Exploring lipin1 as a promising therapeutic target for the treatment of Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive and devastating muscle disease, resulting from the absence of dystrophin. This leads to cell membrane instability, susceptibility to contraction-induced muscle damage, subsequent muscle degeneration, and eventually disability and early death of patients. Currently, there is no cure for DMD. Our recent studies identified that lipin1 plays a critical role in maintaining myofiber stability and integrity. However, lipin1 gene expression levels are dramatically reduced in the skeletal muscles of DMD patients and mdx mice. METHODS: To identify whether increased lipin1 expression could prevent dystrophic pathology, we employed unique muscle-specific mdx:lipin1 transgenic (mdx:lipin1Tg/0) mice in which lipin1 was restored in the dystrophic muscle of mdx mice, intramuscular gene delivery, as well as cell culture system. RESULTS: We found that increased lipin1 expression suppressed muscle degeneration and inflammation, reduced fibrosis, strengthened membrane integrity, and resulted in improved muscle contractile and lengthening force, and muscle performance in mdx:lipin1Tg/0 compared to mdx mice. To confirm the role of lipin1 in dystrophic muscle, we then administered AAV1-lipin1 via intramuscular injection in mdx mice. Consistently, lipin1 restoration inhibited myofiber necroptosis and lessened muscle degeneration. Using a cell culture system, we further found that differentiated primary mdx myoblasts had elevated expression levels of necroptotic markers and medium creatine kinase (CK), which could be a result of sarcolemmal damage. Most importantly, increased lipin1 expression levels in differentiated myoblasts from mdx:lipin1Tg/0 mice substantially inhibited the elevation of necroptotic markers and medium CK levels. CONCLUSIONS: Overall, our data suggest that lipin1 is a promising therapeutic target for the treatment of dystrophic muscles.

Voltage sensor current, SR Ca2+ release, and Ca2+ channel current during trains of action potential-like depolarizations of skeletal muscle fibers.

In skeletal muscle, CaV 1.1 serves as the voltage sensor for both excitation-contraction coupling (ECC) and L-type Ca2+ channel activation. We have recently adapted the technique of action potential (AP) voltage clamp (APVC) to monitor the current generated by the movement of intramembrane voltage sensors (IQ ) during single imposed transverse tubular AP-like depolarization waveforms (IQAP ). We now extend this procedure to monitoring IQAP , and Ca2+ currents during trains of tubular AP-like waveforms in adult murine skeletal muscle fibers, and compare them with the trajectories of APs and AP-induced Ca2+ release measured in other fibers using field stimulation and optical probes. The AP waveform remains relatively constant during brief trains (<1 sec) for propagating APs in non-V clamped fibers. Trains of 10 AP-like depolarizations at 10 Hz (900 ms), 50 Hz (180 ms), or 100 Hz (90 ms) did not alter IQAP amplitude or kinetics, consistent with previous findings in isolated muscle fibers where negligible charge immobilization occurred during 100 ms step depolarizations. Using field stimulation, Ca2+ release did exhibit a considerable decline from pulse to pulse during the train, also consistent with previous findings, indicating that the decline of Ca2+ release during a short train of APs is not correlated to modification of charge movement. Ca2+ currents during single or 10 Hz trains of AP-like depolarizations were hardly detectable, were minimal during 50 Hz trains, and became more evident during 100 Hz trains in some fibers. Our results verify predictions on the behavior of the ECC machinery in response to AP-like depolarizations and provide a direct demonstration that Ca2+ currents elicited by single AP-like waveforms are negligible, but can become more prominent in some fibers during short high-frequency train stimulation that elicits maximal isometric force.

