Genetic correction of dystrophin deficiency and skeletal muscle remodeling in adult MDX mouse via transplantation of retroviral producer cells.

Duchenne muscular dystrophy (DMD) is an X-linked, lethal disease caused by mutations of the dystrophin gene. No effective therapy is available, but dystrophin gene transfer to skeletal muscle has been proposed as a treatment for DMD. We have developed a strategy for efficient in vivo gene transfer of dystrophin cDNA into regenerating skeletal muscle. Retroviral producer cells, which release a vector carrying the therapeutically active dystrophin minigene, were mitotically inactivated and transplanted in adult nude/mdx mice. Transplantation of 3 x 10(6) producer cells in a single site of the tibialis anterior muscle resulted in the transduction of between 5.5 and 18% total muscle fibers. The same procedure proved also feasible in immunocompetent mdx mice under short-term pharmacological immunosuppression. Minidystrophin expression was stable for up to 6 mo and led to alpha-sarcoglycan reexpression. Muscle stem cells could be transduced in vivo using this procedure. Transduced dystrophic skeletal muscle showed evidence of active remodeling reminiscent of the genetic normalization process which takes place in female DMD carriers. Overall, these results demonstrate that retroviral-mediated dystrophin gene transfer via transplantation of producer cells is a valid approach towards the long-term goal of gene therapy of DMD.

Membranes undergoing phase transitions are preferentially hydrolyzed by beta-bungarotoxin.

beta-Bungarotoxin preferentially hydrolyzes choline phospholipids (dilauroyl, dimyristoyl, dipalmitoyl) at their respective gel to liquid crystalline phase transition temperatures. Cholesterol markedly reduces the rate of phospholipid hydrolysis; at 0.33 mol percent cholesterol:phospholipid, the toxin's phospholipase activity is completely inhibited.

Potassium channel toxins.

Many venom toxins interfere with ion channel function. Toxins, as specific, high affinity ligands, have played an important part in purifying and characterizing many ion channel proteins. Our knowledge of potassium ion channel structure is meager because until recently, no specific potassium channel toxins were known, or identified as such. This review summarizes the sudden explosion of research on potassium channel toxins that has occurred in recent years. Toxins are discussed in terms of their structure, physiological and pharmacological properties, and the characterization of toxin binding sites on different subtypes of potassium ion channels.

Identification of two toxins from scorpion (Leiurus quinquestriatus) venom which block distinct classes of calcium-activated potassium channel.

Two polypeptide toxins from scorpion (Leiurus quinquestriatus) venom which block distinct classes of calcium-activated potassium channels have been identified and partially purified. One toxin, at 50-100 ng/ml, blocks apamin-sensitive potassium fluxes in hepatocytes and inhibits [125I]monoiodoapamin binding. The other, more basic, toxin blocks apamin-insensitive potassium fluxes in erythrocytes at 200 ng/ml and, to our knowledge, is the first toxin shown to block the erythrocyte calcium-activated potassium channel with high affinity. The possible co-identity of this latter toxin with charybdotoxin is discussed.

Transcription of the dystrophin gene in Duchenne muscular dystrophy muscle.

Dystrophin is the recently discovered defective gene product in Duchenne and Becker muscular dystrophy (DMD and BMD). Dystrophin transcripts have been amplified and identified in diagnostic needle muscle biopsy samples using the polymerase chain reaction (PCR) procedure. Using 5'- and 3'-primers, dystrophin transcripts can be detected in both DMD and BMD muscle biopsies, on either side of defined deletions within the dystrophin gene.

Calmodulin-binding profiles for nebulin and dystrophin in human skeletal muscle.

