Membrane-bound enzymes of erythrocytes in human muscular dystrophy: (Na+ + K+-ATPase, Ca2+-ATPase, K+- and Ca2+-p-nitrophenylphosphatase.

Four enzyme activities were studied in erythrocyte membranes from patients with Duchenne and congenital myotonic muscular dystrophy. (Na⁺ + K⁺)-stimulated, Mg²⁺-dependent adenosinetriphosphatase, measured in two different media, showed normal activity and ouabain inhibition, as did K⁺-stimulated p-nitrophenylphosphatase. The specific activity of Ca²⁺-stimulated p-nitrophenylphosphatase was twice normal in Duchenne membranes. Ca²⁺-stimulated, Mg²⁺-dependent adenosine-triphosphatase was augmented in membranes from both Duchenne and congenital myotonic muscular dystrophic patients. The cause of the increased activities may be the necessity for compensating an alteration in the calcium metabolism in the dystrophic erythrocytes.Several kinetic parameters of the two Ca²⁺-stimulated enzyme activities were studied in Duchenne and control membranes. Most were not changed, with the exception of the Na⁺-stimulation of Ca²⁺-ATPase. In Duchenne membranes two affinity sites were present with half maximal activating concentrations of 58 ± 4 and 4 ± 1 mM Na⁺. In control membranes only one affinity site was found with K_a = 26 ± 9 mM Na⁺.     

Freeze-fracture studies of erythrocyte plasma membrane in human neuromuscular diseases

Freeze-fracture studies were conducted in erythrocyte plasma membrane from 8 patients with Duchenne muscular dystrophy (DMD), 8 age-matched controls, 3 adult controls, 10 patients with myotonic muscular dystrophy, and 26 other neuromuscular disease controls. There was marked depletion of intramembranous particles in Duchenne dystrophy, whereas intramembranous particle density counts in other neuromuscular diseases were within normal limits. Therefore, the internal molecular architecture of the erythrocyte membrane is abnormal in Duchenne dystrophy, supporting the concept that a membrane defect involving multiple tissues is present in this disorder.     

Erythrocyte surface membrane alterations.

Electrophoretic mobility measurements were made of red blood cells obtained from patients with Duchenne and myotonic muscular dystrophy, from dystrophic mice and chickens, and from corresponding controls. Alterations in the erythrocyte surface electrokinetic properties were found in dystrophic mice and chickens and in many, but not all, patients with muscular dystrophy. The results are consistent with the concept of muscular dystrophy as a systemic membrane disease not limited to muscle.     

Erythrocyte membrane lysophospholipase activity in muscular dystrophy

The membrane lysophospholipase activity of erythrocytes obtained from Duchenne muscular dystrophy patients was lower than that of erythrocytes from age-matched normal boys. On the other hand, the membrane enzyme activity of erythrocytes from myotonic dystrophy patients was not different from that of their age- and sex-matched controls. Dipyridamole (0.1 mM) and glycerol 3-phosphorylcholine (2 mM) had no significant effect on these enzyme activities. These results suggest that membrane lysophospholipid metabolism may be altered in Duchenne muscular dystrophy but not in myotonic dystrophy.     

Erythrocyte membrane abnormalities in human myotonic dystrophy

Membrane-bound enzyme activities and cardiac glycoside binding were determined in red blood cell membrane preparations from patients with myotonic dystrophy and in age matched controls. Na⁺-K⁺-activated ATPase activity was signficantly increased in myotonic patients. [³H]Ouabain binding to erythrocyte membranes was also significantly increased in myotonic dystrophy patients. The Mg²⁺-ATPase (ouabain-insensitive) was, however, unchanged. The K⁺-stimulated paranitrophenyl phosphatase (KPNPPase) activity was markedly enhanced in myotonic patients as compared to controls. The kinetic analysis showed a marked change in V_max of Na⁺-K⁺ ATPase with respect to the activation by Na⁺, K⁺ and ATP. However, the K_m values were the same in control as well as in myotonic groups. The increased erythrocyte membrane Na⁺-K⁺-ATPase activity, KPNPPase and [³H]ouabain binding in myotonic patients supports the hypothesis that generalized membrane abnormality may be involved in pathogenesis of the human myotonic dystrophy.     

Myotonic muscular dystrophy. Time-dependent alterations in erythrocyte membrane fluidity

The muscle cell membrane may be the site of the basic molecular defect in myotonic muscular dystrophy. Many laboratories, including our own, have suggested that this defect may also be manifested in membrane of extraneural tissue. In previous studies, we found that electron spin resonance results suggested an increased membrane fluidity in erythrocyte membranes that had aged two days in buffer, but we and others could find no such changes in fresh erythrocyte membranes. To investigate these findings further, the results of an initial study of the time course of the membrane fluidity changes in erythrocytes in myotonic muscular dystrophy are given in the present report. They suggested that increased membrane fluidity changes in erythrocytes in myotonic muscular dystrophy are given in the present report. They suggested that increased membrane fluidity in myotonic dystrophy is manifested after two days of in vivo ageing and confirm our original findings. These results are discussed in relation to possible effects of metabolic deprivation or of protein-lipid alterations in erythrocytes.     

