Twitch Tension Research Articles

Immobilization with Atrophy Induces De Novo Expression of Neuronal Nicotinic α7 Acetylcholine Receptors in Muscle Contributing to Neurotransmission

Mature acetylcholine receptor (AChR) isoform normally mediates muscle contraction. The hypothesis that α7AChRs up-regulate during immobilization and contribute to neurotransmission was tested pharmacologically using specific blockers to mature (waglerin-1), immature (αA-OIVA), and α7AChRs (methyllycaconitine), and nonspecific muscle AChR antagonist, α-bungarotoxin. Mice were immobilized; contralateral limbs were controls. Fourteen days later, anesthetized mice were mechanically ventilated. Nerve-stimulated tibialis muscle contractions on both sides were recorded, and blockers enumerated above sequentially administered via jugular vein. Data are mean ± standard error. Immobilization (N = 7) induced tibialis muscle atrophy (40.6 ± 2.8 vs. 52.1 ± 2.0 mg; P < 0.01) and decrease of twitch tension (34.8 ± 1.1 vs. 42.9 ± 1.5 g; P < 0.01). Waglerin-1 (0.3 ± 0.05 μg/g) significantly (P = 0.001; N = 9) depressed twitch tension on contralateral (≥97%) versus immobilized side (approximately 45%). Additional waglerin-1 (total dose 1.06 ± 0.12 μg/g or approximately 15.0 × ED50 in normals) could not depress twitch of 80% or greater on immobilized side. Immature AChR blocker, αA-OIVA (17.0 ± 0.25 μg/g) did not change tension bilaterally. Administration of α-bungarotoxin (N = 4) or methyllycaconitine (N = 3) caused 96% or greater suppression of the remaining twitch tension on immobilized side. Methyllycaconitine, administered first (N = 3), caused equipotent inhibition by waglerin-1 on both sides. Protein expression of α7AChRs was significantly (N = 3; P < 0.01) increased on the immobilized side. Ineffectiveness of waglerin-1 suggests that the twitch tension during immobilization is maintained by receptors other than mature AChRs. Because αA-OIVA caused no neuromuscular changes, it can be concluded that immature AChRs contribute minimally to neurotransmission. During immobilization approximately 20% of twitch tension is maintained by up-regulation of α-bungarotoxin- and methyllycaconitine-sensitive α7AChRs.

Impact of E163R cTnT Mutation on Cardiac Mechanics and Energetics in a Murine Model

Introduction: Many of cTnT mutations linked to cardiomyopathies fall the TNT1 domain/N terminal tail region of unresolved high definition structure. This region (∼94-170) of cTnT is critical to Tm binding and contraction regulation. Here, the impact of the E163R mutation in cTnT-TNT1 on contractile function and tension cost was investigated using intact and skinned preparations from WT and transgenic mouse hearts.Methods: Left and right ventricular trabeculae were dissected from non-transgenic wild type (WT) and heterozygous (Δ160E or E163R) mouse hearts and mounted isometrically to record twitch tension or, when skinned, Ca2+ activated force. Myofibrillar ATPase activity was measured by fluorimetric enzyme coupled assay (de Tombe and Stienen, 1995).Results: Myocardium of E163R mice shows: (i) no change of myosin isoform expression (ii) maintained peak isomentric twitch tension at all stimulation frequencies, (iii) prolonged time to peak and time to 50% relaxation, with preserved rate-adaptation of twitch duration, (iv) changes of the short-term interval force relationship and increased occurrence of spontaneous contractions. No significant differences were found in maximum Ca2+ activated tension of E163R and WT skinned trabeculae. However, Ca2+ sensitivity of tension was significantly increased in E163R skinned trabeculae when compared with WT. As to the economy of force maintenance, preliminary experiments suggest an increase of tension cost in trabeculae from E163R hearts. Resting ATPase activity also tended to be higher in E163R preparations. Kinetics of force development and relaxation will be assessed on single myofibrils, isolated from the same hearts.Conclusions: Both primary sarcomeric changes and secondary E-C coupling alterations contribute to mechanical impairment in E163R cTnT mutant myocardium. Supported by: EC Grant n. 241577 (BIG-Heart)

