Biomedical subjects

R E Godt

Publications and source records attributed to R E Godt.

At least 19 recordsLinked to original sources

PubMed2001

Protein osmotic pressure and the state of water in frog myoplasm.

We measured the osmotic pressure of diffusible myoplasmic proteins in frog (Rana temporaria) skeletal muscle fibers by using single Sephadex beads as osmometers and dialysis membranes as protein filters. The state of the myoplasmic water was probed by determining the osmotic coefficient of parvalbumin, a small, abundant diffusible protein distributed throughout the fluid myoplasm. Tiny sections of membrane (3.5- and 12-14-kDa cutoffs) were juxtaposed between the Sephadex beads and skinned semitendinosus muscle fibers under oil. After equilibration, the beads were removed and calibrated by comparing the diameter of each bead to its diameter measured in solutions containing 3-12% Dextran T500 (a long-chain polymer). The method was validated using 4% agarose cylinders loaded with bovine serum albumin (BSA) or parvalbumin. The measured osmotic pressures for 1.5 and 3.0 mM BSA were similar to those calculated by others. The mean osmotic pressure produced by the myoplasmic proteins was 9.7 mOsm (4 degrees C). The osmotic pressure attributable to parvalbumin was estimated to be 3.4 mOsm. The osmotic coefficient of the parvalbumin in fibers is approximately 3.7 mOsm mM(-1), i.e., roughly the same as obtained from parvalbumin-loaded agarose cylinders under comparable conditions, suggesting that the fluid interior of muscle resembles a simple salt solution as in a 4% agarose gel.

Animals↗

PubMed2001

FGF-8 in the ventral pharynx alters development of myocardial calcium transients after neural crest ablation.

Cardiac neural crest ablation results in depressed myocardial calcium transients and elevated proliferation in myocardium at a stage when cardiac neural crest cells are not in contact with the myocardium. To test the hypothesis that cardiac neural crest-derived cells, which migrate into the caudal, ventral pharynx at stage 14, block a signal from the ventral pharynx, we cultured stage 12 chick heart tube or myocardial strips in the presence or absence of ventral pharynx. We found that myocardium cultured with ventral pharynx that had not yet contacted neural crest cells had significantly reduced calcium transients and an increased rate of proliferation. Ventral pharynx from intact embryos at a stage when neural crest-derived cells had reached the pharynx had no effect on myocardial calcium transients. Ventral pharynx from neural crest-ablated embryos continued to suppress myocardial calcium transients at this later stage. Myocardium cultured with FGF-2 also showed a significant reduction in calcium transients. An FGF-2-neutralizing Ab reversed the deleterious effect of the ventral pharynx on myocardial calcium transients and proliferation. We therefore examined the expression of FGF-2 and similar FGFs in the ventral pharynx. Only FGF-8 was expressed in a temporospatial pattern that made it a viable candidate for altering the myocardial calcium transient during stages 14-18. In explant cultures, neutralizing Ab for FGF-8 rescued development of the myocardial calcium transient in neural crest-ablated chick embryos.

Animals↗

PubMed1999

Excessive microtubules are not responsible for depressed force per cross-bridge in cardiac neural-crest-ablated embryonic chick hearts.

Ablation of the cardiac neural crest (CNCA) in embryonic chicks results in a high incidence of persistent truncus arteriosus, a congenital heart defect associated with decreased myocardial contractility. Using left ventricular trabeculae from chicks at embryonic day (ED) 15, we have previously shown that the twitch force of intact preparations is significantly reduced whereas the maximal calcium-activated force of skinned preparations is not significantly different in CNCA and sham-operated animals. We also previously found that the ventricular content of myosin, as well as of actin and tropomyosin, was nearly doubled in ED 15 hearts after CNCA. Since the number of cross-bridges is proportional to the myosin concentration, these data suggest that the force exerted per cross-bridge is decreased in CNCA hearts. We investigated the possibility that the decrease in force per cross-bridge is caused by inhibition of the contractile apparatus by excessive microtubules. To the contrary, we found that the total beta-tubulin content and the fraction of beta-tubulin polymerized in microtubules measured by Western blotting was the same in ventricular muscle strips from CNCA and sham-operated embryos. Furthermore, exposure to microtubule-destabilizing agents did not improve the force-producing capability of the contractile apparatus in CNCA embryos. We conclude that depression of force per cross-bridge in hearts from CNCA embryos is not due to an excess of microtubules.

