Intact Muscle Fibers Research Articles

The effect of high intensity interval training on muscle contractile function 8 weeks following chemically-induced ovarian failure

The negative effects of ovariectomy on muscle contractile function have been well-characterized, however, the effects of gradual ovarian failure (i.e., perimenopausal transition into late-stage menopause) on muscle function over the lifespan have received less attention. Furthermore, whether exercise training can mitigate changes in muscle contractile function associated with menopause is unclear. The objective of this study, using a chemically-induced ovarian failure mouse model (4-vinylcyclohexene diepoxide; VCD), was to investigate time-course changes in muscle contractility of sedentary controls and the potential of high intensity interval training to mitigate any deleterious effects of ovarian failure. Starting at 11wks old, mice were injected with 160mg/kg/day of VCD for 15 days. Twenty-eight (VCD-trained: n=10, VCD-sedentary: n=10, control: n=8) CD1 female mice were used in this study, with the VCD-trained group beginning training at the onset of ovarian failure for a total of 8 weeks. Contractile properties of the plantar flexors were assessed using an in-vivo set-up 8 weeks following the onset of ovarian failure. As well, a fatigue task (repeated maximal isometric contractions until torque decreased by 60%) was performed. Recovery was measured immediately after, and up to 10min following task termination. Upon completion of mechanical testing, mice were sacrificed and intact muscle fibres were isolated from the flexor digitorum brevis, and myoplasmic free Ca2+ (tetanic [Ca2+]i) concentrations were measured across stimulation frequencies of 10-200 Hz and throughout 50 tetanic contractions (70Hz) to replicate our fatigue task. There was no difference in pre-fatigue values across groups for peak twitch torque, peak 100Hz torque, RTD, 10:100Hz torque, tetanic [Ca2+]i during low (10Hz) and high Hz (100Hz) stimulation, and all were reduced similarly immediately following the fatigue task. Repetitions to task failure was similar across groups and tetanic [Ca2+]i during repetitive contractions (n=50) was reduced similarly across groups. As well, all groups recovered similarly across these measures. Torque and RTD did not recover fully by 10min for either measure, while 10Hz, 100Hz and 10:100Hz tetanic [Ca2+]i was recovered immediately following the fatigue task. Given 10Hz torque was relatively maintained following the fatigue task, and there was a ~40% decline in 100Hz torque, the 10:100Hz ratio increased throughout recovery — this, combined with the [Ca2+] data indicate that calcium sensitivity and release are unlikely contributors to impaired force production following the fatigue task across groups. The present study aimed to assess the impact of training on muscle contractility using a mouse model of gradual ovarian failure. Unlike other models of ovarian failure, there does not seem to be any impairment in muscular performance at the joint level in our VCD-mice, and they responded similarly across groups in response to repetitive fatiguing contractions and exercise training. Supported by NSERC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Time course changes in in vivo muscle mechanical function and Ca2+ regulation of force following experimentally induced gradual ovarian failure in mice.

The abrupt cessation of ovarian hormone release is associated with declines in muscle contractile function, yet the impact of gradual ovarian failure on muscle contractility across peri-, early- and late-stage menopause remains unclear. In this study, a 4-vinylcyclohexene diepoxide (VCD)-induced ovarian failure mouse model was used to examine time course changes in muscle mechanical function. Plantar flexors of female mice (VCD: n=10; CON: n=8) were assessed at 40 (early perimenopause), 80 (late perimenopause), 120 (menopause onset) and 176 (late menopause) days post-initial VCD injection. A torque-frequency relationship was established across a range of frequencies (10-200Hz). Isotonic dynamic contractions were elicited against relative loads (10-80% maximal isometric torque) to determine the torque-velocity-power relationship. Mice then performed a fatigue task using intermittent 100Hz isometric contractions until torque dropped by 60%. Recovery of twitch, 10Hz and 100Hz torque were tracked for 10min post-task failure. Additionally, intact muscle fibres from the flexor digitorum brevis underwent a fatigue task (50 repetitions at 70Hz), and 10 and 100Hz tetanic [Ca2+] were monitored for 10min afterward. VCD mice exhibited 16% lower twitch torque than controls across all time points. Apart from twitch torque, 10Hz torque and 10Hz tetanic [Ca2+], where VCD showed greater values relative to pre-fatigue during recovery, no significant differences were observed between control and VCD mice during recovery. These results indicate that gradual ovarian failure has minimal detriments to in vivo muscle mechanical function, with minor alterations observed primarily for low-frequency stimulation during recovery from fatigue.

