Cultures Of Skeletal Myotubes Research Articles

Exercise Inducible Lactate Dehydrogenase B Regulates Mitochondrial Function in Skeletal Muscle

Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate, which are critical fuel metabolites of skeletal muscle particularly during exercise. However, the physiological relevance of LDH remains poorly understood. Here we show that Ldhb expression is induced by exercise in human muscle and negatively correlated with changes in intramuscular pH levels, a marker of lactate production, during isometric exercise. We found that the expression of Ldhb is regulated by exercise-induced peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α). Ldhb gene promoter reporter studies demonstrated that PGC-1α activates Ldhb gene expression through multiple conserved estrogen-related receptor (ERR) and myocyte enhancer factor 2 (MEF2) binding sites. Transgenic mice overexpressing Ldhb in muscle (muscle creatine kinase (MCK)-Ldhb) exhibited increased exercise performance and enhanced oxygen consumption during exercise. MCK-Ldhb muscle was shown to have enhanced mitochondrial enzyme activity and increased mitochondrial gene expression, suggesting an adaptive oxidative muscle transformation. In addition, mitochondrial respiration capacity was increased and lactate production decreased in MCK-Ldhb skeletal myotubes in culture. Together, these results identified a previously unrecognized Ldhb-driven alteration in muscle mitochondrial function and suggested a mechanism for the adaptive metabolic response induced by exercise training.

Fluvastatin-induced alterations of skeletal muscle function in hypercholesterolaemic rats

Although statins, the most widely used drugs in the treatment of hyperlipidaemia, are generally accepted as efficient and safe drugs their side-effects on skeletal muscle have been reported with increasing frequency. The lack of an animal model in which these side effects would consistently be observed is one of the important drawbacks in studying statin associated myopathy. To overcome this and enable the studying of the effects of fluvastatin on skeletal muscles an animal model with high blood cholesterol levels was developed. In these animals cholesterol levels rose more than seven fold (from 1.5 ± 0.1 to 10.7 ± 2.0 mmol/l; n = 15 and 16) with a dramatic increase in low density lipoprotein/high density lipoprotein ratio (from 0.29 ± 0.02 to 1.56 ± 0.17). While the latter was reversed by statin treatment, an elevation in blood creatine kinase (CK) level indicated the presence of muscle wasting. Fibers from m. extensor digitorum longus (EDL) showed significant reduction in cross sectional area in the statin treated groups. Statin treatment also decreased the proliferation and fusion of skeletal myotubes in culture. In line with this, resting intracellular calcium concentration ([Ca(2+)](i)) was reduced in statin treated satellite cells and myotubes. On the other hand, in adult skeletal muscle fibers statin treatment increased resting [Ca(2+)](i) (116 ± 4 nM vs. 151 ± 5 nM; n = 33 and 34) and decreased both twitch and tetanic force both in EDL and m. soleus. In addition, in m. soleus the duration of twitch and tetanic force was shortened. These results clearly indicate that statin administration in these animals results in a myopathy characterized by decreased muscle force and elevated plasma CK level.

Interaction of the Calcium Channel α2/δ1 Subunit with an ATP Synthase in the Plasma Membrane of Muscle Cells