Animals↗

PubMed1999

Parvalbumin concentration and diffusion coefficient in frog myoplasm.

The concentrations and diffusivity of two isoforms of parvalbumin, IVa and IVb, were measured using quantitative SDS PAGE in single fibers from semitendinosus muscles of the frog Rana temporaria. The concentrations of IVa and IVb were 2.9 +/- 0.3 (SEM) and 4.5 +/- 0.5 g l-1 total fiber volume, respectively. The total concentration of parvalbumin (7.4 +/- 0.8 g l-1 total fiber) corresponds to a cytosolic concentration of 0.9 +/- 0.1 mmol l-1 myoplasmic water. Estimates for the transverse and longitudinal diffusion coefficients for parvalbumin at 4 degrees C were obtained in two ways: (1) by diffusion of parvalbumin out of skinned fibers into droplets of relaxing solution, and (2) by diffusion of parvalbumin between two juxtaposed skinned fibers under oil. The transverse diffusion coefficient obtained using the droplet method was significantly lower than that obtained using juxtaposed fibers, but the longitudinal diffusion coefficients obtained from both methods were similar. The juxtaposed fiber method more accurately approximates parvalbumin diffusion in undisturbed myoplasm because no artificial solutions were used and, upon fiber-to-fiber contact, a potentially confounding oil barrier at the interface rapidly disperses. The juxtaposed fiber method yielded values for transverse (4.27 +/- 0.87 x 10(-7) cm2 s-1) and longitudinal (3.20 +/- 0.74 x 10(-7) cm2 s-1) diffusion coefficients that were not significantly different, suggesting that diffusion of parvalbumin in myoplasm is essentially isotropic. The average diffusion coefficient of frog parvalbumin in myoplasm (3.74 +/- 0.81 x 10(-7) cm2 s-1; 4 degrees C) is approximately a third of that estimated for frog parvalbumin diffusing in bulk water into and out of 3% agarose cylinders (10.6 x 10(-7) cm2 s-1; 4 degrees C). The reduced translational mobility of parvalbumin in myoplasm reflects an elevated effective viscosity due to tortuosity and viscous drag imposed by the fixed proteins of the cytomatrix and the numerous diffusible particles of the cytosol.

Animals↗

PubMed1999

A novel role for cardiac neural crest in heart development.

Ablation of premigratory cardiac neural crest results in defective development of the cardiac outflow tract. The purpose of the present study was to correlate the earliest functional and morphological changes in heart development after cardiac neural crest ablation. Within 24 hours after neural crest ablation, the external morphology of the hearts showed straight outflow limbs, tighter heart loops, and variable dilations. Incorporation of bromodeoxyuridine in myocytes, an indication of proliferation, was doubled after cardiac neural crest ablation. The myocardial calcium transients, which are a measure of excitation-contraction coupling, were depressed by 50% in both the inflow and outflow portions of the looped heart tube. The myocardial transients could be rescued by replacing the cardiac neural crest. The cardiac jelly produced by the myocardium was distributed in an uneven, rather than uniform, pattern. An extreme variability in external morphology could be attributed to the uneven distribution of cardiac jelly. In the absence of cardiac neural crest, the myocardium was characterized by somewhat disorganized myofibrils that may be a result of abnormally elevated proliferation. In contrast, endocardial development appeared normal, as evidenced by normal expression of fibrillin-2 protein (JB3 antigen) and normal formation of cushion mesenchyme and trabeculae. The signs of abnormal myocardial development coincident with normal endocardium suggest that the presence of cardiac neural crest cells is necessary for normal differentiation and function of the myocardium during early heart development. These results indicate a novel role for neural crest cells in myocardial maturation.

Animals↗

PubMed1998

Alterations of myocardial contraction associated with a structural heart defect in embryonic chicks.