Beta-Adrenergic Response in Human HCM Myocardium: Effects of Ranolazine

Background: Rest or exercise obstruction is present in two thirds of patients with hypertrophic cardiomyopathy (HCM) and is a major determinant of symptoms and disability. Hypercontractile upper septum is the main pathophysiological determinant of obstruction, thus negative inotropic interventions such as dysopiramide or beta-blockers are the only available options to pharmacologically treat obstruction, commonly with partial efficacy. We have previously demonstrated that ranolazine ameliorates diastolic function in trabeculae from septal samples of obstructive HCM patients undergoing myectomy (Coppini et al, Circulation 2013). Methods: Patch clamp studies and intracellular Ca2+ recordings were performed in isolated myocytes from myectomy samples of obstructive HCM patients; intact trabeculae were used for mechanical measurements. Myocardial specimens from non-failing non-hypertrophic patients or patients with secondary hypertrophy were used as controls. Results: Dysopiramide (Dys) reduced twitch tension in a dose dependent manner and 5µM Dys accelerated contraction kinetics in HCM trabeculae. Isoproterenol 10−7 mol/L (Iso) determined a significant potentiation of twitch amplitude and an accelleration of contraction kinetics (both time to peak and relaxation). Changes induced by Iso in control trabeculae were similar. Interestingly, Iso caused APD prolongation in HCM cardiomyocytes instead of the shortening observed in control cells. This was likely related to the unbalance between depolarizing and repolarizing currents, including increased Late-Na+ current (INaL). The INaL blocker Ranolazine 10 µM (Ran) applied on top of Iso (Iso+Ran) markedly reduced isometric twitch tension of HCM trabeculae, while Ran alone showed no negative inotropic effect. Contraction kinetics in Iso+Ran were still significantly faster compared to baseline. Conclusions: Beta adrenergic stimulation may enhance septal contractility and determine obstruction in HCM. Ranolazine, by reducing septal tension at peak exercise but not at rest, may represent a safe therapeutic option for obstruction.

E-C Coupling Alterations and Spontaneous Activity in Mice Carrying Cardiac Troponin T Mutations

Ca2+ handling abnormalities are an early-onset pathogenic element in HCM. Here we characterize pro-arrhythmogenic changes in E-C coupling that occur in intact trabeculae and cardiomyocytes from cTnT mutant mouse models of HCM (R92Q, E163R and Δ160E) and test the effects of specific pharmacological interventions. Compared to WT, R92Q trabeculae ([Ca2+]o 2 mM, 30°C) showed (i) preserved peak isometric twitch tension and prolonged relaxation kinetics associated to decreased SERCA levels, (ii) faster mechanical restitution, further accelerated by isoproterenol (Iso) 100nM, (iii) decreased Ca2+-recirculation fraction markedly increased by Iso (iv) frequent after-contractions or regular spontaneous beats during stimulation pauses that increased in response to Iso. Compared to WT, R92Q cardiomyocytesshowed (i) prolonged action potentials associated with ionic current remodeling, (ii) slower rate of Ca2+transient decay, (iii) elevated diastolic [Ca2+]i, (iv) spontaneous Ca2+ waves during stimulation pauses. In R92Q preparations, the late-Na+ current blocker Ranolazine (Ran 10 μM) (i) reduced the rate of spontaneous beats and spontaneous Ca2+ waves, (ii) hastened Ca2+ transient kinetics and reduced diastolic Ca2+, (iii) reduced and reversed the acceleration of mechanical restitution and the increase in Ca2+ recirculation fraction induced by Iso.Compared to R92Q, occurrence of spontaneous contractions was similar in E163R but less pronounced in Δ160E. Iso and Ran showed similar effects in all three mouse models, in spite of some quantitative differences. The results are consistent with those recently reported in human HCM myocytes (Coppini et al, Circulation 2013) and suggest that remodeling and dysfunction of NCX and RyR2 contribute to the pro-arrhythmogenic E-C coupling abnormalities observed in HCM.

Presynaptic neuromuscular action of a methanolic extract from the venom of Rhinella schneideri toad

BackgroundRhinella schneideri, previously known as Bufo paracnemis, is a common toad in many regions of Brazil. Its venom exerts important cardiovascular effects on humans and other animals. Although this toad venom has been the subject of intense investigations, little is known about its neuromuscular activity.MethodsThe neurotoxicity of a methanolic extract of R. schneideri venom was tested on mouse phrenic nerve-diaphragm (PND) preparations mounted for conventional twitch tension recording – in response to indirect stimulation – and for electrophysiological measurements.ResultsVenom extract (50 μg/mL) increased the muscle twitch tension in PND preparations but did not significantly alter the resting membrane potential values. Electrophysiological evaluations showed that the extract (50 μg/mL) significantly augmented the frequency of miniature end-plate potential (from 38 ± 3.5 to 88 ± 15 after 60 minutes; n = 5; p < 0.05) and quantal content (from 128 ± 13 to 272 ± 34 after five minutes; n = 5; p < 0.05). Pretreatment with ouabain (1 μg/mL) for five minutes prevented the increase in quantal content (117 ± 18 and 154 ± 33 after five and 60 minutes, respectively).ConclusionThese results indicate that the methanolic extract of R. schneideri venom acts primarily presynaptically to enhance neurotransmitter release in mouse phrenic-diaphragm preparations.