Nebulin and dystrophin are two high-molecular-mass skeletal muscle proteins that have both been associated with the defective gene in Duchenne muscular dystrophy, although the function of neither protein is known. Other high-molecular-mass, calmodulin-binding proteins have recently been implicated in regulating calcium release from skeletal muscle. Western blots of human skeletal muscle biopsy samples were probed with biotinylated calmodulin; nebulin was identified as a prominent high-molecular-mass calmodulin-binding protein but dystrophin did not bind detectable amounts of biotinylated calmodulin. Dystrophin was absent in a Duchenne muscle biopsy.

High affinity binding of [125I]monoiodoapamin to isolated guinea-pig hepatocytes.

The bee venom neurotoxin apamin has been labelled with 125I and its binding to isolated guinea-pig hepatocytes measured under physiological conditions. A single saturable component of [125I]monoiodoapamin binding with a Kd of 350 pM and Bmax of 0.99 fmol/mg dry wt was identified. Native apamin displaced labelled apamin with a Kd of 376 pM which is broadly in keeping with the concentrations found to inhibit K loss from guinea-pig hepatocytes. These observations, together with the binding found in other tissues, suggest that specific binding of labelled apamin is particularly associated with apamin-sensitive, Ca-activated K-channels.

Dystrophin abnormalities in polymyositis and dermatomyositis.

The expression of dystrophin in muscle biopsies from nine cases of polymyositis, ten cases of juvenile dermatomyositis and three adults with dermatomyositis was studied by Western blot analysis and immunocytochemistry. Five antibodies corresponding to different N- and C-terminal regions of the dystrophin gene were used. Sixteen of the 22 cases (73%) showed an abnormality in the expression of dystrophin on Western blot analysis, either with a reduced molecular weight protein or a reduced amount. Immunostaining was abnormal in 11 out of 19 cases (58%) and showed varying degrees of discontinuity or loss of sarcolemmal staining. Immunolabelling of these areas with antibodies to beta-spectrin was normal implying that the changes were not caused by a loss of the sarcolemma. These results show that secondary changes in the expression of dystrophin can occur in the absence of an abnormality in the corresponding gene and that dystrophin cannot be used in isolation as a diagnostic marker for muscular dystrophy.

Manifesting carriers of Xp21 muscular dystrophy; lack of correlation between dystrophin expression and clinical weakness.

Ten females presenting with muscle weakness and a raised serum creatine kinase revealed abnormalities in the expression of dystrophin in their muscle biopsies and were diagnosed as manifesting carriers of Xp21 Duchenne/Becker muscular dystrophy. Seven cases, aged 3-22 yr at the time of biopsy, had a variable proportion of dystrophin-deficient fibres and an abnormal expression on immunoblot. These were confidently diagnosed as manifesting carriers. Results in the remaining three cases, aged 8-10 yr, were less clear-cut. Dystrophin expression on immunoblots was slightly reduced and some unevenness and reduction of immunolabelling was seen on sections, but dystrophin-deficient fibres were not a feature of these cases. The weakness in the ten carriers ranged from minimal to severe and there was no correlation between the degree of weakness and the number of dystrophin-deficient fibres. Two minimally weak girls had a high proportion of dystrophin-deficient fibres. Our results show that analysis of dystrophin expression is useful for the differential diagnosis of carriers of Xp21 dystrophy and autosomal muscular dystrophy, but that dystrophin expression does not correlate directly with the degree of clinical weakness.

Early induction by crotoxin of biphasic frequency changes and giant miniature endplate potentials in frog muscle.