Ablation of cardiac neural crest at stages 8-10 produces a structural heart defect (persistent truncus arteriosus, PTA) in embryonic chicks. PTA is associated with decreased myocardial contractility, as indicated by decreased left ventricular ejection fraction. We compared the force of small ventricular strips from normal and defective chick hearts. In intact muscle, ablation of the neural crest leads to a 30-50% decrease in twitch force at any level of extracellular Ca2+ (0.45-20 mM) at embryonic days (ED) 7 and 15, relative to sham-operated controls. These differences could reflect defects at the level of the contractile apparatus and/or in the excitation-contraction coupling process. To distinguish changes of the contractile apparatus, we used detergent skinned preparations. The maximal Ca(2+)-activated force (Fmax) at ED15 was not significantly different between control and experimental embryos. At ED 7, however, Fmax was reduced by 36% in experimental preparations. Electron-micrographs showed that the organization and orientation of the myofibrils was similar in experimental and control ventricles. At ED 14, however, the average myofibrillar diameter was significantly increased in experimental ventricles. The content of the major myofibrillar proteins (myosin heavy chain, actin, and tropomyosin), determined from polyacrylamide gel electrophoresis and Coomassie Blue staining, normalized to total protein, was not statistically different in experimental and control ventricles at ED7. At ED15, however, content of these proteins was doubled in experimental ventricles. These data suggest a possible defect of the contractile apparatus at both ED 7 and 15, since the ratio of Fmax/myosin is reduced in the experimental hearts.

Animals↗

PubMed1998

Role of cardiac neural crest cells in cardiovascular development.

The discovery in the chick embryo that a specific region of the neural crest, termed the cardiac neural crest, is essential for septation of the cardiac outflow tract and for aortic arch artery development has led to the classification of a whole series of human cardiac defects as neural crest-associated. Recently, several mouse genetic models have been effectively employed to yield new insights into the relationship between cardiac neural crest and structural heart development. In all the animal models of neural crest-related heart defects, prenatal mortality is too high to be attributed to structural defects of the heart alone, and there are obvious signs of severe cardiac dysfunction. The evidence indicates that poor viability is from impaired cardiac excitation-contraction coupling and contractile function at the myocyte level. The continued study of experimental and genetically defined models with neural crest-associated heart defects will prove useful in identifying the common pathways by which the neural crest contributes to normal heart development.

Animals↗

PubMed1997

Neural crest is involved in development of abnormal myocardial function.

Around 85% of embryos homozygous for the splotch (Sp2H) allele (Sp2H/Sp2H), a Pax3 mutation, develop persistent truncus arteriosus (PTA), a defect related to the cardiac neural crest. These embryos die by 14.5 days post coitum. In an investigation of the cause of lethality in these embryos, we used digital video imaging microscopy to examine beating embryonic hearts in situ at 13.5 dpc. The hearts of Sp2H/Sp2H embryos with PTA clearly showed poor function when compared with normal litter mates. Contractile force was examined in detergent-skinned ventricular muscle strips from Sp2H/Sp2H embryos at ages 12.5 and 13.5 dpc. There was no significant difference in the maximum force or in myosin content between Sp2H/Sp2H and control groups, indicating no significant dysfunction of the contractile apparatus in hearts from Sp2H/Sp2H embryos. Ca2+ transients were examined in enzymatically-dissociated ventricular myocytes and were significantly reduced in defective hearts, indicating that reduced cardiac function in Sp2H/Sp2H embryos with PTA was due to impaired excitation-contraction (EC) coupling. Ca2+ currents were examined using the perforated patch clamp technique. The magnitude of the Ca2+ current was found to be reduced by approximately 3.2-fold in Sp2H/Sp2H hearts with PTA compared to normal. Since the sarcoplasmic reticulum is sparse or absent in the embryonic heart, the impaired EC coupling was due to the reduction in Ca2+ current. These observations suggest that neural crest abnormalities result in a defect in EC coupling, causing depressed myocardial function and death in utero from cardiac failure. Interestingly, Sp2H/Sp2H hearts without PTA had normal EC coupling. These results indicated that impaired EC coupling was secondary to the Pax3 mutation. The findings in this report indicate an important role for the neural crest in the development of normal myocardial function, and represent the first demonstration of impaired excitation-contraction coupling in a genetically-defined embryonic mammalian model of a cardiac structural defect.

