Stiffness and tension during and after sudden length changes of glycerinated single insect fibrillar muscle fibres

Rapid length changes were applied (within 0.2 ms or 0.4 ms) to single isometrically contracted glycerol extracted muscle fibres of the dorsal longitudinal muscle ofLethocerus maximus suspended in an Ca²⁺ and ATP containing solution at 20–23‡ C. Force transients and the fibre stiffness were measured during and after rapid length changes. At length changesbelow 0.5% of the initial fibre length (∼ 2.4 Μm sarcomere length) the mechanical transients were characterized as follows: (1) After stretch and after release the force regains at least partly the value of tension before the length change within a quick phase of tension recovery. The quick phase induced by stretch was nearly completed within 1–2 ms. (2) A pulse in length of 1.5 ms duration, i.e., a stretch followed by a release to the initial length or a release followed by a stretch to the initial length, was applied to the fibre. The force transient induced by this procedure regains after the second length change the value of the isometric tension before the procedure. (3) The stiffness was constant during each length change of the “pulse” and was equal during the first and the second length changes. These findings are predicted by the muscle contraction model of Huxley and Simmons (1971): The identical force before and after a length pulse may indicate that the rotation of cross bridges after the first length change is followed by a rotation into the original position after the second length change. The constancy of the stiffness during the length changes may indicate a Hookean elastic element of the cross bridge. The similarity of the stiffness during the first and the second length changes, i.e., before and after the quick phase, gives evidence that the quick phases after stretch and after release are not accompanied by a change in the net number of attached cross bridges. If stretches ofmore than 0.5% of the initial length were applied, the mechanical transient of the muscle fibre changed as follows: (1) An ultra fast tension decay phase (duration < 0.4 ms) was observed in addition to the slower decay phase induced by the smaller stretches. (2) If the initial stretch was followed by a release to the initial length, no fast recovery phase was observed, which returns the force to the value before the stretch. The reduced tension value persists for a longer period in time than 10 ms. (3) If the muscle was stretched and released repetitively an ultra fast quick phase was induced only by the first stretch. (4) The stiffness increased during stretch, but was found to be the same in the isometrically contracting muscle and after the quick tension decay phase following a large stretch. These findings indicate that the contraction model of Huxley and Simmons has to be extended by a further process additional to cross bridge rotation in case of large stretches (> 0.5%L ini). The findings are taken to indicate a rapid detachment and reattachment of overstrained cross bridges, i.e., a cross bridge slippage induced by large stretches.     

Phase transition in force during ramp stretches of skeletal muscle.

Active glycerinated rabbit psoas fibers were stretched at constant velocity (0.1-3.0 lengths/s) under sarcomere length control. As observed by previous investigators, force rose in two phases: an initial rapid increase over a small stretch (phase I), and a slower, more modest rise over the remainder of the stretch (phase II). The transition between the two phases occurred at a critical stretch (LC) of 7.7 +/- 0.1 nm/half-sarcomere that is independent of velocity. The force at critical stretch (PC) increased with velocity up to 1 length/s, then was constant at 3.26 +/- 0.06 times isometric force. The decay of the force response to a small step stretch was much faster during stretch than in isometric fibers. The addition of 3 mM vanadate reduced isometric tension to 0.08 +/- 0.01 times control isometric tension (P0), but only reduced PC to 0.82 +/- 0.06 times P0, demonstrating that prepowerstroke states contribute to force rise during stretch. The data can be explained by a model in which actin-attached cross-bridges in a prepowerstroke state are stretched into regions of high force and detach very rapidly when stretched beyond this region. The prepowerstroke state acts as a mechanical rectifier, producing large forces during stretch but small forces during shortening.     

History-dependent mechanical properties of permeabilized rat soleus muscle fibers.

