Isometric Contractile Properties Research Articles

Sexual Dimorphism of Skeletal Muscle in a Mouse Model of Breast Cancer: A Functional and Molecular Analysis.

Breast cancer incidence in men is statistically rare; however, given the lack of screening in males, more advanced stages at initial diagnosis result in lower 5-year survival rates for men with breast cancer compared to women. A sexual dimorphism, with respect to the effect of tumor growth on cachexia incidence and severity, has also been reported across cancer types. The purpose of this study was to examine the sexual dimorphism of breast cancer as it pertains to skeletal muscle function and molecular composition. Using female and male transgenic PyMT mice, we tested the hypothesis that the isometric contractile properties and molecular composition of skeletal muscle would be differentially affected by breast tumors. PyMT tumor-bearing mice of each sex, corresponding to maximal tumor burden, were compared to their respective controls. RNA sequencing of skeletal muscle revealed different pathway alterations that were exclusive to each sex. Further, differentially expressed genes and pathways were substantially more abundant in female tumor mice, with only minimal dysregulation in male tumor mice, each compared to their respective controls. These differences in the transcriptome were mirrored in isometric contractile properties, with greater tumor-induced dysfunction in females than male mice, as well as muscle wasting. Collectively, these data support the concept of sexually dimorphic responses to cancer in skeletal muscle and suggest that these responses may be associated with the clinical differences in breast cancer between the sexes. The identified sex-dependent pathways within the muscle of male and female mice provide a framework to evaluate therapeutic strategies targeting tumor-associated skeletal muscle alterations.

Direct measurement of human skeletal muscle specific tension and fiber length

Skeletal muscle’s isometric contractile properties are one of the classic structure-function relationships in all of physiology allowing for extrapolation of single fiber mechanical properties to whole muscle properties based on the muscle’s optimal fiber length and physiological cross-sectional area (PCSA). However, this relationship has only been validated in small animals and then extrapolated to human muscles which are much larger in terms of length and PCSA. We leveraged a unique surgical technique in which an intact, living, human gracilis muscle is transferred from the thigh to the arm, restoring elbow flexion after brachial plexus injury. During this surgery we directly measured subject specific gracilis muscle force-length relationship in situ and properties ex vivo. We hypothesized that the human gracilis would have a functional fiber length of about 23 cm, based on the anatomical literature, and a specific tension of 225 kPa, based on the physiological literature. Data are provided as mean±standard deviation of the mean. Twelve subjects (7 males, average age 54±12 years). In situ passive and active tension were directly measured using a buckle force transducer (BFT) placed on the distal gracilis tendon. Passive tension was measured in each of four joint configurations (JC) prior to muscle stimulation, and, to produce active tension, the anterior branch of the obturator nerve was stimulated. The muscle was progressively lengthened by extending the knee and abducting the hip. Since we could not measure optimal fiber length directly in our subjects during isometric testing we used a functional surrogate for optimal fiber length, the full-width at half-maximum (FWHM) defined from their force vs. length relationship. The FWHM is the width of the active force vs. muscle length curve at half the maximum force. A linear relationship between FWHM and optimal fiber length has been established across several mammalian species and muscles. Thus, to calculate FWHM, we fit each patient’s calculated fully active force vs. muscle length data to a quadratic curve and then calculated width at half the calculated maximum force from the equation. Optimal fiber length was then calculated from the previously established linear relationship between FWHM and optimal fiber length. Each subject’s PCSA was calculated from their muscle volume and optimal fiber length.From these experimental data we established a human muscle fiber-specific tension of 171±84 kPa (n=12). We also determined the average gracilis optimal fiber length was 12.9±3.3 cm (n=12). Surprisingly, these fiber lengths were about half of the previously reported optimal fascicle lengths of 23 cm. Thus, the long gracilis muscle appears to be composed of relatively short fibers acting in parallel that may not have been appreciated based on traditional anatomical methods. We thus suggest that the value of 171 kPa be considered the best current estimate of human muscle specific tension. This work was supported the Department of Veterans Affairs grant 1 I01 RX002462 and, in part, by Research Career Scientist Award Number IK6 RX003351 from the United States (U.S.) Department of Veterans Affairs Rehabilitation R&D (Rehab RD) Service. Pilot projects leading to this protocol were supported by the Shirley Ryan AbilityLab Catalyst Grant Program, National Science Foundation Graduate Research Fellowship under Grant No. 1255833 and the Mayo Clinic Graduate School. This is the full abstract presented at the American Physiology Summit 2023 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.

Direct intraoperative measurement of isometric contractile properties in living human muscle.