1. Following the addition of crotoxin (250 nM) at the frog neuromuscular junction, there was an initial fall in frequency of miniature endplate potentials (m.e.p.ps), followed by a secondary rise which was characterized by the appearance of large spontaneous potentials (giants, g.m.e.p.ps) and an occasional large potential of the burst type. 2. In the presence of 2-(4-phenylpiperidino)cyclohexanol (AH5183, vesicamol), an inhibitor of vesicular acetylcholine uptake, the frequency of g.m.e.p.ps induced by crotoxin was reduced. 3. The characteristic changes in m.e.p.p. frequency and amplitude distribution were absent with crotoxin in Sr-EGTA Ringer. In the presence of high concentrations of Mn (3.6 or 5.4 mM with 0.9 mM Ca), the crotoxin-induced initial fall and the onset of the secondary rise in m.e.p.p. and g.m.e.p.p. frequencies were slower. The timing of these phases was unaffected by Ca concentrations ranging from 6.3 to 0.9 mM. 4. High concentrations of Mn ions partially inhibited the phospholipase A2 activity of crotoxin on artificial phospholipid membranes. This also supports the involvement of the Ca-dependent phospholipase A2 subunit in both phases of the physiological action of the toxin. 5. G.m.e.p.ps were associated with a moderate increase in m.e.p.p. frequency (2-3 s-1) and were of a time-course similar to that of m.e.p.ps. They persisted after washing with medium lacking Ca ions and in the presence of Ca-Mn Ringer that blocked evoked responses. 6. It is concluded that crotoxin, acting through its phospholipase A2 subunit, produces specific disturbances of synaptic exocytosis and vesicle formation in the axolemma of the motor nerve terminal which lead to biphasic changes in m.e.p.p. frequency and the onset of large spontaneous potentials.

Effects of potassium channel toxins from Leiurus quinquestriatus hebraeus venom on responses to cromakalim in rabbit blood vessels.

1. The effects of fractionated Leiurus quinquestriatus hebraeus venom on cromakalim-induced 86Rb+ efflux in rabbit aortic smooth muscle were examined. 2. Crude venom (0.1-30 micrograms ml-1) produced a concentration-dependent decrease of 1 microM cromakalim-induced 86Rb+ response. The maximum blocking activity attainable was approximately 60%. 3. Fractionation of crude venom by gel permeation chromatography and subsequent chromatography on a cation ion-exchange column, produced two fractions (X and XI), active in the 86Rb+ blocking assay. 4. Fraction XII contained charybdotoxin (approximately 85% pure). After a final high performance liquid chromatography (h.p.l.c.) purification step, the purified toxin failed to inhibit the cromakalim-stimulated 86Rb+ efflux although it was a potent inhibitor of A23187-induced K+ flux in human erythrocytes and the large conductance calcium-activated potassium channel in rabbit portal vein smooth muscle. 5. Subsequent purification of fraction X by h.p.l.c. yielded a minor peak which contained 86Rb+ blocking activity. This subfraction was also capable of inhibiting apamin-sensitive, angiotensin II-stimulated K+ flux in guinea-pig hepatocytes. 6. It is concluded that the potassium channel opened by cromakalim in rabbit aortic smooth muscle is not blocked by charybdotoxin but by another distinct toxin in the venom of Leiurus quinquestriatus hebraeus.

Effects of charybdotoxin, a blocker of Ca2+-activated K+ channels, on motor nerve terminals.

1. The contribution of Ca2+-activated K+ currents (IK,Ca) to the control of electrical excitability of motor nerve terminals and the control of acetylcholine release was assessed by studying the effects of the specific K(Ca) channel blocking toxins charybdotoxin and apamin. Electrical activity of the terminal regions of motor nerves was assessed by extracellular recording from an electrode placed in the perineural sheaths of nerves in the mouse triangularis sterni and frog cutaneous pectoris preparations. Acetylcholine release was monitored by intracellular recording of endplate potentials (e.p.ps). 2. Charybdotoxin (20-300 nM), but not apamin (10 nM-2.5 microM), selectively reduced the amplitude of an IK,Ca unmasked by prior blockade of the delayed rectifier K+ current with 3,4-diaminopyridine (3,4-DAP). 3. In the combined presence of 3,4-DAP and charybdotoxin, large Ca2+-dependent plateau responses developed, but only moderate and transient increases in acetylcholine release occurred. 4. In the absence of 3,4-DAP, charybdotoxin did not alter the electrical activity of, or the transmitter release from motor nerve terminals. 5. A possible role of the charybdotoxin-sensitive IK,Ca in the control of transmitter release is discussed.

