Skeletal Muscle Isoform Research Articles

Impact of anti-S2 Peptides on myosin S2 Isoforms and HCM Mutants

Myosin subfragment-2 (S2) is an unstable coiled coil that influences the binding of myosin heads to actin. Instability in the myosin S2 could underlie one of the causes for hyper-contractility in Hypertrophic Cardiomyopathy (HCM). Supporting this hypothesis, HCM mutations E924K and E930del in myosin S2 reduced myosin S2 coiled coil stability compared to wild type as measured by Forester's Resonance Energy Transfer (FRET) and Gravitational Force Spectroscopy (GFS). To modulate the stability of the S2, anti-S2 peptides called stabilizer and destabilizer peptides because they are designed to increase and decrease the stability of the MYH7 S2 coiled coil, were tested on a range of human S2 peptide sequences. The anti-S2 peptides were tested on myosin S2 peptides across MYH11 (smooth), MYH7 (cardiac), and MYH2 (skeletal) with GFS and FRET. The results demonstrated that the mechanical stability was increased by stabilizer and decreased by destabilizer across the cardiac and skeletal myosin S2 peptides but not for smooth muscle isoform. Dose-response curves for FRET assays indicated that both peptides bound at nanomolar concentrations to wild type cardiac and skeletal, but not smooth muscle isoforms. The wild type cardiac S2 peptide required more force to unzip than skeletal, smooth, or mutant peptides in the absence of stabilizer peptide. Effectively, the stabilizer peptide improved the stability of both HCM mutants (E924K and E930del). The stabilizer peptide enhanced dimer formation in mutant cardiac, and skeletal myosin S2 peptides, and destabilizer increased flexibility of cardiac and skeletal myosin S2 wild type peptide. This study demonstrates that the structural stability of myosin S2 can be altered by designed small peptides as well as mutations. These anti-S2 peptides share similar specificities and high affinities for the tested striated muscle isoforms but not the smooth muscle myosin isoform.

Identification of a novel pathogenic mutation of the MYH3 gene in a family with distal arthrogryposis type 2B.

Distal arthrogryposis (DA) type 2B (DA2B) is an autosomal dominant congenital disorder, characterized by camptodactyly, thumb adduction, ulnar deviation and facial features, including small mouth, down‑slanting palpebral fissure and slight nasolabial fold. It has been reported that four genes are associated with DA2B, including troponin I, fast‑twitch skeletal muscle isoform, troponin T3, fast skeletal, myosin heavy chain 3 (MYH3) and tropomyosin 2, which are all associated with embryonic limb morphogenesis and skeletal muscle contraction. In the present study, three affected family members and five unaffected individuals were identified through clinical and radiological assessment. Genomic DNA was obtained from the three patients, which then underwent whole‑exome sequencing, and candidate mutations were verified by Sanger sequencing in all available family members and 100 healthy volunteers. Then, the spatial models of embryonic MYH were further constructed. In the clinic, the three patients recruited to the present study were diagnosed with DA2B. Mutation analysis indicated that there was a novel heterogeneous missense mutation c.2506 A>G (p.K836E) in the MYH3 gene among the affected individuals, which was highly conserved and was not identified in the unaffected family members and healthy controls. Furthermore, protein modeling revealed that the altered position interacted with regulatory light chain. Thus, the present study identified a novel pathogenic mutation of the MYH3 gene in a Chinese family with DA2B, which expanded the mutational spectrum of MYH3 and provided additional information regarding the association between mutation locations and different types of DA.

Substituted cysteine scanning in D1-S6 of the sodium channel hNav1.4 alters kinetics and structural interactions of slow inactivation

