Frank de Paoli

Protective effects of lactic acid on force production in rat skeletal muscle

1 During strenuous exercise lactic acid accumulates producing a reduction in muscle pH. In addition, exercise causes a loss of muscle K+ leading to an increased concentration of extracellular K+ ([K+]o). Individually, reduced pH and increased [K+]o have both been suggested to contribute to muscle fatigue. 2 To study the combined effect of these changes on muscle function, isolated rat soleus muscles were incubated at a [K+]o of 11 mm, which reduced tetanic force by 75 %. Subsequent addition of 20 mm lactic acid led, however, to an almost complete force recovery. A similar recovery was observed if pH was reduced by adding propionic acid or increasing the CO2 tension. 3 The recovery of force was associated with a recovery of muscle excitability as assessed from compound action potentials. In contrast, acidification had no effect on the membrane potential or the Ca2+ handling of the muscles. 4 It is concluded that acidification counteracts the depressing effects of elevated [K+]o on muscle excitability and force. Since intense exercise is associated with increased [K+]o, this indicates that, in contrast to the often suggested role for acidosis as a cause of muscle fatigue, acidosis may protect against fatigue. Moreover, it suggests that elevated [K+]o is of less importance for fatigue than indicated by previous studies on isolated muscles.

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 μS/cm2, P < 0.01) but not with changes in potassium conductance (405 ± 20 to 455 ± 30 μS/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 μM 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.

Additive protective effects of the addition of lactic acid and adrenaline on excitability and force in isolated rat skeletal muscle depressed by elevated extracellular K

During strenuous exercise, extracellular K+ ([K+]o) is increased, which potentially can reduce muscle excitability and force production. In addition, exercise leads to accumulation of lactate and H+ and increased levels of circulating catecholamines. Individually, reduced pH and increased catecholamines have been shown to counteract the depressing effect of elevated K+. This study examines (i) whether the effects of addition of lactic acid and adrenaline on the excitability of isolated muscles are caused by separate mechanisms and are additive and (ii) whether the effect of adding lactic acid or increasing CO2 is related to a reduction of intra‐ or extracellular pH. Rat soleus muscles were incubated at a [K+]o of 15 mm, which reduced tetanic force by 85%. Subsequent addition of 20 mm lactic acid or 10−5m adrenaline led to a small recovery of force, but when added together induced an almost complete force recovery. Compound action potentials showed that the force recovery was associated with recovery of muscle excitability. The improved excitability after addition of adrenaline was associated with increased Na+–K+ pump activity resulting in hyperpolarization and an increase in the chemical Na+ gradient. In contrast, addition of lactic acid had no effect on the membrane potential or the Na+–K+ pump activity, but most likely increased excitability via a reduction in intracellular pH. It is concluded that the protective effects of acidosis and adrenaline on muscle excitability and force took place via different mechanisms and were additive. The results suggest that circulating catecholamines and development of acidosis during exercise may improve the tolerance of muscles to elevated [K+]o.

Regulation of ClC-1 and KATP channels in action potential–firing fast-twitch muscle fibers

Action potential (AP) excitation requires a transient dominance of depolarizing membrane currents over the repolarizing membrane currents that stabilize the resting membrane potential. Such stabilizing currents, in turn, depend on passive membrane conductance (Gm), which in skeletal muscle fibers covers membrane conductances for K+ (GK) and Cl− (GCl). Myotonic disorders and studies with metabolically poisoned muscle have revealed capacities of GK and GCl to inversely interfere with muscle excitability. However, whether regulation of GK and GCl occur in AP-firing muscle under normal physiological conditions is unknown. This study establishes a technique that allows the determination of GCl and GK with a temporal resolution of seconds in AP-firing muscle fibers. With this approach, we have identified and quantified a biphasic regulation of Gm in active fast-twitch extensor digitorum longus fibers of the rat. Thus, at the onset of AP firing, a reduction in GCl of ∼70% caused Gm to decline by ∼55% in a manner that is well described by a single exponential function characterized by a time constant of ∼200 APs (phase 1). When stimulation was continued beyond ∼1,800 APs, synchronized elevations in GK (∼14-fold) and GCl (∼3-fold) caused Gm to rise sigmoidally to ∼400% of its level before AP firing (phase 2). Phase 2 was often associated with a failure to excite APs. When AP firing was ceased during phase 2, Gm recovered to its level before AP firing in ∼1 min. Experiments with glibenclamide (KATP channel inhibitor) and 9-anthracene carboxylic acid (ClC-1 Cl− channel inhibitor) revealed that the decreased Gm during phase 1 reflected ClC-1 channel inhibition, whereas the massively elevated Gm during phase 2 reflected synchronized openings of ClC-1 and KATP channels. In conclusion, GCl and GK are acutely regulated in AP-firing fast-twitch muscle fibers. Such regulation may contribute to the physiological control of excitability in active muscle.