Different magnitude of resistance to non-depolarizing muscle relaxants in dexamethasone-treated rat diaphragm associated with altered acetylcholine receptor expression.

The aim of this study was to investigate the influence of chronic dexamethasone (Dex) administration on rat diaphragm sensitivity to non-depolarizing muscle relaxants (NDMRs) and muscular nicotinic acetylcholine receptor (nAChR) expression, which may help direct future administration of NDMRs. Adult male Sprague-Dawley rats were randomized to receive a daily intraperitoneal injection of Dex (600 μg/kg body mass) or an equivalent volume of saline (N = 20 in each group) for 14 days. We evaluated isometric twitch tensions of nerve-hemidiaphragm preparations elicited by indirect supramaximal stimulation at 0.1 Hz. Real-time quantitative PCR was performed to determine the mRNA expression of two nAChR subunits (ε-subunit and γ-subunit) in the diaphragm. Dex administration markedly (P < 0.01) increased the 50% twitch depression (IC50) of the three NDMRs. The IC50 ratio, which standardized the magnitudes of the resistance, was the largest for atracurium, with the second largest for vecuronium and the smallest for rocuronium (P < 0.01). The ε- and γ-subunit mRNAs were both upregulated with an increased γ/ε ratio in rats exposed to Dex. The results indicated that chronic Dex administration induces hyposensitivity to NDMRs, the degree of which depends on the kind of neuromuscular blocker, and is associated with increased nAChR expression.

Evidence for the Role of Camkinase II and Synapsin I in the Restoration of Neurotransmission in Botulinum Neurotoxin a Intoxicated Nerve Terminals

At the active zones in the nerve terminals, presynaptic Ca2+ is recognized as an important second messenger that regulates neurotransmission. However, the precise role of Ca2+ in the regulation of exocytosis still remains to be elusive. Synapsin I (SYN1) is a member of the synapsin family (SYN1/2/3) of synaptic vesicle phosphoproteins that is phosphorylated by Ca2+/calmodulin dependent protein kinase II (CaMKII) at the Serine 603. At the motor nerve terminals (MNT), SYN I regulates synaptic transmission and the depolarization signal mediated rise in Ca2+ influx stimulates CaMKII dependent phosphorylation of SYN1. Botulinum neurotoxin A (BoNT/A) selectively proteolyzes SNAP-25 to inhibit acetylcholine (ACh) release and causes life-threatening neuroparalysis. In our efforts to develop therapeutic strategies to counteract BoNT/A at its primary site of action, the MNT, we discovered that mouse MNT express transient receptor potential vanilloid 1 (TRPV1) channel protein and that capsaicin (TRPV1 agonist) antagonized the neuroparalytic effects of BoNT/A. Capsaicin, when injected post BoNT/A exposure, significantly accelerated recovery from neuroparalysis by restoring ACh release and muscle functions. Capsaicin treatment also decreased the total duration of paralysis by 50% and synergistically increased the effects of 3,4 diaminopyridine (blocks K+ channels and prolongs action potential) on ACh release measured by stimulus evoked twitch tension in BoNT/A poisoned nerve muscle preparations. Further, BoNT/A treatment, in vitro, decreased the expression of CaMKII and SYN1 and the phosphorylation of SYN1 in cholinergic Neuro 2a cells. Capsaicin treatment, post BoNT/A, reversed this. We hypothesize that at the MNT, capsaicin stimulated Ca2+ influx via TRPV1 triggers exocytosis by facilitating synaptic transmission via CaMKII mediated phosphorylation of SYN1.