Lipin1 plays complementary roles in myofibre stability and regeneration in dystrophic muscles.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by dystrophin mutations, leading to the loss of sarcolemmal integrity, and resulting in progressive myofibre necrosis and impaired muscle function. Our previous studies suggest that lipin1 is important for skeletal muscle regeneration and myofibre integrity. Additionally, we discovered that mRNA expression levels of lipin1 were significantly reduced in skeletal muscle of DMD patients and the mdx mouse model. To understand the role of lipin1 in dystrophic muscle, we generated dystrophin/lipin1 double knockout (DKO) mice, and compared the limb muscle pathology and function of wild-type B10, muscle-specific lipin1 deficient (lipin1Myf5cKO ), mdx and DKO mice. We found that further knockout of lipin1 in dystrophic muscle exhibited a more severe phenotype characterized by increased necroptosis, fibrosis and exacerbated membrane damage in DKO compared to mdx mice. In barium chloride-induced muscle injury, both lipin1Myf5cKO and DKO showed prolonged regeneration at day 14 post-injection, suggesting that lipin1 is critical for muscle regeneration. In situ contractile function assays showed that lipin1 deficiency in dystrophic muscle led to reduced specific force production. Using a cell culture system, we found that lipin1 deficiency led to elevated expression levels of necroptotic markers and medium creatine kinase, which could be a result of sarcolemmal damage. Most importantly, restoration of lipin1 inhibited the elevation of necroptotic markers in differentiated primary lipin1-deficient myoblasts. Overall, our data suggests that lipin1 plays complementary roles in myofibre stability and muscle function in dystrophic muscles, and overexpression of lipin1 may serve as a potential therapeutic strategy for dystrophic muscles. KEY POINTS: We identified that lipin1 mRNA expression levels are significantly reduced in skeletal muscles of Duchenne muscular dystrophy patients and mdx mice. We found that further depletion of lipin1 in skeletal muscles of mdx mice induces more severe dystrophic phenotypes, including enhanced myofibre sarcolemma damage, muscle necroptosis, inflammation, fibrosis and reduced specific force production. Lipin1 deficiency leads to elevated expression levels of necroptotic markers, whereas restoration of lipin1 inhibits their expression. Our results suggest that lipin1 is functionally complementary to dystrophin in muscle membrane integrity and muscle regeneration.

A randomized pilot trial of a mobile delivered brief motivational interviewing and behavioral economic alcohol intervention for emerging adults.

OBJECTIVE: Mobile health (mHealth) interventions show potential to broaden the reach of efficacious alcohol brief motivational interventions (BMIs). However, efficacy is mixed and may be limited by low participant attention and engagement. The present study examined the feasibility, acceptability, and preliminary efficacy of a live text-message delivered BMI in a pilot randomized clinical trial. METHOD: Participants were 66 college students (63.6% women; 61.9% White; Mage = 19.95, SD = 1.66) reporting an average of 11.88 (SD = 8.74) drinks per week, 4.42 (SD = 3.59) heavy drinking episodes (HDEs), and 8.44 (SD = 5.62) alcohol-related problems in the past month. Participants were randomized to receive either (a) education or (b) an alcohol BMI plus behavioral economic substance-free activity session (SFAS), each followed by 4 weeks of mini sessions. All sessions were administered via live text-message. Participants completed assessments postintervention (after the 4th mini session) and at 3-month follow-up. RESULTS: 90.9% completed both initial full-length sessions and at least two of the four mini sessions with 87.9% retention at 3-month follow-up. Participants found the interventions useful, interesting, relevant, and effective, with no between-group differences. There were no statistically significant group differences in drinks per week or alcohol-related problems at follow-up, but BMI + SFAS participants reported fewer past-month HDEs than those who received education. CONCLUSIONS: Live text-messaging to deliver the BMI + SFAS is feasible and well-received. The preliminary efficacy results should be interpreted cautiously due to the small sample size but support further investigation. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Plateau potentials contribute to myotonia in mouse models of myotonia congenita.

