BAM 10 (4), 2000

Table of Contents

Articles       151        Calmodulin kinase-mediated phosphorylation of phospholamban in skeletal muscle sarcoplasmic reticulum.
                                  A critical reappraisal of the  state of the problem at the light of new findings with human normal and diseased muscle
                                  A Margreth, A Pallanca, and E Damiani    [Full text pdf 907Kb]

                    159        Cell death in cultured adult rat cardiomyocytes: use of the comet assay to distinguish apoptosis from necrosis
                                  M Trevor Page, PJE Quintana, JA Ligutti, and RA Sabbadini   [Full text pdf 232Kb]

                    171        A multicenter double-blind randomized trial of deflazacort versus prednisone in duchenne muscular dystrophy:
                                  analysis after 2 years
                                  MD Bonifati, G Ruzza, P Bonometto, A Berardinelli, K Gorni, S Orcesi, G Lanzi, M Ermani, and C Angelini    
                                  [Full text pdf 53.6Kb]

                    177        Altered lactate kinetics from exercising muscle in Hereditary Spastic Paraplegia
                                  G Siciliano, L Pasquali, ML Manca, E Pastorini, C Patrono, FM Santorelli, C Casali, and L Murri   
                                  [Full text pdf 33.3Kb]

                    181        Suppression of motor innervation induces fiber diversity in rat soleus muscle
                                  L Stevens, T Toursel, AM Lenfant, M Falempin, and Y Mounier  [Full text pdf 272Kb] 
  

                    191        Caveolae in the muscle overworked in an extended position
                                  A Jakubiec-Puka, and D Biral    [Full text pdf 471Kb]

                    197        Transforming growth factor-b2 in muscle fibers with rimmed vacuoles
                                  N Murakami, K Koishi, I Nonaka, and IS McLennan    [Full text pdf 525Kb]

                    201        Are we making progress in defining the role and regulation of myogenic satellite cells?
                                  MV Dodson     [Full text pdf 13.4Kb]
 
 
 
 
 

Calmodulin Kinase-Mediated Phosphorylation of Phospholamban in Skeletal Muscle Sarcoplasmic Reticulum. A Critical Reappraisal of the State of the Problem at the Light of New Findings with Human Normal and Diseased Muscle

Alfredo Margreth, Alessandra Pallanca, and Ernesto Damiani

NRC Unit for Muscle Biology and Physiopathology, Department of Experimental Biomedical Sciences, University of Padova, Padova, Italy

The Ca²⁺-transport system of the sarcoplasmic reticulum (SR) of mammalian hind-limb slow-twitch muscles has unique regulatory features, that have long been attributed to the special differentiating influence from the particular frequency and pattern of discharge of the innervating a motorneurons in the anterior horns of the spinal cord. This review summarizes the molecular mechanisms underlying such specific effects, focusing on phosphorylation of phospholamban (PLB) and of SR Ca²⁺-ATPase by calmodulin kinase II (CaM K II). Regarding specifically the hypothesis according to which the neural control over the expression of fiber-type specific gene products in skeletal muscle SR encompasses the expression of PLB gene, there is divergent evidence, when the problem is examined in a wide range of mammalian species, from small rodent species to the human species. The discrepancy is underlined by observations, that the protein level of expression of PLB is virtually zero in rat slow-twitch muscle, and that it increases with animal body size; and with a consequent lack of correlation with the total amount of slow-twitch muscle Ca²⁺-ATPase isoform found to be relatively immutable across different mammalian species. The experimental evidence appearing to be in conflict with the paradigma, is particularly striking in the case of human skeletal muscle having a mixed fiber composition. Most interestingly, transitions of Ca²⁺-ATPase isoforms in human skeletal muscle fibers, under pathohological conditions leading to the appearance of intermediate fibers, were found not to be accompanied by down-regulation either of PLB or of CaM K II, suggesting that in such muscle expression of PLB and of the fast-twitch Ca²⁺-ATPase isoform may be not mutually exclusive, or not in the absolute sense predicted by theory. The outstanding property that seems to link human skeletal and cardiac SR together, is the high PLB/Ca²⁺-ATPase ratio. A common property with rabbit slow-twitch muscle SR appears to be the presence of a highly active, PLB-dedicated form of CaM K II. The postulated, complex interplay beween intracellular Ca²⁺ fluxes and the activity state of CaM K II, while adding interesting new facets to the regulatory features of SR Ca²⁺-transport, inevitably invites to a number of questions, regarding the exact correlation between such mechanisms and the E-C characteristics and twitch properties of the muscle, depending also on the animal species.