Sequences in the myosin A rod interact with UNC-89/obscurin and the zinc-finger protein UNC-98 during thick filament assembly and M-line formation in C. elegans striated muscle.

The M-line of striated muscle is a complex structure that anchors myosin-containing thick filaments and also participates in signaling and proteostasis. While the physical associations among many M-line components have been defined, the mechanism of thick filament attachment is not completely understood. In Caenorhabditis elegans, myosin A is essential for viability and forms the site of M-line attachment at the center of the filament, whereas myosin B forms the filament arms. Using a mutant myosin A that forms ectopic filaments, we examined interactions between myosin A and M-line proteins in intact muscle cells. Ectopic myosin A recruits the giant kinase UNC-89/obscurin, a presumed scaffolding protein, in an interaction that requires the zinc-finger protein UNC-98, but not UNC-82/NUAK, UNC-97/PINCH, or UNC-96. In myosin A mutants, UNC-89/obscurin patterning is highly defective in embryos and adults. A chimeric myosin containing 169 residues of the myosin A C-terminal rod, coincident with the UNC-98/ZnF binding site, is sufficient for colocalization of UNC-89/obscurin and UNC-98/ZnF in M-line structures whereas a myosin chimera lacking these residues colocalizes with UNC-89/obscurin in M-lines that lack UNC-98. Thus, at least two myosin A rod regions contribute independently to M-line organization. We hypothesize that these M-line-organizing functions correspond to the essential "filament initiation function" performed by this isoform.

Ca2+ and force during dynamic contractions in mouse intact skeletal muscle fibers

Muscle fiber force production is determined by the excitation frequency of motor nerves, which induce transient increases in cytoplasmic free Ca2+ concentration ([Ca2+]i) and the force-generating capacity of the actomyosin cross-bridges. Previous studies suggest that, in addition to altered cross-bridge properties, force changes during dynamic (concentric or eccentric) contraction might be affected by Ca2+-dependent components. Here we investigated this by measuring [Ca2+]i and force in mouse muscle fibers undergoing isometric, concentric, and eccentric contractions. Intact single muscle fibers were dissected from the flexor digitorum brevis muscle of mice. Fibers were electrically activated isometrically at 30–100 Hz and after reaching the isometric force plateau, they were actively shortened or stretched. We calculated the ratio (relative changes) in force and [Ca2+]i attained in submaximal (30 Hz) and near-maximal (100 Hz) contractions under isometric or dynamic conditions. Tetanic [Ca2+]i was similar during isometric, concentric and eccentric phases of contraction at given stimulation frequencies while the forces were clearly different depending on the contraction types. The 30/100 Hz force ratio was significantly lower in the concentric (44.1 ± 20.3%) than in the isometric (50.3 ± 20.4%) condition (p = 0.005), whereas this ratio did not differ between eccentric and isometric conditions (p = 0.186). We conclude that the larger force decrease by decreasing the stimulation frequency during concentric than during isometric contraction is caused by decreased myofibrillar Ca2+ sensitivity, not by the decreased [Ca2+]i.

Sporadic inclusion body myositis-derived myotube culture revealed muscle cell-autonomous expression profiles.

