Nogo-A knockdown inhibits hypoxia/reoxygenation-induced activation of mitochondrial-dependent apoptosis in cardiomyocytes.

Programmed cell death of cardiomyocytes following myocardial ischemia increases biomechanical stress on the remaining myocardium, leading to myocardial dysfunction that may result in congestive heart failure or sudden death. Nogo-A is well characterized as a potent inhibitor of axonal regeneration and plasticity in the central nervous system, however, the role of Nogo-A in non-nervous tissues is essentially unknown. In this study, Nogo-A expression was shown to be significantly increased in cardiac tissue from patients with dilated cardiomyopathy and from patients who have experienced an ischemic event. Nogo-A expression was clearly associated with cardiomyocytes in culture and was localized predominantly in the endoplasmic reticulum. In agreement with the findings from human tissue, Nogo-A expression was significantly increased in cultured neonatal rat cardiomyocytes subjected to hypoxia/reoxygenation. Knockdown of Nogo-A in cardiomyocytes markedly attenuated hypoxia/reoxygenation-induced apoptosis, as indicated by the significant reduction of DNA fragmentation, phosphatidylserine translocation, and caspase-3 cleavage, by a mechanism involving the preservation of mitochondrial membrane potential, the inhibition of ROS accumulation, and the improvement of intracellular calcium regulation. Together, these data demonstrate that knockdown of Nogo-A may serve as a novel therapeutic strategy to prevent the loss of cardiomyocytes following ischemic/hypoxic injury.

Myocardial S-adenosylhomocysteine hydrolase is important for adenosine production during normoxia.

The coronary vasodilator adenosine can be formed in the heart by breakdown of AMP or S-adenosylhomocysteine (SAdoHcy). The purpose of this study was to get insight into the relative importance of these routes of adenosine formation in both the normoxic and the ischemic heart. A novel HPLC method was used to determine myocardial adenosine and SAdoHcy. Accumulation of SAdoHcy was induced in isolated rat hearts by perfusion with L-homocysteine thiolactone or L-homocysteine. The release of adenosine, inosine, hypoxanthine, xanthine and uric acid was determined. Additional in vitro experiments were performed to determine the kinetic parameters of S-adenosylhomocysteine hydrolase. During normoxia the thiolactone caused a concentration-dependent increase in SAdoHcy. At 2000 microM of the thiolactone an SAdoHcy accumulation of 0.49 nmol/min per g wet weight was found during normoxia. L-Homocysteine (200 microM) caused an increase of 0.37 and 4.17 nmol SAdoHcy/min per g wet weight during normoxia and ischemia, respectively. The adenosine concentration in ischemic hearts was significantly lower when homocysteine was infused (6.2 vs. 11.5 nmol/g; P less than 0.05). Purine release was increased 4-fold during ischemia. The Km for hydrolysis of SAdoHcy was about 12 microM. At in vitro conditions favoring near-maximal SAdoHcy synthesis (72 microM adenosine, 1.8 mM homocysteine), the synthesis rate in homogenates was 10 nmol/min per g wet weight. From the combined in vitro and perfusion studies, we conclude that S-adenosylhomocysteine hydrolase can contribute significantly to adenosine production in normoxic rat heart, but not during ischemia.

Myocardial xanthine oxidase/dehydrogenase.

High-energy phosphates in heart muscle deprived of oxygen are rapidly broken down to purine nucleosides and oxypurines. We studied the role of xanthine oxidase/dehydrogenase (EC 1.2.3.2/EC 1.2.1.37) in this process with novel high-pressure liquid chromatographic techniques. Under various conditions, including ischemia and anoxia, the isolated perfused rat heart released adenosine, inosine and hypoxanthine, and also substantial amounts of xanthine and urate. Allopurinol, an inhibitor of xanthine oxidase, greatly enhanced the release of hypoxanthine. From the purine release we calculated that the rat heart contained about 18 mU xanthine oxidase per g wet weight. Subsequently, we measured a xanthine oxidase activity of 9 mU/g wet wt. in rat-heart homogenate. When endogenous low molecular weight inhibitors were removed by gel-filtration, the activity increased to 31 mU/g wet wt. Rat myocardial xanthine oxidase seems to be present mainly in the dehydrogenase form, which upon storage at -20 degrees C is converted to the oxidase form.

Spontaneous and propagated contractions in rat cardiac trabeculae.

