ABSTRACT
The troponin complex is a key regulator of muscle contraction. Multiple variants in skeletal troponin encoding genes result in congenital myopathies. TNNC2 has been implicated in a novel congenital myopathy, TNNI2 and TNNT3 in distal arthrogryposis (DA), and TNNT1 and TNNT3 in nemaline myopathy (NEM). Variants in skeletal troponin encoding genes compromise sarcomere function, e.g., by altering the Ca2+ sensitivity of force or by inducing atrophy. Several potential therapeutic strategies are available to counter the effects of variants, such as troponin activators, introduction of wild-type protein through AAV gene therapy, and myosin modulation to improve muscle contraction. The mechanisms underlying the pathophysiological effects of the variants in skeletal troponin encoding genes are incompletely understood. Furthermore, limited knowledge is available on the structure of skeletal troponin. This review focusses on the physiology of slow and fast skeletal troponin and the pathophysiology of reported variants in skeletal troponin encoding genes. A better understanding of the pathophysiological effects of these variants, together with enhanced knowledge regarding the structure of slow and fast skeletal troponin, will direct the development of treatment strategies.
Subject(s)
Myotonia Congenita/metabolism , Troponin/metabolism , Animals , Humans , Muscle Contraction , Myotonia Congenita/genetics , Myotonia Congenita/physiopathology , Sarcomeres/metabolism , Troponin/chemistry , Troponin/geneticsABSTRACT
Non-dystrophic myotonias have been linked to loss-of-function mutations in the ClC-1 chloride channel or gain-of-function mutations in the Nav1.4 sodium channel. Here, we describe a family with members diagnosed with Thomsen's disease. One novel mutation (p.W322*) in CLCN1 and one undescribed mutation (p.R1463H) in SCN4A are segregating in this family. The CLCN1-p.W322* was also found in an unrelated family, in compound heterozygosity with the known CLCN1-p.G355R mutation. One reported mutation, SCN4A-p.T1313M, was found in a third family. Both CLCN1 mutations exhibited loss-of-function: CLCN1-p.W322* probably leads to a non-viable truncated protein; for CLCN1-p.G355R, we predict structural damage, triggering important steric clashes. The SCN4A-p.R1463H produced a positive shift in the steady-state inactivation increasing window currents and a faster recovery from inactivation. These gain-of-function effects are probably due to a disruption of interaction R1463-D1356, which destabilizes the voltage sensor domain (VSD) IV and increases the flexibility of the S4-S5 linker. Finally, modelling suggested that the p.T1313M induces a strong decrease in protein flexibility on the III-IV linker. This study demonstrates that CLCN1-p.W322* and SCN4A-p.R1463H mutations can act alone or in combination as inducers of myotonia. Their co-segregation highlights the necessity for carrying out deep genetic analysis to provide accurate genetic counseling and management of patients.
Subject(s)
Chloride Channels/genetics , Mutation/genetics , Myotonia Congenita/genetics , Myotonia/genetics , NAV1.4 Voltage-Gated Sodium Channel/genetics , Female , Genetic Testing/methods , Humans , Male , Middle Aged , Myotonia Congenita/metabolism , NAV1.4 Voltage-Gated Sodium Channel/metabolism , PedigreeABSTRACT
Troponin C (TnC) is a critical regulator of skeletal muscle contraction; it binds Ca2+ to activate muscle contraction. Surprisingly, the gene encoding fast skeletal TnC (TNNC2) has not yet been implicated in muscle disease. Here, we report 2 families with pathogenic variants in TNNC2. Patients present with a distinct, dominantly inherited congenital muscle disease. Molecular dynamics simulations suggested that the pathomechanisms by which the variants cause muscle disease include disruption of the binding sites for Ca2+ and for troponin I. In line with these findings, physiological studies in myofibers isolated from patients' biopsies revealed a markedly reduced force response of the sarcomeres to [Ca2+]. This pathomechanism was further confirmed in experiments in which contractile dysfunction was evoked by replacing TnC in myofibers from healthy control subjects with recombinant, mutant TnC. Conversely, the contractile dysfunction of myofibers from patients was repaired by replacing endogenous, mutant TnC with recombinant, wild-type TnC. Finally, we tested the therapeutic potential of the fast skeletal muscle troponin activator tirasemtiv in patients' myofibers and showed that the contractile dysfunction was repaired. Thus, our data reveal that pathogenic variants in TNNC2 cause congenital muscle disease, and they provide therapeutic angles to repair muscle contractility.
