The relationship between ATP hydrolysis and active force in compressed and swollen skinned muscle fibers of the rabbit

We investigated whether the inhibition of force generation observed in compressed muscle fibers is accompanied by a coupled reduction in hydrolytic activity. Isometric force and rates of ATP hydrolysis (ATPase) were measured as functions of the relative width of chemically skinned skeletal muscle fiber segments immersed in relaxing (pCa>8) and activating (pCa 4.9) salt solutions. Osmotic radial compression of the fiber segment was produced (with little or no affect on striation spacing) by adding Dextran T500 to the bathing media. ADP as a product of ATP hydrolysis in fibers undergoing 10–15 min contractions was measured using high pressure liquid chromatography. Compression of the (initially swollen) fiber segment with dextran produced a slight (4%) increase in average active force and then, with further compression, a sharp decrease (with maximum around in situ width). With compression, the average ATPase of the fiber decreased monotonically, and with extreme compression (with 0.22 g dextran per ml), ATPase fell to a fifth of its level determined in dextran-free solution while force was abolished. The time course of active force development was described by the sum of two exponential functions, the faster of which characterized the rate of rise. Fiber compression (0.14 g dextran per ml) reduced the rate of rise of force ten-fold compared to that in dextran-free solution. Hindrance of cross movement is proposed to account for the inhibition of active force generation and (coupled) ATPase in compressed fibers.     

The Drosophila projectin mutant,bent D,has reduced stretch activation and altered indirect flight muscle kinetics

Projectin is a ca. 900 kDa protein that is a member of the titin protein superfamily. In skeletal muscle titins are involved in the longitudinal reinforcement of the sarcomere by connecting the Z-band to the M-line. In insect indirect flight muscle (IFM), projectin is believed to form the connecting filaments that link the Z-band to the thick filaments and is responsible for the high relaxed stiffness found in this muscle type. The Drosophila mutant bent ^D (bt ^D ) has been shown to have a breakpoint close to the carboxy-terminal kinase domain of the projectin sequence. Homozygotes for bt ^D are embryonic lethal but heterozygotes (bt ^D /+) are viable. Here we show that bt ^D /+ flies have normal flight ability and a slightly elevated wing beat frequency (bt ^D /+ 223 ± 13 Hz; +/+203 ± 5 Hz, mean ± SD; P < 0.01). Electron microscopy of bt ^D /+ IFM show normal ultrastructure but skinned fiber mechanics show reduced stretch activation and oscillatory work. Although bt ^D /+ IFM power output was at wild-type levels, maximum power was achieved at a higher frequency of applied length perturbation (bt ^D /+ 151 ± 6 Hz; +/+ 102 ± 14 Hz; P < 0.01). Results were interpreted in the context of a viscoelastic model of the sarcomere and indicate altered cross-bridge kinetics of the power-producing step. These results show that the bt ^D mutation reduces oscillatory work in a way consistent with the proposed role of the connecting filaments in the stretch activation response of IFM.     

Physical and biological properties of a novel siloxane adhesive for soft tissue applications