Sarcomere length measurement by microscopic and laser diffraction techniques in trabeculae of rat heart, superfused with Krebs-Henseleit solution at 21 degrees C, showed spontaneous local sarcomere shortening after electrically stimulated twitches. The contractions originated in a region of several hundred micrometers throughout the width of the muscle close to the end of the preparation that was damaged by dissection. The contractions propagated at a constant velocity along the trabeculae. The velocity of propagation increased from 0 to 10 mm/s in proportion to the number of stimuli (3-30) in a train of electrically evoked twitches at 2 Hz and at an external calcium ion concentration ([Ca++]o) of 1.5 mM. At a constant number of stimuli (n), the velocity of propagation increased from 0 to 15 mm/s with [Ca++]o increasing from 1 to 7 mM. In addition, increase of n and [Ca++]o led to an increase of the extent of local sarcomere shortening during the spontaneous contractions, and the occurrence of multiple contractions. Spontaneous contractions with much internal shortening and a high velocity of propagation frequently induced spontaneous synchronized contractions and eventually arrhythmias. Propagation of spontaneous contractions at low and variable velocity is consistent with the hypothesis that calcium leakage into damaged cells causes spontaneous calcium release from the overloaded sarcoplasmic reticulum in the damaged cells. This process propagates as a result of diffusion of calcium into adjacent cells, which triggers calcium release from their sarcoplasmic reticulum. We postulate that the propagation velocity depends on the intracellular calcium ion concentration, with increases with n and [Ca++]o.

A model of propagating calcium-induced calcium release mediated by calcium diffusion.

The effect of sudden local fluctuations of the free sarcoplasmic [Ca++]i in cardiac cells on calcium release and calcium uptake by the sarcoplasmic reticulum (SR) was calculated with the aid of a simplified model of SR calcium handling. The model was used to evaluate whether propagation of calcium transients and the range of propagation velocities observed experimentally (0.05-15 mm s(-1)) could be predicted. Calcium fluctuations propagate by virtue of focal calcium release from the SR, diffusion through the cytosol (which is modulated by binding to troponin and calmodulin and sequestration by the SR), and subsequently induce calcium release from adjacent release sites of the SR. The minimal and maximal velocities derived from the simulation were 0.09 and 15 mm s(-1) respectively. The method of solution involved writing the diffusion equation as a difference equation in the spatial coordinates. Thus, coupled ordinary differential equations in time with banded coefficients were generated. The coupled equations were solved using Gear's sixth order predictor-corrector algorithm for stiff equations with reflective boundaries. The most important determinants of the velocity of propagation of the calcium waves were the diastolic [Ca++]i, the rate of rise of the release, and the amount of calcium released from the SR. The results are consistent with the assumptions that calcium loading causes an increase in intracellular calcium and calcium in the SR, and an increase in the amount and rate of calcium released. These two effects combine to increase the propagation velocity at higher levels of calcium loading.

Altered contractile function in heart failure.

The syndrome of congestive heart failure (CHF) is an entity of ever increasing clinical significance. CHF is characterized by a steady decrease in cardiac pump function which is eventually lethal. The mechanisms that underlie the decline in cardiac function are incompletely understood. End-stage CHF often involves the general loss of functional myocytes, a hyperplasia of the extracellular matrix, ventricular chamber remodeling, and decreased myocyte function. This review article focuses on the latter aspect of CHF, mechanisms of decreased myocyte function. Recent data from studies on human myocardial tissue obtained in the setting of cardiac transplantation or from studies that employed experimental animal models of CHF have suggested depressed myocyte function. The mechanisms that may be involved in the decline of myocyte contractile function include alterations in (i) calcium handling, (ii) myofilament function, and (iii) the cytoskeleton. At present, however, it is not known how or to what degree these alterations in cellular processes contribute to the decline of in vivo cardiac pump function in CHF. Accurate knowledge regarding the cellular processes that participate in the development of CHF is critical to the development of innovative strategies aimed to combat CHF.

Synergistic effect of nifedipine and propranolol on adenosine (catabolite) release from ischemic rat heart.

