Department of Animal Science and Institute of Rare Earth for Biological Aplication, Chonbuk National University, Deokjin-gu, Jeonju-si, Jeollabuk-do, Republic of Korea.
In Vitro Cell Dev Biol Anim. 2012 Jun;48(6):359-68. doi: 10.1007/s11626-012-9513-0. Epub 2012 May 31.
The current study was conducted to evaluate the functions of μ-calpain (CAPN1), calpastatin, HSPs (heat shock proteins), and caspases during myogenesis and cell death induced by sodium azide (NaN(3)) hypoxia. The cell samples were divided into three groups: satellite cells formed at confluent monolayer (stage 1), stage 1 cells fusion into myotubes on d eight post-differentiation (stage 2), and stage 2 cells treated with 1 mM NaN(3) for 24 h (stage 3). Real-time RT-PCR showed that stage 2 cells had increased CAPN1, calpastatin, caspase 7, and CARD9 (Caspase activation and recruitment domain 9) mRNA expressions compared to stage 1 cells (*p < 0.05). By Western blotting caspase 3, caspase 7, caspase 8, and caspase 9 protein levels increased in cells at stage 2 compared to cells at stage 1 (*p < 0.05). Real-time RT-PCR showed that stage 3 cells had increased CAPN1, calpastatin, caspase 7, HSP70 (70 kDA heat shock proteins), and HSP90 (90 kDA heat shock proteins-alpha) and decreased CARD9 mRNA expression compared to stage 2 cells (*p < 0.05). Stage 3 samples had increase caspase 7 and caspase 12 activities compared to stage 2 samples, and by Western blotting protein levels of both HSP70 and HSP90 expressions, increased significantly under hypoxia condition (*p < 0.05). Here, we conclude that CAPN1, calpastatin, caspase 3, caspase 7, caspase 8, and CARD9 have important roles for satellite cell myogenesis; and that caspase 7, 12, HSP70, and HSP90 are involved in the process of apoptotic cell death under hypoxia conditions and we speculate that these proteins may be involved in early postmortem proteolysis and meat tenderization.
本研究旨在评估 μ-钙蛋白酶 (CAPN1)、钙蛋白酶抑制剂、热休克蛋白 (HSPs) 和半胱天冬酶在卫星细胞形成融合肌管和细胞凋亡过程中的作用，凋亡由叠氮化钠 (NaN3) 诱导缺氧引起。细胞样本分为三组：在汇合单层形成的卫星细胞 (第 1 阶段)、第 1 阶段细胞融合形成肌管 (第 2 阶段)，第 2 阶段细胞用 1mMNaN3 处理 24 小时 (第 3 阶段)。实时 RT-PCR 显示，与第 1 阶段细胞相比，第 2 阶段细胞的 CAPN1、钙蛋白酶抑制剂、半胱天冬酶 7 和 Caspase 激活和募集域 9 (CARD9) mRNA 表达增加 (*p < 0.05)。通过 Western blot 分析，与第 1 阶段细胞相比，第 2 阶段细胞中的半胱天冬酶 3、半胱天冬酶 7、半胱天冬酶 8 和半胱天冬酶 9 蛋白水平升高 (*p < 0.05)。实时 RT-PCR 显示，与第 2 阶段细胞相比，第 3 阶段细胞的 CAPN1、钙蛋白酶抑制剂、半胱天冬酶 7、热休克蛋白 70 (70kDa 热休克蛋白) 和热休克蛋白 90 (90kDa 热休克蛋白-α) 表达增加，CARD9mRNA 表达减少 (*p < 0.05)。与第 2 阶段样本相比，第 3 阶段样本中的半胱天冬酶 7 和半胱天冬酶 12 活性增加，通过 Western blot 分析，在缺氧条件下 HSP70 和 HSP90 的蛋白水平显著增加 (*p < 0.05)。本研究表明，CAPN1、钙蛋白酶抑制剂、半胱天冬酶 3、半胱天冬酶 7、半胱天冬酶 8 和 CARD9 对半突细胞的肌发生有重要作用；半胱天冬酶 7、12、HSP70 和 HSP90 参与缺氧条件下的细胞凋亡过程，我们推测这些蛋白可能参与死后早期的蛋白水解和肉嫩化过程。