Slow inactivation in voltage-gated Na+ channels (Navs) plays an important physiological role in excitable tissues (muscle, heart, nerves) and mutations that disrupt Nav slow inactivation can result in pathophysiologies (myotonia, arrhythmias, epilepsy). While the molecular mechanisms responsible for slow inactivation remain elusive, previous studies have suggested a role for the pore-lining D1-S6 helix. The goals of this research were to determine if (1) cysteine substitutions in D1-S6 affect gating kinetics and (2) methanethiosulfonate ethylammonium (MTSEA) accessibility changes in different kinetic states. Site-directed mutagenesis in the human skeletal muscle isoform hNav1.4 was used to substitute cysteine for eleven amino acids in D1-S6 from L433 to L443. Mutants were expressed in HEK cells and recorded from with whole-cell patch clamp. All mutations affected one or more baseline kinetics of the sodium channel, including activation, fast inactivation, and slow inactivation. Substitution of cysteine (for nonpolar residues) adjacent to polar residues destabilized slow inactivation in G434C, F436C, I439C, and L441C. Cysteine substitution without adjacent polar residues enhanced slow inactivation in L438C and N440C, and disrupted possible H-bonds involving Y437:D4 S4-S5 and N440:D4-S6. MTSEA exposure in closed, fast-inactivated, or slow-inactivated states in most mutants had little-to-no effect. In I439C, MTSEA application in closed, fast-inactivated, and slow-inactivated states produced irreversible reduction in current, suggesting I439C accessibility to MTSEA in all three kinetic states. D1-S6 is important for Nav gating kinetics, stability of slow-inactivated state, structural contacts, and state-dependent positioning. However, prominent reconfiguration of D1-S6 may not occur in slow inactivation.

Molecular Insights into Calcium Dependent Regulation of Ryanodine Receptor Calcium Release Channels.

Ryanodine receptor calcium release channels (RyRs) play central roles in controlling intracellular calcium concentrations in excitable and non-excitable cells. RyRs are located in the sarcoplasmic or endoplasmic reticulum, intracellular Ca2+ storage compartment, and release Ca2+ during cellular action potentials or in response to other cellular stimuli. Mammalian cells express three structurally related isoforms of RyR. RyR1 and RyR2 are the major RyR isoforms in skeletal and cardiac muscle, respectively, and RyR3 is expressed in various tissues along with the other two isoforms. A prominent feature of RyRs is that the Ca2+ release channel activities of RyRs are regulated by calcium ions; therefore, intracellular Ca2+ release controls positive- and negative-feedback phenomena through the RyRs. RyR channel activities are also regulated by Ca2+ indirectly, i.e. through Ca2+ binding proteins at both cytosolic and sarco/endoplasmic reticulum luminal sides. Here, I summarize Ca2+-dependent feedback regulation of RyRs including recent progress in the structure/function aspects.

Proteomic analysis of the skeletal muscles from dysferlinopathy patients

Dysferlinopathy is an autosomal recessive disease caused by pathogenic variants in DYSF gene. We compared muscle protein extracts from dysferlinopathy patients and control subjects to identify new biomarkers of this myopathy. We reviewed the medical records from January 2002 to October 2016. Eight vastus lateralis muscle samples from five dysferlinopathy patients and three control subjects were selected. We separated proteins/peptides from all eight muscle protein extracts using two-dimensional electrophoresis (2DE). Data were acquired from liquid chromatography-mass spectrometry protein fragmentation patterns after comparing the spot volumes. Western blotting revealed total dysferlin loss in the dysferlinopathy patients but normal expression in the control subjects. 2DE indicated somewhat diverse protein constellations between the dysferlinopathy and control groups. Image analysis showed that 80 spots were differently expressed between two dysferlinopathy and one control samples. We selected 44 spots with consistently different volume between dysferlinopathy and control groups. Liquid chromatography-mass spectrometry indicated 26 differently expressed proteins. Western blotting revealed that creatine kinase M-type, carbonic anhydrase III (muscle specific) and desmin were significantly elevated in dysferlinopathy muscle. Additionally, four proteins (myosin light chain 1/3, skeletal muscle isoform; lamin A/C; ankyrin repeat domain 2; and eukaryotic translation initiation factor 5A-1) were inconsistently elevated in the dysferlinopathy samples. We confirmed the usefulness of the classic biomarker and have newly identified the altered expression of proteins in the skeletal muscles of dysferlinopathy patients.

The loss of slow skeletal muscle isoform of troponin T in spindle intrafusal fibres explains the pathophysiology of Amish nemaline myopathy.

