The role of passive structures in force enhancement of skeletal muscles following active stretch

We recently found that force enhancement following active stretch in skeletal muscles is accompanied by an increase in passive force following deactivation (J. Exp. Biol. 205 (2002) 1275). However, it is not known if this increase in passive force contributes to the force enhancement observed in the active muscle, and if it is observed at all muscle lengths. The purposes of this study were to quantify the amount of passive force increase as a function of muscle lengths, and to determine if this passive force contributes to the force enhancement observed in the active muscle. Experiments were performed on cat soleus (n = 24) using techniques published previously (J. Biomech. 30(9) (1997) 865). Conceptually, tests involved comparisons of force enhancement and passive force increase for a variety of stretch tests in soleus. Furthermore, in one test, activation of the soleus was interrupted for 1s in the force-enhanced state, and soleus was then re-activated. We found that total force enhancement and passive force increase were positively correlated for all test conditions, that passive force increase following stretch of the active soleus only occurred at muscle lengths corresponding to the descending limb of the force-length relationship, that increases in passive force for a given stretch magnitude became greater at long muscle lengths, and that upon reactivation, there was a remnant passive force enhancement. We conclude from these results that the passive force enhancement following stretch of an active muscle contributes to the total force enhancement, that this passive contribution increases with increasing muscle length, and that there must be at least one other factor than passive force increase that contributes to the total force enhancement, as the passive force increase was always smaller than the total force enhancement. A by-product of this investigation was that we observed a shift in the passive force-length relationship that was dependent on muscle activation, stretch magnitude and muscle length. Therefore, the passive force-length relationship is not a constant property of skeletal muscle, but depends critically on the muscle's contractile history.     

The effects of muscle stretching and shortening on isometric forces on the descending limb of the force-length relationship

The purpose of this study was to examine the effects of stretching and shortening on the isometric forces at different lengths on the descending limb of the force-length relationship. Cat soleus (N = 10) was stretched and shortened by various amounts on the descending limb of the force-length relationship, and the steady-state forces following these dynamic contractions were compared to the isometric forces at the corresponding muscle lengths. We found a shift of the force-length relationship to greater force values following muscle stretching, and to smaller force values following muscle shortening. Shifts in both directions critically depended on the magnitude of stretching/shortening and the final muscle length. We confirm recent findings that the steady-state isometric force following some stretch conditions clearly exceeded the maximal isometric forces at optimum muscle length, and that force enhancement was associated with an increase in the passive force, i.e., a passive force enhancement. When the passive force enhancement was subtracted from the total force enhancement, forces following stretch were always equal to or smaller than the isometric force at optimum muscle length. Together, these findings led to the conclusions: (a). that force enhancement is composed of an "active and a "passive" component; (b). that the "passive" component of force enhancement allows for forces greater than the maximal isometric forces at the muscle's optimum length; and (c). that force enhancement and force depression are critically affected by muscle length and stretch/shortening amplitude.     

Modeling the oscillation dynamics of activated airway smooth muscle strips

When strips of activated airway smooth muscle are stretched cyclically, they exhibit force-length loops that vary substantially in both position and shape with the amplitude and frequency of the stretch. This behavior has recently been ascribed to a dynamic interaction between the imposed stretch and the number of actin-myosin interactions in the muscle. However, it is well known that the passive rheological properties of smooth muscle have a major influence on its mechanical properties. We therefore hypothesized that these rheological properties play a significant role in the force-length dynamics of activated smooth muscle. To test the plausibility of this hypothesis, we developed a model of the smooth muscle strip consisting of a force generator in series with an elastic component. Realistic steady-state force-length loops are predicted by the model when the force generator obeys a hyperbolic force-velocity relationship, the series elastic component is highly nonlinear, and both elastic stiffness and force generation are adjusted so that peak loop force equals isometric force. We conclude that the dynamic behavior of airway smooth muscle can be ascribed in large part to an interaction between connective tissue rheology and the force-velocity behavior of contractile proteins.     

