Rat Rotator Cuff Tear RNASeq

Myosteatosis is the pathological accumulation of lipid that occurs in conjunction with atrophy and fibrosis following skeletal muscle injury or disease. Little is known about the mechanisms by which lipid accumulates in myosteatosis, but many studies have demonstrated the degree of lipid infiltration negatively correlates with muscle function and regeneration. Our goal was to identify biochemical pathways that lead to muscle dysfunction and lipid accumulation in injured rotator cuff muscles, a model that demonstrates severe myosteatosis. Adult rats were subjected to a massive tear to the rotator cuff musculature. After a period of either 0 (healthy control), 10, 30, or 60 days, muscles were prepared for RNA sequencing, shotgun lipidomics, metabolomics, biochemical measures, electron microscopy, and muscle fiber contractility. Following rotator cuff injury, there was a decrease in muscle fiber specific force production that was lowest at 30d. There was a dramatic time dependent increase in triacylglyceride content. Interestingly, genes related to not only triacylglyceride synthesis, but also lipid oxidation were largely downregulated over time. Using bioinformatics techniques, we identified that biochemical pathways related to mitochondrial dysfunction and reactive oxygen species were considerably increased in muscles with myosteatosis. Long chain acyl-carnitines and L-carnitine, precursors to beta-oxidation, were depleted following rotator cuff tear. Electron micrographs showed injured muscles displayed large lipid droplets within mitochondria at early time points, and an accumulation of peripheral segment mitochondria at all time points. Several markers of oxidative stress were elevated following rotator cuff tear. The results from this study suggest that the accumulation of lipid in myosteatosis is not a result of canonical lipid synthesis, but occurs due to decreased lipid oxidation in mitochondria. A failure in lipid utilization by mitochondria would ultimately cause an accumulation of lipid even in the absence of increased synthesis. Further study will identify whether this process is required for the onset of myosteatosis.肌肉脂肪变性（Myosteatosis）是指骨骼肌损伤或疾病后伴随萎缩与纤维化发生的病理性脂质蓄积。目前对肌肉脂肪变性的脂质蓄积机制尚不清楚，但多项研究已证实，脂质浸润程度与肌肉功能及再生能力呈负相关。本研究旨在明确损伤肩袖肌肉中导致肌肉功能障碍与脂质蓄积的生化通路，该模型可表现出严重的肌肉脂肪变性。实验采用成年大鼠，对其肩袖肌肉造成大型撕裂伤。分别于造模后0天（健康对照组）、10天、30天及60天采集肌肉样本，进行RNA测序、鸟枪脂质组学、代谢组学、生化检测、电子显微镜观察及肌纤维收缩力测定。肩袖损伤后，肌纤维比力产生量逐渐下降，并于30天时降至最低。三酰甘油含量随时间推移显著升高。有趣的是，随着时间推移，不仅三酰甘油合成相关基因，脂质氧化相关基因的表达也显著下调。通过生物信息学分析，我们发现肌肉脂肪变性样本中，线粒体功能障碍与活性氧相关的生化通路显著激活。作为β-氧化前体的长链酰基肉碱与L-肉碱，在肩袖撕裂后出现耗竭。电子显微镜图像显示，损伤肌肉在早期时点的线粒体内可见大型脂滴，且各时点均出现外周段线粒体的蓄积。肩袖撕裂后，多项氧化应激标志物水平均出现升高。本研究结果表明，肌肉脂肪变性中的脂质蓄积并非经典脂质合成途径所致，而是源于线粒体脂质氧化能力下降。即使脂质合成未增加，线粒体脂质利用障碍最终仍会导致脂质蓄积。后续研究将明确该过程是否为肌肉脂肪变性发病的必要条件。