Key words: Ca²⁺-calmodulin dependent protein kinase II, skeletal muscle, sarcoplasmic reticulum, sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA), phospholamban.

Basic Appl Myol 10 (4): 151-157, 2000

Address correspondence to:

Prof. A. Margreth, Department of Experimental Biomedical Sciences, University of Padova, viale G. Colombo 3, 35121 Padova, Italy, phone 049 8276037, fax 049 8276040, Email margreth@civ.bio.unipd.it.

Cell Death in Cultured Adult Rat Cardiomyocytes: Use of the Comet Assay to Distinguish Apoptosis from Necrosis

M. Trevor Page⁽¹⁾, Penelope J.E. Quintana⁽²⁾, Joseph A. Ligutti⁽¹⁾, and Roger A. Sabbadini⁽¹⁾

(1) Department of Biology and (2) Graduate School of Public Health,San Diego State University

Apoptosis is a form of programmed cell death that has only recently been implicated in the pathogenesis of cardiac diseases, including ischemia and reperfusion damage and in response to cytokines and other apoptotic triggers. Methods to study apoptosis range from measures of early changes in morphology to late events such as DNA laddering. In studying apoptosis in the heart, the application of some of these techniques is limited due to low sensitivity of detection and difficulties in quantification, especially when working with adherent cell cultures of cardiomyocytes. We present here results on the modification of the single cell gel electrophoresis or comet assay in order to assess apoptosis of adult cardiomyocytes in primary cell cultures. The comet assay quantifies DNA fragmentation in individual cell nuclei embedded in agarose and then electrophoresed. We present data on TNFa and sphingosine-induced apoptosis in adult rat cardiomyocytes as assessed by the comet assay. By using a combination of the alkaline, neutral and subcellular versions of the comet assay, we were able to accurately differentiate apoptosis from necrosis in cardiomyocytes. We conclude that the comet assay is a sensitive and useful method for the detection of apoptosis in cardiomyocytes.

Key words: apoptosis, cardiomyocytes, comet assay, necrosis, sphingosine, TNFa.

Basic Appl Myol 10 (4): 159-170, 2000

Address correspondence to:

Roger A. Sabbadini, Ph.D., Department of Biology, San Diego State University, 5500 Campanile Dr., San Diego, CA 92182 4614, U.S.A., tel. (619) 594 6272, fax (619) 594 0626, Email rsabba@sunstroke.sdsu.edu.

A Multicenter Double-Blind Randomized Trial of Deflazacort Versus Prednisone in Duchenne Muscular Dystrophy: Analysis after 2 Years

Marco D. Bonifati, Giampietro Ruzza, Piero Bonometto, Angela Berardinelli⁽¹⁾, Kseniya Gorni⁽¹⁾, Simona Orcesi⁽¹⁾, Giovanni Lanzi⁽¹⁾, Mario Ermani, and Corrado Angelini

Neuromuscular Center, Department of Neurology, University of Padova, Padova, and (1) Department of Child Neurology, Istituto Mondino, University of Pavia,Pavia, Italy

We have conducted a double-blind, randomized, multicentric trial in 18 Duchenne muscular dystrophy (DMD) boys, whose age ranged from 5.2 to 14.6 years (mean 7.3 yrs) for treatment with either deflazacort (0.9 mg/kg/day) or prednisone (0.75 mg/kg/day). To reduce side effects after one year the treatment schedule was switched to an alternate day regimen. The two groups were randomized and stratified on the basis of age and functional score at the onset of treatment.We followed the patients every 3 months in the first year and then every four months, evaluating MRC scale in four limb muscles, two in the right upper limb (deltoid and triceps) and two in the right lower limb (ileopsoas and quadriceps femoris) and performance of four functions (walking for 10 meters, climbing stairs, Gowers’ manoeuvre, and rising from a chair). We evaluated the differences in MRC and functional score with respect to baseline. Statistical significance was calculated by the Mann-Whitney test. Side effects were monitored by a questionnaire and by routine blood examination (serum creatine kinase, glucose, ions, hematocrit and complete blood count) and weight and height were recorded at each visit. Change in body weight was evaluated with the student t-test. After 24 months there were 2 drop out patients in the deflazacort group and 3 in the prednisone group for loss of independent ambulation. The two steroids were equally effective improving motor function and functional performances in the first 6 months and then we observed a slow down in the course of the disease.At 9 months, the average weight increase respect to baseline value was 5% (2 Kg) in the deflazacort group and 18% in the prednisone group (p< 0.005) and after 24 months it was 19% in the deflazacort group and 41% in the prednisone group. Only 3 patients on deflazacort had an increase in body weight that exceeded 20% with respect to baseline, but all the remaining patients in the prednisone group had an increase in body weight of over 20%.Two fractures occurred in the deflazacort group. Bone formation and growth evaluated with X-ray of the left hand for bone age did not appear different in the two groups. Eye examination revealed a slight cataract in 3 patients in the deflazacort group and in 1 in the prednisone group. Other minor and slight side effects such as behavioural changes, increased appetite and cushingoid appearance were observed in both groups.Steroid treatment with deflazacort appears to cause less side effects than prednisone, particularly on weight gain, which could be important to maximize motor performances and to avoid long term complications such as spinal deformity and respiratory fatigue.