Fatty acid activation and transfer in blood cells of patients with muscular dystrophy.

Two membrane-bound enzymes, concerned in repair of erythrocyte membranes, have been investigated in patients with muscular dystrophy. The activation of long-chain fatty acids is normal in erythrocytes from Duchenne patients, but increases two-fold in cells from myotonic dystrophy patients (congenital form). This alteration is not present in leucocytes. In all leucocytes tested palmitate was the preferred substrate while palmitoleate and linoleate were activated at a lower rate. In the erythrocytes the 3 fatty acids were activated at the same rate. Carnitine palmitoyltransferase was not significantly altered in erythrocytes of both groups of patients.     

Erythrocyte ghosts (Na++K+) ATPase activity in duchenne's dystrophy and myotonia

Summary In Duchenne muscular dystrophy the activity of (Na⁺+K⁺) ATPase in erythrocyte ghosts is reduced and its reaction to ouabain is paradoxical both in low sodium and high sodium systems. No such changes were seen in a case of Becker dystrophy, in limb-girdle dystrophy, and in neurogenic atrophy of muscles. In myotonic dystrophy and congenital myotonia the activity of ATPase and its inhibition by ouabain were depressed.The investigations were carried out as part of agreement with NIH, Bethesda, USA, No. 05-002-1     

Epiretinal membrane: a treatable cause of visual disability in myotonic dystrophy type 1

A wide range of ocular abnormalities have been documented to occur in patients with myotonic dystrophy type 1. The objectives of this study were to investigate the macular and optic nerve morphology using optical coherence tomography in patients with myotonic dystrophy type 1. A total of 30 myotonic dystrophy type 1 patients and 28 controls were recruited for participation. All participants underwent a thorough ophthalmologic examination, including spectral-domain optical coherence tomography of the macula and retinal nerve fibre layer. Images were reviewed by a retinal specialist ophthalmologist, masked to the diagnosis of the participants. Average macular thickness was significantly greater in the myotonic dystrophy group compared to controls [327.3 μm vs. 308.5 μm (p < 0.001)]. Macular thickness was significantly greater (p < 0.005) in five of the nine macular regions. The increase in macular thickness was due to the increased prevalence of epiretinal membranes in the myotonic dystrophy patient group (p = 0.0002): 48.2 % of myotonic dystrophy patient eyes had evidence of epiretinal membrane, compared with 12.5 % of control eyes. Examination revealed that 56.7 % of myotonic dystrophy patients had an epiretinal membrane in at least one eye. Visual acuity was reduced due to the presence of epiretinal membrane in six patient eyes and none of the control eyes. The presence of an epiretinal membrane was significantly correlated with increasing age in the patient group. We report an increased prevalence of epiretinal membrane in the myotonic dystrophy type 1 group. This may be a previously under-recognised form of visual impairment in this group. Epiretinal membranes can be treated surgically. We suggest that, in addition to a comprehensive clinical examination, optical coherence tomography examination is implemented as part of an ophthalmological assessment for the myotonic dystrophy type 1 patient with reduced visual acuity.     

In vivo synthesis of phosphatidylcholine in rat brain via the phospholipid methylation pathway

We examined the in vivo synthesis of brain phosphatidylcholine (PC) by the methylation of phosphatidylethanolamine (PE). [3H-methyl]methionine was infused i.c.v., by indwelling cannula, and brain samples were taken 0.5-18 h thereafter and assayed for [3H]PC, as well as for its biosynthetic intermediates [3H]phosphatidylmonomethylethanolamine ([3H]PMME) and [3H]phosphatidyldimethylethanolamine ([3H]PDME), and for [3H]lysophosphatidylcholine ([3H]LPC) and S-[3H]adenosylmethionine ([3H]SAM). Most of the [3H]PC (79-94%) was present ipsilateral to the infusion site, indicating that the radioactivity in the [3H]PC was primarily of intracerebral origin, and not taken up from the blood. Moreover, only very low levels of [3H]PC were attained in brains of animals receiving [3H]methionine i.p. and these levels were symmetrically distributed. [3H]PMME and [3H]PDME turned over with apparent half-lives of 2.2 h and 2.4 h. In contrast, the accumulation of brain [3H]PC was biphasic, suggesting the existence of two pools, the more labile of which turned over rapidly (t1/2 = 5 h) and was formed for as long as [3H]PMME and [3H]PDME are present in the brain, and another, which was distinguishable only at 18 h after the [3H]methionine infusion. (The latter pool may have been synthesized from [3H]choline that was released via the hydrolysis of some of the brain [3H]PC previously formed by the methylation of PE.) Subcellular fractionation of brain tissue obtained after in vivo labelling with [3H]methionine revealed that mitochondrial PC had the highest specific radioactivity (dpm per mumol total lipid phosphorus), and myelin the least. These observations affirm that rat brain does synthesize PC in vivo by methylating PE, and the technique provides an experimental system which may be useful for examining the physiological regulation of this process.     