Subject(s)
Calcium , Molecular Dynamics Simulation , Muscle Contraction , Myotonia Congenita , Sarcomeres , Troponin C , Binding Sites , Calcium/chemistry , Calcium/metabolism , Humans , Myotonia Congenita/genetics , Myotonia Congenita/metabolism , Sarcomeres/chemistry , Sarcomeres/genetics , Sarcomeres/metabolism , Troponin C/chemistry , Troponin C/genetics , Troponin C/metabolismABSTRACT
BACKGROUND: Congenital myopathy (CM) is a group of clinically and genetically heterogeneous muscle disorders, characterized by muscle weakness and hypotonia from birth. Currently, no definite treatment exists for CM. A de novo mutation in Tropomyosin 3-TPM3(E151G) was identified from a boy diagnosed with CM, previously TPM3(E151A) was reported to cause CM. However, the role of TPM3(E151G) in CM is unknown. METHODS: Histopathological, swimming behavior, and muscle endurance were monitored in TPM3 wild-type and mutant transgenic fish, modelling CM. Gene expression profiling of muscle of the transgenic fish were studied through RNAseq, and mitochondria respiration was investigated. RESULTS: While TPM3(WT) and TPM3(E151A) fish show normal appearance, amazingly a few TPM3(E151G) fish display either no tail, a crooked body in both F0 and F1 adults. Using histochemical staining for the muscle biopsy, we found TPM3(E151G) displays congenital fiber type disproportion and TPM3(E151A) resembles nemaline myopathy. TPM3(E151G) transgenic fish dramatically swimming slower than those in TPM3(WT) and TPM3(E151A) fish measured by DanioVision and T-maze, and exhibit weaker muscle endurance by swimming tunnel instrument. Interestingly, L-carnitine treatment on TPM3(E151G) transgenic larvae significantly improves the muscle endurance by restoring the basal respiration and ATP levels in mitochondria. With RNAseq transcriptomic analysis of the expression profiling from the muscle specimens, it surprisingly discloses large downregulation of genes involved in pathways of sodium, potassium, and calcium channels, which can be rescued by L-carnitine treatment, fatty acid metabolism was differentially dysregulated in TPM3(E151G) fish and rescued by L-carnitine treatment. CONCLUSIONS: These results demonstrate that TPM3(E151G) and TPM3(E151A) exhibit different pathogenicity, also have distinct gene regulatory profiles but the ion channels were downregulated in both mutants, and provides a potential mechanism of action of TPM3 pathophysiology. Our results shed a new light in the future development of potential treatment for TPM3-related CM.
Subject(s)
Carnitine/metabolism , Myotonia Congenita/metabolism , Tropomyosin/genetics , Animals , Animals, Genetically Modified , Muscle, Skeletal/metabolism , Tropomyosin/chemistry , Tropomyosin/metabolism , Zebrafish/abnormalities , Zebrafish/metabolismABSTRACT
BACKGROUND: Myotonia congenita (MC) is a common channelopathy affecting skeletal muscle and which is due to pathogenic variants within the CLCN1 gene. Various alterations in the function of the channel have been reported and we here illustrate a novel one. METHODS: A patient presenting the symptoms of myotonia congenita was shown to bear a new heterozygous missense variant in exon 9 of the CLCN1 gene (c.1010 T > G, p.(Phe337Cys)). Confocal imaging and patch clamp recordings of transiently transfected HEK293 cells were used to functionally analyze the effect of this variant on channel properties. RESULTS: Confocal imaging showed that the F337C mutant incorporated as well as the WT channel into the plasma membrane. However, in patch clamp, we observed a smaller conductance for F337C at -80 mV. We also found a marked reduction of the fast gating component in the mutant channels, as well as an overall reduced voltage dependence. CONCLUSION: To our knowledge, this is the first report of a mixed alteration in the biophysical properties of hClC-1 consisting of a reduced conductance at resting potential and an almost abolished voltage dependence.