Sporadic inclusion body myositis (sIBM) is a muscle disease in older people and is characterized by inflammatory cell invasion into intact muscle fibers and rimmed vacuoles. The pathomechanism of sIBM is not fully elucidated yet, and controversy exists as to whether sIBM is a primary autoimmune disease or a degenerative muscle disease with secondary inflammation. Previously, we established a method of collecting CD56-positive myoblasts from human skeletal muscle biopsy samples. We hypothesized that the myoblasts derived from these patients are useful to see the cell-autonomous pathomechanism of sIBM. With these resources, myoblasts were differentiated into myotubes, and the expression profiles of cell-autonomous pathology of sIBM were analyzed. Myoblasts from three sIBM cases and six controls were differentiated into myotubes. In the RNA-sequencing analysis of these "myotube" samples, 104 differentially expressed genes (DEGs) were found to be significantly upregulated by more than twofold in sIBM, and 13 DEGs were downregulated by less than twofold. For muscle biopsy samples, a comparative analysis was conducted to determine the extent to which "biopsy" and "myotube" samples differed. Fifty-three DEGs were extracted of which 32 (60%) had opposite directions of expression change (e.g., increased in biopsy vs decreased in myotube). Apolipoprotein E (apoE) and transmembrane protein 8C (TMEM8C or MYMK) were commonly upregulated in muscle biopsies and myotubes from sIBM. ApoE and myogenin protein levels were upregulated in sIBM. Given that enrichment analysis also captured changes in muscle contraction and development, the triggering of muscle atrophy signaling and abnormal muscle differentiation via MYMK or myogenin may be involved in the pathogenesis of sIBM. The presence of DEGs in sIBM suggests that the myotubes formed from sIBM-derived myoblasts revealed the existence of muscle cell-autonomous degeneration in sIBM. The catalog of DEGs will be an important resource for future studies on the pathogenesis of sIBM focusing on primary muscle degeneration.

Oxidative behaviour, aggregation and structural properties of silver carp myofibrillar proteins with different molecular states subjected to freeze–thaw process

SummarySilver carp myofibrillar proteins (MP) with different moleculars states were constructed by the addition of NaCl and fracture degree of muscle fibres. Results showed that salt‐dissolved MP (P+) under unfrozen and oxidised state showed higher oxidation degree with higher carbonyl (3.42 nmol mg−1) and dityrosine contents (25.98 a.u.) (P < 0.05) compared with saltless‐polymeric MP (P) and salt‐induced swelling MP (F+) due to addition of salt and broken muscle fibres. After oxidative and freeze–thaw process, P+ group also exhibited more cross‐linking as evidenced by turbidity (0.73), solubility (64.96%) and SDS‐PAGE, but unordered aggregates in P group increased probably due to severe frozen denaturation. Moreover, F+ group of salt‐induced swelling MP from intact muscle fibres suffered less structural damages with higher fluorescence intensity (668.41 a.u.) and higher α‐helix contents (15.56%). Overall, Fish‐ Hua of salt‐dissolved MP had a higher oxidation level and NaCl contributed to improve freeze–thaw stability of MP during freeze–thaw process.

Effect of artemisinin combined with allicin on improving cardiac function, fibrosis and NF-κB signaling pathway in rats with diabetic cardiomyopathy

Myocardial fibrosis and inflammation cause cardiac hypertrophy, arrhythmias, and heart failure in diabetics, a leading cause of mortality. Since it’s complicated, no drug treats diabetic cardiomyopathy. This research examined the effects of artemisinin and allicin on heart function, myocardial fibrosis, and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway in diabetic cardiomyopathy rats. A total of 50 rats were separated into 5 groups, 10 of which were the control group. 40 rats received 65 μg/g streptozotocin intraperitoneally. 37 of 40 animals fit the investigation. The artemisinin, allicin, and artemisinin/allicin groups each included nine animals. The artemisinin group received 75 mg/kg of artemisinin, the allicin group received 40 mg/kg of allicin, and the combination group received equal dosages of artemisinin and allicin gavage for four weeks. After the intervention, in each group cardiac functions, myocardial fibrosis, and NF-κB signaling pathway protein expression were assessed. All of the examined groups had greater levels of LVEDD, LVESD, LVEF, FS, E/A, and the NF-κB pathway proteins: NF-κB p65 and p-NF-κB p65 than the normal group, except for the combination group. Artemisinin and allicin did not vary statistically. Compared to the model group, the artemisinin, allicin, and combined groups showed various degrees of improvement from the pathological pattern, with more intact muscle fibers, neater arrangement, more normal cell morphology, artemisinin and allicin alleviated cardiac dysfunction and decreased myocardium fibrosis in diabetic cardiomyopathy rats by inactivating the NF-κB signaling cascade.