It has long been accepted that myotonia (muscle stiffness) in patients with muscle channelopathies is due to myotonic discharges (involuntary firing of action potentials). In a previous study, we identified a novel phenomenon in myotonic muscle: development of plateau potentials, transient depolarizations to near -35 mV lasting for seconds to minutes. In the current study we examined whether plateau potentials contribute to myotonia. A recessive genetic model (ClCadr mice) with complete loss of muscle chloride channel (ClC-1) function was used to model severe myotonia congenita with complete loss of ClC-1 function and a pharmacologic model using anthracene-9-carboxylic acid (9 AC) was used to model milder myotonia congenita with incomplete loss of ClC-1 function. Simultaneous measurements of action potentials and myoplasmic Ca2+ from individual muscle fibers were compared to recordings of whole muscle force generation. In ClCadr muscle both myotonia and plateau potentials lasted 10s of seconds to minutes. During plateau potentials lasting 1-2 min, there was a gradual transition from high to low intracellular Ca2+, suggesting a transition in individual fibers from myotonia to flaccid paralysis in severe myotonia congenita. In 9 AC-treated muscles, both myotonia and plateau potentials lasted only a few seconds and Ca2+ remained elevated during the plateau potentials, suggesting plateau potentials contribute to myotonia without causing weakness. We propose, that in myotonic muscle, there is a novel state in which there is contraction in the absence of action potentials. This discovery provides a mechanism to explain reports of patients with myotonia who suffer from electrically silent muscle contraction lasting minutes.

A structural mechanism of nuclear receptor biased agonism.

Efforts to decrease the adverse effects of nuclear receptor (NR) drugs have yielded experimental agonists that produce better outcomes in mice. Some of these agonists have been shown to cause different, not just less intense, on-target transcriptomic effects; however, a structural explanation for such agonist-specific effects remains unknown. Here, we show that partial agonists of the NR peroxisome proliferator-associated receptor γ (PPARγ), which induce better outcomes in mice compared to clinically utilized type II diabetes PPARγ-binding drugs thiazolidinediones (TZDs), also favor a different group of coactivator peptides than the TZDs. We find that PPARγ full agonists can also be biased relative to each other in terms of coactivator peptide binding. We find differences in coactivator-PPARγ bonding between the coactivator subgroups which allow agonists to favor one group of coactivator peptides over another, including differential bonding to a C-terminal residue of helix 4. Analysis of all available NR-coactivator structures indicates that such differential helix 4 bonding persists across other NR-coactivator complexes, providing a general structural mechanism of biased agonism for many NRs. Further work will be necessary to determine if such bias translates into altered coactivator occupancy and physiology in cells.

The role of action potential changes in depolarization-induced failure of excitation contraction coupling in mouse skeletal muscle.

Excitation-contraction coupling (ECC) is the process by which electrical excitation of muscle is converted into force generation. Depolarization of skeletal muscle resting potential contributes to failure of ECC in diseases such as periodic paralysis, intensive care unit acquired weakness and possibly fatigue of muscle during vigorous exercise. When extracellular K+ is raised to depolarize the resting potential, failure of ECC occurs suddenly, over a narrow range of resting potentials. Simultaneous imaging of Ca2+ transients and recording of action potentials (APs) demonstrated failure to generate Ca2+ transients when APs peaked at potentials more negative than -30mV. An AP property that closely correlated with failure of the Ca2+ transient was the integral of AP voltage with respect to time. Simultaneous recording of Ca2+ transients and APs with electrodes separated by 1.6mm revealed AP conduction fails when APs peak below -21mV. We hypothesize propagation of APs and generation of Ca2+ transients are governed by distinct AP properties: AP conduction is governed by AP peak, whereas Ca2+ release from the sarcoplasmic reticulum is governed by AP integral. The reason distinct AP properties may govern distinct steps of ECC is the kinetics of the ion channels involved. Na channels, which govern propagation, have rapid kinetics and are insensitive to AP width (and thus AP integral) whereas Ca2+ release is governed by gating charge movement of Cav1.1 channels, which have slower kinetics such that Ca2+ release is sensitive to AP integral. The quantitative relationships established between resting potential, AP properties, AP conduction and Ca2+ transients provide the foundation for future studies of failure of ECC induced by depolarization of the resting potential.

Episodic future thinking as a brief alcohol intervention for heavy drinking college students: A pilot feasibility study.