Force enhancement above the initial isometric force on the descending limb of the force-length relationship

Edman et al. (J. General Physiol. 80 (1982) 769) observed in single fibres of frog that the steady-state forces following active fibre stretch were greater than the purely isometric force obtained at the length from which the stretch was initiated. Operating on the descending limb of the force-length relationship, such a result can only be explained within the framework of the sarcomere length non-uniformity theory, if some fibre segments shortened during the fibre stretch. However, such a result was not found, leaving Edman's observation unexplained. Force enhancement above the initial isometric force has not been investigated systematically in whole muscle, and therefore it is not known whether this property is also part of whole muscle mechanics. The purpose of this study was to test if the steady-state forces following active stretch of cat semitendinosus were greater than the corresponding purely isometric forces at the muscle length from which the stretch was started. Cat semitendinosus was stretched by various amounts on the descending limb of the force-length relationship, and the steady-state forces following these stretches were compared to the corresponding isometric forces at the initial and final muscle lengths. In 109 of 131 tests, the steady-state forces following stretching were greater than the isometric forces at the initial muscle lengths. Force enhancement increased with increasing amounts of stretching, and force enhancement above the initial isometric force was more likely to occur following stretches of great compared to small amplitude. Passive forces following active muscle stretching were often significantly greater than the passive forces at the same muscle length following an isometric contraction or a passive stretching of the muscle. This observation was made consistently at the longest muscle lengths tested. It appears, therefore, that there is a passive force that accounts for part of the force enhancement above the isometric force at the initial muscle length, and that provides increased passive force when a muscle is actively, rather than passively, stretched at long muscle lengths. We conclude that cat semitendinosus demonstrates steady-state force enhancement above the corresponding purely isometric force at the initial muscle length on the descending limb of the force-length relationship for many contractile conditions, and that a unique, and so far undetected, passive, parallel element contributes to this force enhancement, particularly at long muscle lengths where muscle is assumed to be most vulnerable to injuries associated with sarcomere length instability.     

Activation of G protein-coupled estrogen receptor induces endothelium-independent relaxation of coronary artery smooth muscle

Estrogens can either relax or contract arteries via rapid, nongenomic mechanisms involving classic estrogen receptors (ER). In addition to ERα and ERβ, estrogen may also stimulate G protein-coupled estrogen receptor 1 (GPER) in nonvascular tissue; however, a potential role for GPER in coronary arteries is unclear. The purpose of this study was to determine how GPER activity influenced coronary artery reactivity. In vitro isometric force recordings were performed on endothelium-denuded porcine arteries. These studies were augmented by RT-PCR and single-cell patch-clamp experiments. RT-PCR and immunoblot studies confirmed expression of GPER mRNA and protein, respectively, in smooth muscle from either porcine or human coronary arteries. G-1, a selective GPER agonist, produced a concentration-dependent relaxation of endothelium-denuded porcine coronary arteries in vitro. This response was attenuated by G15, a GPER-selective antagonist, or by inhibiting large-conductance calcium-activated potassium (BK(Ca)) channels with iberiotoxin, but not by inhibiting NO signaling. Last, single-channel patch-clamp studies demonstrated that G-1 stimulates BK(Ca) channel activity in intact smooth muscle cells from either porcine or human coronary arteries but had no effect on channels isolated in excised membrane patches. In summary, GPER activation relaxes coronary artery smooth muscle by increasing potassium efflux via BK(Ca) channels and requires an intact cellular signaling mechanism. This novel action of estrogen-like compounds may help clarify some of the controversy surrounding the vascular effects of estrogens.     

