Ca2+ levels in myotubes grown from the skeletal muscle of dystrophic (mdx) and normal mice.

1. Myotubes were grown in culture from normal (C57BL/ScSn) and mdx mice and the cytosolic [Ca2+] was monitored through development (5‐21 days in culture) using fura‐2 loaded via ionophoresis. Simultaneous measurements of the membrane potential and cytosolic [Ca2+] were made in normal and mdx myotubes before, during and after stimulation by action potentials elicited following anode break excitation. All experiments were undertaken at 22 degrees C. All data are expressed as means +/‐ S.E.M. 2. A new method was developed which enabled accurate determination of the fluorescence characteristics of fura‐2 in murine skeletal muscle fibres. In the under in vitro conditions by 14.60 +/‐ 0.05, 9.40 +/‐ 0.15 and 6.90 +/‐ 0.43% respectively. 3. The resting cytosolic [Ca2+] in the mdx myotubes was consistently higher than in the normal myotubes throughout the developmental period measured. Overall, the resting cytosolic [Ca2+] in mdx myotubes (134 +/‐ 9 nM, n = 22) was twofold higher than in normal myotubes (66 +/‐ 6 nM, n = 26). After stimulation (one to three action potentials) the cytosolic [Ca2+] of both mdx and normal myotubes remained elevated. The mdx myotubes (236 +/‐ 55 nM, n = 5) again had approximately double the cytosolic [Ca2+] of normal myotubes (109 +/‐ 19 nM, n = 9). 4. The time course and amplitude of the Ca2+ responses measured in the mdx and normal myotubes after action potential stimulation were variable. Two categories of Ca2+ response were observed in mdx and normal myotubes, the first consisted of a small, slow rise in [Ca2+] that remained elevated and the second consisted of a rapid (time to peak 7.4 +/‐ 1.5 ms) (n = 8) rise in [Ca2+] with amplitudes in the range 61‐773 nM and a [Ca2+] decay rate constant of 4.35 +/‐ 1.57 s‐1 (n = 8) (range 0.96‐15 s‐1). 5. In conclusion, the elevated cytosolic [Ca2+] reported here through development of cultured mdx myotubes suggests that this genetic disorder results in a defect which compromises the ability of the myotubes to strictly regulate cytosolic [Ca2+]. The results are consistent with the presence of functionally abnormal Ca2+ channels recently reported in mdx myotubes.

[1]  D. Jones,et al.  Muscle damage in mdx mice , 1991, Nature.

[2]  J. Huard,et al.  Dystrophin expression in myotubes formed by the fusion of normal and dystrophic myoblasts , 1991, Muscle & nerve.

[3]  H. Jockusch,et al.  Decreased osmotic stability of dystrophin-less muscle cells from the mdx mouse , 1991, Nature.

[4]  W. Denetclaw,et al.  Increased activity of calcium leak channels in myotubes of Duchenne human and mdx mouse origin. , 1990, Science.

[5]  D. A. Williams,et al.  Resting calcium concentrations in isolated skeletal muscle fibres of dystrophic mice. , 1990, The Journal of physiology.

[6]  J. Lansman,et al.  Calcium entry through stretch-inactivated ion channels in mdx myotubes , 1990, Nature.

[7]  K. Campbell,et al.  Association of dystrophin and an integral membrane glycoprotein , 1989, Nature.

[8]  Y. Amagai,et al.  Calcium action potential and prolonged afterhyperpolarization in developing myotubes of a mouse clonal myogenic cell line. , 1989, The Japanese journal of physiology.

[9]  R. Steinhardt,et al.  Increased protein degradation results from elevated free calcium levels found in muscle from mdx mice , 1988, Nature.

[10]  S. Baylor,et al.  Fura‐2 calcium transients in frog skeletal muscle fibres. , 1988, The Journal of physiology.

[11]  R. Hodges,et al.  The Duchenne muscular dystrophy gene product is localized in sarcolemma of human skeletal muscle , 1988, Nature.

[12]  D. Schiffer,et al.  Free cytoplasmic Ca++ at rest and after cholinergic stimulus is increased in cultured muscle cells from Duchenne muscular dystrophy patients , 1988, Neurology.

[13]  S. Dimauro,et al.  RAPID COMMUNICATION Immunocytochemical Study of Dystrophin in Muscle Culturesfrom Patients with Duchenne Muscular Dystrophy and Unaffected Control Patients , 2022 .

[14]  Eric P. Hoffman,et al.  Dystrophin: The protein product of the duchenne muscular dystrophy locus , 1987, Cell.

[15]  D. Bers,et al.  The effect of temperature and ionic strength on the apparent Ca-affinity of EGTA and the analogous Ca-chelators BAPTA and dibromo-BAPTA. , 1987, Biochimica et biophysica acta.

[16]  L. Duchen,et al.  The mutant mdx: inherited myopathy in the mouse. Morphological studies of nerves, muscles and end-plates. , 1987, Brain : a journal of neurology.

[17]  R. Fink,et al.  Calcium and strontium activation of single skinned muscle fibres of normal and dystrophic mice. , 1986, The Journal of physiology.

[18]  Marinos C. Dalakas,et al.  Muscle biopsy — a practical approach , 1986, The Ulster Medical Journal.

[19]  E. Neher,et al.  The Ca signal from fura‐2 loaded mast cells depends strongly on the method of dye‐loading , 1985, FEBS letters.

[20]  R. Tsien,et al.  A new generation of Ca2+ indicators with greatly improved fluorescence properties. , 1985, The Journal of biological chemistry.

[21]  D. Stephenson,et al.  Calcium‐activated force responses in fast‐ and slow‐twitch skinned muscle fibres of the rat at different temperatures. , 1981, The Journal of physiology.

[22]  D. Fambrough,et al.  Electrical properties of normal and dysgenib mouse skeletal muscle in culture , 1973, Journal of cellular physiology.