The pathogenic mechanism and the neuromuscular reflex-related phenotype (e.g. tremors accompanied by clonus) of Amish nemaline myopathy, as well as of other recessively inherited TNNT1 myopathies, remain to be clarified. The truncated slow skeletal muscle isoform of troponin T (ssTnT) encoded by the mutant TNNT1 gene is unable to incorporate into myofilaments and is degraded in muscle cells. By contrast to extrafusal muscle fibres, spindle intrafusal fibres of normal mice contain a significant level of cardiac TnT and a low molecular weight splice form of ssTnT. Intrafusal fibres of ssTnT-knockout mice have significantly increased cardiac TnT. Rotarod and balance beam tests have revealed abnormal neuromuscular co-ordination in ssTnT-knockout mice and a blunted response to a spindle sensitizer, succinylcholine. The loss of ssTnT and a compensatory increase of cardiac TnT in intrafusal nuclear bag fibres may increase myofilament Ca2+ -sensitivity and tension, impairing spindle function, thus identifying a novel mechanism for the development of targeted treatment. A nonsense mutation at codon Glu180 of TNNT1 gene causes Amish nemaline myopathy (ANM), a recessively inherited disease with infantile lethality. TNNT1 encodes the slow skeletal muscle isoform of troponin T (ssTnT). The truncated ssTnT is unable to incorporate into myofilament and is degraded in muscle cells. The symptoms of ANM include muscle weakness, atrophy, contracture and tremors accompanied by clonus. An ssTnT-knockout (KO) mouse model recapitulates key features of ANM such as atrophy of extrafusal slow muscle fibres and increased fatigability. However, the neuromuscular reflex-related symptoms of ANM have not been explained. By isolating muscle spindles from ssTnT-KO and control mice aiming to examine the composition of myofilament proteins, we found that, in contrast to extrafusal fibres, intrafusal fibres contain a significant level of cardiac TnT and the low molecular weight splice form of ssTnT. Intrafusal fibres from ssTnT-KO mice have significantly increased cardiac TnT. Rotarod and balance beam tests revealed impaired neuromuscular co-ordination in ssTnT-KO mice, indicating abnormality in spindle functions. Unlike the wild-type control, the beam running ability of ssTnT-KO mice had a blunted response to a spindle sensitizer, succinylcholine. Immunohistochemistry detected ssTnT and cardiac TnT in nuclear bag fibres, whereas fast skeletal muscle TnT was detected in nuclear chain fibres, and cardiac α-myosin was present in one of the two nuclear bag fibres. The loss of ssTnT and a compensatory increase of cardiac TnT in nuclear bag fibres would increase myofilament Ca2+ -sensitivity and tension, thus affecting spindle activities. This mechanism provides an explanation for the pathophysiology of ANM, as well as a novel target for treatment.

AMP-activated protein kinase complexes containing the β2 regulatory subunit are up-regulated during and contribute to adipogenesis.

AMP-activated protein kinase (AMPK) is a heterotrimer of α-catalytic and β- and γ-regulatory subunits that acts to regulate cellular and whole-body nutrient metabolism. The key role of AMPK in sensing energy status has led to significant interest in AMPK as a therapeutic target for dysfunctional metabolism in type 2 diabetes, insulin resistance and obesity. Despite the actions of AMPK in the liver and skeletal muscle being extensively studied, the role of AMPK in adipose tissue and adipocytes remains less well characterised. Small molecules that selectively influence AMPK heterotrimers containing specific AMPKβ subunit isoforms have been developed, including MT47-100, which selectively inhibits complexes containing AMPKβ2. AMPKβ1 and AMPKβ2 are the principal AMPKβ subunit isoforms in rodent liver and skeletal muscle, respectively, yet the contribution of specific AMPKβ isoforms to adipose tissue function, however, remains largely unknown. This study therefore sought to determine the contribution of AMPKβ subunit isoforms to adipocyte biology, focussing on adipogenesis. AMPKβ2 was the principal AMPKβ isoform in 3T3-L1 adipocytes, isolated rodent adipocytes and human subcutaneous adipose tissue, as assessed by the contribution to total cellular AMPK activity. Down-regulation of AMPKβ2 with siRNA inhibited lipid accumulation, cellular adiponectin levels and adiponectin secretion during 3T3-L1 adipogenesis, whereas down-regulation of AMPKβ1 had no effect. Incubation of 3T3-L1 cells with MT47-100 selectively inhibited AMPK complexes containing AMPKβ2 whilst simultaneously inhibiting cellular lipid accumulation as well as cellular levels and secretion of adiponectin. Taken together, these data indicate that increased expression of AMPKβ2 is an important feature of efficient adipogenesis.