Biaxial vasoactivity of porcine coronary artery

The passive mechanical properties of blood vessel mainly stem from the interaction of collagen and elastin fibers, but vessel constriction is attributed to smooth muscle cell (SMC) contraction. Although the passive properties of coronary arteries have been well characterized, the active biaxial stress-strain relationship is not known. Here, we carry out biaxial (inflation and axial extension) mechanical tests in right coronary arteries that provide the active coronary stress-strain relationship in circumferential and axial directions. Based on the measurements, a biaxial active strain energy function is proposed to quantify the constitutive stress-strain relationship in the physiological range of loading. The strain energy is expressed as a Gauss error function in the physiological pressure range. In K(+)-induced vasoconstriction, the mean ± SE values of outer diameters at transmural pressure of 80 mmHg were 3.41 ± 0.17 and 3.28 ± 0.24 mm at axial stretch ratios of 1.3 and 1.5, respectively, which were significantly smaller than those in Ca(2+)-free-induced vasodilated state (i.e., 4.01 ± 0.16 and 3.75 ± 0.20 mm, respectively). The mean ± SE values of the inner and outer diameters in no-load state and the opening angles in zero-stress state were 1.69 ± 0.04 mm and 2.25 ± 0.08 mm and 126 ± 22°, respectively. The active stresses have a maximal value at the passive pressure of 80-100 mmHg and at the active pressure of 140-160 mmHg. Moreover, a mechanical analysis shows a significant reduction of mean stress and strain (averaged through the vessel wall). These findings have important implications for understanding SMC mechanics.     

Simulated microgravity effects on the rat carotid and femoral arteries: role of contractile protein expression and mechanical properties of the vessel wall.

The goal of this study was to determine the effects of microgravity on myofilament protein expression and both passive and active length-force relationships in carotid and femoral arteries. Microgravity was simulated by 20-day hindlimb unweighting (HU) in Wistar male rats, and carotid and femoral artery segments were isolated from both HU and control (CTL) rats for Western blot and length-force analysis. Western blots revealed that HU significantly decreased myosin light chain-20 (MLC-20) protein levels in both carotid and femoral arteries and decreased myosin heavy chain (MHC) in femoral artery. alpha-Actin levels were not altered by HU treatment in either artery. Length-force analysis demonstrated that HU did not change either passive or active length-force relationships in the femoral artery. HU-treated arterial rings developed significantly less force to 100 mM K(+) than CTL, but optimal lengths were identical. In the carotid artery, length-active force curves were identical for both CTL and HU; however the length-passive force curve for HU-treated rings exhibited a steeper slope than CTL, suggesting decreased compliance of the artery wall. In conclusion, our data suggest that the HU-induced decreases in both MLC-20 and MHC in femoral artery are responsible for the decreased contraction to 100 mM K(+) in HU-treated femoral artery rings. In the carotid artery, the HU-induced decrease in vessel wall compliance may counter any decrease in contractility caused by the decreased MLC-20 levels.     

Adaptation of active tone in the mouse descending thoracic aorta under acute changes in loading

Arteries can adapt to sustained changes in blood pressure and flow, and it is thought that these adaptive processes often begin with an altered smooth muscle cell activity that precedes any detectable changes in the passive wall components. Yet, due to the intrinsic coupling between the active and passive properties of the arterial wall, it has been difficult to delineate the adaptive contributions of active smooth muscle. To address this need, we used a novel experimental–computational approach to quantify adaptive functions of active smooth muscle in arterial rings excised from the proximal descending thoracic aorta of mice and subjected to short-term sustained circumferential stretches while stimulated with various agonists. A new mathematical model of the adaptive processes was derived and fit to data to describe and predict the effects of active tone adaptation. It was found that active tone was maintained when the artery was adapted close to the optimal stretch for maximal active force production, but it was reduced when adapted below the optimal stretch; there was no significant change in passive behavior in either case. Such active adaptations occurred only upon smooth muscle stimulation with phenylephrine, however, not stimulation with KCl or angiotensin II. Numerical simulations using the proposed model suggested further that active tone adaptation in vascular smooth muscle could play a stabilizing role for wall stress in large elastic arteries.     

