Intracellular pH during ischemia in skeletal muscle: relationship to membrane potential,extracellular pH,tissue lactic acid and ATP

Intracellular pH (pH_i) was measured with double-barrelled microelectrodes during 4 h of complete tourniquet ischemia in rabbit gastrocnemius muscle (group I). The pH_i was related to extracellular pH (pH_e), membrane potential (E_m), tissue lactic acid (LA) and ATP. A fall in pH_i from 7.00±0.03 to 6.60±0.05 occurred during 4 h of ischemia, with a slight pH-drop (0.07 pH units) during the initial hour and a more pronounced drop of 0.13 pH units during the last hour of ischemia. These changes were paralleled by a considerable decrease in pH_e from 7.30±0.01 to 6.36±0.05 and a sixfold increase of tissue LA. The buffering capacity during the 4 h of ischemia was estimated to 81.9±5.6 mmol H⁺/l×pH. In parallel with the reduction in pH_e, the resting membrane potential decreased from –90 mV and stabilised at around –60 mV after 2 h of ischemia. A less negative cell interior would favour H⁺ extrusion since the E_m-E_H ⁺ gradient was unchanged at about –70 mV during the entire period of ischemia. This could contribute to muscle fiber buffering during ischemia.In another set of experiments (group II) the muscular glycogen reserve was reduced 20 min prior to a 4 h period of ischemia. Thereby an ischemic state was created where ATP levels decreased to 30% of initial, in contrast to the unaltered ATP content in group I. In the low-ATP group II the E_m-E_H ⁺ gradient decreased to 58% of initial and the buffering capacity was slightly but significantly lower (64.6±4 mmol H⁺/l×pH) than in the high-ATP group I. Significant correlation coefficients were obtained between the ATP loss and the E_m-E_H ⁺ gradient (r=0.861,P<0.001). These data suggest that maintenance of the transmembrane H⁺ gradient and to a certain degree the buffer capacity depend on the energy-state of the muscle cell.     

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⁺×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 carbon dioxide on extracellular potassium accumulation and volume in isolated frog spinal cord

A 6–10-fold increase inpCO₂ in the superfusing Ringer solution increased the volume of the extracellular space (ECS) and changed the spatial distribution and amplitude of the extracellular K⁺ accumulation which resulted from dorsal root stimulation. Using the increase in tetraethylammonium concentration ([TEA⁺]) resulting from iontophoretic injection of that ion in the extracellular fluid as an indication of the volume of the ECS, it was found that in highpCO₂ the ECS volume in spinal dorsal horn increased by more than 60%. In addition, in the presence of raisedpCO₂ we also observed the following: (1) The rate of diffusion of TEA⁺ into the dorsal horn increased. (2) The accumulation of K⁺ ovoked by single or tetanic stimulation of the dorsal root was less. (3) The clearance of K⁺ was slowed down. (4) The regions where K⁺ accumulated were more restricted. (5) The K⁺ evoked depolarization of the primary afferent fibres decreased. (6) In contrast to TEA⁺, the rate of diffusion of K⁺ into the dorsal horn decreased. The effects of an increase inpCO₂ on K⁺ accumulation and clearance appear to result from an increase in ECS volume and a possible decrease in glial electrical coupling which interferes with glial spatial buffering of K⁺.     

Fixed acid and carbon dioxide bohr effects as functions of hemoglobin-oxygen saturation and erythrocyte pH in the blood of the frog,Rana temporaria

Using a thin film, dynamic recording technique, the pH sensitivity of the oxygen equilibrium (Bohr effect) of whole blood in the frogRana temporaria, and its dependence on CO₂ and fixed acids and on plasma and erythrocyte pH values were measured. Under standard conditions (20°C,P _{CO 2}=14.7 mm Hg, pH=7.65) the oxygen equilibrium could be described by a P₅₀ value of 38 mm Hg andn ₅₀ of 1.8. Hill plots of the oxygen equilibria showed increased cooperativity in oxygen binding with increasing saturation (n ₂₀ 1.2,n ₈₀ 4.0). Values of the fixed acid and CO₂ Bohr factors ({ie7-1} and {ie7-2}, respectively) were similar at specific saturations (S_{20, 50, 80}) but showed saturation dependence with high values occurring at high saturation. The same statements also hold for the intracellular Bohr factors (derived from the relation between blood P₅₀ and erythrocyte pH) although the values of both {ie7-3} and {ie7-4} now were greater than those related to blood pH.     

