cardiorespiratory symptoms and dysregulation of the intrinsic cardiac innervation. Recently, it has been recognized that post-acute sequelae of acute respiratory infections (ARIs) due to other pathogens can also be a cause of long-term vegetative and somatic symptoms. Understanding cardiac innervation and modulation can help to recognize and treat long COVID and long non-COVID-19 (coronavirus disease 2019) ARIs. This analysis explores the historical foundations of cardiac neuromodulation and its contemporary relevance. By focusing on this concept, we aim to bridge the gap between historical understanding and modern applications. This will illuminate the complex interplay between cardiac function, neural modulation, cardiovascular health, and disease management in the context of long-term cardiorespiratory symptoms and dysregulation of intrinsic cardiac innervations.

Magnesium Sulfate and Its Versatility in Anesthesia: A Comprehensive Review.

In the field of general anesthesia, magnesium sulfate (MgSO4) has become a valuable adjunct because it provides a range of benefits that enhance and optimize conventional aesthetic procedures. This review highlights the various intra-anesthetic benefits of MgSO4 while examining its complex function in the treatment using anesthesia. Magnesium inhibits the release of acetylcholine at the motor endplate and blocks calcium channels at presynaptic nerve terminals. This reduces the amplitude of endplate potential and the excitability of muscle fibers, which increases the potency of a neuromuscular blockade by nondepolarizing neuromuscular blockers. This activity may lessen the need for primary muscle relaxants. Moreover, its capacity to potentially reduce the total amount of main aesthetic agents needed emphasizes its function in maximizing anesthesia dosage, ensuring sufficient depth while perhaps potentially reducing adverse effects linked with increased dosages. MgSO4's adaptable qualities present a viable path for improving anesthetic outcomes, possibly improving patient safety and improving surgical results.

Genetically engineered HEK cells as a valuable tool for studying electroporation in excitable cells

Electric pulses used in electroporation-based treatments have been shown to affect the excitability of muscle and neuronal cells. However, understanding the interplay between electroporation and electrophysiological response of excitable cells is complex, since both ion channel gating and electroporation depend on dynamic changes in the transmembrane voltage (TMV). In this study, a genetically engineered human embryonic kidney cells expressing NaV1.5 and Kir2.1, a minimal complementary channels required for excitability (named S-HEK), was characterized as a simple cell model used for studying the effects of electroporation in excitable cells. S-HEK cells and their non-excitable counterparts (NS-HEK) were exposed to 100 µs pulses of increasing electric field strength. Changes in TMV, plasma membrane permeability, and intracellular Ca2+ were monitored with fluorescence microscopy. We found that a very mild electroporation, undetectable with the classical propidium assay but associated with a transient increase in intracellular Ca2+, can already have a profound effect on excitability close to the electrostimulation threshold, as corroborated by multiscale computational modelling. These results are of great relevance for understanding the effects of pulse delivery on cell excitability observed in context of the rapidly developing cardiac pulsed field ablation as well as other electroporation-based treatments in excitable tissues.

Modulation of sarcopenia phenotypes by glutathione peroxidase 4 overexpression in mice.

