Frog Skeletal Muscle Research Articles

Quantal Properties of Voltage-Dependent Ca2+ Release in Frog Skeletal Muscle Persist After Reduction of [Ca2+] in the Sarcoplasmic Reticulum.

In skeletal muscle, the Ca2+ release flux elicited by a voltage clamp pulse rises to an early peak that inactivates rapidly to a much lower steady level. Using a double pulse protocol the fast inactivation follows an arithmetic rule: if the conditioning depolarization is less than or equal to the test depolarization, then decay (peak minus steady level) in the conditioning release is approximately equal to suppression (unconditioned minus conditioned peak) of the test release. This is due to quantal activation by voltage, analogous to the quantal activation of IP3 receptor channels. Two mechanisms are possible. One is the existence of subsets of channels with different sensitivities to voltage. The other is that the clusters of Ca2+-gated Ryanodine Receptor (RyR) β in the parajunctional terminal cisternae might constitute the quantal units. These Ca2+-gated channels are activated by the release of Ca2+ through the voltage-gated RyR α channels. If the RyR β were at the basis of quantal release, it should be modified by strong inhibition of the primary voltage-gated release. This was attained in two ways, by sarcoplasmic reticulum (SR) Ca2+ depletion and by voltage-dependent inactivation. Both procedures reduced global Ca2+ release flux, but SR Ca2+ depletion reduced the single RyR current as well. The effect of both interventions on the quantal properties of Ca2+ release in frog skeletal muscle fibers were studied under voltage clamp. The quantal properties of release were preserved regardless of the inhibitory maneuver applied. These findings put a limit on the role of the Ca2+-activated component of release in generating quantal activation.

High Time Resolution Analysis of Voltage-Dependent and Voltage-Independent Calcium Sparks in Frog Skeletal Muscle Fibers.

In amphibian skeletal muscle calcium (Ca2+) sparks occur both as voltage-dependent and voltage-independent ligand-activated release events. However, whether their properties and their origin show similarities are still in debate. Elevated K+, constant Cl– content solutions were used to initiate small depolarizations of the resting membrane potential to activate dihydropyridine receptors (DHPR) and caffeine to open ryanodine receptors (RyR) on intact fibers. The properties of Ca2+ sparks observed under control conditions were compared to those measured on depolarized cells and those after caffeine treatment. Calcium sparks were recorded on intact frog skeletal muscle fibers using high time resolution confocal microscopy (x-y scan: 30 Hz). Sparks were elicited by 1 mmol/l caffeine or subthreshold depolarization to different membrane potentials. Both treatments increased the frequency of sparks and altered their morphology. Images were analyzed by custom-made computer programs. Both the amplitude (in ΔF/F0; 0.259 ± 0.001 vs. 0.164 ± 0.001; n = 24942 and 43326, respectively; mean ± SE, p < 0.001) and the full width at half maximum (FWHM, in μm; parallel with fiber axis: 2.34 ± 0.01 vs. 1.92 ± 0.01, p < 0.001; perpendicular to fiber axis: 2.08 ± 0.01 vs. 1.68 ± 0.01, p < 0.001) of sparks was significantly greater after caffeine treatment than on depolarized cells. 9.8% of the sparks detected on depolarized fibers and about one third of the caffeine activated sparks (29.7%) overlapped with another one on the previous frame on x-y scans. Centre of overlapping sparks travelled significantly longer distances between consecutive frames after caffeine treatment then after depolarization (in μm; 1.66 ± 0.01 vs. 0.95 ± 0.01, p < 0.001). Our results suggest that the two types of ryanodine receptors, the junctional RyRs controlled by DHPRs and the parajunctional RyRs are activated independently, using alternate ways, with the possibility of cooperation between neighboring release channels.