The effect of heat stress on skeletal muscle contractile properties

An elevated heat-shock protein (HSP) content protects cells and tissues, including skeletal muscles, from certain stressors. We determined if heat stress and the elevated HSP content that results is correlated with protection of contractile characteristics of isolated fast and slow skeletal muscles when contracting at elevated temperatures. To elevate muscle HSP content, one hindlimb of Sprague-Dawley rats (21-28days old, 70-90g) was subjected to a 15min 42°C heat-stress. Twenty-four hours later, both extensor digitorum longus (EDL) and soleus muscles were removed, mounted in either 20°C or 42°C Krebs-Ringer solution, and electrically stimulated. Controls consisted of the same muscles from the contra-lateral (non-stressed) hindlimbs as well as muscles from other (unstressed) animals. Isolated muscles were twitched and brought to tetanus every 5min for 30min. As expected, HSP content was elevated in muscles from the heat-stressed limbs when compared with controls. Regardless of prior treatment, both EDL and soleus twitch tensions were lower at 42°C when compared with 20°C. In addition, when incubated at 42°C, both muscles showed a drop in twitch tension between 5 and 30min. For tetanic tension, both muscles also showed an increase in tension between 5 and 30min when stimulated at 20°C regardless of treatment but when stimulated at 42°C no change was observed. No protective effect of an elevated HSP content was observed for either muscle. In conclusion, although heat stress caused an elevation in HSP content, no protective effects were conferred to isolated contracting muscles.

Dexamethasone-induced hyposensitivity to rocuronium in rat diaphragm associated with muscle-fiber transformation

The aim of the current study was to investigate the effect of chronic dexamethasone (Dex) administration on rat diaphragm function and sensitivity to rocuronium and muscle‑fiber transformation. Adult male Sprague‑Dawley rats were randomized to receive a daily intraperitoneal injection of Dex to evaluate whether alterations in diaphragm function and susceptibility to rocuronium would be induced. In addition, diaphragm contractile properties, histopathology and isometric twitch tensions of nerve‑hemidiaphragm preparations were evaluated. Dex administration led to impaired diaphragm force generation, increased fatigue resistance and a prolonged half‑relaxation time, as well as time‑to‑peak tension. Dex treatment led to desensitization of the rat diaphragm to rocuronium, as demonstrated by a shift of the rocuronium concentration‑twitch tension curves to the right. Histochemical analysis of adenosine triphosphatase revealed that the distribution and cross‑sectional area of type II fibers were decreased in rats exposed to Dex. The present study indicates that chronic Dex treatment induced alterations in muscle function and that susceptibility to rocuronium is associated with muscle fiber‑type transformation, which may aid in directing future administration of muscle relaxants.

Myocardial contractility in the stress echo lab: from pathophysiological toy to clinical tool

Up-regulation of Ca2+ entry through Ca2+ channels by high rates of beating is involved in the frequency-dependent regulation of contractility: this process is crucial in adaptation to exercise and stress and is universally known as force-frequency relation (FFR). Disturbances in calcium handling play a central role in the disturbed contractile function in myocardial failure. Measurements of twitch tension in isolated left-ventricular strips from explanted cardiomyopathic hearts compared with non-failing hearts show flat or biphasic FFR, while it is up-sloping in normal hearts. Starting in 2003 we introduced the FFR measurement in the stress echo lab using the end-systolic pressure (ESP)/End-systolic volume index (ESVi) ratio (the Suga index) at increasing heart rates. We studied a total of 2,031 patients reported in peer-reviewed journals: 483 during exercise, 34 with pacing, 850 with dobutamine and 664 during dipyridamole stress echo. We demonstrated the feasibility of FFR in the stress echo lab, the clinical usefulness of FFR for diagnosing latent contractile dysfunction in apparently normal hearts, and residual contractile reserve in dilated idiopathic and ischemic cardiomyopathy. In 400 patients with left ventricular dysfunction (ejection fraction 30 ± 9%) with negative stress echocardiography results, event-free survival was higher (p < 0.001) in patients with ΔESPVR (the difference between peak and rest end-systolic pressure-volume ratio, ESPVR) ≥ 0.4 mmHg/mL/m2. The prognostic stratification of patients was better with FFR, beyond the standard LV ejection fraction evaluation, also in the particular settings of severe mitral regurgitation or diabetics without stress-induced ischemia. In the particular setting of selection of heart transplant donors, the stress echo FFR was able to correctly select 34 marginal donor hearts efficiently transplanted in emergency recipients. Starting in 2007, we introduced an operator-independent cutaneous sensor to monitor the FFR: the force is quantified as the sensed pre-ejection myocardial vibration amplitude. We demonstrated that the sensor-derived force changes at increasing heart rates are highly related with both max dP/dt in animal models, and with the stress echo FFR in 220 humans, opening a new window for pervasive cardiac heart failure monitoring in telemedicine systems.

