Evidence for dysfunction in the regulation of cytosolic Ca2+ in excitation-contraction uncoupled dysgenic muscle (original) (raw)
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Effects of external calcium deprivation on single muscle fibers
1967
Deprivation of external calcium causes sudden potentiation of the twitch response of single muscle fibers. The potentiation was 64 4-8 %. Potentiation is simultaneous with membrane depolarization occurring after Ca +÷ removal. This depolarization amounted to 9 4-2 my. Ca ++ removal also alters the action potential. 3 rain after calcium withdrawal, action potential amplitude fell by 36 4-3 my; maximum rates of rise and fall of the spike decreased by 55 4-5 and 63 4-5 % respectively. Changes in shape of the A. P. differ from those seen with other potentiators of the twitch response, such as Zn ++. After short exposure to calcium-free media, potassium-induced contractures show potentiation of peak tension. The S-shaped curve relating potassium contracture tension to log [K]o shifts to the left after such treatment. Calcium deprivation also increased the rate of relaxation of the contractures. This effect depends on the duration of calcium deprivation, and is probably related to the effect of calcium lack on the membrane. The change in relaxation occurred immediately after calcium deprivation, and was reversed by sudden readmission of calcium. Relaxation of twitch and tetanus responses also were affected by Ca lack, but not as rapidly as potassium contractures. The results suggest that external calcium is not directly involved in the process responsible for tension development, supporting the view that this process is mediated by translocation of intracellular calcium. The relaxation process, however, appears to be rapidly affected by deprivation of external calcium. Normally the contractile response of muscle fibers is triggered by the lowering of the fiber m e m b r a n e potential to a threshold value at which the excitationcontraction coupling (ECC) process begins to be effective (1-4). T h e hypothesis which postulated t h a t calcium ions entering the fibers from the extracellular space upon depolarization of the m e m b r a n e activated the contractile material (5, 6) appears to be inadequate on the basis of theoretically calculated diffusion delays (4, 7, 8). However, the presence in the interior of the fiber of the system of transverse tubules which open out directly into the external space could reduce such delays (9, 10). a~77
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 [Ca2l] 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 'C. All data are expressed as means + S.E.M.
The Journal of Physiology, 2004
The mdx mouse, a model of the human disease Duchenne muscular dystrophy, has skeletal muscle fibres which display incompletely understood impaired contractile function. We explored the possibility that action potential-evoked Ca(2+) release is altered in mdx fibres. Action potential-evoked Ca(2+)-dependent fluorescence transients were recorded, using both low and high affinity Ca(2+) indicators, from enzymatically isolated fibres obtained from extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles of normal and mdx mice. Fibres were immobilized using either intracellular EGTA or N-benzyl-p-toluene sulphonamide, an inhibitor of the myosin II ATPase. We found that the amplitude of the action potential-evoked Ca(2+) transients was significantly decreased in mdx mice with no measured difference in that of the surface action potential. In addition, Ca(2+) transients recorded from mdx fibres in the absence of EGTA also displayed a marked prolongation of the slow decay phase. Model simulations of the action potential-evoked transients in the presence of high EGTA concentrations suggest that the reduction in the evoked sarcoplasmic reticulum Ca(2+) release flux is responsible for the decrease in the peak of the Ca(2+) transient in mdx fibres. Since the myoplasmic Ca(2+) concentration is a critical regulator of muscle contraction, these results may help to explain the weakness observed in skeletal muscle fibres from mdx mice and, possibly, Duchenne muscular dystrophy patients.
Early effects of denervation on Ca2+-handling proteins in skeletal muscle
International Journal of Molecular Medicine, 2004
The adaptive response of skeletal muscle fibres depends on a variety of biological factors including loading conditions and neuromuscular activity. An extreme type of atrophy-inducing change in contractile activity is represented by the physical disconnection between the motor nerve and its respective fibre unit. Since fibre type alterations have a striking effect on the Ca 2+-regulatory apparatus, we have investigated the fate of a key Ca 2+-pump and essential Ca 2+binding proteins in extensor digitorum longus specimens two weeks after nerve crash or complete denervation. In contrast to increased levels of sarcalumenin, immunoblotting revealed that the expression of the fast SERCA1 Ca 2+-ATPase isoform is drastically decreased and fast calsequestrin is slightly reduced. Analysis of myosin heavy chain isoforms agreed with this result and showed a fast-to-slow fibre type shifting process following denervation. Hence, changes in muscle activity appear to have a profound effect on the abundance and isoform expression pattern of Ca 2+-handling elements.
The Journal of physiology, 1993
1. Single skeletal muscle fibres were enzymatically isolated from the flexor digitorum brevis muscles (FDB) of dystrophic mdx and control C57BL/10 mice aged 3-9 weeks. In this age range the majority (> 95%) of the mdx fibres were morphologically normal. 2. There was no significant difference between the resting membrane potential (RMP) of mdx and control mice, -71.2 +/- 1.21 (n = 26) and -70.6 +/- 1.15 mV (n = 42), respectively. 3. At RMP more negative than -60 mV the resting calcium (recorded with fura-2, free acid ionophoresed into cell) in the dystrophic mdx cells was not significantly different from the normal animals, 45.7 +/- 4.1 (n = 10) and 46.2 +/- 3.9 nM (n = 9), respectively. 4. The resting cytosolic calcium concentration was measured simultaneously with the RMP. At RMP between -60 to -17 mV there was an increase in the resting calcium concentration in both mdx and control ranging from 79.3 to 252 nM. This increase was most probably due to the activation of the slow ca...