Characterization of Ca(2+)-activated 86Rb+ fluxes in rat C6 glioma cells: a system for identifying novel IKCa-channel toxins.

1. The pharmacological characteristics of a putative Ca2+ activated K+ channel (IKCa channel) in rat glioma C6 cells were studied in the presence of the Ca2+ ionophore, ionomycin and various K+ channel blockers, 86Rb+ being used as a radioisotopic tracer for K+. 2. The resting 86Rb+ influx into C6 cells was 318 +/- 20 pmol s-1. The threshold for ionomycin activation of 86Rb+ influx was approx. 100 nM. At ionomycin concentrations above the activation threshold, the initial rate of 86Rb+ influx was proportional to ionophore concentration. Ionomycin-activated 86Rb+ flux was saturable (EC50 = 0.62 +/- 0.03 microM) and was not inhibited by ouabain. 3. Intracellular Ca2+ increased within 30 s from a basal level of 42 +/- 2 nM to 233 +/- 17 nM, after addition of 2 microM ionomycin. During this period, intracellular pH fell from 7.03 +/- 0.04 to 6.87 +/- 0.03 and the cell hyperpolarized from -34 +/- 10 mV to -76 +/- 2 mV. 4. Single channel conductance measurements on inside-out patches in physiological K+ solutions identified a 14 +/- 3 pS CA(2+)-activated K+ current between -25 mV and +50 mV. In symmetrical (100 mM) K+, the single channel conductance was 26 pS. 5. Externally applied quinine (IC50 = 0.12 +/- 0.34 mM) and tetraethylammonium chloride (IC50 = 10 +/- 1.9 mM) inhibited 86Rb+ influx into C6 cells in a concentration-dependent manner. Charybdotoxin (IC50 = 0.5 +/- 0.02 nM) and iberiotoxin (IC50 = 800 +/- 150 nM), as well as the crude venoms from the scorpions Leiurus quinquestriatus and Mesobuthus tamulus, also inhibited 86Rb+ influx. In contrast, apamin and toxin I had no inhibitory effects on 86Rb+ flux. A screen of fractions from cation exchange h.p.l.c. of Mesob. tamulus venom revealed the presence of at least four charybdotoxin-like peptides. One of these was iberiotoxin; the other three are novel toxins. 6. The ionomycin-activated 86Rb+ influx into rat C6 glioma cells has proved to be a valuable pharmacological assay for the screening of toxins and crude venoms which modify intermediate conductance, Ca2+ activated K+ channel activity.

Activation of a K+ conductance by bradykinin and by inositol-1,4,5-trisphosphate in rat glioma cells: involvement of intracellular and extracellular Ca2+.

Extracellular application of bradykinin and injection of inositol-1,4,5-trisphosphate (Ins-P3) induced a hyperpolarization in polyploid rat glioma cells. Ins-1,4,5-P3 and Ins-2,4,5-P3 were effective but not Ins-4,5-P2, Ins-1,3,4,5-P4 and Ins-1,3,4,5,6-P5. The reversal potential of the hyperpolarizing response induced by bradykinin or by Ins-P3 increased to a comparable degree with increasing the extracellular K+ concentration. Certain blockers of K+ channels, for example charybdotoxin (5-50 nM), Ba2+ (5-20 mM), 4-aminopyridine (5-10 mM) and quinidine (0.1-0.5 mM) reversibly suppressed the membrane potential response to bradykinin or to Ins-P3; however, apamin (1 microM) and D-tubocurarine (0.5 mM) had no effect. Intracellular injection of EGTA made the glioma cells unresponsive to bradykinin. Superfusion of the cells with Ca2(+)-free medium gradually and reversibly abolished the response to bradykinin, but only slightly reduced the effect of Ins-P3. The Ca2+ channel blockers Co2+ (1-5 mM), Mn2+ (2-6 mM) and nifedipine (1-20 microM), but not desmethoxyverapamil (100 microM) inhibited the hyperpolarizing effect of bradykinin. The hyperpolarization induced by Ins-P3, however, was not influenced by Mn2+ (1-5 mM) or by Co2+ (7 mM). Injection of Ca2+ into the glioma cells induced a hyperpolarization susceptible to Ba2+ and quinidine. Treatment of glioma cells with an activator or with inhibitors of protein kinase C or with pertussis toxin did not affect the response to bradykinin. Incubation of the cells with the Ca2+ ionophore A23187 (0.1-1 microM) made the cells unresponsive to bradykinin and, somewhat less, to Ins-P3. At these concentrations the Ca2+ ionophore primarily depletes intracellular Ca2+ stores. In summary, bradykinin, via B2-receptors (blocked by [Thi5,8, D-Phe7]-bradykinin) activates a K+ conductance in glioma cells following a rise of cytosolic Ca2+ activity most likely due to Ins-P3-mediated release of Ca2+ from internal stores. Entry of extracellular Ca2+ appears also to be involved in this process.

