Active Tension In Response Research Articles

Empagliflozin, an SGLT2i, relaxes coronary smooth muscle at pharmacological concentrations

Morbidity and mortality due to heart failure with preserved ejection fraction (HFpEF) is increasing and there are no universally recognized strategies for treatment. Sodium-glucose co-transporter type 2 inhibitors (SGLT2i), such as Empagliflozin, have been shown to reduce myocardial stiffness and improve coronary microvascular performance, but mechanisms are unclear. Previous studies in rabbit aorta and rat mesentery suggest Empagliflozin relaxes smooth muscle through activation of K+ channels. This study investigated the effects of therapeutic and pharmacological concentrations of Empagliflozin (10-300 μM) on the isometric tension of left circumflex (CFX) coronary arteries from Yorkshire swine. We tested the hypothesis that Empagliflozin would relax coronary smooth muscle via a concentration-dependent manner, involving K+ channels. CFX arteries were dissected, cleaned of adventitia, cut into 3 mm rings (n = 16), and mounted in organ baths containing Krebs-Henseleit buffer (37 °C) for isometric tension studies. Each CFX segment was adjusted to optimal preload (~3 g passive tension) as determined by <10 % change in active tension in response to 60 mM KCl. In order to determine whether Empagliflozin caused relaxation and, if so, by what mechanism, rings were contracted with either 30 mM K+ or the thromboxane mimetic U46619 (1 μM). Once tension plateaued, Empagliflozin (10-300 μM) was added in increasing concentrations. Therapeutic concentrations of Empagliflozin (10-30 μM), elicited no significant relaxation. Pharmacologic concentrations of Empagliflozin (≥100 μM) caused significant relaxation from ~15 ± 11% to ~45 ± 10% in response to 100 μM vs. 300 μM Empagliflozin, respectively. Relaxation was not different between rings contracted with K+ or U46619, suggesting membrane hyperpolarization via K+ channels is not involved. Our results represent the first study of the effects of Empagliflozin on coronary smooth muscle reactivity. Data support the idea that relaxation of coronary smooth muscle by Empagliflozin occurs at supra-pharmacologic concentrations in vitro, but K+ channel activation appears to be an unlikely mechanism. This project was supported by National Institutes of Health grant R01 HL158723. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Procedures for obtaining muscle physiology parameters during a gracilis free-functioning muscle transfer in adult patients with brachial plexus injury

A complete understanding of muscle mechanics allows for the creation of models that closely mimic human muscle function so they can be used to study human locomotion and evaluate surgical intervention. This includes knowledge of muscle–tendon parameters required for accurate prediction of muscle forces. However, few studies report experimental data obtained directly from whole human muscle due to the invasive nature of these experiments. This article presents an intraoperative, in vivo measurement protocol for whole muscle–tendon parameters that include muscle–tendon unit length, sarcomere length, passive tension, and active tension in response to external stimulation. The advantage of this protocol is the ability to obtain these rare experimental data in situ in addition to muscle volume and weight since the gracilis is also completely removed from the leg. The entire protocol including the surgical steps for gracilis harvest takes ~ 3 h. Actual testing of the gracilis where experimental data is measured takes place within a 30-min window during surgery.

Assembly of skeletal muscle cells on a Si-MEMS device and their generative force measurement

We have fabricated a simple Si-MEMS device consisting of a microcantilever and a base to measure active tension generated by skeletal muscle myotubes derived from murine myoblast cell line C2C12. We have developed a fabrication process for integration of myotubes onto the device. To position myotubes over the gap between the cantilever and the base without damage due to mechanical peeling or the use of an enzymatic reaction, we cultured myotubes on poly-N-isopropylacrylamide (PNIPAAm) as a sacrifice layer. By means of immune staining of alpha-actinin, it was confirmed that a myotube micropatterned onto the device bridged the gap between the cantilever and the base. After 7d differentiation, the myotube was actuated by electrical stimulation. The active tension generated by the myotube was evaluated by measuring the bending of the cantilever using image processing. On twitch stimulation, the myotube on the device contracted and generated active tension in response to the electrical signals. On tetanus tension measurement, approximately 1.0 microN per single myotube was obtained. The device developed here can be used in wide area of in vitro skeletal muscle studies, such as drug screening, physiology, regenerative medicine, etc.

