Electrical and Mechanical Responses in Deep Abdominal Extensor Muscles of Crayfish and Lobster

Electrical and mechanical studies have been made of the deep abdominal extensor muscles, medial (DEAM) and lateral (DEAL), of crayfish and lobster. The medial muscle responds to direct (intracellular) and indirect stimulation with a transient membrane depolarization which exhibits the properties of a propagated non-decremental action potential but does not overshoot the zero level. The amplitude is about 30 mv in crayfish and 50 mv in lobster. It is followed by a fast all-or-none twitch whose duration at 20°C is 30 to 50 msec. and whose developed tension is 500 gm/cm² or about half the tetanic value. Membrane potential is K⁺-dependent and immersion in high K⁺ induces a brief transient tension rise as in other twitch-type muscles. The action potential and twitch are normal even if all external Na⁺ is replaced with sucrose but vary with external Ca⁺⁺, the action potential increasing 8 to 10 mv for a twofold increase in Ca⁺⁺. The lateral muscle (DEAL) is much slower and responds to intracellular stimulation only with an electrotonic or a local response. Mechanical responses and relaxation speeds are slow with minimal duration of contraction of 0.5 to 2 seconds. Immersion in high K solutions induces large maintained tensions. Sarcomere length in the fast DEAM is uniform and about 2 µ at rest, but in the DEAL speed is less and sarcomere length is greater averaging about 4.5 µ but with a mixed population of fibers.     

The role of calcium in excitation-contraction coupling of lobster muscle

Potassium contractures were induced in lobster muscle bundles under conditions which produced varying KCl fluxes into the fibers. The presence or absence of chloride fluxes during depolarization by high concentrations of potassium, had no effect on the tensions developed. The curve relating tension to the membrane potential had a typical sigmoid shape with an apparent "threshold" for tension at -60 mv. Soaking the muscles in low (0.1 mM) calcium salines for 30 min completely eliminated the potassium contractures but the caffeine contractures were only slightly reduced under these conditions. The potassium contracture could be completely restored in less than 2 min by return of the calcium ions to the saline. Evidence is presented for independent, superficial, and deep calcium sites; the superficial sites appear to be involved in the coupling mechanisms associated with potassium contractures. These sites are highly selective for Ca⁺⁺, and attempts to substitute either Cd⁺⁺, Co⁺⁺, Mg⁺⁺, Ba⁺⁺, or Sr⁺⁺ for Ca⁺⁺ were unsuccessful. However, K⁺ appeared to compete with Ca⁺⁺ for these sites, and the evoked tension could be reduced by prestimulation of the muscle fibers with high K⁺ salines. The results of studies on the influx of ⁴⁵Ca during potassium contractures were compatible with the view of muscle activation by the entry of extracellular calcium.     

Effect of External Calcium and of Temperature on Contraction in Snake Muscle Fibers

The effect of external calcium and of temperature on the contractile responses has been studied in voltage clamped snake twitch muscle fibers. Increasing [Ca⁺⁺]_o from 0.2 to 7.0 mM raised contractile threshold by 15–20 mV, the latter coinciding with the appearance of delayed rectification. The duration of contracture, the rates of rise and decay of tension depended on the level of depolarization and [Ca⁺⁺]_o. The minimum duration of repolarization necessary to restore the contractile response was much shorter in high [Ca⁺⁺]_o. When the bathing solution was cooled to 10 from 20°C the time-course of contracture was markedly prolonged and the outward current was reduced without significant change in maximum tension. The threshold for contraction tended to be somewhat lower at the lower temperature. The contractile repriming was much slower at low temperature. However, reduction in temperature slowed the rate of recovery much less at low [Ca⁺⁺]_o than at normal [Ca⁺⁺]_o.     