Episodic future thinking (EFT), an exercise that involves cognitive simulation of future events, has demonstrated proximal effects on alcohol demand and delayed reward discounting (DRD). However, few studies have investigated EFT's potential to reduce alcohol use and increase positive behaviors outside of the laboratory. This study is the first to pilot an academic goal-relevant EFT (A-EFT) intervention for heavy drinking college students. The primary goals were to evaluate the feasibility and acceptability of A-EFT in this population. A secondary goal was to evaluate between- and within-condition changes from baseline to post-intervention and 1-month assessments. Participants were 45 undergraduates (73% women, 53% White, 27% Black) who reported at least two past-month heavy drinking episodes (4/5 drinks for women/men). Participants were randomized to a brief in-person A-EFT intervention or to a vivid memory task (VMT) control. In support of feasibility and acceptability, recruitment and retention rates were over 80% and participants rated the A-EFT intervention as enjoyable, personally relevant, and interesting. In support of initial efficacy, participants assigned to A-EFT increased the amount of time spent studying compared to controls (d = 1.16) at 1-month assessment. Participants in the A-EFT group reported moderate effect size reductions in alcohol consumption, and a similar size increase in protective drinking strategies. Demand intensity decreased moderately at post-intervention for participants in the A-EFT condition but there was little change in DRD. The current study provides support for the feasibility and acceptability of an academic goal-relevant EFT intervention and for further research to establish treatment efficacy. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Acetylcholine receptor subunit expression in Huntington's disease mouse muscle.

Huntington's disease (HD) causes neurological impairments, as well as muscle dysfunction, including smaller neuromuscular junctions (NMJs). This study assessed the expression levels of the subunits of the nicotinic acetylcholine receptor (nAChR) in muscles of the R6/2 mouse model of HD. Based on our previous findings of reduced NMJ size in R6/2 mice, it was hypothesized that muscles from R6/2 mice would also show an altered expression pattern of nAChR subunits compared to wild-type (WT) mice. Therefore, the mRNA levels of nAChR subunits were quantified in R6/2 and WT mouse muscles using qRT-PCR. Denervated muscles from WT mice served as positive controls for alterations in nAChR expression. Although some changes in nAChR subunit expression occurred in R6/2 muscles, the expression levels closely resembled WT. However, the expression of nAChR subunit-ε (Chrne) was significantly decreased in R6/2 muscles relative to WT. This study demonstrates that only minor changes in nAChR subunit expression occurs in R6/2 mouse muscles and that reduction in Chrne expression may be related to a reduction in NMJ size in R6/mice.

Into the spotlight: RGK proteins in skeletal muscle.

The RGK (Rad, Rem, Rem2 and Gem/Kir) family of small GTPases are potent endogenous inhibitors of voltage-gated Ca2+ channels (VGCCs). While the impact of RGK proteins on cardiac physiology has been investigated extensively, much less is known regarding their influence on skeletal muscle biology. Thus, the purpose of this article is to establish a basis for future investigation into the role of RGK proteins in regulating the skeletal muscle excitation-contraction (EC) coupling complex via modulation of the L-type CaV1.1 VGCC. The pathological consequences of elevated muscle RGK protein expression in Type II Diabetes, Amyotrophic Lateral Sclerosis (ALS), Duchenne's Muscular Dystrophy and traumatic nerve injury are also discussed.

The mechanism underlying transient weakness in myotonia congenita.

In addition to the hallmark muscle stiffness, patients with recessive myotonia congenita (Becker disease) experience debilitating bouts of transient weakness that remain poorly understood despite years of study. We performed intracellular recordings from muscle of both genetic and pharmacologic mouse models of Becker disease to identify the mechanism underlying transient weakness. Our recordings reveal transient depolarizations (plateau potentials) of the membrane potential to -25 to -35 mV in the genetic and pharmacologic models of Becker disease. Both Na+ and Ca2+ currents contribute to plateau potentials. Na+ persistent inward current (NaPIC) through NaV1.4 channels is the key trigger of plateau potentials and current through CaV1.1 Ca2+ channels contributes to the duration of the plateau. Inhibiting NaPIC with ranolazine prevents the development of plateau potentials and eliminates transient weakness in vivo. These data suggest that targeting NaPIC may be an effective treatment to prevent transient weakness in myotonia congenita.