High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System.

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches involve either differentiating myogenic progenitor cells into immature myotubes or the short-term ex vivo culture of isolated individual muscle fibers. A key benefit of ex vivo culture over in vitro is the retention of the complex cellular architecture and contractile characteristics. Here, we detail an experimental protocol for the isolation of intact flexor digitorum brevis muscle fibers from mice and their subsequent ex vivo culture. In this protocol, muscle fibers are embedded in a fibrin-based and basement membrane matrix hydrogel to immobilize the fibers and maintain their contractile function. We then describe methods to assess the muscle fiber contractile function using an optics-based, high-throughput contractility system. The embedded muscle fibers are electrically stimulated to induce contractions, after which their functional properties, such as sarcomere shortening and contractile velocity, are assessed using optics-based quantification. Coupling muscle fiber culture with thissystem allows for high-throughput testing of the effects of pharmacological agents on contractile function and ex vivo studies of genetic muscle disorders. Finally, this protocol can also be adapted to study dynamic cellular processes in muscle fibers using live-cell microscopy.

Titin activates myosin filaments in skeletal muscle by switching from an extensible spring to a mechanical rectifier

Titin is a molecular spring in parallel with myosin motors in each muscle half-sarcomere, responsible for passive force development at sarcomere length (SL) above the physiological range (>2.7 μm). The role of titin at physiological SL is unclear and is investigated here in single intact muscle cells of the frog (Rana esculenta), by combining half-sarcomere mechanics and synchrotron X-ray diffraction in the presence of 20 μM para-nitro-blebbistatin, which abolishes the activity of myosin motors and maintains them in the resting state even during activation of the cell by electrical stimulation. We show that, during cell activation at physiological SL, titin in the I-band switches from an SL-dependent extensible spring (OFF-state) to an SL-independent rectifier (ON-state) that allows free shortening while resisting stretch with an effective stiffness of ~3 pN nm-1 per half-thick filament. In this way, I-band titin efficiently transmits any load increase to the myosin filament in the A-band. Small-angle X-ray diffraction signals reveal that, with I-band titin ON, the periodic interactions of A-band titin with myosin motors alter their resting disposition in a load-dependent manner, biasing the azimuthal orientation of the motors toward actin. This work sets the stage for future investigations on scaffold and mechanosensing-based signaling functions of titin in health and disease.

Titin function in the active sarcomere studied in frog muscle fibers upon inhibition of myosin motors by para-nitro-blebbistatin.

Titin spans the whole half-sarcomere, through the I-band connecting the Z-line to the myosin filament tip, and through the A-band, associated to myosin filament, up to M-line. Titin in the I-band transmits the stress also without attached myosin motors and is responsible for the passive force developed when a skeletal muscle fibre is stretched at rest. However, the passive stiffness in the range of physiological sarcomere lengths (SL, <2.7 µm) is too low to account for a putative role of I-band titin as mechanical link able to sustain mechanosensing in myosin filaments and prevent development of sarcomere inhomogeneity during contraction. Here we define titin mechanics during tetanic stimulation of intact frog muscle fibers using 20 μM para-nitro-blebbistatin (which fully inhibits myosin ATPase in vitro) to suppress the myosin-based mechanical responses. Small-angle X-ray fiber diffraction at the European Synchrotron ESRF demonstrates that the resting structure of thick filament is fully preserved upon tetanic stimulation. Under these conditions we use stepwise changes in force (10-50 pN per half-thick filament, htf) to elicit the lengthening transient that defines titin dynamic stiffness in situ at 2.3–3.0 µm SL. We show that, upon tetanic stimulation titin switches from the OFF-state characterised by a large, SL-dependent, extensibility, to the ON-state in which it opposes stretching with an effective stiffness one-two order of magnitude larger (∼3 pN·nm−1·htf−1) and independent of SL. The results demonstrate that, in the active sarcomere working at physiological lengths, I-band titin is an efficient mechanical link between the Z-line and the myosin filament tip, a condition for playing a role in mechanosensing-based myosin filament activation and for opposing to development of SL inhomogeneity during contraction. Supported by ECRF, University of Florence and MUR (Italy), ESRF (France) and EJP-RD.