Heterozygous variants in MYBPC1 are associated with an expanded neuromuscular phenotype beyond arthrogryposis.

Encoding the slow skeletal muscle isoform of myosin binding protein-C, MYBPC1 is associated with autosomal dominant and recessive forms of arthrogryposis. The authors describe a novel association for MYBPC1 in four patients from three independent families with skeletal muscle weakness, myogenic tremors, and hypotonia with gradual clinical improvement. The patients carried one of two de novo heterozygous variants in MYBPC1, with the p.Leu263Arg variant seen in three individuals and the p.Leu259Pro variant in one individual. Both variants are absent from controls, well conserved across vertebrate species, predicted to be damaging, and located in the M-motif. Protein modeling studies suggested that the p.Leu263Arg variant affects the stability of the M-motif, whereas the p.Leu259Pro variant alters its structure. In vitro biochemical and kinetic studies demonstrated that the p.Leu263Arg variant results in decreased binding of the M-motif to myosin, which likely impairs the formation of actomyosin cross-bridges during muscle contraction. Collectively, our data substantiate that damaging variants in MYBPC1 are associated with a new form of an early-onset myopathy with tremor, which is a defining and consistent characteristic in all affected individuals, with no contractures. Recognition of this expanded myopathic phenotype can enable identification of individuals with MYBPC1 variants without arthrogryposis.

Expression of troponin subunits in the rat renal afferent arteriole.

Vascular smooth muscle cells of the renal afferent arteriole are unusual in that they must be able to contract very rapidly in response to a sudden increase in systemic blood pressure in order to protect the downstream glomerular capillaries from catastrophic damage. We showed that this could be accounted for, in part, by exclusive expression, at the protein level, of the "fast" (B) isoforms of smooth muscle myosin II heavy chains in the afferent arteriole, in contrast to other vascular smooth muscle cells such as the rat aorta and efferent arteriole which express exclusively the "slow" (A) isoforms (Shiraishi et al. (2003) FASEB. J. 17, 2284-2286). As contraction of the more rapidly contracting striated (skeletal and cardiac) muscles is regulated by the thin filament-associated troponin (Tn) system, we hypothesized that Tn or a Tn-like system may exist in afferent arteriolar cells and contribute to the unusually rapid contraction of this tissue in response to increased intraluminal pressure. We examined the expression of TnC (Ca2+ -binding subunit), TnI (inhibitory subunit), and TnT (tropomyosin-binding subunit) in vascular smooth muscle cells of the rat renal afferent arteriole at the mRNA level. Fast-twitch skeletal muscle and slow-twitch skeletal muscle/cardiac TnC isoforms and slow-twitch skeletal muscle and cardiac TnI isoforms were detected by reverse transcription-polymerase chain reaction (RT-PCR) and confirmed by cDNA sequencing. Furthermore, cardiac and slow-twitch skeletal muscle TnI isoforms, but not fast-twitch skeletal muscle TnI, were detected in isolated afferent arterioles at the protein level by proximity ligation assay. Finally, striated muscle myosin II heavy chain expression was identified in isolated rat afferent arterioles by RT-PCR. We conclude that, in addition to Ca2+ -mediated phosphorylation of myosin II regulatory light chains, contraction of the afferent arteriole may be regulated by a mechanism normally associated with the much more rapidly contracting cardiac and skeletal muscles, which involves Ca2+ binding to TnC, leading to alleviation of inhibition of the actomyosin MgATPase by TnI and tropomyosin and rapid contraction of the vessel.

Morpholino-induced exon skipping stimulates cell-mediated and humoral responses to dystrophin in mdx mice.