Platelet abnormalities in muscular dystrophy

Platelets which have complex membranes and calcium shifts similar to those in muscles were investigated in 14 patients with muscular dystrophy and 20 suitable controls. In 4 Duchenne and one limb-girdle dystrophy aggregations were done and found to be depressed with adrenaline and ADP. Electron microscopic and chemical examinations revealed an increased number of dense bodies, changed permeability and/or binding of cations and elevated intracellular calcium in all the 9 cases of Duchenne dystrophy while the 2 limb-girdle and 3 myotonic dystrophies varied. A two phase polymer separation system applied to fixed platelets of all patients and controls showed no abnormality of surface negative charge.     

Risk Factors for Cerebral Infarction in Duchenne Muscular Dystrophy: Review With our 2 Cases

Background: Although the incidence of cerebral infarction is higher in Duchenne muscular dystrophy (.75 per 100) than in the general population (7.5-11.4 per 100 000), only 18 cases have been reported, and prevention and management guidelines for infarction in this disorder remain lacking. Patients and Methods: We encountered 2 cases of Duchenne muscular dystrophy with cerebral infarction. To clarify risk factors for such infarction in Duchenne muscular dystrophy, we reviewed 20 cases, including our 2 patients. Results: Age at onset of infarction ranged from 4 to 31 years (n = 19). Most patients were 16-21 years old (14 of 19; 73.7%). Eighteen patients (90%) had dilated cardiomyopathy (DCM), showing a higher frequency than in the age-matched general Duchenne muscular dystrophy population. Left ventricular ejection fraction (LVEF) ranged from 10.2% to 42% (median, 20%; n = 9). Detectable cardiac thrombus and atrial fibrillation were rare (2 of 17; 11.8%, and 1 of 17; 5.9%, respectively). Conclusions: Presence of DCM with low LVEF seems to be the strongest risk factor for cerebral infarction in Duchenne muscular dystrophy.     

Third case of Duchenne muscular dystrophy and West syndrome: Expanding the spectrum of the DMD neuropsychiatric phenotype

Duchenne muscular dystrophy is an X-Linked neuromuscular disorder, and the most common muscular dystrophy. Neuropsychiatric phenotype associated to DMD gene mutations include now low IQ scores, epilepsy, autism, and attention deficit disorder. These have been observed with higher frequency in mutations that disrupt the short isoforms Dp71 and Dp140. West syndrome has been previously reported in two unrelated patients with Duchenne muscular dystrophy. Here, we report the third patient with West syndrome who had a novel hemizygous nonsense pathogenic variant in the exon 8 of the DMD gene c.811C>T, p.(Gln271*), suggesting West syndrome as part of the neuropsychiatric spectrum in Duchenne muscular dystrophy.     

Evidence against a generalized membrane defect in dystrophic mice platelets

The response of the membrane-bound enzyme AChE to changes in temperatures was investigated to test the applicability of the “generalized membrane defect” hypothesis proposed for human myotonic and Duchenne muscular dystrophies to the two forms of muscular dystrophy expressed in mice. For intact platelets from homozygous normal and dystrophic mice of both strains, a break (Tc) occurred in the Arrhenius plot of AChE activity at approximately 22 C. Solubilization of membrane-bound AChE by Triton X-100 produced a nonlinear Arrhenius plot over the temperature range (7.7 C to 37 C) in normal and dystrophic mice of both strains. However, in the presence of phospholipase A₂ + C and Triton X-100, a linear Arrhenius plot was produced indicating that the membrane-bound enzyme is normally modulated by a bulk lipid domain as well as by a tightly bound (immobilized) phospholipid domain. The temperature response of platelet AChE from normal and dystrophic mice of both strains was not significantly different. These results showing normal temperature kinetics of AChE do not lend support to the theory of a membrane defect in the platelets of dystrophic mice.     