Ca 2+ sensitisation of force production by noradrenaline in femoral conductance and resistance arteries from rats with postinfarction congestive heart failure

In this study we tested the hypothesis that arterial myofilament Ca 2+ sensitivity and/or the Ca 2+ sensitising effect of noradrenaline (NA) is enhanced in post-infarction congestive heart failure (CHF), which could contribute to the high peripheral vascular resistance in this condition. Femoral skeletal muscle resistance and conductance arteries (mean lumen diameters of 159 and 519 μm) from rats with CHF and sham-operated control rats were used. Isometric tension development and intracellular free calcium concentration ([Ca 2+] i) were measured simultaneously in isolated vessel segments using wire myography and the FURA-2 fluorescence technique. In conductance and resistance arteries, the resting levels of [Ca 2+] i and tension in physiological saline solution (PSS) and active tension in response to single doses of 125 mM K + (KPSS) were unaffected by CHF. During cumulative application of extracellular Ca 2+ to arteries depolarised with 125 mM K + or activated with 30 μM NA, [Ca 2+] i and vessel wall tension were similar in CHF and control rats. However, the conductance arteries showed significantly higher calcium sensitivity than resistance arteries in these experiments. We conclude that an abnormality in the sensitivity of the contractile apparatus to Ca 2+, or in NA-induced Ca 2+ sensitisation in arterial vascular smooth muscle cells is unlikely to contribute to the ubiquitously elevated vascular resistance associated with CHF. However, our data demonstrate significant differences in vascular Ca 2+ handling, myofilament Ca 2+ sensitivity and tension development between resistance and conductance arteries, regardless of CHF.

Regional differences in the response of feline esophageal smooth muscle to stretch and cholinergic stimulation.

There are no objective differences in neural elements that explain regional differences in neural influences along the smooth muscle (SM) esophageal body (EB). Regional differences in muscle properties are present in the lower esophageal sphincter (LES). This study examines whether regional differences in SM properties exist along the EB and are reflected in length-tension relationships and responses to cholinergic excitation. Circular SM strips from feline EB at 1 cm (EB1) and 3 cm (EB3) above LES and from clasp and sling muscle bundles of LES were assessed in normal and calcium-free solutions with and without bethanechol stimulation. Neural inhibition was assessed by electrical field stimulation (EFS). EB3 developed significantly higher tension in response to stretch and to bethanechol than did EB1. The relaxation response to EFS in bethanechol-precontracted strips was less in EB3 than in EB1. In LES, clasp developed higher resting tension than sling but less active tension in response to bethanechol. EFS-induced relaxations of sling and clasp tissues precontracted by bethanechol were not different. In calcium-free solution, length-tension differences between EB3 and EB1 persisted, but those of LES clasp and sling were abolished. Therefore, regional myogenic differences exist in feline EB circular SM as well as in LES and may contribute to the nature of esophageal contraction.

Length dependence of tension generation in rat skinned cardiac muscle: role of titin in the Frank-Starling mechanism of the heart.