Development of a biological scaffold engineered using the extracellular matrix secreted by skeletal muscle cells

The performance of implantable biomaterials derived from decellularized tissue, including encouraging results with skeletal muscle, suggests that the extracellular matrix (ECM) derived from native tissue has promising regenerative potential. Yet, the supply of biomaterials derived from donated tissue will always be limited, which is why the in-vitro fabrication of ECM biomaterials that mimic the properties of tissue is an attractive alternative. Towards this end, our group has utilized a novel method to collect the ECM that skeletal muscle myoblasts secrete and form it into implantable scaffolds. The cell derived ECM contained several matrix constituents, including collagen and fibronectin that were also identified within skeletal muscle samples. The ECM was organized into a porous network that could be formed with the elongated and aligned architecture observed within muscle samples. The ECM material supported the attachment and in-vitro proliferation of cells, suggesting effectiveness for cell transplantation, and was well tolerated by the host when examined in-vivo. The results suggest that the ECM collection approach can be used to produce biomaterials with compositions and structures that are similar to muscle samples, and while the physical properties may not yet match muscle values, the in-vitro and in-vivo results indicate it may be a suitable first generation alternative to tissue derived biomaterials.     

Effect of hyperthyroidism on the contractile and histochemical properties of fast and slow twitch skeletal muscle in the rat

Male Wistar rats were made hyperthyroid by intraperitoneal injections on alternate days for 1–6 weeks, of 200 g/kg triiodothyronine (T₃). The effects of this treatment on the contractile properties of the soleus, a slow twitch and the extensor digitorum longus (EDL), a fast twitch skeletal muscle, were studied in vivo in the anaesthetized animal. Post mortem, serial frozen sections of both muscles were stained histochemically for myosin ATPase, succinic dehydrogenase and phosphorylase. Muscle fibres were classified as either slow twitch (SO), fast twitch oxidative glycolytic (FOG) or fast twitch glycolytic (FG).Elevation of plasma T₃ levels is associated with progressive alterations in the muscle fibre populations of both muscles. In the soleus there is conversion of SO to FOG fibres while in the EDL the main effect is FG to FOG conversion. There are also marked changes, mainly confined to the soleus muscle, in contractile properties; progressive increases in isometric twitch and tetanic tension and in the rates of contraction and relaxation during both twitch and tetanus.The effect of T₃ on slow muscle contractility may be related to its effect on muscle phenotype. However, changes in the former precede detectable fibre population changes. T₃ may influence properties such as the Ca²⁺ binding activity of sarcoplasmic reticulum of existing slow twitch fibres before the later changes associated with the interconversion of fibre types occur.     

Nucleotide diphosphates activate the ATP-sensitive potassium channel in mouse skeletal muscle

Patch-clamp techniques were used to study the effects of internal nucleotide diphosphates on the K_ATP channel in mouse skeletal muscle. In inside-out patches, application of GDP (100 M) and ADP (100 M) reversibly increased the channel activity. In the presence of internal Mg²⁺ (1 mM), low concentrations of ADP (<300 M) enhanced channel activity and high concentrations of ADP (>300 M) limited channel opening while GDP activated the channel at all concentrations tested. In the absence of internal Mg²⁺, ADP decreased channel activity at all concentrations tested while GDP had no noticeable effect at submillimolar concentrations and inhibited channel activity at millimolar concentrations. GDP [S] (100 M), which behaved as a weak GDP agonist in the presence of Mg²⁺, stimulated ADP-evoked activation whereas it inhibited GDP-evoked activation. The K⁺ channel opener pinacidil was found to activate the K_ATP channel but only in the presence of internal GDP, ADP and GDP [S]. The results are discussed in terms of the existence of multiple nucleotide binding sites, in charge of the regulation of the K_ATP channel.     

