ClC-3 is required for LPA-activated Cl- current activity and fibroblast-to-myofibroblast differentiation

To determine the effects of chloride channel 3 (ClC-3) knockdown and overexpression on lysophosphatidic acid (LPA)- and volume-regulated anion channel Cl(-) currents (I(Cl,LPA) and I(Cl,VRAC), respectively), cell differentiation, and cell volume regulation, a short hairpin RNA (shRNA) expression system based on a mouse U6 promoter was used to knock down ClC-3 in human corneal keratocytes and human fetal lung fibroblasts. ClC-3 overexpression was achieved by electroporating full-length ClC-3, within a pcDNA3.1 vector, into these two cell lines. RT-PCR and Western blot analysis were used to detect ClC-3 mRNA and protein levels. Whole cell perforated patch-clamp recording was used to measure I(Cl,LPA) and I(Cl,VRAC) currents, and fluorescence-activated cell sorting analysis was used to measure cell volume regulation. ClC-3 knockdown significantly decreased I(Cl,LPA) and I(Cl,VRAC) activity in the presence of transforming growth factor-beta(1) (TGF-beta(1)) compared with controls, whereas ClC-3 overexpression resulted in increased I(Cl,LPA) activity in the absence of TGF-beta(1). ClC-3 knockdown also resulted in a reduction of alpha-smooth muscle actin (alpha-SMA) protein levels in the presence of TGF-beta(1), whereas ClC-3 overexpression increased alpha-SMA protein expression in the absence of TGF-beta(1). In addition, keratocytes transfected with ClC-3 shRNA had a significantly blunted regulatory volume decrease response following hyposmotic stimulation compared with controls. These data confirm that ClC-3 is important in VRAC function and cell volume regulation, is associated with the I(Cl,LPA) current activity, and participates in the fibroblast-to-myofibroblast transition.     

Regulation of intracellular Cl- concentration through volume-regulated ClC-3 chloride channels in A10 vascular smooth muscle cells

We previously found that antisense oligonucleotide specific to ClC-3 (ClC-3 antisense) prevented rat aortic smooth muscle cell proliferation, which was related to cell volume regulation. In the present study, we further characterized the regulation of intracellular Cl(-) concentrations ([Cl(-)](i)) via volume-regulated ClC-3 Cl(-) channels in an embryo rat aortic vascular smooth muscle cell line (A10 cell) and ClC-3 cDNA-transfected A10 cells (ClC-3-A10) using multiple approaches including [Cl(-)](i) measurement, whole cell patch clamp, and application of ClC-3 antisense and intracellular dialysis of an anti-ClC-3 antibody. We found that hypotonic solution decreased [Cl(-)](i) and evoked a native I(Cl.vol) in A10 cells. The responses of [Cl(-)](i) and I(Cl.vol) to hypotonic challenge were enhanced by expression of ClC-3, and inhibited by ClC-3 antisense. The currents in A10 (I(Cl.vol)) and in ClC-3-A10 cells (I(Cl.ClC-3)) were remarkably inhibited by intracellular dialysis of anti-ClC-3 antibody. Reduction in [Cl(-)](i) and activation of I(Cl.vol) and I(Cl.ClC-3) in A10 and ClC-3-A10 cells, respectively, were significantly inhibited by activation of protein kinase C (PKC) by phorbol-12,13-dibutyrate (PDBu) and inhibition of tyrosine protein kinase by genistein. Sodium orthovanadate (vanadate), a protein-tyrosine phosphatase inhibitor, however, enhanced the cell swelling-induced reduction in [Cl(-)](i), accompanied by the activation of I(Cl.vol) and I(Cl.ClC-3) in a voltage-independent manner. Our results suggest that the volume-regulated ClC-3 Cl(-) channels play important role in the regulation of [Cl(-)](i) and cell proliferation of vascular smooth muscle cells.     

Analysis of the interaction between properdin and factor B, components of the alternative-pathway C3 convertase of complement.