Subject(s)
Chloride Channels/genetics , Mutation, Missense , Myotonia Congenita/genetics , Action Potentials , Cell Membrane/metabolism , Cell Membrane/physiology , Chloride Channels/metabolism , HEK293 Cells , Humans , Ion Channel Gating , Myotonia Congenita/metabolism , Protein TransportABSTRACT
In myotonia, reduced Cl- conductance of the mutated ClC-1 channels causes hindered muscle relaxation after forceful voluntary contraction due to muscle membrane hyperexcitability. Repetitive contraction temporarily decreases myotonia, a phenomena called "warm up." The underlying mechanism for the reduction of hyperexcitability in warm-up is currently unknown. Since potassium displacement is known to reduce excitability in, for example, muscle fatigue, we characterized the role of potassium in native myotonia congenita (MC) muscle. Muscle specimens of ADR mice (an animal model for low gCl- conductance myotonia) were exposed to increasing K+ concentrations. To characterize functional effects of potassium ion current, the muscle of ADR mice was exposed to agonists and antagonists of the big conductance Ca2+-activated K+ channel (BK) and the voltage-gated Kv7 channel. Effects were monitored by functional force and membrane potential measurements. By increasing [K+]0 to 5 mM, the warm-up phenomena started earlier and at [K+]0 7 mM only weak myotonia was detected. The increase of [K+]0 caused a sustained membrane depolarization accompanied with a reduction of myotonic bursts in ADR mice. Retigabine, a Kv7.2-Kv7.5 activator, dose-dependently reduced relaxation deficit of ADR myotonic muscle contraction and promoted the warm-up phenomena. In vitro results of this study suggest that increasing potassium conductivity via activation of voltage-gated potassium channels enhanced the warm-up phenomena, thereby offering a potential therapeutic treatment option for myotonia congenita.
Subject(s)
Chloride Channels/genetics , Large-Conductance Calcium-Activated Potassium Channels/metabolism , Myotonia Congenita/metabolism , Potassium/metabolism , Animals , Chlorides/metabolism , KCNQ Potassium Channels/metabolism , Large-Conductance Calcium-Activated Potassium Channels/agonists , Large-Conductance Calcium-Activated Potassium Channels/antagonists & inhibitors , Male , Membrane Potentials , Mice , Muscle Contraction , Mutation , Myotonia Congenita/genetics , Myotonia Congenita/physiopathology , Potassium Channel Blockers/pharmacologyABSTRACT
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.
Subject(s)
Isometric Contraction/physiology , Morpholines/pharmacology , Myotonia Congenita/genetics , Myotonia Congenita/metabolism , Pyrroles/pharmacology , TRPV Cation Channels/antagonists & inhibitors , TRPV Cation Channels/deficiency , Animals , Anthracenes/pharmacology , Isometric Contraction/drug effects , Mice , Mice, Knockout , Morpholines/therapeutic use , Muscle, Skeletal/drug effects , Muscle, Skeletal/physiology , Myotonia Congenita/prevention & control , Pyrroles/therapeutic useABSTRACT
In 1970, the study of the pathomechanisms underlying myotonia in muscle fibers isolated from myotonic goats highlighted the importance of chloride conductance for skeletal muscle function; 20 years later, the human ClC-1 chloride channel has been cloned; last year, the crystal structure of human protein has been solved. Over the years, the efforts of many researchers led to significant advances in acknowledging the role of ClC-1 in skeletal muscle physiology and the mechanisms through which ClC-1 dysfunctions lead to impaired muscle function. The wide spectrum of pathophysiological conditions associated with modification of ClC-1 activity, either as the primary cause, such as in myotonia congenita, or as a secondary adaptive mechanism in other neuromuscular diseases, supports the idea that ClC-1 is relevant to preserve not only for skeletal muscle excitability, but also for skeletal muscle adaptation to physiological or harmful events. Improving this understanding could open promising avenues toward the development of selective and safe drugs targeting ClC-1, with the aim to restore normal muscle function. This review summarizes the most relevant research on ClC-1 channel physiology, associated diseases, and pharmacology.