Skeletal muscle's isometric contractile properties are one of the classic structure-function relationships in all of biology allowing for extrapolation of single fibre mechanical properties to whole muscle properties based on the muscle's optimal fibre length and physiological cross-sectional area (PCSA). However, this relationship has only been validated in small animals and then extrapolated to human muscles, which are much larger in terms of length and PCSA. The present study aimed to measure directly the in situ properties and function of the human gracilis muscle to validate this relationship. We leveraged a unique surgical technique in which a human gracilis muscle is transferred from the thigh to the arm, restoring elbow flexion after brachial plexus injury. During this surgery, we directly measured subject specific gracilis muscle force-length relationship in situ and properties ex vivo. Each subject's optimal fibre length was calculated from their muscle's length-tension properties. Each subject's PCSA was calculated from their muscle volume and optimal fibre length. From these experimental data, we established a human muscle fibre-specific tension of 171kPa. We also determined that average gracilis optimal fibre length is 12.9cm. Using this subject-specific fibre length, we observed an excellent fit between experimental and theorical active length-tension curves. However, these fibre lengths were about half of the previously reported optimal fascicle lengths of 23cm. Thus, the long gracilis muscle appears to be composed of relatively short fibres acting in parallel that may not have been appreciated based on traditional anatomical methods. KEY POINTS: Skeletal muscle's isometric contractile properties represent one of the classic structure-function relationships in all of biology and allow scaling single fibre mechanical properties to whole muscle properties based on the muscle's architecture. This physiological relationship has only been validated in small animals but is often extrapolated to human muscles, which are orders of magnitude larger. We leverage a unique surgical technique in which a human gracilis muscle is transplanted from the thigh to the arm to restore elbow flexion after brachial plexus injury, aiming to directly measure muscles properties in situ and test directly the architectural scaling predictions. Using these direct measurements, we establish human muscle fibre-specific tension of ∼170kPa. Furthermore, we show that the gracilis muscle actually functions as a muscle with relatively short fibres acting in parallel vs. long fibres as previously assumed based on traditional anatomical models.

Electrical stimulation to promote muscle and motor unit force and endurance after spinal cord injury.

Fatigue is a common feature of paralysed skeletal muscle, hindering performance when subjected to functional electrical stimulation (ES) for movement. We asked whether (1) 20Hz ES for 5% of each day (2.5s on and 2.5s off for 3h) increases tibialis anterior and medial gastrocnemius muscle and motor unit (MU) endurance after paralysis by hemisection and deafferentation (HSDA), and (2) muscle length or loading affects their isometric contractile properties. The daily 5% ES increased muscle endurance, largely independent of muscle length or loading, but to a lesser extent than the daily 50% ES (2.5s on and 2.5s off for 24h). The former was effective in counteracting the decline and slowing of muscle force promoted by the 50% ES. The altered muscle properties were confirmed at the MU level in final experiments once the properties had plateaued. Fast-fatigable MUs were converted to fatigue-intermediate and -resistant MUs that finally comprised ∼80% as compared to ∼10% of the total MU number in the daily 5% ES and the control normal groups, respectively. We conclude that the daily 5% ES regimen counteracts the fatigue of paralysed muscle without compromising contractile force, and thereby, is effective in conditioning muscle for effective movement. KEY POINTS: We asked whether 20Hz electrical stimulation (ES) for 5% of each day (2.5s on and 2.5s off for 3 h; 5% ES) preserves medial gastrocnemius and tibialis anterior muscle and MU isometric contractile forces and increases their endurance after paralysis. Daily 5% ES promoted increased muscle endurance irrespective of the muscle length or loading but to a lesser extent than daily 50% ES (20Hz ES 2.5s on and 2.5s off for 24 h). 5% ES was effective in counteracting decline and slowing of muscle force that resulted from 50% ES. Motor units (MUs) were converted from fast fatigable to fatigue intermediate and resistant MUs, comprising ∼80% as compared to ∼10% in the control normal groups. We conclude that the 5% ES regimen counteracts the fatigue of paralysed muscle without compromising contractile force, and thereby is effective in conditioning the muscle for effective movement.

Effects of long-term treatment with dietary theobromine on rat skeletal muscles.