The dynamics of Ca2+ within the sarcoplasmic reticulum of frog skeletal muscle. A simulation study

In skeletal muscle, Ca2+ release from the sarcoplasmic reticulum (SR) triggers contraction. In this study we develop a two compartment model to account for the Ca2+ dynamics in frog skeletal muscle fibers. The two compartments in the model correspond to the SR and the cytoplasm, where the myofibrils are placed. We use a detailed model for the several Ca2+ binding proteins in the cytoplasm in line with previous models. As a new feature, Ca2+ binding sites within the SR, attributed to calsequestrin, are modeled based on experimentally obtained properties. The intra SR Ca2+ buffer shows cooperativity, well represented by a Hill equation with parameters that depend on the initial [Ca2+] in the SR ([Ca2+]SR). The number of total sites as well as the [Ca2+]SR of half saturation are reduced as the resting [Ca2+]SR is reduced, on the other hand the Hill number is not changed. The buffer power remained roughly constant.The release process is activated by a voltage dependent mechanism that increases the Ca2+ permeability of the SR. We use the permeability time course and amplitude experimentally obtained during a voltage clamp pulse to drive the simulations. This model successfully reproduces the SR and cytoplasmic transients observed.Additionally, we simulate [Ca2+] SR transients in the case of high concentration of extrinsic Ca2+ buffers added to the cytoplasm to explore what properties of the permeability are necessary to account for the experimentally observed [Ca2+]SR transients. The main novelty of the model, the intra SR Ca2+ buffer, is crucial for reproducing the experimental observations and it would be of use in future theoretical studies of excitation contraction coupling in skeletal muscle.

The regulation of Akt and FoxO transcription factors during dehydration in the African clawed frog (Xenopus laevis).

The African clawed frog (Xenopus laevis) naturally tolerates severe dehydration using biochemical adaptation, one of which is the elevation of antioxidant defenses during whole-body dehydration. The present study investigated the role and regulation of a pathway known to regulate oxidative stress response, the Akt-FoxO signaling pathway, in clawed frog skeletal muscle, responding to medium (15%) and high (30%) dehydration. Protein levels of total and phosphorylated Akt, FoxO1, and FoxO3 were assessed via immunoblotting, in addition to the levels of the E3 ubiquitin ligase known to be associated with muscle atrophy, MAFbx. Akt activity/phosphorylation in addition to its total protein levels were decreased in the skeletal muscle during dehydration, and this corresponded with decreases in the relative phosphorylation of FoxO1 and FoxO3 as well on several residues. Akt is an inhibitor of FoxO1 and FoxO3 activity via phosphorylation, suggesting that FoxO activities were increased during dehydration stress. Furthermore, MAFbx showed decreased protein expression during high dehydration as well, suggesting that the clawed frog may exhibit some natural resistance to skeletal muscle atrophy during severe dehydration conditions. In addition to identifying that the suppression of Akt could lead to an activation of FoxO transcription factors in X. laevis during dehydration, these investigations suggest that X. laevis dehydration may implicate FoxO1 and FoxO3 in controlling skeletal muscle atrophy in X. laevis exposed to dehydration. This study implicates the Akt signaling pathway, its regulation of FoxO transcription factors, and FoxO-controlled targets, in stress adaptation against dehydration.

Contracting striated muscle has a dynamic I-band spring with an undamped stiffness 100 times larger than the passive stiffness.

Fast sarcomere-level mechanics in contracting intact fibres from frog skeletal muscle reveal an I-band spring with an undamped stiffness 100 times larger than the known static stiffness. This undamped stiffness remains constant in the range of sarcomere length 2.7-3.1µm, showing the ability of the I-band spring to adapt its length to the width of the I-band. The stiffness and tunability of the I-band spring implicate titin as a force contributor that, during contraction, allows weaker half-sarcomeres to equilibrate with in-series stronger half-sarcomeres, preventing the development of sarcomere length inhomogeneity. This work opens new possibilities for the detailed in situ description of the structural-functional basis of muscle dysfunctions related to mutations or site-directed mutagenesis in titin that alter the I-band stiffness. Force and shortening in the muscle sarcomere are due to myosin motors from thick filaments pulling nearby actin filaments toward the sarcomere centre. Thousands of serially linked sarcomeres in muscle make the shortening (and the shortening speed) macroscopic, while the intrinsic instability of in-series force generators is likely prevented by the cytoskeletal protein titin that connects the thick filament with the sarcomere end, working as an I-band spring that accounts for the rise of passive force with sarcomere length (SL). However, current estimates of titin stiffness, deduced from the passive force-SL relation and single molecule mechanics, are much smaller than what is required to avoid the development of large inhomogeneities among sarcomeres. In this work, using 4kHz stiffness measurements on a population of sarcomeres selected along an intact fibre isolated from frog skeletal muscle contracting at different SLs (temperature 4°C), we measure the undamped stiffness of an I-band spring that at SL>2.7µm attains a maximum constant value of ∼6pNnm-1 per half-thick filament, two orders of magnitude larger than expected from titin-related passive force. We conclude that a titin-like dynamic spring in the I-band, made by an undamped elastic element in-series with damped elastic elements, adapts its length to the SL with kinetics that provide force balancing among serially linked sarcomeres during contraction. In this way, the I-band spring plays a fundamental role in preventing the development of SL inhomogeneity.