Comparison of regulated passive membrane conductance in action potential-firing fast- and slow-twitch muscle

In several pathological and experimental conditions, the passive membrane conductance of muscle fibers (Gm) and their excitability are inversely related. Despite this capacity of Gm to determine muscle excitability, its regulation in active muscle fibers is largely unexplored. In this issue, our previous study (Pedersen et al. 2009. J. Gen. Physiol. doi:10.1085/jgp.200910291) established a technique with which biphasic regulation of Gm in action potential (AP)-firing fast-twitch fibers of rat extensor digitorum longus muscles was identified and characterized with temporal resolution of seconds. This showed that AP firing initially reduced Gm via ClC-1 channel inhibition but after ∼1,800 APs, Gm rose substantially, causing AP excitation failure. This late increase of Gm reflected activation of ClC-1 and KATP channels. The present study has explored regulation of Gm in AP-firing slow-twitch fibers of soleus muscle and compared it to Gm dynamics in fast-twitch fibers. It further explored aspects of the cellular signaling that conveyed regulation of Gm in AP-firing fibers. Thus, in both fiber types, AP firing first triggered protein kinase C (PKC)-dependent ClC-1 channel inhibition that reduced Gm by ∼50%. Experiments with dantrolene showed that AP-triggered SR Ca2+ release activated this PKC-mediated ClC-1 channel inhibition that was associated with reduced rheobase current and improved function of depolarized muscles, indicating that the reduced Gm enhanced muscle fiber excitability. In fast-twitch fibers, the late rise in Gm was accelerated by glucose-free conditions, whereas it was postponed when intermittent resting periods were introduced during AP firing. Remarkably, elevation of Gm was never encountered in AP-firing slow-twitch fibers, even after 15,000 APs. These observations implicate metabolic depression in the elevation of Gm in AP-firing fast-twitch fibers. It is concluded that regulation of Gm is a general phenomenon in AP-firing muscle, and that differences in Gm regulation may contribute to the different phenotypes of fast- and slow-twitch muscle.

Regulation of Na+-K+ homeostasis and excitability in contracting muscles: implications for fatigue.

The performance of skeletal muscles depends on their ability to initiate and propagate action potentials along their outer membranes in response to motor signals from the central nervous system. This excitability of muscle fibres is related to the function of Na+ and K+ and Cl- channels and to steep chemical gradients for the ions across the cell membranes, i.e., the sarcolemma and T-tubular membranes. At rest, the chemical gradients for Na+ and K+ are maintained within close limits by the action of the Na+-K+ pump. During contractile activity, however, the muscles lose K+, which causes an increase in the concentration of K+ in the extracellular compartments of the body, the magnitude of which depends on the intensity of the exercise and the size of the muscle groups involved. Since the ensuing reduction in the chemical K+ gradient can have adverse effects on muscle excitability, it has repeatedly been suggested that, during intense exercise, the loss of K+ from muscle fibres can contribute to the complex set of mechanisms that leads to the development of muscle fatigue. In this review, aspects of the regulation of Na+-K+ homeostasis and excitability in contracting muscles is discussed within this context, together with the implications for the contractile function of skeletal muscles.

Lactate per se improves the excitability of depolarized rat skeletal muscle by reducing the Cl − conductance

Studies on rats have shown that lactic acid can improve excitability and function of depolarized muscles. The effect has been related to the ensuing reduction in intracellular pH causing inhibition of muscle fibre Cl− channels. However, since several carboxylic acids with structural similarities to lactate can inhibit muscle Cl− channels it is possible that lactate per se can increase muscle excitability by exerting a direct effect on these channels. We therefore examined the effects of lactate on the function of intact muscles and skinned fibres together with effects on pH and Cl− conductance (Gcl). In muscles where extracellular compound action potentials (M‐waves) and tetanic force response to excitation were reduced by (mean ±s.e.m.) 82 ± 4% and 83 ± 2%, respectively, by depolarization with 11 mm extracellular K+, both M‐waves and force exhibited an up to 4‐fold increase when 20 mm lactate was added. This effect was present already at 5 mm and saturated at 15 mm lactate, and was associated with a 31% reduction in GCl. The effects of lactate were completely blocked by Cl− channel inhibition or use of Cl−‐free solutions. Finally, both experiments where effects of lactate on intracellular pH in intact muscles were mimicked by increased CO2 tension and experiments with skinned fibres showed that the effects of lactate could not be related to reduced intracellular pH. It is concluded that addition of lactate can inhibit ClC‐1 Cl− channels and increase the excitability and contractile function of depolarized rat muscles via mechanisms not related to a reduction in intracellular pH.