Plastic changes of skeletal muscles, such as hypertrophy and atrophy, are dependent on physiological activities and regulated by a variety of signaling pathways, including cyclic adenosine monophosphate (cAMP) pathway. The cAMP inducing agents, such as the β2-adrenergic agonist clenbuterol, are known to induce muscle hypertrophy, and has been reported to induce slow-to-fast transitions in rat soleus muscle. Theobromine, one of the active components of cacao, functions as an inhibitor of phosphodiesterase and increases cAMP. This study hypothesized that theobromine, like clenbuterol, can induce muscle hypertrophy and influence contractile properties. Male Wistar rats were fed a normal diet or a diet containing 0.05% theobromine for 20weeks. Using biochemical, anatomical, and physiological techniques, effects of dietary theobromine on skeletal muscles (soleus, extensor digitorum longus, plantaris, and gastrocnemius) were examined. There were no significant differences in body weight, serum levels of proteins and lipids, muscle weights, dry/wet ratio of muscle weights, mitochondrial oxidation enzyme activity of muscles, isometric contractile properties of muscles, and muscle fatigue between control and theobromine-fed rats. Quantitative analysis of mRNA, however, revealed upregulation of myosin heavy chain 2x and myogenic differentiation 1, as previously reported in clenbuterol-treated muscles. The long-term theobromine (0.05%) diet in rats had no effect in inducing muscle hypertrophy and in changing contractile properties, although it had some similar effects of clenbuterol on muscle gene expression.

Early movement restriction deteriorates motor function and soleus muscle physiology

Children with low physical activity and interactions with environment experience atypical sensorimotor development and maturation leading to anatomical and functional disorganization of the sensorimotor circuitry and also to enduring altered motor function. Previous data have shown that postnatal movement restriction in rats results in locomotor disturbances, functional disorganization and hyperexcitability of the hind limb representations in the somatosensory and motor cortices, without apparent brain damage. Due to the reciprocal interplay between the nervous system and muscle, it is difficult to determine whether muscle alteration is the cause or the result of the altered sensorimotor behavior (Canu et al., 2019). In the present paper, our objectives were to evaluate the impact of early movement restriction leading to sensorimotor restriction (SMR) during development on the postural soleus muscle and on sensorimotor performance in rats, and to determine whether changes were reversed when typical activity was resumed. Rats were submitted to SMR by hind limb immobilization for 16 h / day from birth to postnatal day 28 (PND28). In situ isometric contractile properties of soleus muscle, fiber cross sectional area (CSA) and myosin heavy chain content (MHC) were studied at PND28 and PND60. In addition, the motor function was evaluated weekly from PND28 to PND60. At PND28, SMR rats presented a severe atrophy of soleus muscle, a decrease in CSA and a force loss. The muscle maturation appeared delayed, with persistence of neonatal forms of MHC. Changes in kinetic properties were moderate or absent. The Hoffmann reflex provided evidence for spinal hyperreflexia and signs of spasticity. Most changes were reversed at PND60, except muscle atrophy. Functional motor tests that require a good limb coordination, i.e. rotarod and locomotion, showed an enduring alteration related to SMR, even after one month of ‘typical’ activity. On the other hand, paw withdrawal test and grip test were poorly affected by SMR whereas spontaneous locomotor activity increased over time. Our results support the idea that proprioceptive feedback is at least as important as the amount of motor activity to promote a typical development of motor function. A better knowledge of the interplay between hypoactivity, muscle properties and central motor commands may offer therapeutic perspectives for children suffering from neurodevelopmental disorders.

Mechanics of dystrophin deficient skeletal muscles in very young mice and effects of age.

The MDX mouse is an animal model of Duchenne muscular dystrophy, a human disease marked by an absence of the cytoskeletal protein, dystrophin. We hypothesized that 1) dystrophin serves a complex mechanical role in skeletal muscles by contributing to passive compliance, viscoelastic properties, and contractile force production and 2) age is a modulator of passive mechanics of skeletal muscles of the MDX mouse. Using an in vitro biaxial mechanical testing apparatus, we measured passive length-tension relationships in the muscle fiber direction as well as transverse to the fibers, viscoelastic stress-relaxation curves, and isometric contractile properties. To avoid confounding secondary effects of muscle necrosis, inflammation, and fibrosis, we used very young 3-wk-old mice whose muscles reflected the prefibrotic and prenecrotic state. Compared with controls, 1) muscle extensibility and compliance were greater in both along fiber direction and transverse to fiber direction in MDX mice and 2) the relaxed elastic modulus was greater in dystrophin-deficient diaphragms. Furthermore, isometric contractile muscle stress was reduced in the presence and absence of transverse fiber passive stress. We also examined the effect of age on the diaphragm length-tension relationships and found that diaphragm muscles from 9-mo-old MDX mice were significantly less compliant and less extensible than those of muscles from very young MDX mice. Our data suggest that the age of the MDX mouse is a determinant of the passive mechanics of the diaphragm; in the prefibrotic/prenecrotic stage, muscle extensibility and compliance, as well as viscoelasticity, and muscle contractility are altered by loss of dystrophin.