A simple model describing the elastic properties of human umbilical arterial smooth muscle

Elastic properties of cylindrical segments of 71 normal double and 4 single human umbilical arteries were studied. This specimen is rich in vascular smooth muscle in comparison with other great arteries. Outer diameter versus intraluminal pressure characteristic curves were taken with decreasing pressures in vitro in different contraction states. Tangential force and circumferential incremental elastic modulus were computed. A sum of 222 curves was analysed. The investigations showed that if we take tangential force and outer radius values measured at the same pressure levels but in different contraction states, then a similar proportional change in tangential force will induce a similar proportional passive change in the outer radius, to some extent independently of the degree of active tangential shortening of the segment. For example to induce a 10% passive decrease of the outer radius from values measured at 100 mm Hg intraluminal pressure, tangential force had to be decreased by 76.7 +/- 9.9% in single relaxed arteries, and by 79.6 +/- 0.8% in normal double relaxed segments. These values corresponded to intraluminal pressure levels of 26.4 +/- 4.9 mm Hg and 23.1 +/- 0.9 mm Hg, respectively. In 30% active spontaneous shortening to reach the same 10% passive decrease in outer radius from the value measured at 100 mm Hg, tangential force had to be decreased by 75.2 +/- 1.9% which corresponded to 29.6 +/- 20 mm Hg. The same values in 5-HT induced contraction, 30% active shortening were 76.7 +/- 2.0% and 28.4 +/- 2.6 mm Hg, respectively. In addition to the similarity of relative changes in tangential force, the pressure levels were to some extent also similar. These data suggest that elastic elements in the human umbilical arterial smooth muscle may be organized in such a way as to ensure similar prestretch of similar elastic elements at similar pressures independently of the degree of active shortening of the circumference.     

Stretch-induced,steady-state force enhancement in single skeletal muscle fibers exceeds the isometric force at optimum fiber length

Stretch-induced force enhancement has been observed in a variety of muscle preparations and on structural levels ranging from single fibers to in vivo human muscles. It is a well-accepted property of skeletal muscle. However, the mechanism causing force enhancement has not been elucidated, although the sarcomere-length non-uniformity theory has received wide support. The purpose of this paper was to re-investigate stretch-induced force enhancement in frog single fibers by testing specific hypotheses arising from the sarcomere-length non-uniformity theory. Single fibers dissected from frog tibialis anterior (TA) and lumbricals (n=12 and 22, respectively) were mounted in an experimental chamber with physiological Ringer's solution (pH=7.5) between a force transducer and a servomotor length controller. The tetantic force-length relationship was determined. Isometric reference forces were determined at optimum length (corresponding to the maximal, active, isometric force), and at the initial and final lengths of the stretch experiments. Stretch experiments were performed on the descending limb of the force-length relationship after maximal tetanic force was reached. Stretches of 2.5-10% (TA) and 5-15% lumbricals of fiber length were performed at 0.1-1.5 fiber lengths/s. The stretch-induced, steady-state, active isometric force was always equal or greater than the purely isometric force at the muscle length from which the stretch was initiated. Moreover, for stretches of 5% fiber length or greater, and initiated near the optimum length of the fiber, the stretch-enhanced active force always exceeded the maximal active isometric force at optimum length. Finally, we observed a stretch-induced enhancement of passive force. We conclude from these results that the sarcomere length non-uniformity theory alone cannot explain the observed force enhancement, and that part of the force enhancement is associated with a passive force that is substantially greater after active compared to passive muscle stretch.     

Altered biomechanical properties of carotid arteries in two mouse models of muscular dystrophy.