Effect of ATP on the Calcium Efflux in Dialyzed Squid Giant Axons

Dialysis perfusion technique makes it possible to control the internal composition of squid giant axons. Calcium efflux has been studied in the presence and in the virtual absence (<5 µM) of ATP. The mean calcium efflux from axons dialyzed with 0.3 µM ionized calcium, [ATP]_i > 1,000 µM, and bathed in artificial seawater (ASW) was 0.24 ± 0.02 pmol·cm^-2·s^-1 (P/CS) (n = 8) at 22°C. With [ATP]_i < 5 µM the mean efflux was 0.11 ± 0.01 P/CS (n = 15). The curve relating calcium efflux to [ATP]_i shows a constant residual calcium efflux in the range of 1–100 µM [ATP]_i. An increase of the calcium efflux is observed when [ATP]_i is >100 µM and saturates at [ATP]_i > 1,000 µM. The magnitude of the ATP-dependent fraction of the calcium efflux varies with external concentrations of Na⁺, Ca⁺⁺, and Mg⁺⁺. These results suggest that internal ATP changes the affinity of the calcium transport system for external cations.     

Juxtaposition of the changes in intracellular calcium and force during staircase potentiation at 30 and 37°C

Ca²⁺ entry during the action potential stimulates muscle contraction. During repetitive low frequency stimulation, skeletal muscle undergoes staircase potentiation (SP), a progressive increase in the peak twitch force induced by each successive stimulus. Multiple mechanisms, including myosin regulatory light chain phosphorylation, likely contribute to SP, a temperature-dependent process. Here, we used the Ca²⁺-sensitive fluorescence indicators acetoxymethyl (AM)-furaptra and AM-fura-2 to examine the intracellular Ca²⁺ transient (ICT) and the baseline Ca²⁺ level at the onset of each ICT during SP at 30 and 37°C in mouse lumbrical muscle. The stimulation protocol, 8 Hz for 8 s, resulted in a 27 ± 3% increase in twitch force at 37°C and a 7 ± 2% decrease in twitch force at 30°C (P < 0.05). Regardless of temperature, the peak rate of force production (+df/dt) was higher in all twitches relative to the first twitch (P < 0.05). Consistent with the differential effects of stimulation on twitch force at the two temperatures, raw ICT amplitude decreased during repetitive stimulation at 30°C (P < 0.05) but not at 37°C. Cytosolic Ca²⁺ accumulated during SP such that baseline Ca²⁺ at the onset of ICTs occurring late in the train was higher (P < 0.05) than that of those occurring early in the train. ICT duration increased progressively at both temperatures. This effect was not entirely proportional to the changes in twitch duration, as twitch duration characteristically decreased before increasing late in the protocol. This is the first study identifying a changing ICT as an important, and temperature-sensitive, modulator of muscle force during repetitive stimulation. Moreover, we extend previous observations by demonstrating that contraction-induced increases in baseline Ca²⁺ coincide with greater +df/dt but not necessarily with higher twitch force.     

Mechanisms for Intracellular Calcium Regulation in Heart : I. Stopped-Flow Measurements of Ca++ Uptake by Cardiac Mitochondria

Initial velocities of energy-dependent Ca⁺⁺ uptake were measured by stopped-flow and dual-wavelength techniques in mitochondria isolated from hearts of rats, guinea pigs, squirrels, pigeons, and frogs. The rate of Ca⁺⁺ uptake by rat heart mitochondria was 0.05 nmol/mg/s at 5 µM Ca⁺⁺ and increased sigmoidally to 8 nmol/mg/s at 200 µM Ca⁺⁺. A Hill plot of the data yields a straight line with slope n of 2, indicating a cooperativity for Ca⁺⁺ transport in cardiac mitochondria. Comparable rates of Ca⁺⁺ uptake and sigmoidal plots were obtained with mitochondria from other mammalian hearts. On the other hand, the rates of Ca⁺⁺ uptake by frog heart mitochondria were higher at any Ca⁺⁺ concentrations. The half-maximal rate of Ca⁺⁺ transport was observed at 30, 60, 72, 87, 92 µM Ca⁺⁺ for cardiac mitochondria from frog, squirrel, pigeon, guinea pig, and rat, respectively. The sigmoidicity and the high apparent K_m render mitochondrial Ca⁺⁺ uptake slow below 10 µM. At these concentrations the rate of Ca⁺⁺ uptake by cardiac mitochondria in vitro and the amount of mitochondria present in the heart are not consistent with the amount of Ca⁺⁺ to be sequestered in vivo during heart relaxation. Therefore, it appears that, at least in mammalian hearts, the energy-linked transport of Ca⁺⁺ by mitochondria is inadequate for regulating the beat-to-beat Ca⁺⁺ cycle. The results obtained and the proposed cooperativity for mitochondrial Ca⁺⁺ uptake are discussed in terms of physiological regulation of intracellular Ca⁺⁺ homeostasis in cardiac cells.     