Both nifedipine a calcium antagonist, and propranolol a beta-adrenergic blocker, are used as protective agents of the ischemic myocardium. In the clinical setting, the combination of the two drugs is used successfully although several case reports indicate potential dangers of the combination. For this reason we decided to study the combined effect of nifedipine and DL-propranolol in the isolated rat heart made ischemic for a short period of time. Apex displacement was taken as a measure of contractility. Release of the AMP catabolites adenosine, inosine, (hypo)xanthine and uric acid was used as a marker of ATP breakdown. Contractility during ischemia was not affected by the drugs. DL-Propranolol (30 or 150 micrograms/l) had no effect on ischemic myocardial purine release, while nifedipine (15 micrograms/l) reduced purine release during ischemia by 33% (P less than 0.02). The combination of 15 micrograms/l nifedipine and 150 micrograms/l DL-propranolol decreased purine release by 53% (P less than 0.005 vs. nifedipine). We conclude from these results that propranolol has a synergistic effect, adding to the beneficial action of nifedipine on ischemic myocardium.

Nifedipine reduces adenine nucleotide breakdown in ischemic rat heart.

An ATP-sparing effect has been demonstrated for a number of calcium antagonists. Nifedipine probably has a similar action, but data supporting this view are limited. Therefore we decided to study the effect of nifedipine on high-energy phosphate (and carbohydrate) metabolism in the ischemic rat heart. Langendorff preparations were made ischemic for less than 15 min. The reduction in coronary flow was 60 or 70%. Apex displacement during ischemia, a measure of contractility, was comparable for nifedipine-treated and untreated hearts. Ischemia caused a considerable release of the AMP catabolites adenosine, inosine and (hypo)xanthine, and of lactate. Nifedipine (10-100 micrograms/l) prevented this in a dose-dependent way. The highest dose reduced the release of purines and lactate by 90% (P less than 0.01) and 60% (P less than 0.001), respectively. The drug acted in a similar way during reperfusion. Due to ischemia, the adenylate energy charge (ATP + 0.5 ADP)/(ATP + ADP + AMP), decreased 15% (P less than 0.001); nifedipine at a concentration of 100 micrograms/l prevented this decrease (P less than 0.05). We conclude that nifedipine exerts a beneficial effect on myocardial adenine nucleotide metabolism during ischemia and reperfusion.

Measurements of active myocardial tension under a wide range of physiological loading conditions.

Active tension developed while cardiac muscle shortens has been studied extensively under afterloaded isotonic or isovelocity conditions. However, these are not true in vivo loading conditions. To obtain more physiological loading, we controlled sarcomere length to follow the time courses that we observed previously in a beating canine left ventricle. Sarcomere length was measured by laser diffraction in 12 rat cardiac trabeculae, superfused with Krebs-Henseleit solution (25 degrees C; [Ca] = 1.5 mM). Force was measured by a silicon strain gauge. Sarcomere length time courses were scaled slightly in time to account for temperature and species differences. We examined the relationships between active tension and sarcomere length under loading observed over a wide range of left ventricular preloads and afterloads, and at two sites. Under all loading conditions, active tension was not isotonic but declined steadily throughout the ejection period. While there were major differences in peak tension dependent on loading conditions and the incidence of 'pre-ejection' sarcomere shortening, these factors did not influence the relationship between sarcomere length and peak active tension. This study provides excellent illustrations of the potential differences in stress (1) within a ventricular wall, and (2) under different operating conditions. Moreover, it provides data for developing models of fiber contraction to be synthesized into a whole heart for predicting potential differences in stress at all sites and under all loading conditions.

Determinants of velocity of sarcomere shortening in mammalian myocardium.