Myotonia is a neuromuscular condition that causes problems with the relaxation of muscles following voluntary movements. One type of myotonia is Becker disease, also called recessive myotonia congenita. This is a genetic condition that causes muscle stiffness as a result of involuntary muscle activity. Patients may also suffer transient weakness for a few seconds or as long as several minutes after initiating a movement. The cause of these bouts of temporary weakness is still unclear, but there are hints that it could be linked to the muscle losing its excitability, the ability to respond to the stimuli that make it contract. However, this is at odds with findings that show that muscles in Becker disease are hyperexcitable. Muscle excitability depends on the presence of different concentrations of charged ions (positively charged sodium, calcium and potassium ions and negatively charged chloride ions) inside and outside of each muscle cells. These different concentrations of ions create an electric potential across the cell membrane, also called the 'membrane potential'. When a muscle cell gets stimulated, proteins on the cell membrane known as ion channels open. This allows the flow of ions between the inside and the outside of the cell, which causes an electrical current that triggers muscle contraction. To better understand the causes behind this muscle weakness, Myers et al. used mice that had either been genetically manipulated or given drugs to mimic Becker disease. By measuring both muscle force and the electrical currents that drive contraction, Myers et al. found that the mechanism underlying post-movement weakness involved a transient change in the concentrations of positively charged ions inside and outside the cells. Further experiments showed that proteins that regulate the passage of both sodium and calcium in and out of the cell ­ called sodium and calcium channels ­ contributed to this change in concentration. In addition, Myers et al. discovered that using a drug called ranolazine to stop sodium ions from entering the cell eliminated transient weakness in live mice. These findings suggest that in Becker disease, muscles cycle rapidly between being hyperexcited or not able to be excited, and that targeting the flow of sodium ions into the cell could be an effective treatment to prevent transient weakness in myotonia congenita. This study paves the way towards the development of new therapies to treat Becker disease as well as other muscle ion channel diseases with transient weakness such as periodic paralysis.

Psychometric evaluation of the Reward Probability Index in emerging adult drinkers.

OBJECTIVE: Diminished access to environmental rewards is an established risk factor for addiction and a focus of many effective treatment approaches. Nevertheless, there is inconsistency in measurement approaches and a need for a psychometrically sound measure. The Reward Probability Index (RPI; Carvalho, Behavior Therapy, 42, 2011, pp. 249-262) is a 20-item self-report rating scale that measures access to and ability to experience psychosocial reward. METHOD: The current studies sought to evaluate the psychometric properties of the RPI in 2 samples of emerging adult heavy drinkers. RESULTS: In Study 1, exploratory factor analysis in a sample of 393 college student drinkers supported a 2-factor model of the RPI (Reward Probability and Environmental Suppressors) after removal of redundant items, and corresponding subscales demonstrated good internal consistency. In Study 2, confirmatory factor analysis with 602 emerging adult drinkers recruited from the community supported the 2-factor model as best fitting after removal of one poor indicator, although absolute fit was only adequate. This 2-factor model demonstrated configural, metric, and scalar invariance across non-college and college subgroups as well as Black and White subgroups. Study 2 also demonstrated that the revised RPI subscales showed significant associations with measures of substance-free activity participation and enjoyment, anhedonia, and depressive symptoms. Furthermore, the study revealed the RPI Environmental Suppressors subscale predicted alcohol-related problems (ß = .25, p < .001) beyond demographic covariates, weekly drinking, and depressive symptoms. CONCLUSIONS: These studies provide evidence for the validity of the RPI as an efficient measure of access to reward among emerging adult heavy drinkers. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

A mouse model of Huntington's disease shows altered ultrastructure of transverse tubules in skeletal muscle fibers.

