REVIEW         115        High performance liquid chromatography in the analysis and separation of contractile proteins.
                                       L Dalla Libera      [Full text pdf 238Kb]

ARTICLES     119        Coexpression of Myosin Heavy Chain 2b with Myosin Heavy Chain 1- Fact or Artefact?
                                      I Erzen, J Sketelj, O Agbulut, D Angaut-Petit and GS Butler-Browne      [Full text pdf 1.7Mb]

                        127        The Effect of Therapeutic Electrical Stimulation in Children with Diplegic Cerebral Palsy as
                                      Measured by Gait Analysis
                                      S Liron-Keshet, E Tirosh, J Mizrahi, O Verbitsky, E Isakov, R Marmur and S Rabino
                                      [Full text pdf 141Kb]

                        133        Evaluation of the Torque Developed by the Elbow Flexors in Patients with Neuromuscular Diseases
                                      J Rozman, M Bunc and A Zupan      [Full text pdf 680Kb]

                        139        Latissimus Dorsi Vascular Delay Improves Muscle Function for Use in Cardiomyoplasty
                                      K Rossini, E Giardini, A Donà, A El Messlemani and F Mazzoleni      [Full text pdf 1.17Mb]

                        143        Cardiomyocyte Apoptosis Induced in Langendorff Preparation of Isolated Guinea-Pig Heart
                                      Perfused with Krebs-Henseleit Solution Deprived of Glucose, with and without Oxygen Supply
                                      J Tong, Y Cao, Y Hotta, N Ishikawa, T Takeo, H Nishimaki, K Masuda, C Long Yang, K Kasai,
                                      H Ikeda and G Itoh      [Full text pdf 2.58Mb]
 

High Performance Liquid Chromatography in the Analysis and Separation of Contractile Proteins

Luciano Dalla Libera

C.N.R. Institute of Neuroscience, Unit for Muscle Biology and Pathophysiology, Department of Biomedical Sciences University of Padova, Padova, Italy

High performance liquid chromatography (HPLC) utilizing a support of small rigid particles of uniform size has been shown to provide a rapid method for separation of many types of molecules of biological interest. Myosin, the major component of the thick filaments of muscles, is a large asymmetric molecule containing two heavy chains molecular weight about 200000) and two each of two different classes of light chains (molecular weight range 16000-27000). Since myosin light and heavy chains are currently used as a useful markers in studying both diseased and normal muscle tissues, it is interesting to apply HPLC to the study of these proteins. Using a 330-Å-pore C-18 column we were able to separate each other myosin light chains. Furthermore, the light chains were digested with S. aureus V8 protease and the fragments obtained were separated on a 100-Å-pore C-18 column. By this approach we were able to demonstrate differences in the peptide maps of light chains characterized either by high degree of homology or by the same apparent molecular weight and apparent isoelectric point. The use of the HPLC chromatographic column filled with hydroxylapatite made possible the purification of the whole myosin molecule (molecular weight about 500000) in non denaturing conditions from relatively complex mixtures.
Key words: HPLC, hydroxylapatite, myosin, peptide mapping.
 
 
 
 
 

Coexpression of Myosin Heavy Chain 2b with Myosin Heavy Chain 1- Fact or Artefact?

Ida Erzen, Janez Sketelj(1), Onnik Agbulut(2), Denise Angaut-Petit(3), and Gillian S. Butler-Browne(2)

Institute of Anatomy and (1) Institute of Pathophysiology, Medical Faculty, Ljubljana, Slovenia, (2) CNRS URA 2115, Faculté de Medécine, Paris, and (3) Laboratoire de Neurobiologie Cellulaire et Moléculaire, CNRS, Gif-sur-Yvette Cedex, France

In skeletal muscle, pure fibres expressing one myosin heavy chain (MyHC) isoform, and intermediate fibres, expressing two and exceptionally three MyHCs have been described. When skeletal muscle adapts its fibre type profile to changed functional demands MyHC isoform transformation follows the pathway: MyHC-1 * MyHC-2a * MyHC-2x/d * MyHC-2b. Therefore, in hybrid fibres only successive isoforms from the proposed pathway should coexist. However, jump fibres in which MyHC-1 is co-expressed with MyHC-2x/d have been described recently. The present study describes possible coexpression of MyHC-1 with MyHC-2b in transforming as well as in normal control mouse and rat muscle fibres. The study is only descriptive and provides not sufficient proof to exclude the possible artefact resulting from unknown technical reasons.
Key words: coexpression, mouse, myosin heavy chains, rat.
 