At the basis of the Frank-Starling mechanism is the intrinsic ability of cardiac muscle to produce active tension in response to stretch. Titin, a giant filamentous molecule involved in passive tension development, is intimately associated with the thick filament in the sarcomere. Titin may therefore contribute to active tension development by modulating the thick filament structure when the muscle is elongated. Rat skinned right ventricular trabeculae were used. Passive tension at a sarcomere length (SL) of 2.0 to 2.4 micrometer was decreased after treatment of the preparation with trypsin (0.25 microgram/mL) for 13 minutes in the relaxed state at 20 degrees C. This mild trypsin treatment degraded titin without affecting other major contractile proteins. The sarcomere structure was little affected by brief contractions in the trypsin-treated preparations. When SL was adjusted to the slack SL (1.9 micrometer), active tension was unaffected by trypsin under partial (pCa 5.55) and maximal (pCa 4.8) activation. At longer SLs, however, active tension was significantly (P<0.01) decreased after trypsin treatment at either pCa. The increase in active tension on reduction of interfilament lattice spacing, produced by dextran T-500 (molecular weight approximately 500 000), was not influenced by trypsin (SL 1.9 micrometer). In trypsin-treated preparations, the increase in active tension as a function of muscle diameter was nearly the same for lengthening and osmotic compression at the slack SL. The length-dependent activation in cardiac muscle, an underlying mechanism of the Frank-Starling law of the heart, is at the myofilament level, predominantly modulated by titin and interfilament lattice spacing changes.

Calcium sensitivity and agonist-induced calcium sensitization in small arteries of young and adult spontaneously hypertensive rats.

The sensitivity of the myofilaments to Ca2+ is increased during agonist-induced contraction of vascular smooth muscle. Given the important contribution of vascular tone to the elevation of peripheral resistance observed in genetic hypertension, we have investigated whether alterations in myofilament Ca2+ sensitivity occur in small arteries from spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto (WKY) controls during the developmental and established phases of hypertension. Segments of mesenteric, renal, and femoral artery with an average lumen diameter <300 microm from 5- or 20-week-old rats were mounted in a wire myograph. Morphological measurements were made and the vessels permeabilized with Staphylococcus aureus alpha-toxin. Dose-response curves to increasing concentrations of Ca2+ were obtained and the ability of 100 nmol/L endothelin-1 (ET-1) or 10 micromol/L norepinephrine (NE) in the presence of 10 micromol/L GTP to enhance tension in response to low Ca2+ (pCa6.7) was determined. Systolic, diastolic, and mean blood pressures were higher in SHR than in WKY at 5 and 20 weeks. The media thickness:lumen diameter ratio was increased in mesenteric and femoral arteries from SHR compared with WKY at 5 and 20 weeks. There was no difference in media thickness:lumen diameter ratio in renal arteries or between 5- and 20-week animals in any vascular bed. The pCa curves were not different in mesenteric, renal, or femoral arteries from hypertensive compared with normotensive rats or between age groups, except in femoral arteries at 20 weeks, which exhibited a greater sensitivity to Ca2+ in SHR. Tension developed in response to maximal Ca2+ (pCa5.0) was greater in permeabilized mesenteric arteries from SHR compared with WKY at 20 weeks of age only; media stress was again similar in both strains but increased in older animals compared with younger animals in mesenteric arteries from WKY. The submaximal contraction induced by pCa6.7 was greater in femoral and renal than mesenteric arteries. GTP (10 micromol/L) augmented the tension developed to pCa6.7 in mesenteric arteries at 5 and 20 weeks and in renal arteries at 20 weeks. Addition of 100 nmol/L ET-1 or 10 micromol/L NE in the continued presence of GTP markedly increased tension in mesenteric arteries at 5 and 20 weeks. In renal arteries, 10 micromol/L NE enhanced Ca2+ sensitivity in the presence of GTP in SHR at 5 and 20 weeks and WKY at 5 weeks. In femoral arteries, there was a tendency for ET-1 and NE to increase Ca2+ sensitivity, but this increase was significant in WKY at 20 weeks (ET-1) and SHR at 5 weeks (NE) only. We have demonstrated that the sensitivity of the myofilaments to Ca2+ and ET-1- or NE-induced Ca2+ sensitization is not different in permeabilized small mesenteric, renal, or femoral arteries from SHR compared with WKY controls. Only in SHR mesenteric arteries at 20 weeks of age was there evidence of increased active tension in response to maximal Ca2+, despite structural differences, consistent with increased muscle mass in femoral arteries from SHR. We conclude that it is unlikely that a ubiquitous abnormality of the sensitivity of the contractile apparatus to Ca2+ or agonist-induced Ca2+ sensitization in vascular smooth muscle underlies the elevated total peripheral resistance associated with hypertension.