An X-Ray Diffraction Study on Early Structural Changes in Skeletal Muscle Contraction

Structural changes in frog skeletal muscle were studied using x-ray diffraction with a time resolution of 0.53–1.02 ms after a single electrical stimulus at 8°C. Tension began to drop at 6 ms (latency relaxation), reached a minimum at 8 ms, and then twitch tension developed. The intensity of the meridional reflection at 1/38.5 nm⁻¹, from troponin molecules on the thin filament, began to increase at 4–5 ms and reached a maximum at ~12 ms. The meridional reflections based on the myosin 43-nm repeat began to decrease when the tension began to develop. The peak position of the third-order myosin meridional reflection began to shift toward the higher angle at ~5 ms, reached a maximum shift (0.02%) at 10 ms, and then moved toward the lower angle. The intensity of the second actin layer line at 1/18 nm⁻¹ in the axial direction, which was measured at 12°C, began to rise at 5 ms, whereas the latency relaxation started at 3.5 ms. These results suggest that 1), the Ca²⁺-induced structural changes in the thin filament and a structural change in the thick filament have already taken place during latency relaxation; and 2), the Ca²⁺ regulation of the thin filament is highly cooperative.     

Chronic Lead Exposure Increases Blood Pressure and Myocardial Contractility in Rats

We investigated the cardiovascular effects of lead exposure, emphasising its direct action on myocardial contractility. Male Wistar rats were sorted randomly into two groups: control (Ct) and treatment with 100 ppm of lead (Pb) in the drinking water. Blood pressure (BP) was measured weekly. At the end of the treatment period, the animals were anaesthetised and haemodynamic parameters and contractility of the left ventricular papillary muscles were recorded. Blood and tissue samples were properly stored for further biochemical investigations. Statistical analyses were considered to be significant at p<0.05. The lead concentrations in the blood reached approximately 13 µg/dL, while the bone was the site of the highest deposition of this metal. BP in the Pb-treated group was higher from the first week of lead exposure and remained at the same level over the next four weeks. Haemodynamic evaluations revealed increases in systolic (Ct: 96±3.79 vs. Pb: 116±1.37 mmHg) and diastolic blood pressure (Ct: 60±2.93 vs. Pb: 70±3.38 mmHg), left ventricular systolic pressure (Ct: 104±5.85 vs. Pb: 120±2.51 mmHg) and heart rate (Ct: 307±10 vs. Pb: 348±16 bpm). Lead treatment did not alter the force and time derivatives of the force of left ventricular papillary muscles that were contracting isometrically. However, our results are suggestive of changes in the kinetics of calcium (Ca⁺⁺) in cardiomyocytes increased transarcolemmal Ca⁺⁺ influx, low Ca⁺⁺ uptake by the sarcoplasmic reticulum and high extrusion by the sarcolemma. Altogether, these results show that despite the increased Ca⁺⁺ influx that was induced by lead exposure, the myocytes had regulatory mechanisms that prevented increases in force, as evidenced in vivo by the increased systolic ventricular pressure.     

Intracellular Calcium Binding and Release in Frog Heart

The capacities and affinities of intracellular calcium-binding sites have been studied in frog ventricles, in which the concentration of Ca⁺⁺ in the sarcoplasm can be controlled as a result of treatment with EDTA. The total calcium content of calcium-depleted and nondepleted muscles at rest and muscles generating considerable tension was 0.8, 1.4, and 5.4 µmol/g of muscle, respectively. Net movement of calcium into or out of the cells occurred without change in tension when the sarcoplasmic concentration of Ca⁺⁺ was either of two values, less than 10^-7 M or approximately 5 x 10^-7 M. These data can be explained by the presence of two groups of intracellular calcium sinks which compete with the contractile proteins, one with a capacity of about 0.6 µmol/g and an affinity constant greater than 10⁷ M^-1 and a second with a capacity of 4.0 µmol/g and an affinity constant of about 2 x 10⁶ M^-1. The higher affinity calcium is released by anoxia, oligomycin, or abrupt changes in sarcoplasmic Ca⁺⁺. Muscles soaked in Sr-Ringer's contain electron densities in the sarcoplasmic reticulum and to a lesser extent in the mitochondria.     