Our laboratory previously showed lipid hydroperoxides and oxylipin levels are elevated in response to loss of skeletal muscle innervation and are associated with muscle pathologies. To elucidate the pathological impact of lipid hydroperoxides, we overexpressed glutathione peroxidase 4 (GPx4), an enzyme that targets reduction of lipid hydroperoxides in membranes, in adult CuZn superoxide dismutase knockout (Sod1KO) mice that show accelerated muscle atrophy associated with loss of innervation. The gastrocnemius muscle from Sod1KO mice shows reduced mitochondrial respiration and elevated oxidative stress (F2 -isoprostanes and hydroperoxides) compared to wild-type (WT) mice. Overexpression of GPx4 improved mitochondrial respiration and reduced hydroperoxide generation in Sod1KO mice, but did not attenuate the muscle loss that occurs in Sod1KO mice. In contrast, contractile force generation is reduced in EDL muscle in Sod1KO mice relative to WT mice, and overexpression of GPx4 restored force generation to WT levels in Sod1KO mice. GPx4 overexpression also prevented loss of muscle contractility at the single fibre level in fast-twitch fibres from Sod1KO mice. Muscle fibres from Sod1KO mice were less sensitive to both depolarization and calcium at the single fibre level and exhibited a reduced activation by S-glutathionylation. GPx4 overexpression in Sod1KO mice rescued the deficits in both membrane excitability and calcium sensitivity of fast-twitch muscle fibres. Overexpression of GPx4 also restored the sarco/endoplasmic reticulum Ca2+ -ATPase activity in Sod1KO gastrocnemius muscles. These data suggest that GPx4 plays an important role in preserving excitation-contraction coupling function and Ca2+ homeostasis, and in maintaining muscle and mitochondrial function in oxidative stress-induced sarcopenia. KEY POINTS: Knockout of CuZn superoxide dismutase (Sod1KO) induces elevated oxidative stress with accelerated muscle atrophy and weakness. Glutathione peroxidase 4 (GPx4) plays a fundamental role in the reduction of lipid hydroperoxides in membranes, and overexpression of GPx4 improves mitochondrial respiration and reduces hydroperoxide generation in Sod1KO mice. Muscle contractile function deficits in Sod1KO mice are alleviated by the overexpression of GPx4. GPx4 overexpression in Sod1KO mice rescues the impaired muscle membrane excitability of fast-twitch muscle fibres and improves their calcium sensitivity. Sarco/endoplasmic reticulum Ca2+ -ATPase activity in Sod1KO muscles is decreased, and it is restored by the overexpression of GPx4. Our results confirm that GPx4 plays an important role in preserving excitation-contraction coupling function and Ca2+ homeostasis, and maintaining muscle and mitochondrial function in oxidative stress-induced sarcopenia.

Epileptic Channelopathies and Neuromuscular Disorders in Newborns: A Narrative Review.

Neonates can have ion channel abnormalities known as channelopathies, which can impact any organ system. These abnormalities cause seizures, which can result in developmental delays and lead to early death. For a child's long-term neurodevelopment, early identification as a channelopathy is essential to avoid any brain damage. Therefore, this review aims to focus on early diagnostic criteria. Since it might be difficult for doctors to interpret the presenting symptoms of channelopathies, a thorough diagnostic examination that follows a methodical step-by-step procedure is essential. Skeletal muscle fiber and neuron excitability depend on voltage-gated sodium channels. It is now known that mutations in voltage-gated sodium channel genes can cause a growing variety of fatal or debilitating pediatric neurological diseases. Episodic paralysis, myotonia, newborn hypotonia, respiratory impairment, laryngospasm/stridor, congenital myasthenia, and myopathy are examples of muscle phenotypes. There may be a connection between sodium channel malfunction and abrupt infant death, according to recent findings. Numerous epilepsy syndromes and complex encephalopathies are among the manifestations of different channelopathies that are becoming more widely recognized.

Retigabine suppresses loss of force in mouse models of hypokalaemic periodic paralysis.

Recurrent episodes of weakness in periodic paralysis are caused by intermittent loss of muscle fibre excitability, as a consequence of sustained depolarization of the resting potential. Repolarization is favoured by increasing the fibre permeability to potassium. Based on this principle, we tested the efficacy of retigabine, a potassium channel opener, to suppress the loss of force induced by a low-K+ challenge in hypokalaemic periodic paralysis (HypoPP). Retigabine can prevent the episodic loss of force in HypoPP. Knock-in mutant mouse models of HypoPP (Cacna1s p.R528H and Scn4a p.R669H) were used to determine whether pre-treatment with retigabine prevented the loss of force, or post-treatment hastened recovery of force for a low-K+ challenge in an ex vivo contraction assay. Retigabine completely prevents the loss of force induced by a 2 mM K+ challenge (protection) in our mouse models of HypoPP, with 50% inhibitory concentrations of 0.8 ± 0.13 μM and 2.2 ± 0.42 μM for NaV1.4-R669H and CaV1.1-R528H, respectively. In comparison, the effective concentration for the KATP channel opener pinacidil was 10-fold higher. Application of retigabine also reversed the loss of force (rescue) for HypoPP muscle maintained in 2 mM K+. Our findings show that retigabine, a selective agonist of the KV7 family of potassium channels, is effective for the prevention of low-K+ induced attacks of weakness and to enhance recovery from an ongoing loss of force in mouse models of type 1 (Cacna1s) and type 2 (Scn4a) HypoPP. Substantial protection from the loss of force occurred in the low micromolar range, well within the therapeutic window for retigabine.