The aim of this study was to investigate the adhesive properties of an in-house aminopropyltrimethoxysilane-methylenebisacrylamide (APTMS-MBA) siloxane system and compare them with a commercially available adhesive, n-butyl cyanoacrylate (nBCA). The ability of the material to perform as a soft tissue adhesive was established by measuring the physical (bond strength, curing time) and biological (cytotoxicity) properties of the adhesives on cartilage. Complementary physical techniques, X-ray photoelectron spectroscopy, Raman and infrared imaging, enabled the mode of action of the adhesive to the cartilage surface to be determined. Adhesion strength to cartilage was measured using a simple butt joint test after storage in phosphate-buffered saline solution at 37 degrees C for periods up to 1 month. The adhesives were also characterised using two in vitro biological techniques. A live/dead stain assay enabled a measure of the viability of chondrocytes attached to the two adhesives to be made. A water-soluble tetrazolium assay was carried out using two different cell types, human dermal fibroblasts and ovine meniscal chondrocytes, in order to measure material cytotoxicity as a function of both supernatant concentration and time. IR imaging of the surface of cartilage treated with APTMS-MBA siloxane adhesive indicated that the adhesive penetrated the tissue surface marginally compared to nBCA which showed a greater depth of penetration. The curing time and adhesion strength values for APTMS-MBA siloxane and nBCA adhesives were measured to be 60 s/0.23 MPa and 38 min/0.62 MPa, respectively. These materials were found to be significantly stronger than either commercially available fibrin (0.02 MPa) or gelatin resorcinol formaldehyde (GRF) adhesives (0.1 MPa) (P < 0.01). Cell culture experiments revealed that APTMS-MBA siloxane adhesive induced 2% cell death compared to 95% for the nBCA adhesive, which extended to a depth of approximately 100-150 microm into the cartilage surface. The WST-1 assay demonstrated that APTMS-MBA siloxane was significantly less cytotoxic than nBCA adhesive as an undiluted conditioned supernatant (P < 0.001). These results suggest that the APTMS-MBA siloxane may be a useful adhesive for medical applications.     

The metabolic response to prolonged walking in fed and fasted men

Summary Six healthy men walked 37 km (23 miles) per day over a 3-lap course for each of 4 consecutive days. Subjects were allowed breakfast and an unrestricted diet was consumed after completion of the walk, but no food was consumed during or between laps. At a later date the same subjects walked over the same course after an overnight fast and without breakfast. Completion time for each lap was 139±1 min (mean ±SE) and exercise intensity was equivalent to 17±1% . Mean 24h energy intake was 14.5±0.8 MJ during the fed walk. Estimated daily energy expenditure was 12.0 MJ. Blood glucose concentration fell significantly on the first, third and fourth days of the fed walk, but no subject became hypoglycaemic. Glucose concentration did not fall during the fasted walk and was significantly higher pre-exercise and at the end of laps one and three when compared to the first day of the fed walk. Blood alanine concentration fell significantly after the end of the first lap of each day of the fed walk but not during the fasted walk. Blood lactate levels did not change during the course of either walk. Plasma free fatty acid, glycerol and blood 3-hydroxybutyrate concentrations were unchanged during the passage of the first lap on each day of the fed walk, but all three had increased significantly by the end of the first lap of the fasted walk. The elevations from rest to the end of each day's walking for these three metabolites were of similar magnitude when comparing each day of the fed walk and the fed and fasted walks. The present experiment indicates that feeding a mixed diet can affect the pattern of substrate mobilization in a similar manner to that seen during more strenuous exercise. It also appears that the pattern of fuel substrate mobilization is very similar from one day to the next providing dietary intake is similar.     

The use of a deuterium tracer technique to follow the fate of fluids ingested by human subjects: effects of drink volume and tracer concentration and content

