Role of extracellular [Ca2+] in fatigue of isolated mammalian skeletal muscle

The possiblerole of altered extracellular Ca²⁺concentration([Ca²⁺]_o)in skeletal muscle fatigue was tested on isolated slow-twitch soleusand fast-twitch extensor digitorum longus muscles of the mouse. Thefollowing findings were made. 1) Achange from the control solution (1.3 mM[Ca²⁺]_o)to 10 mM[Ca²⁺]_o,or to nominally Ca²⁺-freesolutions, had little effect on tetanic force in nonfatigued muscle.2) Almost complete restoration oftetanic force was induced by 10 mM[Ca²⁺]_oin severely K⁺-depressed muscle(extracellular K⁺ concentration of10-12 mM). This effect was attributed to a 5-mV reversal of theK⁺-induced depolarization andsubsequent restoration of ability to generate action potentials(inferred by using the twitch force-stimulation strength relationship).3) Tetanic force depressed bylowered extracellular Na⁺concentration (40 mM) was further reduced with 10 mM[Ca²⁺]_o.4) Tetanic force loss at elevatedextracellular K⁺ concentration (8 mM) and lowered extracellular Na⁺concentration (100 mM) was partially reversed with 10 mM[Ca²⁺]_oor markedly exacerbated with low[Ca²⁺]_o.5) Fatigue induced by using repeatedtetani in soleus was attenuated at 10 mM[Ca²⁺]_o(due to increased resting and evoked forces) and exacerbated at low[Ca²⁺]_o.These combined results suggest, first, that raised[Ca²⁺]_oprotects against fatigue rather than inducing it and, second, that aconsiderable depletion of[Ca²⁺]_oin the transverse tubules may contribute to fatigue.

     

Positive inotropism in mammalian skeletal muscle in vitro during and after fatigue

We tested the hypothesis that positive inotropic factors decrease fatigue and improve recovery from fatigue in mammalian skeletal muscle in vitro. To induce fatigue, we stimulated mouse soleus and extensor digitorum longus (EDL) to perform isometric tetanic contractions (50 impulses x s(-1) for 0.5 s) at 6 contractions x min(-1) for 60 min in soleus and 3 contractions x min(-1) for 20 min in EDL. Muscles were submerged in Krebs-Henseleit bicarbonate solution (Krebs) at 27 degrees C gassed with 95% nitrogen - 5% carbon dioxide (anoxia). Before and for 67 min after the fatigue period, muscles contracted at 0.6 contractions x min(-1) in 95% oxygen - 5% carbon dioxide (hyperoxia). We added a permeable cAMP analog (N6, 2'-O-dibutyryladenosine 3':5'-cyclic monophosphate at 10(-3) mol x L(-1) (dcAMP)), caffeine (2 x 10(-3) mol x L(-1), or Krebs as vehicle control at 25 min before, during, or at the end of the fatigue period. In soleus and EDL, both challenges added before fatigue significantly increased developed force but only caffeine increased developed force when added during the fatigue period. At the end of fatigue, the decrease in force in challenged muscles was equal to or greater than in controls so that the force remaining was the same or less than in controls. EDL challenged with dcAMP or caffeine at any time recovered more force than controls. In soleus, caffeine improved recovery except when added before fatigue. With dcAMP added to soleus, recovery was better after challenges at 10 min and the end of the fatigue period. Thus, increased intracellular concentrations of cAMP and (or) Ca2+ did not decrease fatigue in either muscle but improved recovery from fatigue in EDL and, in some conditions, in soleus.     

Stimulation pulse characteristics and electrode configuration determine site of excitation in isolated mammalian skeletal muscle: implications for fatigue.

