A finite element model of the electrically stimulated human thigh: changes due to denervation and training

Abstract:  The complete denervation of muscles leads to changes in the muscle fibers as well as in the surrounding tissue. Concerning excitability the most important changes are reductions in fiber diameter, in muscle cross-sectional area, and in electrical conductivity of the muscle tissue. These changes can be partially reversed by intensive electrical stimulation.
Evaluation of a 3D finite element axial symmetric model of the human thigh shows that the training leads to a reduction in threshold values between 17 and 51 percent, depending on the position of the fiber in the thigh. Single parameter variation clarifies the influence of each of the different factors. The electrode position was found to be most effective with the electrodes as far apart from each other as possible. Due to (i) comparatively higher changes in potentials at the distal electrode; and (ii) variations in sodium channel dynamics, lowest threshold values can be reached with a hyperpolarizing first phase of the biphasic impulse at the distal electrode. The tissue of the denervated muscle is known to be highly inhomogeneous. Simulations demonstrate that the related irregularities in the field can actually initiate fiber activation.
3D finite element simulations show the overall positive effects of FES on muscle tissue, especially an improved excitability of the muscle fibers. Furthermore the method gives an insight into the relations between potential distribution, electrode position, geometric effects, and muscle fiber activation that cannot be obtained by measurements.     

Körperliches Training und zelluläre Anpassung des Muskels

Resistance training and to a lesser extent endurance training are capable of enhancing protein synthesis in skeletal muscle via various signaling pathways. Additionally, the expression of muscle fiber types responds to different regimes of training stimuli and immobilization as characterized by changes in myosin heavy chain isoforms (I?IIA?IIX). Eccentric resistance training has been shown to be highly efficient in inducing sarcomeric protein assembly in the longitudinal orientation of muscle cells. However, concentric contractions lead to a hypertrophic response (increased fiber diameter) in muscle which can still be activated in old age. The central signaling pathway to mediate the elevation of protein synthesis in response to training is the mTOR pathway, which is also stimulated by free amino acids. Moreover, adaptation to endurance training is mediated by the calcium-calcineurin-NFATc1 pathway which is strongly activated by the calcium transients involved in the muscle contraction process. High contraction frequency and long duration of training sessions are essential for activation and maintenance of fiber type I expression as well as for induction of transformation of type II into type I fibers. Endurance training sessions should therefore be longer than 30 min and dominated by periods of high frequency contractions. A further factor in the muscular response to training includes the recruitment and integration of satellite cells into muscle fibers. Satellite cells can respond to muscular stretch, activity and injury with increased proliferation and can later be integrated into muscle fibers. Therefore, new myonuclei are available to enhance mRNA synthesis and protein expression in muscle cells. New understanding of the cellular mechanisms of signal transduction in muscle in response to training, bed rest and ageing will help to optimize training and interventions in an ageing population.     

Fiber-type composition and fiber size of the human cricopharyngeal muscle and the pharyngeal constrictor muscle

Background:  Despite a similar density of nicotinic acetylcholine receptors, the upper esophageal sphincter is sensitive to partial neuromuscular block, whereas the pharyngeal constrictor muscle is more resistant. In order to postulate possible mechanisms behind this difference in pharmacological response, basic knowledge of morphological and physiological features of these muscles is needed. The aim of this study was to compare the muscle fiber-type composition, the size and the morphology of the muscle fibers of the cricopharyngeal muscle, the main component of the upper esophageal sphincter, with that of the pharyngeal constrictor muscle.
Methods:  Muscle specimens were obtained from five patients undergoing surgery with laryngectomy. Muscle fiber type was determined by myosin heavy chain immunohistochemistry and the muscle fiber cross-sectional area was measured for each fiber type by planimetry. Morphology of muscle fibers was evaluated by histochemistry.
Results:  The muscle fiber cross-sectional area was generally smaller in the cricopharyngeal muscle compared with the pharyngeal constrictor muscle ( P <  0.001). The composition of fiber types showed a large interindividual variability with no distinct difference between the studied muscles. Aberrant histological features were common in both the cricopharyngeal muscle and the pharyngeal constrictor muscle.
Conclusion:  The main morphological difference between the neuromuscular blocking agents sensitive cricopharyngeal muscle and the more resistant pharyngeal constrictor muscle is a uniformly smaller size of contributing fiber types in the cricopharyngeal muscle than in the pharyngeal constrictor muscle. The muscle fiber-type composition does not differ between the two studied muscles.     

