Cell culture

Satellite cells from extensor digitorum longus (EDL) muscles were isolated by Drs. O.

Dorchies and S. Wagner in the laboratory of Professor P. Poindron (University of Strasbourg, France).

Myogenesis can be divided in 3 different parts (see introduction, 1.1.1):

- proliferation, which involve myoblasts (figure 2.1.A). These cells are small and mononucleated,

- differentiation, where confluent myoblasts align each other and fuse. The resulting cells are called myotubes (figure 2.1.B). They are multinucleated and can be hundreds of micrometers tall.

- fiber formation, where myotubes become mature and functional fibers (figure 2.1.C).

This maturation needs innervation.

Cultures of purified myoblasts were prepared in Petri dishes (Falcon, Becton Dickinson) and maintained at 37°C in a water-saturated atmosphere of 95% air / 5% CO2.

They were obtained as described previously (Pinset & Montarras, 1998) with minor modifications. Briefly, 3-week old mdx C57Bl/10 and control C57Bl/10 mice were killed by cervical dislocation, EDL muscle was aseptically removed bilaterally, cleaned of their tendinous ends and adhering connective tissue, and minced into small pieces of approximately 1 mm³. Individual muscles from 2 mice were pooled. After the muscle tissues were rinsed in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco), digestion was performed by 3 successive rounds of incubation in 10 ml of DMEM supplemented with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, pH 7.4), 10% fetal calf serum (FCS) (Invitrogen, Life Technologies), and 0.15% pronase (Roche), at 37°C. After the first incubation (5 min) the tissues were triturated with a serologic pipette and the supernatant, containing mainly blood cells and connective debris, was discarded. The second and third incubations lasted 20 min each with frequent triturations. The supernatants were collected, pooled, filtered through a 40 µm mesh cell strainer (Falcon, Becton Dickinson) and centrifuged at 300 x g for 10 min. The cell pellets were resuspended in growth medium (see below) and the suspensions plated onto collagen type I-coated (1 µg/cm²; Sigma) 100 mm

diameter Petri dishes, one dish per original muscle. Growth medium is composed of a 1:1 (v/v) mixture of DMEM and MCDB202 (CryoBioSystem), supplemented with 20% FCS, 2%

Ultroser^® SF (Biosepra SA), antibiotics (Ciproxin, Bayer) and NaHCO3 from concentrated solution (Gibco) to get 2.6 g/l in complete medium. Myoblasts were plated on coated dishes and grown for approximately 2 days in growth medium. For the experiments cells were induced to fuse by changing the growth medium to differentiation medium (DMEM supplemented with 1.7% FCS, 3.3% horse serum (HS, Sigma), 10 µg/ml insulin (Fluka) and Ciproxin).

(B) (C) (A)

Figure 2.1: (A) myoblasts, (B) myotubes and (C) fiber (isolated from flexor digitorus brevis muscle). Bar = 50 µm

2.2 Aequorin

2.2.1 Plasmids and amplification

The aequorin plasmids were gifts from Professor T. Pozzan, University of Padova, Italy. Cells were transfected with a pcDNAI expression vector containing a cDNA encoding aequorin for Ca²⁺ measurement, fused with the synaptosome-associated protein of 25 kDa (SNAP-25) to measure subsarcolemmal Ca²⁺ (pm[Ca²⁺]) (Brini et al., 1999), the human mitochondrial cytochrome c oxidase subunit VIII to measure mitochondrial Ca²⁺ (m[Ca²⁺]) (Robert et al., 2001) and calsequestrin to measure SR Ca²⁺ (sr[Ca²⁺]). Cytosolic aequorin is not tagged with any specific sequence. By this way the aequorin is localized in the cytosol (figure 2.2).

To amplify these pcDNAI-based plasmids which carry the supF suppressor tRNA gene, we used the MC1061/P3 bacteria (Invitrogen, Life Technologies). The P3 plasmid presents in these bacteria carries a wild-type kanamycin-resistance gene, amber mutant

versions of ampicillin and tetracyclin-resistance genes. Once transformed, the presence of the supF suppressor tRNA gene confers to the bacteria the ampicillin and tetracyclin resistance by the translational suppression of the defective genes. The plasmids were amplified as follows.