Denervated (1-10 days) rat epitrochlearis muscles were isolated, and basal and insulin-stimulated protein and glucose metabolism were studied. Although basal rates of glycolysis and glucose transport were increased in 1-10-day-denervated muscles, basal glycogen-synthesis rates were unaltered and glycogen concentrations were decreased. Basal rates of protein degradation and synthesis were increased in 1-10-day-denervated muscles. The increase in degradation was greater than that in synthesis, resulting in muscle atrophy. Increased rates of proteolysis and glycolysis were accompanied by elevated release rates of leucine, alanine, glutamate, pyruvate and lactate from 3-10-day-denervated muscles. ATP and phosphocreatine were decreased in 3-10-day-denervated muscles. Insulin resistance of glycogen synthesis occurred in 1-10-day denervated muscles. Insulin-stimulated glycolysis and glucose transport were inhibited by day 3 of denervation, and recovered by day 10. Inhibition of insulin-stimulated protein synthesis was observed only in 3-day-denervated muscles, whereas regulation by insulin of net proteolysis was unaffected in 1-10-day-denervated muscles. Thus the results demonstrate enhanced glycolysis, proteolysis and protein synthesis, and decreased energy stores, in denervated muscle. They further suggest a defect in insulin's action on protein synthesis in denervated muscles as well as on glucose metabolism. However, the lack of concurrent changes in all insulin-sensitive pathways and the absence of insulin-resistance for proteolysis suggest multiple and specific cellular defects in insulin's action in denervated muscle.     

Chloride currents from the transverse tubular system in adult mammalian skeletal muscle fibers

Chloride fluxes are the main contributors to the resting conductance of mammalian skeletal muscle fibers. ClC-1, the most abundant chloride channel isoform in this preparation, is believed to be responsible for this conductance. However, the actual distribution of ClC-1 channels between the surface and transverse tubular system (TTS) membranes has not been assessed in intact muscle fibers. To investigate this issue, we voltageclamped enzymatically dissociated short fibers using a two-microelectrode configuration and simultaneously recorded chloride currents (I(Cl)) and di-8-ANEPPS fluorescence signals to assess membrane potential changes in the TTS. Experiments were conducted in conditions that blocked all but the chloride conductance. Fibers were equilibrated with 40 or 70 mM intracellular chloride to enhance the magnitude of inward I(Cl), and the specific ClC-1 blocker 9-ACA was used to eliminate these currents whenever necessary. Voltage-dependent di-8-ANEPPS signals and I(Cl) acquired before (control) and after the addition of 9-ACA were comparatively assessed. Early after the onset of stimulus pulses, di-8-ANEPPS signals under control conditions were smaller than those recorded in the presence of 9-ACA. We defined as attenuation the normalized time-dependent difference between these signals. Attenuation was discovered to be I(Cl) dependent since its magnitude varied in close correlation with the amplitude and time course of I(Cl). While the properties of I(Cl), and those of the attenuation seen in optical records, could be simultaneously predicted by model simulations when the chloride permeability (P(Cl)) at the surface and TTS membranes were approximately equal, the model failed to explain the optical data if P(Cl) was precluded from the TTS membranes. Since the ratio between the areas of TTS membranes and the sarcolemma is large in mammalian muscle fibers, our results demonstrate that a significant fraction of the experimentally recorded I(Cl) arises from TTS contributions.     

ClC-2 chloride channels contribute to HTC cell volume homeostasis

Membrane Cl(-) channels play an important role in cell volume homeostasis and regulation of volume-sensitive cell transport and metabolism. Heterologous expression of ClC-2 channel cDNA leads to the appearance of swelling-activated Cl(-) currents, consistent with a role in cell volume regulation. Since channel properties in heterologous models are potentially modified by cellular background, we evaluated whether endogenous ClC-2 proteins are functionally important in cell volume regulation. As shown by whole cell patch clamp techniques in rat HTC hepatoma cells, cell volume increases stimulated inwardly rectifying Cl(-) currents when non-ClC-2 currents were blocked by DIDS (100 microM). A cDNA closely homologous with rat brain ClC-2 was isolated from HTC cells; identical sequence was demonstrated for ClC-2 cDNAs in primary rat hepatocytes and cholangiocytes. ClC-2 mRNA and membrane protein expression was demonstrated by in situ hybridization, immunocytochemistry, and Western blot. Intracellular delivery of antibodies to an essential regulatory domain of ClC-2 decreased ClC-2-dependent currents expressed in HEK-293 cells. In HTC cells, the same antibodies prevented activation of endogenous Cl(-) currents by cell volume increases or exposure to the purinergic receptor agonist ATP and delayed HTC cell volume recovery from swelling. These studies provide further evidence that mammalian ClC-2 channel proteins are functional and suggest that in HTC cells they contribute to physiological changes in membrane Cl(-) permeability and cell volume homeostasis.     