Effect of Cooking on Physicochemical and Microstructural Properties of Chicken Breast Meat

The effect of cooking on pH, juiciness, instrumental colour and microstructural properties of chicken breast meat was investigated. Industrial skinless chicken breast meat samples were purchased, frozen and sliced into dimensions , thawed and cooked by air frying (AF), baking (BK), deep fat frying (DF) and grilling (GR) at 170, 180 and 1900C for 0, 4, 8, 12 and 16 min. The pH value of the cooked samples increased from 6.05 to 6.25. Cooking methods, temperatures and times each resulted to increase in pH. The results of objective sensory instrumental analyses showed that cooking decreased significantly (p &lt; 0.05) juiciness of cooked chicken breast meat. Samples cooked by BK had the highest juiciness value of 24.91%, while DF cooked samples had the least value of 13.89%.The instrumental analyses increased L*, a*, b* values and browning index. The temperature and time of cooking showed similar effects on juiciness and instrumental colour. Short cooking time (8 min) and 1700C resulted in higher juiciness and best appetizing appearance to the consumers. The microstructure studies showed that raw chicken breast meat had an intact muscle fibres and bundles, but cooking caused disintegration of muscle fibres, perimysial – collagen shrinkage and it resulted to drier samples with big cracks/ voids and big surface damages, particularly in AF, BK and GR cooked products at 1900C for 8 min.

Sarcomere dynamics revealed by a myofilament integrated FRET-based biosensor in live skeletal muscle fibers

The sarcomere is the functional unit of skeletal muscle, essential for proper contraction. Numerous acquired and inherited myopathies impact sarcomere function causing clinically significant disease. Mechanistic investigations of sarcomere activation have been challenging to undertake in the context of intact, live skeletal muscle fibers during real time physiological twitch contractions. Here, a skeletal muscle specific, intramolecular FRET-based biosensor was designed and engineered into fast skeletal muscle troponin C (TnC) to investigate the dynamics of sarcomere activation. In transgenic animals, the TnC biosensor incorporated into the skeletal muscle fiber sarcomeres by stoichiometric replacement of endogenous TnC and did not alter normal skeletal muscle contractile form or function. In intact single adult skeletal muscle fibers, real time twitch contractile data showed the TnC biosensor transient preceding the peak amplitude of contraction. Importantly, under physiological temperatures, inactivation of the TnC biosensor transient decayed significantly more slowly than the Ca2+ transient and contraction. The uncoupling of the TnC biosensor transient from the Ca2+ transient indicates the biosensor is not functioning as a Ca2+ transient reporter, but rather reports dynamic sarcomere activation/ inactivation that, in turn, is due to the ensemble effects of multiple activating ligands within the myofilaments. Together, these findings provide the foundation for implementing this new biosensor in future physiological studies investigating the mechanism of activation of the skeletal muscle sarcomere in health and disease.

Effects of mutant lamins on nucleo-cytoskeletal coupling in Drosophila models of LMNA muscular dystrophy.

The nuclei of multinucleated skeletal muscles experience substantial external force during development and muscle contraction. Protection from such forces is partly provided by lamins, intermediate filaments that form a scaffold lining the inner nuclear membrane. Lamins play a myriad of roles, including maintenance of nuclear shape and stability, mediation of nuclear mechanoresponses, and nucleo-cytoskeletal coupling. Herein, we investigate how disease-causing mutant lamins alter myonuclear properties in response to mechanical force. This was accomplished via a novel application of a micropipette harpooning assay applied to larval body wall muscles of Drosophila models of lamin-associated muscular dystrophy. The assay enables the measurement of both nuclear deformability and intracellular force transmission between the cytoskeleton and nuclear interior in intact muscle fibers. Our studies revealed that specific mutant lamins increase nuclear deformability while other mutant lamins cause nucleo-cytoskeletal coupling defects, which were associated with loss of microtubular nuclear caging. We found that microtubule caging of the nucleus depended on Msp300, a KASH domain protein that is a component of the linker of nucleoskeleton and cytoskeleton (LINC) complex. Taken together, these findings identified residues in lamins required for connecting the nucleus to the cytoskeleton and suggest that not all muscle disease-causing mutant lamins produce similar defects in subcellular mechanics.