The calcium channel α2/δ1 subunit is expressed prior to Cav1.1 and is localized at the leading edges of young skeletal myotubes, suggesting that it may interact with other cellular components. To address this issue, multi protein complexes were isolated from skeletal muscle of P0 mice and analyzed with blue native PAGE and Western blots. These studies showed that the α2/δ1 subunit is found in a large complex of ∼670 kDa, which was also present in cultures of skeletal myotubes and C2C12 cells. This complex was isolated and further analyzed with mass spectroscopy. Mascot search results identified the β subunit of the ATP synthase (ATP5β) in the ∼670 kDa complex, which is normally present in the mitochondrial membrane. However, the presence of ATP5β and α2/δ1 subunit in the plasma membrane was confirmed by biotin labeling of membrane proteins. Immunocytochemistry analysis of skeletal myotubes in culture showed co-localization of α2/δ1 subunit and ATP5β in some cells. To determine a functional significance of the interaction between α2/δ1 subunit and ATP5β, calcium transients were electrically evoked and recorded from myotubes using confocal microscopy. Exposure of myotubes to an ATP5β monoclonal antibody resulted in a significantly faster decay of calcium transients after a train of stimuli. This effect was less pronounced when myotubes were stimulated with a single pulse. These experiments provide the first direct evidence suggesting that the α2/δ1 subunit may be able to form complexes with proteins other than the Cav1.1 and suggest new signaling roles of α2/δ1 in muscle cells. In addition, they show that a molecule involved in the synthesis of ATP in the inner mitochondrial membrane can be also localized in the plasma membrane. Supported by the Muscular Dystrophy Association.

Dissociation of charge movement from calcium release and calcium current in skeletal myotubes by gabapentin.

The skeletal muscle L-type calcium channel or dihydropyridine receptor (DHPR) plays an integral role in excitation-contraction (E-C) coupling. Its activation initiates three sequential events: charge movement (Q(r)), calcium release, and calcium current (I(Ca,L)). This relationship suggests that changes in Q(r) might affect release and I(Ca,L). Here we studied the effect of gabapentin (GBP) on the three events generated by DHPRs in skeletal myotubes in culture. GBP specifically binds to the alpha(2)/delta(1) subunit of the brain and skeletal muscle DHPR. Myotubes were stimulated with a protocol that included a depolarizing prepulse to inactivate voltage-dependent proteins other than DHPRs. Gabapentin (50 microM) significantly increased Q(r) while decreasing the rate of rise of calcium transients. Gabapentin also reduced the maximum amplitude of the I(Ca,L) (as we previously reported) without modifying the kinetics of activation. Exposure of GBP-treated myotubes to 10 microM nifedipine prevented the increase of Q(r) promoted by this drug, indicating that the extra charge recorded originated from DHPRs. Our data suggest that GBP dissociates the functions of the DHPR from the initial voltage-sensing step and implicates a role for the alpha(2)/delta(1) subunit in E-C coupling.

Inhibition of CapZ during myofibrillogenesis alters assembly of actin filaments.

The actin filaments of myofibrils are highly organized; they are of a uniform length and polarity and are situated in the sarcomere in an aligned array. We hypothesized that the barbed-end actin-binding protein, CapZ, directs the process of actin filament assembly during myofibrillogenesis. We tested this hypothesis by inhibiting the actin-binding activity of CapZ in developing myotubes in culture using two different methods. First, injection of a monoclonal antibody that prevents the interaction of CapZ and actin disrupts the non-striated bundles of actin filaments formed during the early stages of myofibril formation in skeletal myotubes in culture. The antibody, when injected at concentrations lower than that required for disrupting the actin filaments, binds at nascent Z-disks. Since the interaction of CapZ and the monoclonal antibody are mutually exclusive, this result indicates that CapZ binds nascent Z-disks independent of an interaction with actin filaments. In a second approach, expression in myotubes of a mutant form of CapZ that does not bind actin results in a delay in the appearance of actin in a striated pattern in myofibrils. The organization of alpha-actinin at Z-disks also is delayed, but the organization of titin and myosin in sarcomeres is not significantly altered. We conclude that the interaction of CapZ and actin is important for the organization of actin filaments of the sarcomere.

