Troponin T nuclear localization and its role in aging skeletal muscle.
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ABSTRACT:
SUBMITTER: Zhang T
PROVIDER: S-EPMC3592954 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Age-related muscle mass and strength decline (sarcopenia) impairs the performance of daily living activities and can lead to mobility disability/limitation in older adults. Biological pathways in muscle that lead to mobility problems have not been fully elucidated. Immunoglobulin G (IgG) infiltration in muscle is a known marker of increased fiber membrane permeability and damage vulnerability, but whether this translates to impaired function is unknown. Here, we report that IgG1 and IgG4 are abundantly present in the skeletal muscle (vastus lateralis) of ~ 50% (11 out of 23) of older adults (> 65 years) examined. Skeletal muscle IgG1 was inversely correlated with physical performance (400 m walk time: r = 0.74, p = 0.005; SPPB score: r = - 0.73, p = 0.006) and muscle strength (r = - 0.6, p = 0.05). In a murine model, IgG was found to be higher in both muscle and blood of older, versus younger, C57BL/6 mice. Older mice with a higher level of muscle IgG had lower motor activity. IgG in mouse muscle co-localized with cardiac troponin T (cTnT) and markers of complement activation and apoptosis/necroptosis. Skeletal muscle-inducible cTnT knockin mice also showed elevated IgG in muscle and an accelerated muscle degeneration and motor activity decline with age. Most importantly, anti-cTnT autoantibodies were detected in the blood of cTnT knockin mice, old mice, and older humans. Our findings suggest a novel cTnT-mediated autoimmune response may be an indicator of sarcopenia.
| S-EPMC9616986 | biostudies-literature
Project description:Loss of strength in human and animal models of aging can be partially attributed to a well-recognized decrease in muscle mass; however, starting at middle-age, the normalized force (force/muscle cross-sectional area) in the knee extensors and single muscle fibers declines in a curvilinear manner. Strength is lost faster than muscle mass and is a more consistent risk factor for disability and death. Reduced expression of the voltage sensor Ca(2+) channel α1 subunit (Cav1.1) with aging leads to excitation-contraction uncoupling, which accounts for a significant fraction of the decrease in skeletal muscle function. We recently reported that in addition to its classical cytoplasmic location, fast skeletal muscle troponin T3 (TnT3) is fragmented in aging mice, and both full-length TnT3 (FL-TnT3) and its carboxyl-terminal (CT-TnT3) fragment shuttle to the nucleus. Here, we demonstrate that it regulates transcription of Cacna1s, the gene encoding Cav1.1. Knocking down TnT3 in vivo downregulated Cav1.1. TnT3 downregulation or overexpression decreased or increased, respectively, Cacna1s promoter activity, and the effect was ablated by truncating the TnT3 nuclear localization sequence. Further, we mapped the Cacna1s promoter region and established the consensus sequence for TnT3 binding to Cacna1s promoter. Systemic administration of BDA-410, a specific calpain inhibitor, prevented TnT3 fragmentation, and Cacna1s and Cav1.1 downregulation and improved muscle force generation in sedentary old mice.
| S-EPMC4854922 | biostudies-literature
Project description:Troponin T (TnT) plays a major role in striated muscle contraction. We recently demonstrated that the fast skeletal muscle TnT3 isoform is localized in the muscle nucleus, and either its full-length or COOH-terminus leads to muscle cell apoptosis. Here, we further explored the mechanism by which it enters the nucleus and promotes cytotoxicity. Amino acid truncation and substitution showed that its COOH-terminus contains a dominant nuclear/nucleolar localization sequence (KLKRQK) and the basic lysine and arginine residues might play an important role in the nuclear retention and nucleolar enrichment of KLKRQK-DsRed fusion proteins. Deleting this domain or substituting lysine and arginine residues (KLAAQK) resulted in a dramatic loss of TnT3 nuclear and nucleolar localization. In contrast, the GATAKGKVGGRWK domain-DsRed construct localized exclusively in the cytoplasm, indicating that a nuclear exporting sequence is possibly localized in this region. Additionally, we identified a classical DNA-binding leucine zipper domain (LZD) which is conserved among TnT isoforms and species. Deletion of LZD or KLKRQK sequence significantly reduced cell apoptosis compared to full-length TnT3. We conclude that TnT3 contains both a nuclear localization signal and a DNA-binding domain, which may mediate nuclear/nucleolar signaling and muscle cell apoptosis.
