Long-term study of Ca2+ homeostasis and of survival in collagenase-isolated muscle fibres from normal and mdx mice
Long-term study of Ca2+ homeostasis and of survival in collagenase-isolated muscle fibres from normal and mdx mice
复制标题
DOI：
10.1113/jphysiol.2002.020487
复制
发表时间：
2002-08-01
影响因子：
5.5
通讯作者：
Gillis, JM
中科院分区：
文献类型：
--
作者：
De Backer, F;Vandebrouck, C;Gillis, JM
MDX小鼠的骨骼肌缺乏肌营养不良蛋白，这为研究细胞内钙离子在纤维退化中的作用提供了可能性。用胶原酶分离3月龄MDX小鼠和正常小鼠的趾短屈肌，培养6d(d0~d5)，观察其存活情况。同时研究了细胞内[Ca~(2+)]、被动Mn2+内流(指示Ca~(2+)内流)和机械敏感/电压非依赖性钙通道的活动。从DO到D3，正常纤维的存活率稳步下降，而MDX纤维从D1到D2出现加速的纤维死亡。这可能会大大减少，但不能通过将外部[Ca~(2+)]降低10倍来消除。在d0-d5期间，MDX和正常纤维均表现出一过性的Mn2+内流和钙通道活动增加，在d1达到高峰，d3-d4消失。MDX纤维的增幅总是明显更大。总共在6天的时间里，对68根MDX纤维和62根正常小鼠的纤维进行了130对[Ca~(2+)](I)和Mn2+内流的测量。在90%的纤维中，[Ca~(2+)](I)保持在25-85 nm范围内，而Mn2+内流的变化超过10倍。MDX小鼠的纤维中Mn2+内流的中位数比对照C57小鼠的纤维中高45%。然而，正常小鼠和mdx小鼠的纤维内[Ca~(2+)](I)浓度之间并无显著差异。在培养液中加入25-75 nm的钙离子载体(4-bromo-A23187)并不影响两种类型纤维的胞浆[Ca~(2+)]水平，但如预期的那样显著增加了Mn2+的内流速率。因此，在MDX和正常纤维中，钙稳态是同样强劲的。在d1时，其余10%的纤维显示高水平的Mn2+内流和/或升高的[Ca~(2+)](I)超过100 nm。这并不影响正常纤维的存活率，但可能是MDX纤维死亡率增加的原因。
Skeletal muscles of the mdx mouse lack dystrophin offering the possibility to study the role of intracellular Ca2+ ions in fibre degeneration. Flexor digitorum brevis muscles of 3-month-old mdx and normal mice were dissociated with collagenase; fibres were maintained in culture for 6 days (do to d5) and their survival was assessed. Cytosolic [Ca2+], passive Mn2+ influx (indicative of Ca2+ influx) and activity of mechanosensitive/voltage-independent Ca2+ channels were studied over the same period. Survival of normal fibres declined steadily from do to d3, but an acceleration of fibre death occurred in mdx fibres from d1 to d2. This could be greatly reduced but not abolished by lowering external [Ca2+] 10-fold. In the d0-d5 period,both mdx and normal fibres showed transient increases of Mn2+ influx and activity of the Ca2+ channels; these peaked at d1 and disappeared by d3-d4. Increases were always significantly larger in mdx fibres. Altogether, over the 6 days, 130 paired measurements of [Ca2+](i) and Mn2+ influx were made on 68 fibres from mdx and 62 fibres from normal mice. In 90 % of the fibres, [Ca2+](i) remained within the 25-85 nM limits while Mn2+ influx varied more than 10-fold. The median for Mn2+ influx was 45 % greater in fibres from mdx mice than in fibres from control C57 mice. However, there was no significant difference between [Ca2+](i) medians in fibres from normal and mdx mice. Addition of 25-75 nm of a Ca2+ ionophore (4-bromo-A23187) to the medium did not affect the level of cytosolic [Ca2+] in both types of fibres, while markedly increasing the rate of Mn2+ influx, as expected. Thus, Ca2+ homeostasis was equally robust in mdx and normal fibres. The remaining 10 % of the fibres showed, at d1, high levels of Mn2+ influx and/or elevated [Ca2+](i) above 100 nm. This did not affect survival of normal fibres but was probably responsible of the increased death rate in mdx fibres.