Key words: Deflazacort, Prednisone, Duchenne Dystrophy, trial.

Basic Appl Myol 10 (4): 171-175, 2000

Address correspondence to:

Corrado Angelini, Neuromuscular Center, Department of Neurology, via Giustiniani 5, University of Padova, 35121 Padova, tel. 0039 049 8213610, fax 0039 049 8751770, Email cmusc@ux1.unipd.it.

Altered Lactate Kinetics from Exercising Muscle in Hereditary Spastic Paraplegia

Gabriele Siciliano, Livia Pasquali, Maria L Manca, Elena Pastorini, Clara Patrono⁽¹⁾, Filippo M Santorelli⁽¹⁾, Carlo Casali⁽¹⁾ and Luigi Murri

Department of Neurosciences, Neurological Clinics, University of Pisa, Pisa, and (1) Bambino Gesù Hospital, Roma, Italy

Hereditary spastic paraplegia (HSP) is a group of familial neurodegenerative disorders characterized by progressive lower limb spasticity and weakness due to degeneration of corticospinal axons. These disorders are classified both genetically, according to the mode of inheritance, and clinically, as pure and complicated forms. Recently the discovery that paraplegin, the defective protein in autosomal recessive HSP-SPG7, localizes in mitochondria allowed to foster the hypothesis that some mitochondrial dysfunctions can play a pathogenic role in HSP.The aim of our study was to indirectly evaluate oxidative metabolism in contracting muscle, by assessing the anaerobic lactate threshold in 7 patients (5 M and 2 F, mean age 48.0± 13.9 yrs) affected by HSP, both autosomal dominant or sporadic, during an incremental bicycle exercise.Analysis of venous lactate curve showed that lactate levels were significantly higher than in controls (peak normalised lactate: 378.8 vs. 271%, p<0.01). Furthermore, an early threshold of lactate was detected only in HSP patients.Even if other factors such as chronic spasticity or muscle deconditioning have to be taken into account in the interpretation of our data, these results suggest possible occurrence of mitochondrial involvement in skeletal muscle of HSP patients.

Key words: exercise test, lactate, mitochondria, paraplegin.

Basic Appl Myol 10 (4): 177-180, 2000

Address correspondence to:

Dr. Gabriele Siciliano, MD, PhD, Department of Neurosciences, Neurological Clinics, Via Roma 67, 56126 Pisa (I), tel. 39 050 993046/993334, fax 39 050 554808, Email gsicilia@neuro.med.unipi.it.

Suppression of Motor Innervation Induces Fiber Diversity in Rat Soleus Muscle

Laurence Stevens, Thierry Toursel, Anne Marie Lenfant, Maurice Falempin, and Yvonne Mounier

Laboratoire de Plasticité Neuromusculaire, Université des Sciences et Technologies de Lille, Villeneuve d’Ascq Cedex, France

Rat soleus muscle was submitted to 15 days of spinal cord ventral root section (deefferentation) in order to specifically disturb motor innervation. The electrophoretic analysis of the deefferented soleus muscle showed a reduction in myosin heavy chain (MHC) 1 (53% versus 92%) and new expressions in MHC 2D/X (12%) and MHC 2B (22%) relative concentrations, while MHC 2A composition did not evolve significantly. Single skinned fibers from control and deefferented soleus were identified according to their MHC composition and their functional properties (calcium/strontium activations and maximal shortening velocities) were established. Control soleus contained 72% of slow (S) fibers expressing MHC 1 and slow functional properties, and 28% of hybrid fast (HF) fibers, coexpressing MHC 1 with MHC 2A predominantly, and presenting fast functional properties. In deefferented soleus, four fiber types were discriminated: S and HF, and new fast types expressing MHC 2A, and/or MHC 2D/X plus MHC 2B. Moreover, deefferented soleus fibers were very much atrophied and presented alterations in Ca activation properties, the effects being more marked on the fast fibers. All the data support the use, on the rat, of deefferented muscle as a model for motoneuron disease.