Animals↗

PubMed1997

Effect of cardiac neural crest ablation on contractile force and calcium uptake and release in chick heart.

Cardiac neural crest ablation (CNCA) in the chick embryo at stages 8-10 results in reduced contractility of the heart that can be observed as early as stage 14. We found that intact trabeculae from embryonic day (E) 15 experimental animals after CNCA display an approximately 50% decrease in twitch force relative to sham-operated E15 control animals. In control and CNCA trabeculae skinned in Triton X-100 and bathed in our standard solutions, neither maximum Ca(2+)-activated force nor Ca2+ sensitivity of the contractile apparatus was significantly different. CNCA resulted in a marked reduction in the magnitude of the Ca2+ transient in trabeculae, estimated using fura 2 acetoxymethyl ester. CNCA had no effect on the half-time of Ca2+ loading by the sarcoplasmic reticulum (SR) of saponin skinned trabeculae at fixed Ca2+. However, it slightly reduced the Ca2+ sensitivity of Ca2+ uptake by the SR. Its most dramatic effect was to essentially abolish Ca(2+)-induced Ca2+ release from the SR. These effects on Ca2+ metabolism explain, in part, the decrease in the intracellular Ca2+ transient and myocardial contractility observed with CNCA.

Animals↗

PubMed1996

Influence of physiological L(+)-lactate concentrations on contractility of skinned striated muscle fibers of rabbit.

These experiments investigated the effects of physiological concentrations of L(+)-lactate on the contractility of chemically skinned rabbit fast-twitch psoas, slow-twitch soleus, and cardiac muscles at pH 7.L(+)-Lactate depressed maximal calcium-activated force (Fmax) of all muscles studied within the range of 5-20 (slow-twitch muscle) or 5-25 mM (fast-twitch and cardiac muscles). Fmax of fast-twitch fibers was inhibited to the greatest degree (9% in K2 creatine phosphate solutions). In all of these muscle types, Fmax returned to control levels as L(+)-lactate was increased to 30-50 mM. Substitution of neither D-lactate nor propionate for L(+)-lactate significantly altered Fmax. In addition, with the exception of fast-twitch muscle (where the Hill coefficient decreased), L(+)-lactate concentrations, which maximally inhibited Fmax, did not affect the force vs. pCa relationship of muscles tested. These results demonstrate that L(+)-lactate significantly contributes to the depression of muscle function noted during lactic acidosis, directly inhibiting Fmax of the contractile apparatus. This contribution is maximal in fast-twitch muscle where L(+)-lactate is responsible for as much as one-third of the depressant effect on Fmax of the contractile apparatus noted during lactic acidosis.

Animals↗

PubMed1996

Relationship between short-range stiffness and yielding in type-identified, chemically skinned muscle fibers from the cat triceps surae muscles.