Changes in Textural Quality and Water Retention of Spiced Beef under Ultrasound-Assisted Sous-Vide Cooking and Its Possible Mechanisms.

The present study investigated the effects of ultrasound (28 kHz, 60 W at 71 °C for 37 min) combined with sous-vide cooking (at 71 °C for 40, 60, 80, 100, 120 min) on the textural quality, water distribution, and protein characteristics of spiced beef. Results showed that the spiced beef treated with conventional cooking (CT) had the highest cooking loss (41.31%), but the lowest value of shear force (8.13 N), hardness (55.66 N), springiness (3.98 mm), and chewiness (64.36 mJ) compared to ultrasound-assisted sous-vide (USV) and sous-vide cooking (SV) groups. Compared with long-time thermal treatment, USV heating within 100 min enhanced the water retention of spiced beef by maintaining the lower values of cooking loss (16.64~25.76%), T2 relaxation time (242.79~281.19 ms), and free water content (0.16~2.56%), as evident by the intact muscle fibers. Moreover, the USV group had relatively lower carbonyl content, but higher sulfhydryl content compared to CT and SV groups. More protein bands coupled with a minor transformation from α-helixes to β-turns and random coils occurred in USV40~USV80. In conclusion, these results indicated that USV treatment within 100 min positively affected the textural quality and water retention of spiced beef by moderate protein oxidation.

Sphingomyelinase activity promotes atrophy and attenuates force in human muscle fibres and is elevated in heart failure patients.

BackgroundActivation of sphingomyelinase (SMase) as a result of a general inflammatory response has been implicated as a mechanism underlying disease‐related loss of skeletal muscle mass and function in several clinical conditions including heart failure. Here, for the first time, we characterize the effects of SMase activity on human muscle fibre contractile function and assess skeletal muscle SMase activity in heart failure patients.MethodsThe effects of SMase on force production and intracellular Ca2+ handling were investigated in single intact human muscle fibres. Additional mechanistic studies were performed in single mouse toe muscle fibres. RNA sequencing was performed in human muscle bundles exposed to SMase. Intramuscular SMase activity was measured from heart failure patients (n = 61, age 69 ± 0.8 years, NYHA III‐IV, ejection fraction 25 ± 1.0%, peak VO2 14.4 ± 0.6 mL × kg × min) and healthy age‐matched control subjects (n = 10, age 71 ± 2.2 years, ejection fraction 60 ± 1.2%, peak VO2 25.8 ± 1.1 mL × kg × min). SMase activity was related to circulatory factors known to be associated with progression and disease severity in heart failure.ResultsSphingomyelinase reduced muscle fibre force production (−30%, P < 0.05) by impairing sarcoplasmic reticulum (SR) Ca2+ release (P < 0.05) and reducing myofibrillar Ca2+ sensitivity. In human muscle bundles exposed to SMase, RNA sequencing analysis revealed 180 and 291 genes as up‐regulated and down‐regulated, respectively, at a FDR of 1%. Gene‐set enrichment analysis identified ‘proteasome degradation’ as an up‐regulated pathway (average fold‐change 1.1, P = 0.008), while the pathway ‘cytoplasmic ribosomal proteins’ (average fold‐change 0.8, P < 0.0001) and factors involving proliferation of muscle cells (average fold‐change 0.8, P = 0.0002) where identified as down‐regulated. Intramuscular SMase activity was ~20% higher (P < 0.05) in human heart failure patients than in age‐matched healthy controls and was positively correlated with markers of disease severity and progression, and with several circulating inflammatory proteins, including TNF‐receptor 1 and 2. In a longitudinal cohort of heart failure patients (n = 6, mean follow‐up time 2.5 ± 0.2 years), SMase activity was demonstrated to increase by 30% (P < 0.05) with duration of disease.ConclusionsThe present findings implicate activation of skeletal muscle SMase as a mechanism underlying human heart failure‐related loss of muscle mass and function. Moreover, our findings strengthen the idea that SMase activation may underpin disease‐related loss of muscle mass and function in other clinical conditions, acting as a common patophysiological mechanism for the myopathy often reported in diseases associated with a systemic inflammatory response.

