Isolated Frog Skeletal Muscle Fibres Research Articles

Confocal microscopy study of membrane organelles of the skeletal muscle fiber in the process of Zenker’s (spreading) necrosis

Using laser confocal microscopy and some vital fluorescent dyes (acridine orange, RH 414, DiOC6(3), rhodamine 123, fluorescein dextran), changes of the T-system and cellular acidic organelles were studied during spreading (Zenker’s) necrosis of isolated frog skeletal muscle fibers. The most characteristic of the initial stages of development of Zenker’s necrosis is the formation of numerous vacuoles as a result of local T-system swellings. The vacuole length can reach tens of micrometers. They are located both near nuclear poles and between myofibrils. Until the moment of contraction knot separation, the vacuoles preserve their connections with normal T-tubules and under certain conditions (glycerol influx to the fiber) are reversible. The vacuoles deform nuclei and cisternae of the sarcoplasmic reticulum. Acidic cell organelles accumulating acridine orange (lysosomes, late endosomes, trans-Golgi cisternae) are located in the immediate vicinity both of normal and of vacuolated T-tubules. In the course of the development of the pathological process, the size and number of acidic organelles increases and they tend to be clustered. Vacuolation of the T-system during necrosis was not accompanied by vacuole content acidification. At late stages of necrosis, alterations of nuclei and sarcoplasmic reticulum were observed. The role of cellular acidic organelles and of the T-system vacuolation in development of various muscle pathologies is discussed.

Effect of Microwave Electromagnetic Field on Skeletal Muscle Fibre Activity

The aim of the present study was to investigate the influence of microwave irradiation on fatiguing activity of isolated frog skeletal muscle fibres. The changes in the electrical and mechanical activity were used as criteria for the exposure effects. Repetitive suprathreshold stimulation with interstimu-lus interval of 200ms for 3min was applied. Intracellular (ICAP) and extracellular (ECAP) action potentials and twitch contractions (Tw) of muscle fibres after 1 hour microwave exposure (2.45GHz, 20mW/cm 2 power density) were compared with those recorded after one hour sham exposure (control). The duration of uninterrupted activity in the trial (endurance time; ET) was not significantly affected by microwave field exposure. After microwave irradiation, the ICAP amplitude was higher, the rising time was shorter, and the resting membrane potential was more negative compared to controls. There was a slower rate of parameters changes during ET in potentials obtained from irradiated fibres. Microwave exposure increased the propagation velocity of excitation, the ECAP and Tw amplitudes, as well as shortened their time parameters. We concluded that a 2.45GHz microwave field possesses a stimulating effect on muscle fibre activity, which is in part due to its specific, non-thermal properties. The microwave induced-changes in muscle fibre activity may reduce development of skeletal muscle fatigue.

Reversible depression of action potentials and force production in frog single muscle fibres by calmodulin-inhibitors.

The effects of the calmodulin-inhibitors trifluoperazine, thioridazine and zaldaride maleate on the responses to electrical stimulation in isolated frog skeletal muscle fibres were investigated. All three drugs initially reduced the amplitude of the action potentials but potentiated twitch force. This was followed by a total loss of action potentials and force production. However, the resting membrane potential was not changed. The effects were completely reversible upon removal of the drugs. These results suggest that an intact calmodulin system is required for normal function of the sarcolemmal sodium channels of frog skeletal muscle.

Nystatin-, mycoheptin- and levorin-induced conductance in the membrane of frog skeletal muscle fibres.