Adaptation of skeletal muscle to resistance training

This paper was prepared in partial fulfillment for Doctoral Degree in Physical Therapy at Texas Woman's University in Houston, TX. Resistance training is frequently used in rehabilitation to improve musculoskeletal function. The increased ability of skeletal muscle to generate force following resistance training results from two important changes: 1) the adaptation of the muscle fiber, and 2) the extent to which the motor unit can activate the muscle (neural adaption). The purpose of this article is to provide a review of research investigating the effects of resistance training on muscle fibers and on nervous system input. Muscle fiber adaptations caused by resistance training include increased cross-sectional area of the muscle (hypertrophy, hyperplasia, or both), selective hypertrophy of fast twitch fibers, decreased or maintained mitochondrial number and capillary density of muscle, and possible changes in energy sources. Changes in nervous system input resulting from resistance training include recruitment of an increased number and firing rate of motor units, increased reflex potentiation, and improved synchronization. An understanding of the adaptations occurring in muscle in response to resistance training provides a fundamental basis for which appropriate clinical exercise training programs can be developed for the rehabilitation of patients. J Orthop Sports Phys Ther 1990;12(6):248-255.     

A novel approach to simulate Hodgkin-Huxley-like excitation with COMSOL Multiphysics

Abstract:  A proof of concept for the evaluation of external nerve and muscle fiber excitation with the finite element software COMSOL Multiphysics, formerly known as FEMLAB, is presented. This software allows the simultaneous solution of fiber excitation by 1D models of the Hodgkin-Huxley type which are embedded in a volume conductor where the electric field is mainly dominated by the electrode currents. This way the presented bidomain model includes the interaction between electrode currents and transmembrane currents during the excitation process. Especially for direct muscle fiber stimulation (cardiac muscle, denervated muscle) the effects from secondary currents from large populations of excited fibers seem to be significant. The method has many applications, for example, the relation between stimulus parameters and fiber recruitment can be analyzed.     

Knee loading dynamically alters intramedullary pressure in mouse femora

Dynamic mechanical loads have been known to stimulate bone formation. Many biophysical factors such as number of daily loading cycles, bone strain, strain-induced interstitial fluid flow, molecular transport, and modulation of intramedullary pressure have been considered as potential mediators in mechanotransduction of bone. Using a knee loading modality that enhances anabolic responses in mouse hindlimb, we addressed a question: Do oscillatory loads applied to the knee induce dynamic alteration of intramedullary pressure in the femoral medullary cavity? To answer this question, mechanical loads were applied to the knee with a custom-made piezoelectric loader and intramedullary pressure in the femoral medullary cavity was measured with a fiber optic pressure sensor. We observed that in response to sinusoidal forces of 0.5 Hz and 10 Hz, pressure amplitude increased up to 4-N loads and reached a plateau at 130 Pa. This amplitude significantly decreased with a loading frequency above 20 Hz. To confirm alteration of intramedullary pressure, real-time motion of microparticles in a glass tube inserted to the femoral medullary cavity ex vivo was visualized. Taken together, these data reveal that knee loading dynamically alters intramedullary pressure as a function of loading intensities and frequencies.     