Synchronization Modulation of Na/K Pumps Induced Membrane Potential Hyperpolarization in Both Physiological and Hyperkalemic Conditions.

The capability of the synchronization modulation (SM) technique in enhancing the function of Na/K pumps has been demonstrated in various cells and tissues, including cardiomyocytes, a monolayer of cultured MDCK kidney cells, peripheral blood vessels, and frog skeletal muscles. This study characterized the membrane potential hyperpolarization induced by SM in both physiological and high [K+]o conditions on single skeletal muscle fibers. The results showed that SM could consistently induce membrane potential hyperpolarization by a few millivolts, and this hyperpolarization was not possible in the presence of ouabain. In contrast, the same electrical pulses but with random frequencies, constant frequencies, or synchronization with backward-modulation could not hyperpolarize the membrane potential. Prolonged field application and higher field intensity enhanced the effects of SM-induced hyperpolarization. Finally, the effect of SM was tested on skeletal muscle fibers incubated in a solution with high external potassium. Results showed that the SM electric field could hyperpolarize the membrane potential even if the external K+ concentration was higher than the normal, which implied the therapeutic effects of the SM electric field on the hyperkalemic situation.

Molecular game theory for a toxin-dominant food chain model.

Animal toxins that are used to subdue prey and deter predators act as the key drivers in natural food chains and ecosystems. However, the predators of venomous animals may exploit feeding adaptation strategies to overcome toxins their prey produce. Much remains unknown about the genetic and molecular game process in the toxin-dominant food chain model. Here, we show an evolutionary strategy in different trophic levels of scorpion-eating amphibians, scorpions and insects, representing each predation relationship in habitats dominated by the paralytic toxins of scorpions. For scorpions preying on insects, we found that the scorpion α-toxins irreversibly activate the skeletal muscle sodium channel of their prey (insect, BgNaV1) through a membrane delivery mechanism and an efficient binding with the Asp/Lys-Tyr motif of BgNaV1. However, in the predatory game between frogs and scorpions, with a single point mutation (Lys to Glu) in this motif of the frog's skeletal muscle sodium channel (fNaV1.4), fNaV1.4 breaks this interaction and diminishes muscular toxicity to the frog; thus, frogs can regularly prey on scorpions without showing paralysis. Interestingly, this molecular strategy also has been employed by some other scorpion-eating amphibians, especially anurans. In contrast to these amphibians, the Asp/Lys-Tyr motifs are structurally and functionally conserved in other animals that do not prey on scorpions. Together, our findings elucidate the protein-protein interacting mechanism of a toxin-dominant predator-prey system, implying the evolutionary game theory at a molecular level.

A study of the mechanisms of excitation-contraction coupling in frog skeletal muscle based on measurements of [Ca2+] transients inside the sarcoplasmic reticulum.

