Passively Stretched Research Articles

Inhibition and potentiation of the mechanoreflex following pharmacological modulation of TRPC6 in male rats

We determined the role played by the transient receptor potential canonical 6 (TRPC6) in evoking the mechanical component of the exercise pressor reflex in male decerebrated Sprague-Dawley rats. Quadruple-labelled immunohistochemistry were used in dorsal root ganglion cells to identify the cells that innervate the triceps surae muscles (DiI), the cells that express TRPC6 and the cells that are (Nf200) or are not myelinated (peripherin) (n=4). In addition, static contraction of triceps surae muscles was evoked by electrically stimulating the left tibial nerve before and after injecting into the superficial epigastric artery the TRPC6 antagonists, BI-749327 (n = 11; 12μg.kg−1) or SAR7334 (n = 11; 7μg.kg−1), or the TRPC6 positive modulator, C20 (n = 11; 18μg.kg−1). Similar experiments were conducted while the triceps surae muscles were passively stretched (n = 8 to 12), a maneuver that isolates the mechanical component of the reflex. Blood pressure, tension, renal sympathetic nerve activity (RSNA) and blood flow were recorded throughout the experiments. The neurons innervating the triceps-surae muscles were positive to TRPC6 antigen and co-expressed the neurofilament 200kDa. These cells were not positive for peripherin. While the TRPC6 blockers decreased the pressor response to static contraction (-32 to -42%; P < 0.05) and passive stretch (-35 to 52%; P < 0.05), the positive modulator increased both of these responses (55 to 65%; P < 0.05). In addition, BI-749327 decreased the peak and integrated RSNA responses to both static contraction (-39 to -43%; P < 0.05) and passive stretch (-56 to -62%; P < 0.05) whereas C20 increased these responses to stretch only (55 to 235%; P < 0.05). All of these results were not replicated when the drugs were injected intravenously or when the vehicle of the drug was injected into the superficial epigastric artery. Collectively, our results showed that TRPC6 play a key role in evoking the mechanical component of the exercise pressor reflex in male rats. Supported by NIH grants HL 161160, HL 156594 and HL 156513. 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.

Inhibition and potentiation of the exercise pressor reflex by pharmacological modulation of TRPC6 in male rats.

We determined the role played by the transient receptor potential canonical 6 (TRPC6) channel in evoking the mechanical component of the exercise pressor reflex in male decerebrated Sprague-Dawley rats. TRPC6 channels were identified by quadruple-labelled (DiI, TRPC6, neurofilament-200 and peripherin) immunohistochemistry in dorsal root ganglion (DRG) cells innervating the triceps surae muscles (n=12). The exercise pressor reflex was evoked by statically contracting the triceps surae muscles before and after injection of the TRPC6 antagonist BI-749327 (n=11; 12μgkg-1 ) or SAR7334 (n=11; 7μgkg-1 ) or the TRPC6 positive modulator C20 (n=11; 18μgkg-1 ). Similar experiments were conducted while the muscles were passively stretched (n=8-12), a manoeuvre that isolated the mechanical component of the reflex. Blood pressure, tension, renal sympathetic nerve activity (RSNA) and blood flow were recorded. Of the DRG cells innervating the triceps surae muscles, 85% stained positive for the TRPC6 antigen, and 45% of those cells co-expressed neurofilament-200. Both TRPC6 antagonists decreased the reflex pressor responses to static contraction (-32 to -42%; P < 0.05) and to passive stretch (-35 to -52%; P < 0.05), whereas C20 increased these responses (55-65%; P < 0.05). In addition, BI-749327 decreased the peak and integrated RSNA responses to both static contraction (-39 to -43%; P < 0.05) and passive stretch (-56 to -62%; P < 0.05), whereas C20 increased the RSNA to passive stretch only. The onset latency of the decrease or increase in RSNA occurred within 2s of the onset of the manoeuvres (P < 0.05). Collectively, our results show that TRPC6 plays a key role in evoking the mechanical component of the exercise pressor reflex. KEY POINTS: The exercise pressor reflex plays a key role in the sympathetic and haemodynamic responses to exercise. This reflex is composed of two components, namely the mechanoreflex and the metaboreflex. The receptors responsible for evoking the mechanoreflex are poorly documented. A good candidate for this function is the transient receptor potential canonical 6 (TRPC6) channel, which is activated by mechanical stimuli and expressed in dorsal root ganglia of rats. Using two TRPC6 antagonists and one positive modulator, we investigated the role played by TRPC6 in evoking the mechanoreflex in decerebrated rats. Blocking TRPC6 decreased the renal sympathetic and the pressor responses to both contraction and stretch, the latter being a manoeuvre that isolates the mechanoreflex. In contrast, the positive modulator increased the pressor reflex to contraction and stretch, in addition to the sympathetic response to stretch. Our results provide strong support for a role played by the TRPC6 channel in evoking the mechanoreflex.