Extracellular calcium dependence of contracture and modulation by serotonin in buccal muscle E1 of Aplysia

Serotonin [5-hydroxytryptamine (5-HT)] enhances acetyl choline (ACh)-elicited contractures of Aplysia buccal muscles E1 and I5. The possible role of external calcium in regulating the magnitude of ACh contracture in the presence and absence of 5-HT was investigated. Superfusion of E1 with zero calcium medium caused ACh contractures to fail within one to two minutes. Recovery of ACh contracture upon restoring normal medium occurred within two to four minutes. In the absence of 5-HT, ACh contracture decreased proportionally to external [Ca⁺⁺] in the concentration range of 0–10 mM; however, the amount of enhancement of of ACh contracture following 5-HT treatment did not decrease with external [Ca⁺⁺] as long as [Ca⁺⁺] was above a threshold concentration that varied from preparation to preparation. For most preparations, the enhancement of ACh contracture by 5-HT was dependent on the presence of external calcium during 5-HT treatment. Calcium influx into muscles E1 and I5 increased approximately two and a half fold in the presence of 10^?6 M 5-HT. A model in which 5-HT brings about calcium “loading” of an ACh releasable intracellular storage site is discussed.     

Tension in Isolated Frog Muscle Fibers Induced by Hypertonic Solutions

The effect of hypertonic solutions on the tension of isolated twitch muscle fibers of the frog has been investigated. Increased tonicity up to about 1.7 times normal (1.7 T) caused a very small, graded, maintained tension increase. Above about 1.7 T a large, transient contracture response was superimposed on the small tension. The contracture response was graded with tonicity and reached a maximum at 2.5 T of 108 ± 25 mN·mm² a third of the maximum tetanic tension in isotonic solution. Contracture tension developed with a delay which decreased with increased tonicity. The contracture threshold was lower and the delay shorter in small fibers than in large. Contractures were obtained equally well in depolarized as in polarized fibers. They were completely suppressed by 0.1–0.5 mM tetracaine. The possible mechanism responsible for the tension-inducing effect of hypertonic solutions is discussed in terms of the close similarity between the properties of these contractures and those caused by caffeine, and it is suggested that the effect is due to a release of calcium from internal stores.     

Lanthanum in heart cell culture. Effect on calcium exchange correlated with its localization

Correlation of the localization of La⁺⁺⁺ with its effects on Ca⁺⁺ exchange in cultured rat heart cells is examined with the use of a recently developed technique. 75% of cellular Ca⁺⁺ is exchangeable and is completely accounted for by two kinetically defined phases. The rapidly exchangeable phase has a t ½ = 1.15 min and accounts for 1 1 mmoles Ca⁺⁺/kg wet cells or 43% of the exchangeable Ca⁺⁺ (cells perfused with [Ca⁺⁺]_o = 1 mM) Phase 2 has a t ½ = 19.2 min and accounts for 1.5 mmoles Ca⁺⁺/kg wet cells or 57% of the exchangeable Ca⁺⁺. 0.5 mM [La⁺⁺⁺]_o displaces 0 52 mmoles Ca⁺⁺/kg wet cells—all from phase 1—and almost completely abolishes subsequent Ca⁺⁺ influx and efflux The presence of La⁺⁺⁺ in the washout converts the washout pattern to a single phase system with a t ½ = 124 min. The effects upon Ca⁺⁺ exchange are coincident with abolition of contractile tension but regenerative depolarization of the tissue is maintained Electron microscope localization of the La⁺⁺⁺ places it exclusively in the external lamina or basement membrane of the cells. The study indicates that negatively charged sites in the basement membrane play a crucial role in the E-C coupling process in heart muscle     