Maximal unloaded velocity of shortening of cardiac muscle (Vo) depends on the level of activation of the contractile filaments. We have tested the hypothesis that this dependence may be caused by viscous resistance of the muscle to length changes. Twitch force (Fo) and sarcomere shortening were studied in trabeculae dissected from the right ventricle of rat myocardium, superfused with modified Krebs-Henseleit solution at 25 degrees C. Sarcomere length (SL) was measured by laser diffraction techniques; force was measured by a silicon strain gauge; velocity of sarcomere shortening was measured using the "isovelocity release" technique. Vo and Fo at slack SL were a sigmoidal function of [Ca2+]o, but Vo was more sensitive to [Ca2+]o (Km: 0.44 +/- 0.04 mM) than isometric twitch force (Km: 0.68 +/- 0.03 mM). At [Ca2+]o = 1.5 mM, Vo was independent of SL above 1.9 microns, but depended on SL at lower [Ca2+]o and always depended on SL < 1.9 microns. A constant relation was observed between Vo and Fo, irrespective whether Fo was altered by variation of [Ca2+]o or SL above slack length. Visco-elastic properties of unstimulated muscles were studied at SL = 2.0 microns by small linear length changes at varied velocities up to 40 microns/s. The force response to stretch, after correction for the contribution of static parallel elasticity, consisted of an exponential increase of force (tau = 4 ms) and an exponential decline after the stretch. This response would be expected from an arrangement of a viscous element in series with an elastic element. Viscous force increased in proportion to stretch velocity by 0.2-0.5% of Fo/micron/s up to 15 microns/s, while the calculated stiffness of the elastic component was 25-45 N.mm-3, suggesting that the most likely structural candidate for this visco-elastic element is titin. Dynamic stiffness at 500 Hz was proportional to instantaneous force during shortening and was 12% of stiffness at maximal twitch force when shortening occurred at Vo. This suggests that the number of active force generators, even at maximal activation, is strongly reduced during shortening at Vo. The observed relation between Vo and Fo could be explained by a model in which shortening velocity of the cardiac sarcomere depends on the level of activation and hence on the number of cross bridges supporting the viscous load.

The effects of sarcomere length and Ca++ on force and velocity of shortening in cardiac muscle.

The mechanism(s) underlying the effects of varied calcium concentration and of varied sarcomere length on force development and on the velocity of shortening in cardiac muscle were investigated. Sarcomere dynamics were investigated in thin trabeculae from rat heart with laser diffraction techniques; force was measured with silicon strain gauge 10 kHz. The unloaded velocity of sarcomere shortening was measured with the use of the 'isovelocity' technique. After study of intact muscles, superfused with modified Krebs-Henseleit solution at 25 degrees C, preparations were skinned with relaxing solution containing Triton X-100 and investigated at varied free Ca++. Force increased in all intact muscles continually with sarcomere length from 1.6-2.4 microns; the relation between force and sarcomere length was convex toward the ordinate at high Ca++0 and convex toward the abscissa at low Ca++0. Similar relations between force and sarcomere length were found in skinned trabeculae. Unloaded velocity of shortening (V0) was independent of time between 50 ms and 150 ms following onset of the twitch. V0 increased, in this period with increasing sarcomere length from 1.6 to 1.9 microns from 0 to 13 micron/s; above that length the velocity was constant. V0 increased at a sarcomere length of 2.00 microns with increasing Ca++0 to a maximum at Ca++0 = 1.2 mM above which V0 remained constant though force increased by 100%. These results suggest that the force-sarcomere length relation in cardiac muscle can be explained on the basis of length dependent activation of the contractile filaments to Ca++. Whether the different responses of force and of unloaded velocity of shortening to variations in sarcomere length and in Ca++ concentration are consistent with the hypothesis that force development and unloaded velocity of shortening are controlled by different mechanisms is discussed.

Rheology of myocardium. The relation between force, velocity, sarcomere length and activation in rat cardiac muscle.

The relations between force, shortening velocity and sarcomere length (F-V-SL) during cardiac contraction, underlie Starling's Law of the Heart. F-V-SL were investigated in isolated, intact and skinned trabeculae and myocytes from rat heart. SL and V were measured with laser diffraction techniques; F was measured with a silicon strain gauge. The "ascending" F-SL relation appeared to result from both length dependent sensitivity of the contractile system to activator calcium ions and the presence of restoring forces (Fr), residing in the collagen skeleton of the muscle. Fr increased exponentially with decreasing SL below slack length to 25% of maximal twitch force (Ft) at SL = 1.60 microns. V was inversely proportional to the load and attained a maximum at zero load (Vo). Vo increased with factors that increased F: [Ca++], SL, and time during the twitch. Vo reached a maximum and remained constant (13.5 microns/s) when F attained or exceeded 50% of its maximum value. Viscous force in the passive muscle increased with V to a maximum of 4% of Ft at V = 40 microns/s. The relation between Vo and these factors could be predicted by a model of contraction in which the measured visco-elastic properties of myocardium were incorporated, while the truly unloaded maximal velocity of sarcomere shortening was assumed to be independent of the level of activation of the contractile filaments. A model of the cardiac cycle which explains the relation between Frank's and Starling's laws is presented.