Cytokine and Chemokine Release from Immortalised Human Myotubes in Culture: Effect of Contractions and Comparison with Isolated Intact Mature Adult Murine Muscle Fibres

Skeletal muscle releases a number of signalling molecules, including immunomodulatory cytokines and chemokines which can act in an autocrine, paracrine and endocrine fashion. For example, neuromuscular integrity is dependent on efficient and appropriate signalling from muscle to nerve and vice versa and when such signalling fails then tissue deterioration occurs. The aim of this study was to characterise the pattern and time course of release of pro‐ and anti‐ inflammatory cytokines and chemokines from cultured immortalised human myotubes at rest and following contractions compared with release from isolated viable intact mouse muscle fibres. Immortalised human myoblasts were fused to mature into myotubes for 7 days (Donna et al, 2003). Alternatively, single muscle fibres were isolated from the flexor digitorum brevis (FDB) muscle of adult C57Bl/6 mice (Pye et al, 2007). Cells and isolated fibres were subjected to electrical field stimulation with trains of biphasic square wave pulses of 2 ms duration for 0.5 s repeated every 5 s at 50 Hz and 30 V per well of fibres for a total stimulation time of 15 min. Media from all samples was analysed for cytokine content at 15 minutes and 24 hours following contractions using a multiplexed cytokine assay (Luminex) and data were standardised to protein content of the well. At 15 minutes following contractions of isolated muscle fibres, a significant increase in a number of cytokines and chemokines were seen in the media, including IL‐5, IL‐6, IL‐10 and G‐CSF although this was not fully replicated in 2D cultured myotubes. At 24 hours following contractions, IL‐6 content was significantly increased in the media of both isolated fibres and 2D cultured myotubes. Thus, data demonstrate some commonality of cytokines/chemokines released but future studies will examine release from more physiological 3D muscle constructs as well as the effects of such release on neuronal cell structure and function.Pye et al, J Physiol. 2007 May 15; 581(Pt 1): 309–318.Donna et al, Mol Cancer Res. 2003 (1) (9) 643‐653.

Passive and active mechanical properties of titin studied in intact frog muscle fibers upon inhibition of myosin motors by para-nitro-blebbistatin

In the striated muscle thousands of serially linked half-sarcomeres (the force generating units) make macroscopic the shortening but also imply instability, with lengthening of the weaker half-sarcomeres caused by shortening of the stronger ones. The giant protein titin spans the whole half-sarcomere, through the I-band, connecting the Z-line at the end of the sarcomere to the tip of the thick filament and through the A-band, associated to thick filament, up to M-line at the centre of the sarcomere.

Lipocalin 2 Influences Bone and Muscle Phenotype in the MDX Mouse Model of Duchenne Muscular Dystrophy.