The effects of the polyene antibiotics nystatin (2 x 10(-5)-10(-4) mol/l), mycoheptin (1.3 x 10(-6)-10(-5) mol/l) and levorin (10(-8)-5 x 10(-5) mol/l) on isolated frog skeletal muscle fibres and whole sartorius muscles of the frog have been investigated. Cation conductance was measured under current clamp conditions using a double sucrose-gap technique. Cation effluxes were studied by means of flame emission photometry. All three antibiotics increased the cation conductance and efflux rates; however, differences between the polyenes were found in the steady state values of induced cation transport at a given concentration. The values of both induced conductance gA infinity and efflux rate constants KA formed the following sequence: levorin > mycoheptin > nystatin, demonstrating a correlation with the order of antifungal activities. The dose-response curves of lg polyene-induced cation transport against lg of antibiotic concentration in our experiments had slope values which were much lower than those in bilayers: 1.7 and 1.3 for nystatin and mycoheptin, respectively, whereas the aromatic heptaene levorin had an even smaller concentration dependence. The decline in the equilibrium conductance caused by nystatin- and mycoheptin removal was very fast (during the first minute tau = 0.74 and 2.39 min, respectively). In contrast, levorin-induced conductance was irreversible. It is proposed that the processes which limit the rate of channel formation are different in biological and model membranes.

Non-homogeneous Ca release in isolated frog skeletal muscle fibres.

We have examined the spatial distribution of [Ca2+]i during tetanic stimulation in frog skeletal muscle cells using a fluorescence imaging method. We have found a completely unexpected pattern of Ca release: Ca is released forming gradients composed of spots of very significant and slow fluctuations of calcium release. Our findings could be explained if the calcium release process in skeletal muscle is influenced significantly by [Ca2+]i, such as in cardiac muscle, and suggests that the SR/Ca release control can include the established voltage-dependent plus a cardiac-like process of calcium-induced Ca release and a Ca release inhibition by Ca.

Volume changes during contraction of isolated frog muscle fibers.

A microscope objective and electronic imaging system were used to determine how isolated frog skeletal muscle fibers adjust their volume during an isometric tetanus. Cross-sectional area and volume of the middle third of a fiber increased rapidly with the development of active tension, which indicates that contraction produced components of force perpendicular to the long axis. The extreme ends are known to shorten whether or not the middle of a fiber is isometric or stretched. Shortening of the ends may shift water towards the middle, which could account for the volume changes we observed. The cytoskeletal matrices of muscle evidently adjust rapidly during contraction to maintain a dynamic equilibrium between the axial and radial forces that stabilize the whole cell. The Z disks have been shown to expand during active, but not passive, tension development. Z disks might be the elastic elements of the muscle cytoskeleton primarily involved in rapid balancing of the radial components of active force.

The effect of the benzothiazepine diltiazem on force and Ca2+ current in isolated frog skeletal muscle fibres.

1. The action of the D-cis and L-cis isomers of the benzothiazepine derivative diltiazem on isometric force and L-type Ca2+ inward current in short muscle fibres from toe muscles of the frog (Rana esculenta and R. temporaria) was investigated under voltage clamp control with two internal microelectrodes. The experiments were performed at 10 degrees C in TEA-sulphate solution, in which the concentration of ionized Ca2+ was about 4 mM. 2. In the presence of diltiazem (D-cis, 0.25-30 microM; L-cis, 5-100 microM) normal Ca2+ currents and phasic contractures could be elicited by a depolarizing voltage step. However, after long-lasting depolarizations both the Ca2+ channel and the force controlling system (voltage sensor) remained refractory for minutes instead of seconds as under control conditions, i.e. they were 'paralysed'. L-cis diltiazem was about 20 times less effective in comparison with the D-cis isomer. 3. Speed of restoration of force and Ca2+ current was retarded by up to two orders of magnitude, depending on the applied concentration of diltiazem. 4. The steady state potential dependence of force restoration (V0.5 = -49 mV; the potential at which half of the voltage sensors are restored) and that of Ca2+ current restoration (V0.5 = -57 mV) were not altered by diltiazem. 5. The time course of the transition to paralysis, which was quite slow under control conditions, could be accelerated by diltiazem in a concentration-dependent manner. 6. Paralysis developed at the same speed when the fibre was depolarized from -90 mV to -30 mV or to +30 mV. 7. The voltage dependence of Ca2+ current inactivation (inactivation curve; conditioning pulses of 2 min duration) was shifted by about 14 mV to more negative potentials (V0.5 = -31 mV; V'0.5 = -45 mV) under the influence of 1 microM-D-cis diltiazem. It is assumed that no steady state was reached during the conditioning pulse. 8. The voltage dependence of force activation (V0.5 = -45 mV) was not altered by diltiazem. 9. Since the effects of both isomers of diltiazem could only be disclosed during the adjustment of a new steady state and since the drugs acted in a similar way on the inactivation kinetics of force and Ca2+ current, it is concluded that, in depolarized fibres, the drug facilitates the transition to a stabilized inactivated form of the voltage sensor/Ca2+ channel molecule.

