Abstract：
OBJECTIVE: Parvalbumin (PV) is a primary calcium buffer in mouse fast skeletal muscle fibers. Previous work showed that PV ablation has a limited impact on cytosolic Ca2+ ([Ca2+]cyto) transients and contractile response, while it enhances mitochondrial density and mitochondrial matrix-free calcium concentration ([Ca2+]mito). Here, we aimed to quantitatively test the hypothesis that mitochondria act to compensate for PV deficiency.
METHODS: We determined the free Ca2+ redistribution during a 2 s 60 Hz tetanic stimulation in the sarcoplasmic reticulum, cytosol, and mitochondria. Via a reaction-diffusion Ca2+ model, we quantitatively evaluated mitochondrial uptake and storage capacity requirements to compensate for PV lack and analyzed possible extracellular export.
RESULTS: [Ca2+]mito during tetanic stimulation is greater in knock-out (KO) (1362 ± 392 nM) than in wild-type (WT) (855 ± 392 nM), p < 0.05. Under the assumption of a non-linear intramitochondrial buffering, the model predicts an accumulation of 725 μmoles/L fiber (buffering ratio 1:11 000) in KO, much higher than in WT (137 μmoles/L fiber, ratio 1:4500). The required transport rate via mitochondrial calcium uniporter (MCU) reaches 3 mM/s, compatible with available literature. TEM images of calcium entry units and Mn2+ quenching showed a greater capacity of store-operated calcium entry in KO compared to WT. However, levels of [Ca2+]cyto during tetanic stimulation were not modulated to variations of extracellular calcium.
CONCLUSIONS: The model-based analysis of experimentally determined calcium distribution during tetanic stimulation showed that mitochondria can act as a buffer to compensate for the lack of PV. This result contributes to a better understanding of mitochondria\'s role in modulating [Ca2+]cyto in skeletal muscle fibers.
METHODS: We determined the free Ca2+ redistribution during a 2 s 60 Hz tetanic stimulation in the sarcoplasmic reticulum, cytosol, and mitochondria. Via a reaction-diffusion Ca2+ model, we quantitatively evaluated mitochondrial uptake and storage capacity requirements to compensate for PV lack and analyzed possible extracellular export.
RESULTS: [Ca2+]mito during tetanic stimulation is greater in knock-out (KO) (1362 ± 392 nM) than in wild-type (WT) (855 ± 392 nM), p < 0.05. Under the assumption of a non-linear intramitochondrial buffering, the model predicts an accumulation of 725 μmoles/L fiber (buffering ratio 1:11 000) in KO, much higher than in WT (137 μmoles/L fiber, ratio 1:4500). The required transport rate via mitochondrial calcium uniporter (MCU) reaches 3 mM/s, compatible with available literature. TEM images of calcium entry units and Mn2+ quenching showed a greater capacity of store-operated calcium entry in KO compared to WT. However, levels of [Ca2+]cyto during tetanic stimulation were not modulated to variations of extracellular calcium.
CONCLUSIONS: The model-based analysis of experimentally determined calcium distribution during tetanic stimulation showed that mitochondria can act as a buffer to compensate for the lack of PV. This result contributes to a better understanding of mitochondria\'s role in modulating [Ca2+]cyto in skeletal muscle fibers.
摘要：
目的：小白蛋白（PV）是小鼠快速骨骼肌纤维中的主要钙缓冲剂。以前的工作表明，肺静脉消融对细胞溶质Ca2+（[Ca2+]细胞）瞬变和收缩反应的影响有限，同时增强线粒体密度和线粒体基质中的无钙浓度（[Ca2]mito）。这里,我们旨在定量检验线粒体可以补偿PV缺乏的假设。
方法：我们确定了肌浆网中2s60Hz强直刺激期间的游离Ca2再分布，胞质溶胶,和线粒体.通过反应扩散Ca2+模型，我们定量评估了线粒体摄取和储存能力需求,以弥补PV缺乏,并分析了可能的细胞外输出.
结果：[Ca2+]mito在强直刺激期间的敲除(KO)(1362±392nM)大于野生型(WT)(855±392nM),p<0.05。在非线性线粒体内缓冲的假设下，该模型预测在KO中积累725μmoles/L纤维(缓冲比1:11000)，远高于WT(137μmoles/L纤维，比率1：4500）。通过线粒体钙单质转运蛋白（MCU）所需的转运速率达到3mM/s，与现有文献兼容。钙进入单元和Mn2淬灭的TEM图像显示，与WT相比，KO中存储操作的钙进入能力更高。然而,强直性刺激期间[Ca2]细胞的水平不受细胞外钙变化的调节。
结论：对强直性刺激过程中实验确定的钙分布的基于模型的分析表明，线粒体可以充当缓冲剂来补偿PV的缺乏。这一结果有助于更好地理解线粒体在调节骨骼肌纤维[Ca2+]细胞中的作用。
方法：我们确定了肌浆网中2s60Hz强直刺激期间的游离Ca2再分布，胞质溶胶,和线粒体.通过反应扩散Ca2+模型，我们定量评估了线粒体摄取和储存能力需求,以弥补PV缺乏,并分析了可能的细胞外输出.
结果：[Ca2+]mito在强直刺激期间的敲除(KO)(1362±392nM)大于野生型(WT)(855±392nM),p<0.05。在非线性线粒体内缓冲的假设下，该模型预测在KO中积累725μmoles/L纤维(缓冲比1:11000)，远高于WT(137μmoles/L纤维，比率1：4500）。通过线粒体钙单质转运蛋白（MCU）所需的转运速率达到3mM/s，与现有文献兼容。钙进入单元和Mn2淬灭的TEM图像显示，与WT相比，KO中存储操作的钙进入能力更高。然而,强直性刺激期间[Ca2]细胞的水平不受细胞外钙变化的调节。
结论：对强直性刺激过程中实验确定的钙分布的基于模型的分析表明，线粒体可以充当缓冲剂来补偿PV的缺乏。这一结果有助于更好地理解线粒体在调节骨骼肌纤维[Ca2+]细胞中的作用。