Chloride channel inhibition improves neuromuscular function under conditions mimicking neuromuscular disorders.

The skeletal muscle Cl- channels, the ClC-1 channels, stabilize the resting membrane potential and dampen muscle fibre excitability. This study explored whether ClC-1 inhibition can recover nerve-stimulated force in isolated muscle under conditions of compromised neuromuscular transmission akin to disorders of myasthenia gravis and Lambert-Eaton syndrome. Nerve-muscle preparations were isolated from rats. Preparations were exposed to pre-or post-synaptic inhibitors (ω-agatoxin, elevated extracellular Mg2+ , α-bungarotoxin or tubocurarine). The potential of ClC-1 inhibition (9-AC or reduced extracellular Cl- ) to recover nerve-stimulated force under these conditions was assessed. ClC-1 inhibition recovered force in both slow-twitch soleus and fast-twitch EDL muscles exposed to 0.2µmol/L tubocurarine or 3.5mmol/L Mg2+ . Similarly, ClC-1 inhibition recovered force in soleus muscles exposed to α-bungarotoxin or ω-agatoxin. Moreover, the concentrations of tubocurarine and Mg2+ required for reducing force to 50% rose from 0.14±0.02µmol/L and 4.2±0.2mmol/L in control muscles to 0.45±0.03µmol/L and 4.7±0.3mmol/L in muscles with 9-AC respectively (P<.05, paired T test). Inhibition of acetylcholinesterase (neostigmine) and inhibition of voltage-gated K+ channels (4-AP) relieve symptoms in myasthenia gravis and Lambert-Eaton syndrome, respectively. Neostigmine and 9-AC additively increased the tubocurarine concentration required to reduce nerve-stimulated force to 50% (0.56±0.05µmol/L with 9-AC and neostigmine) and, similarly, 4-AP and 9-AC additively increased the Mg2+ concentration required to reduce nerve-stimulated force to 50% (6.5±0.2mmol/L with 9-AC and 4-AP). This study shows that ClC-1 inhibition can improve neuromuscular function in pharmacological models of compromised neuromuscular transmission.

Effects of extracellular potassium on calcium handling and force generation in a model of excitation-contraction coupling in skeletal muscle

It is well-established that extracellular potassium (Ko+) accumulation reduces muscle fiber excitability, however the effects of Ko+ on the excitation–contraction coupling (ECC) pathway are less understood. In vivo and in vitro studies following fatiguing stimulation protocols are limited in their ability to capture the effects of Ko+ on force production in combination with other simultaneously changing factors. To address this, a computational model of ECC for slow and fast twitch muscle is presented to explore the relative contributions of excitability-induced and metabolic-induced changes in force generation in response to increasing K+o. The model incorporates mechanisms previously unexplored in modelling studies, including the effects of extracellular calcium on excitability, calcium-dependent inhibition of calcium release, ATP-dependent ionic pumping, and the contribution of ATP hydrolysis to intracellular phosphate accumulation rate. The model was able to capture the frequency-dependent biphasic Force-K+o response observed experimentally. Force potentiation for moderately elevated K+o was driven by increased action potential duration, myoplasmic calcium potentiation, and phosphate accumulation rate, while attenuation of force at higher K+o was due to action potential failure resulting in reduced calcium release. These results suggest that altered calcium release and phosphate accumulation work together with elevated Ko+ to affect force during sustained contractions.

Can a chloride channel blocker mitigate muscle fatigue?

Can a chloride channel blocker mitigate muscle fatigue?

Markov and Eyring Modeling Predict Differential Contributions of Homologous Mutations in Hypokalemic Periodic Paralysis to Skeletal Muscle Fiber Excitability

Hypokalemic periodic paralysis is caused by mutations in the S4 region of voltage-gated sodium or calcium channels found in skeletal muscle. These mutations elicit a non-canonical omega current observed as a proton- or cationic leak through the voltage sensor domain in these channels. S4 mutations also produce defects in channel activation and inactivation. Here, we compared the impact of gating defects for homologous S4:R2 hNaV1.4 mutations on the excitability of muscle fibers. For domain I, R222G was identified in an index family as the clinical outcome of this mutation has not previously been described.