Carbohydrate composition of erythrocyte membranes and glycosidase activities in serum in patients with myotonic dystrophy, limb-girdle dystrophy and congenital myotonia

A number of abnormalities in cell membrane function, including cells other than muscle cells, have been described in patients with inherited muscular diseases such as myotonic dystrophy and congenital myotonia. The basic molecular defects are, however, still unknown. The complex carbohydrates of membrane-bound glycoconjugates are of vital importance for the normal performance of the cell membrane. In this study the concentrations of the three major carbohydrates (sialic acid, galactose and hexosamines) of the erythrocyte membrane were therefore determined in patients with myotonic dystrophy, limb-girdle dystrophy and congenital myotonia. The activities of relevant glycosidases in serum were also assayed. In each of the three diseases pertinent changes of the carbohydrate pattern were found. In patients with myotonic dystrophy the sialic acid and in patients with limb-girdle dystrophy the hexosamine concentration was significantly reduced (P less than 0.0005). The sialic acid, galactose and hexosamine concentrations were all significantly increased in patients with congenital myotonia. No increase of the neuraminidase (sialidase) activity was found in sera from patients with myotonic dystrophy. In patients with limb-girdle dystrophy, the activities of serum hexosaminidases were normal. These results support the contention that certain inherited muscular diseases may represent generalized membrane disorders, and suggests that disturbances of membrane-bound glycoproteins and/or glycolipids might be of importance in the pathogenesis of some of these disorders.     

Freeze fracture studies of muscle plasma membrane in human muscular dystrophy

Summary Freeze fracture analysis of intramembranous particle density in skeletal muscle plasma membrane from 7 patients with Duchenne muscular dystrophy (DMD), 5 patients with facioscapulohumeral muscular dystrophy (FSH) and 5 patients with myotonic dystrophy (MyD) were carried out. Marked deplction of intramembranous particles including orthogonal arrays were significantly decreased in FSH. No abnormalities were noted in MyD.This work was supported by a Research Center Grant from the Muscular Dystrophy Association of America and by grants NS-08075, NS-14471 and 5 M01 RR00040 from the U.S. Public Health Service     

Erythrocyte shape in Duchenne muscular dystrophy.

In a controlled and blind study we were not able to identify any abnormality of erythrocytes of eight patients with Duchenne muscular dystrophy compared with seven approximately age-matched unaffected siblings (six males, one female step-sibling). Erythrocyte morphology was found to be very sensitive to various types of cell treatment. At this time, erythrocyte morphology, evaluated by the techniques used, should not be considered an established diagnostic test for the Duchenne muscular dystrophy patient or carrier.     

Computer method for the analysis of evoked motor unit potentials. 2. Duchenne, limb-girdle, facioscapulohumeral and myotonic muscular dystrophies.

Single motor unit potentials recorded from surface electrodes over the extensor digitorum brevis muscle and evoked by stimulation of the anterior tibial nerve at the ankle were obtained by a computer subtraction method. Their latencies, durations, amplitudes, and areas were measured in control subjects and patients with Duchenne, limb-girdle, facioscapulohumeral, and myotonic muscular dystrophy. Lateral popliteal motor nerve conduction velocities were also recorded. In the muscular dystrophies there was a significant increase in both the latencies and durations of motor unit potentials, the latter in notable contrast with the findings of conventional needle electromyography. Fastest motor conduction velocities were significantly reduced in the limb-girdle, facioscapulohumeral, and myotonic muscular dystrophy patients, while the shortest distal motor latencies were significantly prolonged in these patients and those with Duchenne muscular dystrophy. The results support the presence of a definitive neurogenic influence in the muscular dystrophies.     

Activity limitations in patients with neuromuscular disorders: A responsiveness study of the ACTIVLIM questionnaire

Recently, a self-reported scale of activity limitations, the ACTIVLIM questionnaire, was developed and validated in patients with neuromuscular disorders (NMD). The purpose of this study was to investigate its sensitivity to change. One hundred thirty-two patients with NMD (mean age, range: 31, 6–80) were assessed twice, with 21 ± 4 months in between, using the ACTIVLIM questionnaire. Mean score change, effect size, standardized response, mean paired t-test and an individual-level statistical approach were calculated for groups of patients according to their self-rated functional status evolution and for three main diagnostic groups (Ambulant and wheelchair-bound Duchenne muscular dystrophy patients, myotonic dystrophy patients, and patients with Charcot–Marie–Tooth neuropathy). The responsiveness indices showed that the change in activity measures was higher in patients who reported deteriorated functional status and in patients with Duchenne muscular dystrophy. The ACTIVLIM questionnaire showed a good sensitivity to change and could be useful in research settings to characterize the disease course of NMD.     

Na+ + K+] Mg2+-ATPase of muscle plasma membranes in Duchenne muscular dystrophy

The activity of [Na+ + K+] Mg2+-ATPase of muscle surface membrane was investigated in 20 cases of the Duchenne type of progressive muscular dystrophy; it was found to be diminished and to have a changed reactivity to ouabain. There was nothing like it in cases of limb-girdle dystrophy and neurogenic muscular atrophies investigated for the purpose of comparisons, whereas in some cases of myotonic dystrophy and myotonia congenita the activity of the ATPase was indeed depressed, but the response to ouabain invariably remained normal.