The effect of the phenylalkylamine D888 (devapamil) on force and Ca2+ current in isolated frog skeletal muscle fibres.

1. The effects of the (+)- and the (-)-isomer of the phenylalkylamine derivative D888 (desmethoxyverapamil or devapamil) on isometric force and slow Ca2+ inward current were investigated in short toe muscle fibres of the frog (Rana temporaria). The experiments were performed under voltage-clamp conditions with two flexible internal glass microelectrodes at 10 degrees C in a TEA sulphate solution containing approximately 4 mM-free Ca2+. 2. In the presence of 0.05-5 microM-(-)-D888 a normal phasic contracture could be induced by a depolarizing voltage step. When depolarization was maintained for some minutes the force-controlling system turned into a stabilized inactivated state (paralysis) from which it recovered upon repolarization within minutes instead of seconds. With the (+)-isomer (0.5-20 microM), a similarly retarded restoration was observed. However, it proved to be less effective than the (-)-isomer. 3. D888 caused a shift to more negative potentials of the S-shaped curve, which describes the voltage dependence of force restoration in the steady state (restoration time 15 min). The potential of half-maximum restoration in the absence of the drug (V = -35.8 mV) changed as follows. (-)-D888: -56 mV (0.05 microM), -69 mV (0.2 microM), -77.5 mV (0.5 microM), and -82 mV (5 microM); (+)-D888: -55.8 mV (0.5 microM), -76.5 mV (5 microM), and -85 mV (20 microM). 4. On the assumption that D888 binds only to the inactivated form of the voltage sensor of force control in the T-tubular membrane (modulated receptor hypothesis) the data presented in paragraph 3 allowed an estimation of the drug-receptor dissociation constants. The KD values ascertained in this way, 1.71 nM for the (-)-isomer and 12.9 nM for the (+)-isomer, are in fair agreement with those obtained from [3H]D888 binding studies by other authors. 5. A comparison between equal concentrations of the two isomers regarding their effect on the speed of restoration and the time needed to transform the sensor into the paralysed state suggests that the differences in the dissociation constants are mainly due to a greater dissociation rate of the (+)-isomer from the sensor. 6. The restoration of the Ca2+ channel was retarded by D888 to a similar extent as that of the voltage sensor. This parallel action on both systems indicates structural similarities between the voltage sensor and the Ca2+ channel. 7. It is concluded that D888 'stabilizes' the inactivated state of the voltage sensor and the Ca2+ channel in a way similar to D600, but with a higher potency. Both isomers of D888 showed an antagonistic action and differed only in their potency.

The apamin-sensitive potassium current in frog skeletal muscle: its dependence on the extracellular calcium and sensitivity to calcium channel blockers

Slow outward potassium currents were recorded in isolated frog skeletal muscle fibres using the double mannitol-gap voltage-clamp technique. Detubulated fibres failed to generate a slow outward current, and apamin had no effect on the remaining current. The maximum blocking effect of organic and inorganic Ca2+-channel blockers on the slow outward channels of intact fibres was larger than that of apamin. Apamin failed to induce an additional block when applied after Ca2+-channel blockers. In a low-Ca2+ solution (OCa, EGTA 1 mM) the slow outward current was slightly increased and the blocking effect of apamin was enhanced. A Ca2+-rich solution (Ca2+ X 10) increased the slow outward current and the blocking effect of apamin was drastically reduced. It is concluded that the apamin-sensitive current which is a component of the slow outward K+ current is located in the tubular membrane. Its activation seems barely dependent on the Ca2+ influx via the slow inward Ca2+ current. Apamin-receptor binding appears to be dependent on the extracellular Ca2+ concentration. Blockade of slow outward current by Ca2+-channel blockers is likely to be the result of a direct action on the slow K+ permeability rather than a consequence of Ca2+ channel inhibition.