Dietary nitrate supplementation increases diaphragm peak power in old mice.

Respiratory muscle function declines with ageing, contributing to breathing complications in the elderly. Here we report greater in vitro respiratory muscle contractile function in old mice receiving supplemental NaNO3 for 14days compared with age-matched controls. Myofibrillar protein phosphorylation, which enhances contractile function, did not change in our study - a finding inconsistent with the hypothesis that this post-translational modification is a mechanism for dietary nitrate to improve muscle contractile function. Nitrate supplementation did not change the abundance of calcium-handling proteins in the diaphragm of old mice, in contrast with findings from the limb muscles of young mice in previous studies. Our findings suggest that nitrate supplementation enhances myofibrillar protein function without affecting the phosphorylation status of key myofibrillar proteins. Inspiratory muscle (diaphragm) function declines with age, contributing to ventilatory dysfunction, impaired airway clearance, and overall decreased quality of life. Diaphragm isotonic and isometric contractile properties are reduced with ageing, including maximal specific force, shortening velocity and peak power. Contractile properties of limb muscle in both humans and rodents can be improved by dietary nitrate supplementation, but effects on the diaphragm and mechanisms behind these improvements remain poorly understood. One potential explanation underlying the nitrate effects on contractile properties is increased phosphorylation of myofibrillar proteins, a downstream outcome of nitrate reduction to nitrite and nitric oxide. We hypothesized that dietary nitrate supplementation would improve diaphragm contractile properties in aged mice. To test our hypothesis, we measured the diaphragm function of old (24months) mice allocated to 1mm NaNO3 in drinking water for 14days (n=8) or untreated water (n=6). The maximal rate of isometric force development (∼30%) and peak power (40%) increased with nitrate supplementation (P<0.05). There were no differences in the phosphorylation status of key myofibrillar proteins and abundance of Ca2+-release proteins in nitrate vs. control animals. In general, our study demonstrates improved diaphragm contractile function with dietary nitrate supplementation and supports the use of this strategy to improve inspiratory function in ageing populations. Additionally, our findings suggest that dietary nitrate improves diaphragm contractile properties independent of changes in abundance of Ca2+-release proteins or phosphorylation of myofibrillar proteins.

Abstract 69: Cardiac Fibrosis Induced by Mkk6-p38 Activation Alters Myocardial Stiffness and Myofilament Function

Heart failure with preserved ejection fraction (HFpEF) accounts for at least half of the patients with heart failure, but to date there are no proven therapies for these patients. Cardiac fibrosis and increased collagen content plays an important role in the pathophysiology of HFpEF. Studies in human HFpEF patients have indicated increased cardiac fibrosis, myocardial stiffness and myofilament calcium sensitivity. However, it is unclear how and if cardiac fibrosis directly alters myofilament function. In this study we used a new model of cardiac fibrosis genetically induced by the constitutive activation of MKK6-p38 (MKK6-Tg) signaling specifically in resident cardiac fibroblasts to determine the effect of fibrosis on myofilament stiffness and function. The MKK6-Tg mice develop interstitial and perivascular fibrosis in the heart accompanied with severe diastolic dysfunction, although systolic function remains normal. We measured the passive stiffness and isometric contractile properties of demembranated papillary muscle preparations. Our results show an increase in passive stiffness in demembranated papillary muscles from MKK6-Tg mice compared to NTG littermates when stretched to 24% from sarcomere length of 2.0 μM. Passive tension at SL=2.3 was significantly increased in MKK6-Tg mice (28±4 vs. 13±3 mN/mm 2 , P&lt;0.05) compared to NTG mice, which was ascribed to changes in Titin isoform as well as extracellular matrix composition. The MKK6-Tg mice also show an increase in Ca 2+ sensitivity (pCa 50 =5.72±0.05 vs. 5.56±0.02, P&lt;0.05). This was associated with an increased rate of force redevelopment in MKK6-Tg mice compared to NTG littermates (k TR =35±8 vs. 22±3 s -1 ). Towards understanding the mechanism of these myofilament changes we performed phosphate affinity electrophoresis and saw decreased phosphorylation of both Troponin I and Troponin T in MKK6-Tg hearts. There was no change in troponin I phosphorylation level at Ser23/24, indicating role of none-PKA mediated phosphorylation in mechanical changes seen in this model.