Subject(s)
Chloride Channels/metabolism , Chlorides/metabolism , Muscle, Skeletal/metabolism , Animals , Humans , Myotonia Congenita/metabolismABSTRACT
Myotonia congenita and hypokalemic periodic paralysis type 2 are both rare genetic channelopathies caused by mutations in the CLCN1 gene encoding voltage-gated chloride channel CLC-1 and the SCN4A gene encoding voltage-gated sodium channel Nav1.4. The patients with concomitant mutations in both genes manifested different unique symptoms from mutations in these genes separately. Here, we describe a patient with myotonia and periodic paralysis in a consanguineous marriage pedigree. By using whole-exome sequencing, a novel F306S variant in the CLCN1 gene and a known R222W mutation in the SCN4A gene were identified in the pedigree. Patch clamp analysis revealed that the F306S mutant reduced the opening probability of CLC-1 and chloride conductance. Our study expanded the CLCN1 mutation database. We emphasized the value of whole-exome sequencing for differential diagnosis in atypical myotonic patients.
Subject(s)
Chloride Channels/genetics , Hypokalemic Periodic Paralysis/complications , Hypokalemic Periodic Paralysis/genetics , Myotonia Congenita/complications , Myotonia Congenita/genetics , NAV1.4 Voltage-Gated Sodium Channel/genetics , Adolescent , Adult , Aged , Amino Acid Sequence , China , Chloride Channels/chemistry , Chloride Channels/metabolism , Consanguinity , Conserved Sequence , Diagnosis, Differential , Female , HEK293 Cells , Humans , Hypokalemic Periodic Paralysis/metabolism , Male , Middle Aged , Models, Molecular , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation , Myotonia Congenita/metabolism , NAV1.4 Voltage-Gated Sodium Channel/metabolism , Pedigree , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Exome Sequencing , Young AdultABSTRACT
Myotonia congenita (MC) is a rare disorder characterized by stiffness and weakness of the limb and trunk muscles. Mutations in the SCN4A gene encoding the alpha-subunit of the voltage-gated sodium channel Nav1.4 have been reported to be responsible for sodium channel myotonia (SCM). The Nav1.4 channel is expressed in skeletal muscles, and its related channelopathies affect skeletal muscle excitability, which can manifest as SCM, paramyotonia and periodic paralysis. In this study, the missense mutation p.V445M was identified in two individual families with MC. To determine the functional consequences of having a mutated Nav1.4 channel, whole-cell patch-clamp recording of transfected Chinese hamster ovary cells was performed. Evaluation of the transient Na+ current found that a hyperpolarizing shift occurs at both the activation and inactivation curves with an increase of the window currents in the mutant channels. The Nav1.4 channel's co-expression with the Navß4 peptide can generate resurgent Na+ currents at repolarization following a depolarization. The magnitude of the resurgent currents is higher in the mutant than in the wild-type (WT) channel. Although the decay kinetics are comparable between the mutant and WT channels, the time to the peak of resurgent Na+ currents in the mutant channel is significantly protracted compared with that in the WT channel. These findings suggest that the p.V445M mutation in the Nav1.4 channel results in an increase of both sustained and resurgent Na+ currents, which may contribute to hyperexcitability with repetitive firing and is likely to facilitate recurrent myotonia in SCM patients.
Subject(s)
Mutation, Missense , Myotonia Congenita/genetics , Myotonia Congenita/physiopathology , NAV1.4 Voltage-Gated Sodium Channel/physiology , Amino Acid Sequence , Animals , Asian People , CHO Cells , Channelopathies/genetics , Channelopathies/metabolism , Channelopathies/physiopathology , Cricetulus , Female , Humans , Male , Myotonia Congenita/metabolism , NAV1.4 Voltage-Gated Sodium Channel/chemistry , NAV1.4 Voltage-Gated Sodium Channel/genetics , NAV1.4 Voltage-Gated Sodium Channel/metabolism , Patch-Clamp Techniques , PedigreeABSTRACT
This letter describes progress towards an M4 PAM preclinical candidate inspired by an unexpected aldehyde oxidase (AO) metabolite of a novel, CNS penetrant thieno[2,3-c]pyridine core to an equipotent, non-CNS penetrant thieno[2,3-c]pyrdin-7(6H)-one core. Medicinal chemistry design efforts yielded two novel tricyclic cores that enhanced M4 PAM potency, regained CNS penetration, displayed favorable DMPK properties and afforded robust in vivo efficacy in reversing amphetamine-induced hyperlocomotion in rats.
