Muscle Invivo Research Articles

Evaluation of Functional Erythropoietin Receptor Status in Skeletal Muscle In Vivo: Acute and Prolonged Studies in Healthy Human Subjects

BackgroundErythropoietin receptors have been identified in human skeletal muscle tissue, but downstream signal transduction has not been investigated. We therefore studied in vivo effects of systemic erythropoietin exposure in human skeletal muscle.Methodology/Principal FindingsThe protocols involved 1) acute effects of a single bolus injection of erythropoietin followed by consecutive muscle biopsies for 1–10 hours, and 2) a separate study with prolonged administration for 16 days with biopsies obtained before and after. The presence of erythropoietin receptors in muscle tissue as well as activation of Epo signalling pathways (STAT5, MAPK, Akt, IKK) were analysed by western blotting. Changes in muscle protein profiles after prolonged erythropoietin treatment were evaluated by 2D gel-electrophoresis and mass spectrometry. The presence of the erythropoietin receptor in skeletal muscle was confirmed, by the M20 but not the C20 antibody. However, no significant changes in phosphorylation of the Epo-R, STAT5, MAPK, Akt, Lyn, IKK, and p70S6K after erythropoietin administration were detected. The level of 8 protein spots were significantly altered after 16 days of rHuEpo treatment; one isoform of myosin light chain 3 and one of desmin/actin were decreased, while three isoforms of creatine kinase and two of glyceraldehyd-3-phosphate dehydrogenase were increased.Conclusions/SignificanceAcute exposure to recombinant human erythropoietin is not associated by detectable activation of the Epo-R or downstream signalling targets in human skeletal muscle in the resting situation, whereas more prolonged exposure induces significant changes in the skeletal muscle proteome. The absence of functional Epo receptor activity in human skeletal muscle indicates that the long-term effects are indirect and probably related to an increased oxidative capacity in this tissue.

Effect of Repair Tightness on in Vivo Muscle Function After Acute and Chronic Rotator Cuff Repair

Effect of Repair Tightness on in Vivo Muscle Function After Acute and Chronic Rotator Cuff Repair

Reliability, Validity, and Precision of a Handheld Myometer for Assessing in Vivo Muscle Stiffness

Reliability, Validity, and Precision of a Handheld Myometer for Assessing in Vivo Muscle Stiffness

Insulin and GH Signaling in Human Skeletal Muscle In Vivo following Exogenous GH Exposure: Impact of an Oral Glucose Load

IntroductionGH induces acute insulin resistance in skeletal muscle in vivo, which in rodent models has been attributed to crosstalk between GH and insulin signaling pathways. Our objective was to characterize time course changes in signaling pathways for GH and insulin in human skeletal muscle in vivo following GH exposure in the presence and absence of an oral glucose load.MethodsEight young men were studied in a single-blinded randomized crossover design on 3 occasions: 1) after an intravenous GH bolus 2) after an intravenous GH bolus plus an oral glucose load (OGTT), and 3) after intravenous saline plus OGTT. Muscle biopsies were taken at t = 0, 30, 60, and 120. Blood was sampled at frequent intervals for assessment of GH, insulin, glucose, and free fatty acids (FFA).ResultsGH increased AUCglucose after an OGTT (p<0.05) without significant changes in serum insulin levels. GH induced phosphorylation of STAT5 independently of the OGTT. Conversely, the OGTT induced acute phosphorylation of the insulin signaling proteins Akt (ser473 and thr308), and AS160.The combination of OGTT and GH suppressed Akt activation, whereas the downstream expression of AS160 was amplified by GH.We Concluded the Following1) A physiological GH bolus activates STAT5 signaling pathways in skeletal muscle irrespective of ambient glucose and insulin levels 2) Insulin resistance induced by GH occurs without a distinct suppression of insulin signaling proteins 3) The accentuation of the glucose-stimulated activation of AS 160 by GH does however indicate a potential crosstalk between insulin and GH.Trial RegistrationClinicalTrials.gov NCT00477997