Human ClC-3 is not the swelling-activated chloride channel involved in cell volume regulation

Volume regulation is essential for normal cell function. A key component of the cells' response to volume changes is the activation of a channel, which elicits characteristic chloride currents (I(Cl, Swell)). The molecular identity of this channel has been controversial. Most recently, ClC-3, a protein highly homologous to the ClC-4 and ClC-5 channel proteins, has been proposed as being responsible for I(Cl, Swell). Subsequently, however, other reports have suggested that ClC-3 may generate chloride currents with characteristics clearly distinct from I(Cl, Swell). Significantly different tissue distributions for ClC-3 have also been reported, and it has been suggested that two isoforms of ClC-3 may be expressed with differing functions. In this study we generated a series of cell lines expressing variants of ClC-3 to rigorously address the question of whether or not ClC-3 is responsible for I(Cl, Swell). The data demonstrate that ClC-3 is not responsible for I(Cl, Swell) and has no role in regulatory volume decrease, furthermore, ClC-3 is not activated by intracellular calcium and fails to elicit chloride currents under any conditions tested. Expression of ClC-3 was shown to be relatively tissue-specific, with high levels in the central nervous system and kidney, and in contrast to previous reports, is essentially absent from heart. This distribution is also inconsistent with the previous proposed role in cell volume regulation.     

Quantitative analysis of the voltage-dependent gating of mouse parotid ClC-2 chloride channel

Various ClC-type voltage-gated chloride channel isoforms display a double barrel topology, and their gating mechanisms are thought to be similar. However, we demonstrate in this work that the nearly ubiquitous ClC-2 shows significant differences in gating when compared with ClC-0 and ClC-1. To delineate the gating of ClC-2 in quantitative terms, we have determined the voltage (V(m)) and time dependence of the protopore (P(f)) and common (P(s)) gates that control the opening and closing of the double barrel. mClC-2 was cloned from mouse salivary glands, expressed in HEK 293 cells, and the resulting chloride currents (I(Cl)) were measured using whole cell patch clamp. WT channels had I(Cl) that showed inward rectification and biexponential time course. Time constants of fast and slow components were approximately 10-fold different at negative V(m) and corresponded to P(f) and P(s), respectively. P(f) and P(s) were approximately 1 at -200 mV, while at V(m) > or = 0 mV, P(f) approximately 0 and P(s) approximately 0.6. Hence, P(f) dominated open kinetics at moderately negative V(m), while at very negative V(m) both gates contributed to gating. At V(m) > or = 0 mV, mClC-2 closes by shutting off P(f). Three- and two-state models described the open-to-closed transitions of P(f) and P(s), respectively. To test these models, we mutated conserved residues that had been previously shown to eliminate or alter P(f) or P(s) in other ClC channels. Based on the time and V(m) dependence of the two gates in WT and mutant channels, we constructed a model to explain the gating of mClC-2. In this model the E213 residue contributes to P(f), the dominant regulator of gating, while the C258 residue alters the V(m) dependence of P(f), probably by interacting with residue E213. These data provide a new perspective on ClC-2 gating, suggesting that the protopore gate contributes to both fast and slow gating and that gating relies strongly on the E213 residue.     

Hyposmotic activation of ICl,swell in rabbit nonpigmented ciliary epithelial cells involves increased ClC-3 trafficking to the plasma membrane.