Caffeine and excitation-contraction coupling in the guinea pig taenia coli

The effects of caffeine (0.2–10 mM) on the electrical and mechanical activities of guinea pig taenia coli were investigated with the double sucrose-gap method. Caffeine evoked a small tension with a latency of 20–30 sec, then phasic contraction developed and finally relaxation. The initial tension development also appeared in the Na-free solution without any marked changes in the membrane potential and membrane resistance. The phasic contraction disappeared in the Na-free solution. The relaxation in the presence of caffeine was accompanied by depolarization block of the spike generation. The minimum concentration of Ca ion needed to evoke the tension development by the caffeine was 10^-7 M. Caffeine also potentiated the twitch tension below a concentration of 5 mM either in the Na-free solution or at low temperature (5°C). NO₃ ^- and Br^- showed a similar response to caffeine on the potentiation of the twitch tension at low temperature.     

Der Sauerstoffverbrauch phasischer und tonischer Skelettmuskeln des Frosches in Lösungen mit hohem K+- und variiertem Ca++-Gehalt

The O₂-uptake of phasic and tonic muscles fromRana temporaria was measured under the influence of high extra-cellular concentrations of K⁺ by means of the Pt-electrode together with simultaneous registration of muscles-mechanics. The O₂-uptake in isotonic KCl coincides with the amount of tension (tetanus and contracture); at relaxation the stimulation of respiration ceases; in spite of the permanent depolarization also the “resting respiration” decreases. If isotonic KCl is replaced by Ringer, respiration remains at an elevated state; so-called after-contractures expressing a plastic tonus reveal no additional O₂-uptake. In muscles deprived of Ca⁺⁺ the development of tonus and increase of respiration is strongly reduced, whereas both parameters increase considerably on Ca-enriched muscles. By removing of Ca⁺⁺ in Ca-free Ringer a slight increase in O₂-uptake occurs; in phasic muscles because of twitches, in tonic muscles because of weak contractures (Ca-withdrawal contracture). After addition of Ca⁺⁺ the increase of respiration occurs before mechanical effects become observable. A further increase of Ca-concentration produces a slow and relatively weak tonus, at which O₂-uptake increases transitorily, but decreases soon in spite of the developping contracture (rigor). Oxygen uptake is related to mechanics but not to depolarization of the membrane. The latter is linked with both processes by means of coupling reactions, whereby Ca⁺⁺ plays an important role. In addition to the electro-mechanical coupling, the existence of a direct Ca⁺⁺-dependent electro-respiratory coupling may be assumed.     

Studies of cardiac muscle with a high permeability to calcium produced by treatment with ethylenediaminetetraacetic acid

Thin strips of frog ventricle were isolated and bathed for 15 min in a solution containing 140 mM KCl, 5 mM Na₂ATP, 3 mM EDTA, and 10 mM Tris buffer at pH 7.0. The muscle was then exposed to contracture solutions containing 140 mM KCl, 5 mM Na₂ATP, 1 mM MgCl₂, 10 mM Tris, 3 mM EGTA, and CaCl₂ in amounts to produce concentrations of free calcium from 10^-4.8 M to 10^-9 M. The muscles developed some tension at approximately 10^-8 M, and maximum tension was achieved in 10^-5 M Ca⁺⁺. They relaxed in Ca⁺⁺ concentrations less than 10^-8 M. The development of tension by the EDTA-treated muscles was normalized by comparison with twitch tension at a stimulation rate of 9 per min before exposure to EDTA. In 10^-5 M Ca⁺⁺ tension was always several times the twitch tension and was greater than the contracture tension of a frog ventricular strip in KCl low Na-Ringer. Tension equal to half-maximum was produced at approximately 10^-6.2 M Ca⁺⁺. Intracellular recording of membrane potential indicated that after EDTA treatment the resting potential of cells in Ringer solution with 10^-5 M Ca or less was between 5 and 20 mv. Contracture solutions did not produce tension without prior treatment with EDTA. The high permeability of the membrane produced by EDTA was reversed and the normal resting and action potentials restored in 1 mM Ca-Ringer. Similar studies of EDTA-treated rabbit right ventricular papillary muscle produced a similar tension vs. Ca⁺⁺ concentration relation, and the high permeability state reversed with exposure to normal Krebs solution.     