Real-time Monitoring of Force Response Measured in Mechanically Stimulated Tissue-Engineered Cartilage

Abstract:  Mechanical stimulation improves tissue-engineered cartilage development both in terms of biochemical composition and structural properties. However, the link between the compositional changes attributed to mechanical stimulation and the changing structural properties of the engineered cartilage is poorly understood. We hypothesize that transient events associated with construct stiffening can be documented and used to understand milestones in construct development. To do this, we designed and built a mechanical stimulation bioreactor that can continuously record the force response of the engineered construct in real time. This study documents the transient changes of the stiffness of tissue-engineered cartilage constructs over the first 14 days of their development under cyclic loading. Compressive strain stimulation (15%, 1 Hz) was applied to poly(ethylene glycol) (PEG) hydrogels seeded with primary articular chondrocytes. The average compressive modulus of strain-stimulated constructs was 12.7 ± 1.45 kPa after 2 weeks, significantly greater ( P  < 0.01) than the average compressive moduli of both unstimulated constructs (10.7 ± 0.94 kPa) and nonviable stimulated constructs (11.2 ± 0.91 kPa). The system was able to document that nearly all of the stiffness increase occurred over the last 2 days of the experiment, where live-cell constructs demonstrated a rapid 20% increase in force response. The system's ability to track significant increases in stiffness over time was also confirmed by Instron testing. These results present a novel view of the early mechanical development of tissue-engineering cartilage constructs and suggest that the real-time monitoring of force response may be used to noninvasively track the development of engineered tissue.     

Low-dose estrogen treatment suppresses periosteal bone formation in response to mechanical loading

Estrogen and exercise influence cortical bone formation. Both affect bone during growth, but with complex interactions. We hypothesized that estrogen reduces the osteogenic response caused by exercise at the periosteal surface of bone, while it enhances bone formation on the endocortical surface. To test our hypothesis, 16 young (8 weeks old) male Sprague-Dawley rats were randomized into two groups: (1) low-dose 17- ethynylestradiol treatment + bone loading (EE₂) or (2) vehicle-treated + bone loading (vehicle). We applied controlled loading to the right ulna at a peak force of 17 N, 2 min/day, 3 days/week for 5 weeks to simulate exercise. The left nonloaded ulna served as an internal control for loading. Mechanical loading increased cortical area (7.7%) and bone mineral content (8%) in the vehicle-treated group (P < 0.05) but only slightly increased cortical area in the EE₂ group (P = 0.08). Histomorphometry showed 1 week of mechanical loading increased periosteal bone formation rate by 29% in the vehicle group and this response was reduced (P < 0.05) to only 15% in the EE₂ group. At the endocortical surface, there were no differences in the loading response between the vehicle and EE₂-treated groups. We conclude low-dose EE₂ suppresses the mechanical loading response on the periosteal surface of long bones, but had no effect on the loading response at the endocortical bone surface in growing male rats.     

The nephrotoxicity risk in rats subjected to heavy muscle activity

When the body is exposed to insults, the kidneys exhibit adaptive changes termed renal cytoresistance, characterized by cholesterol accumulation in the membranes of the tubule cells. However, heavy muscle activity has not yet been accepted as one of the stressors that could lead to cytoresistance. In order to study the renal functional characteristics of animals exposed to heavy muscle activity, rats were subjected to exhaustive treadmill exercise for 5 days and their data was compared to those of sedentary controls. It was found that in exercised rats, blood lactate, muscle citrate synthase and proximal tubule peroxynitrite levels were all elevated, suggesting the presence of oxidative stress in the proximal tubule segments. However, mean arterial pressure, renal blood flow, glomerular filtration rate, fractional excretion of sodium and potassium, and organic anion excretion remained normal. Despite unchanged blood cholesterol levels, cholesterol loading in the proximal tubule segments, especially the free form, and decreased lactate dehydrogenase release from cytoresistant proximal tubule segments indicated the development of renal cytoresistance. However, this resistance did not seem to have protected the kidneys as expected because organic anion accumulation associated with glycosuria and proteinuria, in addition to the elevated urinary cholesterol levels, all imply the presence of an impaired glomerular permeability and reabsorption in the proximal tubule cells. Therefore, we suggest that in response to heavy muscle activity the tubular secretion may remain intact, although cytoresistance in the proximal tubule cells may affect the tubular reabsorptive functions and basolateral uptake of substances. Thus, this differential sensitivity in the cytoresistance should be taken into account during functional evaluation of the kidneys.