Optical evidence for a chloride conductance in the T-system of frog skeletal muscle

T-system action potentials were recorded optically from intact frog skeletal muscle fibers stained with the non-penetrating potentiometric dye NK-2367. The effect of chloride removal on the falling phase of the radially propagating tubular action potential was studied to determine whether a chloride conductance located in the T-system membranes contributes to tubular repolarization during activity. Our results show that, in chloride-free Ringer, repolarization of the tubular action potential is significantly slowed. Moreover, the late phase of tubular repolarization is characterized by a large afterpotential, which is highly temperature-dependent and appears as a secondary peak above 10° C. The optical data were compared with predicted T-system action potentials generated from a radial cable equivalent circuit model of the T-system, in which the effects of a distributed tubular leak conductance were tested. Results of this analysis are consistent with the proposal that some of the outward repolarization current during the T-system action potential is drawn across a chloride conductance located in the T-system membranes.     

Intracellular pH and buffer power of type 1 and 2 fibres from skeletal muscle ofXenopus laevis

Intracellular pH (pH_i) and buffering power of type 1 and type 2 fibres from the iliofibularis muscle of the clawed frog,Xenopus laevis, have been measured using pH-sensitive microelectrodes. In phosphate buffered Ringer's solution (extracellular pH 7.25, 20–22°C), mean pH_i and its variance were similar in the two fibre types (6.86±SD 0.15±SEM 0.03,n=24, type 1, and 6.86±SD 0.12±SEM 0.03,n=15, type 2). On changing to Ringer's solution containing CO₂ and HCO ₃ ^– (extracellular pH 7.25, 20–22°C), pH_i became more acid in both fibre types. Although H⁺ ions were not at electrochemical equilibrium across the surface membrane, active transport did not return pH_i to its original value during exposure to CO₂. The buffering powers calculated from the changes in pH_i were not significantly different, 41.6 mmol·l^–1 per pH unit (±SEM 4.0,n=17) for type 1 and 49.3 mmol·l per pH unit (±SEM 7.2,n=11) for type 2 fibres. Thus differences in the mechanical properties of these fibre types are not due simply to a difference of the intracellular pH or buffering of resting fibres. Other possible explanations are discussed for the changes in some contractile properties that occur when pH_i is acidified.     

Use of selective toxins to separate surface and tubular sodium currents in frog skeletal muscle fibers

The interaction between toxin from the venom of the scorpionTityus serrulatus and sodium channels in skeletal muscle membranes from the frogCaudiverbera caudiverbera was studied. Sodium current from cut sartorius muscle fibers is a complex signal in which early and late components are difficult to separate. External application of Tityus toxin initially blocked the early component in a voltage-dependent manner. Longer exposure to the toxin induced a complete blockade of the two components of the inward current. Application of tetrodotoxin to fibers pretreated with Tityus toxin at submaximal concentrations allowed the observation of the two distinct components of the inward current. Binding of¹²⁵I-labelled toxin to highly purified membrane fractions from the same muscle was used to establish the presence of high affinit receptors both in the transverse-tubular and in the surface membrane.     

Recovery dynamics of skeletal muscle oxygen uptake during the exercise off-transient

The time course of muscle recovery from contractions (i.e., muscle off-kinetics), measured directly at the site of O₂ exchange, i.e., in the microcirculation, is unknown. Whereas biochemical models based upon creatine kinase flux rates predict slower off- than on-transients [Kushmerick, M.J., 1998. Comp. Biochem. Physiol. B: Biochem. Mol. Biol.] whole muscle data [Krustrup, et al. J. Physiol.] suggest on–off symmetry.