Excitation-Contraction Coupling in Zebrafish Ventricular Myocardium Is Regulated by Trans-Sarcolemmal Ca2+ Influx and Sarcoplasmic Reticulum Ca2+ Release

Zebrafish (Danio rerio) have become a popular model in cardiovascular research mainly due to identification of a large number of mutants with structural defects. In recent years, cardiomyopathies and other diseases influencing contractility of the heart have been studied in zebrafish mutants. However, little is known about the regulation of contractility of the zebrafish heart on a tissue level. The aim of the present study was to elucidate the role of trans-sarcolemmal Ca²⁺-flux and sarcoplasmic reticulum Ca²⁺-release in zebrafish myocardium. Using isometric force measurements of fresh heart slices, we characterised the effects of changes of the extracellular Ca²⁺-concentration, trans-sarcolemmal Ca²⁺-flux via L-type Ca²⁺-channels and Na⁺-Ca²⁺-exchanger, and Ca²⁺-release from the sarcoplasmic reticulum as well as beating frequency and β-adrenergic stimulation on contractility of adult zebrafish myocardium. We found an overall negative force-frequency relationship (FFR). Inhibition of L-type Ca²⁺-channels by verapamil (1 μM) decreased force of contraction to 22±7% compared to baseline (n=4, p<0.05). Ni²⁺ was the only substance to prolong relaxation (5 mM, time after peak to 50% relaxation: 73±3 ms vs. 101±8 ms, n=5, p<0.05). Surprisingly though, inhibition of the sarcoplasmic Ca²⁺-release decreased force development to 54±3% in ventricular (n=13, p<0.05) and to 52±8% in atrial myocardium (n=5, p<0.05) suggesting a substantial role of SR Ca²⁺-release in force generation. In line with this finding, we observed significant post pause potentiation after pauses of 5 s (169±7% force compared to baseline, n=8, p<0.05) and 10 s (198±9% force compared to baseline, n=5, p<0.05) and mildly positive lusitropy after β-adrenergic stimulation. In conclusion, force development in adult zebrafish ventricular myocardium requires not only trans-sarcolemmal Ca²⁺-flux, but also intact sarcoplasmic reticulum Ca²⁺-cycling. In contrast to mammals, FFR is strongly negative in the zebrafish heart. These aspects need to be considered when using zebrafish to model human diseases of myocardial contractility.     

Calcium Efflux from Internally Dialyzed Squid Giant Axons

Calcium efflux has been studied in squid giant axons under conditions in which the internal composition was controlled by means of a dialysis perfusion technique. The mean calcium efflux from axons dialyzed with 0.3 µM calcium and 5 mM ATP was 0.26 pmol/cm²·s at 22°C. The curve relating the Ca efflux with the internal Ca concentration had a slope of about one for [Ca]_i lower than 0.3µM and a slope smaller than one for higher concentrations. Under the above conditions replacement of [Na]_o and [Ca]_o by Tris and Mg causes an 80% fall in the calcium efflux. When the axons were dialyzed with a medium free of ATP and containing 2 mM cyanide plus 5µg/ml oligomycin, analysis of the perfusion effluent gave values of 1–4 µM ATP. Under this low ATP condition, replacement of external sodium and calcium causes the same drop in the calcium efflux. The same effect was observed at higher [Ca]_i, (80 µM). These results suggest that the Na-Ca exchange component of the calcium efflux is apparently not dependent on the amounts of ATP in the axoplasm. Axons previously depleted of ATP show a significant transient drop in the calcium efflux when ATP is added to the dialysis medium. This effect probably represents the sequestering of calcium by the mitochondrial system. The consumption of calcium by the mitochondria of the axoplasm in dialyzed axons was determined to be of the order of 6.0 x 10^-7 mol Ca⁺⁺/mg of protein with an initial rate of 2.6 x 10^-8 mol Ca⁺⁺/min·mg of protein. Axons dialyzed with 2 mM cyanide after 8–10-min delays show a rise in the calcium efflux in the presence of "normal" amounts of exogenous ATP. This effect seems to indicate that cyanide, per se, can release calcium ions from internal sources.     