Effects of apamin on the outward potassium current of isolated frog skeletal muscle fibres.

The effect of apamin, a polypeptidic toxin from bee venom which is a specific blocker of certain Ca2+-dependent K+ channels, has been tested (50-100nM) on voltage clamped single skeletal muscle fibres of the frog. The results have shown the existence of an inhibitory effect of the toxin on the slow outward K+ current which suggests the existence of a Ca2+-sensitive component of the slow K+ permeability in the plasma membrane of the frog muscle fibre.

Birefringence signal and early mechanical changes at normal and increased tonicities in frog skeletal muscle.

Simultaneous measurements of the time course of early mechanical events and the early large birefringence signal were performed during activation of isolated frog skeletal muscle fibres bathed in iso- and hypertonic media. A piezo-electric transducer was used which had a resonance frequency of 3.3 kHz and a sensitivity of 740 mV/mN (0.5 mV noise peak to peak) and a compliance of 0.6 mu/mN. The delay from stimulus onset to the onset of the birefringence signal was estimated to be 0.78 +/- 0.03 ms in normal Ringer solution at room temperature (20-23 degrees C). The beginning of latency relaxation appeared subsequent to the birefringence signal after a delay of 0.36 +/- 0.03 ms. Increasing the tonicity retarded the time course of both birefringence signal and latency relaxation. In solutions of twofold greater tonicity an increase in tension (denoted pre-relaxation contraction) was observed to precede latency relaxation. Pre-relaxation contraction became more prominent with increasing tonicity and appeared to coincide with its onset with the onset of the birefringence signal. The pre-relaxation contraction was shown not to be a stimulus artifact and was not reduced in amplitude by D 600 or calcium-poor solutions. Lowering the temperature to 2.2 degrees C in normal Ringer solution delayed the onsets of the birefringence signal and latency relaxation, and increased the interval between the two signals but did not result in a tension increase before latency relaxation. The coincidence of the birefringence signal onset with the onset of pre-relaxation contraction suggests a common aetiology. The aetiology remains speculative but seems to require hypertonicity for expression of the mechanical signal.

Changes in intracellular Ca2+ induced by shortening imposed during tetanic contractions.

Calcium transients, monitored by aequorin, and force were recorded simultaneously during tetanic contractions of isolated frog skeletal muscle fibers. Quick length changes were applied to the fibers during contractions at sarcomere lengths on the descending limb of the length-tension relationship. Previous experiments showed that regulatory Ca2+ binding sites are apparently saturated during a plateau of tetanic force development at these sarcomere lengths. However, quick releases of greater than 4 to 5% of fiber length produced a momentary fall in the calcium transient that followed a time course similar to the redevelopment of force. The fall in the Ca2+ transient after a release was maximum at striation spacings about half way along the descending limb (2.6-2.7 microns), which suggests it is not related to an increase in the number of Ca2+ binding sites distributed uniformly along the filaments. The effect was absent or barely detectable when highly stretched fibers were released during contraction. The fall in the Ca2+ transient was unrelated to the time during a tetanus that a release was made or to the velocity of the release. One explanation of these results is that complexes between actin and myosin are broken by a sudden reduction of length, and as they reform during the recovery of force the affinity of troponin for Ca2+ increases. Quick stretch had no effect on the rapid decay of Ca2+ transients, but stretch increased peak force and slowed relaxation for almost a second after the end of stimulation. Evidently the decrease in the rate of relaxation produced by stretch is unrelated to changes in the amount of Ca2+ released or the rate of Ca2+ removal, which supports suggestions that the kinetics of muscle relaxation are determined by more than one mechanism. The apparent increase in the overall duration of mechanical activity after stretch probably results from the longitudinal inhomogeneity in the duration of activity - known to occur during relaxation - coupled with the decreased compliance of stretched fibers.

Prolonged action potential of frog skeletal muscle membrane in Ca-free EGTA solution.