Potassium channel Shaker play a protective role against cardiac aging in Drosophila.

Potassium channels, which are the most diverse group of the ion channel family, play an important role in the repolarization of cardiomyocytes. Recent studies showed that potassium channels, such as KCNQ and HERG/eag, play an important role in regulating adult heart function through shaping the action potential and maintaining the rhythm of cardiac contraction. The potassium channel protein Shaker is the first voltage-gated potassium channel found in Drosophila to maintain the electrical excitability of neurons and muscle cells, but its role in adult cardiac function is still unclear. In this study, Drosophila was used as a model to study the role of Shaker channel in the maintenance of cardiac function under stress and aging. The incidence of heart failure was observed in shaker mutant after external electrical pacing, which simulates cardiac stress. Additionally, The cardiac-specific driver hand4.2 Gal4 was used to specifically knock down the expression of the potassium channel shaker in Drosophila. The cardiac parameter was analyzed at 1, 3, 5 weeks of age on cardiac specific knockdown of shaker using Drosophila adult cardiac physiological assay. The results showed that the mutation of shaker gene seriously affect the cardiac function under stress, demonstrated by significant increase in heart failure rate under electrical stimulation. In addition, cardiac specific knockdown of shaker increased the incidence of arrhythmias in Drosophila at the age of 5 weeks. Cardiac-specific knockdown of shaker reduces life span. Therefore, the results of this study suggest a vital role of the potassium channel shaker in maintaining normal cardiac function during aging.

Next-generation sequencing application to investigate skeletal muscle channelopathies in a large cohort of Italian patients

Non-dystrophic myotonias and periodic paralyses are a heterogeneous group of disabling diseases classified as skeletal muscle channelopathies. Their genetic characterization is essential for prognostic and therapeutic purposes; however, several genes are involved. Sanger-based sequencing of a single gene is time-consuming, often expensive; thus, we designed a next-generation sequencing panel of 56 putative candidate genes for skeletal muscle channelopathies, codifying for proteins involved in excitability, excitation-contraction coupling, and metabolism of muscle fibres. We analyzed a large cohort of 109 Italian patients with a suspect of NDM or PP by next-generation sequencing. We identified 24 patients mutated in CLCN1 gene, 15 in SCN4A, 3 in both CLCN1 and SCN4A, 1 in ATP2A1, 1 in KCNA1 and 1 in CASQ1. Eight were novel mutations: p.G395Cfs*32, p.L843P, p.V829M, p.E258E and c.1471+4delTCAAGAC in CLCN1, p.K1302R in SCN4A, p.L208P in ATP2A1 and c.280–1G>C in CASQ1 genes. This study demonstrated the utility of targeted next generation sequencing approach in molecular diagnosis of skeletal muscle channelopathies and the importance of the collaboration between clinicians and molecular geneticists and additional methods for unclear variants to make a conclusive diagnosis.

MUSCLE FUNCTION & HOMEOSTASIS / MOLECULAR THERAPEUTIC APPROACHES: P.73 Depletion of ATP limits membrane excitability of skeletal muscle by increasing both ClC1-open probability and membrane conductance

Activation of skeletal muscle contractions require that action potentials can be excited and propagated along the muscle fibers. It is known that muscle fiber excitability is regulated during repeated firing of action potentials by cellular signalling systems that control the function of ion channels that determine the resting membrane conductance (Gm). In fast-twitch muscle, prolonged firing of action potentials triggers a marked increase in Gm, reducing muscle fiber excitability and causing action potential failure.