Role of Constituents of Optison in Optison-Mediated Gene Transfection Enhancement in Skeletal Muscle In Vivo

The mechanism by which Optison (an albumin-shelled, octafluoropropane gas-filled microbubble contrast agent; Amersham Health, Amersham, England) enhances gene transfection in skeletal muscle in vivo with or without ultrasound (US) is unclear. The possible mechanisms were investigated by experimenting with different constituents, both with and without US. Plasmid DNA (10 μg) encoding green fluorescent protein was mixed with Optison or its constituents dissolved in saline (in an equivalent concentration as in Optison) and injected into the tibialis anterior muscle of mice with or without adjunct US (1 MHz, 2 W/cm², 30 seconds, and 20% duty cycle). The efficiencies of green fluorescent protein transgene expression were determined under different experimental conditions: (1) plasmid plus saline as a negative control; (2) plasmid plus Optison as a positive control; (3) plasmid plus heat-treated Optison (without microbubbles); (4) plasmid plus human serum albumin; (5) plasmid plus N-acetyltryptophan; and (6) plasmid plus caprylic acid. Transfection efficiency was assessed by counting the maximum number of green fluorescent protein-positive fibers. Tissue damage was assessed by measuring the damaged area on serial sections. Heat-treated Optison with or without US and albumin with US showed similarly high levels of transgene expression as Optison in mouse muscle without substantially increased tissue damage. N-Acetyltryptophan and caprylic acid had no effect on the delivery of plasmid green fluorescent protein into mouse muscle but instead showed the potential to increase tissue damage. These data suggest that US and albumin separately potentiate transfection in this model. The combination of albumin and perfluoropropane is highly effective, which probably explains why Optison is so effective.

Regulation of Glucose Uptake by Endothelin-1 in Human Skeletal Muscle in Vivo and in Vitro

Context: Expression of the vasoconstrictor and proinflammatory peptide endothelin (ET)-1 is increased in insulin-resistant (IR) subjects. Objective: The aim of this study was to investigate whether ET-1 regulates skeletal muscle glucose uptake in IR subjects in vivo and in cultured human skeletal muscle cells. Design and Participants: Eleven subjects participated in three protocols using brachial artery infusion of: A) BQ123 (10 nmol/min) and BQ788 (10 nmol/min) (ETA and ETB receptor antagonist, respectively), followed by coinfusion with insulin (0.05 mU/kg/min); B) insulin alone; and C) insulin followed by coinfusion with ET-1 (20 pmol/min). Main Outcome Measures: Forearm blood flow (FBF) and forearm glucose uptake (FGU) were determined. Glucose uptake and molecular signaling were determined in cultured skeletal muscle cells. Results: ETA/ETB receptor blockade increased FGU by 63% (P < 0.05). Coadministration of insulin caused a further 2-fold increase in FGU (P < 0.001). ETA/ETB receptor blockade combin...

Myostatin Directly Regulates Skeletal Muscle Fibrosis

Skeletal muscle fibrosis is a major pathological hallmark of chronic myopathies in which myofibers are replaced by progressive deposition of collagen and other extracellular matrix proteins produced by muscle fibroblasts. Recent studies have shown that in the absence of the endogenous muscle growth regulator myostatin, regeneration of muscle is enhanced, and muscle fibrosis is correspondingly reduced. We now demonstrate that myostatin not only regulates the growth of myocytes but also directly regulates muscle fibroblasts. Our results show that myostatin stimulates the proliferation of muscle fibroblasts and the production of extracellular matrix proteins both in vitro and in vivo. Further, muscle fibroblasts express myostatin and its putative receptor activin receptor IIB. Proliferation of muscle fibroblasts, induced by myostatin, involves the activation of Smad, p38 MAPK and Akt pathways. These results expand our understanding of the function of myostatin in muscle tissue and provide a potential target for anti-fibrotic therapies.

