Xp21 dystrophin and 6q dystrophin-related protein. Comparative immunolocalization using multiple antibodies.

A protein of Mr 400 K and slightly lower Mr than Xp21 dystrophin was detected in skeletal muscle from patients with Duchenne muscular dystrophy by three antibodies raised against the midrod and C-terminal portions of chicken dystrophin, and by antibodies to dystrophin-related protein. Immunocytochemistry showed continuous sarcolemmal staining of Duchenne muscle with these antibodies. Subcellular localization to the inner face of the plasma membrane of Duchenne muscle was demonstrated by immunoelectron microscopy using the model of a Duchenne patient deleted for most of the dystrophin gene. Other antibodies were specific for Xp21 dystrophin. In conclusion, a dystrophin homologue that may be identical to the previously described dystrophin-related protein (DRP)1 is expressed in Duchenne muscle with intracellular distribution similar to Xp21 dystrophin in normal muscle.

A homologue of dystrophin is expressed at the neuromuscular junctions of normal individuals and DMD patients, and of normal and mdx mice. Immunological evidence.

Polyclonal and monoclonal antibodies, which recognize different regions and epitopes of the dystrophin molecule, bind to a protein of Mr 400,000 which is present in extracts of mdx muscle from regions which contain neuromuscular junctions (NMJ) and is absent from those which do not. This NMJ-associated homologue of dystrophin has at least 2 epitopes which are different to usual Xp21 form of dystrophin expressed along the sarcolemma of muscle fibres in normal muscles. This protein is also expressed at the NMJ of a DMD patient who lacks the first 52 exons of the Xp21 dystrophin gene and it must therefore be translated from a different gene transcript.

Assay of serum cardiac myosin heavy chain fragments in patients with acute myocardial infarction: determination of infarct size and long-term follow-up.

To evaluate the correlation between myosin heavy chain release and the necrosis mass, serum levels of myosin heavy chain fragments were determined serially in 55 patients with acute myocardial infarction. Eight of these patients were successfully treated with thrombolytic agents: the others were not treated. The same myosin titration was applied to the sera of 25 dogs with an experimental myocardial infarction. Six of the dogs were successfully treated with thrombolytic agents. The time courses of the myosin concentrations are typical and monophasic for all patients with a noncomplex myocardial infarction. The values for the kinetic parameters of myosin release are comparable to those previously reported. We have now determined that cumulative myosin release significantly correlates with cumulative creatine kinase (CK), CK-MB, and lactate dehydrogenase release, as well as with thallium-201 distribution, as determined for different patient groups. Thrombolytic treatment does not seem to qualitatively upset myosin kinetics. The results obtained in dogs with or without thrombolysis conclusively indicate that myosin release is a quantitative index of the necrosis mass. From a practical point of view, a few serial determinations of serum levels of myosin heavy chains are enough to estimate the necrosed mass in patients with acute myocardial infarction. More generally, serum myosin titration could be useful in detecting any cardiac disturbance involving myocardial injury resulting in membrane leakage of cardiac cells.

Distribution pattern of alpha and beta myosin in normal and diseased human ventricular myocardium.

All fibers in three normal, four dilated, and two ischemic human ventricles were classified according to their myosin content using three sets of monoclonal antibodies each specific for one myosin heavy chain isoform (alpha, beta and beta'). Numerous fibers contained only beta myosin heavy chain (denoted as beta fibers), others contained either alpha and beta, or beta and beta' myosin heavy chain (denoted as alpha beta and beta beta' fibers, respectively). The percentages of alpha beta fibers were systematically determined along the walls of seven homologous regions of the ventricular myocardium. In all ventricles, there was an alpha beta-fiber transmural gradient, with less alpha beta fiber in the subendocardium than in the subepicardium. More alpha beta fibers were found in the right than in the left ventricular wall but there was no difference between the mid-portion and the apex of the free wall of each ventricle. The diseased ventricles contained a lower alpha beta fiber percentage than the normal hearts. beta beta' fibers were very rare in the normal ventricles (less than 5%) and almost inexistent in pathological hearts. The correlation between the mean alpha beta fiber percentages of the diseased hearts and their cardiac indices (r = 0.88, P less than 0.05) suggests that the small amount of alpha myosin distributed in a large number of ventricular fibers could play a role in the contractile performance of the heart. In conclusion, this study provides evidence for 1) an alpha beta fiber transmural gradient, and 2) a lower alpha myosin ratio in diseased than in normal human ventricle.

[Evaluation of the efficacy of intravenous fibrinolytic treatment in the acute phase of myocardial infarction. Value of the determination of human plasma cardiac myosin]. / Mesure de l'efficacité d'un traitement fibrinolytique par voie intraveineuse à la phase aiguë de l'infarctus du myocarde. Apport du dosage de la myosine cardiaque humaine plasmatique.