1. Transient, stretch-evoked force responses of chemically skinned muscle fibers from the cat hindlimb were investigated. The purpose of these experiments was to determine the exent to which short-range stiffness, the apparent stiffness exerted by the fiber over the first 0.5% of length change, is higher in type I than type II muscle fibers. Fibers were obtained from soleus and vastus intermedius muscles, which contain predominantly type I fibers, the LGm, a compartment of the lateral gastrocnemius muscle that contains predominantly type II fibers, and LG3, a compartment of mixed type. 2. Beyond a short range of approximately 1% of muscle length during a 0.5 muscle length/s (ML/s) stretch, most fibers exhibited an abrupt decrease in apparent stiffness or yield. Fibers from the muscles containing predominantly type S (slow twitch, or type I) fibers, soleus and vastus intermedius, exhibited such a pronounced decline in apparent stiffness that force declined as well during continued stretch. Most of the fibers from the LG3 compartment could be divided into two distinct groups depending upon whether or not they showed a force yield at the stretch velocity of 0.5 ML/s. 3. The short-range stiffness measured over the first 0.5% of stretch was greater for fibers showing force yield than for those that did not at matched initial forces and normalized stretch amplitudes. This result is consistent with the hypothesis that the same mechanism that endows the fiber with high short-range stiffness is also responsible for a greater extent of yielding. 4. Fibers from soleus were found to exhibit a force yield over a 200-fold range of velocities (0.01-2 ML/s). In contrast, most fibers from the LGm compartment showed only an increase in extent of yield with stretch velocity. Some of these fibers eventually yielded in force, but only when they were stretched at velocities > 2 ML/s. The proposed relationship between high short-range stiffness and yielding was supported by the finding that short-range stiffness increased sharply in the range of velocities where the fiber showed the greatest increase in extent of yield. 5. After the physiological experiments, fibers were subjected to SDS gel electrophoresis. Two distinct patterns of bands in the low molecular weight range were found to correspond to the two types that were identified on the basis of their dynamic mechanical properties. Fibers that did not yield at 0.5 ML/s showed a band pattern very similar to that of rabbit psoas (type II) fibers. 6. These results support the hypothesis that type I fibers are specialized in presenting a high short-range stiffness for effective postural control in advance of reflex mechanisms and that this property results from intrinsic properties of the fiber and is not due to differences in the dimensions of type I and II fibers. Yielding serves to protect the fiber from damaging levels of force during lengthening contractions. The importance of these transient properties to the mechanical behavior of muscle during ongoing movements is suggested by the observation that high stiffness followed by yielding is repeated with little alteration during successive stretches.

Animals↗

PubMed1995

The effect of 2,3-butanedione 2-monoxime (BDM) on ventricular trabeculae from the avian heart.

2,3-butanedione 2-monoxime (BDM, 3-30 mM) decreased twitch force of intact ventricular trabeculae isolated from 19-day embryonic chick hearts in a dose-dependent manner. The responses to BDM were rapid and reversible. In an attempt to determine the cellular basis for the inhibitory effect of BDM, experiments were carried out on skinned muscle fibres and isolated myocytes. In trabeculae skinned with Triton X-100, BDM depressed maximum calcium activated force (Fmax) with an IC50 of 14 mM. At 3 mM BDM, the proportional decrease in twitch force in intact tissue was similar to that of Fmax in skinned tissue. At higher BDM concentrations (10 and 30 mM), however, the proportional decrease in twitch force was greater than that of Fmax. BDM (up to 10 mM) had no effect on the normalized force-pCa relationship. In saponin-skinned preparations, BDM (3 and 30 mM) released calcium from the fully loaded sarcoplasmic reticulum to a slightly greater extent in the absence of calcium (pCa 8.5) than in the presence of a fixed level of free calcium (pCa 5.5). Whole cell patch clamping of freshly isolated chick myocytes demonstrated that BDM caused a dose-dependent decrease in the T- and L-type calcium current. Therefore, at low BDM concentrations (3 mM), the decrease in twitch force can be ascribed predominantly to depression of the contractile apparatus while, at higher concentrations of BDM, there is an additional inhibitory effect of BDM on excitation-contraction coupling.

Animals↗

PubMed1995

Approximating the isometric force-calcium relation of intact frog muscle using skinned fibers.

In previous papers we used estimates of the composition of frog muscle and calculations involving the likely fixed charge density in myofibrils to propose bathing solutions for skinned fibers, which best mimic the normal intracellular milieu of intact muscle fibers. We tested predictions of this calculation using measurements of the potential across the boundary of skinned frog muscle fibers bathed in this solution. The average potential was -3.1 mV, close to that predicted from a simple Donnan equilibrium. The contribution of ATP hydrolysis to a diffusion potential was probably small because addition of 1 mM vanadate to the solution decreased the fiber actomyosin ATPase rate (measured by high-performance liquid chromatography) by at least 73% but had little effect on the measured potential. Using these solutions, we obtained force-pCa curves from mechanically skinned fibers at three different temperatures, allowing the solution pH to change with temperature in the same fashion as the intracellular pH of intact fibers varies with temperature. The bath concentration of Ca2+ required for half-maximal activation of isometric force was 1.45 microM (22 degrees C, pH 7.18), 2.58 microM (16 degrees C, pH 7.25), and 3.36 microM (5 degrees C, pH 7.59). The [Ca2+] at the threshold of activation at 16 degrees C was approximately 1 microM, in good agreement with estimates of threshold [Ca2+] in intact frog muscle fibers.