Permeabilized rat soleus muscle fibers were subjected to repeated triangular length changes (paired ramp stretches/releases, 0.03 l(0), +/- 0.1 l(0) s(-1) imposed under sarcomere length control) to investigate whether the rate of stiffness recovery after movement increased with the level of Ca(2+) activation. Actively contracting fibers exhibited a characteristic tension response to stretch: tension rose sharply during the initial phase of the movement before dropping slightly to a plateau, which was maintained during the remainder of the stretch. When the fibers were stretched twice, the initial phase of the response was reduced by an amount that depended on both the level of Ca(2+) activation and the elapsed time since the first movement. Detailed analysis revealed three new and important findings. 1) The rates of stiffness and tension recovery and 2) the relative height of the tension plateau each increased with the level of Ca(2+) activation. 3) The tension plateau developed more quickly during the second stretch at high free Ca(2+) concentrations than at low. These findings are consistent with a cross-bridge mechanism but suggest that the rate of the force-generating power-stroke increases with the intracellular Ca(2+) concentration and cross-bridge strain.     

The effect of sarcomere length and stretching on the rate of ATP splitting in glycerinated rabbit psoas muscle fibers.

The effect of sarcomere length and stretching on the tension and the rate of ATP splitting was studied using small fiber bundles from glycerinated rabbit psoas muscle. The rate of ATP slitting was determined by measuring ADP production, while the tension development in response to a contracting solution (at pCa 5.3) was recorded in the same preparation. The isometric tension developed by the preparation decreased when the sarcomere length was increased. The decrease of tension development was accompanied by a decrease in the rate of ATP splitting. If a preparation exerting steady isometric tension was stretched by 5--10% at a velocity of 0.1 mm/s, the rate of ATP splitting was increased after stretching, while the steady isometric tension attained after stretching was also higher than the initial value. The extent of the excess ATP splitting caused by stretching decreased with increasing sarcomere length. These results suggest that the rate of the interaction cycle between actin and myosin molecules may increase as a result of stretching.     

Stretch induced tension rise in a molluscan smooth muscle skinned by freeze drying

Summary The anterior byssus retractor muscle (ABRM) ofMytilus edulis was skinned by freeze drying. Tension transients in response to quick length steps were recorded during isometric contraction induced in ATP salt solution containing 2×10^–6 M Ca²⁺. These transients consisted of four phases similar to those described by Huxley (1974) in skeletal muscle. Under certain conditions (stretch amplitude not larger than 0.6% L_O), and in particular in the presence of cyclic AMP, we observed a delayed tension rise following a quick stretch (stretch activation) which appears to be similar to the stretch activation of insect flight muscle (Jewell and Rüegg 1966).     

ATP causes a structural change in retinal rod outer segments: Disc swelling Is not involved

Mg2+-ATP was found to produce a 15 to 30% drop in the turbidity of suspensions of broken retinal rod outer segments from the toad Bufo marinus, prepared by washing or flotation in sucrose. This in vitro process has a half-time of about two minutes and appears to be irreversible. It is not affected by the bleaching of rhodopsin. Direct measurements show that the drop in turbidity is not due to swelling of the disc internal space measured in outer segments recovered by centrifugation. Instead, the total packed volume of the outer segments increases following incubation in Mg2+-ATP. Under the specific conditions of these experiments, the total pellet volume increase was 26 +/- 22% (13 experiments) when corrected for the percent of rhodopsin recovered in the centrifugal pellet. The magnitude of the ATP effect on turbidity suggests that the majority of the discs are involved in some kind of structural change. Vanadium in the +5 oxidation state (vanadate) is an inhibitor of the Mg2+-ATP effect on turbidity at a half-maximal concentration of 0.2 to 0.4 microM, and inhibition is rapidly reversed by norepinephrine, which complexes vanadate. A Mg2+-ATPase activity in extensively washed outer segment membranes, previously shown to be activated as much as twofold following light exposure of the membranes, is not sensitive to vanadate at the concentrations which block the ATP-dependent change of turbidity.     