 
 
 
 

The Effect of Therapeutic Electrical Stimulation in Children with Diplegic Cerebral Palsy as Measured by Gait Analysis

Shiri Liron-Keshet, Emanuel Tirosh, Joseph Mizrahi(1), Oleg Verbitsky(1), Eli Isakov(2), Rachel Marmur(1) and Sheri Rabino

Institute for Child Development, Bnai-Zion Medical Center, Haifa, (1) Department of Biomedical Engineering, Technion, Israel Institute of Technology, Haifa, and (2) Kinesiology Laboratory, Loewenstein Hospital, Sackler School of Medicine, Tel Aviv University, Israel

The present work was designed as a controlled longitudinal study to test the hypothesis that therapeutic electrical stimulation (TES) to the lower limb muscles of children with cerebral palsy (CP) can improve the quality of gait of these children. Diplegic CP was selected for this study, so that one leg served as the study leg and the other as a control. Four diplegic CP children (mean age 7.7 ? 2 years) took part. TES was delivered to the quadriceps and dorsiflexors of the right legs in 20 minutes sessions, 4 times a week for a total period of six to ten weeks. Gait assessment was made on a 3 m walkway, fitted with parallel bars. Stride length, knee and ankle range and average stride velocity were measured and compared before and after TES treatment and between the stimulated and the unstimulated legs. Results indicated that although the effect of electrical stimulation varied from one subject to the other, an overall improvement in gait quality occurred as reflected by the positive changes in the following walking variables. Stride length and Stride velocity improved in three out of the four subjects, although some of the changes were not statistically significant. Range of knee motion generally improved in all four subjects. Also the ankle range of motion improved in three subjects. Of particular interest was the surprising lack of preferential effect of TES on the stimulated (Rt) leg as compared to the other (Lt) leg.
Key words: diplegic cerebral palsy, therapeutic electrical stimulation, gait analysis.
 
 
 
 
 

Evaluation of the Torque Developed by the Elbow Flexors in Patients with Neuromuscular Diseases

Janez Rozman, Matja Bunc(1) and Anton Zupan(2)

ITIS d. o. o. Ljubljana, Center for Implantable Technology and Sensors, (1) School of Medicine, Institute of Pathophysiology, and (2) Rehabilitation Institute, Ljubljana, Republic of Slovenia

In planning the optimum treatment for patients with neuromuscular diseases (NMD), it is essential to know as much as possible about their functional state. Assessment of the strength of certain muscles is the most direct measure of motor deficiency. In the development of normative data needed for patients with NMD, the use of torque measurements is required. Forty-nine patients (31 men and 18 women, mean age 33 ± 8,9) were included in the study. Five groups of patients, each having one of five different NMDs, were formed. We tested unilaterally the biceps brachii muscle that normally generates the highest torque. For this purpose an electronic brace enabling isometric measurements of torque during elbow flexion was designed. The patients produced 3 maximum voluntary elbow flexions that lasted about 3 s and separated by a pause of about 3 s. Force development was rapid with continuous build-up and isometric. About 15 s later the patients produced the last maximum voluntary elbow flexion, keeping it as stable as possible for a period of 30 s. Patients with mitochondrial myopathy (MM), having the longest mean time to maximum torque (1191.7 ms), elicited the highest mean torque in both short (1.34 Nm) as well as in 30 s-long maximum elbow flexions. Patients with facioscapulohumeral muscular dystrophy (MD-FSH), having a mean time to maximum torque (537.66 ms) about half as short, elicited the lowest mean torque in both the short (0.29 Nm) as well as in 30 s-long maximum elbow flexions. Patients with Becker muscular dystrophy (MD-B), having a mean time to maximum torque (1090.5 ms) about twice as long as patients with MD-FSH, elicited a higher mean torque in both short (0.82 Nm) and 30 s-long elbow flexions. Finally, patients with limb-girdle muscular dystrophy (MD-RM) and spinal muscular atrophy type 3 (SMA3), having a similar mean time to maximum torque (472.39 ms for patients with MD-RM and 505.92 ms for patients with SMA3), also elicited similar torque in both short (0.45 Nm for patients with MD-RM and 0.65 Nm for patients with SMA3) and 30 s-long maximum elbow flexions. The results of the study show that the methodology developed to quantitatively measure the torque developed by elbow flexions in patients with NMD enables the characteristics and natural course of NMD to be more objectively documented. Accordingly, the optimum treatment for patients with NMD could be restored.
Key words: elbow flexors, electronic brace, neuromuscular disease, torque.
 