Key points

• The cholesterol loading and decreased LDH release from PTSs isolated from exhausted rats indicate the heavy muscle activity induced renal cytoresistance.
• Heavy muscle activity-induced renal cytoresistance did not preserve the kidney functions.
• Organic anion accumulation as well as failure in the absorptive capacity of the tubule cells suggest the presence of some biochemical changes and elevated vulnerability of kidneys against nephrotoxic agents in rats subjected to heavy muscle activity.

Key words: Exercise, proximal tubule, cytoresistance, nephrotoxicity     

Skeletal muscle adaptations following spinal cord contusion injury in rat and the relationship to locomotor function: a time course study.

Experimental spinal cord injury (SCI) via contusion of moderate severity results in residual locomotor deficits, including a lack of coordination and trunk stability. Given that muscle contractile properties and fiber composition adapt to reduced neural input and/or weight bearing, contusion-induced locomotor deficits may reflect changes in hindlimb skeletal muscle. Therefore, we examined muscle adaptations during early (1 week), intermediate (3 week), and late (10 week) stages of motor recovery after moderate SCI. Forty-two Sprague Dawley rats underwent SCI via 1.1mm cord displacement with the OSU impact device or served as age and weight-matched or laminectomy controls. Subsets of rats had soleus (SOL) in vitro physiological testing or SOL and extensor digitorum longus (EDL) myosin heavy chain (MHC) fiber type analysis. At 1 week post-SCI during paralysis/paresis, a significant decrease in wet weight occurred in the plantaris, medial/lateral gastrocnemius (MG/LG), tibialis anterior, and SOL. Changes in contractile properties of the SOL did not accompany muscle wet weight changes. By 3 weeks, the loss of weight-bearing activity early after SCI induced significant decreases in SOL peak twitch and peak tetanic tension as well as significantly greater IIx MHC expression in the EDL. By 10 weeks post-SCI, after several weeks of weight supported stepping, muscle wet weight, contractile properties and MHC composition returned to baseline levels except for MG/LG atrophy. Thus, muscle plasticity appears to be extremely sensitive to locomotor deficits and their resolution after moderate spinal cord contusion.     

Use of premedication for intubation in tertiary neonatal units in the United Kingdom

Background:  Endotracheal intubation and laryngoscopy are frequently performed procedures in neonatal intensive care. These procedures represent profoundly painful stimuli and have been associated with laryngospasm, bronchospasm, hemodynamic changes, raised intracranial pressure and an increased risk of intracranial hemorrhage. These adverse changes can cause significant neonatal morbidity but may be attenuated by the use of suitable premedication.
Aims:  To evaluate current practices for premedication use prior to elective intubation in UK tertiary neonatal units.
Methods:  Telephone questionnaire survey of all 50 tertiary neonatal units in the UK.
Results:  Ninety percent of units report the routine use of sedation prior to intubation and 82% of units routinely use a muscle relaxant. Morphine was the most commonly used sedative and suxamethonium was the most commonly used muscle relaxant. Approximately half of the units also used atropine during intubation. Seventy seven percent of units had a written policy for premedication. Ten percent of the units did not routinely use any sedatives or muscle relaxants for elective intubation.
Conclusions:  In comparison with data from a 1998 survey, our study demonstrated an increase in the number of units that have adopted a written policy for premedication use, and in the number routinely using premedication drugs for elective intubation. There remains little consensus as to which drugs should be used and in what dose.     

The Relationship Between Activity Pattern and Muscle Adaptation in Skeletal Muscle

Muscle is highly plastic in terms of size (maximum force), speed, maximum power, and endurance. Well‐controlled studies in animals have shown that the adult skeletal muscle fiber has a remarkable ability to modify its gene expression so that with long‐term substantial changes in the daily activity pattern the contractile phenotype can be modified across the whole spectrum of fiber type found in control muscle. The contractile phenotype in this context includes the isoform content of myosin and therefore the maximum velocity of shortening, the mitochondrial content and therefore the specific force and aerobic capacity (endurance), and the calcium handling proteins and therefore the speed of activation and relaxation. With voluntary exercise in human subjects, similar responses are observed, although the degree of transformation is restricted by the practical limitations of exercise dosing to changes in mitochondrial activity and muscle size rather than the more profound changes in contractile protein isoform that can be induced with artificial activation over a substantial proportion of the day.     