We examined whether electrical field stimulation with varying characteristics could excite isolated mammalian skeletal muscle through different sites. Supramaximal (20-V, 0.1-ms) pulse stimulation with transverse wire or parallel plate electrodes evoked similar forces in nonfatigued slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles from mice. d-tubocurarine shifted the twitch force-stimulation strength relationship toward higher pulse strengths with both electrode configurations in soleus muscle, suggesting that weaker pulses excite muscle via neuromuscular transmission. With wire stimulation, movement of the recording electrode along the muscle caused a delay between the stimulus artifact and the peak of the action potential, consistent with action potential propagation along the sarcolemma. TTX abolished all contractions evoked with 20-V, 0.1-ms pulses, suggesting that excitation occurred via voltage-dependent Na+ channels and, hence, muscle action potentials. TTX did not prevent force development with > or = 0.4-ms pulses in soleus or 1-ms pulses in EDL muscle. Furthermore, myoplasmic Ca2+ (i.e., the fura 2 ratio) and sarcomere shortening were greater during tetanic stimulation with 2.0-ms than with 0.5-ms pulses in flexor digitorum brevis fibers from rats. TTX prevented all shortening and Ca2+ release with 0.5-ms, but not 2.0-ms, pulses, indicating that longer pulses can directly trigger Ca2+ release. Hence, proper interpretation of mechanistic studies requires precise understanding of how muscles are excited; otherwise, incorrect conclusions can be made. Using this new understanding, we showed that disrupted propagation of action potentials along the surface membrane is a major cause of fatigue in soleus muscle that is focally and continuously stimulated at 125 Hz.     

Kinetics of sealing for transient electropores in isolated mammalian skeletal muscle cells

Permeabilization of the plasma membrane by electrical forces (electroporation) can be either transient or stable. Although the exact molecular mechanics have not yet been described, electroporation is believed to initiate primarily in the lipid bilayer. To better understand the kinetics of membrane permeabilization, we sought to determine the time constants for spontaneous transient pore sealing. By using isolated rat flexor digitorum brevis skeletal muscle cells and a two-compartment diffusion model, we found that pore sealing times (tau p) after transient electroporation were approximately 9 min. tau p was not significantly dependent on the imposed transmembrane potential. We also determined the transmembrane potential (delta Vm) thresholds necessary for transient and stable electroporation in the skeletal muscle cells. delta VmS ranging between 340 mV and 480 mV caused a transient influx of magnesium, indicating the existence of spontaneously sealing pores. An imposed delta Vm of 540 mV or greater led to complete equilibration of the intracellular and extracellular magnesium concentrations. This finding suggests that stable pores are created by the larger imposed transmembrane potentials. These results may be useful for understanding nerve and skeletal muscle injury after an electrical shock and for developing optimal strategies for accomplishing transient electroporation, particularly for gene transfection and cell transformation.     

A1 receptor activation decreases fatigue in mammalian slow-twitch skeletal muscle in vitro.

To test the hypothesis that adenosine improves skeletal muscle cell function, we exposed curarized mouse soleus and extensor digitorum longus (EDL) to a range of concentrations of adenosine (10(-9) M to 10(-5) M). Muscles contracted in Krebs-Henseleit bicarbonate buffer (27 degrees C, 95% O2 and 5% CO2) for 500 ms at 50 Hz once every 90 s. Soleus fatigued significantly less with adenosine present at concentrations of 10(-8) M and higher than with the Krebs-Henseleit vehicle control. Adenosine significantly improved force generation or delayed fatigue of EDL only with the initial adenosine challenge. To investigate the receptor population involved, we exposed soleus to agonists specific for A1 receptors (N6-cyclopentyladenosine, CPA), or A2 receptors (CGS 21680 hydrochloride, CGS), or A3 receptors (N6-benzyl-5'-N-ethylcarboxamidoadenosine, BNECA). CPA (A1) significantly decreased fatigue compared with the Krebs-Henseleit vehicle control at concentrations of 10(-9) M and higher. Muscles exposed to the A2 and A3 agonists did not differ from a Krebs-Henseleit plus methanol control. Phenylephrine (10(-6) M), an alpha-adrenergic agonist that increases the concentration of inositol triphosphate (IP3), significantly improved developed force in soleus. Neither a permeable cAMP analog, 8-bromo-cAMP (10(-5) M), nor a beta, agonist, isoproterenol (10(-6) M), had an effect on force generation in the soleus when compared with a saline control. Thus adenosine slowed fatigue in slow-twitch skeletal muscle through A1 receptors.     