[Ca2+] transients inside the sarcoplasmic reticulum (SR) were recorded in frog skeletal muscle twitch fibers under voltage clamp using the low affinity indicator Mag Fluo 4 (loaded in its AM form) with the purpose of studying the effect on Ca2+ release of extrinsic Ca2+ buffers (i.e. BAPTA) added at high concentration to the myoplasm. When the extrinsic Ca2+ buffer is added to the myoplasm, part of the released Ca2+ binds to it, reducing the Ca2+ signal reported by a myoplasmic indicator. This, in turn, hinders the quantification of the amount of Ca2+ released. Monitoring release by measuring [Ca2+] inside the SR avoids this problem. The application of extrinsic buffers at high concentration reduced the resting [Ca2+] in the SR ([Ca2+]SR) continuously from a starting value close to 400μM reaching the range of 100μM in about half an hour. The effect of reducing resting [Ca2+]SR on the Ca2+ permeability of the SR activated by voltage clamp depolarization to 0mV was studied in cells where the myoplasmic [Ca2+] ([Ca2+]myo) transients were simultaneously recorded with Rhod2. The Ca2+ release flux was calculated from [Ca2+]myo and divided by [Ca2+]SR to obtain the permeability. Peak permeability was significantly reduced, from 0.026 ± 0.005ms-1 at resting [Ca2+]SR = 372 ± 5μM to 0.021 ± 0.004ms-1 at resting [Ca2+]SR = 120 ± 16μM (n = 4, p = 0.03). The time averaged permeability was not significantly changed (0.009 ± 0.003 and 0.010 ± 0.003ms-1, at the higher and lower [Ca2+]SR respectively). Once the cells were equilibrated with the high buffer intracellular solution, the change in [Ca2+]SR (Δ[Ca2+]SR) in response to voltage clamp depolarization (0mV, 200ms) in 20mM BAPTA was significantly lower (Δ[Ca2+]SR = 30.2 ± 3.5μM from resting [Ca2+]SR = 88.8 ± 13.6μM, n = 5) than in 40mM EGTA (Δ[Ca2+]SR = 72.2 ± 10.4μM from resting [Ca2+]SR = 98.2 ± 15.6μM, n = 4) suggesting that a Ca2+ activated component of release was suppressed by BAPTA.

Properties of Oscillating Electrical Pulse Induced Na/K Pump Current on Single Frog Skeletal Muscle

Properties of Oscillating Electrical Pulse Induced Na/K Pump Current on Single Frog Skeletal Muscle

Force and Power of a Synthetic Myosin II-Based Machine

The function as a collective motor of skeletal muscle myosin II is studied in vitro with a synthetic bio-machine, consisting of an ensemble of myosin motors interacting with a single actin filament attached with the correct polarity to a bead trapped in the focus of a Dual Laser Optical Tweezers (DLOT, Bianco et al. Biophys. J.101:866-874, 2011). The motor ensemble provides the condition for cyclic interactions with the actin filament, allowing the development of steady force and filament sliding. The mechanical outputs of the machine are measured by means of the DLOT, which acts as a force transducer (range 0.5-200 pN and resolution ∼0.3 pN), and a piezoelectric nano-positioner carrying the support for the myosin motors, which acts as a length transducer (range 1-75.000 nm and resolution ∼1 nm). Here is reported the performance of a first version of the machine, consisting of an ensemble of myosin motors purified from frog skeletal muscle randomly adsorbed on the surface of a chemically etched single-mode optical fibre with diameter 4 µm. Isometric and isotonic contractions are reproduced by the motor ensemble in solution with physiological [ATP] (2 mM) and temperature 21 °C. Following a drop in force from the maximum isometric value (F0) to a lower value (F), the actin filament slides at a constant velocity (V) which is larger the smaller the force, as expected from the in vivo force-velocity relation. Up to five F-V points for each interaction can be determined, allowing the definition of the maximum power at F ∼0.3 F0 (V ∼2 µm/s) and demonstrating the unequalled ability of the synthetic machine to define the power of native and engineered myosin II motors from striated muscle. Supported by IIT-SEED, Genova and ECRF, 2015 (Italy).

İndoksakarb'ın İzole Kurbağa İskelet Kası Üzerine Etkisi

Amaç: Sodyum kanal blokeri insektisitlerden indoksakarb, tarım alanlarında böceklerin kontrolünde yaygın olarak kullanılır ve böceklerde beslenmeyi durdurup, koordinasyonu bozarak, felç ve ölüme yol açarak etkisini gösterir. Bu çalışmada, indoksakarbın kurbağa iskelet kası üzerine etkisinin biyofiziksel, biyokimyasal ve histolojik yöntemler kullanılarak araştırılması amaçlanmıştır. Gereç ve Yöntemler: On altı Rana ridibunda türü kurbağa kontrol ve deney olmak üzere iki gruba bölünmüş (her grupta n=8) ve sartorius kasları izole edilmiştir. Deney grubundaki izole kaslar 1 saat 10 μM indoksakarbla inkübe edilmiştir. Her bir grupta bileşik kas aksiyon potansiyeli ve kas kontraksiyonu kayıt edilmiş, katalaz (CAT) aktivitesi ve malondialdehit (MDA) düzeyi ölçülmüş ve kas örnekleri transmisyon elektron mikroskop ile incelenmiştir. Bulgular: Aksiyon potansiyelinin genliği ve kontraksiyon kuvveti deney grubunda kontrole göre düşük bulunur iken (p<0,05), aksiyon potansiyelinin süresi deney grubunda kontrole göre daha yüksek bulunmuştur (p<0,05). Kontraksiyon süresi açısından deney grubu ile kontrol grubu arasında fark gözlenmemiştir (p>0,05). CAT ve MDA düzeyleri deney grubunda kontrole göre önemli miktarda artmıştır (p<0,05). Elektron mikroskobik olarak hem deney hem de kontrol grubundaki kas liflerinde normal morfolojik özellikler gözlenmiştir. Sonuç: Bu sonuçlar indoksakarbın hedef olmayan canlılarda iskelet kasında toksik etkiye yol açabileceğini düşündürmüştür.