The Insulin-Sensitizing Effect of Rosiglitazone in Type 2 Diabetes Mellitus Patients Does Not Require Improved in Vivo Muscle Mitochondrial Function

Our objective was to investigate whether improved in vivo mitochondrial function in skeletal muscle and intramyocellular lipids (IMCLs) contribute to the insulin-sensitizing effect of rosiglitazone. Eight overweight type 2 diabetic patients (body mass index = 29.3 +/- 1.1 kg/m(2)) were treated with rosiglitazone for 8 wk. Before and after treatment, insulin sensitivity was determined by a hyperinsulinemic euglycemic clamp. Muscular mitochondrial function (half-time of phosphocreatine recovery after exercise) and IMCL content were measured by magnetic resonance spectroscopy. Insulin sensitivity improved after rosiglitazone (glucose infusion rate: 19.9 +/- 2.8 to 24.8 +/- 2.1 micromol/kg.min; P < 0.05). In vivo mitochondrial function (phosphocreatine recovery half-time: 23.8 +/- 3.5 to 20.0 +/- 1.7 sec; P = 0.23) and IMCL content (0.93 +/- 0.18% to 1.37 +/- 0.40%; P = 0.34) did not change. Interestingly, the changes in PCr half-time correlated/tended to correlate with changes in fasting insulin (R(2) = 0.50; P = 0.05) and glucose (R(2) = 0.43; P = 0.08) levels. Changes in PCr half-time did not correlate with changes in glucose infusion rate (R(2) = 0.08; P = 0.49). The rosiglitazone-enhanced insulin sensitivity does not require improved muscular mitochondrial function.

Intracellular Critical PO2 Measured in Human Skeletal Muscle In Vivo

Under some conditions, intracellular oxygen tension (PO2) may decrease to the point where oxygen availability limits respiration. Information about this critical PO2 and its determinants in vivo is sparse. PURPOSE: To determine the critical PO2 of human skeletal muscle, and its relationship to cellular energy consumption. METHODS: Interleaved proton and phosphorus magnetic resonance spectroscopy were used to quantify deoxymyoglobin and phosphorous metabolites in the tibialis anterior muscle of 6 subjects during 15 minutes of leg ischemia. Critical PO2 was determined as the PO2 at the phosphocreatine inflection point during ischemia. The rate of ATP consumption was determined from the linear slope of PCr depletion following the inflection point. RESULTS: The time to PCr inflection point was 282 ± 112 s (mean±SE). The corresponding critical PO2 was 0.47±0.27 torr, with a range of 0.21–0.96. PO2crit was not associated with the resting rate of ATP consumption (r =0.17), but was directly correlated to the rate of myoglobin desaturation (r =0.93) and inversely correlated to the time required to reach the PCr inflection point (r = −0.74). CONCLUSION: These data suggest that the relatively wide range of PO2crit in these subjects is attributable to inter‐subject differences in the ability of the muscle to maintain ATP production in the face of an ischemic insult.Support NIH AG21094, K02 AG023582

Transcriptional Control of the Pgc-1α Gene in Skeletal Muscle In Vivo

Endurance exercise promotes mitochondrial biogenesis in skeletal muscle possibly by inducing peroxisome proliferator-activated receptor gamma coactivator 1alpha gene transcription through activation of the p38 mitogen-activated protein kinase pathway. A novel imaging analysis revealed the importance of activating transcription factor 2 and histone deacetylase 5/myocyte enhancer factor 2 in the regulation of the Pgc-1alpha gene in skeletal muscle in vivo.