Residual force enhancement is reduced in permeabilized fiber bundles from mdm muscles

Residual Force enhancement (RFE) is defined as the increase in steady state force after active stretch compared with force after isometric contraction at the same final length. Recent studies have demonstrated a pivotal role for titin in RFE. In myofibrils stretched beyond overlap of thick and thin filaments, Ca2+ activated myofibrils had a higher titin-based force and a steeper increase with force with stretch than passively stretched myofibrils at the same average sarcomere length. These results suggest higher titin-based stiffness in active myofibrils, perhaps due to N2A titin-actin interactions in active wild type (WT) muscle. Mdm mice carry a small deletion in titin that includes 53 amino acids in Ig83 in the N2A region. It has been proposed that the mdm mutation prevents N2A titin-actin interactions so that active mdm muscles are more compliant than WT muscles. This decrease in active muscle stiffness should be associated with a reduction in RFE. Based on this rationale, we investigated RFE in permeabilized soleus (SOL) and extensor digitorum longus (EDL) fiber bundles from 20-30 day old mdm mice (n=5), harvested at a young age to diminish the effects of degeneration and regeneration, and 30-60 day old WT mice (n=5). Contractile stress and RFE of EDL (mdm n=19 and WT n=20) and SOL fibers (WT n=19 and mdm n=16) was analyzed using JMP and Graph pad (PRISM) software. Statistics included Shapiro-Wilk (normality), Levene's and Bartlett (homogeneity of variances), Welch's one-way ANOVA (unequal variances), Dunnett's T3 (multiple comparisons), and T-tests with Welch's correction (unpaired). To investigate differences in RFE between genotypes, we performed isometric contractions, as well as active and passive stretch from an average sarcomere length of 2.6 - 3.0 µm on each fiber. This stretch amplitude, within the physiological range, resulted in no observable breakage or slipping of fibers during experiments. We predicted that mdm fiber bundles would show reduced RFE compared to WT fibers. Contractile stress when activated at 2.6 µm was significantly greater in WT EDL (Welch's one-way ANOVA; P<0.05) bundle and SOL (Welch's one-way ANOVA; P<0.05) compared to fiber bundles. Our results showed that mdm muscles exhibit significantly lower RFE than WT for both SOL (Welch's one-way ANOVA; P<0.0001) and EDL (Welch's one-way ANOVA; P<0.0001). This result is consistent with previous observations in single myofibrils and intact muscles. The data support the hypothesis that titin contributes to RFE. We suggest that RFE is reduced in mdm muscles due to impaired Ca2+ dependent titin-actin interactions, resulting from the small deletion in titin that includes 53 amino acids in Ig83 in the N2A region.

Diagnosing Muscle Atrophy by Use of a Comprehensive Method of Assessing the Elastic Properties of Muscle During Passive Stretching.

OBJECTIVE. The purpose of this study was to apply a new method for quantitatively assessing atrophied muscles by use of sonoelastography. SUBJECTS AND METHODS. B-mode sonography and shear-wave elastography of the gastrocnemius muscle (GM) were performed on 12 patients and 32 healthy control volunteers during passive stretching induced by ankle rotation from 40° plantar flexion to 30° dorsiflexion. The GM length and corresponding elastic moduli were measured consecutively per frame. The elastic modulus-length curve was created by plotting the elastic modulus against the GM length during passive stretching. Three physiologically significant parameters-slack elasticity modulus (E0), slack length (l0), and passive elastic coefficient (k)-were estimated from the measured elasticity modulus-length curve to quantify the elastic properties of the GM. RESULTS. All results showed a smooth exponential curve. In the state of relaxation, atrophied GM was softer than GM of healthy control subjects. However, when the muscles were passively stretched from a state of slack to tension, k was significantly greater for the patients with atrophied GMs than for the control subjects. Furthermore, the diagnostic accuracy of k for muscle atrophy was greater than that of E0. CONCLUSION. Increased elastic stiffness may be considered a positive finding for the characterization of muscle atrophy. Because of its high diagnostic accuracy and reproducibility, the elastic modulus-length curve can provide new insights into the diagnosis of muscle disease and allows monitoring of muscle function in rehabilitation.