Increased sodium channel use-dependent inhibition by a new potent analogue of tocainide greatly enhances in vivo antimyotonic activity

Although the sodium channel blocker, mexiletine, is the first choice drug in myotonia, some myotonic patients remain unsatisfied due to contraindications, lack of tolerability, or incomplete response. More therapeutic options are thus needed for myotonic patients, which require clinical trials based on solid preclinical data. In previous structure-activity relationship studies, we identified two newly-synthesized derivatives of tocainide, To040 and To042, with greatly enhanced potency and use-dependent behavior in inhibiting sodium currents in frog skeletal muscle fibers. The current study was performed to verify their potential as antimyotonic agents. Patch-clamp experiments show that both compounds, especially To042, are greatly more potent and use-dependent blockers of human skeletal muscle hNav1.4 channels compared to tocainide and mexiletine. Reduced effects on F1586C hNav1.4 mutant suggest that the compounds bind to the local anesthetic receptor, but that the increased hindrance and lipophilia of the N-substituent may further strengthen drug-receptor interaction and use-dependence. Compared to mexiletine, To042 was 120 times more potent to block hNav1.4 channels in a myotonia-like cellular condition and 100 times more potent to improve muscle stiffness in vivo in a previously-validated rat model of myotonia. To explore toxicological profile, To042 was tested on hERG potassium currents, motor coordination using rotarod, and C2C12 cell line for cytotoxicity. All these experiments suggest a satisfactory therapeutic index for To042. This study shows that, owing to a huge use-dependent block of sodium channels, To042 is a promising candidate drug for myotonia and possibly other membrane excitability disorders, warranting further preclinical and human studies.

Differential effects of contractile potentiators on action potential-induced Ca2+ transients of frog and mouse skeletal muscle fibres.

Muscle fibres, isolated from frog tibialis anterior and mouse flexor digitorum brevis (FDB) were loaded with the fast dye MagFluo-4 to study the effects of potentiators caffeine, nitrate, Zn2+ and perchlorate on Ca2+ transients elicited by single action potentials. Overall, the potentiators doubled the transients amplitude and prolonged by about 1.5-fold their decay time. In contrast, as shown here for the first time, nitrate and Zn2+, but not caffeine, activated a late, secondary component of the transient rising phase of frog but not mouse, fibres. The rise time was increased from 1.9ms in normal solution (NR) to 3.3ms (nitrate) and 4.4ms (Zn2+). In NR, a single exponential, fitted the rising phase of calcium transients of frog (τ1=0.47ms) and mouse (τ1=0.28ms). In nitrate and Zn2+ only frog transients showed a secondary exponential component, τ2=0.72ms (nitrate) and 0.94ms, (Zn2+). We suggest that nitrate and Zn2+ activate a late slower component of the ΔF/F signals of frog but not of mouse fibres, possibly promoting Ca2+ induced Ca2+ release at level of the RyR3, that in frog muscle fibres are localized in the para-junctional region of the triads and are absent in mouse FDB muscle fibres.

Excitation contraction uncoupling by high intracellular [Ca2+] in frog skeletal muscle: a voltage clamp study.