| S-EPMC3667714 | biostudies-literature
Project description:Time-resolved changes in the conformation of troponin in the thin filaments of skeletal muscle were followed during activation in situ by photolysis of caged calcium using bifunctional fluorescent probes in the regulatory and the coiled-coil (IT arm) domains of troponin. Three sequential steps in the activation mechanism were identified. The fastest step (1,100 s(-1)) matches the rate of Ca(2+) binding to the regulatory domain but also dominates the motion of the IT arm. The second step (120 s(-1)) coincides with the azimuthal motion of tropomyosin around the thin filament. The third step (15 s(-1)) was shown by three independent approaches to track myosin head binding to the thin filament, but is absent in the regulatory head. The results lead to a four-state structural kinetic model that describes the molecular mechanism of muscle activation in the thin filament-myosin head complex under physiological conditions.
| S-EPMC3970506 | biostudies-literature
Project description:BackgroundMuscle weakness is a common symptom in numerous diseases and a regularly occurring problem associated with ageing. Prolonged low-frequency force depression (PLFFD) is a form of exercise-induced skeletal muscle weakness observed after exercise. Three different intramuscular mechanisms underlying PLFFD have been identified: decreased sarcoplasmic reticulum Ca2+ release, decreased myofibrillar Ca2+ sensitivity, and myofibrillar dysfunction. We here used these three forms of PLFFD as models to study the effectiveness of a fast skeletal muscle troponin activator, CK-2066260, to mitigate muscle weakness.MethodsExperiments were performed on intact single muscle fibres or fibre bundles from mouse flexor digitorum brevis, which were stimulated with electrical current pulses, while force and the free cytosolic [Ca2+ ] ([Ca2+ ]i ) were measured. PLFFD was induced by three different stimulation protocols: (i) repeated isometric contractions at low intensity (350 ms tetani given every 5 s for 100 contractions); (ii) repeated isometric contractions at high intensity (250 ms tetani given every 0.5 s for 300 contractions); and (iii) repeated eccentric contractions (350 ms tetani with 20% length increase given every 20 s for 10 contractions). The extent and cause of PLFFD were assessed by comparing the force-[Ca2+ ]i relationship at low (30 Hz) and high (120 Hz) stimulation frequencies before (control) and 30 min after induction of PLFFD, and after an additional 5 min of rest in the presence of CK-2066260 (10 μM).ResultsProlonged low-frequency force depression following low-intensity and high-intensity fatiguing contractions was predominantly due to decreased sarcoplasmic reticulum Ca2+ release and decreased myofibrillar Ca2+ sensitivity, respectively. CK-2066260 exposure resulted in marked increases in 30 Hz force from 52 ± 16% to 151 ± 13% and from 6 ± 4% to 98 ± 40% of controls with low-intensity and high-intensity contractions, respectively. Following repeated eccentric contractions, PLFFD was mainly due to myofibrillar dysfunction, and it was not fully reversed by CK-2066260 with 30 Hz force increasing from 48 ± 8% to 76 ± 6% of the control.ConclusionsThe fast skeletal muscle troponin activator CK-2066260 effectively mitigates muscle weakness, especially when it is caused by impaired activation of the myofibrillar contractile machinery due to either decreased sarcoplasmic reticulum Ca2+ release or reduced myofibrillar Ca2+ sensitivity.