Purpose

We tested the hypothesis that the slowed recovery blood flow (Qm) kinetics profile in the spinotrapezius muscle [Ferreira et al., 2006. J. Physiol.] was associated with a slowed muscle recovery compared with that seen at the onset of contractions (time constant, τ  23 s, Behnke et al., 2002. Resp. Physiol.), i.e., on–off asymmetry.

Methods

Measurements of capillary red blood cell flux and microvascular pressure of O₂ (P_O2mv) were combined to resolve the temporal profile of muscle across the moderate intensity contractions-to-rest transition.

Results

Muscle decreased from an end-contracting value of 7.7 ± 0.2 ml/100 g/min to 1.7 ± 0.1 ml/100 g/min at the end of the 3 min recovery period, which was not different from pre-stimulation . Contrary to our hypothesis, muscle in recovery began to decrease immediately (i.e., time delay <2 s) and demonstrated rapid first-order kinetics (τ, 25.5 ± 2.6 s) not different (i.e., symmetrical to) to those during the on-transient. This resulted in a systematic increase in microvascular P_O2 during the recovery from contractions.

Conclusions

The slowed Qm kinetics in recovery serves to elevate the ratio and thus microvascular P_O2. Whether this Qm response is obligatory to the rapid muscle kinetics and hence speeds the repletion of high-energy phosphates by maximizing conductive and diffusive O₂ flux is an important question that awaits resolution.     

Carbon dioxide sensitivity of the central chemosensitive mechanisms

Summary In urethane-anaesthetised adult albino rats ventral surface of the brainstem was stimulated chemically by increasing the local CO₂ concentration and electrically. Two areas were demarcated on the ventral surface of the brainstem, one which showed an increase in pulmonary ventilation on chemical and electrical stimulation, and another which showed a decrease in pulmonary ventilation and sometimes even respiratory arrest. EEG activity recorded from the area from where increased pulmonary ventilation was obtained showed a synchronous slow wave activity during chemical stimulation and inhalation of a CO₂-air mixture. This area is situated 0.5–1 mm lateral to the mid-line extending up to the rootlets of the VIIth to IXth cranial nerves. The response increased proportionately on increasing the strength of the chemical stimulus, till it reached a plateau. In carotid body denervated and chronic hypoxic animals, the magnitude of the responses was shown to be increased, probably due to increased sensitivity of the central chemosensitive mechanisms.     

Effects of enflurane on excitation-contraction coupling in frog skeletal muscle fibers

The effect of enflurane on the excitation-contraction (E-C) coupling of frog skeletal muscle fiber was studied.Low (19.13±0.70 mg%) and high (108.52±4.52 mg%) concentrations of enflurane did not cause substantial changes of the resting membrane potential but a high concentration of enflurane reduced the peak of action potentials and prolonged the duration measured at 50% peak amplitude.During the twitch response, enflurane reduced the peak response of light signal measured with calcium-sensitive photoprotein, aequorin, butpeak tension was not diminished by a low concentration of enflurane. A high concentration of enflurane remarkably inhibited both light signal and tension.During tetanus, a low concentration of enflurane partially abolished light signal and tension. At the high concentration, this inhibitory effect was pronuunced and action potentials were only observed in the initial phase of tetanic stimulation.Enflurane enhanced the increase of light signal observed in rapid cooling contracture.In glycerinated muscle fiber, enflurane shifted the pCa-tension relation to the left in low calcium and suppressed maximally activated tension at high calcium concentration.Inhibitory effect of enflurane on light signal in twitch response without abolition of action potential suggests that enflurane might inhibit E-C coupling in frog skeletal muscle fiber (but it enhances direct calcium release from SR induced by caffeine) and increase calcium sensitivity of contractile element.     