Divalent cations permeation in a Ca2+ non-conducting skeletal muscle dihydropyridine receptor mouse model

In response to excitation of skeletal muscle fibers, trains of action potentials induce changes in the configuration of the dihydropyridine receptor (DHPR) anchored in the tubular membrane which opens the Ca2+ release channel in the sarcoplasmic reticulum membrane. The DHPR also functions as a voltage-gated Ca2+ channel that conducts L-type Ca2+ currents routinely recorded in mammalian muscle fibers, which role was debated for more than four decades. Recently, to allow a closer look into the role of DHPR Ca2+ influx in mammalian muscle, a knock-in (ki) mouse model (ncDHPR) carrying mutation N617D (adjacent to domain II selectivity filter E) in the DHPRα1S subunit abolishing Ca2+ permeation through the channel was generated [Dayal et al., 2017]. In the present study, the Mn2+ quenching technique was initially intended to be used on voltage-clamped muscle fibers from this mouse to determine whether Ca2+ influx through a pathway distinct from DHPR may occur to compensate for the absence of DHPR Ca2+ influx. Surprisingly, while N617D DHPR muscle fibers of the ki mouse do not conduct Ca2+, Mn2+ entry and subsequent quenching did occur because Mn2+ was able to permeate and produce L-type currents through N617D DHPR. N617D DHPR was also found to conduct Ba2+ and Ba2+ currents were strongly blocked by external Ca2+. Ba2+ permeation was smaller, current kinetics slower and Ca2+ block more potent than in wild-type DHPR. These results indicate that residue N617 when replaced by the negatively charged residue D is suitably located at entrance of the pore to trap external Ca2+ impeding in this way permeation. Because Ba2+ binds with lower affinity to D, Ba2+ currents occur, but with reduced amplitudes as compared to Ba2+ currents through wild-type channels. We conclude that mutations located outside the selectivity filter influence channel permeation and possibly channel gating in a fully differentiated skeletal muscle environment.

Depletion of ATP Limits Membrane Excitability of Skeletal Muscle by Increasing Both ClC1-Open Probability and Membrane Conductance

Activation of skeletal muscle contractions require that action potentials can be excited and propagated along the muscle fibers. Recent studies have revealed that muscle fiber excitability is regulated during repeated firing of action potentials by cellular signaling systems that control the function of ion channel that determine the resting membrane conductance (Gm). In fast-twitch muscle, prolonged firing of action potentials triggers a marked increase in Gm, reducing muscle fiber excitability and causing action potential failure. Both ClC-1 and KATP ion channels contribute to this Gm rise, but the exact molecular regulation underlying their activation remains unclear. Studies in expression systems have revealed that ClC-1 is able to bind adenosine nucleotides, and that low adenosine nucleotide levels result in ClC-1 activation. In three series of experiments, this study aimed to explore whether ClC-1 is also regulated by adenosine nucleotides in native skeletal muscle fibers, and whether the adenosine nucleotide sensitivity of ClC-1 could explain the rise in Gm muscle fibers during prolonged action potential firing. First, whole cell patch clamping of mouse muscle fibers demonstrated that ClC-1 activation shifted in the hyperpolarized direction when clamping pipette solution contained 0 mM ATP compared with 5 mM ATP. Second, three-electrode Gm measurement during muscle fiber stimulation showed that glycolysis inhibition, with 2-deoxy-glucose or iodoacetate, resulted in an accelerated and rapid >400% Gm rise during short periods of repeated action potential firing in both fast-twitch and slow-twitch rat, and in human muscle fibers. Moreover, ClC-1 inhibition with 9-anthracenecarboxylic acid resulted in either an absence or blunted Gm rise during action potential firing in human muscle fibers. Third, Gm measurement during repeated action potential firing in muscle fibers from a murine McArdle disease model suggest that the rise in Gm was accelerated in a subset of fibers. Together, these results are compatible with ClC-1 function being regulated by the level of adenosine nucleotides in native tissue, and that the channel operates as a sensor of skeletal muscle metabolic state, limiting muscle excitability when energy status is low.

Fructose Consumption Increases Blood Pressure and Induces Changes in Renal Microvascular Function