In mammalian nonpigmented ciliary epithelial (NPE) cells, hyposmotic stimulation leading to cell swelling activates an outwardly rectifying Cl(-) conductance (I(Cl,swell)), which, in turn, results in regulatory volume decrease. The aim of this study was to determine whether increased trafficking of intracellular ClC-3 Cl channels to the plasma membrane could contribute to the I(Cl,swell) following hyposmotic stimulation. Our results demonstrate that hyposmotic stimulation reversibly activates an outwardly rectifying Cl(-) current that is inhibited by phorbol-12-dibutyrate and niflumic acid. Transfection with ClC-3 antisense, but not sense, oligonucleotides reduced ClC-3 expression as well as I(Cl,swell). Intracellular dialysis with 2 different ClC-3 antibodies abolished activation of I(Cl,swell). Immunofluorescence microscopy showed that hyposmotic stimulation increased ClC-3 immunoreactivity at the plasma membrane. To determine whether this increased expression of ClC-3 at the plasma membrane could be due to increased vesicular trafficking, we examined membrane dynamics with the fluorescent membrane dye FM1-43. Hyposmotic stimulation rapidly increased the rate of exocytosis, which, along with ICl,swell, was inhibited by the phosphoinositide-3-kinase inhibitor wortmannin and the microtubule disrupting agent, nocodazole. These findings suggest that ClC-3 channels contribute to I(Cl,swell) following hyposmotic stimulation through increased trafficking of channels to the plasma membrane.     

Ion-deficient environment induces the expression of basolateral chloride channel, ClC-3-like protein, in gill mitochondrion-rich cells for chloride uptake of the tilapia Oreochromis mossambicus

Gill mitochondrion-rich (MR) cells contain different molecules to carry out functionally distinct mechanisms. To date, the putative mechanism of Cl(-) uptake through the basolateral chloride channel, however, is less understood. To clarify the Cl(-)-absorbing mechanism, this study explored the molecular and morphological alterations in branchial MR cells of tilapia acclimated to seawater (SW), freshwater (FW), and deionized water (DW). Scanning electron microscopic observations revealed that three subtypes of MR cells were exhibited in gill filament epithelia of tilapia. Furthermore, in DW-acclimated tilapia, the subtype I (ion-absorbing subtype) of MR cells predominantly occurred in gill filament as well as lamellar epithelia. Whole-mount double immunofluorescent staining revealed that branchial ClC-3-like protein and Na(+)/K(+)-ATPase (NKA), the basolateral marker of MR cells, were colocalized in tilapia. In SW-acclimated tilapia, all MR cells of gill filament epithelia exhibited faint fluorescence of ClC-3-like protein. In contrast, only some MR cells in gill filament epithelia of FW and DW tilapia expressed basolateral ClC-3-like protein; however, the fluorescence was more intense in FW and DW tilapia than in SW fish. In hyposmotic groups, the number of MR cells immunopositive for ClC-3-like protein was significantly higher in DW-exposed tilapia. Meanwhile, in gill lamellar epithelia of DW tilapia, all MR cells (subtype I) were ClC-3-like protein immunopositive. Double immunostaining of ClC-3-like protein and Na(+)/Cl(-) cotransporter (NCC) revealed that basolateral ClC-3-like protein and apical NCC were colocalized in some MR cells in FW and DW tilapia. Moreover, both mRNA and protein amounts of branchial ClC-3-like protein were significantly higher in DW-acclimated tilapia. To identify whether the expression of branchial ClC-3-like protein responded to changes in environmental [Cl(-)], tilapia were acclimated to artificial waters with normal [Na(+)]/[Cl(-)] (control), lower [Na(+)] (low Na), or lower [Cl(-)] (low Cl). Immunoblotting of crude membrane fractions for gill ClC-3-like protein showed that the protein abundance was evidently enhanced in tilapia acclimated to the low-Cl environment compared with the other groups. Our findings integrated morphological and functional classifications of ion-absorbing MR cells and indicated that ion-deficient water elevated the numbers of subtype I MR cells in both filament and lamellar epithelia of gills with positive ClC-3-like protein immunostaining and increased the expression levels of ClC-3-like protein. This study is the first to illustrate the exhibition of a basolateral chloride channel potentially responsible for Cl(-) absorption in the ion-absorbing subtype of gill MR cells of tilapia.     