Raising the intracellular [Ca2+] ([Ca2+]i) was previously found to produce uncoupling between the electrical depolarization of the transverse tubules and contraction in skinned muscle fibers. Here we study the effect of elevated [Ca2+]i in voltage clamped cut fibers of frog skeletal muscle to establish how the charge movement, a measure of the activation of the dihydropyridine receptors (DHPR)-voltage sensors, and Ca2+ release, a consequence of the opening of the ryanodine receptor (RyR)-release channels, were affected. [Ca2+]i was raised by various procedures (pharmacological release from the sarcoplasmic reticulum, application of high [Ca2+]i intracellular solution, permeabilization of the plasma membrane by a Ca2+ ionophore) all of which produced impairment of excitation-contraction coupling. The charge movement was reduced from 20.2±1.24 to 9.9±0.94 nC/μF meanwhile the Ca2+ release flux was reduced from 13.5+0.7 to 2.2±0.3μM/ms (n=33). This suggests that a significant fraction of the DHPRs that remained functional, could not activate RyRs, and were therefore presumably disconnected. These results are broadly consistent with the original reports in skinned fibers. Uncoupling was prevented by the addition to the intracellular solution of the protease inhibitor leupeptin. In approximately 40% of the uncoupled cells we observed that the [Ca2+]i transient continued to rise after the voltage clamp pulse was turned off. This loss of control by membrane voltage suggests that the uncoupled release channels might have another mechanism of activation, likely by Ca2+.

Presynaptic Molecular Determinants of Quantal Size.

The quantal hypothesis for the release of neurotransmitters at the chemical synapse has gained wide acceptance since it was first worked out at the motor endplate in frog skeletal muscle in the 1950’s. Considering the morphological identification of synaptic vesicles (SVs) at the nerve terminals that appeared to be homogeneous in size, the hypothesis proposed that signal transduction at synapses is mediated by the release of neurotransmitters packed in SVs that are individually uniform in size; the amount of transmitter in a synaptic vesicle is called a quantum. Although quantal size—the amplitude of the postsynaptic response elicited by the release of neurotransmitters from a single vesicle—clearly depends on the number and sensitivity of the postsynaptic receptors, accumulating evidence has also indicated that the amount of neurotransmitters stored in SVs can be altered by various presynaptic factors. Here, I provide an overview of the concepts and underlying presynaptic molecular underpinnings that may regulate quantal size.

An early post-traumatic reaction of lymph-heart striated muscle fibers in adult frog Rana temporaria during the first postoperative week: An electron microscopic and autoradiographic study.

According to the current opinion, lymph-heart striated muscle represents a specialized type of skeletal muscles in frogs. Here, we studied muscle fibers in mechanically damaged lymph hearts during the first postoperative week using electron-microscopic autoradiography. We present evidence that both, the satellite cells and pre-existing muscle fibers bordering the site of injury, contribute directly to the lymph-heart muscle regeneration. Several muscle fibers located in the vicinity of the damaged area displayed features of nuclear and sarcoplasmic activation. We also observed ultrastructural changes indicating activation of a few satellite cells, namely decondensation of chromatin, enlargement of nuclei and nucleoli, appearance of free ribosomes and rough endoplasmic reticulum tubules in the cytoplasm. Electron-microscopic autoradiography showed that 4 h after single (3)H-thymidine administration on the seventh day after injury not only the activated satellite cells, but also some nuclei of myofibers bordering the injured zone are labeled. We showed that both, the myonuclei of fibers displaying the signs of degenerative/reparative processes in the sarcoplasm and the myonuclei of the fibers enriched with highly organized myofibrils, can re-enter into the S-phase. Our results indicate that the nuclei of lymph-heart myofibers can reactivate DNA synthesis during regenerative myogenesis, unlike the situation in regenerating frog skeletal muscle where myogenic cells do not synthesize DNA at the onset of myofibrillogenesis.

Compartment calcium model of frog skeletal muscle during activation

Skeletal muscle contraction is triggered by a rise in calcium (Ca2+) concentration in the myofibrillar space. The objective of this study was to develop a voltage dependent compartment model of Ca2+ dynamics in frog skeletal muscle fibers. The compartment model corresponds to the myofibrillar space (MS) and a calcium store, the sarcoplasmic reticulum (SR). Ca2+ is released from the SR to the MS based on the voltage and is able to bind to several proteins in the MS. We use a detailed model to account for voltage dependent Ca2+ release and inactivation. With this model, we are able to match previous experimental data for Ca2+ release and binding to proteins for an applied (fixed) voltage. We explore the sensitivity of parameters in the model and illustrate the importance of inactivation of the SR; during a long depolarization, the SR must be inactivated in order to achieve realistic Ca2+ concentrations in the MS. A Hodgkin Huxley type model was also developed to describe voltage at the surface membrane using electrophysiological data from previous experiments. This voltage model was then used as the time dependent voltage to determine Ca2+ release from the SR. With this fully coupled model, we were able to match previous experimental results for Ca2+ concentrations for a given applied current. Additionally, we examined simulated Ca2+ concentrations in the case of twitch and tetanus, corresponding to different applied currents. The developed model is robust and reproduces many aspects of voltage dependent calcium signaling in frog skeletal muscle fibers. This modeling framework provides a platform for future studies of excitation contraction coupling in skeletal muscle fibers.