Exon skipping is a promising genetic therapeutic strategy for restoring dystrophin expression in the treatment of Duchenne muscular dystrophy (DMD). The potential for newly synthesized dystrophin to trigger an immune response in DMD patients, however, is not well established. We have evaluated the effect of chronic phosphorodiamidate morpholino oligomer (PMO) treatment on skeletal muscle pathology and asked whether sustained dystrophin expression elicits a dystrophin-specific autoimmune response. Here, two independent cohorts of dystrophic mdx mice were treated chronically with either 800 mg/kg/month PMO for 6months (n = 8) or 100 mg/kg/week PMO for 12 weeks (n = 11). We found that significant muscle inflammation persisted after exon skipping in skeletal muscle. Evaluation of humoral responses showed serum-circulating antibodies directed against de novo dystrophin in a subset of mice, as assessed both by Western blotting and immunofluorescent staining; however, no dystrophin-specific antibodies were observed in the control saline-treated mdx cohorts (n = 8) or in aged (12-month-old) mdx mice with expanded 'revertant' dystrophin-expressing fibers. Reactive antibodies recognized both full-length and truncated exon-skipped dystrophin isoforms in mouse skeletal muscle. We found more antigen-specific T-cell cytokine responses (e.g. IFN-g, IL-2) in dystrophin antibody-positive mice than in dystrophin antibody-negative mice. We also found expression of major histocompatibility complex class I on some of the dystrophin-expressing fibers along with CD8+ and perforin-positive T cells in the vicinity, suggesting an activation of cell-mediated damage had occurred in the muscle. Evaluation of complement membrane attack complex (MAC) deposition on the muscle fibers further revealed lower MAC deposition on muscle fibers of dystrophin antibody-negative mice than on those of dystrophin antibody-positive mice. Our results indicate that de novo dystrophin expression after exon skipping can trigger both cell-mediated and humoral immune responses in mdx mice. Our data highlights the need to further investigate the autoimmune response and its long-term consequences after exon-skipping therapy. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

SAT-013 Dihydrotestosterone Does Not Modulate Key Lipogenesis Regulator in Skeletal Muscle from Female Mice

Polycystic ovary syndrome (PCOS) affects 10% of women and is hallmarked by hyperandrogenemia (HA), which is also a component of non-alcoholic fatty liver disease (NAFLD). In this project, we aimed to investigate how androgen and androgen receptor (AR) impact a key player of lipogenesis, sterol regulatory element-binding protein 1 (SREBP1). To this end, skeletal muscle from lean, female wild type mice and mice administered dihydrotestosterone (DHT) for one month were extracted and analyzed to determine the effect of low dose DHT on lipogenic cytosolic protein and gene expression. We performed Western blot and real-time quantitative polymerase chain reaction (qRT-PCR) analysis of lipogenic intermediates in homogenates of an energy storage tissue. Skeletal muscle was used as a control. Low-dose DHT lowered the active form of cytosolic SREBP-2 in skeletal muscle. However, FAS and p-ACC protein and mRNA expression levels were unchanged in skeletal muscle. The results indicate that SREBP-2 is the primary SREBP isoform in skeletal muscle but that it is not playing a role in DHT-induced lipogenic gene expression or NAFLD. Thank you to HUCM, JHUSOM, and Georgetown University Medical Center (GUMC) for supporting this work.

Structures of the heart specific SERCA2a Ca2+-ATPase.

The sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) performs active reuptake of cytoplasmic Ca2+ and is a major regulator of cardiac muscle contractility. Dysfunction or dysregulation of SERCA2a is associated with heart failure, while restoring its function is considered as a therapeutic strategy to restore cardiac performance. However, its structure has not yet been determined. Based on native, active protein purified from pig ventricular muscle, we present the first crystal structures of SERCA2a, determined in the CPA-stabilized E2-AlF4- form (3.3Å) and the Ca2+-occluded [Ca2]E1-AMPPCP form (4.0Å). The structures are similar to the skeletal muscle isoform SERCA1a pointing to a conserved mechanism. We seek to explain the kinetic differences between SERCA1a and SERCA2a. We find that several isoform-specific residues are acceptor sites for post-translational modifications. In addition, molecular dynamics simulations predict that isoform-specific residues support distinct intramolecular interactions in SERCA2a and SERCA1a. Our experimental observations further indicate that isoform-specific intramolecular interactions are functionally relevant, and may explain the kinetic differences between SERCA2a and SERCA1a.