Subject(s)
Aldehyde Oxidase/metabolism , Myotonia Congenita/metabolism , Receptor, Muscarinic M4/metabolism , Animals , Drug Discovery , Humans , Rats , Structure-Activity RelationshipABSTRACT
SH3 and cysteine-rich domain-containing protein 3 (STAC3) is an essential component of the skeletal muscle excitation-contraction coupling (ECC) machinery, though its role and function are not yet completely understood. Here, we report 18 patients carrying a homozygous p.(Trp284Ser) STAC3 variant in addition to a patient compound heterozygous for the p.(Trp284Ser) and a novel splice site change (c.997-1G > T). Clinical severity ranged from prenatal onset with severe features at birth, to a milder and slowly progressive congenital myopathy phenotype. A malignant hyperthermia (MH)-like reaction had occurred in several patients. The functional analysis demonstrated impaired ECC. In particular, KCl-induced membrane depolarization resulted in significantly reduced sarcoplasmic reticulum Ca2+ release. Co-immunoprecipitation of STAC3 with CaV 1.1 in patients and control muscle samples showed that the protein interaction between STAC3 and CaV 1.1 was not significantly affected by the STAC3 variants. This study demonstrates that STAC3 gene analysis should be included in the diagnostic work up of patients of any ethnicity presenting with congenital myopathy, in particular if a history of MH-like episodes is reported. While the precise pathomechanism remains to be elucidated, our functional characterization of STAC3 variants revealed that defective ECC is not a result of CaV 1.1 sarcolemma mislocalization or impaired STAC3-CaV 1.1 interaction.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Substitution , Malignant Hyperthermia/genetics , Myotonia Congenita/genetics , Adaptor Proteins, Signal Transducing/chemistry , Adolescent , Calcium/metabolism , Child , Child, Preschool , Excitation Contraction Coupling , Female , Genetic Predisposition to Disease , Humans , Infant , Large-Conductance Calcium-Activated Potassium Channel alpha Subunits , Male , Malignant Hyperthermia/etiology , Malignant Hyperthermia/metabolism , Myotonia Congenita/complications , Myotonia Congenita/metabolism , Pedigree , Phenotype , Protein Binding , Protein Transport , Sarcoplasmic Reticulum/metabolism , Severity of Illness Index , Exome Sequencing , Young AdultABSTRACT
Calcium release from internal stores is a quintessential event in excitation-contraction coupling in cardiac and skeletal muscle. The ryanodine receptor Ca2+ release channel is embedded in the internal sarcoplasmic reticulum Ca2+ store, which releases Ca2+ into the cytoplasm, enabling contraction. Ryanodine receptors form the hub of a macromolecular complex extending from the extracellular space to the sarcoplasmic reticulum lumen. Ryanodine receptor activity is influenced by the integrated effects of associated co-proteins, ions, and post-translational phosphor and redox modifications. In healthy muscle, ryanodine receptors are phosphorylated and redox modified to basal levels, to support cellular function. A pathological increase in the degree of both post-translational modifications disturbs intracellular Ca2+ signalling, and is implicated in various cardiac and skeletal disorders. This review summarises our current understanding of the mechanisms linking ryanodine receptor post-translational modification to heart failure and skeletal myopathy and highlights the challenges and controversies within the field.