Downregulation of ClC-2 by JAK2

JAK2 (Janus kinase-2) is activated by cell shrinkage and may thus participate in cell volume regulation. Cell volume regulatory ion channels include the small conductance Cl(-) channels ClC-2. The present study thus explored whether JAK2 influences ClC-2 activity. To this end, ClC-2 was expressed in Xenopus oocytes with or without wild type JAK2, active (V617F)JAK2 or inactive (K882E)JAK2 and the Cl(-) channel activity determined by dual electrode voltage clamp. Expression of ClC-2 was followed by a marked increase of cell membrane conductance. The conductance was significantly decreased following coexpression of JAK2 or (V617F)JAK2, but not by coexpression of (K882E)JAK2. Exposure of the oocytes expressing ClC-2 together with (V617F)JAK2 to the JAK2 inhibitor AG490 (40 μM) resulted in a gradual increase of the conductance. According to chemiluminescence JAK2 decreased the channel protein abundance in the cell membrane. The decline of conductance in ClC-2 and (V617F)JAK2 coexpressing oocytes following inhibition of channel protein insertion by brefeldin A (5 μM) was similar in oocytes expressing ClC-2 with (V617F)JAK2 and oocytes expressing ClC-2 alone, indicating that (V617F)JAK2 might slow channel protein insertion into rather than accelerating channel protein retrieval from the cell membrane. In conclusion, JAK2 down-regulates ClC-2 activity and thus counteracts Cl(-) exit, an effect which may impact on cell volume regulation.     

Basolateral chloride current in human airway epithelia

Electrolyte transport by airway epithelia regulates the quantity and composition of liquid covering the airways. Previous data indicate that airway epithelia can absorb NaCl. At the apical membrane, cystic fibrosis transmembrane conductance regulator (CFTR) provides a pathway for Cl(-) absorption. However, the pathways for basolateral Cl(-) exit are not well understood. Earlier studies, predominantly in cell lines, have reported that the basolateral membrane contains a Cl(-) conductance. However, the properties have varied substantially in different epithelia. To better understand the basolateral Cl(-) conductance in airway epithelia, we studied primary cultures of well-differentiated human airway epithelia. The basolateral membrane contained a Cl(-) current that was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The current-voltage relationship was nearly linear, and the halide selectivity was Cl(-) > Br(-) > I(-). Several signaling pathways increased the current, including elevation of cellular levels of cAMP, activation of protein kinase C (PKC), and reduction of pH. In contrast, increasing cell Ca(2+) and inducing cell swelling had no effect. The basolateral Cl(-) current was present in both cystic fibrosis (CF) and non-CF airway epithelia. Likewise, airway epithelia from wild-type mice and mice with disrupted genes for ClC-2 or ClC-3 all showed similar Cl(-) currents. These data suggest that the basolateral membrane of airway epithelia possesses a Cl(-) conductance that is not due to CFTR, ClC-2, or ClC-3. Its regulation by cAMP and PKC signaling pathways suggests that coordinated regulation of Cl(-) conductance in both apical and basolateral membranes may be important in controlling transepithelial Cl(-) movement.     

N-glycosylation of the Xenopus laevis ClC-5 protein plays a role in cell surface expression, affecting transport activity at the plasma membrane

