【作者】 王敏；

【导师】 佟晓永；

【作者基本信息】 重庆大学 ， 药学， 2024， 硕士

【摘要】 目的:骨骼肌作为人体最丰富的组织,在维持机体运动和稳态等生命过程中发挥重要作用。骨骼肌萎缩是指肌肉质量或纤维大小的减少以及相关机能丧失。肌浆网/内质网钙ATP酶（sarcoplasmic/endoplasmic reticulum calcium-ATPase,SERCA）是细胞中唯一能将胞浆Ca²⁺(intracellular Ca²⁺,[Ca²⁺]_I)泵入肌浆网/内质网中的酶,用于维持细胞钙稳态。SERCA2是骨骼肌中SERCA的主要亚型。骨骼肌萎缩的易感因素如衰老、废用、糖尿病等病理条件下生成的活性氧自由基（ROS）可造成SERCA2活性关键位点第674位半胱氨酸(Cys⁶⁷⁴,C674)不可逆氧化,导致SERCA2功能障碍。本研究旨在阐明SERCA2功能障碍是否引起骨骼肌萎缩并探讨其调控机制。方法:（1）使用SERCA2 C674S敲入（SERCA2 C674S knock-in,SKI）杂合子小鼠,用于模拟病理情况下C674巯基的部分不可逆氧化导致的SERCA2功能障碍;（2）检测SKI小鼠和其同窝野生型（wild-type,WT）小鼠骨骼肌萎缩相关病理改变,包括四肢水平和竖直网格悬挂时间,腓肠肌、胫骨前肌及比目鱼肌重量与体重之比,肌纤维面积;（3）在SKI小鼠和WT小鼠以及SERCA2b-S674腺病毒转染的小鼠成肌细胞系C2C12细胞中检测:[Ca²⁺]_I,线粒体Ca²⁺(mitochondrial Ca²⁺,[Ca²⁺]_m),线粒体膜电位（ΔΨ_m）,钙调控相关蛋白（STIM1、ORAI1、VDAC1、MCU、MICU1）、线粒体生物发生相关蛋白（PGC-1α、TFAM）、线粒体分裂融合相关蛋白（OPA1、MFN2、Drp1、Fis1、MFF）、氧化磷酸化相关蛋白（ATP6、Mt-COX II、Mt-COX IV、ND1）、自噬相关蛋白（Fox O1、PINK1、Parkin、Beclin1、P62、LC3B）的表达水平;（4）通过PGC-1α激动剂烟酰胺核糖（nicotinamide riboside,NR）的体内外干预探究PGC-1α与Fox O1的调控关系;（5）通过NR、Fox O1抑制剂AS1842856（AS）、SERCA激动剂CDN1163的体内干预,检测对蛋白表达和骨骼肌萎缩相关病理现象的影响。结果:（1）SERCA2功能障碍导致小鼠四肢握力下降,肌肉重量和肌纤维面积减少;（2）SERCA2功能障碍增加[Ca²⁺]_I和[Ca²⁺]_m水平,降低ΔΨ_m,干扰线粒体生物发生、线粒体分裂融合和氧化磷酸化等相关蛋白的表达,干扰线粒体功能;（3）SERCA2功能障碍减少调控线粒体功能的关键调节因子PGC-1α的表达,进而促进Fox O1入核,激活细胞自噬;（4）NR、AS、CDN1163分别从改善线粒体功能、抑制Fox O1诱导的自噬、激活SERCA2的角度逆转SKI小鼠肌萎缩病理现象。结论:SERCA2功能障碍干扰骨骼肌Ca²⁺稳态,降低ΔΨ_m,引起线粒体膜去极化,干扰线粒体功能并激活Fox O1的活性,增加自噬水平,从而促进骨骼肌萎缩。激活PGC-1α、抑制Fox O1活性或激活SERCA2功能均能改善SERCA2功能障碍引起的骨骼肌萎缩。更多还原