Subject(s)
Calcium/metabolism , Heart Failure/metabolism , Myotonia Congenita/metabolism , Protein Processing, Post-Translational , Ryanodine Receptor Calcium Release Channel/metabolism , Animals , Calcium Signaling , Excitation Contraction Coupling/physiology , Heart Failure/genetics , Heart Failure/pathology , Humans , Muscle Contraction/physiology , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Myocardium/metabolism , Myocardium/pathology , Myotonia Congenita/genetics , Myotonia Congenita/pathology , Phosphorylation , Reactive Oxygen Species/metabolism , Ryanodine Receptor Calcium Release Channel/genetics , Sarcoplasmic Reticulum/metabolismABSTRACT
INTRODUCTION: The gain-of-function mutations that underlie sodium channel myotonia (SCM) and paramyotonia congenital (PMC) produce differing clinical phenotypes. We used muscle velocity recovery cycles (MVRCs) to investigate membrane properties. METHODS: MVRCs and responses to trains of stimuli were compared in patients with SCM (n = 9), PMC (n = 8), and normal controls (n = 26). RESULTS: The muscle relative refractory period was reduced in SCM, consistent with faster recovery of the mutant sodium channels from inactivation. Both SCM and PMC showed an increased early supernormality and increased mean supernormality following multiple conditioning stimuli, consistent with slowed sodium channel inactivation. Trains of fast impulses caused a loss of amplitude in PMC, after which only half of the muscle fibers recovered, suggesting that the remainder stayed depolarized by persistent sodium currents. DISCUSSION: The differing effects of mutations on sodium channel function can be demonstrated in human subjects in vivo using this technique. Muscle Nerve 57: 586-594, 2018.
Subject(s)
Membrane Potentials , Muscle Fibers, Skeletal/metabolism , Myotonia Congenita/metabolism , Adult , Aged , Case-Control Studies , Female , Humans , Male , Middle Aged , Myotonia Congenita/physiopathology , Myotonic Disorders/metabolism , Myotonic Disorders/physiopathology , Refractory Period, Electrophysiological , Young AdultABSTRACT
Excitation-contraction (EC) coupling in skeletal muscle requires functional and mechanical coupling between L-type voltage-gated calcium channels (CaV1.1) and the ryanodine receptor (RyR1). Recently, STAC3 was identified as an essential protein for EC coupling and is part of a group of three proteins that can bind and modulate L-type voltage-gated calcium channels. Here, we report crystal structures of tandem-SH3 domains of different STAC isoforms up to 1.2-Å resolution. These form a rigid interaction through a conserved interdomain interface. We identify the linker connecting transmembrane repeats II and III in two different CaV isoforms as a binding site for the SH3 domains and report a crystal structure of the complex with the STAC2 isoform. The interaction site includes the location for a disease variant in STAC3 that has been linked to Native American myopathy (NAM). Introducing the mutation does not cause misfolding of the SH3 domains, but abolishes the interaction. Disruption of the interaction via mutations in the II-III loop perturbs skeletal muscle EC coupling, but preserves the ability of STAC3 to slow down inactivation of CaV1.2.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Calcium Channels, L-Type/metabolism , Animals , Binding Sites/physiology , Calcium/metabolism , Calcium Signaling/physiology , Cleft Palate/metabolism , Crystallography, X-Ray/methods , Excitation Contraction Coupling/physiology , Humans , Malignant Hyperthermia/metabolism , Membrane Proteins/metabolism , Muscle, Skeletal/metabolism , Mutation/genetics , Myotonia Congenita/metabolism , Protein Isoforms/metabolism , Rabbits , Ryanodine Receptor Calcium Release Channel/metabolism , Xenopus laevis/metabolismABSTRACT
Maintenance of muscle structure and function depends on the precise organization of contractile proteins into sarcomeres and coupling of the contractile apparatus to the sarcoplasmic reticulum (SR), which serves as the reservoir for calcium required for contraction. Several members of the Kelch superfamily of proteins, which modulate protein stability as substrate-specific adaptors for ubiquitination, have been implicated in sarcomere formation. The Kelch protein Klhl31 is expressed in a muscle-specific manner under control of the transcription factor MEF2. To explore its functions in vivo, we created a mouse model of Klhl31 loss of function using the CRISPR-Cas9 system. Mice lacking Klhl31 exhibited stunted postnatal skeletal muscle growth, centronuclear myopathy, central cores, Z-disc streaming, and SR dilation. We used proteomics to identify several candidate Klhl31 substrates, including Filamin-C (FlnC). In the Klhl31-knockout mice, FlnC protein levels were highly upregulated with no change in transcription, and we further demonstrated that Klhl31 targets FlnC for ubiquitination and degradation. These findings highlight a role for Klhl31 in the maintenance of skeletal muscle structure and provide insight into the mechanisms underlying congenital myopathies.