Role of fibronectin in the inhibitory effect of TGF-β on choline acetyltransferase activity in co-cultures of spinal cord neurons and myotubes

We have recently shown that the enhanced expression of choline acetyltransferase (ChAT) activity in co-cultures of spinal cord motoneurons and muscle cells was blocked by transforming growth factor-β (TGF-β) ( Dev. Brain Res., 57, 129–137, 1990). This study was performed to investigate the role of fibronectin in this effect. TGF-β increased fibronectin level about 2-fold in extracellular matrix of spinal cord cells and skeletal myotubes in culture. Addition of a synthetic polypeptide that competitively inhibits fibronectin binding to its cell surface receptor recovered the TGF-β-induced suppression of ChAT activity in co-cultures. The polypeptide did not affect ChAT activity in cultures of spinal cord cells alone or in co-cultures without TGF-β. These results indicate that TGF-β inhibits the stimulation of ChAT activity in spinal cord neurons in co-culture through a change in the composition and/or amount of fibronectin in the extracellular matrix at neuromuscular contacts.

Characterization of the relation between sodium channels and electrical activity in cultured rat skeletal myotubes: regulatory aspects

The relation among sodium channel density, frequency of electrical activity and maximal rate of rise of the action potential was studied in developing and mature rat skeletal myotubes in culture. The number of tetrodotoxin (TTX)-sensitive Na-channels was determined by measurements of the amount of [ 3H]saxitoxin (STX) bound to the cultures, and electrical properties were recorded with intracellular microelectrodes. The EC 50 for TTX-induced decreases in maximal STX-binding, frequency and rate of rise of action potentials was in the range 8–20 nM. The 3 variables increased in parallel with age in culture to reach peak values at age 7–8 days, and then decreased in parallel until 10–12 days in culture. The age-related increase in Na-channel density was decreased, but not abolished, by prevention of myoblast fusion. Treatment with the Ca 2+ ionophore, A23187, down-regulated, and blockade of Ca-channels with verapamil up-regulated the number of Na-channels. Na-channel density was also increased by chronic treatment with TTX and elevated external [K +], which eleminated spontaneous electrical and contractile activity. Parallel effects were observed on frequency and rate of rise of action potentials. Up-regulation of Na-channels was prevented by simultaneous treatment of myotubes with inhibitors of protein synthesis. We conclude that electrical and mechanical activity of cultured myotubes regulate de novo synthesis of Na-channels through alterations in the level of cytosolic Ca 2+.

Alternative splicing of the neural cell adhesion molecule gene generates variant extracellular domain structure in skeletal muscle and brain.

Myotube mRNA isoforms of the neural cell adhesion molecule (N-CAM) contain a novel sequence block termed muscle-specific domain 1 (MSD1), which is inserted within the extracellular coding region. Here, we report a characterization of the genomic organization of MSD1 and its pattern of expression within cellular N-CAM RNA and polypeptide species. S1 nuclease protection analyses and sequence analysis of an N-CAM human genomic clone containing MSD1 sequences indicated that MSD1 is comprised of three discrete exons of 15, 48, and 42 bp, designated MSD1a, MSD1b, and MSD1c, respectively. Although the MSD1a exon was present in a small proportion of mRNAs from both brain and muscle cells, the entire MSD1 sequence occurred predominantly in mRNAs from differentiated myotube cells. In addition, antiserum raised to a synthetic, MSD1b-encoded peptide sequence was found to stain the cell surface of human skeletal myotubes in culture, whereas myoblasts, fibroblasts, and neural cells were negative. MSD1a, MSD1b, and MSD1c sequences thus arise collectively in N-CAM mRNA and polypeptide isoforms as a result of muscle tissue-specific and developmentally regulated alternative mRNA splicing events. In addition, the occurrence of brain and muscle mRNAs containing only MSD1a indicate that alternative splicing may occur within the MSD region itself to generate further diversity.

Effects of carbamylcholine on membrane potential and Na-K pump activity of cultured rat skeletal myotubes.