Loss of the Slow Skeletal Muscle Isoform of Troponin T Impairs Motor Coordination in Mice

The loss of the slow skeletal muscle isoform of troponin T (ssTnT) encoded by TNNT1 causes lethal myopathies with symptoms including abnormal neuromuscular reflexes. It has been a challenging question that how the loss of one isoform of TnT results in extremely severe phenotypes while the fast isoform of TnT is present in the patient muscles. We investigated ssTnT knockout (KO) mice for the role of ssTnT in muscle spindles that are a mechanosensors critical to normal neuromuscular function. Immunohistological and spindle protein content studies indicated that unlike extrafusal muscle fibers, adult ssTnT-KO mouse spindle intrafusal fibers express high levels of cardiac TnT, which is significantly increased in intrafusal fibers of ssTnT-KO mice. Impaired motor coordination was observed in ssTnT-KO mice with slower walking during a balance beam performance test as compared with age/sex-matched wildtype (WT) control (p<0.05). An interesting observation from the balance beam performance test is that subcutaneous injection of succinylcholine (SCh), a muscle relaxant believed to alter spindle activities, significantly increased the time for WT mice to walk across the balance beam (p<0.01) whereas SCh treatment produced a trend for decreased time across the beam in ssTnT-KO mice (p=0.10). Taken together, our data suggest that the loss of ssTnT in the KO mice impairs spindle intrafusal fiber contractility, identifying a novel therapeutic target. The attenuated effect of SCh treatment on motor coordination in the ssTnT-KO mice may be due to the potentially adaptive increased expression of cardiac TnT in spindle intrafusal fibers. Further testing is currently underway to assessing the hypothesized adaptation of spindle intrafusal fiber TnT isoform expression and function in ssTnT-KO mice during postnatal development.

Discovery of a Novel Nav1.7 Inhibitor From Cyriopagopus albostriatus Venom With Potent Analgesic Efficacy

Spider venoms contain a vast array of bioactive peptides targeting ion channels. A large number of peptides have high potency and selectivity toward sodium channels. Nav1.7 contributes to action potential generation and propagation and participates in pain signaling pathway. In this study, we describe the identification of μ-TRTX-Ca2a (Ca2a), a novel 35-residue peptide from the venom of Vietnam spider Cyriopagopus albostriatus (C. albostriatus) that potently inhibits Nav1.7 (IC50 = 98.1 ± 3.3 nM) with high selectivity against skeletal muscle isoform Nav1.4 (IC50 > 10 μM) and cardiac muscle isoform Nav1.5 (IC50 > 10 μM). Ca2a did not significantly alter the voltage-dependent activation or fast inactivation of Nav1.7, but it hyperpolarized the slow inactivation. Site-directed mutagenesis analysis indicated that Ca2a bound with Nav1.7 at the extracellular S3–S4 linker of domain II. Meanwhile, Ca2a dose-dependently attenuated pain behaviors in rodent models of formalin-induced paw licking, hot plate test, and acetic acid-induced writhing. This study indicates that Ca2a is a potential lead molecule for drug development of novel analgesics.

Thermal unfolding of homodimers and heterodimers of different skeletal-muscle isoforms of tropomyosin

We applied differential scanning calorimetry (DSC) to investigate the structural properties of three isoforms of tropomyosin (Tpm), α, β, and γ, expressed from different genes in human skeletal muscles. We compared specific features of the thermal unfolding of αα, ββ, and γγ Tpm homodimers, as well as of αβ and γβ Tpm heterodimers. The results show that the thermal stability of γγ homodimer is much higher than that of αα homodimer which, in turn, is much more thermostable than the ββ homodimer. The stability of the γβ Tpm heterodimer is much lower than that of the γγ homodimer, and its thermal unfolding is quite different from that for γγ and ββ homodimers, whereas the unfolding of the αβ heterodimer is roughly similar to that of the αα homodimer.