Paradoxical neostigmine-induced TOFfade: On the role of presynaptic cholinergic and adenosine receptors

Neuromuscular transmission is clinically monitored using the train-of-four ratio (TOFratio), which is the quotient between twitch tension produced by the fourth (T4) and by the first (T1) stimulus within a train-of-four stimulation at 2Hz. Neostigmine causes a paradoxical depression of the TOFratio (TOFfade) that is amplified by intra-arterial atropine in cats. This led us to question the usefulness of the TOFratio as a sole testing element to control neostigmine-induced reversal of neuromuscular transmission block caused by non-depolarizing agents. We hypothesized that the inhibition of cholinesterase activity might increase acetylcholine bioavailability and consequently cholinoceptor activation, leading to concomitant adenosine release from nerve endings and skeletal muscle fibers. The involvement of presynaptic muscarinic and adenosine receptors in neostigmine-induced TOFfade in the rat phrenic nerve diaphragm was investigated. Blockade of adenosine A2A receptors with ZM241385 and of muscarinic M2 receptors with methoctramine or atropine amplified neostigmine-induced TOFfade. Notwithstanding TOFfade amplification, the blockade of M2 or A2A receptors increased both T1 and T4 twitch tensions above control during the first 3min of neostigmine application. Beyond that period, the T1 twitch tension returned to baseline, whereas T4 decreased further until the control value with neostigmine alone. Blockade of M1 receptors by pirenzepine did not change neostigmine-induced TOFfade, unless A2A receptors were concurrently blocked with ZM241385. Data indicate that the paradoxical neostigmine-induced fade involves presynaptic mechanisms that regulate transmitter release and synaptic adenosine accumulation, including the activation of adenosine A2A and muscarinic M2 receptors.

Long-chain n-3 DHA reduces the extent of skeletal muscle fatigue in the rat in vivo hindlimb model

Dietary fish oil modifies skeletal muscle membrane fatty acid composition and oxygen efficiency similar to changes in the myocardium. Oxygen efficiency is a key determinant of sustained force in mammalian skeletal muscle. Therefore, in the present study, we tested the effects of a fish-oil diet on skeletal muscle fatigue under the stress of contraction using the rat in vivo autologous perfused hindlimb model. For 8 weeks, male Wistar rats were fed a diet rich in saturated fat (SF), a diet rich in n-6 PUFA or a diet rich in long-chain (LC) n-3 PUFA DHA derived from fish oil. In anaesthetised, mechanically ventilated rats, with their hindlimbs perfused with arterial blood at a constant flow, the gastrocnemius-plantaris-soleus muscle bundle was stimulated via sciatic nerve (2Hz, 6-12V, 0·05ms) to contract repetitively for 30min. Rats fed the n-3 PUFA diet developed higher maximum twitch tension than those fed the SF and n-6 PUFA diets (P<0·05) and sustained twitch tension through more repetitions before the tension declined to 50% of the maximum twitch tension (P<0·05). The n-3 PUFA group used less oxygen for tension developed and produced higher venous lactate concentrations with no difference in glycogen utilisation compared with the SF and n-6 PUFA groups. These results further support that incorporation of DHA into skeletal muscle membranes increases the efficiency of oxygen use over a range of contractile force and this is expressed as a higher sustained force and prolonged time to fatigue.

Neuromuscular blocking activity of pinnatoxins E, F and G

Pinnatoxins are produced by dinoflagellates and belong to the cyclic imine family of toxins. They are fast-acting and highly toxic when administered in vivo in rodent bioassays, causing death by respiratory depression within minutes. Studies have revealed that some cyclic imine toxins cause their toxicity by antagonizing both muscle type and heteromeric and homomeric neuronal nicotinic acetylcholine receptors (nAChRs). Pinnatoxins E, F and G all display potent toxicity in in vivo bioassays, with symptoms of toxicity similar to other cyclic imine toxins. However, very little work has been done on the mechanism of action of these pinnatoxin isomers. Thus the aim of the current study was to investigate the rank order of potency and mechanism of action of pinnatoxins E, F and G. The effects of pinnatoxin E, F and G on in vitro rat hemidiaphragm preparations were investigated using twitch tension and electrophysiological techniques to determine the effects of these toxins on cholinergic transmission at the neuromuscular junction. Pinnatoxins E, F and G all produced concentration-dependent reductions in the nerve evoked twitch response of the rat hemidiaphragm, with IC50 values ranging from 11 to 53 nM and a rank order of potency of F > G > E. Only complete washout of pinnatoxin E was evident, with pinnatoxins F and G displaying slow and incomplete washout profiles. Pinnatoxins F and G also reduced the amplitudes of spontaneous miniature endplate potentials and evoked endplate potentials at the neuromuscular junction, without affecting miniature endplate potential frequency or the resting membrane potential of the muscle fibres. These results show that pinnatoxins E, F and G are all potent neuromuscular blocking agents and cause toxicity by acting as antagonists at muscle type nicotinic acetylcholine receptors.