Tonic contraction and the control of relaxation in a chemically skinned molluscan smooth muscle

The same functional states that characterize the living anterior byssus retractor muscle (ABRM) from Mytilus edulis can be initiated in the saponin-treated (chemically skinned) muscle preparation under controlled biochemical conditions. A tonic contraction was induced if the concentration of free Ca2+ was above approximately 10(7) M in the presence of Mg2+ and ATP. Maximum tension development was achieved at a Ca2+ concentration of approximately 10(4) M. Within these Ca2+ concentrations tension was always associated with the presence of 'active state," as indicated by a high recovery of tension after a quick release in muscle length. Tonic tension, and the associated active state was maintained for hours during these conditions irrespective of variations in both ionic strength and pH. Reduction of the Ca2+ concentration to below threshold for tension initiation during a tonic contraction immediately switched off the active state and relaxation of the muscle preparation resulted. However, the rate of relaxation was extremely low, leaving a substantial fraction of tension in the absence of active state. Both 5-hydroxytryptamine (5-HT) and cAMP accelerated this slow relaxation in the absence of Ca2+. Thus, this state was considered equivalent to the 'catch state" in the living ABRM. In the presence of Ca2+ concentrations above 10(7) M, cAMP did not affect either the maximum tension developed or the Ca2+ sensitivity of the chemically skinned muscle preparation.     

Evidence for a Mg2+-induced conformational change at the ATP-binding site of (Na+ + K+)-ATPase demonstrated with a photoreactive ATP-analogue

1. The 3'-ribosyl ester of ATP with 2-nitro-4-azidophenyl propionic acid has been prepared and its ability to act as a photoaffinity label of (Na+ + K+)-ATPase has been tested. 2. In the dark 3'-O-[3-(2-nitro-4-azidophenyl)-propionyl]adenosine triphosphate (N3-ATP) is a substrate of (Na+ + K+)-ATPase and a competitive inhibitor of ATP hydrolysis. 3. Upon irradiation by ultraviolet light, N3-ATP photolabels the high-affinity ATP-binding site and is covalently attached to the alpha-subunit and an approximately 12000-Mr component. 4. Photolabeling of the alpha-subunit by N3-ATP irreversibly inactivates (Na+ + K+)-ATPase. 5. Photoinactivation is strictly Mg2+-dependent. Na+ enhances the inactivation. ATP or ADP and K+ protect the enzyme against inactivation. 6. Mg2+, in concentrations required for photoinactivation, protects (Na+ + K+)-ATPase against inactivation by tryptic digestion under controlled conditions. 7. It is assumed that a conformational change of the ATP-binding site of (Na+ + K+)-ATPase occurs upon binding of Mg2+ to a low-affinity site.     

Purification and reconstitution of the N,N'-dicyclohexylcarbodiimide-sensitive ATPase complex from spinach chloroplasts.

The dicyclohexylcarbodiimide-sensitive ATPase from spinach chloroplast has been isolated. On sodium dodecyl sulfate gels, seven different polypeptides were seen. Five of these polypeptides coincided with the CF1 subunits, a 7,500-dalton peptide was identified as the proteolipid which interacts with [14C]dicyclohexylcarbodiimide, and there was a 15,500-dalton hydrophobic polypeptide with unknown function. In two-dimentional gels, two additional peptides were resolved, one 17,500 daltons (co-migrating in sodium dodecyl sulfate gels with subunit delta) and one 13,500 daltons (co-migrating with subunit epsilon). Reconstitution was obtained by freezing and thawing the complex with a crude mixture of phospholipids. After reconstitution the complex catalyzed 32P1-ATP exchange (rates of 200 to 400 nmoles x mg-1 x min-1) and ATP formation during acid-to-base transition. These reactions were inhibited by dicyclohexylcarbodiimide and uncouplers. Uncouplers at low concentrations stimulated and at high concentrations inhibited the Mg2+-ATPase activity. ATP hydrolysis and 32P1-ATP exchange were catalyzed by the complex in the presence of either Mg2+ or Mn2+ but not with Ca2+ or Co2+. ATP and GTP were substrates for the exchange reaction but not ADP or CTP.     