Effect of extracellular osmolality on cell volume and resting metabolism in mammalian skeletal muscle

The purpose of the present investigation was to establish an in vitro mammalian skeletal muscle model to study acute alterations in resting skeletal muscle cell volume. Isolated, whole muscles [soleus and extensor digitorum longus (EDL)] were dissected from Long-Evans rats and incubated for 60 min in Sigma medium 199 (1 g of resting tension, bubbled with 95% O(2)-5% O(2), 30 +/- 2 degrees C, and pH 7.4). Medium osmolality was altered to simulate hyposmotic (190 +/- 10 mmol/kg) or hyperosmotic conditions (400 +/- 10 mmol/kg), whereas an isosmotic condition (290 +/- 10 mmol/kg) served as a control. After incubation, relative water content of the muscle decreased with hyperosmotic and increased with hyposmotic condition in both muscle types (P < 0.05). The cross-sectional area of soleus type I and type II fibers increased (P < 0.05) in hyposmotic, whereas hyperosmotic exposure led to no detectable changes. The EDL type II fiber area decreased in the hyperosmotic condition and increased after hyposmotic exposure, whereas no change was observed in EDL type I fibers. Furthermore, exposure to the hyperosmotic condition in both muscle types resulted in decreased muscle ATP and phosphocreatine (P < 0.05) contents and increased creatine and lactate contents (P < 0.05) compared with control and hyposmotic conditions. This isolated skeletal muscle model proved viable and demonstrated that altering extracellular osmolality could cause acute alterations in muscle water content and resting muscle metabolism.     

Increased excitability of acidified skeletal muscle: role of chloride conductance

Generation of the action potentials (AP) necessary to activate skeletal muscle fibers requires that inward membrane currents exceed outward currents and thereby depolarize the fibers to the voltage threshold for AP generation. Excitability therefore depends on both excitatory Na+ currents and inhibitory K+ and Cl- currents. During intensive exercise, active muscle loses K+ and extracellular K+ ([K+]o) increases. Since high [K+]o leads to depolarization and ensuing inactivation of voltage-gated Na+ channels and loss of excitability in isolated muscles, exercise-induced loss of K+ is likely to reduce muscle excitability and thereby contribute to muscle fatigue in vivo. Intensive exercise, however, also leads to muscle acidification, which recently was shown to recover excitability in isolated K(+)-depressed muscles of the rat. Here we show that in rat soleus muscles at 11 mM K+, the almost complete recovery of compound action potentials and force with muscle acidification (CO2 changed from 5 to 24%) was associated with reduced chloride conductance (1731 +/- 151 to 938 +/- 64 microS/cm2, P < 0.01) but not with changes in potassium conductance (405 +/- 20 to 455 +/- 30 microS/cm2, P < 0.16). Furthermore, acidification reduced the rheobase current by 26% at 4 mM K+ and increased the number of excitable fibers at elevated [K+]o. At 11 mM K+ and normal pH, a recovery of excitability and force similar to the observations with muscle acidification could be induced by reducing extracellular Cl- or by blocking the major muscle Cl- channel, ClC-1, with 30 microM 9-AC. It is concluded that recovery of excitability in K(+)-depressed muscles induced by muscle acidification is related to reduction in the inhibitory Cl- currents, possibly through inhibition of ClC-1 channels, and acidosis thereby reduces the Na+ current needed to generate and propagate an AP. Thus short term regulation of Cl- channels is important for maintenance of excitability in working muscle.     

Plasticity in mammalian skeletal muscle.