Following acute myocardial infarction, fragments of human cardiac myosin can be detected in plasma by means of monoclonal antibodies to myosin heavy chains from the left human ventricle. The cumulative amounts of myosin released during the first 9 post-infarction days are proportional to the size of the infarct (Tao Ming et al., in the press). The purpose of our study was to evaluate the effectiveness of a fibrinolytic treatment administered in the acute phase of myocardial infarction by measuring in the plasma, the circulating fragments of human cardiac myosin. Three groups of patients with acute myocardial infarction were investigated: 13 patients (group A) received a conventional treatment; 8 patients (group B) were treated with intravenous streptokinase without success, i.e. with persistence of the coronary occlusion; 9 patients (group C) were successfully treated with intravenous streptokinase, resulting in recanalization of the coronary artery. We found that the cumulative amount of myosin released during the first 9 post-infarction days was significantly lower in patients successfully treated with streptokinase [group C: (3.8 +/- 2.3) 10(3) ng/ml/day]. There was no difference in cumulative release of myosin between control patients [group A: (7.0 +/- 3.3) 10(3) ng/ml/day] and patients with unsuccessful fibrinolytic treatment [group B: (10.0 +/- 4.1) 10(3) ng/ml/day]. These results were unrelated to the localisation of the infarct. It is concluded that measuring the cumulative amounts of myosin released is a means of evaluating the effectiveness of fibrinolytic therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of alpha- and beta-myosin heavy chains in the ventricular fibers of the postnatal developing rat.

Four monoclonal antibodies, two raised against alpha-myosin heavy chain (MHC) and two against beta-MHC, have been used to investigate in situ the fiber distribution of alpha- and beta-MHC in rat cardiac ventricles during postnatal development. Eighteen ventricles from 2-day-old to 1-year-old rats were analyzed. Three fiber populations were determined according to their immunofluorescent labeling: one with only alpha-MHC, one only beta-MHC, and one with mixed alpha- and beta-MHC. Large variations in the proportions of these three fiber populations according to age indicate that: (1) alpha-MHC are expressed in all fibers until the second month; they then disappear in a small endocardial fiber population and in a few apparently conductive fibers around the vessels. (2) beta-MHC are also first expressed in all fibers and then disappear gradually from epicardium to endocardium between the second and fourth weeks, except in the conductive fibers; they reappear during the second month sequentially from endocardium to epicardium; and they are then expressed in almost all fibers, except in a small epicardial fiber population, proportionally larger in the right ventricle than in the left. Immunological characterization of MHC isolated from a 22-day-old-rat ventricle, using anti-beta immunoaffinity chromatography, suggests that MHC of conductive fibers are probably at least partially in an alpha beta heterodimeric form.

Immunocytochemical analysis of myosin heavy chains in human fetal skeletal muscles.

Quadriceps muscle samples from human fetuses (10 weeks of gestation to term) were studied using immunocytochemical methods with monoclonal antibodies against fetal, adult-slow and adult-fast B myosin heavy chains. The monoclonal antibodies were selected for their virtually exclusive specificity for a particular isomyosin and used to investigate the expression of different myosin heavy chains during fetal development of muscle fibres. Concomitant studies of the myofibrillary ATPase pattern of muscle fibres were carried out. A fetal-specific myosin was persistently expressed during fetal life but at a continuously decreasing rate. Adult-slow myosin was observed in a small pool of muscle fibres, histochemically undifferentiated, in fetuses of 14-16 weeks of gestation. However, adult isomyosins appeared intensively only in the late fetal period, progressively replacing fetal myosin. The genes coding for adult-slow myosin are expressed earlier that those coding for adult-fast myosin. Myosin heavy chains specific for the neonatal period were not demonstrated with the antibodies used in this study. The contribution, provided by the present study, to the knowledge of the sequence of events in the expression of myosin heavy chains during normal muscle development, may allow a better understanding of eventual myosin changes which may occur in genetic muscle disorders.

Levels of ventricular myosin fragments in human sera after myocardial infarction, determined with monoclonal antibodies to myosin heavy chains.

Serum levels of ventricular myosin heavy chains were quantitated in patients with acute myocardial infarction using a competitive radioimmunoassay involving monoclonal antibodies to the b-type myosin heavy chains of a human ventricle. Among the seven antibodies selected for their higher affinity for ventricular myosin heavy chains, only four antibodies detected significant and variable myosin amounts in the serum samples of nineteen patients with acute myocardial infarction; the same antibodies occasionally detected, if at all, low myosin amounts in the sera of patients with no clinical sign of myocardial infarction, and no myosin in the serum of the healthy control subjects. The peak levels of myosin release were observed 4.6 +/- 0.5 days (n = 13, P less than 0.01) after myocardial infarction and correlated rather well with the measured creatine kinase peak levels (the correlation coefficients were between 0.75 and 0.81, P less than 0.01, depending on the monoclonal antibody used for myosin determination). The time courses of myosin release varied according to the complexity of the heart attack observed. It was concluded that the titration of serum myosin was probably of little clinical value for therapeutic intervention during the acute phase of myocardial infarction; it could, however be an effective tool for retroactive detection of an infarct and for late estimation of infarct size.