Pulse pressure variation and stroke volume variation during different loading conditions in a paediatric animal model

Background: Previous studies in adult patients and animal models have demonstrated that pulse pressure variation (PPV) and stroke volume variation (SVV) can be used to predict the response to fluid administration. Currently, little information is available on the performance of these variables in infants and neonates. The aim of our study was to assess whether PPV and SVV can predict fluid responsiveness in an animal model and to investigate the influence of different tidal volumes applied.
Methods: PPV and SVV were monitored by pulse contour analysis in 19 anaesthetized and paralysed piglets during ventilation with tidal volumes ( V _T) of 5, 10 and 15 ml/kg both before and after fluid loading with 25 ml/kg of hydroxy-ethyl starch 6% (HES). Cardiac output was measured by pulmonary artery thermodilution and a positive response to HES infusion was defined as ≥20% increase in the stroke volume index (SVI).
Results: Before HES infusion, PPV and SVV were significantly greater during ventilation with a V _T of 10 and 15 ml/kg than during ventilation with a V _T of 5 ml/kg ( P <0.05). After HES infusion, only ventilation with V _T 15 ml/kg resulted in a significant increase in PPV and SVV. As assessed by receiver operating characteristic curve analysis, SVV during ventilation with V _T 10 ml/kg was the best predictor of a positive response to fluid loading (AUC=0.87).
Conclusions: In this paediatric animal model, we found that SVV during ventilation with 10 ml/kg was a sensitive and specific predictor of the response to fluid loading.     

Response of adipose tissue lipoprotein lipase to the cephalic phase of insulin secretion

Modulation of lipoprotein lipase (LPL) allows a tissue-specific partitioning of triglyceride-derived fatty acids, and insulin is a major modulator of its activity. The present studies were aimed to assess in rats the contribution of insulin to the response of adipose tissue and muscle LPL to food intake. Epididymal and retroperitoneal adipose LPL rose 65% above fasting values as early as 1 h after the onset of a 30-min high-carbohydrate meal, with a second activity peak 1 h later that was maintained for an additional 2 h. Soleus muscle LPL was decreased by 25% between 0.5 and 4 h after meal intake. The essential contribution of insulin to the LPL response to food intake was determined by preventing the full insulin response to meal intake by administration of diazoxide (150 mg/kg body wt, in the meal). The usual postprandial changes in adipose and muscle LPL did not occur in the absence of an increase in insulinemia. However, the early (60 min) increase in adipose tissue LPL was not prevented by the drug, likely because of the maintenance of the early centrally mediated phase of insulin secretion. In a subsequent study, rats chronically implanted with a gastric cannula were used to demonstrate that the postprandial rise in adipose LPL is independent of nutrient absorption and can be elicited by the cephalic (preabsorptive) phase of insulin secretion. Obese Zucker rats were used because of their strong cephalic insulin response. After an 8-h fast, rats were fed a liquid diet ad libitum (orally, cannula closed), sham fed (orally, cannula opened), or fed directly into the stomach via the cannula during 4 h. Insulinemia increased 10-fold over fasting levels in ad libitum- and intragastric-fed rats and threefold in sham-fed rats. Changes in adipose tissue LPL were proportional to the elevation in plasma insulin levels, demonstrating that the cephalic-mediated rise in insulinemia, in the absence of nutrient absorption, stimulates adipose LPL. These results demonstrate the central role of insulin in the postprandial response of tissue LPL, and they show that cephalically mediated insulin secretion is able to stimulate adipose LPL.     