Equine muscular dystrophy with myotonia.

OBJECTIVES: To describe a case of equine muscular dystrophy with myotonia. METHODS: A 5-year-old horse presented with hypertrophy and delayed relaxation of the muscles of the hindlimbs from age 2 months. Testicular atrophy developed from 2 years of age. Action and percussion myotonia was associated with weakness in these muscles, and EMG showed diffuse myotonic discharges and myopathic features. Biopsy of the gluteal muscle showed adipose and connective tissue infiltration, marked variation in muscle fibre size, and moth-eaten, ring and whorled fibres. RESULTS: Injection of apamin, a peptide blocker of calcium-activated potassium channels, which inhibits myotonia in human myotonic dystrophy, was ineffective in blocking myotonic discharges. Discharges promptly abated with 2% lidocaine injection. CONCLUSIONS: Myotonia in this horse is associated with dystrophic changes similar to human myotonic dystrophy, though there are some pharmacological differences.

Characterisation of dystrophin in fetuses at risk for Duchenne muscular dystrophy.

Dystrophin, the product of the Duchenne muscular dystrophy (DMD) gene, was studied in muscle from 16 human fetuses at risk for the disease. Eleven high risk (greater than 95% probability) and 5 low-risk (less than 25% probability) fetuses were studied with antibodies raised to different regions of the protein. All low-risk fetuses showed a similar pattern to that of normal fetuses of a comparable age: using Western blot analysis, a protein was detected of similar size and abundance to that of normal fetuses (i.e. smaller molecular weight than that of adult muscle); immunocytochemistry showed uniform sarcolemmal staining in fetuses older than 18 weeks gestation and differential staining of myotubes at different stages of development (distinguished by size) in younger fetuses (less than 15 weeks gestation). In contrast, Western blot analysis of high-risk fetuses detected low levels of dystrophin in 4 cases; 7 fetuses had no detectable protein. Immunocytochemistry with some dystrophin antibodies showed weak staining of the sarcolemma and around central nuclei in younger fetuses; in older fetuses there was little sarcolemmal staining with any antibody other than occasional positive fibres. These results indicate that careful study of dystrophin in fetuses at risk for DMD can be used to establish the clinical phenotype and provide additional information for future family counselling.

Characterisation of dystrophin in carriers of Duchenne muscular dystrophy.