Early Life Exposure to Chronic Intermittent Hypoxia Primes Increased Susceptibility to Hypoxia-Induced Weakness in Rat Sternohyoid Muscle during Adulthood.

Intermittent hypoxia is a feature of apnea of prematurity (AOP), chronic lung disease, and sleep apnea. Despite the clinical relevance, the long-term effects of hypoxic exposure in early life on respiratory control are not well defined. We recently reported that exposure to chronic intermittent hypoxia (CIH) during postnatal development (pCIH) causes upper airway muscle weakness in both sexes, which persists for several weeks. We sought to examine if there are persistent sex-dependent effects of pCIH on respiratory muscle function into adulthood and/or increased susceptibility to re-exposure to CIH in adulthood in animals previously exposed to CIH during postnatal development. We hypothesized that pCIH would cause long-lasting muscle impairment and increased susceptibility to subsequent hypoxia. Within 24 h of delivery, pups and their respective dams were exposed to CIH: 90 s of hypoxia reaching 5% O2 at nadir; once every 5 min, 8 h per day for 3 weeks. Sham groups were exposed to normoxia in parallel. Three groups were studied: sham; pCIH; and pCIH combined with adult CIH (p+aCIH), where a subset of the pCIH-exposed pups were re-exposed to the same CIH paradigm beginning at 13 weeks. Following gas exposures, sternohyoid and diaphragm muscle isometric contractile and endurance properties were examined ex vivo. There was no apparent lasting effect of pCIH on respiratory muscle function in adults. However, in both males and females, re-exposure to CIH in adulthood in pCIH-exposed animals caused sternohyoid (but not diaphragm) weakness. Exposure to this paradigm of CIH in adulthood alone had no effect on muscle function. Persistent susceptibility in pCIH-exposed airway dilator muscle to subsequent hypoxic insult may have implications for the control of airway patency in adult humans exposed to intermittent hypoxic stress during early life.

Muscle-specific deletion of exons 2 and 3 of the IL15RA gene in mice: effects on contractile properties of fast and slow muscles.

Interleukin-15 (IL-15) is a putative myokine hypothesized to induce an oxidative skeletal muscle phenotype. The specific IL-15 receptor alpha subunit (IL-15Rα) has also been implicated in specifying this contractile phenotype. The purposes of this study were to determine the muscle-specific effects of IL-15Rα functional deficiency on skeletal muscle isometric contractile properties, fatigue characteristics, spontaneous cage activity, and circulating IL-15 levels in male and female mice. Muscle creatine kinase (MCK)-driven IL-15Rα knockout mice (mIl15ra(fl/fl)/Cre(+)) were generated using the Cre-loxP system. We tested the hypothesis that IL-15Rα functional deficiency in skeletal muscle would increase resistance to contraction-induced fatigue, cage activity, and circulating IL-15 levels. There was a significant effect of genotype on the fatigue curves obtained in extensor digitorum longus (EDL) muscles from female mIl15ra(fl/fl)/Cre(+) mice, such that force output was greater during the repeated contraction protocol compared with mIl15ra(fl/fl)/Cre(-) control mice. Muscles from female mIl15ra(fl/fl)/Cre(+) mice also had a twofold greater amount of the mitochondrial genome-specific COXII gene compared with muscles from mIl15ra(fl/fl)/Cre(-) control mice, indicating a greater mitochondrial density in these skeletal muscles. There was a significant effect of genotype on the twitch:tetanus ratio in EDL and soleus muscles from mIl15ra(fl/fl)/Cre(+) mice, such that the ratio was lower in these muscles compared with mIl15ra(fl/fl)/Cre(-) control mice, indicating a pro-oxidative shift in muscle phenotype. However, spontaneous cage activity was not different and IL-15 protein levels were lower in male and female mIl15ra(fl/fl)/Cre(+) mice compared with control. Collectively, these data support a direct effect of muscle IL-15Rα deficiency in altering contractile properties and fatigue characteristics in skeletal muscles.

The β2 -adrenoceptor agonist terbutaline recovers rat pharyngeal dilator muscle force decline during severe hypoxia.

