Department of Kinesiology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
J Gen Physiol. 2013 Mar;141(3):297-308. doi: 10.1085/jgp.201210918. Epub 2013 Feb 11.
The increase in isometric twitch force observed in fast-twitch rodent muscles during or after activity, known universally as potentiation, is normally associated with myosin regulatory light chain (RLC) phosphorylation. Interestingly, fast muscles from mice devoid of detectable skeletal myosin light chain kinase (skMLCK) retain a reduced ability to potentiate twitch force, indicating the presence of a secondary origin for this characteristic feature of the fast muscle phenotype. The purpose of this study was to assess changes in intracellular cytosolic free Ca(2+) concentration (Ca(2+)) after a potentiating stimulus in mouse lumbrical muscle (37°C). Lumbricals were loaded with the Ca(2+)-sensitive fluorescent indicators fura-2 or furaptra to detect changes in resting and peak, respectively, intracellular Ca(2+) levels caused by 2.5 s of 20-Hz stimulation. Although this protocol produced an immediate increase in twitch force of 17 ± 3% (all data are n = 10) (P < 0.01), this potentiation dissipated quickly and was absent 30 s afterward. Fura-2 fluorescence signals at rest were increased by 11.1 ± 1.3% (P < 0.01) during potentiation, indicating a significant increase in resting Ca(2+). Interestingly, furaptra signals showed no change to either the amplitude or the duration of the intracellular Ca(2+) transients (ICTs) that triggered potentiated twitches during this time (P < 0.50). Immunofluorescence work showed that 77% of lumbrical fibers expressed myosin heavy chain isoform IIx and/or IIb, but with low expression of skMLCK and high expression of myosin phosphatase targeting subunit 2. As a result, lumbrical muscles displayed no detectable RLC phosphorylation either at rest or after stimulation. We conclude that stimulation-induced elevations in resting Ca(2+), in the absence of change in the ICT, are responsible for a small-magnitude, short-lived potentiation of isometric twitch force. If operative in other fast-twitch muscles, this mechanism may complement the potentiating influence of myosin RLC phosphorylation.
在活动期间或之后，观察到快速收缩鼠肌肉中的等长抽搐力增加，普遍称为增强，通常与肌球蛋白调节轻链 (RLC) 磷酸化有关。有趣的是，缺乏可检测的骨骼肌肌球蛋白轻链激酶 (skMLCK) 的快速肌肉仍然保留了降低的增强抽搐力的能力，表明这种快速肌肉表型特征存在次要来源。本研究的目的是评估在 37°C 下，在增强刺激后小鼠蚓状肌细胞内胞质游离 Ca(2+) 浓度 (Ca(2+)) 的变化。用 Ca(2+) 敏感荧光指示剂 fura-2 或 furaptra 加载蚓状肌，以检测 20-Hz 刺激 2.5 s 引起的静息和峰值细胞内 Ca(2+) 水平的变化。尽管该方案立即使抽搐力增加了 17 ± 3%（所有数据 n = 10）（P < 0.01），但这种增强很快消散，30 秒后消失。增强过程中，fura-2 荧光信号静息时增加了 11.1 ± 1.3%（P < 0.01），表明静息 Ca(2+) 显著增加。有趣的是，furaptra 信号在这段时间内触发增强抽搐的细胞内 Ca(2+) 瞬变 (ICT) 的幅度或持续时间都没有变化（P < 0.50）。免疫荧光工作表明，77%的蚓状肌纤维表达肌球蛋白重链同工型 IIx 和/或 IIb，但 skMLCK 表达低，肌球蛋白磷酸酶靶向亚单位 2 表达高。因此，蚓状肌在静息或刺激后都没有检测到 RLC 磷酸化。我们得出结论，刺激引起的静息 Ca(2+) 升高，而 ICT 没有变化，是等长抽搐力的小幅度、短暂增强的原因。如果在其他快速收缩肌肉中起作用，这种机制可能补充肌球蛋白 RLC 磷酸化的增强作用。