AMP-activated Protein Kinase α2 Activity Is Not Essential for Contraction- and Hyperosmolarity-induced Glucose Transport in Skeletal Muscle

To examine the role of AMP-activated protein kinase (AMPK) in muscle glucose transport, we generated muscle-specific transgenic mice (TG) carrying cDNAs of inactive alpha2 (alpha2i TG) and alpha1 (alpha1i TG) catalytic subunits. Extensor digitorum longus (EDL) muscles from wild type and TG mice were isolated and subjected to a series of in vitro incubation experiments. In alpha2i TG mice basal alpha2 activity was barely detectable, whereas basal alpha1 activity was only partially reduced. Known AMPK stimuli including 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), rotenone (a Complex I inhibitor), dinitrophenol (a mitochondrial uncoupler), muscle contraction, and sorbitol (producing hyperosmolar shock) did not increase AMPK alpha2 activity in alpha2i TG mice, whereas alpha1 activation was attenuated by only 30-50%. Glucose transport was measured in vitro using isolated EDL muscles from alpha2i TG mice. AICAR- and rotenone-stimulated glucose transport was fully inhibited in alpha2i TG mice; however, the lack of AMPK alpha2 activity had no effect on contraction- or sorbitol-induced glucose transport. Similar to these observations in vitro, contraction-stimulated glucose transport, assessed in vivo by 2-deoxy-d-[(3)H]glucose incorporation into EDL, tibialis anterior, and gastrocnemius muscles, was normal in alpha2i TG mice. Thus, AMPK alpha2 activation is essential for some, but not all, insulin-independent glucose transport. Muscle contraction- and hyperosmolarity-induced glucose transport may be regulated by a redundant mechanism in which AMPK alpha2 is one of multiple signaling pathways.

Impact of Stretch-Shortening Cycle Rest Interval on in Vivo Muscle Performance

Overuse and overtraining models have implicated both metabolic and mechanical disturbances as contributors to muscle damage and performance decrement but have produced equivocal results. The purpose of the present study was to investigate the impact of rest interval between sets of stretch-shortening cycles (SSC) on static and dynamic muscle performance Animals were randomly assigned to groups (N = 8 per group) of 10-s, 1-min, or 5-min rest between sets of isometric contractions (10-s, 1-min, or 5-min CON), or SSC (10-s, 1-min, or 5-min INJ). The dorsiflexor muscles were exposed in vivo to either seven sets of 10 SSC (500 degrees . s) or seven sets of isometric contractions. Performance was characterized by isometric exertions and positive, negative, and net work, at pretest, during the sets of SSC, and 48 h postexposure The isometric force at 48 h after the 10-s and 5-min INJ groups were statistically different from the 1-min group (P < 0.05), whereas there was no difference in the CON groups. Negative work of the INJ groups were statistically lower at 48 h than pretest values (P < 0.05), whereas there was no change in positive work. Of the real-time parameters, there was a difference in minimum force and positive work (P < 0.05) with treatment with the 10-s INJ group being most affected. SSC conducted at shorter work-rest cycles resulted in a more profound isometric force decrement 48 h postexposure, and in real-time changes in isometric prestretch force and positive work. These results indicate that short rest intervals between athletic or vocational tasks of heightened physical exertion (i.e., high intensity) may adversely affect performance and increase injury susceptibility.

Resting Potential–dependent Regulation of the Voltage Sensitivity of Sodium Channel Gating in Rat Skeletal Muscle In Vivo

Normal muscle has a resting potential of −85 mV, but in a number of situations there is depolarization of the resting potential that alters excitability. To better understand the effect of resting potential on muscle excitability we attempted to accurately simulate excitability at both normal and depolarized resting potentials. To accurately simulate excitability we found that it was necessary to include a resting potential–dependent shift in the voltage dependence of sodium channel activation and fast inactivation. We recorded sodium currents from muscle fibers in vivo and found that prolonged changes in holding potential cause shifts in the voltage dependence of both activation and fast inactivation of sodium currents. We also found that altering the amplitude of the prepulse or test pulse produced differences in the voltage dependence of activation and inactivation respectively. Since only the Nav1.4 sodium channel isoform is present in significant quantity in adult skeletal muscle, this suggests that either there are multiple states of Nav1.4 that differ in their voltage dependence of gating or there is a distribution in the voltage dependence of gating of Nav1.4. Taken together, our data suggest that changes in resting potential toward more positive potentials favor states of Nav1.4 with depolarized voltage dependence of gating and thus shift voltage dependence of the sodium current. We propose that resting potential–induced shifts in the voltage dependence of sodium channel gating are essential to properly regulate muscle excitability in vivo.