Mutations in the gene encoding ClC-5 lead to X-linked hypercalciuric nephrolithiasis (XLHN), characterized by proteinuria, hypercalciuria, and phosphaturia. In renal proximal tubule cells, ClC-5 was identified as an important player in endocytosis, which ensures reabsorption of filtered protein. However, the recent finding that ClC-5 is a Cl(-)/H(+) antiporter and not a Cl(-) channel as long thought points to the lack of understanding of its functional role. Also, little biochemical data are available about ClC-5 and its post-translational modifications have not been investigated. Here, we examined the role of N-glycosylation of xClC-5 in the Xenopus oocyte expression system by comparing wild-type (WT) xClC-5 and N-glycosylation site mutants. We found that xClC-5 is N-glycosylated on asparagines 169 and 470, which are the only N-glycosylated sites. xClC-5 mutants have an increased susceptibility to polyubiquitination and proteasomal degradation; however, without a notable impact on the expression level. Using a cross-linking reagent, we showed that xClC-5 assembles into protein complexes, independent of its N-glycosylation. Voltage-clamp measurements showed a reduced conductance in the presence of tunicamycin and with xClC-5 N-glycosylation site mutants. Using immunocytochemistry, we localized xClC-5 mainly in intracellular compartments, and found that its cell surface pool is reduced in the absence of N-glycans. We further examined the plasma membrane retrieval of WT and mutant xClC-5 in the presence of Brefeldin A (BFA), and found that the non-glycosylated mutant was retrieved more than five times faster than the WT protein. We conclude that N-glycosylation enhances cell surface expression of xClC-5, increasing its plasma membrane transport activity.     

Oxidation induces ClC-3-dependent anion channels in human leukaemia cells

To test for redox regulation of anion channels in erythroid cells, we exposed K562 cells to oxidants and measured changes in transmembrane Cl(-) currents using patch-clamp, and in intracellular Cl(-) content using the Cl(-) selective dye MQAE. Oxidation with tert-butylhydroperoxide or H(2)O(2) produced a plasma membrane anion permeability with a permselectivity of NO(3)(-)>lactate(-)>gluconate(-). The permeability increase was paralleled by insertion of ClC-3 protein into the plasma membrane as evident from immunofluorescence microscopy and surface biotinylation. Down-regulation of ClC-3 protein by RNA interference as assessed by immunoblotting decreased the oxidation-stimulated permeability. In conclusion, oxidation induces surface expression of ClC-3 and activation of a ClC-3-dependent anion permeability in K562 cells.     

Aging-associated down-regulation of ClC-1 expression in skeletal muscle: phenotypic-independent relation to the decrease of chloride conductance

In order to clarify the mechanism underlying the reduction of resting membrane chloride conductance (gCl) during aging, the levels of mRNA encoding the principal skeletal muscle chloride channel, ClC-1, were measured. Total RNA samples isolated from tibialis anterior muscles of aged (24-29 months old) and adult (3-4 months old) rats were examined for ClC-1 expression using Northern blot analysis, and macroscopic gCl was recorded from extensor digitorum longus muscle fibers from each adult and aged rat in vitro using a two intracellular microelectrode technique. Although interindividual variability was observed, aged rats exhibited a parallel reduction of both gCl and ClC-1 mRNA expression as compared to adult rats. A linear correlation exists between individual values of ClC-1 mRNA and gCl. These results provide evidence that ClC-1 is the main determinant of sarcolemmal gCl and demonstrate that the decrease of gCl observed during aging is associated with a down-regulation of ClC-1 expression in muscle.     

AMP-activated protein kinase activation prevents denervation-induced decline in gastrocnemius GLUT-4.

This study was designed to determine whether the reductions in GLUT-4 seen in 3-day-denervated muscles can be prevented through chemical activation of 5'-AMP-activated protein kinase (AMPK). Muscle AMPK can be chemically activated in rats using subcutaneous injections with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). In this study, the tibial nerve was sectioned on one side; the other was sham operated but without nerve section. Acute injections of AICAR resulted in significantly increased AMPK activity in denervated gastrocnemius but not soleus muscles. Acetyl-CoA carboxylase activity, a reporter of AMPK activation, declined in both gastrocnemius and soleus in both denervated and contralateral muscles. Three days after denervation, GLUT-4 levels were significantly decreased by approximately 40% in gastrocnemius muscles and by approximately 30% in soleus muscles. When rats were injected with AICAR (1 mg/g body wt) for 3 days, the decline in GLUT-4 levels was prevented in denervated gastrocnemius muscles but not in denervated soleus muscles. The extent of denervation-induced muscle atrophy was similar in AICAR-treated vs. saline-treated rats. These studies provide evidence that some effects of denervation may be prevented by chemical activation of the appropriate signaling pathways.     