Muscular dystrophy is characterized by skeletal muscle weakness and wasting, but little is known about possible alterations to the vasculature. Many muscular dystrophies are caused by a defective dystrophin-glycoprotein complex (DGC), which plays an important role in mechanotransduction and maintenance of structural integrity in muscle cells. The DGC is a group of membrane-associated proteins, including dystrophin and sarcoglycan-delta, that helps connect the cytoskeleton of muscle cells to the extracellular matrix. In this paper, mice lacking genes encoding dystrophin (mdx) or sarcoglycan-delta (sgcd-/-) were studied to detect possible alterations to vascular wall mechanics. Pressure-diameter and axial force-length tests were performed on common carotid arteries from mdx, sgcd-/-, and wild-type mice in active (basal) and passive smooth muscle states, and functional responses to three vasoactive compounds were determined at constant pressure and length. Apparent biomechanical differences included the following: mdx and sgcd-/- arteries had decreased distensibilities in pressure-diameter tests, with mdx arteries exhibiting elevated circumferential stresses, and mdx and sgcd-/- arteries generated elevated axial loads and stresses in axial force-length tests. Interestingly, however, mdx and sgcd-/- arteries also had significantly lower in vivo axial stretches than did the wild type. Accounting for this possible adaptation largely eliminated the apparent differences in circumferential and axial stiffness, thus suggesting that loss of DGC proteins may induce adaptive biomechanical changes that can maintain overall wall mechanics in response to normal loads. Nevertheless, there remains a need to understand better possible vascular adaptations in response to sustained altered loads in patients with muscular dystrophy.     

Increased arterial load alters aortic structural and functional properties during embryogenesis

As in the adult dorsal aorta, the embryonic dorsal aorta is an important determinant of cardiovascular function, and increased stiffness may have secondary effects on cardiac and microcirculatory development. We previously showed that acutely and chronically increased arterial load via vitelline artery ligation (VAL) increases systemic arterial stiffness. To test the hypothesis that local dorsal aortic stiffness also increases, we measured aortic pulse-wave velocity (PWV) and assessed the active and passive properties (stress and strain) of isolated aortic segments. PWV along the dorsal aorta increased acutely and chronically after VAL. Analysis of isolated aortic active properties suggests that load-exposed aortas experienced higher stress, but not strain, at similar intraluminal pressures. When smooth muscle tone was relaxed, strain decreased in VAL vessels, whereas stress became similar to control vessels. Immunohistochemical analysis revealed that although aortic smooth muscle alpha-actin content was similar between groups, more cell layers expressed smooth muscle alpha-actin, and myocyte cell shape was markedly rounder in VAL embryos. Additionally, aortic and perivascular collagen type I and III content significantly increased in load-exposed VAL vessels. Increased production of these proteins is consistent with the observed increase in aortic PWV and decreased strain in VAL passive aortic segments. Thus the embryonic dorsal aorta is sensitive to increased arterial load and adapts by altering its material properties via changes in collagen content.     

Passive and active mechanical properties of the superficial and deep digital flexor muscles in the forelimbs of anesthetized Thoroughbred horses

The superficial (SDF) and deep digital flexor (DDF) muscles are critical for equine forelimb locomotion. Knowledge of their mechanical properties will enhance our understanding of limb biomechanics. Muscle contractile properties derived from architectural-based algorithms may overestimate real forces and underestimate shortening capacity because of simplistic assumptions regarding muscle architecture. Therefore, passive and active (=total - passive) force-length properties of the SDF and DDF muscles were measured directly in vivo. Muscles from the right forelimbs of four Thoroughbred horses were evaluated during general anesthesia. Limbs were fixed to an external frame with the muscle attached to a linear actuator and load cell. Each muscle was stretched from an unloaded state to a range of prefixed lengths, then stimulated while held at that length. The total force did not exceed 4000 N, the limit for the clamping device. The SDF and DDF muscles produced 716+/-192 and 1577+/-203 N maximum active isometric force (F(max)), had ascending force-length ranges (R(asc)) of 5.1+/-0.2 and 9.1+/-0.4 cm, and had passive stiffnesses of 1186+/-104 and 1132+/-51 N/cm, respectively. The values measured for F(max) were much smaller than predicted based on conservative estimates of muscle specific tension and muscle physiological cross-sectional area. R(asc) were much larger than predicted based on muscle fiber length estimates. These data suggest that accurate prediction of the active mechanical behavior of architecturally complex muscles such as the equine DDF and SDF requires more sophisticated algorithms.     

Reports of the length dependence of fatigue are greatly exaggerated.