Exercise Stimulates Pgc-1α Transcription in Skeletal Muscle through Activation of the p38 MAPK Pathway

Peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1alpha) promotes mitochondrial biogenesis and slow fiber formation in skeletal muscle. We hypothesized that activation of the p38 mitogen-activated protein kinase (MAPK) pathway in response to increased muscle activity stimulated Pgc-1alpha gene transcription as part of the mechanisms for skeletal muscle adaptation. Here we report that a single bout of voluntary running induced a transient increase of Pgc-1alpha mRNA expression in mouse plantaris muscle, concurrent with an activation of the p38 MAPK pathway. Activation of the p38 MAPK pathway in cultured C2C12 myocytes stimulated Pgc-1alpha promoter activity, which could be blocked by the specific inhibitors of p38, SB203580 and SB202190, or a dominant negative p38. Furthermore, the p38-mediated increase in Pgc-1alpha promoter activity was enhanced by increased expression of the downstream transcription factor ATF2 and completely blocked by ATF2DeltaN, a dominant negative ATF2. Skeletal muscle-specific expression of a constitutively active activator of p38, MKK6E, in transgenic mice resulted in enhanced Pgc-1alpha and cytochrome oxidase IV protein expression in fast-twitch skeletal muscles. These findings suggest that contractile activity-induced activation of the p38 MAPK pathway promotes Pgc-1alpha gene expression and skeletal muscle adaptation.

Measurement of Membrane Potential and Intracellular Ca2+ of Arteriolar Endothelium and Smooth Muscle in Vivo

We have developed an intensity analysis technique for fluorescence microscopy that allows us to measure, in real time, the diameter and the membrane potential or intracellular calcium ([Ca2+]i) of in vivo arteriolar endothelium or smooth muscle. Cheek pouch arterioles of anesthetized hamsters were luminally or abluminally labeled with Di-8-ANEPPS, a voltage-sensitive dye, or Fura PE3, a calcium indicator. The peak fluorescence intensities of the images were used to locate the endothelium or smooth muscle. The changes in membrane potential or [Ca2+]i were determined based on the ratiometric analysis of fluorescence intensity of the endothelium or smooth muscle. Membrane depolarization of the smooth muscle using KCl caused a decrease in the ratio of emission, 620 nm/560 nm (∼6 mV/% ratio). The ratio of excitation, 340 nm/380 nm, increased with increasing free Ca2+. Methacholine, a muscarinic receptor agonist, caused arteriolar dilation (12.2 ± 0.9 μm). It produced hyperpolarization of the endothelium and smooth muscle (2.8 ± 0.6% and 2.3 ± 0.3% in ratio). Methacholine also induced an increase in [Ca2+]i (11.0 ± 1.1% in ratio) of the endothelium. In contrast, methacholine caused a biphasic change in [Ca2+]i of the smooth muscle, a rapid reduction (−3.4 ± 0.2% in ratio) followed by a prolonged increase (2.4 ± 0.2% in ratio). These results demonstrate that the peak intensity analysis can be used to determine in real time the changes in membrane potential or [Ca2+]i of in vivo endothelium or smooth muscle.