| S-EPMC7749611 | biostudies-literature
Project description:Skeletal muscle has emerged as one of the most important tissues involved in regulating systemic metabolism. The gastrocnemius is a powerful skeletal muscle composed of predominantly glycolytic fast-twitch fibers that are preferentially lost among old age. This decrease in gastrocnemius muscle mass is remarkable during aging; however, the underlying molecular mechanism is not fully understood. Strikingly, there is a ~70% decrease in Cisd2 protein, a key regulator of lifespan in mice and the disease gene for Wolfram syndrome 2 in humans, within the gastrocnemius after middle age among mice. A proteomics approach was used to investigate the gastrocnemius of naturally aged mice, and this was compared to the autonomous effect of Cisd2 on gastrocnemius aging using muscle-specific Cisd2 knockout (mKO) mice as a premature aging model. Intriguingly, dysregulation of calcium signaling and activation of UPR/ER stress stand out as the top two pathways. Additionally, the activity of Serca1 was significantly impaired and this impairment is mainly attributable to irreversibly oxidative modifications of Serca. Our results reveal that the overall characteristics of the gastrocnemius are very similar when naturally aged mice and the Cisd2 mKO mice are compared in terms of pathological alterations, ultrastructural abnormalities, and proteomics profiling. This suggests that Cisd2 mKO mouse is a unique model for understanding the aging mechanism of skeletal muscle. Furthermore, this work substantiates the hypothesis that Cisd2 is crucial to the gastrocnemius muscle and suggests that Cisd2 is a potential therapeutic target for muscle aging.
| S-EPMC5770874 | biostudies-literature
Project description:As muscle contraction requires ATP and Ca(2+), skeletal muscle function is highly dependent on communication between two major intracellular organelles: mitochondria and sarcoplasmic reticulum (SR). In adult skeletal muscle, mitochondria located within the I-band of the sarcomere are connected to the SR by small ?10 nm electron dense tethers that bridge the outer mitochondrial membrane to the region of SR that is ?130 nm from the site of Ca(2+) release. However, the molecular composition of tethers and their precise impact on mitochondrial Ca(2+) uptake in skeletal muscle is unclear. Mitofusin-2 (Mfn2) is a transmembrane GTPase present in both mitochondria and ER/SR membranes that forms trans-dimers and participates in mitochondrial fusion. Here we evaluated the role Mfn2 plays in mitochondrial morphology, localization, and functional SR-mitochondrial Ca(2+) crosstalk in adult skeletal muscle. Compared to a non-targeting (CTRL) siRNA, in vivo electroporation of 400 nM Mfn2 siRNA (Mfn2 KD) into mouse footpads resulted in a marked acute reduction (67±3%) in Mfn2 protein levels in flexor digitorum brevis (FDB) muscles that occurred without a change in other key Ca(2+) regulatory proteins. Electron microscopy analyses revealed that Mfn2 knockdown resulted in a change in mitochondria morphology and mis-localization of some mitochondria from the I-band to the A-band region of the sarcomere. To assess the role of Mfn2 in SR-mitochondrial crosstalk, we measured mitochondrial Ca(2+) uptake and myoplasmic Ca(2+) transients with rhod-2 and mag-fluo-4, respectively, during repetitive high frequency tetanic stimulation (5×100 Hz tetani, 500 ms/tetani, 0.2 duty cycle) in CTRL and Mfn2 KD fibers. Mitochondrial Ca(2+) uptake during repetitive tetanic stimulation was significantly reduced (40%) in Mfn2 KD FDB fibers, which was accompanied by a parallel elevation in the global electrically evoked myoplasmic Ca(2+) transient. Mfn2 KD also resulted in a reduction of the mitochondrial membrane potential, which contributed to the observed decrease in activity-dependent mitochondrial Ca(2+) uptake. Consistent with this idea, a similar decrease in mitochondrial Ca(2+) uptake was also observed in wild type fibers following a comparable reduction in mitochondrial membrane potential induced by acute exposure to a low concentration (50 nM) of carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). In addition, both global and mitochondrial Ca(2+) transients during repetitive tetanic stimulation were similarly reduced by both slow (EGTA) and fast (BAPTA) Ca(2+) chelating agents. Together, these results indicate that Mfn2 promotes proper mitochondrial morphology, localization, and membrane potential required for optimal activity-dependent mitochondrial Ca(2+) uptake and buffering of the global myoplasmic Ca(2+) transient in adult skeletal muscle.