Relative force depression associated with muscle fatigue is reported to be greater when assessed at short vs. long muscle lengths. This appears to be due to a rightward shift in the force-length relationship. This rightward shift may be caused by stretch of in-series structures, making sarcomere lengths shorter at any given muscle length. Submaximal force-length relationships (twitch, double pulse, 50 Hz) were evaluated before and after repetitive contractions (50 Hz, 300 ms, 1/s) in an in situ preparation of the rat medial gastrocnemius muscle. In some experiments, fascicle lengths were measured with sonomicrometry. Before repetitive stimulation, fascicle lengths were 11.3 +/- 0.8, 12.8 +/- 0.9, and 14.4 +/- 1.2 mm at lengths corresponding to -3.6, 0, and 3.6 mm where 0 is a reference length that corresponds with maximal active force for double-pulse stimulation. After repetitive stimulation, there was no change in fascicle lengths; these lengths were 11.4 +/- 0.8, 12.6 +/- 0.9, and 14.2 +/- 1.2 mm. The length dependence of fatigue was, therefore, not due to a stretch of in-series structures. Interestingly, the rightward shift that was evident when active force was calculated in the traditional way (subtraction of the passive force measured before contraction) was not seen when active force was calculated by subtracting the passive force that was associated with the fascicle length reached at the peak of the contraction. This calculation is based on the assumption that passive force decreases as the fascicles shorten during a fixed-end contraction. This alternative calculation revealed similar postfatigue absolute active force depression at all lengths. In relative terms, a length dependence of fatigue was still evident, but this was greatly diminished compared with that observed when active force was calculated with the traditional method.     

Length-tension relationships of small arteries, veins, and lymphatics from the rat mesenteric microcirculation

The passive and active length-tension relationships of isolated rat mesenteric lymphatics ( approximately 150 microm ID), and adjacent small arteries ( approximately 240 microm) and veins ( approximately 275 microm) were compared under isometric conditions using a wire myograph. About 60% of the lymphatic vessels developed spontaneous contractions in physiological saline solution at nominal preload. To maximally activate smooth muscle, 145 mM K(+) + 5 x 10(-5) M norepinephrine was used for arteries, and 145 mM K(+) + 1 x 10(-6) M substance P was used for lymphatics and veins. In response, arteries exhibited monotonic force development to a plateau level, whereas lymphatics and veins showed biphasic force development, consisting of a transient force peak followed by partial relaxation to a plateau over approximately 5 min. The passive and the active length-tension curves were similar in shape among all three vessels. However, the maximal active tension of arteries (3.4 +/- 0.42 mN/mm) was significantly greater than peak active tension (0.59 +/- 0.04 mN/mm) or plateau tension (0.20 +/- 0.04 mN/mm) in small veins and greater than peak active tension (0.34 +/- 0.02 mN/mm) or plateau tension (0.21 +/- 0.02 mN/mm) in lymphatics. Maximal active medial wall stress was similar between lymphatics and veins but was approximately fivefold higher in small arteries. For lymphatics, the pressure calculated from the optimal preload was significantly higher than that found previously in isobaric studies of isolated lymphatics, suggesting the capacity to operate at higher than normal pressures for increased responsiveness. Our results represent the first mechanical comparisons of arterial, venous, and lymphatic vessels in the same vasculature.     

Characterization of the passive mechanical properties of spine muscles across species

Passive mechanical properties differ between muscle groups within a species. Altered functional demands can also shift the passive force-length relationship. The extent that passive mechanical properties differ within a muscle group (e.g. spine extensors) or between homologous muscles of different species is unknown. It was hypothesized that multifidus, believed to specialize in spine stabilization, would generate greater passive tensile stresses under isometric conditions than erector spinae, which have more generalized functions of moving and stabilizing the spine; observing greater multifidus moduli in different species would strengthen this hypothesis. Permeabilized fibre bundles (n = 337) from the multifidus and erector spinae of mice, rats, and rabbits were mechanically tested. A novel logistic function was fit to the experimental data to fully characterize passive stress and modulus. Species had the greatest effect on passive muscle parameters with mice having the largest moduli at all lengths. Rats generated less passive stress than rabbits due to a shift of the passive force-length relationship towards longer muscle lengths. Rat multifidus generated slightly greater stresses than erector spinae, but no differences were observed between mouse muscles. The secondary objective was to determine the parameters required to simulate the passive force-length relationship. Experimental data were compared to the passive muscle model in OpenSim. The default OpenSim model, optimized for hindlimb muscles, did not fit any of the spine muscles tested; however, the model could accurately simulate experimental data after adjusting the input parameters. The optimal parameters for modelling the passive force-length relationships of spine muscles in OpenSim are presented.     