Effects of Insulin on Subcellular Localization of Hexokinase II in Human Skeletal Muscle in Vivo

The phosphorylation of glucose to glucose-6-phosphate, catalyzed by hexokinase, is the first committed step in glucose uptake into skeletal muscle. Two isoforms of hexokinase, HKI and HKII, are expressed in human skeletal muscle, but only HKII expression is regulated by insulin. HKII messenger RNA, protein, and activity are increased after 4 h of insulin infusion; however, glucose uptake is stimulated much more rapidly, occurring within minutes. Studies in rat muscle suggest that changes in the subcellular distribution of HKII may be an important regulatory factor for glucose uptake. The present studies were undertaken to determine if insulin causes an acute redistribution of HKII activity in human skeletal muscle in vivo. Muscle biopsies (vastus lateralis muscle) were performed before and at the end of 30 min insulin infusion, performed using the euglycemic clamp technique. Muscle biopsies were subfractionated into soluble and particulate fractions to determine if insulin acutely changes the subcellular distribution of HKII. Insulin decreased HKII activity in the soluble fraction from 2.20 +/- 0.31 to 1.40 +/- 0.18 pmoles/(min[chempt]micrograms) and increased HKII activity in the particulate fraction from 3.02 +/- 0.46 to 3.45 +/- 0.46 pmoles/(min[chempt]micrograms) (P < 0.01 for both). These changes in HKII activity were correlated with changes in HKII protein, as determined by immunoblot analysis (r = 0.53, P = 0.05). Insulin had no effect on the subcellular distribution of HKII activity, which was primarily restricted to the soluble fraction. These studies are consistent with the conclusion that, in vivo in human skeletal muscle, insulin changes the subcellular distribution of HKII within 30 min.

A Novel, Rapid, and Highly Sensitive Mass Assay for Phosphatidylinositol 3,4,5-Trisphosphate (PtdIns(3,4,5)P3) and Its Application to Measure Insulin-stimulated PtdIns(3,4,5)P3 Production in Rat Skeletal Muscle in Vivo

The pivotal role of phosphatidylinositol 3-kinase (PI 3-kinase) in signal transduction has been well established in recent years. Receptor-regulated forms of PI 3-kinase are thought to phosphorylate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) at the 3-position of the inositol ring to give the putative lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4, 5)P3). Cellular levels of PtdIns(3,4,5)P3 are currently measured by time-consuming procedures involving radiolabeling with high levels of 32PO4, extraction, and multiple chromatography steps. To avoid these lengthy and hazardous procedures, many laboratories prefer to assay PI 3-kinase activity in cell extracts and/or appropriate immunoprecipitates. Such approaches are not readily applied to measurements of PtdIns(3,4,5)P3 in extracts of animal tissues. Moreover, they can be misleading since the association of PI 3-kinases in molecular complexes is not necessarily correlated with the enzyme's activity state. Direct measurements of PtdIns(3,4,5)P3 would also be desirable since its concentration may be subject to additional control mechanisms such as activation or inhibition of the phosphatases responsible for PtdIns(3,4,5)P3 metabolism. We now report a simple, reproducible isotope dilution assay which detects PtdIns(3,4,5)P3 at subpicomole sensitivity, suitable for measurements of both basal and stimulated levels of PtdIns(3,4,5)P3 obtained from samples containing approximately 1 mg of cellular protein. Total lipid extracts, containing PtdIns(3,4,5)P3, are first subjected to alkaline hydrolysis which results in the release of the polar head group Ins(1,3,4,5)P4. The latter is measured by its ability to displace [32P]Ins(1,3,4,5)P4 from a highly specific binding protein present in cerebellar membrane preparations. We show that this assay solely detects PtdIns(3,4,5)P3 and does not suffer from interference by other compounds generated after alkaline hydrolysis of total cellular lipids. Measurements on a wide range of cells, including rat-1 fibroblasts, 1321N1 astrocytoma cells, HEK 293 cells, and rat adipocytes, show wortmannin-sensitive increased levels of PtdIns(3,4,5)P3 upon stimulation with appropriate agonists. The enhanced utility of this procedure is further demonstrated by measurements of PtdIns(3,4,5)P3 levels in tissue derived from whole animals. Specifically, we show that stimulation with insulin increases PtdIns(3,4,5)P3 levels in rat skeletal muscle in vivo with a time course which parallels the activation of protein kinase B in the same samples.