Subject(s)
Muscle, Skeletal/metabolism , Myotonia Congenita/genetics , Myotonia Congenita/metabolism , Transcription Factors/deficiency , Animals , Filamins/genetics , Filamins/metabolism , MEF2 Transcription Factors/metabolism , Mice , Mice, Knockout , Muscle, Skeletal/pathology , Myotonia Congenita/pathology , Transcription Factors/metabolism , UbiquitinationABSTRACT
The flow of intracellular calcium (Ca2+) is critical for the activation and regulation of important biological events that are required in living organisms. As the major Ca2+ repositories inside the cell, the endoplasmic reticulum (ER) and the sarcoplasmic reticulum (SR) of muscle cells are central in maintaining and amplifying the intracellular Ca2+ signal. The morphology of these organelles, along with the distribution of key calcium-binding proteins (CaBPs), regulatory proteins, pumps, and receptors fundamentally impact the local and global differences in Ca2+ release kinetics. In this review, we will discuss the structural and morphological differences between the ER and SR and how they influence localized Ca2+ release, related diseases, and the need for targeted genetically encoded calcium indicators (GECIs) to study these events.
Subject(s)
Calcium Signaling , Malignant Hyperthermia/genetics , Myotonia Congenita/genetics , Sarcoplasmic Reticulum/metabolism , Tachycardia/genetics , Animals , Calcium Channels/genetics , Calcium Channels/metabolism , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Humans , Malignant Hyperthermia/metabolism , Myotonia Congenita/metabolism , Sarcoplasmic Reticulum/genetics , Tachycardia/metabolismABSTRACT
Muscle contraction upon nerve stimulation relies on excitation-contraction coupling (ECC) to promote the rapid and generalized release of calcium within myofibers. In skeletal muscle, ECC is performed by the direct coupling of a voltage-gated L-type Ca2+ channel (dihydropyridine receptor; DHPR) located on the T-tubule with a Ca2+ release channel (ryanodine receptor; RYR1) on the sarcoplasmic reticulum (SR) component of the triad. Here, we characterize a novel class of congenital myopathy at the morphological, molecular, and functional levels. We describe a cohort of 11 patients from 7 families presenting with perinatal hypotonia, severe axial and generalized weakness. Ophthalmoplegia is present in four patients. The analysis of muscle biopsies demonstrated a characteristic intermyofibrillar network due to SR dilatation, internal nuclei, and areas of myofibrillar disorganization in some samples. Exome sequencing revealed ten recessive or dominant mutations in CACNA1S (Cav1.1), the pore-forming subunit of DHPR in skeletal muscle. Both recessive and dominant mutations correlated with a consistent phenotype, a decrease in protein level, and with a major impairment of Ca2+ release induced by depolarization in cultured myotubes. While dominant CACNA1S mutations were previously linked to malignant hyperthermia susceptibility or hypokalemic periodic paralysis, our findings strengthen the importance of DHPR for perinatal muscle function in human. These data also highlight CACNA1S and ECC as therapeutic targets for the development of treatments that may be facilitated by the previous knowledge accumulated on DHPR.
