Marcello Cantini

Extremely low frequency pulsed electromagnetic fields increase cell proliferation in lymphocytes from young and aged subjects.

The effect of the in vitro exposure to extremely low frequency pulsed electromagnetic fields (PEMFs) on the proliferation of human lymphocytes from 24 young and 24 old subjects was studied. The exposure to PEMFs during a 3-days culture period or during the first 24 hours was able to increase phytohaemagglutinin-induced lymphocyte proliferation in both groups. Such effect was greater in lymphocytes from old people which showed a markedly reduced proliferative capability and, after PEMF exposure, reached values of 3H-TdR incorporation similar to those of young subjects. The relevance of these data for the understanding and the reversibility of the proliferative defects in cells from aged subjects and for the assessment of risk related to the environmental exposure to PEMFs has to be considered.

Alteration in Calcium Handling at the Subcellular Level in mdx Myotubes

In this study, we have tested the hypothesis that augmented [Ca2+] in subcellular regions or organelles, which are known to play a key role in cell survival, is the missing link between Ca2+ homeostasis alterations and muscular degeneration associated with muscular dystrophy. To this end, different targeted chimeras of the Ca2+-sensitive photoprotein aequorin have been transiently expressed in subcellular compartments of skeletal myotubes of mdx mice, the animal model of Duchenne muscular dystrophy. Direct measurements of the [Ca2+] in the sarcoplasmic reticulum, [Ca2+] sr , show a higher steady state level at rest and a larger drop after KCl-induced depolarization inmdx compared with control myotubes. The peaks in [Ca2+] occurring in the mitochondrial matrix ofmdx myotubes are significantly larger than in controls upon KCl-induced depolarization or caffeine application. The augmented response of mitochondria precedes the alterations in the Ca2+ responses of the cytosol and of the cytoplasmic region beneath the membrane, which become significant only at a later stage of myotube differentiation. Taking into account the key role played by mitochondria Ca2+ handling in the control of cell death, our data suggest that mitochondria are potential targets of impaired Ca2+ homeostasis in muscular dystrophy.

Macrophage-released Factor Stimulates Selectively Myogenic Cells in Primary Muscle Culture

Myolibers arc reconstituted by the proliferation and fusion of muscle precursor cells when skeletal muscle is injured. One of the critical events is the peak accumulation of macrophages after 48 hours at the damage site before the satellite cell proliferation. In addition to their well-known role as a scavenger cell, there is now direct evidence of a mitogenic role of macrophages in regenerating muscle. We have utilized an in vitro model to directly investigate and prove that macrophages increase myoblast growth not only of satellite cells, but also of primary myoblasts. Rat muscle cells were cultured in the presence or absence of exudate macrophages obtained by peritoneal washing after thioglycollate broth injection. Macrophage cocullure increases several times the myoblasts/myotubes yield. This effect is particularly evident in muscle culture conditions in which fibroblast growth is predominant over myoblast proliferation, suggesting a myoblast selective mitogenic effect of macrophages. The results are confirmed by quantitative analyses of both DNA and skeletal muscle-specific-contractile proteins by gel electrophoresis and immunocytochemistry. Experiments with macrophage-conditioned media show this effect is mediated by soluble factors. This growth factor-like activity, which has been shown to be acid-stable and heatlabile, exerts its effects not only on specialized satellite cells during muscle regeneration, but also has a broader mitotic activity on all myogenic cells. In view of the role of muscle regeneration in muscle diseases and of the perspectives offered by gene therapy via myoblasts, we strongly believe that our results open new opportunities in removing many of the clinical constraints associated with repair and cell transplantation.

Extremely low frequency pulsed electromagnetic fields increase interleukin-2 (IL-2) utilization and IL-2 receptor expression in mitogen-stimulated human lymphocytes from old subjects

The effects of the exposure of mitogen‐stimulated human lymphocytes from aged subjects to low‐frequency pulsed electromagnetic fields (PEMFs) were studied by measuring the production of interleukin‐2 (IL‐2) and the expression of IL‐2 receptor. PEMF‐exposed cultures that presented increased [3H]thymidine incorporation showed lower amounts of IL‐2 in their supernatants, but higher percentages of IL‐2 receptor‐positive cells and of T‐activated lymphocytes. Taken together, these data suggest that PEMFs were able to modulate mitogen‐induced lymphocyte proliferation by provoking an increase in utilization of IL‐2, most likely acting on the expression of its receptor on the plasma membrane.

Macrophage-secreted myogenic factors: a promising tool for greatly enhancing the proliferative capacity of myoblasts in vitro and in vivo.

