Department of Anesthesiology, Pharmacology & Therapeutics, University of British Columbia (UBC), Vancouver, BC, Canada.
UBC Centre for Heart Lung Innovation, St. Paul's Hospital, Vancouver, BC, Canada.
J Cachexia Sarcopenia Muscle. 2022 Feb;13(1):544-560. doi: 10.1002/jcsm.12879. Epub 2021 Dec 19.
Muscular dystrophy (MD) causes muscle wasting and is often lethal in patients due to a lack of proven therapies. In contrast, mouse models of MD are notoriously mild. We have previously shown severe human-like muscle pathology in mdx [Duchenne MD (DMD)] and dysferlin-deficient limb-girdle MD type 2B (LGMD2B) mice by inactivating the gene encoding for apolipoprotein E (ApoE), a lipid transporter synthesized by the liver, brain and adipocytes to regulate lipid and fat metabolism. Having recently established that human DMD is a novel type of primary genetic dyslipidaemia with elevated cholesterol, we sought to determine whether cholesterol could exacerbate the muscle wasting process observed in severe rodent MD.
Severe mdx and dysferlin knock-out mice lacking ApoE were treated with ezetimibe (15 mg/kg/day), a clinically approved drug exhibiting few pleiotropic effects. In separate studies, dietary cholesterol was raised (from 0.2% to 2% cholesterol) in combination with experimental micro-injury and direct cholesterol injection assays. Muscles were assessed histologically for changes in collagen and adipocyte infiltration and both transcriptomic and cellular changes by RNA-seq and fluorescence-activated cell sorting analysis.
Treatment of severe DMD and LGMD2B mice with ezetimibe completely prevented clinical signs of ambulatory dysfunction (0% incidence vs. 33% for vehicle treatment; P < 0.05). Histological analyses revealed that ezetimibe-reduced fibro-fatty infiltration up to 84% and 63% in severely affected triceps (P ≤ 0.0001) and gastrocnemius (P ≤ 0.003) muscles, resulting in a respective 1.9-fold and 2.2-fold retention of healthy myofibre area (P ≤ 0.0001). Additionally, raising dietary cholesterol and thus concentrations of plasma low-density lipoprotein-associated cholesterol (by 250%; P < 0.0001) reduced overall survivability (by 100%; P < 0.001) and worsened muscle damage in the LGMD2B triceps by 767% (P < 0.03). Micro-pin-induced mechanical injury in LGMD2B mice fed a high cholesterol diet exacerbated muscle damage by 425% (P < 0.03) and increased macrophage recruitment (by 98%; P = 0.03) compared with those injured on a chow diet. Parallel RNA-seq analyses revealed that injury in cholesterol-fed mice also modulated the expression of 3671 transcripts (1953 up-regulated), with fibrogenic, inflammatory and programmed cell death-associated pathways among the most enriched. Mice lacking dysferlin also displayed heightened muscle necrosis (by 123%; P < 0.0001) following a direct intramuscular injection of cholesterol compared with control mice.
Cholesterol exacerbates rodent MD. Specific inhibition of cholesterol absorption with ezetimibe may safely attenuate human MD severity and delay death.
肌肉萎缩症（MD）导致肌肉消耗，由于缺乏经过验证的治疗方法，患者通常因此致命。相比之下，MD 的小鼠模型通常较为轻微。我们之前通过使载脂蛋白 E（ApoE）基因失活，在 mdx [杜氏肌营养不良症（DMD）]和肌营养不良蛋白 2B 型肢带型 MD（LGMD2B）缺陷小鼠中观察到严重的类似人类的肌肉病理学，ApoE 是一种由肝脏、大脑和脂肪细胞合成的脂质转运蛋白，用于调节脂质和脂肪代谢。最近我们发现人类 DMD 是一种新型原发性血脂异常，胆固醇升高，我们试图确定胆固醇是否会加剧严重啮齿动物 MD 中观察到的肌肉消耗过程。
用依泽替米贝（ezetimibe）（15mg/kg/天）治疗缺乏 ApoE 的严重 mdx 和肌营养不良蛋白缺陷小鼠，依泽替米贝是一种具有很少多效性作用的临床批准药物。在单独的研究中，通过提高饮食中的胆固醇（从 0.2%提高到 2%胆固醇），并结合实验性微损伤和直接胆固醇注射检测，来研究胆固醇对肌肉的影响。通过组织学分析评估胶原蛋白和脂肪细胞浸润的变化，通过 RNA-seq 和荧光激活细胞分选分析评估转录组和细胞变化。
用依泽替米贝治疗严重的 DMD 和 LGMD2B 小鼠可完全预防运动功能障碍的临床症状（依泽替米贝治疗组的发生率为 0%，而对照组为 33%；P<0.05）。组织学分析显示，依泽替米贝减少了高达 84%和 63%的纤维脂肪浸润，分别为严重影响的三头肌（P≤0.0001）和比目鱼肌（P≤0.003），从而使健康肌纤维面积分别保留了 1.9 倍和 2.2 倍（P≤0.0001）。此外，提高饮食中的胆固醇水平，从而提高与血浆低密度脂蛋白相关的胆固醇浓度（增加 250%；P<0.0001），降低了整体存活率（减少 100%；P<0.001），并使 LGMD2B 三头肌的肌肉损伤恶化了 767%（P<0.03）。在喂食高胆固醇饮食的 LGMD2B 小鼠中，微针引起的机械损伤使肌肉损伤恶化了 425%（P<0.03），并增加了巨噬细胞募集（增加 98%；P=0.03），与在普通饮食中受伤的小鼠相比。平行的 RNA-seq 分析显示，在胆固醇喂养的小鼠中，损伤还调节了 3671 个转录本的表达（1953 个上调），其中纤维化、炎症和程序性细胞死亡相关途径最为丰富。与对照小鼠相比，缺乏肌营养不良蛋白的小鼠在肌肉内直接注射胆固醇后，肌肉坏死也增加了 123%（P<0.0001）。
胆固醇会加剧啮齿动物 MD。用依泽替米贝特异性抑制胆固醇吸收可能安全地减轻人类 MD 的严重程度并延迟死亡。