Impact of ejection on magnitude and time course of ventricular pressure-generating capacity.

This study focuses on elucidating how ventricular afterloading conditions affect the time course of change of left ventricular pressure (LVP) throughout the cardiac cycle, with particular emphasis on revealing specific limitations in the time-varying elastance model of ventricular dynamics. Studies were performed in eight isolated canine hearts ejecting into a simulated windkessel afterload. LVP waves measured (LVPm) during ejection were compared with those predicted (LVPpred) according to the elastance theory. LVPm exceeded LVPpred from a time point shortly after the onset of ejection to the end of the beat. The instantaneous difference between LVPm and LVPpred increased steadily as ejection proceeded and reached between 45 and 65 mmHg near end ejection. This was in large part due to an average 35-ms prolongation of the time to end systole (tes) in ejecting compared with isovolumic beats. The time constant of relaxation was decreased on ejecting beats so that, despite the marked prolongation of tes, the overall duration of ejecting contractions was not greater than that of isovolumic beats. The results demonstrate a marked ejection-mediated enhancement and prolongation of ventricular pressure-generating capacity during the ejection phase of the cardiac cycle with concomitant acceleration of relaxation. None of these factors are accounted for by the time-varying elastance theory.

Force and velocity of sarcomere shortening in trabeculae from rat heart. Effects of temperature.

The effect of temperature on the force-sarcomere velocity relation (20 degrees, 25 degrees, and 30 degrees C) and maximum velocity of sarcomere shortening (Vo; range 15 degrees-35 degrees C) was studied in trabeculae from rat heart. Sarcomere length and Vo were measured by laser diffraction techniques. Sarcomere length and sarcomere velocity, determined from each of the first-order diffraction lines, differed by less than 4%. Slack sarcomere length in the trabeculae appeared to be 1.9 microns. Isovelocity release techniques were used to obtain sarcomere velocity and Vo directly. Sarcomere velocity was measured at SL = 1.9-2.0 microns for elimination of contributions of parallel elastic force and restoring force to the external load of the sarcomeres. Peak twitch force development (Fo) was maximal (Fo-max) at 25 degrees C at [Ca2+]o = 1.5 mM. Lowering of the temperature below 25 degrees C led to development of spontaneous sarcomere activity and depression of Fo; both responses could be prevented by the addition of 0.5 mM procaine. Increase of temperature above 25 degrees C reduced twitch duration and Fo. Hill's rectangular hyperbola fitted the force-velocity data if the load during shortening was less than 70% of Fo. Vo appeared to be independent of the level of activation at all temperatures when Fo was maintained above 90% of Fo-max, either by an increase of [Ca2+]o (to 3.0 mM) or by paired pulse stimulation. Vo increased with increasing temperature; the parameter a, calculated from force-velocity relations measured at 20 degrees, 25 degrees, and 30 degrees C, decreased with increasing temperature. The Arrhenius plot of Vo was studied in detail over a wider temperature range (15 degrees-35 degrees C) and in smaller temperature increments. The relation was linear between 18 degrees and 33 degrees C; the observed Q10, defined as the ratio of Vo measured at temperature (T) over Vo at T-10 degrees C, was 4.6 A Q10 of 4.6 for Vo is consistent with the reported temperature dependence of rat cardiac actin-activated myosin ATPase, which suggests that the same reaction step may limit the activity of the enzyme in vitro and during shortening of the cardiac sarcomeres at zero external load.

Effect of adenosine on contractile state and oxygen consumption in isolated rat hearts.

We studied the effects of adenosine on oxygen consumption and contractile state in 17 isolated, crystalloid-perfused, isovolumically contracting rat heart preparations at constant coronary flow. In 10 experiments we determined adenosine-contractile state dose-response relationships in three groups of hearts using two different perfusates and in the presence and absence of adrenergic blockade. Adenosine consistently reduced contractile state in a dose-dependent fashion, reducing the ventricular pressure developed at a constant ventricular volume by 24% on average at its maximal effect. An adenosine concentration of 111 microM on average produced 50% of the maximal effect. In seven experiments we determined the end-systolic pressure-volume and oxygen consumption-pressure-volume area (MVO2-PVA) relationships at two calcium concentrations (1.5 and 0.75 mM) and with adenosine 400 microM (1.5 mM Ca2+). Contractile state was indexed by the developed pressure at a ventricular volume of 0.3 ml (P0.3). Compared with 1.5 mM Ca2+, mean P0.3 was reduced by 38% with 0.75 mM Ca2+ and by 18% with adenosine. Whereas the MVO2-PVA slopes did not change, the mean MVO2 intercept was reduced by 22% with 0.75 mM Ca2+ and by 13% with adenosine. The MVO2 intercept, which represents the oxygen consumed by the unloaded heart, was directly related to P0.3. This relationship, which represents the oxygen cost of contractility, was not affected by adenosine. We conclude that at constant coronary flow and perfusion pressure adenosine reduces myocardial contractility and the oxygen consumed for excitation-contraction coupling. However, adenosine does not affect the slope of the MVO2-PVA relation or the oxygen cost of contractility.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of calcium and EMD-53998 on oxygen consumption in isolated canine hearts.

