Division of Vascular Surgery; Department of Surgery, University of Pittsburgh Medical Center, Magee Women's Hospital, 200 Lothrop Street, Pittsburgh, PA, 15213, USA.
McGowan Institute for Regenerative Medicine, University of Pittsburgh Medical Center, Bridgeside Point, Pittsburgh, PA, 15213, USA.
Mol Med. 2020 Jul 8;26(1):69. doi: 10.1186/s10020-020-00190-2.
We previously showed that the autophagy inhibitor chloroquine (CQ) increases inflammatory cleaved caspase-1 activity in myocytes, and that caspase-1/11 is protective in sterile liver injury. However, the role of caspase-1/11 in the recovery of muscle from ischemia caused by peripheral arterial disease is unknown. We hypothesized that caspase-1/11 mediates recovery in muscle via effects on autophagy and this is modulated by CQ.
C57Bl/6 J (WT) and caspase-1/11 double-knockout (KO) mice underwent femoral artery ligation (a model of hind-limb ischemia) with or without CQ (50 mg/kg IP every 2nd day). CQ effects on autophagosome formation, microtubule associated protein 1A/1B-light chain 3 (LC3), and caspase-1 expression was measured using electron microscopy and immunofluorescence. Laser Doppler perfusion imaging documented perfusion every 7 days. After 21 days, in situ physiologic testing in tibialis anterior muscle assessed peak force contraction, and myocyte size and fibrosis was also measured. Muscle satellite cell (MuSC) oxygen consumption rate (OCR) and extracellular acidification rate was measured. Caspase-1 and glycolytic enzyme expression was detected by Western blot.
CQ increased autophagosomes, LC3 consolidation, total caspase-1 expression and cleaved caspase-1 in muscle. Perfusion, fibrosis, myofiber regeneration, muscle contraction, MuSC fusion, OCR, ECAR and glycolytic enzyme expression was variably affected by CQ depending on presence of caspase-1/11. CQ decreased perfusion recovery, fibrosis and myofiber size in WT but not caspase-1/11KO mice. CQ diminished peak force in whole muscle, and myocyte fusion in MuSC and these effects were exacerbated in caspase-1/11KO mice. CQ reductions in maximal respiration and ATP production were reduced in caspase-1/11KO mice. Caspase-1/11KO MuSC had significant increases in protein kinase isoforms and aldolase with decreased ECAR.
Caspase-1/11 signaling affects the response to ischemia in muscle and effects are variably modulated by CQ. This may be critically important for disease treated with CQ and its derivatives, including novel viral diseases (e.g. COVID-19) that are expected to affect patients with comorbidities like cardiovascular disease.
我们之前的研究表明，自噬抑制剂氯喹（CQ）可增加肌细胞中炎症性裂解的半胱天冬酶-1 活性，而 caspase-1/11 在非感染性肝损伤中具有保护作用。然而，caspase-1/11 在由外周动脉疾病引起的肌肉缺血再灌注中的作用尚不清楚。我们假设 caspase-1/11 通过对自噬的影响来介导肌肉的恢复，而 CQ 可以调节这种作用。
C57Bl/6J（WT）和 caspase-1/11 双重敲除（KO）小鼠接受股动脉结扎（后肢缺血模型），同时给予或不给予 CQ（50mg/kg，每隔一天腹腔注射一次）。通过电子显微镜和免疫荧光检测 CQ 对自噬体形成、微管相关蛋白 1A/1B-轻链 3（LC3）和 caspase-1 表达的影响。激光多普勒灌注成像每 7 天记录一次灌注。21 天后，在胫骨前肌中进行原位生理测试，评估最大力收缩，还测量肌纤维大小和纤维化。测量肌肉卫星细胞（MuSC）耗氧量（OCR）和细胞外酸化率。通过 Western blot 检测 caspase-1 和糖酵解酶的表达。
CQ 增加了肌肉中的自噬体、LC3 凝聚、总 caspase-1 表达和裂解的 caspase-1。CQ 对灌注、纤维化、肌纤维再生、肌肉收缩、MuSC 融合、OCR、ECAR 和糖酵解酶的表达的影响因 caspase-1/11 的存在而不同。CQ 降低了 WT 小鼠的灌注恢复、纤维化和肌纤维大小，但对 caspase-1/11KO 小鼠没有影响。CQ 降低了整体肌肉的最大力，减少了 MuSC 中的肌细胞融合，这些影响在 caspase-1/11KO 小鼠中更为严重。CQ 降低了 caspase-1/11KO 小鼠的最大呼吸和 ATP 产生。caspase-1/11KO MuSC 的蛋白激酶同工酶和醛缩酶显著增加，ECAR 减少。
caspase-1/11 信号通路影响肌肉对缺血的反应，CQ 对其作用的调节存在差异。这对于用 CQ 及其衍生物治疗的疾病可能至关重要，包括预计会影响患有心血管疾病等合并症的新型病毒性疾病（如 COVID-19）。
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