Muscular design in the equine interosseus muscle

We studied the forelimb interosseus muscle in horses, Equus caballus, to determine the muscular properties inherent in its function. Some authors have speculated that the equine interosseus contains muscle fibers at birth only to undergo loss of these fibers through postnatal ontogeny. We describe the muscle fibers in eight interosseus specimens from adult horses. These fibers were studied histochemically using myosin ATPase studies and immunocytochemically using several antibodies directed against type I and type II myosin heavy chain antibodies. We determined that 95% of the fibers were type I, presumed slow-twitch fibers. All fibers exhibited normal morphological appearance in terms of fiber diameter and cross-sectional area, suggesting that the muscles are undergoing normal cycles of recruitment. SDS-PAGE studies of myosin heavy chain isoforms were consistent with these observations of primarily slow-twitch muscle. Fibers were determined to be approximately 800 microm long when studied using nitric acid digestion protocols. Short fiber length combined with high pinnation angles suggest that the interosseus muscle is able to generate large amounts of force but can produce little work (measured as pulling the distal tendon proximally). While the equine interosseus muscle has undergone a general reduction of muscle content during its evolution, it remains composed of a significant muscular component that likely contributes to forelimb stability and elastic storage of energy during locomotion.     

Effects of actinomycin D on fibrillation activity in denervated skeletal muscles of the rat

The effects of actinomycin D on fibrillation activity, acetylcholine sensitivity and resting membrane potential of denervated muscles of the rat was studied. Actinomycin D (0.7 mg/kg I.V.) administered 1 day after denervation delays the appearance of fibrillation for approximately 3 days. If this drug is given 5–7 days after denervation, it is also capable of blocking the already established fibrillation but fails to suppress extrajunctional cholinergic receptors and to reverse the fall in resting potential. The mechanical responses of denervated muscles are unaffected by actinomycin D. These results suggest that in fibrillation a genetic induction of newly formed RNA and protein is involved. It is also suggested that these molecules probably have a more rapid turnover than those required for the formation of extrasynaptic receptors in denervated muscle.     

Modification of the dystrophic phenotype after transient neonatal denervation: role of MHC isoforms.

While it recently has been demonstrated that it is possible to modify the phenotypic expression of murine dystrophy (dy/dy) (i.e., prevent myofiber loss) by subjecting the extensor digitorum longus (EDL) muscle of 14-day-old dy/dy mice to transient neonatal denervation (Moschella and Ontell, 1987), the mechanism responsible for this phenomenon has not been determined. Since it has been suggested that the effects of dystrophy vary according to fiber type, the fiber type frequency in 100-day-old normal (+/+) and dy/dy EDL muscles subjected to transient neonatal denervation has been determined by immunohistochemical analysis of their myosin heavy chain (MHC) composition. This frequency has been compared with that found in the EDL muscles of 14- and 100-day-old unoperated +/+ and dy/dy mice, in order to determine whether the reinnervation of transiently denervated neonatal muscle results in a preponderance of fibers of the type that might be spared dystrophic deterioration. In unoperated dy/dy muscle there is a progressive decrease in the frequency and in the absolute number of fibers that express MHC2B, with 100-day-old dy/dy muscles having approximately 32% of the number of myofibers fibers containing MHC2B as is found in age-matched +/+ muscles. The number of fibers containing the other fast isoforms (MHC2A and MHC2X) is similar in +/+ and dy/dy muscles at this age, indicating that fibers with MHC2B are most affected by the dystrophic process. Reinnervation following transient neonatal denervation of both the +/+ and the dy/dy EDL muscles results in a similar decrease (approximately 62%) in the number of myofibers containing MHC2B and an increase in myofibers containing the other fast MHC isoforms (MHC2A and MHC2X). The selective effect of dy/dy on fibers containing MHC2B and the sparing of myofibers in transiently denervated dy/dy muscle (which contains a reduced frequency of fibers containing MHC2B) are consistent with, although not direct proof of, the hypothesis that alterations in the fiber type may play a role in the failure of myofibers in transiently denervated dy/dy muscles to undergo dystrophic deterioration. Evidence is presented suggesting that neurons that supply myofibers containing MHC2B may be at a selective disadvantage in their ability to reinnervate neonatally denervated muscles.