Hypoxia-induced pulmonary arterial contraction appears to be dependent on myosin light chain phosphorylation.

The signal transduction pathway of hypoxic pulmonary arterial contraction has not been elucidated. Phosphorylation of the 20-kDa myosin light chain (MLC20) is thought to be essential for vascular muscle contraction. However, there are reports that smooth muscle will contract in response to nonphysiological stimuli such as phorbol esters without the involvement of MLC20 phosphorylation. The purpose of this study was to determine if hypoxia-induced pulmonary arterial contraction is dependent on MLC20 phosphorylation. Isolated rat pulmonary and carotid (for comparative purposes) arterial strips were contracted with 80 mM KCl to establish maximum active tension in response to membrane depolarization. The strips were then stimulated with one of the following: 30 mM KCl, 1 microM phenylephrine, 0.01 microM angiotensin II, 1 microM phorbol 12-myristate 13-acetate (PMA), or hypoxia (95% N2-5% CO2). In some experiments ML-9, a myosin light chain kinase inhibitor, or calphostin C, a protein kinase C (PKC) inhibitor, was introduced into the bath before hypoxia. Isometric tension was recorded as a function of time. Muscle strips were freeze-clamped (liquid N2) at various time points during the course of responses to the various stimuli. MLC20 phosphorylation levels were measured by ureaglycerol gel electrophoresis followed by Western blot procedure. Results show that increased MLC20 phosphorylation correlates with initiation of pulmonary arterial smooth muscle contraction in response to all agonists with the exception of PMA, a known activator of PKC. The MLC20 phosphorylation levels correlate with tension development in response to hypoxia, and ML-9 abolished the hypoxic contractions. In contrast, hypoxia relaxed carotid arterial muscle, and there was a corresponding decrease in the MLC20 phosphorylation level. In conclusion, hypoxia appears to result in MLC20 phosphorylation-mediated contraction in conduit pulmonary arterial muscle and in MLC20 dephosphorylation-mediated relaxation in systemic arterial muscle.

Morphological and functional alterations of mesenteric small resistance arteries in early renal hypertension in rats

We investigated the structure and reactivity of small resistance arteries of two-kidney, one-clip (2K,1C) and one-kidney, one-clip (1K,1C) Goldblatt hypertensive rats within 4-6 wk of development of hypertension. Blood vessels from the mesenteric vascular bed with lumen diameter less than 300 microns were mounted on a wire myograph. The media of the vessel wall was significantly increased and lumen diameter was decreased in 2K,1C and 1K,1C rats. External diameter of blood vessels was reduced in both 2K,1C and 1K,1C rats, whereas cross-sectional area of the wall was increased significantly in 1K,1C rats. Wall tension in response to KCl was significantly lower in 2K,1C and 1K,1C hypertensive rats, whereas tension in response to norepinephrine (NE) was reduced in 1K,1C hypertensive rats but was similar in 2K,1C rats and controls. Active tension in response to arginine vasopressin (AVP) was similar in all groups. As a consequence of the reduced lumen circumference of small arteries, effective pressure in response to NE was similar in hypertensive and control rats, whereas effective pressure in response to AVP was exaggerated in the hypertensive rats. The sensitivity to NE and AVP was similar in all groups. These results show the rapid development of functional and structural changes in small resistance arteries in renal hypertensive rats within 4-6 wk of hypertension, with significant reduction in external and lumen diameters, increased media width, and increased media-to-lumen ratio, which enhance vascular reactivity to vasoconstrictors, in particular NE and AVP.