While it has been recognized for many years that different limb muscles belonging to the same mammal may have markedly differing contractile characteristics, it is only comparatively recently that it has been demonstrated that these differences depend upon the motor innervation. By appropriately changing the peripheral nerve innervating a mammalian skeletal muscle, it is possible to change dramatically the contractile behaviour of the reinnervated muscle. The manner by which the motor innervation determines the nature of a muscle fibre's contractile machinery is not completely understood, but it appears that the number and pattern of motor nerve impulses reaching the muscle play an important role. The biochemical changes occurring within muscle fibres whose contractile properties have been modified by altered motor innervation include the synthesis of different contractile proteins.     

Slow charge movement in mammalian skeletal muscle

Voltage-dependent charge movements were measured in the rat omohyoid muscle with the three-microelectrode voltage-clamp technique. Contraction was abolished with hypertonic sucrose. The standard (ON-OFF) protocol for eliciting charge movements was to depolarize the fiber from -90 mV to a variable test potential (V) and then repolarize the fiber to -90 mV. The quantity of charge moved saturated at test potentials of approximately 0 mV. The steady state dependence of the amount of charge that moves as a function of test potential could be well fitted by the Boltzmann relation: Q = Qmax/(1 + exp[-(V - V)/k]), where Qmax is the maximum charge that can be moved, V is the potential at which half the charge moves, and k is a constant. At 15 degrees C, these values were Qmax = 28.5 nC/microF, V = -34.2 mV, and k = 8.7 mV. Qmax, k, and V exhibited little temperature dependence over the range 7-25 degrees C. "Stepped OFF" charge movements were elicited by depolarizing the fiber from -90 mV to a fixed conditioning level that moved nearly all the mobile charge (0 mV), and then repolarizing the fiber to varying test potentials. The sum of the charge that moved when the fiber was depolarized directly from -90 mV to a given test potential and the stepped OFF charge that moved when the fiber was repolarized to the same test potential had at all test potentials a value close to Qmax for that fiber. In nearly all cases, the decay phase of ON, OFF, and stepped OFF charge movements could be well fitted with a single exponential. The time constant, tau decay, for an ON charge movement at a given test potential was comparable to tau decay for a stepped OFF charge movement at the same test potential. Tau decay had a bell-shaped dependence on membrane potential: it was slowest at a potential near V (the midpoint of the steady state charge distribution) and became symmetrically faster on either side of this potential. Raising the temperature from 7 to 15 degrees C caused tau decay to become faster by about the same proportion at all potentials, with a Q10 averaging 2.16. Raising the temperature from 15 to 25 degrees C caused tau decay to become faster at potentials near V, but not at potentials farther away.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of potassium and chloride permeability over the surface and T-tubule membranes of mammalian skeletal muscle

Summary The distribution of K and Cl permeability,P _K andP _Cl, over the surface and T-tubule membranes of red rat sternomastoid fibers has been determined. Membrane potential,V _m, was recorded with 3-m KCl-filled glass microelectrodes. Changes inV _m with changes in [K]_o or [Cl]_o were used to estimateP _Cl/P _K in normal and detubulated preparations. The results show that the T-tubule membrane has a highP _Cl and is therefore different from the T-tubule membrane of amphibian fibers. Analysis of the time course of depolarization when [K]_o was raised (in SO₄ solutions) showed thatP _K was distributed over the surface and T-tubule membranes. Two observations suggested that T-tubuleP _Cl was higher than the surfaceP _Cl. Firstly, in normal fibers, the depolarization caused by an increase in [K]_o was 3.5 times greater in SO₄ solutions than in Cl solutions. In marked contrast, the depolarization in glycerol-treated fibers was independent of [Cl]_o. Secondly, the rapid change inV _m when [Cl]_o was changed was reduced by 80% after glycerol treatment. Both observations suggest thatP _Cl was low in glycerol-treated fibers.P _Cl/P _K was calculated from theV _m data using Goldman, Hodgkin and Katz equations for Na and K or for Na, K, and Cl. In normal fibersP _Cl/P _K=4.5 and in glycerol-treated fibersP _Cl/P _K=0.28. Since it is unlikely that glycerol treatment would increaseP _K, the reduction in the ratio must follow the loss of Cl permeability channels in the T-tubule membrane.