Adiponectin alleviates contractile dysfunction of genioglossus in rats exposed to chronic intermittent hypoxia

Background Genioglossal dysfuntion takes an important role in pathogenesis of obstructive sleep apnea hypopnea syndrome (OSAHS) in which chronic intermittent hypoxia (CIH) is the major pathological origin. Recent studies have suggested genioglossal injury induced by CIH might be improved by adiponectin. The aim of this study was to investigate the effects of adiponectin on genioglossus contractile properties in rats exposed to CIH. Methods Thirty-nine healthy male Wistar rats were randomly divided into three groups: normal control (NC), CIH and adiponectin supplement (CIH+Ad) with 13 rats in each. Rats in NC were kept breathing normal air, while rats in CIH and CIH+Ad experienced the same CIH environment eight hours per day for 35 successive days. Rats in CIH+Ad were given intravenous adiponectin of 10 μg twice a week for 30 successive days. Rats in the NC and CIH were injected with normal saline as a control. After 35 days' CIH exposure, the levels of serum adiponectin and genioglossus contractile properties were compared. Results Serum adiponectin level was significantly lower in CIH than in NC (1210 ng/ml vs. 2236 ng/ml). Serum adiponectin level in CIH+Ad (1844 ng/ml) was significantly higher than CIH but lower than NC. Twitch tension, time to peak tension, half relaxation time and tetanic tension were significantly lower in CIH than NC and improved in CIH+Ad. All mean tetanic fatigue indices decreased more rapidly in the first 20 seconds than during the subsequent 100 seconds. Tetanic fatigue indices in NC and CIH+Ad were significantly higher compared to CIH. Conclusions CIH could lead to hypoadiponectinaemia, impaired genioglossus contractile properties and decreased fatigue resistance in rats. Such changes could be partially offset by supplementation of adiponectin.

Nonsurgically induced disuse muscle atrophy and neuromuscular dysfunction upregulates alpha7 acetylcholine receptors

Previous models of muscle disuse have invariably used surgical methods that require the repetitive application of plaster casts. A method of disuse atrophy that does not require such repetitive applications is described herein. Modified plastic pipette tubing was applied to a single hindlimb (mouse), from thigh to foot, resulting in immobilization of the knee in the extension position, and the ankle in the plantar flexion position. This method resulted in the loss of soleus muscle to 11%, 22%, 39%, and 45% of its original mass at 3, 7, 14, and 21 days, respectively, in association with a significant decrease of tibialis twitch (25%) and tetanic tensions (26%) at 21 days, compared with the contralateral side and (or) sham-immobilized controls. Immunohistochemical analysis of the soleus using fluorescent α-bungarotoxin revealed a significant increase in the number of synapses per unit area (818 + 31 compared with 433 + 16/mm(2)) and an increase in muscle fibers per unit area (117 compared with 83/mm(2)), most likely related to the atrophy of muscle fibers bringing synapses closer. A 3-fold increase in alpha7 acetylcholine receptor (α7AChR) protein expression, along with increased expression of α1AChR subunit in the immobilized side compared with the contralateral side was observed. The physiology and pharmacology of the novel finding of upregulation of α7AChRs with disuse requires further study.

Autologous Pump‐Perfused Rat Hind Limb Preparation for Investigating Muscle Function and Metabolism In Vivo

Oxygen delivery, underpinned by vascular tone, is the principle limiting factor in the study of skeletal muscle physiology, particularly during muscle contraction. The aim of this study was to develop an autologous perfused rat hind limb preparation for the study of skeletal muscle contractile function. Adult Wistar rats were surgically prepared using a by-pass system for pump-controlled arterial blood flow to, and venous return from the hind limb during periods of quiescence and twitch contraction of the gastrocnemius-plantaris-soleus muscle bundle. During rest, hind limb perfusion pressure (102±5mmHg) was not different to systemic arterial pressure (99±4mmHg). Hind limb pressure was responsive to vasoconstrictors and vasodilators (±50mmHg). The arterial PO2 (100±3mmHg), O2 saturation, and acid-base balance (pH: 7.42±0.01) contributed to resting hind limb (a-v)O2 difference (4.8±0.5mL/100mL) and VO2 (0.31±0.03μmol/g/min wet weight). Repetitive isometric twitch tension (1Hz, 0.05ms, 10minutes) was best maintained at a flow rate of 2mL/min (VO2 increased fivefold during muscle contraction) and efficiency of oxygen use increased from 0.27±0.08-0.52±0.07N/μmol/min. The autologous rat hind limb provided resting vascular tone allowing maintenance of perfusion pressure at flows within the physiological range. Oxygen delivery supported repetitive twitch contractions and facilitated measurement of active metabolism.