1. We measured changes in resting membrane potential (Em) and Na-K pump activity, assayed by ouabain-sensitive 86Rb uptake, in response to carbamylcholine (CCh) and its continued presence in single rat skeletal myotubes in culture. 2. CCh caused immediate depolarization from control Em (-80 to -85 mV) to near 0 followed by repolarization of varying degrees depending on the age of the culture and temperature of the recording medium; repolarization of Em was most apparent by culture age 8-9 days in vitro (DIV), Em reaching values as high as -60 mV by 5-10 min after peak depolarization at 37 degrees C. 3. Input resistance, which decreased during CCh depolarization, increased only slightly during the initial phase of repolarization and then remained essentially unchanged during the major component of membrane repolarization in the presence of CCh. 4. Ouabain, given before CCh, prevented repolarization of Em and, when given after repolarization had begun, reversed it and caused Em to return to about -7 mV. 5. Na-K pump activity was decreased in myotubes in which Em did not repolarize or did so only slightly, and was increased by over 40-50% in myotubes whose Em repolarized by 40-60 mV, even though CCh was still present in the medium. Inhibition of pump activity in non repolarizing myotubes was related to Na influx, inhibition being reversed to stimulation when CCh was administered to myotubes in Na-free medium. 6. Repeated (three or four times) or prolonged (up to 60-min) administration of CCh to myotubes in which repolarization was hardly expressed (age 6-7 DIV) caused increases both in the amount of repolarization and in 86Rb uptake, both being related to the number or duration of CCh exposures. 7. We conclude that repolarization of Em following CCh-induced depolarization of cultured rat skeletal myotubes depends to a large extent on an increase in activity of the electrogenic Na-K pump.

Characterization of Thyroid Hormone Effects on Na-K Pump and Membrane Potential of Cultured Rat Skeletal Myotubes*

The purpose of this study was to characterize the effects of thyroid hormone on the Na-K pump and resting membrane potential (EM) of rat skeletal myotubes in culture. Myotubes were obtained from fetal (19-21 day) or neonatal rats (1-2 day) by serial trypsinization and maintained in culture for up to 10 days. Cells were treated with T4 or T3 on day 6 or 7, and measurements were made of EM, [3H]ouabain binding, and ouabain-sensitive 86Rb uptake at various times thereafter. Hormone treatment increased the values of all three variables within 24 h, plateau levels being attained by 48-72 h. Cycloheximide and actinomycin D totally blocked the effects of thyroid hormone when added together to the cells, thus suggesting that protein synthesis is necessary for the effects of these hormones. Scatchard analysis showed that the new receptors have lower ouabain affinity than those in control. Blockade of spontaneously occurring action potentials with tetrodotoxin, which blocks voltage-dependent Na channels, or Na/H antiporter with amiloride, abolished the hormone effects seen after 24 h and significantly reduced those obtained after 48 h of hormone treatment. The results demonstrate that thyroid hormone-induced increased amount and activity of the electrogenic Na-K pump in cultured myotubes occurs, at least in part, in response to an initial effect to increase Na influx. Moreover, the findings are consistent with the concept that the Na-K pump plays an important role in regulation of EM in this preparation.

Nerve growth factor supports growth of rat skeletal myotubes in culture

Effects of nerve growth factor (NGF) were examined on the growth of rat skeletal myotubes in culture and the expression of NaK pump activity in this preparation. We found NGF to cause an immediate increase in electrogenic NaK pump activity as determined by electrogenic component of membrane potential (Em) and ouabain-sensitive 86Rb uptake. When given chronically, NGF was able to replace serum as an essential supplement for development of cultured myotubes. Thus, when maintained in a serum-free, basal nutrient medium (DMEM), myotubes progressively deteriorated as indicated by morphological appearance, Em and the number of [ 3H]ouabain binding sites compared with myotubes grown in normal, serum-supplemented growth medium (GM). In contrast, the presence of NGF in DMEM completely prevented the deterioration of these properties, their values actually exceeding those in GM. These findings demonstrate a trophic effect of NGF on bioelectric properties of neonatal mammalian muscle cells.