The Journal of Physiology, 2008
Intracellular calcium signals regulate multiple cellular functions. They depend on release of Ca 2+ from cellular stores into the cytosol, a process that in many types of cells appears to be tightly controlled by changes in [Ca 2+ ] within the store. In contrast with cardiac muscle, where depletion of Ca 2+ in the sarcoplasmic reticulum is a crucial determinant of termination of Ca 2+ release, in skeletal muscle there is no agreement regarding the sign, or even the existence of an effect of SR Ca 2+ level on Ca 2+ release. To address this issue we measured Ca 2+ transients in mouse flexor digitorum brevis (FDB) skeletal muscle fibres under voltage clamp, using confocal microscopy and the Ca 2+ monitor rhod-2. The evolution of Ca 2+ release flux was quantified during long-lasting depolarizations that reduced severely the Ca 2+ content of the SR. As in all previous determinations in mammals and non-mammals, release flux consisted of an early peak, relaxing to a lower level from which it continued to decay more slowly. Decay of flux in this second stage, which has been attributed largely to depletion of SR Ca 2+ , was studied in detail. A simple depletion mechanism without change in release permeability predicts an exponential decay with time. In contrast, flux decreased non-exponentially, to a finite, measurable level that could be maintained for the longest pulses applied (1.8 s). An algorithm on the flux record allowed us to define a quantitative index, the normalized flux rate of change (NFRC), which was shown to be proportional to the ratio of release permeability P and inversely proportional to Ca 2+ buffering power B of the SR, thus quantifying the 'evacuability' or ability of the SR to empty its content. When P and B were constant, flux then decayed exponentially, and NFRC was equal to the exponential rate constant. Instead, in most cases NFRC increased during the pulse, from a minimum reached immediately after the early peak in flux, to a time between 200 and 250 ms, when the index was no longer defined. NFRC increased by 111% on average (in 27 images from 18 cells), reaching 300% in some cases. The increase may reflect an increase in P, a decrease in B, or both. On experimental and theoretical grounds, both changes are to be expected upon SR depletion. A variable evacuability helps maintain a constant Ca 2+ output under conditions of diminishing store Ca 2+ load.
Spatially segregated control of Ca2+ release in developing skeletal muscle of mice
The Journal of Physiology, 1999
1. Confocal laser scanning microscopy was used to monitor Ca¥ signals in primary-cultured myotubes, prepared from forelimbs of wild-type or ryanodine receptor type 3 (RyR3) knockout mice. Myotubes loaded with the acetoxymethyl ester (AM) form of fluo_3 were imaged at rest or under whole-cell patch clamp. 2. Discrete Ca¥ release events were detected in intact wild-type and RyR3-knockout myotubes. They showed almost no difference in amplitude and width, but were substantially different in duration. In wild-type myotubes (660 events, 57 cells) the amplitude was 1·27 (0·85, 1·97) (median (25%, 75%)) units of resting fluorescence, the full width at halfmagnitude (FWHM) was 1·4 (0·9, 2·3) ìm, and the full duration at half-magnitude (FDHM) was 25·3 (9·6, 51·7) ms. In RyR3-knockout myotubes (655 events, 83 cells) the amplitude was 1·30 (0·84, 2·08), FWHM was 1·63 (1·02, 2·66) ìm, and FDHM was 43·6 (23·6, 76·9) ms. 3. Depolarization under voltage clamp of both wild-type and RyR3-knockout myotubes produced substantial Ca¥ release devoid of discrete Ca¥ events. Discrete events were still present but occurred without correlation with the applied pulse, largely at locations where the pulse did not elicit release. 4. The local correspondence between voltage control and absence of discrete events implies that the functional interaction with voltage sensors suppresses the mechanism that activates discrete events. Because it applies whether RyR3 is present or not, it is this exclusion by voltage of other control mechanisms, rather than isoform composition, that primarily determines the absence of discrete Ca¥ events in adult mammalian muscle.
The network of calcium regulation in muscle
Acta biochimica Polonica, 2003
In this review the molecular characteristics and reaction mechanisms of different Ca(2+) transport systems associated with various membranes in muscle cells will be summarized. The following topics will be discussed in detail: a brief history of early observations concerning maintenance and regulation of cellular Ca(2+) homeostasis, characterization of the Ca(2+) pumps residing in plasma membranes and sarco(endo)plasmic reticulum, mitochondrial Ca(2+) transport, Ca(2+)-binding proteins, coordinated expression of Ca(2+) transport systems, a general background of muscle excitation-contraction coupling with emphasis to the calcium release channels of plasma membrane and sarcoplasmic reticulum, the structure and function of dihydropyridine and ryanodine receptors of skeletal and cardiac muscles, and finally their disposition in various types of muscles.