Adenosine Triphosphatases↗

PubMed1993

Influence of ionic strength on contractile force and energy consumption of skinned fibers from mammalian and crustacean striated muscle.

Increased ionic strength decreases maximal calcium-activated force (Fmax) of skinned muscle fibers via mechanisms that are incompletely understood. In detergent-skinned fibers from either rabbit (psoas) or lobster (leg or abdomen), Fmax in KCl-containing solutions was less than in potassium methanesulfonate (KMeSO3), which we showed previously was the least deleterious salt for adjusting ionic strength. In either salt, lobster fibers were considerably less sensitive to elevated ionic strength than rabbit fibers. Trimethylamine N-oxide (TMAO, a zwitterionic osmolyte found in high concentration in cells of salt-tolerant animals) increased Fmax, especially in high KCl solutions. In this regard, TMAO was more effective than a variety of other natural or synthetic zwitterions. In rabbit fibers, increasing ionic strength decreases Fmax but has little effect on contractile ATPase rate measured simultaneously using a linked-enzyme assay. Thus high salt increases the tension-cost of contraction (i.e. ratio ATPase/Fmax). At both high and low salt, TMAO decreases tension-cost. Given a simple two-state model of the cross-bridge cycle, these data indicate that ionic strength and TMAO affect the apparent detachment rate constant. High ionic strength KCl solutions extract myosin heavy- and light-chains, and troponin C from rabbit fibers. This extraction is virtually abolished by TMAO. Natural zwitterions, such as TMAO, have been shown to protect proteins against destabilization by high salt or other denaturatants. Our data indicate that, even in the best of salts, destabilization of the actomyosin complex may play a role in the effect of high ionic strength on the contractile process.

Adenosine Triphosphate↗

PubMed1993

Contraction of developing avian heart muscle.

1. Developmental changes in contraction of chick heart show strong similarities with those of the mammalian myocardium. 2. Normalized twitch force of intact trabeculae from chick left ventricle increases most markedly during the 3-day period around the time of hatching. 3. At any age, elevation of extracellular [Ca2+] to 10-20 mM increases twitch force to a maximum. 4. Studies using membrane-free ("skinned") trabeculae demonstrate that the developmental increase in twitch force is paralleled by an increase in the maximal contractile capability of the muscle, that is probably due to proliferation of contractile proteins. 5. At all ages studied, maximal twitch force of intact trabeculae at 10-20 mM extracellular [Ca2+] is similar to maximal Ca(2+)-activated force of the trabeculae after skinning. 6. Calcium sensitivity of the contractile apparatus in chick heart decreases with development in parallel with isoform switching in troponin T. 7. The depressant effect of acidosis on calcium sensitivity of the contractile apparatus increases with development in parallel with isoform switching in troponin I. 8. As in mammalian heart, both acidosis and inorganic phosphate (Pi) depress force generation by the contractile machinery of chick heart.

Animals↗

PubMed1992

Technical problems related to the analysis of the effects of inorganic phosphate on cardiac muscle.