Single skinned muscle fibers in duchenne muscular dystrophy generate normal force

We measured the intrinsic mechanical properties and protein content of single skinned muscle fibers obtained from patients who had Duchenne muscular dystrophy. To check for possible nonspecific changes caused by muscle disease per se, we also studied the properties of muscle fibers obtained from patients exhibiting severe muscle weakness due to polymyositis. Relative to control fibers obtained from 4 patients with normal or nonmyopathic muscle, we found no significant changes in the ability of muscle fibers from the patients with Duchenne muscular dystrophy or polymyositis to generate active tension in response to calcium or resting tension in response to stretch. In addition, we found no significant changes in the concentrations of the major contractile proteins myosin and actin, of the elastic protein titin, or of the structural proteins nebulin and alpha-actinin. In contrast, immunocytochemical studies showed that dystrophin was absent in the biopsy specimens from the patients with Duchenne muscular dystrophy, but localized at the cell membrane in all of the other muscle biopsy specimens used in this study. These results indicate that myofibrils assemble and function normally in Duchenne muscular dystrophy. Therefore, the absence of dystrophin, which is the primary biochemical defect in this disease, leads to clinical weakness by causing the breakdown of muscle fibers that were once capable of generating normal force, while the surviving fibers exhibit normal contractility.

Ontogeny of bladder compliance

AbstractRecent advances in technology have allowed for the diagnosis of many urologic abnormalities through the use of antenatal ultrasonography. There is great debate in the pediatric literature as to whether the hydronephrosis which is diagnosed via antenatal ultrasound is always secondary to obstruction, or in certain cases represents a benign dilatation. Benign dilatations are postulated to be secondary to the increased fetal diuresis and a more compliant fetal urinary tract. In this study we present evidence for a gradual decline in bladder compliance with maturation. A summary of our results demonstrates that bladders obtained from 1 day old rabbits are 2 and 3 times more compliant than bladders of 4–8 week old rabbits. Active tension reaches a peak when the muscle is stretched to 150% of its initial resting length. However, 1 day old bladder tissue exhibits a higher active tension in response to field stimulation when compared to the response of bladder tissue obtained from 4 and 8 week old rabbits. In summary, compliance and active tension decrease with maturation.

A physiological role for titin and nebulin in skeletal muscle.

Production of active force in skeletal muscle results from the interaction of myosin-containing thick filaments with actin-containing thin filaments. These muscles are also passively elastic, producing forces that resist stretch independently of ATP splitting or of interaction between the filaments. The mechanism of this passive elasticity is unknown; suggestions include gap filaments in the region between thick and thin filaments in muscles stretched beyond filament overlap, or intermediate filaments which connect successive Z-disks. Recently, the two exceptionally large proteins titin (also called connectin) and nebulin (originally called band 3) have been implicated in passive elasticity (for review see refs 7, 8). Here, we show that after these proteins are degraded by low doses of ionizing radiation, the ability of single skinned muscle cells to generate both passive tension in response to stretch and active tension in response to calcium is greatly reduced. These effects are accompanied by axial misalignment of thick filaments. Titin and/or nebulin apparently provide axial continuity for the production of resting tension on stretch and also tend to keep the thick filaments centred within the sarcomere during force generation.

Maintenance of tone, role of arachidonate metabolites, and effects of sensitization in guinea pig trachea.

A study was made of the mechanisms underlying production of resting active tension in guinea pig tracheal smooth muscle and the changes with active sensitization to ovalbumin. The same types of tissues were also analyzed as to their responses to arachidonate. Responses for each tissue were expressed in relation to a scale between zero active tension and maximum active tension in response to carbachol. A variety of selective and nonselective inhibitors of cyclooxygenase or 5-lipoxygenase were shown to affect active tension in a manner consistent with the conclusion that a cyclooxygenase product, probably prostaglandin F(PGF2 alpha) and not thromboxanes was chiefly responsible. The inhibition of active tension produced by cyclooxygenase inhibition was shown to be related to the initial active tension, such that tissues with greater resting active tension had greater reductions in tone. No differences of major importance were found as to the mechanisms underlying tone production in control and sensitized tissues. The tension changes in response to exogenous arachidonate were also found to be dependent on the initial level of active tension; when this was low, tension increased, when it was high, tension decreased or did not change. Effects of inhibitors on these responses were again consistent with the conclusion that primarily excitant prostaglandins, not thromboxanes, were produced. Some suggestive evidence for production of excitatory and inhibitory nonprostaglandin metabolites was obtained. No difference of major importance between control and sensitized tissues was observed in the magnitude or underlying mechanism of production of active tension.