Passive stretch regulates skeletal muscle glucose uptake independent of nitric oxide synthase.

Skeletal muscle contraction increases glucose uptake via an insulin-independent mechanism. Signaling pathways arising from mechanical strain are activated during muscle contractions, and mechanical strain in the form of passive stretching stimulates glucose uptake. However, the exact mechanisms regulating stretch-stimulated glucose uptake are not known. Since nitric oxide synthase (NOS) has been implicated in the regulation of glucose uptake during ex vivo and in situ muscle contractions and during exercise, and NO is increased with stretch, we examined whether the increase in muscle glucose uptake during stretching involves NOS. We passively stretched isolated extensor digitorum longus muscles (15 min at ~100-130 mN) from control mice and mice lacking either neuronal NOSµ (nNOSµ) or endothelial NOS (eNOS) isoforms, as well as used pharmacological inhibitors of NOS. Stretch significantly increased muscle glucose uptake appoximately twofold ( P < 0.05), and this was unaffected by the presence of the NOS inhibitors NG-monomethyl-l-arginine (100 µM) or NG-nitro-l-arginine methyl ester (100 µM). Similarly, stretch-stimulated glucose uptake was not attenuated by deletion of either eNOS or nNOSµ isoforms. Furthermore, stretching failed to increase skeletal muscle NOS enzymatic activity above resting levels. These data clearly demonstrate that stretch-stimulated skeletal muscle glucose uptake is not dependent on NOS. NEW & NOTEWORTHY Passive stretching is known to activate muscle glucose uptake through mechanisms that partially overlap with contraction. We report that genetic knockout of endothelial nitric oxide synthase (NOS) or neuronal NOS or pharmacological NOS inhibition does not affect stretch-stimulated glucose uptake. Passive stretch failed to increase NOS activity above resting levels. This information is important for the study of signaling pathways that regulate stretch-stimulated glucose uptake and indicate that NOS should be excluded as a potential signaling factor in this regard.

Does partial titin degradation affect sarcomere length nonuniformities and force in active and passive myofibrils?

The aim of this study was to determine the role of titin in preventing the development of sarcomere length nonuniformities following activation and after active and passive stretch by determining the effect of partial titin degradation on sarcomere length nonuniformities and force in passive and active myofibrils. Selective partial titin degradation was performed using a low dose of trypsin. Myofibrils were set at a sarcomere length of 2.4 µm and then passively stretched to sarcomere lengths of 3.4 and 4.4 µm. In the active condition, myofibrils were set at a sarcomere length of 2.8 µm, activated, and actively stretched by 1 µm/sarcomere. The extent of sarcomere length nonuniformities was calculated for each sarcomere as the absolute difference between sarcomere length and the mean sarcomere length of the myofibril. Our main finding is that partial titin degradation does not increase sarcomere length nonuniformities after passive stretch and activation compared with when titin is intact but increases the extent of sarcomere length nonuniformities after active stretch. Furthermore, when titin was partially degraded, active and passive stresses were substantially reduced. These results suggest that titin plays a crucial role in actively stretched myofibrils and is likely involved in active and passive force production.

Regional increase in ROS within stretched region exacerbates arrhythmias in rat trabeculae with nonuniform contraction.

In diseased hearts, impaired muscle within the hearts is passively stretched by contractions of the more viable neighboring muscle during the contraction phase. We investigated whether in the myocardium with nonuniform contraction such passive stretch regionally generates ROS within the stretched region and exacerbates arrhythmias. In trabeculae from rat hearts, force, intracellular Ca2+, and membrane potential were measured. To assess regional ROS generation, the slope of the change in the 2',7'-dichlorofluorescein fluorescence (DCFslope) was calculated at the each pixel position along the long axis of trabeculae using DCF fluorescence images. Ca2+ waves and arrhythmias were induced by electrical stimulation. A H2O2 (1mmol/L) jet regionally increased the DCFslope within the jet-exposed region. A blebbistatin (10μmol/L) jet caused passive stretch of the muscle within the jet-exposed region during the contraction phase and increased the DCFslope within the stretched region, the velocity of Ca2+ waves, and the number of beats after electrical stimulation (0.2μmol/L isoproterenol), while 3μmol/L diphenyleneiodonium (DPI), NADPH oxidase inhibitor, decreased them.A jet of a solution containing 0.2 mmol/L H2O2 in addition to 10 µmol/L blebbistatin also increased them. A H2O2 jet within the region where Ca2+ waves propagated increased their velocity. In the myocardium with nonuniform contraction, passive stretch of the muscle by contractions of the neighboring muscle regionally increases ROS within the stretched region, and the regional ROS exacerbates arrhythmias by activating the propagation of Ca2+ waves.