Subject(s)
Calcium Channels/genetics , Calcium Channels/metabolism , Myotonia Congenita/genetics , Myotonia Congenita/metabolism , Adolescent , Adult , Calcium/metabolism , Calcium Channels, L-Type , Cells, Cultured , Child , Cohort Studies , Family , Female , Humans , Male , Middle Aged , Muscle Cells/metabolism , Muscle Cells/pathology , Muscle, Skeletal/diagnostic imaging , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Mutation , Myotonia Congenita/diagnostic imaging , Myotonia Congenita/pathology , Phenotype , Sequence Homology, Amino Acid , Young AdultABSTRACT
Mutations in human CLC-1 chloride channel are associated with the skeletal muscle disorder myotonia congenita. The disease-causing mutant A531V manifests enhanced proteasomal degradation of CLC-1. We recently found that CLC-1 degradation is mediated by cullin 4 ubiquitin ligase complex. It is currently unclear how quality control and protein degradation systems coordinate with each other to process the biosynthesis of CLC-1. Herein we aim to ascertain the molecular nature of the protein quality control system for CLC-1. We identified three CLC-1-interacting proteins that are well-known heat shock protein 90 (Hsp90)-associated co-chaperones: FK506-binding protein 8 (FKBP8), activator of Hsp90 ATPase homolog 1 (Aha1), and Hsp70/Hsp90 organizing protein (HOP). These co-chaperones promote both the protein level and the functional expression of CLC-1 wild-type and A531V mutant. CLC-1 biosynthesis is also facilitated by the molecular chaperones Hsc70 and Hsp90ß. The protein stability of CLC-1 is notably increased by FKBP8 and the Hsp90ß inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) that substantially suppresses cullin 4 expression. We further confirmed that cullin 4 may interact with Hsp90ß and FKBP8. Our data are consistent with the idea that FKBP8 and Hsp90ß play an essential role in the late phase of CLC-1 quality control by dynamically coordinating protein folding and degradation.
Subject(s)
Chloride Channels/genetics , HSP90 Heat-Shock Proteins/genetics , Homeodomain Proteins/genetics , Molecular Chaperones/genetics , Tacrolimus Binding Proteins/genetics , Tumor Suppressor Proteins/genetics , Chloride Channels/antagonists & inhibitors , Chloride Channels/metabolism , Cullin Proteins/genetics , Cullin Proteins/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Gene Expression Regulation , Genetic Vectors/chemistry , Genetic Vectors/metabolism , HEK293 Cells , HSP90 Heat-Shock Proteins/metabolism , Homeodomain Proteins/metabolism , Humans , Lentivirus/genetics , Lentivirus/metabolism , Leupeptins/pharmacology , Models, Biological , Molecular Chaperones/metabolism , Myotonia Congenita/genetics , Myotonia Congenita/metabolism , Myotonia Congenita/pathology , Patch-Clamp Techniques , Proteasome Endopeptidase Complex/drug effects , Proteasome Endopeptidase Complex/metabolism , Proteolysis , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Tacrolimus Binding Proteins/metabolism , Transfection , Tumor Suppressor Proteins/metabolism , Ubiquitination/drug effectsABSTRACT
Myotonia congenita is an inherited disease that is characterized by impaired muscle relaxation after contraction caused by loss-of-function mutations in the skeletal muscle ClC-1 channel. We report a novel ClC-1 mutation, T335N, that is associated with a mild phenotype in 1 patient, located in the extracellular I-J loop. The purpose of this study was to provide a solid correlation between T335N dysfunction and clinical symptoms in the affected patient as well as to offer hints for drug development. Our multidisciplinary approach includes patch-clamp electrophysiology on T335N and ClC-1 wild-type channels expressed in tsA201 cells, Western blot and quantitative PCR analyses on muscle biopsies from patient and unaffected individuals, and molecular dynamics simulations using a homology model of the ClC-1 dimer. T335N channels display reduced chloride currents as a result of gating alterations rather than altered surface expression. Molecular dynamics simulations suggest that the I-J loop might be involved in conformational changes that occur at the dimer interface, thus affecting gating. Finally, the gene expression profile of T335N carrier showed a diverse expression of K+ channel genes, compared with control individuals, as potentially contributing to the phenotype. This experimental paradigm satisfactorily explained myotonia in the patient. Furthermore, it could be relevant to the study and therapy of any channelopathy.-Imbrici, P., Altamura, C., Camerino, G. M., Mangiatordi, G. F., Conte, E., Maggi, L., Brugnoni, R., Musaraj, K., Caloiero, R., Alberga, D., Marsano, R. M., Ricci, G., Siciliano, G., Nicolotti, O., Mora, M., Bernasconi, P., Desaphy, J.-F., Mantegazza, R., Camerino, D. C. Multidisciplinary study of a new ClC-1 mutation causing myotonia congenita: a paradigm to understand and treat ion channelopathies.