 
 
 
 

Latissimus Dorsi Vascular Delay Improves Muscle Function for Use in Cardiomyoplasty

Katia Rossini, Elena Giardini(1), Andrea Donà, Abdul El Messlemani and Francesco Mazzoleni(1)

Department of Biomedical Science, and (1) Institute of Plastic Surgery, University of Padova, Italy

The aim of our study was to investigate the effectiveness of vascular delay of a LD muscle flap in an experimental rat model of Dynamic Cardiomyoplasty. We hypothesized that muscle function would be improved by a vascular delay procedure that increases distal muscle perfusion of the Latissimus Dorsi (LD) muscle. In the first group of 6 rats the right LD were subjected to a “vascular delay” procedure, the left LD were tenotomized and resutured in the shortened position it spontaneously attained to mimic transposition effects of cardiomyoplasty, which result in LD distal devascularization and decreased resting tension. After 7 days the LD muscle flaps were studied. In a second experimental group, the right LD muscles of 6 rats were subjected to a vascular delay procedure. One week later both right (vascular delay) and left (control) LD muscles were subjected to a tenotomy procedure. Seven days later the LD flaps were studied. In both group the LD muscles were excised and fixed in liquid nitrogen at resting length. Muscle damage was graded by histological morphometry (H-E) on distal, medial, proximal cryostat sections. Muscle damage in distal vascular delay LD was 9% ± 1.87 (SE), in distal tenotomized LD was 79% ± 9.97, and in distal muscle tenotomized after vascular delay was 41.67% ± 7.03. Muscle damage in medial vascular delay LD was 17% ± 7, in medial tenotomized LD was 30% ± 7.85, and in distal muscle tenotomized after vascular delay was 18.34% ± 4.59. Muscle damage in proximal vascular delay LD was 18%?4.63, in proximal tenotomized LD was 25.42% ± 7.4, and in proximal muscle tenotomized after vascular delay was 12.5% ± 2.81. In conclusion we found that “vascular delay” procedure of LD tenotomized and resutured in the shortened position, that mimics the Dynamic Cardiomyoplasty significantly reduces muscle damage and improves LD muscle flap perfusion and function particularly in the distal portion of muscle.
Key words: dynamic cardiomyoplasty, tenotomy, vascular delay.
 
 
 
 

Cardiomyocyte Apoptosis Induced in Langendorff Preparation of Isolated Guinea-Pig Heart Perfused with Krebs-Henseleit Solution Deprived of Glucose, with and without Oxygen Supply

Jie Tong, Yongtong Cao(1), Yoshihiro Hotta(2), Naohisa Ishikawa(2), Tomohiro Takeo, Haruaki Nishimaki, Kenji Masuda, Chao Long Yang, Kenji Kasai, Hiroshi Ikeda, and Gen Itoh

First Department of Pathology, (1) Department of Surgery, and (2) Department of Pharmacology, Second Aichi Medical University School of Medicine, Japan

Whether or not deprivation of glucose and oxygen can induce cardiomyocyte apoptosis was examined. Four groups of Langendorff preparations of guinea-pig hearts were perfused for 6 h with Krebs-Henseleit (K-H) solution deprived of glucose (group A), deprived of oxygen (group B), and deprived of both glucose and oxygen (group C), then examined for the mode of myocardial cell death. The control was perfused with K-H solution.
Results: In group A, myocardial cell death by apoptosis and necrosis was induced. In group C, the apoptotic lesion induced was exaggerated compared to that produced by glucose deprivation alone. In group A and C, decreases in intracellular ATP (ATPi) and intracellular acidosis were observed. In group B and the control, myocardial cells survived with spotted necrotic foci without myocardial-cell apoptosis. A mild decrease in ATPi was observed. However, intracellular pH did not shift to acidity (7.0-7.2).
Conclusion: Myocardial cell apoptosis was not induced by a 6-h stimulus of oxygen-deprivation (hypoxia) alone, but rather by a 6-h stimulus of glucose-deprivation with or without an oxygen supply. Deprivation of both glucose and oxygen intensified the apoptotic lesion. Apoptosis coincided with a decrease in the levels of ATPi and intracellular acidosis.
Key words: acidosis, apoptosis, ATP, deprivation of glucose and oxygen, Langendorff preparation, TUNEL.