Obstructive sleep apnoea syndrome (OSAS) is a debilitating condition characterized by recurrent occlusions of the pharyngeal airway during sleep accompanied by arterial hypoxaemia. Upper airway muscle dysfunction is implicated in the pathophysiology of OSAS. Pharmacological agents that improve muscle contractile and endurance properties may have therapeutic value. We tested the hypothesis that the β(2) -adrenoceptor agonist terbutaline improves rat sternohyoid muscle performance especially during hypoxic stress. Isometric contractile and endurance properties were examined ex vivo in Krebs solution at 35°C. Muscles were incubated in tissue baths under hyperoxic (95% O(2) /5% CO(2)) conditions in the absence (control) or presence of the β(2) -adrenoceptor agonist terbutaline (1 μM). In additional experiments under hypoxic (95% N(2) /5% CO(2)) conditions, the effects of terbutaline were examined in the presence of the β-adrenoceptor antagonist propranolol (1 μM). Hypoxia significantly impaired sternohyoid force production. Terbutaline completely recovered hypoxic depression of force, an effect that was blocked by co-application with propranolol. The β(2) -adrenoceptor agonist terbutaline completely recovers hypoxic depression of upper airway muscle force. β(2) -adrenoceptor agonists warrant investigation in animal models of OSAS reporting upper airway and diaphragm muscle dysfunction.

Muscle-derived decellularised extracellular matrix improves functional recovery in a rat latissimus dorsi muscle defect model

Craniofacial maxillary injuries represent nearly 30% of all battlefield wounds, often involving volumetric muscle loss (VML). The physical loss of muscle results in functional deficits and cosmetic disfigurement. Although surgical solutions are limited, advances in biomaterials offer great promise for the restoration of form and function following VML. The primary purpose of this study was to determine whether muscle function could be restored in a novel VML rat model using muscle-derived extracellular matrix (M-ECM). Ten percent of the mass of the latissimus dorsi (LD) was excised. Three groups were examined: 1) no repair of defect (DEF), 2) repair with M-ECM and 3) sham (all procedures except muscle excision). Four and 8 weeks post-surgery, the isometric contractile properties of the LD were assessed in situ and selected histological properties were evaluated. The defect resulted in an initial reduction in peak isometric force (Po) of 30%. At 8 weeks the difference between DEF and sham was 20.5%. At the same time, M-ECM was only 8.4% below sham. Although the histological analysis revealed a narrow, but well-formed band of muscle running along the middle of the M-ECM, it was judged to be too small to account for the observed improvement in muscle force. Repair of VML with M-ECM can dramatically improve muscle function independent of muscle regeneration by providing a physical bridge that accommodates force transmission across the injury site. This method of repair may provide an easily translatable surgical method for selected forms of VML.

Sternohyoid and diaphragm muscle form and function during postnatal development in the rat

What is the central question of this study? Co-ordinated activity of the thoracic pump and pharyngeal dilator muscles is critical for maintaining airway calibre and respiratory homeostasis. Whilst postnatal maturation of the diaphragm has been well characterized, surprisingly little is known about the developmental programme in the airway dilator muscles. What is the main finding and its importance? Developmental increases in force-generating capacity and fatigue in the sternohyoid and diaphragm muscles are attributed to a maturational shift in muscle myosin heavy chain phenotype. This maturation is accelerated in the sternohyoid muscle relative to the diaphragm and may have implications for the control of airway calibre in vivo. The striated muscles of breathing, including the thoracic pump and pharyngeal dilator muscles, play a critical role in maintaining respiratory homeostasis. Whilst postnatal maturation of the diaphragm has been well characterized, surprisingly little is known about the developmental programme in airway dilator muscles given that co-ordinated activity of both sets of muscles is needed for the maintenance of airway calibre and effective pulmonary ventilation. The form and function of sternohyoid and diaphragm muscles from Wistar rat pups [postnatal day (PD) 10, 20 and 30] was determined. Isometric contractile and endurance properties were examined in tissue baths containing Krebs solution at 35°C. Myosin heavy chain (MHC) isoform composition was determined using immunofluorescence. Muscle oxidative and glycolytic capacity was assessed by measuring the activities of succinate dehydrogenase and glycerol-3-phosphate dehydrogenase using semi-quantitative histochemistry. Sternohyoid and diaphragm peak isometric force and fatigue increased significantly with postnatal maturation. Developmental myosin disappeared by PD20, whereas MHC2B areal density increased significantly from PD10 to PD30, emerging earlier and to a much greater extent in the sternohyoid muscle. The numerical density of fibres expressing MHC2X and MHC2B increased significantly during development in the sternohyoid. Diaphragm succinate dehydrogenase activity and sternohyoid glycerol-3-phosphate dehydrogenase activity increased significantly with age. Developmental increases in force-generating capacity and fatigue in the sternohyoid and diaphragm muscles are attributed to a postnatal shift in muscle MHC phenotype. The accelerated maturation of the sternohyoid muscle relative to the diaphragm may have implications for the control of airway calibre in vivo.