Strain sensitivity and turnover rate of low force cross-bridges in contracting skeletal muscle fibers in the presence of phosphate.

Inorganic phosphate (Pi) decreases the isometric tension of skinned skeletal muscle fibers, presumably by increasing the relative fraction of a low force quaternary complex of actin, myosin, ADP, and Pi (A.M.ADP.Pi). At the same time, Pi gives rise to a fast relaxing mechanical component as detected by oscillations at 500 Hz. To characterize the dynamic properties of this A.M.ADP.Pi complex, the effect of Pi on the tension response to stretch was investigated with rabbit psoas fibers. A ramp stretch applied in the presence of 20 mM Pi increased tension more than in the control solution (0 mM Pi) but reduced the fast relaxing component to the control level. Thus, a stretch seems to convert the low force, fast relaxing A.M.ADP.Pi complex to a high force, slow relaxing form. However, the Pi-induced enhancement of the tension response was not observed until the fibers were stretched more than 0.4% of their length, suggesting that a critical cross-bridge extension of approximately 4 nm is required for this conversion. The rate constant of the attachment/detachment of this low force complex was estimated from the velocity dependence of the enhancement. It was approximately 10 s-1, in marked contrast to the A.M.ADP.Pi complex under low salt, relaxed conditions (approximately 10,000 s-1). The enhancement of the tension response was not observed when isometric tension was reduced by lowering free calcium, implying that calcium and Pi affect different steps in the actomyosin ATPase cycle during contraction.     

Ca2+- or Mg2+-Dependent Enzymic ATP Hydrolysis Associated with the Microsomal Fraction of Frog Sciatic Nerves

The microsomal fraction of frog sciatic nerves was found to contain Ca2+- or Mg2+-dependent hydrolytic activity toward different nucleoside di- and triphosphates. In the presence of Ca2+ substrate specificity was in the order CTP > UTP > GTP > ATP. When Mg2+ was used, the triphosphates were approximately equally good substrates. ATP hydrolytic activity was very similar with Ca2+ or Mg2+ as the cofactor, whereas Ca2+ was the more potent activator of hydrolysis of the other triphosphates tested. The preparation showed some activity toward the nucleoside diphosphates but none toward the monophosphates or p-nitrophenylphosphate. The enzymic properties of ATP hydrolysis were more closely studied. The hydrolysis was optimal at 18--24 degrees C in the presence of 1 mM-Ca2+ or 1 mM-Mg2+. Ca2+- and Mg2+-ATP hydrolysis displayed pH maxima around 8.0--8.5 and 7.4--8.0, respectively. Vmax values for Ca2+- and Mg2+-ATP hydrolysis similar: approx. 12 mumol Pi per h per mg protein with a Km value of approx. 0.05 mM. The ATP hydrolysis activity was inhibited by NaF but unaffected by ouabain, vanadate, cytochalasin B, and various drugs known to influence ATPase activity of mitochondria. Zn2+ stimulated the ATP hydrolysis activity at low concentrations (10(-6)-10(-5) M) and inhibited it at higher concentrations. The possibility that these observations account for stimulation and inhibition of axonal transport in frog sciatic nerves exposed to similar concentrations of Zn2+ is discussed.     

Stiffness and tension during and after sudden length changes of glycerinated rabbit psoas muscle fibres

A sudden stretch (within 0.3 ms) of glycerol-extracted rabbit psoas fibre bundle suspended in ATP-salt solution caused an immediate tension increase followed by a rapid tension decay (quick phase) which was nearly completed within 3 ms. The quick phase was missing or much reduced in the absence of ATP when the fibres were in rigor. Since the immediate stiffness of the fibres was nearly the same at the onset and at the end of the quick phase, the latter cannot be due to cross-bridges detachment per se. However, it may be ascribed to a conformational change (e.g. rotation) of attached bridges as suggested by Huxley and Simmons. Alternatively it might be explained by a slippage of attached cross-bridges. This mechanism would presuppose fast detachment and reattachment of strongly strained cross-bridges during the quick phase. Evidence for such a process was obtained by analysing the tension transients obtained when fibre bundles subjected to a large stretch were subsequently (within 10 ms) released to the initial length, as well as from stiffness measurements during the sudden length change: The stiffness was not found to be constant either during stretch or during the release. This may be taken to mean that the number of attached cross-bridges does not remain constant even during a rapid length change. In view of these results, the model proposed by Huxley and Simmons might be extended to take account of rapid attachment and detachment of crossbridges.     