Congenital Titinopathy: Comprehensive characterization and pathogenic insights

ObjectiveComprehensive clinical characterization of congenital titinopathy to facilitate diagnosis and management of this important emerging disorder.MethodsUsing massively parallel sequencing we identified 30 patients from 27 families with 2 pathogenic nonsense, frameshift and/or splice site TTN mutations in trans. We then undertook a detailed analysis of the clinical, histopathological and imaging features of these patients.ResultsAll patients had prenatal or early onset hypotonia and/or congenital contractures. None had ophthalmoplegia. Scoliosis and respiratory insufficiency typically developed early and progressed rapidly, whereas limb weakness was often slowly progressive, and usually did not prevent independent walking. Cardiac involvement was present in 46% of patients. Relatives of 2 patients had dilated cardiomyopathy. Creatine kinase levels were normal to moderately elevated. Increased fiber size variation, internalized nuclei and cores were common histopathological abnormalities. Cap‐like regions, whorled or ring fibers, and mitochondrial accumulations were also observed. Muscle magnetic resonance imaging showed gluteal, hamstring and calf muscle involvement. Western blot analysis showed a near‐normal sized titin protein in all samples. The presence of 2 mutations predicted to impact both N2BA and N2B cardiac isoforms appeared to be associated with greatest risk of cardiac involvement. One‐third of patients had 1 mutation predicted to impact exons present in fetal skeletal muscle, but not included within the mature skeletal muscle isoform transcript. This strongly suggests developmental isoforms are involved in the pathogenesis of this congenital/early onset disorder.InterpretationThis detailed clinical reference dataset will greatly facilitate diagnostic confirmation and management of patients, and has provided important insights into disease pathogenesis. Ann Neurol 2018;83:1105–1124

The force and stiffness of myosin motors in the isometric twitch of a cardiac trabecula and the effect of the extracellular calcium concentration.

Fast sarcomere-level mechanics in intact trabeculae, which allows the definition of the mechano-kinetic properties of cardiac myosin in situ, is a fundamental tool not only for understanding the molecular mechanisms of heart performance and regulation, but also for investigating the mechanisms of the cardiomyopathy-causing mutations in the myosin and testing small molecules for therapeutic interventions. The approach has been applied to measure the stiffness and force of the myosin motor and the fraction of motors attached during isometric twitches of electrically paced trabeculae under different extracellular Ca2+ concentrations. Although the average force of the cardiac myosin motor (∼6pN) is similar to that of the fast myosin isoform of skeletal muscle, the stiffness (1.07pNnm-1 ) is 2- to 3-fold smaller. The increase in the twitch force developed in the presence of larger extracellular Ca2+ concentrations is fully accounted for by a proportional increase in the number of attached motors. The mechano-kinetic properties of the cardiac myosin were studied in situ, in trabeculae dissected from the right ventricle of the rat heart, by measuring the stiffness of the half-sarcomere both at the twitch force peak (Tp ) of an electrically paced intact trabecula at different extracellular Ca2+ concentrations ([Ca2+ ]o ), and in the same trabecula after skinning and induction of rigor. Taking into account the contribution of filament compliance to half-sarcomere compliance and the lattice geometry, we found that the stiffness of the cardiac myosin motor is 1.07±0.09pNnm-1 , which is slightly larger than that of the slow myosin isoform of skeletal muscle (0.6-0.8pNnm-1 ) and 2- to 3-fold smaller than that of the fast skeletal muscle isoform. The increase in Tp from 61±4kPa to 93±9kPa, induced by raising [Ca2+ ]o from 1 to 2.5mm at sarcomere length ∼2.2μm, is accompanied by an increase of the half-sarcomere stiffness that is explained by an increase of the fraction of actin-attached motors from 0.08±0.01 to 0.12±0.02, proportional to Tp . Consequently, each myosin motor bears an average force of 6.14±0.52pN independently of Tp and [Ca2+ ]o . The application of fast sarcomere-level mechanics to intact trabeculae to define the mechano-kinetic properties of the cardiac myosin in situ represents a powerful tool for investigating cardiomyopathy-causing mutations in the myosin motor and testing specific therapeutic interventions.