| S-EPMC4300280 | biostudies-literature
Project description:Age-associated changes in gene expression in skeletal muscle of healthy individuals reflect accumulation of damage and compensatory adaptations to preserve tissue integrity. To characterize these changes, RNA was extracted and sequenced from muscle biopsies collected from 53 healthy individuals (22-83 years old) of the GESTALT study of the National Institute on Aging-NIH. Expression levels of 57,205 protein-coding and non-coding RNAs were studied as a function of aging by linear and negative binomial regression models. From both models, 1134 RNAs changed significantly with age. The most differentially abundant mRNAs encoded proteins implicated in several age-related processes, including cellular senescence, insulin signaling, and myogenesis. Specific mRNA isoforms that changed significantly with age in skeletal muscle were enriched for proteins involved in oxidative phosphorylation and adipogenesis. Our study establishes a detailed framework of the global transcriptome and mRNA isoforms that govern muscle damage and homeostasis with age.
| S-EPMC8016876 | biostudies-literature
Project description:The zebrafish mutant silent partner is characterized by a dysmorphic, non-contractile ventricle resulting in an inability to generate normal blood flow. We have identified the genetic lesion in the zebrafish homolog of the slow twitch skeletal/cardiac troponin C gene. Although human troponin C1 (TNNC1) is expressed in both cardiac and skeletal muscle, duplication of this gene in zebrafish has resulted in tissue-specific partitioning of troponin C expression and function. Mutation of the zebrafish paralog tnnc1a, which is expressed predominantly in the heart, results in a loss of contractility and myofibrillar organization within ventricular cardiomyocytes, while skeletal muscle remains functional and intact. We further show that defective contractility in the developing heart results in abnormal atrial and ventricular chamber morphology. Together, our results suggest that tnnc1a is required both for the function and structural integrity of the contractile machinery in cardiomyocytes, helping to clarify potential mechanisms of troponin C-mediated cardiomyopathy.
| S-EPMC2965274 | biostudies-literature
Project description:BackgroundAgeing skeletal muscle undergoes chronic denervation, and the neuromuscular junction (NMJ), the key structure that connects motor neuron nerves with muscle cells, shows increased defects with ageing. Previous studies in various species have shown that with ageing, type II fast-twitch skeletal muscle fibres show more atrophy and NMJ deterioration than type I slow-twitch fibres. However, how this process is regulated is largely unknown. A better understanding of the mechanisms regulating skeletal muscle fibre-type specific denervation at the NMJ could be critical to identifying novel treatments for sarcopenia. Cardiac troponin T (cTnT), the heart muscle-specific isoform of TnT, is a key component of the mechanisms of muscle contraction. It is expressed in skeletal muscle during early development, after acute sciatic nerve denervation, in various neuromuscular diseases and possibly in ageing muscle. Yet the subcellular localization and function of cTnT in skeletal muscle is largely unknown.MethodsStudies were carried out on isolated skeletal muscles from mice, vervet monkeys, and humans. Immunoblotting, immunoprecipitation, and mass spectrometry were used to analyse protein expression, real-time reverse transcription polymerase chain reaction was used to measure gene expression, immunofluorescence staining was performed for subcellular distribution assay of proteins, and electromyographic recording was used to analyse neurotransmission at the NMJ.ResultsLevels of cTnT expression in skeletal muscle increased with ageing in mice. In addition, cTnT was highly enriched at the NMJ region-but mainly in the fast-twitch, not the slow-twitch, muscle of old mice. We further found that the protein kinase A (PKA) RIα subunit was largely removed from, while PKA RIIα and RIIβ are enriched at, the NMJ-again, preferentially in fast-twitch but not slow-twitch muscle in old mice. Knocking down cTnT in fast skeletal muscle of old mice: (i) increased PKA RIα and reduced PKA RIIα at the NMJ; (ii) decreased the levels of gene expression of muscle denervation markers; and (iii) enhanced neurotransmission efficiency at NMJ.ConclusionsCardiac troponin T at the NMJ region contributes to NMJ functional decline with ageing mainly in the fast-twitch skeletal muscle through interfering with PKA signalling. This knowledge could inform useful targets for prevention and therapy of age-related decline in muscle function.
| S-EPMC5659053 | biostudies-literature