Daily muscle stretching enhances blood flow, endothelial function, capillarity, vascular volume and connectivity in aged skeletal muscle.

In aged rats, daily muscle stretching increases blood flow to skeletal muscle during exercise. Daily muscle stretching enhanced endothelium-dependent vasodilatation of skeletal muscle resistance arterioles of aged rats. Angiogenic markers and capillarity increased in response to daily stretching in muscles of aged rats. Muscle stretching performed with a splint could provide a feasible means of improving muscle blood flow and function in elderly patients who cannot perform regular aerobic exercise. Mechanical stretch stimuli alter the morphology and function of cultured endothelial cells; however, little is known about the effects of daily muscle stretching on adaptations of endothelial function and muscle blood flow. The present study aimed to determine the effects of daily muscle stretching on endothelium-dependent vasodilatation and muscle blood flow in aged rats. The lower hindlimb muscles of aged Fischer rats were passively stretched by placing an ankle dorsiflexion splint for 30minday-1 , 5daysweek-1 , for 4weeks. Blood flow to the stretched limb and the non-stretched contralateral limb was determined at rest and during treadmill exercise. Endothelium-dependent/independent vasodilatation was evaluated in soleus muscle arterioles. Levels of hypoxia-induced factor-1α, vascular endothelial growth factor A and neuronal nitric oxide synthase were determined in soleus muscle fibres. Levels of endothelial nitric oxide synthase and superoxide dismutase were determined in soleus muscle arterioles, and microvascular volume and capillarity were evaluated by microcomputed tomography and lectin staining, respectively. During exercise, blood flow to plantar flexor muscles was significantly higher in the stretched limb. Endothelium-dependent vasodilatation was enhanced in arterioles from the soleus muscle from the stretched limb. Microvascular volume, number of capillaries per muscle fibre, and levels of hypoxia-induced factor-1α, vascular endothelial growth factor and endothelial nitric oxide synthase were significantly higher in the stretched limb. These results indicate that daily passive stretching of muscle enhances endothelium-dependent vasodilatation and induces angiogenesis. These microvascular adaptations may contribute to increased muscle blood flow during exercise in muscles that have undergone daily passive stretch.

The Origin of the Force Increase Observed after Active Stretch Beyond Myofilament Overlap in Single Muscle Fibers

At long sarcomere lengths (SLs) beyond myofilament overlap, only passive forces, produced by passive structural elements, are possible. However, Leonard et al. (2010) showed a dramatic increase in force above the passive force when myofibrils were actively stretched beyond actin and myosin filament overlap. It has been suggested that titin's stiffness might increase, or characteristic length decrease, with activation resulting in the increase in force observed when muscles are actively stretched compared to when they are passively stretched. Our purpose was to investigate whether the increase in force after active stretch beyond overlap was caused by a passive component/titin independently of the actin and myosin cross bridges. Therefore muscle fibres were actively stretched to a SL beyond myofilament overlap (5.0 μm) and then deactivated or treated with a cross bridge inhibitor, 2,3-butanedione monoxime (BDM). Fibres were also passively stretched to a SL of 5.0 μm. Results show similar decrease in force, of about 50%, after deactivation and treatment with a BDM solution. However, the forces after deactivation and treatment with BDM are higher than the purely passive stretch force at an average SL of 5.0 μm. The decrease in force after deactivation may be explained assuming that titin is an “activable/deactivable” spring whose stiffness is modulated by activation. However, the decrease in force following BDM exposure cannot be reconciled with a titin based mechanism only. BDM is known to inhibit cross bridge-based force, and thus the decrease in force after BDM exposure is likely associated with cross bridge inhibition. In conclusion, the force increase observed after active stretch beyond myofilament overlap is likely associated with cross-bridge and titin forces.