【Abstract】 Objective:The skeletal muscle,being the most abundant tissue in the human body,plays a crucial role in maintaining both the body’s movement and homeostasis.A decrease in muscle mass and fiber size accompanied by a drop in associated function is known as skeletal muscle atrophy.Sarcoplasmic/endoplasmic reticulum calcium-ATPase（SERCA）is the only enzyme that pumps intracellular Ca²⁺([Ca²⁺]_I)into the sarcoplasmic reticulum/endoplasmic reticulum to maintain calcium homeostasis in cells.The predominant subtype of SERCA in skeletal muscle is SERCA2.As an important site in the regulation of SERCA2 activity,Cys⁶⁷⁴ is inactivated by irreversible oxidation（C674-SO₃H）,which is brought on by reactive oxygen species（ROS）generated under pathological circumstances such as diabetes,aging,and physical inactivity—all of which are risk factors for the atrophy of skeletal muscle.This study attempted to determine whether SERCA2 dysfunction leads to skeletal muscle atrophy and to investigate the regulatory mechanism involved.Methods:1)SERCA2 C674S knock-in（SKI）heterozygote mice were used to simulate the dysfunction of SERCA2 caused by partial irreversible oxidation of the C674thiol group in pathological conditions;2)Pathological alterations associated with skeletal muscle atrophy were detected in both SKI and WT mice,including horizontal and vertical grid hanging time of their limbs,the gastrocnemius,tibialis anterior,and soleus muscle weight ratios to body weight,and the area of their muscle fibers;3)We detected the expression levels of[Ca²⁺]_I,mitochondrial Ca²⁺([Ca²⁺]_m),mitochondrial membrane potential（ΔΨ_m）,calcium regulation-related proteins（STIM1,ORAI1,VDAC1,MCU,MICU1）,mitochondrial biogenesis-related proteins（PGC-1α,TFAM）,mitochondria fission and fusion-related proteins（OPA1,MFN2,Drp1,Fis1,MFF）,oxidative phosphorylation-related proteins（ATP6,Mt-COX II,Mt-COX IV,ND1）and autophagy-related proteins（Fox O1,PINK1,Parkin,Beclin1,P62,LC3B）in WT and SKI mice,as well as in mouse myoblast cell line C2C12 cells transfected with the adenovirus SERCA2b-S674;4)The regulatory relationship between PGC-1αand Fox O1 was investigated through the intervention of PGC-1αagonist nicotinamide riboside（NR）in vivo and in vitro;5)Using in vivo intervention,the effects of NR,the Fox O1 inhibitor AS1842856（AS）,and the SERCA agonist CDN1163 on protein expression and pathological events associated with skeletal muscle atrophy were identified.Results:1)SERCA2 dysfunction led to a decrease in limb grip strength,reduced muscle mass and fiber area in mice;2)SERCA2 dysfunction increased[Ca²⁺]_I and[Ca²⁺]_m levels,reducedΔΨ_m,interfered with the expression of mitochondrial biogenesis,mitochondrial fission and fusion,oxidative phosphorylation and other related proteins,and interfered with mitochondrial function;3)SERCA2 dysfunction reduced the expression of PGC-1α,a key regulator of mitochondrial function,which in turn promoted the entry of Fox O1 into the nucleus and activated autophagy;4)NR,AS,and CDN1163reversed the pathological phenomenon of muscle atrophy in SKI mice from the perspectives of improving mitochondrial function,inhibiting Fox O1-induced autophagy,and activating SERCA2,respectively.Conclusions:SERCA2 dysfunction promoted skeletal muscle atrophy by interfering with skeletal muscle Ca²⁺homeostasis,decreasingΔΨ_m,inducing mitochondrial membrane depolarization,interfering with mitochondrial function,stimulating Fox O1activity,and increasing the level of autophagy.Activation of PGC-1α,inhibition of Fox O1activity,or activation of SERCA2 function can improve skeletal muscle atrophy.更多还原