Deuterium oxide (2H2O) has been added to drinks as a tracer for water to estimate the availability to the body water pool of ingested fluids, but doubts have been raised as to the reliability of the method. The present investigation evaluated the effects of systematic variations in the volume of fluid consumed and the amount and concentration of added tracer on the rate of accumulation of tracer in arterialized blood after ingestion of a labelled drink. Three separate experiments were undertaken. In expt 1, six healthy men ingested on separate occasions 200, 400 and 800 ml of a dilute glucose-electrolyte solution: all test drinks contained the same concentration (40 g l-1) of 2H2O. In expt 2, six healthy men ingested 200, 400 and 800 ml of the same glucose-electrolyte drink: each drink contained 8 g of 2H2O so that the concentration, but not the amount, of 2H2O differed between treatments. In expt 3, six healthy men ingested 400 ml of the same drink on three separate occasions: each drink contained 8, 16 or 32 g of tracer so that amount and concentration of 2H2O both varied. Arterialized venous blood samples were collected for the determination of deuterium (2H) concentration before ingestion of the test drink and at intervals for 120 min after ingestion. All trials for each of the experiments were conducted in the morning after an overnight fast and trials were in randomized order and separated by 7 days. In expt 1, the blood 2H concentration at all time points from 2 min after ingestion of the test drink onwards was higher for the drink containing 32 g 2H2O than for the drink containing 16 g 2H2O, which in turn was higher than after ingestion of the drink containing 8 g of 2H2O. In expt 2, no significant differences between treatments were observed at any time. In expt 3, the rate of 2H accumulation was greater after ingestion of the drink containing 32 g of 2H2O than after either of the other two drinks, and the 2H accumulation rate was greater after ingestion of the drink containing 16 g of 2H2O than after the drink containing 8 g of 2H2O. When data from all three experiments were combined, significant correlations were observed between the rate of accumulation of 2H in the circulation (p.p.m. min-1) and the amount (rs = 0.75, P < 0001) and concentration (rs = 0.69, P < 0001) of 2H2O in the test drink, but there was no relationship (rs = 0.09, P = 0.5) between the rate of 2H accumulation in the blood and the volume of the drink consumed. The results suggest that the rate of tracer accumulation in the blood after ingestion of different volumes of test drinks is not a reliable indication of the availability of the ingested fluid, but that the method gives at least a qualitative measure of the sum of the effects of gastric emptying and intestinal water absorption.     

Use of a conductance (volume) catheter and transient inferior vena caval occlusion for rapid determination of pressure-volume relationships in man

Determination of left ventricular pressure-volume relationships in situ ideally requires both a method for easy measurement of multiple pressure-volume loops and a rapid and reversible means of altering load. We report a technique, previously used in animals, that combines conductance catheter volumes and rapid inferior vena caval occlusion to permit routine measurement of calibrated P-V relationships in man for the first time. An 8F volume catheter with a 3F micromanometer tipped pressure catheter placed through its lumen was advanced to the left ventricular apex through a femoral artery. A thermodilution output catheter was placed through a 9F femoral venous sheath and later replaced with an IVC balloon occlusion catheter, through which a 2.5F bipolar wire was advanced for atrial pacing. A specialized data system facilitated collection, editing, and data analysis at the time of cardiac catheterization. Absolute volume calibration required cardiac output measurement and injection of hypertonic saline. IVC occlusion decreased peak left ventricular pressure by 42 +/- 17 (SD) (P less than .001) mm Hg in 15 patients. Endsystolic pressure-volume relationships (ESPVR) were determined with 5-8 cardiac cycles with an average of r2 of 0.94 +/- 0.05 and were generally reproducible. The slope of the ESPVR demonstrated consistency among a group of normal patients (n = 6), and was significantly lower than the slope derived from a group of patients with ventricular hypertrophy (n = 9). We conclude that left ventricular pressure-volume relationships can be easily and repeatedly determined as part of a routine cardiac catheterization in man.     

Alternating contractility in pulsus alternans studied in the isolated canine heart

We examined pulsus alternans in seven isolated, perfused canine left ventricles ejecting into a simulated arterial impedance. Left ventricular pressure-volume loops were measured during pulsus alternans while filing-source pressure was lowered. In all cases two distinct linear end-systolic pressure-volume relationships (ESPVRs) were noted for the strong and weak beats. The slopes of the ESPVRs of the strong beats were significantly greater than those of the weak beats (mean difference 0.9 +/- 0.6 mm Hg/ml, p less than .01), while the intercepts were not significantly different (mean difference 0.06 +/- 0.5 ml). Diastolic pressure-volume relationships for the strong and weak beats were not significantly different, excluding incomplete relaxation as a cause of pulsus alternans. Although the weak beats had both a smaller preceding end-diastolic volume and a larger end-systolic volume, the presence of two distinct ESPVRS for the strong and weak beats shows there is alternating ventricular chamber contractility in pulsus alternans that is not solely due to the Starling mechanism. The magnitude of alternation in pump function parameters such as pressure and stroke volume during pulsus alternans reflects the complex interactions of alternating contractile state with alternations in preload and afterload.