Blood Lipid is Associated with Skeletal Muscle PPARδ Protein Content after a 10-week Resistance Exercise Training

Peroxisome proliferator activated receptors (PPARs) are main regulators of blood lipid profile while PPARδ is the most abundant PPARs isoform in skeletal muscle. Although skeletal muscle is a major player in lipid metabolism, the association between muscle PPARδ and blood lipid profile is currently unknown. PURPOSE: The purpose of this study was to investigate the association between muscle PPARδ protein content and blood lipid in the context of resistance exercise training. METHODS: Fifteen untrained, healthy young men (n=8) and women (n=7) performed a 10-week progressive whole-body resistance exercise training program. Muscle samples were obtained from the vastus lateralis muscles 24 hours before the first exercise and 24 hours after the last exercise. Blood samples were collected from antecubital veins immediately before the first and the last exercises. Western Blotting was conducted to quantify the PPARδ content, and serum lipid profile was analyzed. RESULTS: While PPARδ protein content was not correlated with blood lipid profile before the training, it was negatively correlated with total cholesterol (R=-0.731, R-square=0.534, P=0.04) and LDL (R=-0.746, R-square=0.557, P=0.033) after the training. CONCLUSION: The results imply that resistance exercise training may enhance the regulation of blood lipid via PPARδ.Table: No title available.

Troponin Subunits are Expressed in the Rat Afferent Arteriole

Vascular smooth muscle cells of the renal afferent arteriole are unusual: they must be able to contract very rapidly in response to a sudden increase in systemic blood pressure, thereby protecting the downstream glomerular capillaries from catastrophic damage. We showed that this could be accounted for in part by exclusive expression, at the protein level, of the “fast” (B) isoforms of smooth muscle myosin II heavy chains in the afferent arteriole, in contrast to other vascular smooth muscle cells such as the rat aorta and efferent arteriole, which express exclusively the “slow” (A) isoforms (Shiraishi M et al (2003) FASEB J 17: 2284–2286). Since contraction of the more rapidly‐contracting striated (skeletal and cardiac) muscles is regulated by the thin filament‐associated troponin (Tn) system, we hypothesized that Tn or a Tn‐like system may exist in afferent arteriolar cells and contribute to the unusually rapid contraction of this tissue in response to increased intraluminal pressure. We examined the expression of TnC (Ca2+‐binding subunit), TnI (inhibitory subunit) and TnT (tropomyosin‐binding subunit) in vascular smooth muscle cells of the rat renal afferent arteriole at the mRNA level. Fast skeletal muscle and slow skeletal muscle/cardiac TnC isoforms and slow skeletal muscle and cardiac TnI isoforms were detected by RT‐PCR and confirmed by cDNA sequencing. Furthermore, cardiac and slow skeletal muscle TnI isoforms, but not fast skeletal muscle TnI were detected in isolated afferent arterioles at the protein level by proximity ligation assay. We conclude that, in addition to Ca2+‐mediated phosphorylation of myosin regulatory light chains, contraction of the afferent arteriole may be regulated by a mechanism normally associated with the much more rapidly contracting cardiac and skeletal muscles, which involves Ca2+ binding to TnC, leading to alleviation of inhibition of the actomyosin MgATPase by TnI and tropomyosin and rapid contraction of the vessel.Support or Funding InformationSupported by the Canadian Institutes of Health ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular Cloning, Characterization, and Temporal Expression Profile of Troponin I Type 1 (TNNI1) Gene in Skeletal Muscle During Early Development of Gaoyou Duck (Anas Platyrhynchos Domestica)

ABSTRACTTNNI1 encodes the slow skeletal muscle isoform of troponin I. In the present study, the basic characteristic and expressing profile of the TNNI1 gene was first explored in Gaoyou ducks. Full-length TNNI1 cDNA of Gaoyou duck was obtained using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cDNA consisted of a 57-base pair (bp) 5′UTR, a 345-bp 3′UTR, and a 564-bp open reading frame. The predicted protein was predicted to be hydrophilic, nonsecretory protein and contained 17 phosphorylation sites. Multiple alignments and phylogenetic tree analyses showed that the predicted protein was relatively conserved in avian. TNNI1 mRNA could be detected in every tissue analyzed at 70 days of age, and the muscle tissues had relatively high expression level, with the highest level seen in leg muscle. The TNNI1 gene was differentially expressed in the breast muscle and leg muscle during embryonic and posthatching development. Our findings reveal the sequence characterization and expression patterns of the TNNI1 gene, which may provide correlative evidence that TNNI1 gene plays an important role in duck muscle fiber development and meat quality.