Key words: atrophy, calcium and strontium activations, deefferentation, maximal shortening velocities, myosin heavy chains, myosin light chains, slow-to-fast transitions.

Basic Appl Myol 10 (4): 181-190, 2000

Address correspondence to:

Dr L. Stevens, Laboratoire de Plasticité Neuromusculaire, Bâtiment SN4, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d’Ascq Cedex, France, fax +33 (0)3 20 43 68 88, tel. +33 (0)3 20 33 70 86, Email Laurence.Stevens@univ-lille1.fr.

Caveolae in the Muscle Overworked in an Extended Position

Anna Jakubiec-Puka, and Donatella Biral⁽¹⁾

Dept. of Cellular Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland and (1) C.N.R. Unit for Muscle Biology and Physiopathology, University of Padova, Padova, Italy

The amount of caveolae increased in the rat leg muscles while electro-stimulated in extension for some hours. In muscles stimulated for the same period in neutral position, or held in extension without stimulation, the amount of caveolae was comparable to the control level; it increased only while the experiments lasted longer. Thus, eccentric contraction accelerates the formation of caveolae in muscle fibres.

Key words: caveolae, dystrophin, dystrophy, eccentric contraction, electro-stimulation, sarcolemma, skeletal muscle.

Basic Appl Myol 10 (4): 191-195, 2000

Address correspondence to:

Dr Anna Jakubiec-Puka, Dept. of Cellular Biochemistry, Nencki Inst. of Experimental Biology, Pasteura 3, 02-093 Warsaw, Poland, phone (48 22) 659 85 71, fax (48 22) 822 53 42.

Transforming Growth Factor-b2 in Muscle Fibers with Rimmed Vacuoles

Nobuyuki Murakami^(1,2), Kyoko Koishi⁽¹⁾, Ikuya Nonaka⁽²⁾, and Ian S. McLennan⁽¹⁾

(1) Department of Anatomy and Structural Biology, University of Otago, Dunedin, New Zealand, and (2) Department of Ultrastructural Research, National Center of Neurology and Psychiatry (NCNP), Kodaira, Tokyo, Japan

Transforming growth factor-b2 (TGF-b2) is associated with tau-containing neurofibrillar tangles in Alzheimer’s disease. We report here that the expressions of tau and TGF-b2 are highly correlated in the vacuolated muscle fibres in human and rat myopathies. This implicates TGF-b2 as a regulator of tau in muscle and neurons.

Key words: chloroquine, rimmed vacuole, skeletal muscle, tau, transforming growth factor-b2.

Basic Appl Myol 10 (4): 197-199, 2000

Address correspondence to:

Ian S. McLennan, Department of Anatomy and Structural Biology, University of Otago, PO. Box 913, Dunedin, New Zealand, tel. (64) 3 479 7364, fax (64) 3 479 7254, Email ian.mclennan@stonebow.otago.ac.nz.

Are We Making Progress in Defining the Role and Regulation of Myogenic Satellite Cells?

Michael V. Dodson

Muscle Growth Biology Laboratory, Department of Animal Sciences, Washington State University, Pullman, USA

A more concerted effort is required in order to expedite our progress in determining the role and regulation of myogenic satellite cells, during muscle development, following some trauma (and hence repair) to a muscle, or during the aging process. Scientists 1) using divergent animal models, 2) studying satellite cells at different levels (i.e. cellular, molecular or comparative), and 3) evaluating satellite cells in applied, clinical or exercise procedures should join together in order to make more efficient gains in knowledge about satellite cells.

Key words: satellite cells, research progress, collaborative linkages.

Basic Appl Myol 10 (4): 201-202, 2000

Address correspondence to:

Michael V. Dodson, Muscle Growth Biology Laboratory, Department of Animal Sciences, Washington State University, Pullman, WA 99164-6310 (USA).