Difference in time-course of relaxant effect of rocuronium between patients with ocular and generalized myasthenia gravis

Objective To compare the time-course of relaxant effect of rocuronium between patients with ocular and generalized myasthenia gravis (MG).Methods Twenty-seven ASA physical status I or Ⅱ patients with MG of both sexes,aged 12-64 yr,with body mass index of 17-26 kg/m2,scheduled for elective extended thymectomy,were divided into 2 groups according to Osserman stage:ocular group (group O,n =10) and generalized group (group G,n =17).Anesthesia was induced with iv injection of fentanyl 2 μg/kg,midazolam 0.05 mg/kg,and propofol 1.5 mg/kg.All patients were tracheal intubated and mechanically ventilated.Anesthesia was maintained with iv infusion of propofol 4-8 mg· kg-1 · h-1 and remifentanil 0.2 μg· kg-1 · min-1.Twitch tension was monitored in the adductor pollicis muscle by train-of-four stimulation of the ulnar nerve (intensity 60 mA,interval 12 s,frequency 2 Hz,wave length 0.2 ms).Rocuronium 0.6 mg/kg was injected intravenously after calibration.Mean arterial pressure (MAP),HR,heart rate variability (HRV) and low frequency and high frequency (LF/HF) ratio was recorded.The onset time of muscle relaxation,time for T1 to recover to 25％,time for T1 to recover to 50％ and recovery index were recorded.Results Compared with the baseline value,there were no significant changes in MAP,HR,HRV,LF/HF ratio at all time points in the two groups (P ＞ 0.05).Compared with group O,there was no significant change in the onset time of muscle relaxation,and the time for T1 to recover to 25％,time for T1 to recover to 50％ and recovery index were significantly prolonged in group G (P ＜ 0.05).Conclusion The duration of rocuronium-induced neuromuscular block is significantly longer in patients with generalized MG than those with ocular MG,while the onset time is comparable between the two groups. Key words: Androstanols; Myasthenia gravis; Neuromuscular blockade

Daily Subacute Paraquat Exposure Decreases Muscle Function and Substantia Nigra Dopamine Level

The use of the herbicide paraquat (1,1'-dimethyl-4,4'-bipyridylium dichloride; PQ) which is widely used in agriculture is known to cause dopaminergic neurotoxicity. However, the mechanisms underlying this effect are not fully understood. This present study investigated the behavioral manifestations, motor coordination, and dopaminergic neurodegeneration following exposure to PQ. Male rats were injected with PQ (10 mg/kg i.p.) daily for three weeks. Rotarod systems were used for measuring locomotor activity and motor coordination. The effects of PQ on dorsiflexor, electrophysiologically-induced muscle contraction were studied. Dopamine concentrations in the ventral mesencephalon were measured by high performance liquid chromatography and the number of dopaminergic neurons in substantia nigra pars compacta was estimated by tyrosine hydroxylase immunohistochemistry. PQ induced difficulty in movement and significant reduction in motor activity and twitch tension at the dorsiflexor skeletal muscle. The number of tyrosine hydroxylase positive neurons was significantly less in the substantia nigra pars compacta and nigral dopamine level was significantly reduced in PQ treated animals (20.4+/-3.4 pg/mg) when compared with control animals (55.0+/-2.4 pg/mg wet tissue). Daily treatment of PQ for three weeks induces selective dopaminergic neuronal loss in the substantia nigra and significant behavioral and peripheral motor deficit effects.