Identification of a nicotinic acetylcholine receptor on neurons using an alpha-neurotoxin that blocks receptor function

An alpha-neurotoxin, Bgt 3.1, that reversibly blocks the ACh response of chick ciliary ganglion neurons has been used to identify 2 classes of high-affinity binding sites on the cells in culture. The first class appears to be the alpha-bungarotoxin binding site on the neurons. The second class of Bgt 3.1 sites is distinct from the alpha-bungarotoxin binding sites and has the properties expected for the functional nicotinic ACh receptor on the cells. Equilibrium binding and kinetic studies indicate a Kd value of 5-6 nM for Bgt 3.1 at the second class of sites. The kinetics and affinity of binding are consistent with those inferred from previous physiological studies for Bgt 3.1 inhibition of receptor function. Bgt 3.1 binding to the sites is completely inhibited by each of the cholinergic ligands ACh, carbachol, nicotine, d-tubocurarine, and trimethaphan, but not by alpha-bungarotoxin. Highest site densities are found in cultures of ciliary and sympathetic ganglion neurons, cell types known to have ganglionic nicotinic ACh receptors. Low levels of sites may be present in cultures of spinal cord and dorsal root ganglion neurons; no binding is found in cultures of skeletal myotubes or cardiac cells when alpha-bungarotoxin is used to block Bgt 3.1 binding to alpha-bungarotoxin sites. These results demonstrate that Bgt 3.1 can be used as a specific probe for the nicotinic ACh receptor on chick autonomic neurons.

Contribution of electrogenic sodium-potassium ATPase to resting membrane potential of cultured rat skeletal myotubes

The contribution of electrogenic Na +-K + ATPase to resting membrane potential (Em) of mature and developing rat skeletal myotubes in culture was determined by examining effects of inhibition of this enzyme on Em. Ouabain, a specific Na +-K + ATPase inhibitor, caused resting Em to decrease within 30 s by 5–8 mV and reach a minimum value of about −60 mV after 5 min. The decrease in Em was not accompanied by a decrease in input resistance for up to 15 min after application. Resting Em was found to be dependent on the temperature of the recording medium with maximum values of Em ranging from −85 to −90 mV at a temperature of 35–37°C and minimum values about −60 mV at 10–15°C. Ouabain (1 mM), added to cultures at low temperature (10–15°C) did not further decrease Em but did prevent the increase in Em that occurs with increasing temperature up to 37°C. Resting Em of cultured myotubes was reduced to about −60 mV by reducing the supply of ATP either with 2,4 dinitrophenol (DNP), which inhibits oxidative phosphorylation or with fluorodinitrobenzene (FDNB), which inhibits creatine phosphokinase. Neither of these compounds, when added to cultures in the presence of ouabain, reduced resting Em to a value lower than that obtained with ouabain alone. The developmental increase in Em which occurs after fusion appeared to be due to an increase in activity of Na +-K + ATPase; at any given age, Em was higher at 35 °C than at 21 °C, and addition of ouabain caused Em to fall to a value of −60 to −65 mV, regardless of age. The appearance of the electrogenic component of Em was found to be dependent to some extent on initial plating density of the cells.

The nitrendipine-sensitive Ca2+ channel in chick muscle cells and its appearance during myogenesis in vitro and in vivo.

The nitrendipine-sensitive Ca2+ channel of chick skeletal myotubes in culture has been studied using both the 45Ca2+ flux technique and [3H]nitrendipine binding experiments. Ca2+ uptake is insensitive to nitrendipine when chick myotubes in culture are polarized. Whereas depolarization reveals a new component of 45Ca2+ influx which is inhibited by nitrendipine. Half-maximal inhibition occurs at a nitrendipine concentration of 0.7 nM. This value is similar to the dissociation constant Kd = 0.4 nM found in [3H]nitrendipine binding experiments. During myogenesis in vitro the nitrendipine receptor is absent in myoblasts and appears in parallel with the fusion process. Two stages of increased binding have been observed in vivo. The first one, which occurs during embryonic life, has the same properties as in the in vitro development. The second stage occurs near hatching and corresponds to a large increase in the number of nitrendipine receptors. This increase is accompanied by a decrease of affinity of nitrendipine for its receptor by a factor of 4 to 10. Chronic denervation produces a further increase in the number of nitrendipine receptors which reaches a factor of about 2 at 15 days of denervation. Results are discussed in relation to the particular localization of these channels in transverse tubules and with the innervation.