Abstract. In this work we set out to determine if the murine macrophage J774 cell line can be used to produce myogenic growth factors. Activated J774 macrophages were grown in serum-free conditions. The macrophage-conditioned medium (MCM) was then used to treat cultures of primary myoblasts and regenerating muscle tissue, in vitro and in vivo respectively. MCM activity in vitro was tested by analyzing the expression of muscle-specific transcription factors, in parallel with the proliferation and differentiation rates of the cells. The macrophage-secreted factors greatly enhanced the proliferative potential of both rat and human primary myoblasts and were found to be highly muscle-specific. In vivo, MCM administration markedly enhanced the regenerative processes in damaged muscles. The ability to produce large amounts of macrophage-secreted myogenic factor(s) in the absence of serum holds great promise for its biochemical characterization and successive application in therapeutic protocols, both for ex vivo gene therapy and for muscle repair.

Direct Monitoring of the Calcium Concentration in the Sarcoplasmic and Endoplasmic Reticulum of Skeletal Muscle Myotubes

Direct monitoring of the free Ca2+ concentration in the sarcoplasmic reticulum (SR) was carried out in rat skeletal myotubes transfected with a specifically targeted aequorin chimera (srAEQ). Myotubes were also transfected with a chimeric aequorin (erAEQ) that we have demonstrated previously is retained in the endoplasmic reticulum (ER). Immunolocalization analysis showed that although both recombinant proteins are distributed in an endomembrane network identifiable with immature SR, the erAEQ protein was retained also in the perinuclear membrane. The difficulty of measuring [Ca2+] in 100–1000 μm range was overcome with the use of the synthetic coelenterazine analogue, coelenterazine n. We demonstrate that the steady state levels of [Ca2+] measured with srAEQ is around 300 μm, whereas that measured with erAEQ is significantly lower, i.e. around 200 μm. The effects of caffeine, high KCl, and nicotinic receptor stimulation, in the presence or absence of external calcium or after blockade of the Ca-ATPase, were investigated with both chimeras. The kinetics of [Ca2+] changes revealed by the erAEQ were similar, but not identical, neither quantitatively nor qualitatively, to those monitored with the srAEQ, indicating that at this stage of muscle development, differences exist between SR and ER in their mechanisms of Ca2+ handling. The functional implications of these findings are discussed.

Gene transfer into satellite cell from regenerating muscle: Bupivacaine allows β-gal transfection and expression in vitro and in vivo

SummaryA large bulk of experimental evidence (15) suggests that myogenic cell transfer can be regarded as a promising therapeutic approach in the cure of inherited pathologies. In particular, it has been shown that primary myoblasts obtained from embryonic or neonatal muscles allows the recovery of the normal phenotype in defective muscle tissues. The utilization of this approach in clinical settings still bears heavy limitations. Apart from the legal and ethical difficulties, the use of muscles obtained from aborted fetus is challenged by a large risk of rejection, due to the incompatibility between donor and recipient. In this context based on the genetic alteration and reimplanting of the patient’s own satellite cells, appears an approach attractive. Myoblasts derived from satellite cells are the obligate candidates for experiments, but the production of sufficient cell numbers is a major problem. Local anesthetics [Bupivacaine (1-n-butyl-DL-piperidine-2-carboxylic acid-2, 6-dimethyl anilide hydrochloride) and related molecules] had been used to induce myofiber damage (and thus satellite cells proliferation) and thereby may represent a tool for increasing the yield of myoblasts from adult muscles (1,9,17). We will show that satellite cells obtained from adult muscles after bupivacaine injection can be transfected in vitro and that the transfected gene is expressed in vitro and in vivo, after reimplantation of the modified myoblasts in recipient muscles.

Enhancing Effect of Low Frequency Pulsed Electromagnetic Fields on Lectin-Induced Human Lymphocyte Proliferation

The effect on cell proliferation of continuous exposure of human peripheral lymphocytes from a total of 19 subjects (14 healthy donors, 1 patient with muscular dystrophy (M.D.) and his 4 siblings) to pulsed extremely-low-frequency, low-intensity electromagnetic fields (PEMFs) was studied. PEWS were not mitogenic by themselves, and a slightly reduced 3H-thymidine (3H-TdR) incorporation in unstimulated lymphocyte cultures was observed. In phytohemagglutinin-stimulated lymphocyte cultures, increased 3H-TdR incorporation was observed in all subjects. The increase was statistically significant at optimal and supraoptimal phytohemagglutinin doses. The subjects belonging to the M.D. family exhibited a PEMF-induced increase of 3H-TdR incorporation higher than that found in healthy donors. The variables that appeared to be important ere the characteristics of the field, the geometry of the microculture system, the dose of the mitogen employed, and the immune status of the lymphocyte donors. The hypothesis that PEW...