Relationship between membrane Cl− conductance and contractile endurance in isolated rat muscles

•  Excitable cells commonly regulate their ability to generate and propagate action potentials through modulating the properties of the membrane ion channels. Recently it was shown that muscle activity is associated with rapid, protein kinase C (PKC)‐dependent ClC‐1 Cl− channel inhibition. •  Here the functional significance of this ClC‐1 inhibition for contractile endurance was determined in isolated slow‐ and fast‐twitch rat muscles. •  Experiments showed that PKC inhibition led to reduced contractile endurance, an effect of PKC that disappeared when extracellular Cl− was removed or ClC‐1 channels were inhibited. •  Experiments where resting Cl− conductance (GCl) was manipulated by reduction of solution Cl− or inhibition of ClC‐1 Cl− channels suggest a biphasic dependency of contractile endurance on GCl with an optimum close to the GCl observed in active muscles. •  The biphasic relation between GCl and endurance seems to reflect that lowered GCl on one side increases muscle excitability but on the other side reduces the ability of the muscle fibres to maintain K+ homeostasis and membrane potential during contractions. •  This study suggested that PKC‐dependent ClC‐1 Cl− channel regulation is important for the maintenance of contractile endurance in working muscle.

Role of physiological ClC-1 Cl− ion channel regulation for the excitability and function of working skeletal muscle

Electrical membrane properties of skeletal muscle fibers have been thoroughly studied over the last five to six decades. This has shown that muscle fibers from a wide range of species, including fish, amphibians, reptiles, birds, and mammals, are all characterized by high resting membrane permeability for Cl− ions. Thus, in resting human muscle, ClC-1 Cl− ion channels account for ∼80% of the membrane conductance, and because active Cl− transport is limited in muscle fibers, the equilibrium potential for Cl− lies close to the resting membrane potential. These conditions—high membrane conductance and passive distribution—enable ClC-1 to conduct membrane current that inhibits muscle excitability. This depressing effect of ClC-1 current on muscle excitability has mostly been associated with skeletal muscle hyperexcitability in myotonia congenita, which arises from loss-of-function mutations in the CLCN1 gene. However, given that ClC-1 must be drastically inhibited (∼80%) before myotonia develops, more recent studies have explored whether acute and more subtle ClC-1 regulation contributes to controlling the excitability of working muscle. Methods were developed to measure ClC-1 function with subsecond temporal resolution in action potential firing muscle fibers. These and other techniques have revealed that ClC-1 function is controlled by multiple cellular signals during muscle activity. Thus, onset of muscle activity triggers ClC-1 inhibition via protein kinase C, intracellular acidosis, and lactate ions. This inhibition is important for preserving excitability of working muscle in the face of activity-induced elevation of extracellular K+ and accumulating inactivation of voltage-gated sodium channels. Furthermore, during prolonged activity, a marked ClC-1 activation can develop that compromises muscle excitability. Data from ClC-1 expression systems suggest that this ClC-1 activation may arise from loss of regulation by adenosine nucleotides and/or oxidation. The present review summarizes the current knowledge of the physiological factors that control ClC-1 function in active muscle.

Extracellular magnesium and calcium reduce myotonia in isolated ClC-1 chloride channel-inhibited human muscle

Introduction: Experimental myotonia induced in rat muscle by ClC‐1 chloride channel‐inhibited has been shown to be related inversely to extracellular concentrations of Mg2+ and Ca2+ ([Mg2+]o and [Ca2+]o) within physiological ranges. Because this implicates a role for [Mg2+]o and [Ca2+]o in the variability of symptoms among myotonia congenita patients, we searched for similar effects of [Mg2+]o and [Ca2+]o on myotonia in human muscle. Methods: Bundles of muscle fibers were isolated from abdominal rectus in patients undergoing abdominal surgery. Myotonia was induced by ClC‐1 inhibition using 9‐anthracene carboxylic acid (9‐AC) and was assessed from integrals of force induced by 5‐Hz stimulation for 2 seconds. Results: Myotonia disappeared gradually when [Mg2+]o or [Ca2+]o were elevated throughout their physiological ranges. These effects of [Mg2+]o and [Ca2+]o were additive and interchangeable. Conclusions: These findings suggest that variations in symptoms in myotonia congenita patients may arise from physiological variations in serum Mg2+ and Ca2+. Muscle Nerve 51: 65–71, 2015