Lipocalin 2 (Lcn2) is an adipokine involved in bone and energy metabolism. Its serum levels correlate with bone mechanical unloading and inflammation, two conditions representing hallmarks of Duchenne Muscular Dystrophy (DMD). Therefore, we investigated the role of Lcn2 in bone loss induced by muscle failure in the MDX mouse model of DMD. We found increased Lcn2 serum levels in MDX mice at 1, 3, 6, and 12 months of age. Consistently, Lcn2 mRNA was higher in MDX versus WT muscles. Immunohistochemistry showed Lcn2 expression in mononuclear cells between muscle fibres and in muscle fibres, thus confirming the gene expression results. We then ablated Lcn2 in MDX mice, breeding them with Lcn2−/− mice (MDXxLcn2−/−), resulting in a higher percentage of trabecular volume/total tissue volume compared to MDX mice, likely due to reduced bone resorption. Moreover, MDXxLcn2−/− mice presented with higher grip strength, increased intact muscle fibres, and reduced serum creatine kinase levels compared to MDX. Consistently, blocking Lcn2 by treating 2-month-old MDX mice with an anti-Lcn2 monoclonal antibody (Lcn2Ab) increased trabecular volume, while reducing osteoclast surface/bone surface compared to MDX mice treated with irrelevant IgG. Grip force was also increased, and diaphragm fibrosis was reduced by the Lcn2Ab. These results suggest that Lcn2 could be a possible therapeutic target to treat DMD-induced bone loss.

Acute exposure to extracellular BTP2 does not inhibit Ca2+ release during EC coupling in intact skeletal muscle fibers.

The inhibitor of store-operated Ca2+ entry (SOCE) BTP2 was reported to inhibit ryanodine receptor Ca2+ leak and electrically evoked Ca2+ release from the sarcoplasmic reticulum when introduced into mechanically skinned muscle fibers. However, it is unclear how effects of intracellular application of a highly lipophilic drug like BTP2 on Ca2+ release during excitation-contraction (EC) coupling compare with extracellular exposure in intact muscle fibers. Here, we address this question by quantifying the effect of short- and long-term exposure to 10 and 20 µM BTP2 on the magnitude and kinetics of electrically evoked Ca2+ release in intact mouse flexor digitorum brevis muscle fibers. Our results demonstrate that neither the magnitude nor the kinetics of electrically evoked Ca2+ release evoked during repetitive electrical stimulation were altered by brief exposure (2 min) to either BTP2 concentration. However, BTP2 did reduce the magnitude of electrically evoked Ca2+ release in intact fibers when applied extracellularly for a prolonged period of time (30 min at 10 µM or 10 min at 20 µM), consistent with slow diffusion of the lipophilic drug across the plasma membrane. Together, these results indicate that the time course and impact of BTP2 on Ca2+ release during EC coupling in skeletal muscle depends strongly on whether the drug is applied intracellularly or extracellularly. Further, these results demonstrate that electrically evoked Ca2+ release in intact muscle fibers is unaltered by extracellular application of 10 µM BTP2 for <25 min, validating this use to assess the role of SOCE in the absence of an effect on EC coupling.

Insect elementary calcium release events measured in honeybee muscle cells

Subcellular calcium variations are involved in physiological and pathological mechanisms. Whereas elementary calcium release events (CREs) have been known for almost three decades in intact muscle cells isolated from vertebrates, they remained not characterized in invertebrates until recently. Dynamic confocal imaging was used on intact skeletal muscle cells isolated enzymatically from the adult honeybee legs to characterize spatio-temporal features of subcellular CREs. The frequency of these insect CREs, measured in x-y time lapse series, was higher than frequencies usually described in vertebrates. Spatial spread at half maximum was larger than in vertebrates and had a slightly ellipsoidal shape, two characteristics that may be related to ultrastructural features specific to invertebrate cells. In line-scan experiments, the histogram of CREs' duration followed a bimodal distribution, supporting the existence of both sparks and embers. Unlike in vertebrates, embers and sparks had similar amplitudes, a difference that could be related to genomic differences and/or excitation-contraction coupling specificities in honeybee skeletal muscle fibers. Arthropods muscle cells show strong genomic, ultrastructural and physiological differences with vertebrates and a comparative analysis may help to better understanding the roles and regulations of CREs. From a toxicological point of view, such a comparison will lead to better anticipating the myotoxicity of new insecticides targeting ryanodine receptors. Recent studies described the effects of these insecticides on macroscopic calcium homeostasis in bee neurons and muscle cells. Here, cyantraniliprole, the most recently approved anthranilic diamide in Europe, triggers calcium transients in bee muscle cell as well. Cyantraniliprole effects on Ca2+ sparks are currently under study.