Upper Airway Dilator Muscle Weakness Following Intermittent and Sustained Hypoxia in the Rat: Effects of a Superoxide Scavenger

Obstructive sleep apnoea syndrome (OSAS) is a common disorder associated with upper airway muscle dysfunction. Agents that improve respiratory muscle performance may have considerable therapeutic value. We examined the effects of acute exposure to sustained and intermittent hypoxia on rat pharyngeal dilator muscle function. Additionally, we sought to test the efficacy of antioxidant treatment in ameliorating or preventing hypoxia-related muscle dysfunction. Isometric contractile and endurance properties of isolated rat sternohyoid muscle bundles were examined at 35 °C in vitro. Muscle bundles were exposed to one of four gas treatments: hyperoxia (control), sustained hypoxia (SH), intermittent hypoxia (IH) or hypoxia/re-oxygenation (HR), in the absence or presence of the superoxide scavenger--Tempol (10 mM). Stress-frequency relationship was determined in response to electrical stimulation (10-100 Hz in increments of 10-20 Hz, train duration: 300 ms). Muscle performance was also assessed during repetitive muscle stimulation (40 Hz, 300 ms every 2 s for 2.5 min). Compared to control, IH and HR treatments significantly decreased sternohyoid muscle force. The negative inotropic effect of the two gas protocols was similar, but both were of lesser magnitude than the effects of SH. SH, but not IH and HR, increased muscle fatigue. Tempol significantly increased sensitivity to stimulation in all muscle preparations and caused a leftward shift in the stress-frequency relationship of IH and SH treated muscles. Tempol did not ameliorate sternohyoid muscle fatigue during SH. We conclude that Tempol increases upper airway muscle sensitivity to stimulation but only modestly ameliorates respiratory muscle weakness during intermittent and sustained hypoxic conditions in vitro. Respiratory muscle fatigue during sustained hypoxia appears unrelated to oxidative stress.

The effects of hypoventilation on the physiological and biochemical properties of hindlimb muscles

The purpose of this study was to determine physiological and biochemical properties of hindlimb muscles after bilateral phrenic nerve denervation (DNV).Male Wistar rats (10 wk) underwent DNV (n=18) by cutting the phrenic nerve at the cervical level under the stereomicroscope or sham surgery (n=18, CON). Analyses were performed 4, 8 and 12 wks after surgery. The myosin heavy chain (MHC) isoform profile and in vitro isometric contractile properties of the soleus (SOL) and extensor digitram longus (EDL) were analyzed.DNV induced changes in SpO2 characteristic of hypoventilation syndromes. After 12 wks, increases in MHC 1 and decreases in MHC 2a (both p&lt;0.05) were observed in the DNV SOL. Increases in MHC 1 (7%), MHC 2a (7%) and MHC 2d (9%) and decreases in MHC 2b (23%) (all p&lt;0.05) were measured in the DNV EDL. After 8 wks and 12 wks of DNV, the peak twitch force of EDL decreased (~30%) and after 12 wks the fatigue index increased (19%) (all p&lt;0.05). However, DNV did not affect the contractile properties of SOL.In conclusion chronically low SpO2 and decreased hindlimb contractile activity induced by DNV resulted in a unique fast‐to‐slow MHC shift in SOL and EDL. This was accompanied by enhanced fatigue resistance and impaired contractile function in the EDL but not the SOL.This work was supported by Grants‐in‐Aid for Scientific Research from the Japanese Ministry of Science and Technology, no 24500628.

Mechanical ventilation impairs sarcomeric protein function in rat diaphragm single fibers

Mechanical ventilation (MV) is a widely used clinical intervention for patients unable to maintain alveolar ventilation. Although a life‐saving measure for patients suffering from respiratory failure, MV promotes diaphragm atrophy and contractile dysfunction. The cellular basis of contractile dysfunction remains unclear. The mechanisms of contractile dysfunction are important to understand because MV‐induced diaphragm weakness can delay the weaning process. We studied triton‐permeabilized diaphragm single fibers from rats after 12 hrs of spontaneous breathing (SB) and MV (n = 4/group). Key parameters of isometric contractile properties were maximal specific tetanic force (sPo), rate constant of tension redevelopment (ktr), and calcium sensitivity (pCa50). In type I diaphragm fibers, sPo did not differ between groups. MV slowed ktr by ~6% and decreased the pCa50 such that calcium concentration eliciting 50% sPo increased by ~20%. In type II diaphragm fibers, MV diminished sPo by ~32% and slowed ktr by ~22%. However, pCa50 did not differ between groups. Together, our data suggest that MV impairs diaphragm sarcomeric protein function as evidenced by diminished specific force and calcium sensitivity, and slowing of cross bridge cycling kinetics in membrane‐permeabilized single fibers.Funding support: NIH R00‐HL098453 (LFF) and HL087839 (SKP)