Huntington's disease (HD) is a fatal and progressive condition with severe debilitating motor defects and muscle weakness. Although classically recognized as a neurodegenerative disorder, there is increasing evidence of cell autonomous toxicity in skeletal muscle. We recently demonstrated that skeletal muscle fibers from the R6/2 model mouse of HD have a decrease in specific membrane capacitance, suggesting a loss of transverse tubule (t-tubule) membrane in R6/2 muscle. A previous report also indicated that Cav1.1 current was reduced in R6/2 skeletal muscle, suggesting defects in excitation-contraction (EC) coupling. Thus, we hypothesized that a loss and/or disruption of the skeletal muscle t-tubule system contributes to changes in EC coupling in R6/2 skeletal muscle. We used live-cell imaging with multiphoton confocal microscopy and transmission electron microscopy to assess the t-tubule architecture in late-stage R6/2 muscle and found no significant differences in the t-tubule system density, regularity, or integrity. However, electron microscopy images revealed that the cross-sectional area of t-tubules at the triad were 25% smaller in R6/2 compared with age-matched control skeletal muscle. Computer simulation revealed that the resulting decrease in the R6/2 t-tubule luminal conductance contributed to, but did not fully explain, the reduced R6/2 membrane capacitance. Analyses of bridging integrator-1 (Bin1), which plays a primary role in t-tubule formation, revealed decreased Bin1 protein levels and aberrant splicing of Bin1 mRNA in R6/2 muscle. Additionally, the distance between the t-tubule and sarcoplasmic reticulum was wider in R6/2 compared with control muscle, which was associated with a decrease in junctophilin 1 and 2 mRNA levels. Altogether, these findings can help explain dysregulated EC coupling and motor impairment in Huntington's disease.

Statistically derived patterns of behavioral economic risk among heavy-drinking college students: A latent profile analysis.

High levels of 3 behavioral economic indices (delay discounting, alcohol demand, and proportionate substance-related reinforcement) are consistently associated with greater alcohol misuse and alcohol-related problems. However, it is unclear whether and how these variables jointly increase the risk for alcohol-related outcomes among college students who engage in heavy episodic drinking (HED; 4/5+ drinks for women/men, respectively). The current study used a person-centered approach to identify similar patterns of behavioral economic domains among heavy-drinking college students and investigate the relationship between these empirically derived classes and alcohol-related outcomes. A sample of 393 college students (60.8% female, 78.9% White/Caucasian) reporting at least 2 heavy drinking episodes in the previous month completed measures of alcohol use and problems, demographics, delay discounting, and alcohol reward value (alcohol demand and proportionate substance-related reinforcement). Latent profile analyses revealed that a 3-class solution provided the best fit to the data: a low reward value, high discounting (LRHD) class (n = 53), a moderate reward value, low discounting (MRLD) class (n = 214), and a high reward value, high discounting (HRHD) class (n = 126). Members of the HRHD class reported significantly greater alcohol consumption, past-month HED episodes, alcohol-related problems, and symptoms of alcohol use disorder than those in the MRLD and LRHD classes. The results suggest that there are 3 constellations of behavioral economic processes and that, consistent with the reinforcer pathology model, students who overvalue alcohol-related reward and discount the future more steeply are at the greatest risk for alcohol misuse and alcohol-related problems. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Loss of membrane integrity drives myofiber death in lipin1-deficient skeletal muscle.

Mutations in lipin1 are suggested to be a common cause of massive rhabdomyolysis episodes in children; however, the molecular mechanisms involved in the regulation of myofiber death caused by the absence of lipin1 are not fully understood. Loss of membrane integrity is considered as an effective inducer of cell death in muscular dystrophy. In this study, we utilized a mouse line with selective homozygous lipin1 deficiency in the skeletal muscle (Lipin1Myf5cKO ) to determine the role of compromised membrane integrity in the myofiber death in lipin1-deficient muscles. We found that Lipin1Myf5cKO muscles had significantly elevated proapoptotic factors (Bax, Bak, and cleaved caspase-9) and necroptotic proteins such as RIPK1, RIPK3, and MLKL compared with WT mice. Moreover, Lipin1Myf5cKO muscle had significantly higher membrane disruptions, as evidenced by increased IgG staining and elevated uptake of Evans Blue Dye (EBD) and increased serum creatine kinase activity in Lipin1Myf5cKO muscle fibers. EBD-positive fibers were strongly colocalized with apoptotic or necroptotic myofibers, suggesting an association between compromised plasma membrane integrity and cell death pathways. We further show that the absence of lipin1 leads to a significant decrease in the absolute and specific muscle force (normalized to muscle mass). Our work indicates that apoptosis and necroptosis are associated with a loss of membrane integrity in Lipin1Myf5cKO muscle and that myofiber death and dysfunction may cause a decrease in contractile force.