Further Characterization of Dopamine Release by Permeabilized PC 12 Cells

Rat pheochromocytoma cells (PC12) permeabilized with staphylococcal alpha-toxin release [3H]dopamine after addition of micromolar Ca2+. This does not require additional Mg2+-ATP (in contrast to bovine adrenal medullary chromaffin cells). We also observed Ca2+-dependent [3H]-dopamine release from digitonin-permeabilized PC12 cells. Permeabilization with alpha-toxin or digitonin and stimulation of the cells were done consecutively to wash out endogenous Mg2+-ATP. During permeabilization, ATP was removed effectively from the cytoplasm by both agents but the cells released [3H]dopamine in response to micromolar Ca2+ alone. Replacement by chloride of glutamate, which could sustain mitochondrial ATP production in permeabilized cells, does not significantly alter catecholamine release induced by Ca2+. However, Mg2+ without ATP augments the Ca2+-induced release. The release was unaltered by thiol-, hydroxyl-, or calmodulin-interfering substances. Thus Mg2+-ATP, calmodulin, or proteins containing -SH or -OH groups are not necessary for exocytosis in permeabilized PC12 cells.     

Dynamic light scattering study of the effect of Mg2+ and ATP on synthetic myosin filaments.

The dynamic light scattering (DLS) method provides us with information about the apparent diffusion coefficient, Dapp, as well as the static scattering intensity, Is, of particles in solution. For long but thin rods with length L and diameter d, the dependence on L and d of Dapp is quite different from that of Is. By means of DLS we studied synthetic myosin filaments of rabbit skeletal muscle in solution at pH 8.3 and 10 degrees C. It appeared that Mg2+ ions induced thickening and lengthening of the filaments, whereas ATP (and ADP) induced thinning and shortening (depolymerization) of the filaments. When ATP was added to the filament preparation in the presence of Mg2+ ions, it was clearly observed that thinning of the filament (or splitting into subfilaments) occurred before shortening (or depolymerization).     

Conformational change accompanying formation of oligomycin-induced Na(+)-bound forms and their conversion to ADP-sensitive phosphoenzymes in Na+,K(+)-ATPase.

Oligomycin reduced the fluorescence intensity of an N-(p-(2-benzimidazoly)phenyl) maleimide (BIPM) probe at Cys-964 of the alpha-chain of pig kidney Na+,K(+)-ATPase with increase in the concentration of Na+ with a Hill coefficient of nh = 0.77 with Kh = 231 mM. The maximum fluorescence decrease was around 80% of the value observed after accumulation of ADP-sensitive phosphoenzyme (E1P) in the presence of 2 M Na+. The addition of Mg2+ and ATP with Na+ or choline chloride to give the same final ligand concentration to the Na(+)-enzyme-oligomycin complex formed with 16 mM Na+ + 1,984 mM choline chloride or 2 M Na+ induced rapid phosphorylation (20 or 21/s) and slower fluorescence decrease (12.1 +/- 1.2 or 10.1 +/- 3.2/s). These additions to the Na(+)-enzyme complex formed under the former or the latter conditions induced slow phosphorylation (13/s) prior to a much slower fluorescence decrease (3.4 +/- 0.3 or 8.6 +/- 0.7/s). The addition of Ca2+ and ATP to these enzyme complexes induced rapid fluorescence changes (21-11/s) followed by one order of magnitude slower rates of phosphorylation (1.5-1.3 s). These data suggest that the decrease in BIPM fluorescence induced by ATP with Ca2+ or with Mg2+, reflects the change of the Na+ binding state before or after the formation of E1P, respectively.     