Studies on the relation between latency relaxation and resting cross-bridges of frog skeletal muscle

Summary Latency relaxation of frog skeletal muscle (LR) was investigated with respect to its relation to resting cross-bridges. A decrease in the initial stiffness of the resting muscle (stiffness of the muscle during the beginning of a length-change) was found, when repeated triangular length-changes were imposed on the muscle. This decrease in the initial stiffness depends on the velocity of the length-change. It is interpreted that the decrease in the initial stiffness reflects a detachment of the resting cross-bridges from their binding-sites. The LR, induced immediately after the offset of the length-changes, i.e. when the cross-bridges are still detached, showed an increased depth, its time course remaining unchanged. There is a strong correlation between the increase in the depth of the LR and the decrease in the initial stiffness. The LR regained its original depth (depth without a preceding length-change) about 5 s after the offset of the length-changes (20°C). It is suggested that the LR of skeletal muscle is not due to a detachment of resting cross-bridges.This work had been given in preliminary form at the 45th meeting of the Deutsche Physiologische Gesellschaft in Vienna, 1975     

Contractile properties of skeletal muscle fibre bundles from mice deficient in carbonic anhydrase II

The function of cytosolic carbonic anhydrase (CA) isozyme II is largely unknown in skeletal muscle. Because of this, we compared the in vitro contractile properties of extensor digitorum longus (EDL) and soleus (SOL) fibre bundles from mice deficient in CA II (CAD) to litter mate controls (LM). Twitch rise, 1/2 relaxation time and peak twitch force at 22°C of fibre bundles from CAD EDL [28.4±1.4 ms, 31.2±2.3 ms, 6.2±1.0 Newton/cm² (N/cm²), respectively] and CAD SOL (54.2±7.5 ms, 75.7±13.8 ms, 2.9±0.5 N/cm², respectively) were significantly higher compared to LM EDL (20.5±2.2 ms, 21.9±3.7 ms, 4.5±0.2 N/cm²) and LM SOL (42.8±3.5 ms, 51.4±2.4 ms, 2.1±0.4 N/cm²). However, in acidic Krebs–Henseleit solution, mimicking the pH, PCO₂, and HCO₃ ^– of arterial blood from CAD mice, twitch rise, 1/2 relaxation time, and peak twitch force of fibre bundles from CAD EDL (19.3±0.7 ms, 19.7±2.3 ms, 4.8±0.8 N/cm²) and CAD SOL (41.4±3.6 ms, 51.9±5.5 ms, 2.2±0.7 N/cm²) were not significantly different from LM fibre bundles in normal Krebs–Henseleit solution (EDL: 19.7±1.1 ms, 21.6±0.6 ms, 4.7±0.2 N/cm²; SOL: 42.5±3.1 ms, 51.8±2.6 ms, 1.8±0.3 N/cm²). A higher pH_i during exposure to acidic bathing solution was maintained by CAD EDL (7.37±0.02) and CAD SOL (7.33±0.05) compared to LM EDL (7.28±0.04) and LM SOL (7.22±0.02). This suggests that the skeletal muscle of CAD mice possesses an improved defense of pH_i against elevated pCO₂. In support of this, apparent non-bicarbonate buffer capacity (in mequiv H⁺ (pH unit)^–1 (kg cell H₂O)^–1) as determined by pH microelectrode was markedly increased in CAD EDL (75.7±4.1) and CAD SOL (85.9±3.3) compared to LM EDL (39.3±4.7) and LM SOL (37.5±3.8). Both latter phenomena may be related to the slowed rate of intracellular acidification seen in CAD SOL in comparison with LM SOL upon an increase in PCO₂ of the bath. In conclusion, skeletal muscle from mice deficient in CA II exhibits altered handling of acid–base challenges and shows normal contractile behavior at normal intracellular pH.     

Stiffness changes and fibre type transitions in rat soleus muscle produced by jumping training

Rat soleus muscles were overloaded with intent to induce a relative increase in fast fibres and modifications in muscular stiffness. The overloading technique was a training period consisting of an 11-week vertical jump programme. The method of controlled releases was used to obtain tension/extension curves characterizing the elastic behaviour of the soleus. Fibre typing was made by myofibrillar adenosine 5-triphosphatase staining. With regard to a control group, training resulted in a relative decrease in type I fibres for the benefit of type II fibres. Training also induced a decrease in muscle stiffness as attested notably by significant differences in maximal extension. These results are interpreted in terms of modifications occurring in the active fraction of the so-called series elastic component.     