Mechanisms of altered skeletal muscle action potentials in the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) patients suffer from progressive and debilitating motor dysfunction for which only palliative treatment is currently available. Previously, we discovered reduced skeletal muscle Cl- channel (ClC-1) and inwardly rectifying K+ channel (Kir) currents in R6/2 HD transgenic mice. To further investigate the role of ClC-1 and Kir currents in HD skeletal muscle pathology, we measured the effect of reduced ClC-1 and Kir currents on action potential (AP) repetitive firing in R6/2 mice using a two-electrode current clamp. We found that R6/2 APs had a significantly lower peak amplitude, depolarized maximum repolarization, and prolonged decay time compared with wild type (WT). Of these differences, only the maximum repolarization was accounted for by the reduction in ClC-1 and Kir currents, indicating the presence of additional ion channel defects. We found that both KV1.5 and KV3.4 mRNA levels were significantly reduced in R6/2 skeletal muscle compared with WT, which explains the prolonged decay time of R6/2 APs. Overall, we found that APs in WT and R6/2 muscle significantly and progressively change during activity to maintain peak amplitude despite buildup of Na+ channel inactivation. Even with this resilience, the persistently reduced peak amplitude of R6/2 APs is expected to result in earlier fatigue and may help explain the motor impersistence experienced by HD patients. This work lays the foundation to link electrical changes to force generation defects in R6/2 HD mice and to examine the regulatory events controlling APs in WT muscle.

TRPV4 Antagonism Prevents Mechanically Induced Myotonia.

OBJECTIVE: Myotonia is caused by involuntary firing of skeletal muscle action potentials and causes debilitating stiffness. Current treatments are insufficiently efficacious and associated with side effects. Myotonia can be triggered by voluntary movement (electrically induced myotonia) or percussion (mechanically induced myotonia). Whether distinct molecular mechanisms underlie these triggers is unknown. Our goal was to identify ion channels involved in mechanically induced myotonia and to evaluate block of the channels involved as a novel approach to therapy. METHODS: We developed a novel system to enable study of mechanically induced myotonia using both genetic and pharmacologic mouse models of myotonia congenita. We extended ex vivo studies of excitability to in vivo studies of muscle stiffness. RESULTS: As previous work suggests activation of transient receptor potential vanilloid 4 (TRPV4) channels by mechanical stimuli in muscle, we examined the role of this cation channel. Mechanically induced myotonia was markedly suppressed in TRPV4-null muscles and in muscles treated with TRPV4 small molecule antagonists. The suppression of mechanically induced myotonia occurred without altering intrinsic muscle excitability, such that myotonia triggered by firing of action potentials (electrically induced myotonia) was unaffected. When injected intraperitoneally, TRPV4 antagonists lessened the severity of myotonia in vivo by approximately 80%. INTERPRETATION: These data demonstrate that there are distinct molecular mechanisms triggering electrically induced and mechanically induced myotonia. Our data indicates that activation of TRPV4 during muscle contraction plays an important role in triggering myotonia in vivo. Elimination of mechanically induced myotonia by TRPV4 inhibition offers a new approach to treating myotonia. ANN NEUROL 2020;88:297-308.

Depressed neuromuscular transmission causes weakness in mice lacking BK potassium channels.