Direct Gene Transfer into Mouse Muscle in Vivo

RNA and DNA expression vectors containing genes for chloramphenicol acetyltransferase, luciferase, and beta-galactosidase were separately injected into mouse skeletal muscle in vivo. Protein expression was readily detected in all cases, and no special delivery system was required for these effects. The extent of expression from both the RNA and DNA constructs was comparable to that obtained from fibroblasts transfected in vitro under optimal conditions. In situ cytochemical staining for beta-galactosidase activity was localized to muscle cells following injection of the beta-galactosidase DNA vector. After injection of the DNA luciferase expression vector, luciferase activity was present in the muscle for at least 2 months.

A Quantitative Evaluation of the Frequency-Response Characteristics of Active Human Skeletal Muscle In Vivo

This paper describes an investigation of the frequency-response characteristics of active human skeletal muscle in vivo over the frequency range 1 Hz to 15 Hz. The applied force, forearm position, and surface electromyograms (from biceps, triceps, and brachioradialis) were recorded simultaneously in four normal adult male subjects for small oscillations of the forearm about a mean position of 90 deg flexion. Two modes of oscillatory behavior are discussed: externally forced oscillations under constant muscle force and voluntary oscillations against an elastic resistance. The observed amplitude and phase relations are presented herein and are compared to the response predicted by a simple model for neuromuscular dynamics. It appears that the small amplitude frequency response of normal skeletal muscle in vivo can be represented by a second order model. The main muscle parameters of this model are a muscular stiffness K, two time constants τ1 and τ2 associated with contraction dynamics, and a time delay τ: typical values of these parameters at moderate contraction levels (approximately 20 percent of maximum voluntary effort) are K = 100 N · m/rad, τ1 and τ2 = 50 ms, and τ = 10 ms. Reflex feedback under forced-oscillation conditions was also examined and may be characterized by a gain parameter (ΔE/Δθ), the ratio of the surface EMG amplitude to the angular displacement of the forearm, and the phase by which the EMG leads muscle stretch. The reflex EMG is observed to lead muscle stretch at all frequencies between 1 Hz and 15 Hz. The muscle stiffness K and the reflex gain parameter (ΔE/Δθ) are approximately proportional to the average force of contraction.

Effect of Epinephrine and Insulin on Adenosine 3'5'-Cyclic Monophosphate - Dependent Protein Kinase in Human Skeletal Muscle In Vivo

Cyclic AMP dependent protein kinase has beeen identified in human skeletal muscle tissue. In crude muscle extracts the enzyme was 3--5 fold activated by cyclic AMP. The cyclic AMP-dependent activity (corresponding to the inactive holoenzyme) was completely inhibited by the heat stable inhibitor of protein kinase. Reciprocal changes of the cyclic AMP-dependent activity in skeletal muscle were observed after administration of epinephrine and insulin in vivo. Infusion of epinephrine in healthy volunteers increased the level of cyclic AMP and decreased the activity of the cyclic AMP-depenent form (i.e. the inactive form) of protein kinase. These changes were reversible after cessation of epinephrine administration. The results are consistent with an activation of protein kinase in vivo due to an epinephrine mediated increase of the concentration of cyclic AMP. I.v. injection of insulin had the opposite effect on the enzyme in skeletal muscle, leading to increased activity of the cyclic AMP-dependent form of protein kinase. Insulin had no effect on the level of cyclic AMP, but promoted a transient increase of cyclic GMP 1 min. after insulin injection. The effect by insulin on protein kinase cannot be related to the level of cyclic AMP or cyclic GMP.