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, was studied in needle biopsy samples taken from the quadriceps muscle of 15 asymptomatic carriers of DMD (13 adults and 2 young girls) and one symptomatic adult carrier. Antibodies to N- and C-terminal regions of dystrophin were used for both Western blot analysis and immunocytochemistry and a monoclonal antibody to beta-spectrin used to assess membrane integrity. All asymptomatic adult carriers showed some abnormality in dystrophin immunostaining but very few negative fibres were present. A clear mosaic of dystrophin positive and negative fibres was seen only in the adult symptomatic carrier and the two young girls. On a Western blot, all carriers studied had dystrophin of normal molecular weight, but most had reduced abundance. In adult carriers, the amount of dystrophin relative to normal controls varied, but it was unrelated to age, serum creatine kinase (CK) levels or to the degree of pathology. Carriers with normal CK showed abnormalities in dystrophin expression. The dystrophin immunoblotting profile of the 2 young girls was very similar to that of their mothers, but the mosaic pattern of immunostaining was not apparent in the older carriers. In conclusion, dystrophin immunostaining and Western blot analysis of biopsy samples from asymptomatic carriers is often abnormal and they may be useful additional aids for establishing carrier status, particularly in younger girls.

Dystrophin and nebulin in the muscular dystrophies.

Skeletal muscle from patients with 5 different forms of muscular dystrophy and from 6 fetuses at high risk (95%) for Duchenne muscular dystrophy (DMD) were probed with specific antibodies for the presence of dystrophin and nebulin. Dystrophin was absent in all 5 patients with DMD and 4 of 6 fetuses at high risk for DMD and present in trace amounts in the remaining two. Dystrophin was also undetectable in one borderline DMD/Becker muscular dystrophy (BMD) case and reduced in 2 of 4 cases of BMD. In contrast, dystrophin was present in all 16 biopsies from 4 other types of muscular dystrophy (congenital, limb girdle, Emery-Dreifuss and facioscapulohumeral). Nebulin profiles varied with the type, severity and duration of the dystrophic process. Nebulin was present in 5 of 6 DMD fetal samples but vastly reduced or absent in all samples of clinically manifest DMD.

Distribution of dystrophin, nebulin and Ricinus communis I (RCA-I)-binding glycoprotein in tissues of normal and mdx mice.

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene locus, appeared as an immunoreactive triplet of polypeptides in striated muscle tissues from normal mice on Western blot analysis. In smooth muscle tissues, an immunoreactive doublet of corresponding molecular weight was detected. No dystrophin was found in normal mouse brain, not even after enrichment for the Triton X-100 insoluble fraction. Dystrophin was absent from all corresponding tissues from the mdx mutant mouse strain which is known to lack dystrophin. The possibility that these immunoreactive bands represent isoforms is discussed. We have also investigated two other high molecular weight proteins which show secondary abnormalities in DMD muscle, namely nebulin and the 370 kDa Ricinus communis I lectin (RCA I)-binding glycoprotein. Nebulin levels were reduced in skeletal muscle from 6-week-old mdx mice but not in oesophagus from the same animals. By contrast, the RCA I-binding 370 kDa glycoprotein which is greatly reduced in DMD skeletal muscle was present in normal amounts in mdx skeletal muscle. These findings show, for the first time, that mdx myopathy differs from DMD myopathy not only morphologically, but also in its secondary biochemical abnormalities.

Charaterization of bumarsin, a 3-hydroxy-3-methylglutaryl-coenzyme reductase inhibitor from Mesobuthus martensii Karsch venom.

Scorpion venoms are rich sources of bioactive peptides and are widely known for their ion channel inhibiting properties. We have isolated, cloned and characterized a venom protein (Bumarsin) from the Chinese scorpion, Mesobuthus martensii Karsch. Bumarsin cDNA encodes a 8132 Da, 72 amino acid mature protein that most probably exists in its native form as a Cys-bridged homodimer. We have identified this novel protein to be an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity. 0.6 µM of Bumarsin inhibits 32% of the HMG-CoA reductase activity, in comparison to 10 µM simvastatin which only inhibits 35% of the activity. RT-PCR and SELDI-TOF mass spectrometric studies demonstrate that bumarsin regulates the expression of both genes and proteins involved in cholesterol homeostasis. Our results suggest that bumarsin may provide a model for the design of novel drugs that can be used to modulate cholesterol homeostasis.