Competition between Li+ and Mg2+ for ATP and ADP in aqueous solution: a multinuclear NMR study

We used 7Li NMR spin-lattice relaxation times and 31P NMR chemical shifts to study the binding of Li+ and Mg2+ to the phosphate moieties of ATP and ADP. To examine the binding of Li+ and Mg2+ to the base and ribose moieties, we used 1H and 13C NMR chemical shifts. The 7Li NMR relaxation times of Li+/Mg2+ mixtures of ATP or ADP increased with increasing concentrations of Mg2+, suggesting competition between the two ions for adenine nucleotides. No significant binding of Li+ and Mg2+ to the base and ribose moieties occurred. At the pH and ionic strength used, 2:1 and 1:1 species of the Li(+)-ATP and Li+-ADP complexes were present, with the 2:1 species predominating. In contrast, 1:1 species predominated for the Mg(2+)-ADP and Mg(2+)-ATP complexes. We calculated the Li(+)-nucleotide binding constants in the presence and absence of Mg2+ and found them to be somewhat greater in the presence of Mg2+. Although competition between Li+ and Mg2+ for ATP and ADP phosphate binding sites in solution is consistent with the 31P chemical shift data, the possibility that the Li+ and Mg2+ form mixed complexes with the phosphate groups of ATP or ADP cannot be ruled out.     

Identification of amino acid residues modified by two ATP analogs in Bacillus subtilis glutamine synthetase.

Bacillus subtilis glutamine synthetase was modified by two ATP analogs, 5'-p-fluorosulfonylbenzoyladenosine (FSBA) and 8-azidoadenosine 5'-triphosphate (8-N3-ATP), each one containing either Mg2+ or Mn2+. The FSBA labeled peptide was monitored by measuring the characteristic absorbance of the 4-carboxybenzenesulfonyl (CBS) part at 243 nm. The 8-N3ATP photolabeled peptide could also be monitored by measuring its absorption at 310 nm. A single CBS-labeled tryptic peptide was obtained, spanning residues 89-91 from the N-terminal of the subunit polypeptide chain, and sequence analysis by Edman degradation revealed that CBS-arginine was at position 91. The amino acids photolabeled by 8-N3ATP at the ATP-binding site in B. subtilis GS were His-186, His-187, and Trp-424. These results suggested that these four amino acids constitute an ATP-binding active site located at the interface between two subunits. The region surrounding Trp-424, which varies among different prokaryotic enzymes, was considered to be involved in a catalytic or regulatory role in B. subtilis GS. Since the same amino acids were labeled when B. subtilis GS was modified with FSBA or 8-N3ATP in the presence of Mn2+ or Mg2+, no conformational difference between B. subtilis GS binding Mn(2+)-ATP and that binding Mg(2+)-ATP was detected by affinity labeling with ATP analogs.     

Effects of excitation-contraction uncoupling by stretch and hypertonicity on metabolism and tension in single frog muscle fibers

Metabolism and tension were examined in single fibers of the semitendinosus muscle of Rana pipiens at 15 degree C after excitation- contraction uncoupling by stretch and hypertonicity. Interrupted tetanic stimulation at 20 HZ for 150 s, of control fibers in isotonic Ringer at a rest sarcomere length (SL) of 2.3 micrometers, resulted in a steadily declining tension, stimulated glycolysis, and significantly reduced fiber phosphocreatine (PCr) and ATP concentrations. Stretching resting muscle fibers to an SL of 4.7 micrometers did not alter metabolite concentrations, but glucose-6-phosphate rose and PCr fell markedly when the stretched fibers were stimulated tetanically, although tension was absent. Immersion of untetanized fibers in 2.5 X isotonic Ringer produced a transient rise in resting tension, an increase in glucose-6-phosphate, and a significant reduction in PCr. During the transient rise in resting tension, PCr consumption per unit of tension-time integral was the same as that in fibers stimulated tetanically in isotonic Ringer. Tetanization of fibers in hypertonic solution did not further alter metabolite concentrations or produce tension. The results indicate that exposure to hypertonicity induces an increase in both tension and consumption of high-energy phosphate bonds (approximately P) in resting fibers, but stretch does not. during tetanic stimulation, stretch interferes with contraction but does not prevent activation, whereas hypertonicity inhibits activation as well as contraction.     