Contributions of Ca2+-Independent Thin Filament Activation to Cardiac Muscle Function

Although Ca²⁺ is the principal regulator of contraction in striated muscle, in vitro evidence suggests that some actin-myosin interaction is still possible even in its absence. Whether this Ca²⁺-independent activation (CIA) occurs under physiological conditions remains unclear, as does its potential impact on the function of intact cardiac muscle. The purpose of this study was to investigate CIA using computational analysis. We added a structurally motivated representation of this phenomenon to an existing myofilament model, which allowed predictions of CIA-dependent muscle behavior. We found that a certain amount of CIA was essential for the model to reproduce reported effects of nonfunctional troponin C on myofilament force generation. Consequently, those data enabled estimation of ΔG_CIA, the energy barrier for activating a thin filament regulatory unit in the absence of Ca²⁺. Using this estimate of ΔG_CIA as a point of reference (∼7 kJ mol⁻¹), we examined its impact on various aspects of muscle function through additional simulations. CIA decreased the Hill coefficient of steady-state force while increasing myofilament Ca²⁺ sensitivity. At the same time, CIA had minimal effect on the rate of force redevelopment after slack/restretch. Simulations of twitch tension show that the presence of CIA increases peak tension while profoundly delaying relaxation. We tested the model’s ability to represent perturbations to the Ca²⁺ regulatory mechanism by analyzing twitch records measured in transgenic mice expressing a cardiac troponin I mutation (R145G). The effects of the mutation on twitch dynamics were fully reproduced by a single parameter change, namely lowering ΔG_CIA by 2.3 kJ mol⁻¹ relative to its wild-type value. Our analyses suggest that CIA is present in cardiac muscle under normal conditions and that its modulation by gene mutations or other factors can alter both systolic and diastolic function.     

Autoradiographic Studies of Intracellular Calcium in Frog Skeletal Muscle

Autoradiographs consisting of a 1000 A thick tissue section and a 1400 A thick emulsion film have been prepared from frog toe muscles labeled with Ca⁴⁵. The muscles had been fixed with an oxalate-containing osmium solution at rest at room temperature, at rest at 4°C, during relaxation following K⁺ depolarization or after prolonged depolarization. From 6 to 39 per cent of K⁺ contracture tension was produced during fixation. The grains in the autoradiographs were always concentrated in the center 0.2 to 0.3 µ of the I band and the region of the overlapping of the thick and thin filaments. The greater the tension produced during fixation, the greater was the concentration in the A band and the smaller the concentration in the I band. Autoradiographs of two muscles fixed by freeze-substitution resembled those of muscles which produced little tension during osmium fixation. Muscles which shortened during fixation produced fewer grains. In the narrow (<2.0 µ) sarcomeres of the shortened muscles, grain density decreased with decreasing sarcomere width. A theoretical analysis of the significance of these grain distributions is proposed and discussed.     

Membrane Potential of Brown Adipose Tissue : A suggested mechanism for the regulation of thermogenesis

Membrane potentials were recorded in isolated segments of interscapular brown adipose tissue from rats. After equilibration at 29°C in Krebs-Ringer bicarbonate buffer a mean value of -51 ± 4 mv (SD) was found. This level could be maintained for up to 5 hr. The mean effective membrane resistance was 1.35 ± 0.45 megohm. The membrane potential was a function of the extracellular potassium concentration. Ouabain (10^-6-10^-3 M) and incubation in K-free buffer produced progressive depolarization. Epinephrine and norepinephrine in concentrations as low as 10^-8 g/ml produced a prompt depolarization. Cooling of the tissue and lowering of the oxygen tension caused a marked and reversible decrease in the membrane potential. In tissue obtained from cold-adapted rats, the membrane potential was considerably diminished. 6Assuming that the membrane potential is some function of the Na permeability of the plasma membrane it is suggested that an increase in the rate of active Na-K transport and ensuing ADP formation might contribute to the increase in respiration seen during exposure to thermogenic stimuli.