The membrane of isolated frog skeletal muscle fibers with or without T-system is depolarized to about -30 mV in a Ca-free solution containing 2 mM EGTA. Under such a condition, the action potential can be produced by a cathodal pulse when the membrane is previously hyperpolarized to -70- -100 mV by a conditioning anodal current. The action potential consists of two different potential components, namely a spike potential and a following slow depolarizing response forming a plateau phase (plateau potential). The membrane conductance during the plateau potential is increased. Both spike and plateau potentials are abolished in the absence of NaCl. TTX blocks a spike potential without affecting a plateau potential, whereas Mn and D-600 act contrarily. The i-v relation shows an anomalous rectification in Na-free solution, suggesting that the K conductance during the plateau phase is decreased. It is suggested that spike and plateau potentials are produced by a movement of sodium ions through sodium and calcium channels of the membrane, respectively.

The influence of free fatty acids on functional properties of isolated skeletal muscles. Octanoate action on membrane resistance.

1. Na-octanoate (in Ringer's solution) affects the membrane resistance of isolated frog skeletal muscle fibres in a biphasic way. Initially there is an increase followed by a more slowly developing decrease. The effect depends on both the concentration of the fatty acid (1-20 mM) and the time of exposure (2-20 min). 2. In muscles exposed either to isotonic K2SO4 or to Ringer's solution with Cl- substituted by methylsulphage, the octanoate-induced resistance drop is the same as in normal Ringer's solution whereas an initial increase is not observed. 3. In Na-free (Tris-) Ringer's solution, only an increase in membrane resistance is caused by octanoate. 4. The results suggest that Na-octanoate decreases the conductance of the resting muscle cell membrane for anions (Cl-1) whereas the permeability for cations (Na+, K+) is increased.

The collapse of the sarcoplasmic reticulum in skeletal muscle.

When various cations, including Ca2+, are in the fixative, both sarcoplasmic reticulum (SR) of whole skeletal muscle and isolated SR vesicles collapse to form pentalaminate "compound membranes" that result from the apparent fusion of the lumenal lamellae of the membranous envelope of the SR. The process may be reversed by subsequently soaking the tissue in 1 M NaCl. An identical morphological phenomenon is observed in unfixed quickly frozen isolated frog skeletal muscle fibers, the cation in that case coming from endogenous sources. The hypothesis is advanced that the collapse is an in vivo process mediated by the sequestration of Ca2+ after contraction. The resulting obliteration of the SR lumen would have the effect of displacing the SR contents into the junctional SR, as well as electrically isolating the free SR from the junctional SR during relaxation. As a consequence, resistive coupling between the plasmalemma and the junctional SR becomes a plausible mechanism for the translation of the action potential into Ca2+ release, since the bulk of the SR membrane capacitance would now remain separated from the plasmalemma during relaxation.

Sarcomere lengthening and tension drop in the latent period of isolated frog skeletal muscle fibers.

A laser diffraction technique has been developed for registering small changes in sarcomere length. The technique is capable of resolving changes as small as 0.2 A in isolated frog skeletal muscle fibers. The small sarcomere lengthening that accompanies the drop in tension in the latent period of contraction was investigated. We suggest this lengthening be named latency elongation (LE). The LE is present in a completely slack fiber and must, therefore, be caused by a forcible lengthening process. Furthermore, the LE is dependent on the existence of an overlap between thin and tick filaments. The rate of elongation and the time interval between stimulation and maximum elongation may vary along the fiber. The maximum elongation was 3-5 A per sarcomere. At any instant the drop in tension is a product of the sum of sarcomere lengthenings along the fiber and the slope stiffness of the series elasticity. The latency relaxation (LR) could be registered in the sarcomere length range from 2.2 mum to 3.6-3.7 mum. The amplitude went through a sharp maximum at 3.0-3.1 mum. In the sarcomere length range from 2.2 to 2.8 mum the delay from onset to maximum LR was nearly proportional to the distance from the Z-line to the overlap zone. A working hypothesis is presented. It is suggested that the LE is caused by a lengthening of the thin filaments.