A drastic increase in fructose consumption over recent decades has been paralleled by a growing prevalence and incidence of hypertension, along with comorbidities, such as diabetes, renal failure, and cardiovascular disease. High fructose corn syrup is a commonly found ingredient in the Western diet. However, the use of fructose as a sweetener has been shown to promote salt‐sensitive hypertension. The mechanisms by which fructose induces an increase in blood pressure and its effect on renal microvascular function are not completely understood. The goal of this study was to understand the mechanisms that lead to fructose‐induced impairment in renal microvascular function and its relation to the development of hypertension. Salt‐resistant Sprague Dawley rats (8 weeks old) were kept on a low salt (LS; 0.4% NaCl) with or without addition of 20% fructose (FT) to the drinking water provided ad libitum. After two weeks control and FT‐treated groups were switched to a high salt (HS; 8% NaCl) diet. Blood pressure was monitored with surgically implanted telemetry devices. Electrolyte balance and changes in renal function were analyzed among experimental groups using urinary (24 hrs collections) and plasma samples. After two weeks on HS, animals were euthanized, and renal microvessels were isolated to assess renal smooth muscle cell (SMC) excitability. We found that FT‐treated animals experienced elevated systolic blood pressure at LS to compare with control, and this difference were significantly higher in HS‐treated groups (107 ± 3 vs 113 ± 3, and 114 ± 4 vs 131 ± 5, mmHg; LS vs LS+FT, and HS vs HS +FT, correspondingly). Furthermore, these changes inversely correlated with the shift in diuresis and electrolyte balance in HS‐treated animals (70 ± 15 vs 39 ± 15 ml/day, 284 ± 91 vs 123 ± 70 Na/Cre ratio, 36 ± 12 vs 14 ± 10 K/Cre ratio, 0.7 ± 0.4 vs 1.4 ± 0.3 Ca/Cre ratio, HS vs HS+FT correspondingly). No changes in albuminuria, compared with controls, were noted in any of the groups. Two‐photon microscopy analysis of the renal microvascular vasomotion revealed a decrease in amplitude of individual SMC Ca2+ activity at the resting, spontaneous state in both LS and HS FT‐treated groups. However, both amplitude and period of Ca2+ fluctuations in SMC was significantly increased when vessels were exposed to ET‐1 in HS+FT group. Moreover, the amplitude of ET‐1‐induced Ca2+ transient in FT‐treated animals were split into two distinct groups with either higher or lower amplitudes relative to control. Overall, renal microvessels exposed to FT revealed a significant decrease in spontaneous vasomotion, which is accompanying by high responsiveness to FT at the HS with significant decrease in frequency of spontaneous events. These effects further promote high sensitivity (above normal) to ET‐1 stimulation, cell damage and finally low responsiveness (below normal) in FT‐treated renal microvessels. In conclusion, these results indicate the detrimental effects of FT on intracellular Ca2+ homeostasis in SMC, renal microvascular function and blood pressure regulation under high dietary salt.Support or Funding InformationNational Institute of Health R35 HL135749 (to A.S.), Michael Keelan, Jr., MD, CVC Research Foundation Grant (to O.P.), and Advancing a Healthier Wisconsin Endowment (to J.D.I.)

Single Amino Acid Changes in the Ryanodine Receptor in the Human Population Have Effects In Vivo on Caenorhabditis elegans Neuro-Muscular Function.

The ryanodine receptor mediates intracellular calcium ion release with excitation of nerve and muscle cells. Ryanodine receptor missense variants cause a number of myopathologies, such as malignant hyperthermia, and have been linked with various neuropathologies, including Alzheimer’s disease. We characterized the consequences of ryanodine receptor variants in vivo. Eight Caenorhabditis elegans strains, with ryanodine receptor modifications equivalent to human myopathic RYR1 variants, were generated by genome editing. In humans, these variants are rare and confer sensitivity to the inhalational anaesthetic halothane when heterozygous. Increased sensitivity to halothane was found in both homozygous and heterozygous C. elegans. Close analysis revealed distinct subtle locomotion defects, due to the different single amino acid residue changes, even in the absence of the external triggering agent. Distinct pre- and postsynaptic consequences of the variants were characterized through the responses to cholinergic pharmacological agents. The range of phenotypes reflects the complexity of the regulatory inputs to the ryanodine receptor and the criticality of the calcium ion channel opening properties, in different cell types and with age. Ryanodine receptors with these single amino acid residue changes still function as calcium ion channels, but with altered properties which are likely to have subtle consequences for human carriers of such variants. The long-term consequences of subtly altered calcium ion signalling could be cumulative and may be focussed in the smaller nerve cells rather than the more robust muscle cells. It was important to assess phenotypes in vivo to properly appreciate consequences for a whole organism.