Halothane induces calcium release from human skinned masseter muscle fibers

BACKGROUND: An increase in masseter muscle tone in response to halothane or succinylcholine anesthesia (or both) can be observed in healthy persons. Thus the authors compared the fiber-type halothane and succinylcholine sensitivities in human masseter and vastus lateralis muscles. METHODS: Masseter and vastus lateralis muscle segments were obtained from 13 and 9 healthy persons, respectively. After chemical skinning of a single fiber and loading the sarcoplasmic reticulum with Ca++ 0.16 microM solution, halothane (0.5-4 vol% bubbled in the incubating solution), succinylcholine (0.1 microM to 10 mM), or both sensitivities were defined as the concentration inducing more than 10% of the maximum tension obtained by application of 16 microM Ca++ solution. The myofilament response to Ca++ was studied with and without halothane by observing the isometric tension of skinned masseter fibers challenged with increasing concentrations of Ca++. Muscle fiber type was determined by the difference in strontium-induced tension measurements. RESULTS: A significant difference in halothane sensitivity was found between type 1 masseter fibers (0.6+/-0.2 vol%; mean +/- SD) versus type 1 (2.7+/-0.6 vol%) and type 2 vastus lateralis muscle (2.5+/-0.4 vol%). Succinylcholine did not induce Ca++ release by the sarcoplasmic reticulum. In the masseter muscle, 0.75 vol% halothane decreased the maximal activated tension by 40% but did not change the Ca++ concentration that yields 50% of the maximal tension. CONCLUSIONS: The very low halothane threshold for Ca++ release from the masseter muscle usually could be counteracted by a direct negative inotropic effect on contractile proteins. However, halothane may increase the sensitivity of the sarcoplasmic reticulum Ca++ release to succinylcholine-induced depolarization, leading to an increase in masseter muscle tone.     

Pharmacological inhibition of myostatin protects against skeletal muscle atrophy and weakness after anterior cruciate ligament tear

Anterior cruciate ligament (ACL) tears are among the most frequent knee injuries in sports medicine, with tear rates in the US up to 250,000 per year. Many patients who suffer from ACL tears have persistent atrophy and weakness even after considerable rehabilitation. Myostatin is a cytokine that directly induces muscle atrophy, and previous studies rodent models and patients have demonstrated an upregulation of myostatin after ACL tear. Using a preclinical rat model, our objective was to determine if the use of a bioneutralizing antibody against myostatin could prevent muscle atrophy and weakness after ACL tear. Rats underwent a surgically induced ACL tear and were treated with either a bioneutralizing antibody against myostatin (10B3, GlaxoSmithKline) or a sham antibody (E1‐82.15, GlaxoSmithKline). Muscles were harvested at either 7 or 21 days after induction of a tear to measure changes in contractile function, fiber size, and genes involved in muscle atrophy and hypertrophy. These time points were selected to evaluate early and later changes in muscle structure and function. Compared to the sham antibody group, 7 days after ACL tear, myostatin inhibition reduced the expression of proteolytic genes and induced the expression of hypertrophy genes. These early changes in gene expression lead to a 22% increase in muscle fiber cross‐sectional area and a 10% improvement in maximum isometric force production that were observed 21 days after ACL tear. Overall, myostatin inhibition lead to several favorable, although modest, changes in molecular biomarkers of muscle regeneration and reduced muscle atrophy and weakness following ACL tear. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2499–2505, 2017

     

The pig-to-primate immune response: relevance for xenotransplantation

Abstract:  Background:  The allotransplantation of some solid organs can be associated with a graft‐vs.‐host (GVH) response from the activity of donor B or T cells. We have investigated whether there is a risk of a GVH response following pig‐to‐primate organ xenotransplantation. Methods:  The responses of 16 pigs (six farm‐housed wild‐type and five wild‐type housed under high herd health conditions [all designated WT], and 5 α1,3‐galactosyltransferase gene‐knockout [GT‐KO] housed under high herd health conditions) to human (n = 6) and baboon (n = 6) peripheral blood mononuclear cells (PBMC) were determined. Assays included flow cytometry, complement‐dependent cytotoxicity, and mixed lymphocyte reaction. Results:  Anti‐primate cytotoxic IgM antibodies were detected in the sera of all pigs, but anti‐primate IgG antibodies were minimal. All pigs demonstrated a cellular proliferative response to primate PBMC that was equivalent to, or greater than, the allo response. The strength of the pig‐to‐primate GVH responses was proportional to the health status of the pigs, those from a high health status herd, particularly from a specific pathogen‐free herd maintained under clean husbandry conditions, where colonization of the gastrointestinal tract may be reduced, having lower responses. Conclusions:  After pig organ transplantation in a primate, if the organ is from an early‐weaned, early‐segregated GT‐KO pig, the strength of a GVH response is likely to be relatively weak. Although not investigated here, any GVH response is likely to be suppressed by the immunosuppressive therapy administered to the recipient to suppress the anti‐donor immune response.     