The effects of bupivacaine injection and oral nitric oxide on extraocular muscle in the rabbit

Nitric oxide (NO) plays a key role in muscle regeneration, which is the primary response, observed during bupivacaine-induced extraocular muscle (EOM) hypertrophy. Our aims were to investigate the effects of bupivacaine injection into the rabbit EOM and the interaction with NO. Superior rectus (SR) muscles of 24 New Zealand albino rabbits were studied. Single muscle twitch tension (SMTT) and tetanic muscle tensions at 50, 75, and 100 Hz were recorded using a 15 V stimuli. The rabbits were equally allocated into three groups. Measurements were performed without any drug treatments in group 1. In groups 2 and 3, bupivacaine, 0.5 ml of a 0.50 % solution, was injected into the EOM, and after 21 days, measurements were performed. Oral isosorbide dinitrate (NO donor) at 20 mg/day was given each day prior to measurements in group 3. SMTTs were 69.9 (66.7-77.6), 187.7 (114.9-252.1) and 204.2 (135.3-311.6) mg in groups 1, 2, and 3 respectively. SMTTs for both groups 2 and 3 were significantly higher than that for group 1 (p < 0.05). Compared with group 1, group 2 exhibited a 3.8-11.7 % increase in the tetanic tensions at 50, 75, and 100 Hz, but none of these differences were statistically significant. The increase was 47.5-137.5 % in group 3 relative to group 2, and the differences were statistically significant except at 100 Hz. The enlargement of the muscle fibers after bupivacaine injection was shown histopathologically. Bupivacaine injection increased the EOM tension in rabbits to some extent. NO augmented the effect of bupivacaine.

Reply from Daniel C. Andersson, Matthew J. Betzenhauser, Steven Reiken, Alisa Umanskaya, Takayuki Shiomi and Andrew R. Marks

We appreciate the comments from Roatta and Farina concerning our paper (Andersson et al. 2012) on the molecular mechanism of adrenergic-induced force potentiation in fast twitch skeletal muscle. Our study explores how modulation of the ryanodine receptor (RyR1), which regulates sarcoplasmic reticulum (SR) Ca2+ release, can affect skeletal muscle force production. The hypothesis we tested was that protein kinase A (PKA)-mediated phosphorylation of the RyR1 enhances twitch force in the fast twitch skeletal muscle in mice. Inotropic effects in skeletal muscle following adrenergic receptor stimulation have been reported in many studies (Cairns & Dulhunty, 1993a; Cairns et al. 1993; Liu et al. 1997). The potentiating effect on isometric twitch force is evident in many different animal species (Bowman, 1980). We and others observe a small, but robust, increase in twitch force (∼10–20%) after adrenergic stimulation (Tashiro, 1973; Cairns & Dulhunty, 1993a; Cairns et al. 1993; Liu et al. 1997; Andersson et al. 2012). More recently, changes in Ca2+ handling have been linked to force potentiation (Cairns & Dulhunty, 1993a,b; Cairns et al. 1993; Liu et al. 1997). Cairns et al. (Cairns & Dulhunty, 1993a,b; Cairns et al. 1993) demonstrated enhanced SR Ca2+ release as the underlying mechanism for increased inotropy. In our paper we show that RyR1 PKA phosphorylation results in increased SR Ca2+ release and is a molecular mechanism underlying fast twitch skeletal muscle inotropy in mice. Indeed, mice harbouring RyR1 that cannot be PKA phosphorylated due to a substitution of an alanine for the serine phosphorylated by PKA in RyR1 fail to respond to catecholamines in terms of increasing the calcium transient or muscle force production (Andersson et al. 2012). Roatta and Farina challenge the view that adrenergic activation increases muscle force development in humans (Roatta & Farina, 2011). They correctly point out that the effect of adrenergic potentiation of skeletal muscle force has been difficult to detect in humans. Moreover, Roatta and Farina note that adrenergic stimulation of slow twitch (type 1) muscle can reduce twitch tension (Tashiro, 1973; Bowman, 1980). With the exception of some postural muscles (e.g. soleus), the mice have predominantly fast twitch fibres (Mathewson et al. 2012). In contrast, human muscles exhibit a substantial mix in fibre type and have considerably more slow twitch fibres, ∼40–70% are type 1 fibres in most human muscles (Johnson et al. 1973). Interestingly, the ability to maintain isometric force production in humans correlates with blood catecholamine levels (French et al. 2007). Therefore, the effect of adrenergic stimulation in human in vivo muscle experiments probably reflects the balance between the force potentiation and reduction depending on the fibre type. The net result of adrenergic stimulation in humans may well depend on the intensity and duration of the stimulation. Proving this in humans may be difficult. Nevertheless, the high degree of evolutionary conservation of the key molecules regulating excitation– contraction coupling strongly argues that the fundamental mechanisms are common in mammalian skeletal muscle.