1 In Duchenne muscular dystrophy (DMD) dysregulation of cytosolic calcium appears to be involved in the degeneration of skeletal muscle fibres. Therefore, we have studied the regulation of the free cytosolic calcium concentration ([Ca2+]) under specific stress conditions in cultured myotubes isolated from the hind limbs of wild-type (C57BL10) and dystrophin-deficient mutant mdx mice. [Ca2+] [Ca2+]c when the cells were exposed to the hypo-osmotic buffer (100 mOsm).5 Incubation of the cell culture for 3-5 days from the onset of induction of myotube formation with the membrane permeable protease inhibitor, calpeptin (50 gM) abolished the rise in [Ca2+]c in mdx myotubes upon exposure to hypo-osmotic shock.6 Treatment of the cell culture for 3-5 days with ac-methylprednisolone (PDN, 10 guM) attenuated the rise in [Ca2+]c following hypo-osmotic stress for both normal and mdx myotubes by about 50%.7 The results described here suggest an increased permeability of mdx myotubes to Ca2+ under specific stress conditions. The ameliorating effect of PDN on [Ca2+]c could explain, at least partly, the beneficial effect of this drug on DMD patients.
1 Increased calcium (Ca2") influx has been incriminated as a potential pathological mechanism in the chronic skeletal muscle degeneration exhibited by Duchenne muscular dystrophy (DMD) patients. We have studied the influence of the glucocorticoid a-methylprednisolone (PDN), the only drug known to have a beneficial effect on the degenerative course of DMD, on Ca2" handling in the C2 skeletal muscle cell line. 2 PDN, when added 3 days (when myoblasts start to fuse into myotubes) after cell seeding, led to a 2 to 4 fold decrease in cellular Ca21 uptake. This decrease was independent of the extracellular Ca2+ concentration applied to cells. The effect took at least 24 h in order to become established (PDN of l0-5 M) and took longer for lower PDN concentrations (ECm of ca. 10-6 M at day 5, 10-6.5 M at day 7 and i0-7 5 M at day 9 in culture). 3 Cellular calcium accumulation was also decreased in PDN-treated myotubes exposed to 45Ca2+-containing medium for 1 to 6 days. 4 No effect of PDN was seen on 45Ca2+ efflux; a decrease in the amount of 45Ca2+released was observed due to the reduction of cellular 45Ca2+ loading.
The effect of hypothyroidism on tension-independent and -dependent heat produced during a twitch and a tetanic contraction of extensor digitorum longus (EDL) and soleus muscle of mice was examined. The amount of heat produced during a twitch and the rate of heat development during a tetanus of EDL and soleus were measured at and above optimal length. The effect of hypothyroidism on force production was less than 30%. Straight lines were used to fit the relation between heat production and force. Hypothyroidism significantly decreases tension-independent heat during contraction of EDL and soleus muscle. Because the tension-independent heat is considered to be related to the Ca2+ cycling, these findings suggest that ATP splitting due to the Ca2+ cycling is reduced in hypothyroid mice. This conclusion was strengthened by the observation that the oxalate-supported Ca2+-uptake activity and Ca2+-loading capacity of muscle homogenates from hypothyroid mice were reduced, respectively, to 51 and to 65% in soleus and to 63 and 73% in EDL muscle as compared with euthyroid mice. The tension-dependent rate of heat development during a tetanus was also decreased in soleus muscle of hypothyroid mice. This suggests a lower rate of ATP hydrolysis related to cross-bridge cycling in this muscle due to the hypothyroid state.
Heat produced by a 1-s isometric tetanus of mouse extensor digitorum longus muscle (EDL; n = 6) and a 1.5-s isometric tetanus of soleus muscle (n = 7) was measured with thermopiles at 20 degrees C, and separated into initial heat (I) and recovery heat (R). In EDL the initial heat was 190 +/- 40 (SD) mJ g-1 and in soleus 52 +/- 9 (SD) mJ g-1. The recovery heat production rate immediately following the tetanus was almost zero in both muscles. It rose in 12 +/- 6 s (EDL) and in 30 +/- 3 s (soleus) to a maximum, to decrease thereafter monoexponentially with a time constant of 30.7 +/- 5.7 s (EDL) and 41.7 +/- 7.2 s (soleus). The measured recovery ratio (R/I) differed between EDL (0.95 +/- 0.14) and soleus (1.54 +/- 0.22). The value for soleus muscles was significantly different from the theoretical value of 1.13. EDL muscles were freeze-clamped at rest (n = 10) and during the recovery phase, 1 min after the onset of the tetanus (n = 10), to determine lactate and creatine phosphate. It was found that no significant amount of net lactate was produced. The amount of creatine phosphate reformed corresponded to the recovery heat produced. The results suggest that metabolic recovery after short tetani of EDL and soleus muscles occurs predominantly through oxidative phosphorylation, but knowledge of respiratory control in the living cell is insufficient to explain its slow onset immediately following contraction and the finding that EDL recovers faster than soleus.
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