Effects of Ca-antagonists on the stretch activation of vascular smooth muscles

Helical or ring strips of cerebral, renal coronary arteries(A) of rabbits, dogs and guinea pigs and saphenous A of aged-SHR (50-60 weeks) developed an active tension in response to quick stretch at a rate of 10 cm/sec to 140 % of the initial muscle length. Cerebral and saphenous A were more responsive to quick stretch than other A. The contraction produced by quick stretch was myogenic and depended not only transmembrane supply of Ca, but also on release of Ca, possibly from the inner surface of the plasma membrane susceptible to chemical skinning. Stretch-induced contraction under control conditions was more resistant to Ca-antagonists(Ca-A) and Ca-withdrawal than the contractions produced by high K and muscle direct electrical stimulation, which were mainly dependent on extracellular Ca. In contrast, the contractile activation due to stretch once enhanced by potentiators of Ca-influx such as cardiac glycosides, hemolysate and vasoactive amines was readily suppressed by Ca-A. Ca-A acts by blocking the entry of Ca and inhibits the refilling of Ca in store sites. This fundamental action explains the cerebral, coronary and other peripheral vasodilating and antihypertensive effects of Ca-A. Contractile activation by quick stretch seems to provide a useful alternative technique to electrical and chemical stimuli studies on myogenic vascular tone.

Length-tension relations of smooth muscle from normal and denervated rat urinary bladders.

Urinary bladders of rats were denervated by bilateral excision of the pelvic ganglion and removed 10 days after the operation. They were filled with 0.75 ml saline and a longitudinal muscle strip was marked out, measured and dissected out. Strips from normal bladders filled with the same volume were used as controls. Denervated bladders were 4-5 times heavier than control bladders. Muscle strips from denervated bladders showed, in contrast to controls, marked phasic spontaneous contractions which were unaffected by tetrodotoxin, indicating a myogenic origin. Active tension in response to AC stimulation was measured at different lengths. In relation to the in situ length (Lin situ) at 0.75 ml the denervated strips had to be stretched to much greater extent than controls in order to reach optimum length (L0) for force development. Furthermore, the denervated strips shortened less in relation to Lin situ than the controls. If active length-tension relations were expressed in relation to L0, the difference between denervated and control strips was abolished. Maximal active force was the same for denervated and control strips. Water content increased significantly in denervated bladders. The results suggest a remodelling of the smooth muscle structure in denervated bladders; the characteristics of the contractile machinery seem, however, to be unaltered.

Norepinephrine stores and contractile force of papillary muscle from the failing human heart.

The relation between cardiac norepinephrine concentration and the functional state of the myocardium was studied in left ventricular papillary muscles removed from patients with congestive heart failure at the time of mitral valve replacement. Myocardial function was assessed by determining the maximum isometric active tension that the papillary muscle could develop in an in vitro system, and by measuring the increase in active tension in response to the norepinephrine-releasing sympathomimetic amine, tyramine. The norepinephrine concentration averaged 0.36 µg./Gm. in 17 muscles. In eight of these a response to tyramine was observed with a 21-per cent increase in active tension, and the norepinephrine concentrations averaged 0.53 µg./Gm.; in the other six muscles tested with tyramine no positive inotropic response was observed, and the norepinephrine concentration was significantly lower, averaging 0.16 µg./Gm. The maximum active tension in 17 muscles averaged 1.78 Gm./mm. 2 , and a significant positive correlation between the maximum tension and the norepinephrine concentration of the individual muscles was observed. In the eight muscles responding to tyramine, the maximum tension observed, 2.34 Gm./mm. 2 , was significantly greater than that observed in the six muscles unresponsive to tyramine, 0.64 Gm./mm. 2 It is concluded that norepinephrine depletion appears to be associated with defective myocardial function although no causal relationship between these two abnormalities has been established.