Thick Filament Compliance in Passively Stretched Skeletal Muscle

Titin is a giant sarcomeric protein (∼2.9MDa to 4.0MDa) that spans half of the sarcomere from the Z disc to the M band. Titin closely interacts with the thick filament inside the A band, and plays important roles in sarcomere development and force generation. In vertebrate muscle, multiple titin isoforms with different molecular weights exist resulting from alternative splicing of a single titin gene. Different sizes of titin lead to different overall stiffnesses of the muscle having multiple effects on muscle function. Titin with different sizes would expect to have different effects on myofilament properties. Here we used the Rbm20 mouse model to study how titins of different sizes, and hence, compliance can affect sarcomere structure with increasing sarcomere length in skeletal muscle using small angle X-ray diffraction. In this Rbm20 mouse model, homozygous mice (KO) expresses the largest titin, and heterozygous mice (HET) expresses an intermediate molecular weight titin compared to titin expressed in wild type mice. The results showed that titin-based passive tension strains thick filaments, and the titin isoform in Rbm20 KO muscle strains the thick filament the least. Our results also showed that the thick filament extensibility, and hence, compliance, in passively stretched muscle is non-linear. Meridional X-ray reflections attributed to myosin binding protein C (MyBP-C) decrease in intensity with increasing sarcomere length, consistent with the notion that MyBP-C interacts with actin and that this interaction is lost when thin filaments are pulled out of the C-zone. Our results also showed that KO muscle with most compliant titin isoform had the largest inter-filament spacing and lowest equatorial intensity ratio at slack length. Supported by R01AR060697 and P41GM103622 from the National Institutes of Health.

Differences in titin segmental elongation between passive and active stretch in skeletal muscle.

Since the 1950s, muscle contraction has been explained using a two-filament system in which actin and myosin exclusively dictate active force in muscle sarcomeres. Decades later, a third filament called titin was discovered. This titin filament has recently been identified as an important regulator of active force, but has yet to be incorporated into contemporary theories of muscle contraction. When sarcomeres are actively stretched, a substantial and rapid increase in force occurs, which has been suggested to arise in part from titin-actin binding that is absent in passively stretched sarcomeres. However, there is currently no direct evidence for such binding within muscle sarcomeres. Therefore, we aimed to determine whether titin binds to actin in actively but not in passively stretched sarcomeres by observing length changes of proximal and distal titin segments in the presence and absence of calcium. We labeled I-band titin with fluorescent F146 antibody in rabbit psoas myofibrils and tracked segmental elongations during passive (no calcium) and active (high calcium) stretch. Without calcium, proximal and distal segments of titin elongated as expected based on their free spring properties. In contrast, active stretch differed statistically from passive stretch, demonstrating that calcium activation increases titin segment stiffness, but not in an actin-dependent manner. The consistent elongation of the proximal segment was contrary to what was expected if titin's proximal segment was attached to actin. This rapid calcium-dependent change in titin stiffness likely contributes to active muscle force regulation in addition to actin and myosin.

Neuro-musculoskeletal simulation of instrumented contracture and spasticity assessment in children with cerebral palsy.

BackgroundIncreased resistance in muscles and joints is an important phenomenon in patients with cerebral palsy (CP), and is caused by a combination of neural (e.g. spasticity) and non-neural (e.g. contracture) components. The aim of this study was to simulate instrumented, clinical assessment of the hamstring muscles in CP using a conceptual model of contracture and spasticity, and to determine to what extent contracture can be explained by altered passive muscle stiffness, and spasticity by (purely) velocity-dependent stretch reflex.MethodsInstrumented hamstrings spasticity assessment was performed on 11 children with CP and 9 typically developing children. In this test, the knee was passively stretched at slow and fast speed, and knee angle, applied forces and EMG were measured. A dedicated OpenSim model was created with motion and muscles around the knee only. Contracture was modeled by optimizing the passive muscle stiffness parameters of vasti and hamstrings, based on slow stretch data. Spasticity was modeled using a velocity-dependent feedback controller, with threshold values derived from experimental data and gain values optimized for individual subjects. Forward dynamic simulations were performed to predict muscle behavior during slow and fast passive stretches.ResultsBoth slow and fast stretch data could be successfully simulated by including subject-specific levels of contracture and, for CP fast stretches, spasticity. The RMS errors of predicted knee motion in CP were 1.1 ± 0.9° for slow and 5.9 ± 2.1° for fast stretches. CP hamstrings were found to be stiffer compared with TD, and both hamstrings and vasti were more compliant than the original generic model, except for the CP hamstrings. The purely velocity-dependent spasticity model could predict response during fast passive stretch in terms of predicted knee angle, muscle activity, and fiber length and velocity. Only sustained muscle activity, independent of velocity, was not predicted by our model.ConclusionThe presented individually tunable, conceptual model for contracture and spasticity could explain most of the hamstring muscle behavior during slow and fast passive stretch. Future research should attempt to apply the model to study the effects of spasticity and contracture during dynamic tasks such as gait.Electronic supplementary materialThe online version of this article (doi:10.1186/s12984-016-0170-5) contains supplementary material, which is available to authorized users.