Competent bacteria MC1061/P3 were placed for 20 minutes in ice. Then bacteria were heat shocked at 37°C for 90 seconds in the presence of 100 ng of the plasmid. After a second incubation in ice for 10 minutes, transformed bacteria were grown in LB medium for 30 minutes at 37°C. Bacteria were seeded in a LB-agar plate containing 25 µg/ml ampicilin and 10 µg/ml tetracycline. After an overnight incubation at 37°C, resistant colonies of bacteria were isolated and further grown in 200 ml LB containing the same antibiotics for 16 hours.

Then, bacterial suspension was centrifuged and the plasmids were purified using the kit Nucleobond® AX500 (Machery-Nagel, Oensingen, CH). The purified plasmids were checked by enzyme digestion.

Figure 2.2: Plasmid map and aequorin targeting sequences.

Mitochondrial aequorin (mtAEQ) is targeted with the cytochrome oxidase subunit VIII (COX), subsarcolemmal aequorin (pmAEQ) with the SNAP-25 sequence and sarcolpasmic reticulum aequorin (srAEQ) with the calsequestrin sequence (CSQ) while cytosolic aequorin (cytAEQ) is not tagged.

2.2.2 Transient transfection

Control and mdx myoblasts were plated at 15,000 cells per cm² on 13 mm Thermanox coverslips (Nalge Nunc International) in 4-well plates. When 80 to 90 % confluent, growth medium was removed and replaced with a serum free medium, Optimem 1 (Gibco). Cells were transfected overnight using Lipofectamine 2000 (Invitrogen, Life Technologies) at a ratio of 1µg DNA per 2µl transfection reagent per well. The DNA-Lipofectamine 2000

complex was prepared in Optimem 1 medium. After overnight incubation this medium was replaced by differentiation medium. Myotubes were used after 3 or 4 days of differentiation.

2.2.3 Calcium measurement

After 3 or 4 days of differentiation, [Ca²⁺] was measured on a population of transfected myotubes as follow. The aequorins were reconstituted in a physiological salt solution (PSS, for composition see 2.8) containing 5 µM coelenterazine (Calbiochem) for at least 1 h before the experiment. Specific conditions are needed for the sarcoplasmic reticulum (SR) and subsarcolemmal targeted aequorin because the removal of intracellular Ca²⁺ is required for the complete aequorin reconstitution (Marsault et al., 1997). Indeed, [Ca²⁺] in these organelles is very high and removal of Ca²⁺ is essential in order to prevent the immediate reconstitution/degradation of aequorin. Thus, SR and subsarcolemmal aequorin was reconstituted in a Ca²⁺ free PSS containing 0.1 mM EGTA in order to decrease the [Ca²⁺] within the cell, while mitochondrial targeted aequorin was reconstituted in 1.2 mM Ca²⁺. Cells were superfused at a rate of 1 ml/min in a custom made 0.5 ml chamber thermostated at 37 °C (MecaTest, Geneva, Switzerland). Emitted luminescence was detected at 466 nm with a photomultiplier apparatus (EMI 9789A, Electron Tubes Limited, UK) and recorded every second using a computer photon-counting board (EMI C660).

Figure 2.3: Schematic picture of the system used for Ca²⁺ measurement

The relationship between recorded counts and [Ca²⁺] is shown in Equation 1 (Alvarez

& Montero, 2002):

[ ]

where L are the recorded photons/s and Lmax the remaining photons which correspond to the total light output during the wholeexperiment minus the photons emitted up to the measured point. KTR and KR are the parameters of the aequorin and n is the number of Ca²⁺ binding sites. For example, at 37°C, KTR= 120, KR= 1.01x10⁷ M^-1 and n= 2.99 for the wild type aequorin. Total light output was obtained by exposing cells to 10 mM CaCl2 after permeabilization with 100 µM digitonin to consume allthe aequorin.

Ca²⁺ concentrations were further analyzed using a software created by R. Pitarelli (Swiss Federal Institute of Technology, Lausanne) under the environment Matlab 7 SP1 (The MathWorks Inc.). Several parameters were determined (figure 2.4).

Figure 2.4: Summary of the analyzed parameters. (AUC: Area Under Curve)

( )

+

2 in = ^TR

Ca M ,

KR−(ratio K× R)

Equation 1

n

L ratio L

/ 1

⎜⎜ max

⎝

⎛ ⎟

⎠

= ⎞