1. We describe how potential artifacts (due to solution composition, buffering capacity of the bathing medium, size of the skinned fiber preparation, permeability of the sarcoplasmic reticulum (SR) vesicles, and proper Kd for Ca2+ of the fluorescent indicator used to measure Ca2+ transport can be avoided in order to determine the effects of inorganic phosphate (Pi, or any other ion) on maximum Ca2+ activated force (Fmax) and Ca2+ sensitivity of skinned cardiac muscle fibers, and Ca(2+)-ATPase activity and uptake properties of isolated cardiac SR-enriched vesicles. 2. To maintain the ionic strength of the bathing medium constant when adding Pi, other ions must be removed. We found that because some salts have a depressant effect on Fmax independent of increased ionic strength (e.g. KCl) while others do not (e.g. Na-acetate), the salt used to adjust ionic strength influences the measured depressant effect of Pi on Fmax. 3. The sensitivity to Ca2+ of the contractile apparatus depends on the sum of the [Na+] and [K+] in the bathing medium. However, we found that the effect of Pi on Ca2+ sensitivity was not significantly influenced by the small changes in the sum of [Na+] and [K+] that were associated with the addition of Pi. 4. The skinned fiber preparations were approximately cylindrical bundles with diameters ranging between 100 and 250 microns. We found that the effect of Pi on Fmax was not influenced by diffusion limitations over this range of bundle diameters. 5. The pH buffering capacity of the bathing solution affects Fmax at pH 6.6. We found that the buffering effect of Pi can influence the mechanical response of skinned fibers independent of a direct effect of Pi on the contractile apparatus when the buffering capacity of the control solution is low. 6. When the Ca(2+)-ATPase of isolated SR vesicles is activated by Ca2+ and MgATP, the vesicles accumulate Ca2+. Unless the vesicles are permeabilized with a Ca2+ ionophore (ionomycin) and the pH adequately buffered, maximum ATPase activity will be underestimated, the broadness of the curve relating Ca2+ to Ca(2+)-ATPase rate overestimated, and the sensitivity to Pi overestimated. 7. The ionic milieu of isolated SR vesicles changes the apparatus dissociation constant (Kd) of Ca(2+)-Fura-2, a fluorescent dye used to quantify SR Ca2+ transport rates. In order to accurately measure the inhibitory effect of Pi on Ca2+ uptake, the influence of Pi on the Kd of fura-2 for Ca2+ must be taken into account.

Animals↗

PubMed1991

Ion-specific and general ionic effects on contraction of skinned fast-twitch skeletal muscle from the rabbit.

We used single fibers from rabbit psoas muscle, chemically skinned with Triton X-100 nonionic detergent, to determine the salts best suited for adjusting ionic strength of bathing solutions for skinned fibers. As criteria we measured maximal calcium-activated force (Fmax), fiber swelling estimated optically, and protein extraction from single fibers determined by polyacrylamide gel electrophoresis with ultrasensitive silver staining. All things considered, the best uni-univalent salt was potassium methanesulfonate, while a number of uni-divalent potassium salts of phosphocreatine, hexamethylenediamine N,N,N',N'-tetraacetic acid, sulfate, and succinate were equally acceptable. Using these salts, we determined that changes in Fmax correlated best with variations of ionic strength (1/2 sigma ci z2i, where ci is the concentration of ion i, and zi is its valence) rather than ionic equivalents (1/2 sigma ci magnitude of zi). Our data indicate that increased ionic strength per sc decreases Fmax, probably by destabilizing the cross-bridge structure in addition to increasing electrostatic shielding of actomyosin interactions.

Animals↗

PubMed1991

Changes in force and calcium sensitivity in the developing avian heart.

The aim of this study was to characterize the development of the contractile properties of intact and chemically skinned muscle from chicken heart and to compare these characteristics with those of developing mammalian heart reported by others. Small trabeculae were dissected from left ventricles of Arbor Acre chickens between embryonic day 7 and young adulthood (7 weeks post-hatching). At all ages, increasing extracellular calcium (0.45-3.6 mM) progressively increased twitch force of electrically stimulated trabeculae. Twitch force at 1.8 mM extracellular calcium, normalized to cross-sectional area, increased to a maximum at 1 day post-hatching, remained constant through 3 weeks post-hatching, but then decreased at 7 weeks post-hatching. The maximal calcium-activated force of trabeculae chemically skinned with Triton X-100 detergent increased to a maximum 2 days before the time of hatching and was not significantly changed up to 7 weeks post-hatching. Over the ages studied, average twitch force in 1.8 mM calcium was between 26 and 66% of maximal calcium-activated force after skinning, suggesting that the contractile apparatus is not fully activated during the twitch in normal Ringer. In skinned trabeculae, the calcium sensitivity of the contractile apparatus was higher in the embryo than in the young adult. These age-dependent changes in calcium sensitivity are correlated with isoform switching in troponin T. A decrease in pH from 7.0 to 6.5 decreased the calcium sensitivity of the contractile apparatus to a greater degree in skinned trabeculae from young adult hearts than in those from embryonic hearts. This change in susceptibility to acidosis is temporally associated with isoform switching in troponin I.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis↗