Local Vascular Hemodynamic Response During and After Passive Stretching

PURPOSE: Only few investigations focused on the peripheral hemodynamic responses to passive limb stretching (PLS), and only in the animal model. Therefore, the aim of this study was to determine in healthy humans the acute effects of PLS on limb blood flow (LBF). Our hypothesis was that peripheral hemodynamic responses would be affected by the PLS, because of the repetitive ischemia/reperfusion cycles induced by the passive muscle tension. METHODS: Twelve healthy individuals (age: 22 ± 3 yrs) underwent PLS of the knee extensors. The PLS procedure (45 s stretch/passive flexion, 15 s release/passive extension) was repeated five times. Measurements of arterial blood velocity and vessel diameter were taken in the passively stretched leg, distal to the inguinal ligament and proximal to the deep superficial femoral bifurcation by ultrasound system. RESULTS: PLS had a significant influence on peripheral hemodynamic. Specifically, LBF responses during the 1st passive flexion of PLS transitory increased with respect to baseline by ∼78% (from 495±110 to 882±121 ml/min; P<0.05). Interestingly, a second hyperemia (to 880±118 ml/min; P<0.05) was recorded during the limb passive extension after the 1st PLS. During the 2nd PLS procedure, the hyperemic response recorded during the passive flexion was markedly reduced with respect to the 1st (608±103 ml/min), while LBF recorded during the passive extension was similar to the 1st PLS (915±131 ml/min). From the 3rd PLS procedure, the hyperemic responses to PLS were negligible, while the peaks of LBF were consistently elevated. CONCLUSION: In agreement with our hypothesis, PLS significantly influenced peripheral circulation in the passively stretched knee extensors. However, the time response of these alterations in the peripheral hemodynamic suggests two different components of this phenomenon. The hyperemic response corresponding to the flexion phase of PLS may be explained by an acute release of nitric oxide derived from the mechanical stretch of the vessels. Afterwards, the response decreased progressively during the 2nd, 3rd, 4th, and 5th PLS procedures. Conversely, the constant hyperemic responses recorded during the passive extensions of PLSs were possibly caused by a reduced ischemia determined by a diminished muscle tension.

Muscle afferent activation causes ventilatory and cardiovascular responses during concurrent hypercapnia in humans

Respiratory and cardiovascular responses to muscle mechanoreflex (passive calf stretch) and metaboreflex activation (local circulatory occlusion) were examined during inhalation of a hypercapnic gas mixture in four trials. These controlled for the effects of central command, metabolite sensitization of muscle afferents and hypercapnia-induced elevation of central respiratory drive. In an isokinetic dynamometer, with circulation through the right leg occluded by inflation of a thigh cuff, 13 participants either rested (control trial; CON) or plantarflexed their ankle at 50% maximal force for 1.5 min (voluntary exercise trial; EX). Thereafter, circulatory occlusion was maintained and the calf passively stretched before return to the resting position. Both trials were performed while breathing air, as well as while breathing a normoxic, hypercapnic (5% CO(2)) gas mixture (CO(2) trial and CO(2)+EX trial). Hypercapnic gas inhalation increased baseline minute ventilation (V), heart rate and mean arterial pressure (+27.67 ± 1.74 l min(-1), +7 ± 0.85 beats min(-1) and +13 ± 3.41 mmHg, respectively; means ± SEM) above control values (9.78 ± 0.86 l min(-1), 62 ± 2.3 beats min(-1) and 88 ± 2.6 mmHg, respectively). Voluntary exercise further increased these variables from baseline during both trials (P < 0.05). During the continued circulatory occlusion after voluntary exercise, mean arterial pressure remained significantly elevated (P < 0.05). Minute ventilation returned to baseline during circulatory occlusion following exercise in the EX trial, but in the CO(2)+EX trial the V remained elevated at end-exercise levels during this period (+7.12 ± 1.13 l min(-1)). Passive stretch caused further increases in V during CO(2)+EX and CO(2) trials but not in CON and EX. These results indicate that in the absence of central command, either muscle metaboreflex and/or mechanoreflex activation stimulates ventilation during concurrent hypercapnia.