Mice lacking functional large-conductance voltage- and Ca2+-activated K+ channels (BK channels) are viable but have motor deficits including ataxia and weakness. The cause of weakness is unknown. In this study, we discovered, in vivo, that skeletal muscle in mice lacking BK channels (BK-/-) was weak in response to nerve stimulation but not to direct muscle stimulation, suggesting a failure of neuromuscular transmission. Voltage-clamp studies of the BK-/- neuromuscular junction (NMJ) revealed a reduction in evoked endplate current amplitude and the frequency of spontaneous vesicle release compared with WT littermates. Responses to 50-Hz stimulation indicated a reduced probability of vesicle release in BK-/- mice, suggestive of lower presynaptic Ca2+ entry. Pharmacological block of BK channels in WT NMJs did not affect NMJ function, surprisingly suggesting that the reduced vesicle release in BK-/- NMJs was not due to loss of BK channel-mediated K+ current. Possible explanations for our data include an effect of BK channels on development of the NMJ, a role for BK channels in regulating presynaptic Ca2+ current or the effectiveness of Ca2+ in triggering release. Consistent with reduced Ca2+ entry or effectiveness of Ca2+ in triggering release, use of 3,4-diaminopyridine to widen action potentials normalized evoked release in BK-/- mice to WT levels. Intraperitoneal application of 3,4-diaminopyridine fully restored in vivo nerve-stimulated muscle force in BK-/- mice. Our work demonstrates that mice lacking BK channels have weakness due to a defect in vesicle release at the NMJ.

Central Role of Subthreshold Currents in Myotonia.

It is generally thought that muscle excitability is almost exclusively controlled by currents responsible for generation of action potentials. We propose that smaller ion channel currents that contribute to setting the resting potential and to subthreshold fluctuations in membrane potential can also modulate excitability in important ways. These channels open at voltages more negative than the action potential threshold and are thus termed subthreshold currents. As subthreshold currents are orders of magnitude smaller than the currents responsible for the action potential, they are hard to identify and easily overlooked. Discovery of their importance in regulation of excitability opens new avenues for improved therapy for muscle channelopathies and diseases of the neuromuscular junction. ANN NEUROL 2020;87:175-183.

Brief Motivational Interventions Are Associated with Reductions in Alcohol-Induced Blackouts Among Heavy Drinking College Students.

BACKGROUND: Alcohol-induced blackouts, a form of anterograde amnesia that restricts the encoding of short-term memories into long-term ones, are among the most severe alcohol-related consequences. College students are at high risk of experiencing alcohol-induced blackouts, and there is a need to determine whether alcohol interventions can effectively reduce blackouts in this population. The current study uses data from 3 randomized clinical trials to examine the effect of various intervention approaches on alcohol-induced blackouts. METHODS: Four interventions were compared over 3 studies: (i) a computerized feedback intervention (electronic Check-Up To Go [e-Chug]; Study 1); (ii) a single-session brief motivational intervention (BMI; Study 1); (iii) a BMI plus behavioral economic session focused on increasing substance-free activities (BMI + Substance-Free Activity Session [SFAS]; Studies 2 and 3); and (iv) a BMI plus supplemental Relaxation Training session (BMI + Relaxation Training; Studies 2 and 3). Studies 1 and 3 also included an assessment-only control condition. For each study, participants reported whether they had experienced an alcohol-induced blackout at each time point; binary logistic regressions examined differential likelihood of experiencing an alcohol-induced blackout over time. RESULTS: Neither the single-session BMI nor e-Chug reduced alcohol-induced blackouts over assessment only; however, participants in the BMI + SFAS or BMI + Relaxation Training condition were significantly less likely to experience an alcohol-induced blackout compared to assessment only at 1-month (Wald = 4.77, odds ratio [OR] = 0.53, p = 0.03) and 6-month follow-ups (Wald = 5.72, OR = 0.52, p = 0.02). Study 2 also revealed a larger effect for the BMI + SFAS over the BMI + Relaxation Training condition at 6 months (Wald = 4.11 OR = 0.22, p = 0.043), although this was not replicated in Study 3. The effects for the 2-session BMIs lasted 6 months, at which point maturation effects diminished differences between assessment-only and intervention conditions. CONCLUSIONS: Two sessions of BMI are a substantial enough dose to result in reductions in alcohol-induced blackouts among college student heavy drinkers.