BACKGROUND: Most positive inotropic agents increase cardiac contractility by increasing the amount of Ca2+ cycled with each beat. The additional amount of oxygen that is consumed by the heart to cycle this additional Ca2+ is believed to reduce myocardial efficiency. On the other hand, it has been suggested that the agent EMD-53998 increases the Ca2+ sensitivity of the contractile proteins without affecting the intracellular Ca2+ transient in cardiac muscle. Therefore, application of this agent may increase cardiac contractility without decreasing myocardial efficiency. The purpose of the present study was to test this hypothesis. METHODS AND RESULTS: We measured myocardial oxygen consumption (MVO2) in six isolated, isovolumically beating blood-perfused canine hearts. The hearts were paced at 120 beats per minute. Contractility was varied in each heart by infusion of either CaCl2 or EMD-53998. With infusion of either agent, MVO2 was a linearly proportional function of contractility. No significant difference between CaCl2 and EMD-53998 could be detected in the interrelation between contractility and MVO2. CONCLUSIONS: We conclude that the "calcium-sensitizing agent" EMD-53998 is a potent positive inotropic agent in the isolated, blood-perfused canine heart. However, EMD-53998 does not provide an energetic advantage over currently used positive inotropic agents.

An internal viscous element limits unloaded velocity of sarcomere shortening in rat myocardium.

1. Peak twitch force (F0) and sarcomere length (SL) were measured in trabeculae that had been dissected from the right ventricle of rat heart and that were superfused with a modified Krebs-Henseleit solution at 25 degrees C. Sarcomere length was measured by laser diffraction techniques. Force was measured with a silicone strain gauge. Unloaded velocity of sarcomere shortening (V0) was measured by the 'isovelocity release' technique. 2. At [Ca2+]o = 1.5 mM and SL below 1.9 microns, V0 increased in proportion to SL, while V0 was independent of SL above 1.9 microns. At [Ca2+]o = 0.5 mM, V0 was proportional to SL up to 2.2 microns. At [Ca2+]o = 0.2 mM, V0 was proportional to SL up to 2.3 microns which is the longest SL that we were able to study in our trabeculae. 3. A unique relationship was observed between V0 and F0, irrespective of whether F0 was altered by variation of [Ca2+]o or sarcomere length above slack length. 4. Passive viscosity (Fv) was measured during the pause between contractions in the presence of 1.5 mM [Ca2+bdo and in the range SL = 2.0-2.1 microns by applying 0.1 micron stretches at various velocities up to v = 30 microns s-1. The force response to stretch, corrected for the contribution of parallel elastic force, showed viscoelastic characteristics with an exponential increase to a maximum (Fv) during stretch and an exponential decline after the end of the stretch. Fv increased, by 0.3%F0 microns-1 s-1, in proportion to v < 5 microns s-1; the increase of Fv was smaller at higher v, suggesting non-Newtonian viscous properties. 5. The time constant of the increase of force during the stretch decreased (tau rise congruent to 7 ms to tau rise congruent to 4 ms) with increases in v (congruent to 4 microns s-1 to v congruent to 10 microns s-1; P = 0.02). The time constant of decay of force at the end of the stretch also decreased with increases in v (tau decay congruent to 8 ms at v congruent to 4 microns s-1 to tau decay congruent to 3 ms at v congruent to 30 microns s-1; P < 0.001). Calculated stiffness of the elastic term of the viscoelastic element was independent of v, i.e. 45-50 N mm-3.(ABSTRACT TRUNCATED AT 400 WORDS)