The Influence of Passive Stretch and NF‐κB Inhibitors on the Morphology of Dystrophic Muscle Fibers

The triangularis sterni (TS) is an expiratory muscle that is passively stretched during inspiration. The magnitude of passive stretch depends upon the location of individual fibers within the TS muscle, with fibers located more caudally being stretched ∼ 5% to 10% more than fibers in the cephalad region. In the mdx mouse model for muscular dystrophy, the TS exhibits severe pathological alterations that are ameliorated by treatment with inhibitors of the NF-κB pathway. The purpose of this study was to assess the influence of passive stretch in vivo on fiber morphology in nondystrophic and mdx TS muscles, and the morphological benefits of treating mdx mice with two distinct NF-κB inhibitors, pyrrolidine dithiocarbamate (PDTC), and ursodeoxycholic acid (UDCA). Transmission electron microscopy revealed Z-line streaming, hypercontraction, and disassociation of the plasma membrane from the basal lamina in mdx fibers. In both nondystrophic and mdx TS muscles, fiber density was larger in more caudal regions. In comparison with nondystrophic TS, fibers in the mdx TS exhibited substantial reductions in diameter throughout all regions. In vivo treatment with either PDTC or UDCA tended to increase fiber diameter in the middle and decrease fiber diameter in the caudal TS, while reducing centronucleation in the middle region. These results suggest that passive stretch induces hypercontraction and plasma membrane abnormalities in dystrophic muscle, and that differences in the magnitude of passive stretch may influence fiber morphology and the actions of NF-κB inhibitors on dystrophic morphology.

Muscle metabolites accentuate muscle sympathetic nerve activity responses to passive muscle stretch

It was speculated that muscle metabolites can sensitize muscle mechanoreceptors, and accentuate muscle sympathetic nerve activity (MSNA) responses to mechanoreceptors stimulation in exercise. However, this hypothesis has not been verified directly in healthy subjects. To test whether the MSNA response to passive stretch could be accentuated with increasing metabolites in the muscles, MSNA, ECG and blood pressure (BP) were recorded in 14 young healthy subjects. Subjects performed static isometric handgrip at 30% maximum voluntary contraction (MVC) until fatigue followed by 4 min post exercise ischemia (PEMI). After 2 min of PEMI, the hand was passively stretched (dorsiflexion). In 10 of the subjects, the stretch protocol was also performed without preceding handgrip or ischemia, ischemia alone, and following non-fatigue handgrip at 30% MVC followed by ischemia. During the PEMI after fatigue handgrip, stretch induced significant increase in MSNA (26.4±2.6 to 30.7±2.8 bursts/min, P=0.002) and mean BP (106±2 to 108±2 mmHg, P=0.01). Stretch performed under the other 3 conditions had no significant effect on MSNA. The present data suggest that muscle metabolites accentuate MSNA response to mechanoreceptors stimulation, probably via sensitizing the mechanoreceptors. Supported by NIH P01 HL077670 and M01 RR010732, and AHA 0565399U, 0635245N.

Muscle sympathetic nerve activity responses to dynamic passive muscle stretch in humans

It is suggested that mechanoreceptors in muscle play an important role in the exercise pressor reflex. However, it has not been verified whether isolated stimulation of the mechanoreceptors can induce responses in muscle sympathetic nerve activity (MSNA) in young healthy individuals. We tested the hypothesis that passive stretch of muscle can evoke an increase in MSNA in healthy individuals. In 12 young subjects, leg calf muscles were passively stretched, or actively contracted for 5 s followed by a 15-25 s (random length) relaxation period. Stretch and contraction were each repeated 25 times. MSNA, heart rate and blood pressure were analysed, and averaged according to the onset of the force on a beat-by-beat basis. At the 1st to the 3rd heart beat from the onset of stretch, MSNA (199 +/- 30%, P < 0.05) as well as heart rate (102.5 +/- 0.7%, P < 0.05) increased transiently but significantly from the prior stretch baseline (100%), followed (from 3rd to 7th beat from the onset of stretch) by a transient increase in mean blood pressure (101.9 +/- 0.3%, P < 0.05) from the baseline. Similar response patterns were observed during active muscle contractions. The present data show that MSNA responses to isolated stimulation of mechanoreceptors are measurable. Because of baroreflex engagement, the magnitude of the response is small and transient, and the haemodynamic consequences using this protocol may be limited.

Muscle sympathetic nerve activity responses to dynamic passive muscle stretch.