Phosphatic metabolites, intracellular pH and free [Mg2+] in single, intact porcine carotid artery segments studied by 31P-NMR

Superfused porcine carotid artery segments (approximately 7 cm lengths) were analyzed by 31P-NMR spectroscopic methods to characterize the 31P spectrum of arterial smooth muscle and to determine the influence of passive stretch (intraluminal pressurization, 95-100 mmHg) on cellular phosphatic metabolite levels, intracellular pH and free magnesium concentration ([Mg2+free]i). Equilibrated, single, intact arteries were studied under steady-state, constant flow conditions at 37 degrees C. Phosphoethanolamine, phosphocholine, inorganic phosphate (Pi), phosphocreatine (PCr) and nucleoside triphosphates (NTP), primarily ATP, were the principle metabolites detected in the 31P-NMR spectrum of intact arterial smooth muscle. The concentration of these metabolites and intracellular pH, as determined from the referenced chemical shift of Pi, were unaffected by pressurization. The PCr:Pi ratios determined for nonpressurized (flaccid) and pressurized arteries were 1.2 +/- 0.1 and 1.3 +/- 0.3, respectively. Intracellular pH averaged 7.02 +/- 0.02 (mean +/- 1 S.D.) for flaccid arteries vs. 7.03 +/- 0.05 for pressurized arteries. The upfield chemical shift of the beta-ATP peak, which has been described in other types of smooth muscle, was also observed in these experiments. Interestingly, pressurization significantly shifted the resonance position of this peak, which was interpreted to represent a change in [Mg2+free]i. The average [Mg2+free]i of flaccid artery preparations was computed to be 0.54 +/- 0.03 x 10(-3) M, as compared to 0.99 +/- 0.10 x 10(-3) M for pressurized arteries. This change in [Mg2+free]i was evident within the first hour following pressurization and persisted thereafter. These findings suggest that altering the resting length of vascular smooth muscle produces a change in [Mg2+free]i. This shift in free Mg2+ levels may act as a metabolic signal triggering a change in vascular smooth muscle metabolism, an effect which has been reported to occur in smooth muscle in response to stretch.     

Reconstitution of the Frank-Starling mechanism in engineered heart tissues

According to the Frank-Starling mechanism, as the heart is stretched, it increases its contraction force. Reconstitution of the Frank-Starling mechanism is an important milestone for producing functional heart tissue constructs. Spontaneously contracting engineered heart tissues (EHTs) were reconstituted by growing dissociated chicken embryo cardiomyocytes in collagen matrices. Twitch and baseline tensions were recorded at precisely controlled levels of tissue strain. The EHTs showed a steep increase in twitch tension from 0.47 +/- 0.02 to 0.91 +/- 0.02 mN/mm2 as they were stretched at a constant rate (2.67% per min) from 86% to 100% of the length at which maximum twitch force was exerted. In response to a sudden stretch (3.3%), the twitch tension increased gradually (approximately 60 s) in a Gd3+-sensitive manner, suggesting the presence of stretch-activated Ca2+ channels. A large difference in baseline tension between lengthening (loading) and shortening (unloading) was also recorded. Disruption of nonsarcomeric actin filaments by cytochalasin D and latrunculin B decreased this difference. A simple mechanical model interprets these results in terms of mechanical connections between myocytes and nonmuscle cells. The experimental results strongly suggest that regulation of twitch tension in EHTs is similar to that of natural myocardium.