Regulation of resting ionic conductances in frog skeletal muscle

The membrane electrical properties and resting ionic conductances of frog semitendinosus muscle fibres were studied in vitro at 25° C with the two-microelectrode cable technique, in the presence of an activator or inhibitor of protein kinase C (PKC) or in the presence of an activator of adenylate cyclase. The PKC activator, 4-phorbol 12,13-dibutyrate (4-PDB), reduced chloride conductance (G _Cl) at concentrations greater than 1 M and did not affect potassium conductance (G _K). At 150 M, the maximum concentration of 4-PDB tested, G _Cl was reduced by 42%. The inactive phorbol ester 4-phorbol 12,13-dibutyrate did not affect G _Cl or G _K. The inhibitory effect of 4-PDB on G _Cl was prevented by pretreatment of the muscle preparation with the PKC inhibitor staurosporine. The adenylate cyclase activator forskolin (1.5–8 M) significantly increased the G _K of the fibres, without affecting G _Cl. Thus, we conclude that frog skeletal muscle G _Cl, unlike rat muscle G _Cl, is relatively insensitive to activators of PKC. Moreover, in frog muscle, protein kinase A is a likely modulator of G _K, but not G _Cl.     

Intracellular free Mg2+ concentration in skeletal muscle fibres of frog and crayfish

The free intracellular Mg²⁺ concentration ([Mg²⁺]_i) was investigated in frog sartorius and crayfish phasic and tonic skeletal muscle fibres, using a new Mg²⁺-sensitive microelectrode based on the ionophore ETH 5214 [Hu et al. (1989) Anal Chem 61:574–576]. In Ringer solution containing 0.5 mmol/l MgCl₂, the mean [Mg²⁺]_i of the frog muscle fibres was 1.3 mmol/l. In phasic crayfish muscle fibres, [Mg²⁺]_i was about twice as high (mean 3.5 mmol/l) as in tonic fibres (mean 1.5 mmol/l), measured in van Harreveld solution containing 1.2 mmol/l MgCl₂. Long-lasting (3–12 h) incubation of frog skeletal muscle fibres in Na⁺-free solution produced a reversible increase of [Mg²⁺]_i by a factor of about 1.7. A tenfold rise of extracellular Mg²⁺ led to an increase in [Mg²⁺]_i in the presence as well as in the absence of Na⁺. In these experiments, mean [Mg²⁺]_i values of 3.2 mmol/l were never exceeded. Thus, [Mg²⁺]_i remained at least 60 times lower than predicted from a passive distribution across the cell membrane. The results suggest the existence of a Na⁺-dependent and a Na⁺-independent Mg²⁺ extrusion mechanism, which is regulated by actual Mg²⁺ concentrations.     

Free calcium in sheep cardiac tissue and frog skeletal muscle measured with Ca2+-selective microelectrodes

Microelectrodes filled with neutral carrier selective to Ca²⁺ were used to measure the free intracellular Ca²⁺ concentration ([Ca²⁺]_i) in sheep cardiac tissue and frog skeletal muscle. Calibration of the electrodes was performed in the presence of a solution resembling the cationic composition of the cytoplasm. [Ca²⁺]_i at rest in normal physiological saline (20–22° C) was 240 nM in Purkinje fibres, 270 nM in ventricular muscle, and 52 nM in skeletal muscle. In Purkinje fibres, elevation of [Ca²⁺]_o from 1.8 mM to 5.4 mM produced a 1.7-fold increase in [Ca²⁺]_i. Elevation of [Ca²⁺]_o from 1.8 mM to 18 mM induced a 2.6-fold increase in [Ca²⁺]_i. Exposure to Na⁺-free solution (Li⁺-substituted) gave rise to elevation of [Ca²⁺]_i by factors of 5.8 and 14 in ventricular muscle and Purkinje fibres, respectively. These latter changes in [Ca²⁺]_i were associated with the development of contractures which reached 34% and 172% of the corresponding twitch tension.