Active force inhibition and stretch-induced force enhancement in frog muscle treated with BDM.

There is evidence that the stretch-induced residual force enhancement observed in skeletal muscles is associated with 1) cross-bridge dynamics and 2) an increase in passive force. The purpose of this study was to characterize the total and passive force enhancement and to evaluate whether these phenomena may be associated with a slow detachment of cross bridges. Single fibers from frog lumbrical muscles were placed at a length 20% longer than the plateau of the force-length relationship, and active and passive stretches (amplitudes of 5 and 10% of fiber length and at a speed of 40% fiber length/s) were performed. Experiments were conducted in Ringer solution and with the addition of 2, 5, and 10 mM of 2,3-butanedione monoxime (BDM), a cross-bridge inhibitor. The steady-state active and passive isometric forces after stretch of an activated fiber were higher than the corresponding forces measured after isometric contractions or passive stretches. BDM decreased the absolute isometric force and increased the total force enhancement in all conditions investigated. These results suggest that total force enhancement is directly associated with cross-bridge kinetics. Addition of 2 mM BDM did not change the passive force enhancement after 5 and 10% stretches. Addition of 5 and 10 mM did not change (5% stretches) or increased (10% stretches) the passive force enhancement. Increasing stretch amplitudes and increasing concentrations of BDM caused relaxation after stretch to be slower, and because passive force enhancement is increased at the greatest stretch amplitudes and the highest BDM concentrations, it appears that passive force enhancement may be related to slow-detaching cross bridges.     

A stepwise method for the isolation of endothelial cells and smooth muscle cells from individual canine coronary arteries

Methods for the stepwise isolation of endothelial cells and smooth muscle cells from individual canine coronary arteries are described. Both cell types can be isolated in pure culture with high yields. Dogs are a common species used in the study of atherosclerosis and coronary artery disease. Capacity to isolate endothelial cells and smooth muscle cells from individual canine coronary arteries should prove useful in the study of coronary artery disease.     

Influence of coronary artery diameter on eNOS protein content

The purpose of this study was to test the hypothesis that the content of endothelial nitric oxide synthase (eNOS) protein (eNOS protein/g total artery protein) increases with decreasing artery diameter in the coronary arterial tree. Content of eNOS protein was determined in porcine coronary arteries with immunoblot analysis. Arteries were isolated in six size categories from each heart: large arteries [301- to 2,500-microm internal diameter (ID)], small arteries (201- to 300-microm ID), resistance arteries (151- to 200-microm ID), large arterioles (101- to 150-microm ID), intermediate arterioles (51- to 100-microm ID), and small arterioles(<50-microm ID). To obtain sufficient protein for analysis from small- and intermediate-sized arterioles, five to seven arterioles 1-2 mm in length were pooled into one sample for each animal. Results establish that the number of smooth muscle cells per endothelial cell decreases from a number of 10 to 15 in large coronary arteries to 1 in the smallest arterioles. Immunohistochemistry revealed that eNOS is located only in endothelial cells in all sizes of coronary artery and in coronary capillaries. Contrary to our hypothesis, eNOS protein content did not increase with decreasing size of coronary artery. Indeed, the smallest coronary arterioles had less eNOS protein per gram of total protein than the large coronary arteries. These results indicate that eNOS protein content is greater in the endothelial cells of conduit arteries, resistance arteries, and large arterioles than in small coronary arterioles.