Halothane Induces Calcium Release from Human Skinned Masseter Muscle Fibers

Background: An increase in masseter muscle tone in response to halothane or succinylcholine anesthesia (or both) can be observed in healthy persons. Thus the authors compared the fiber-type halothane and succinylcholine sensitivities in human masseter and vastus lateralis muscles.

Methods: Masseter and vastus lateralis muscle segments were obtained from 13 and 9 healthy persons, respectively. After chemical skinning of a single fiber and loading the sarcoplasmic reticulum with Ca++ 0.16 [micro sign]M solution, halothane (0.5-4 vol% bubbled in the incubating solution), succinylcholine (0.1 [micro sign]M to 10 mM), or both sensitivities were defined as the concentration inducing more than 10% of the maximum tension obtained by application of 16 [micro sign]M Ca++ solution. The myofilament response to Ca++ was studied with and without halothane by observing the isometric tension of skinned masseter fibers challenged with increasing concentrations of Ca++. Muscle fiber type was determined by the difference in strontium-induced tension measurements.

Results: A significant difference in halothane sensitivity was found between type 1 masseter fibers (0.6 +/- 0.2 vol%; mean +/- SD) versus type 1 (2.7 +/- 0.6 vol%) and type 2 vastus lateralis muscle (2.5 +/- 0.4 vol%). Succinylcholine did not induce Ca++ release by the sarcoplasmic reticulum. In the masseter muscle, 0.75 vol% halothane decreased the maximal activated tension by 40% but did not change the Ca++ concentration that yields 50% of the maximal tension.     

Osteoblast-like cells from estrogen receptor alpha knockout mice have deficient responses to mechanical strain.

INTRODUCTION: In vivo, bones' osteogenic response to mechanical loading involves proliferation of surface osteoblasts. This response is replicated in vitro and involves ERK-mediated activation of the estrogen receptor (ER) alpha and upregulation of estrogen response element activity. This proliferative response can be blocked by selective estrogen receptor modulators and increased by transfection of additional ERalpha. MATERIALS AND METHODS: We have now investigated the mechanisms of ER involvement in osteoblast-like cells' early responses to strain by comparing the responses of primary cultures of these cells derived from homozygous ERalpha knockout (ERKO) mice (ERalpha-/-) with those from their wildtype (ERalpha+/+) and heterozygous (ERalpha+/-) littermates and from ER/beta knockout (BERKO) mice (ERbeta+/+, ERbeta+/-, and ERbeta-/-). RESULTS: Whereas ERalpha+/+, ERalpha+/-, ERbeta+/+, and ERbeta-/- cells proliferate in response to a single 10-minute period of cyclic strain, ERalpha-/- cells do not. Transfection of fully functional, but not mutant, ERalpha rescues the proliferative response to strain in these cells. The strain-related response of ERalpha-/- cells is also deficient in that they show no increased activity of an AP-I driven reporter vector and no strain-related increases in NO production. Their strain-related increase in prostacyclin production is retained. They proliferate in response to fibroblast growth factor-2 but not insulin-like growth factor (IGF)-I or IGF-II, showing the importance of ERalpha in the IGF axis and the ability of ERalpha-/- cells to proliferate normally in response to a mitogenic stimulus that does not require functional ERalpha. CONCLUSIONS: These data indicate ERalpha's obligatory involvement in a number of early responses to mechanical strain in osteoblast-like cells, including those that result in proliferation. They support the hypothesis that reduction in ERalpha expression or activity after estrogen withdrawal results in a less osteogenic response to loading. This could be important in the etiology of postmenopausal osteoporosis.