It is suggested that mechanoreceptors in muscle play an important role in the exercise pressor reflex, however, it has not been verified whether isolated stimulation of the mechanoreceptors can induce responses in muscle sympathetic nerve activity (MSNA) in young healthy individuals. The purpose of present study was to test the hypothesis that passive stretch of muscle can evoke an increase in MSNA in healthy individuals. In 7 young subjects, calf muscles in a leg were passively stretched, or actively contracted for 5 sec followed by a 15–25 sec (random) relaxation period. Stretch and contraction were each repeated 25 times. MSNA, ECG, heart rate, and blood pressure were recorded, analyzed, and averaged according to the onset of the force on a beat-by-beat basis. At the 1st and/or the 2nd heart beat from the onset of stretch, MSNA increased transiently but significantly (7.27±1.87 units/beat) from the prior stretching baseline (4.80±1.43 units/beat, P<0.01), followed (at 3rd to 5th beat from the onset of stretching) by a transient increase in mean blood pressure from the baseline (change 2.48±0.68 mmHg, P<0.05). After end of stretch, another MSNA increase was observed when blood pressure fell. Similar responses were observed during active muscle contractions. The present data can be considered as evidence to support the concept that isolated stimulation of mechanoreceptors can evoke MSNA responses in young healthy humans. Supported by P01 HL077670 and M01 RR010732.

The Effect of Resection on Satellite Cell Activity in Rabbit Extraocular Muscle

A common treatment for motility disorders of the extraocular muscles (EOMs) is a resection procedure in which there is surgical shortening of the muscle. This procedure results in rotation of the globe toward the resected muscle, increased resting tension across the agonist-antagonist pair, and stretching of the elastic components of the muscles. In the rabbit, due to orbital constraints and limited rotation, resection results in more significant stretch of the surgically treated muscle than the antagonist. This surgical preparation allows for the examination of the effects of surgical shortening of one rectus muscle and passive stretch of its ipsilateral antagonist. The insertional 6 mm of the superior rectus muscles of adult rabbits were resected and reattached to the original insertion site. After 7 and 14 days, the animals were injected intraperitoneally with bromodeoxyuridine (BrdU) every 2 hours for 12 hours, followed by a 24-hour BrdU-free period. All superior and inferior rectus muscles from both globes were examined for BrdU incorporation, MyoD expression, neonatal and developmental myosin heavy chain (MyHC) isoform expression, and myofiber cross-sectional area alterations. In the resected muscle and in the passively stretched antagonist muscle, there was a dramatic increase in the number of myofibers positive for neonatal MyHC and in the number of BrdU- and MyoD-positive satellite cells. The addition of BrdU-positive myonuclei increased from 1 per 1000 myofibers in cross sections of control muscles to 2 to 3 per 100 myofibers in the resected muscles. Single myofiber reconstructions showed that multiple BrdU-positive myonuclei were added to individual myofibers. Addition of new myonuclei occurred in random locations along the myofiber length of single fibers. There was no correlation between myofibers with BrdU-positive myonuclei and neonatal MyHC isoform expression. Both active and passive stretch of the rectus muscles produced by strabismus surgery dramatically upregulated the processes of satellite cell activation, integration of new myonuclei into existing myofibers, and concomitant upregulation of immature myosin heavy chain isoforms. Understanding the effects of strabismus surgery on muscle cell biological reactions and myofiber remodeling may suggest new approaches for improving surgical outcomes.

Chronic treatment with agents that stabilize cytosolic IκB-α enhances survival and improves resting membrane potential in MDX muscle fibers subjected to chronic passive stretch

The potential pathogenic role of increased NFkappaB signaling in passively stretched dystrophic skeletal muscle was examined by treating adult mdx mice with an agent that stabilized cytosolic IkappaB-alpha (pyrollidine dithiocarbamate, PDTC)and examining the effects of this treatment on the chronically stretched mdx triangularis sterni (TS) muscle. Daily PDTC treatment significantly increased the number of surviving striated TS fibers regardless of age. TS fibers from untreated mdx mice had significantly lower resting potentials (RPs) than nondystrophic mice. Treatment with GdCl3 to block resting Ca2+ influx had no effect on RP in either nondystrophic or mdx preparations. Daily treatment with PDTC significantly improved the RP regardless of age. These results are consistent with the hypothesis that passive stretch activates an NFkappaB-mediated pathogenic mechanism in dystrophic muscle and suggest that agents which stabilize cytosolic IkappaB-alpha levels may be useful for treating Duchenne and related muscular dystrophies.