The effects of hypoventilation disorder on physiological and biochemical properties of the hindlimb muscles

The purpose of this study was to determine the physiological and biochemical properties of hindlimb muscles after hypoventilation (HPO) induced by bilateral phrenic nerve denervation. Male Wistar rats (10 weeks-old) underwent HPO by the phrenic nerve denervation at the cervical level or sham surgery. Analyses were performed 4, 8, and 12 weeks after the surgery. The myosin heavy chain (MHC) isoform profile and in vitro isometric contractile properties of the soleus (SOL) and extensor digitrum longus muscles (EDL) were analyzed. From the postoperative period, HPO induced characteristic changes in SpO2 such as hypoventilation disorder. After 12 weeks, significant increases in MHC1 and significant decreases in MHC2a were observed in the MHC isoform composition in SOL. Moreover, significant increases in MHC2a and significant decreases in MHC2b were also observed in the MHC isoform composition in EDL muscles in the HPO compared with sham (SHM) group. In our study, the tidal volume after unilateral phrenic nerve denervation decreased by approximately 12%, and that after bilateral phrenic nerve denervation decreased by approximately 35%. We concluded that the reduction in behavioral activity levels in the HPO group may have resulted in changes of the peripheral skeletal muscles as a result of disuse atrophy.

Rapid Onset of Specific Diaphragm Weakness in a Healthy Murine Model of Ventilator-induced Diaphragmatic Dysfunction

Controlled mechanical ventilation is associated with ventilator-induced diaphragmatic dysfunction, which impedes weaning from mechanical ventilation. To design future clinical trials in humans, a better understanding of the molecular mechanisms using knockout models, which exist only in the mouse, is needed. The aims of this study were to ascertain the feasibility of developing a murine model of ventilator-induced diaphragmatic dysfunction and to determine whether atrophy, sarcolemmal injury, and the main proteolysis systems are activated under these conditions. Healthy adult male C57/BL6 mice were assigned to three groups: (1) mechanical ventilation with end-expiratory positive pressure of 2-4 cm H2O for 6 h (n=6), (2) spontaneous breathing with continuous positive airway pressure of 2-4 cm H2O for 6 h (n=6), and (3) controls with no specific intervention (n=6). Airway pressure and hemodynamic parameters were monitored. Upon euthanasia, arterial blood gases and isometric contractile properties of the diaphragm and extensor digitorum longus were evaluated. Histology and immunoblotting for the main proteolysis pathways were performed. Hemodynamic parameters and arterial blood gases were comparable between groups and within normal physiologic ranges. Diaphragmatic but not extensor digitorum longus force production declined in the mechanical ventilation group (maximal force decreased by approximately 40%) compared with the control and continuous positive airway pressure groups. No histologic difference was found between groups. In opposition with the calpains, caspase 3 was activated in the mechanical ventilation group. Controlled mechanical ventilation for 6 h in the mouse is associated with significant diaphragmatic but not limb muscle weakness without atrophy or sarcolemmal injury and activates proteolysis.

Structural and Functional Properties of an Upper Airway Dilator Muscle in Aged Obese Male Rats

Background: Age, obesity and male sex are risk factors for the development of obstructive sleep apnoea syndrome. Objective: We examined structural and functional properties of the sternohyoid muscle in young lean and aged obese male rats. We hypothesized that the aged muscle would be vulnerable to oxidative stress (hypoxia). Methods: Isometric contractile and endurance properties of the sternohyoid muscle were assessed in vitro with or without the superoxide scavenger Tempol (10 mM). Muscle fibre size and density were determined by myosin heavy chain immunofluorescence. Succinate dehydrogenase (SDH) and glycerol-3- phosphate dehydrogenase (GPDH) enzyme activities were determined. Results: Fibre hypertrophy, increased fast twitch (type 2X) fibre density, decreased SDH activity and increased GPDH activity, together with increased force and fatigue, were observed in aged obese muscles compared to young lean muscles. Tempol treatment increased strength and sensitivity to stimulation. Hypoxic depression of force was ameliorated by antioxidant treatment with equivalent effects in young lean and aged obese muscle. Conclusions: We conclude that the rat sternohyoid exhibits indefinite growth and is protected from oxidative stress as the animal ages.