The non‐linear elasticity of the muscle sarcomere and the compliance of myosin motors

Force in striated muscle is due to attachment of the heads of the myosin, the molecular motors extending from the myosin filament, to the actin filament in each half-sarcomere, the functional unit where myosin motors act in parallel. Mechanical and X-ray structural evidence indicates that at the plateau of isometric contraction (force T0), less than half of the elastic strain of the half-sarcomere is due to the strain in the array of myosin motors (s), with the remainder being accounted for by the compliance of filaments acting as linear elastic elements in series with the motor array. Early during the development of isometric force, however, the half-sarcomere compliance has been found to be less than that expected from the linear elastic model assumed above, and this non-linearity may affect the estimate of s. This question is investigated here by applying nanometre-microsecond-resolution mechanics to single intact fibres from frog skeletal muscle at 4 °C, to record the mechanical properties of the half-sarcomere throughout the development of force in isometric contraction. The results are interpreted with mechanical models to estimate the compliance of the myosin motors. Our conclusions are as follows: (i) early during the development of an isometric tetanus, an elastic element is present in parallel with the myosin motors, with a compliance of ∼200 nm MPa(-1) (∼20 times larger than the compliance of the motor array at T0); and (ii) during isometric contraction, s is 1.66 ± 0.05 nm, which is not significantly different from the value estimated with the linear elastic model.

Calcium influx through L-type channels attenuates skeletal muscle contraction via inhibition of adenylyl cyclases

Skeletal muscle contraction is triggered by acetylcholine induced release of Ca2+ from sarcoplasmic reticulum. Although this signaling pathway is independent of extracellular Ca2+, L-type voltage-gated calcium channel (Cav) blockers have inotropic effects on frog skeletal muscles which occur by an unknown mechanism. Taking into account that skeletal muscle fiber expresses Ca+2-sensitive adenylyl cyclase (AC) isoforms and that cAMP is able to increase skeletal muscle contraction force, we investigated the role of Ca2+ influx on mouse skeletal muscle contraction and the putative crosstalk between extracellular Ca2+ and intracellular cAMP signaling pathways. The effects of Cav blockers (verapamil and nifedipine) and extracellular Ca2+ chelator EGTA were evaluated on isometric contractility of mouse diaphragm muscle under direct electrical stimulus (supramaximal voltage, 2ms, 0.1Hz). Production of cAMP was evaluated by radiometric assay while Ca2+ transients were assessed by confocal microscopy using L6 cells loaded with fluo-4/AM. Ca2+ channel blockers verapamil and nifedipine had positive inotropic effect, which was mimicked by removal of extracellular Ca+2 with EGTA or Ca2+-free Tyrode. While phosphodiesterase inhibitor IBMX potentiates verapamil positive inotropic effect, it was abolished by AC inhibitors SQ22536 and NYK80. Finally, the inotropic effect of verapamil was associated with increased intracellular cAMP content and mobilization of intracellular Ca2+, indicating that positive inotropic effects of Ca2+ blockers depend on cAMP formation. Together, our results show that extracellular Ca2+ modulates skeletal muscle contraction, through inhibition of Ca2+-sensitive AC. The cross-talk between extracellular calcium and cAMP-dependent signaling pathways appears to regulate the extent of skeletal muscle contraction responses.

Correction: Head-Head Interactions of Resting Myosin Crossbridges in Intact Frog Skeletal Muscles, Revealed by Synchrotron X-Ray Fiber Diffraction

Correction: Head-Head Interactions of Resting Myosin Crossbridges in Intact Frog Skeletal Muscles, Revealed by Synchrotron X-Ray Fiber Diffraction