Evidence for a functional role of acetylcholinesterase in cultured chick myotubes

This study was undertaken in order to assess the functional role of acetylcholinesterase (AChE) in cultures of chick skeletal muscle cells. Cultures of skeletal myotubes were prepared by mechanical dissociation of limb muscle removed from 11-day-old chick embryos and plating at a concentration of 0.8 × 10 6cells/ml. Cultures incubated for 4–10 days were used for electrophysiological studies with intracellular microelectrodes. Individual myotubes differed with respect to the time course of repolarization following depolarization by acetylcholine (ACh), some cells repolarizing within 2–3 min and others only after 8–10 min. Physostigmine (10 −8–10 −6 M) prolonged or sometimes completely prevented repolarization following ACh-induced depolarization. These results demonstrate that hydrolysis of ACh by AChE in cultured chick skeletal myotubes plays an important role in the repolarization of these cells following ACh-induced depolarization.

Internalization of alpha-bungarotoxin on neurons induced by a neurotoxin that blocks neuronal acetylcholine sensitivity

A protein neurotoxin (Bgt 3.1) present as a minor component in the venom of Bungarus multicinctus has been shown previously to block acetylcholine (ACh) sensitivity on chick ciliary ganglion (CG) neurons in cell culture. Alpha-bungarotoxin (Bgt. 2.2) binds to the neurons but does not block ACh sensitivity; the function of the Bgt. 2.2 binding site is unknown. The present studies demonstrate that Bgt 3.1 can induce the rapid internalization of Bgt 2.2 bound on the surface of CG and sympathetic neurons. The rapid internalization of bound Bgt 2.2 caused by Bgt. 3.1 can be seen with fluoresence microscopy using rhodamine-labeled Bgt 2.2 as the probe and by immunological techniques using anti-Bgt 2.2 antiserum to locate the bound 125I-Bgt 2.2. The rapid internalization is blocked by low temperature or by high concentrations of Bgt 2.2 and is not induced by Bgt 2.2 itself or by small cholinergic ligands. Bound 125I-Bgt 2.2 is released into the medium as degraded material after internalization is induced. Bgt 3.1 does not induce internalization of Bgt 2.2 bound to skeletal myotubes in culture nor does it induce the internalizaton of rhodamine-labeled nerve growth factor bound to sympathetic neurons, suggesting that its effect on neuronally bound Bgt 2.2 might be a specific one. Competition binding studies suggest that Bgt 3.1 may trigger the internalization of bound Bgt 2.2 by direct interaction with a Bgt 2.2 binding site. The effect of Bgt 3.1 on neuronal ACh sensitivity, however, does not depend on internalization of Bgt 2.2 binding sites since full inhibition of ACh sensitivity is still achieved by Bgt 3.1 under conditions where internalization is blocked. Neurons may have more than one class of Bgt 2.2 on the neurons. The internalization of Bgt 2.2 binding sites induced by Bgt 3.1 provides an unusual opportunity to study cellular mechanisms by which neurons can regulate the number and distribution of their surface components.

Induction of acetylcholine receptors on cultured skeletal muscle by a factor extracted from brain and spinal cord.

Extracts of chicken brain and spinal cord increase the total number of acetylcholine receptors and the number of acetylcholine receptors and the number of receptor clusters on uninnervated skeletal myotubes in culture. The active component in these extracts may be unique to neural tissue. Spinal cord cells grown in culture contain the active factor and they secrete it into the medium. Most of the activity is associated with a small molecule possibly a peptide. Such a factor may be responsible for the clustering of receptors at newly formed nerve-muscle synapses.