Targeting of calsequestrin to sarcoplasmic reticulum after deletions of its acidic carboxy terminus

Calsequestrin (CS) is the Ca(2+) binding protein of the junctional sarcoplasmic reticulum (jSR) lumen. Recently, a chimeric CS-HA1, obtained by adding the nine-amino-acid viral epitope hemagglutinin (HA1) to the COOH terminus of CS, was shown to be correctly segregated to the sarcoplasmic reticulum [A. Nori, K. A. Nadalini, A. Martini, R. Rizzuto, A. Villa, and P. Volpe. Am. J. Physiol. 272 (Cell Physiol. 41): C1420-C1428, 1997]. A putative targeting mechanism of CS to jSR implies electrostatic interactions between negative charges on CS and positive charges on intraluminal domains of jSR integral proteins, such as triadin and junctin. To test this hypothesis, 2 deletion mutants of chimeric CS were engineered: CS-HA1DeltaGlu-Asp, in which the 14 acidic residues [-Glu-(Asp)(5)-Glu-(Asp)(7)-] of the COOH-terminal tail were removed, and CS-HA1Delta49(COOH), in which the last, mostly acidic, 49 residues of the COOH terminus were removed. Both mutant cDNAs were transiently transfected in HeLa cells, myoblasts of rat skeletal muscle primary cultures, or regenerating soleus muscle fibers of adult rats. The expression and intracellular localization of CS-HA1 mutants were studied by epifluorescence microscopy with use of antibodies against CS or HA1. CS-HA1 mutants were shown to be expressed, sorted, and correctly segregated to jSR. Thus short or long deletions of the COOH-terminal acidic tail do not influence the targeting mechanism of CS.Calsequestrin (CS) is the Ca2+ binding protein of the junctional sarcoplasmic reticulum (jSR) lumen. Recently, a chimeric CS-HA1, obtained by adding the nine-amino-acid viral epitope hemagglutinin (HA1) to the COOH terminus of CS, was shown to be correctly segregated to the sarcoplasmic reticulum [A. Nori, K. A. Nadalini, A. Martini, R. Rizzuto, A. Villa, and P. Volpe. Am. J. Physiol. 272 ( Cell Physiol. 41): C1420-C1428, 1997]. A putative targeting mechanism of CS to jSR implies electrostatic interactions between negative charges on CS and positive charges on intraluminal domains of jSR integral proteins, such as triadin and junctin. To test this hypothesis, 2 deletion mutants of chimeric CS were engineered: CS-HA1ΔGlu-Asp, in which the 14 acidic residues [-Glu-(Asp)5-Glu-(Asp)7-] of the COOH-terminal tail were removed, and CS-HA1Δ49COOH, in which the last, mostly acidic, 49 residues of the COOH terminus were removed. Both mutant cDNAs were transiently transfected in HeLa cells, myoblasts of rat skeletal muscle primary cultures, or regenerating soleus muscle fibers of adult rats. The expression and intracellular localization of CS-HA1 mutants were studied by epifluorescence microscopy with use of antibodies against CS or HA1. CS-HA1 mutants were shown to be expressed, sorted, and correctly segregated to jSR. Thus short or long deletions of the COOH-terminal acidic tail do not influence the targeting mechanism of CS.

Dystrophin deficient myotubes undergo apoptosis in mouse primary muscle cell culture after DNA damage

Apoptosis has been demonstrated to occur in differentiated myocardial muscle, neonatal skeletal muscle and skeletal myoblasts in response to injury. In this report, we studied differentiated normal and dystrophin deficient murine skeletal muscle cell cultures that have been injured by a pulse of cis-platinum (2 h). Forty-eight hours after DNA damage, dystrophin positive myotubes appeared almost normal though some myoblasts showed DNA fragmentation. On the other hand, dystrophin deficient myotubes presented progressive degeneration via apoptosis detected either by TUNEL or by nuclear morphology. Degeneration of mdx muscle fibers was confirmed by counting both the number of myotubes observed by contrast phase microscopy and myonuclei viewed by immunoreaction for MyoD. A 6-fold decrease in the number of muscle cells was observed in the dystrophin-deficient cell culture compared to the parental culture (P < 0.001). Direct evidence of degenerating myotubes displaying MyoD- and TUNEL-positive nuclei was obtained. Like myoblasts, differentiated dystrophin deficient myotubes were able to degenerate via apoptosis, showing that mature dystrophin deficient cells are fragile and undergo apoptosis when subjected to a mild injury which would normally be repaired in parental cells.