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October 13, 2014
1:00 PM - 2:30 PM
Room Room 231-232
Autophagy Dysfunctions and Anesthesia Related Complications in Muscular Dystrophy
Aki Kashiwagi, M.D., Ph.D., Sachiko Hosokawa, Ph.D., Ryusuke Ueki, M.D., Ph.D., Yusuke Norimatsu, M.D, Ph.D., Jeevendra Martyn, M.D., Shingo Yasuhara, M.D., Ph.D.
Massachusetts General Hospital, Shriners Hospital for Children, Harvard Medical School, Boston, Massachusetts, United States
Background: Duchenne muscular dystrophy (DMD) patients are affected by progressive muscle weakness associated with constantly upregulated myofiber damage and regeneration. DMD patients at advanced stages often receives minor and major orthopedic surgeries to correct the deformities caused by muscle mass loss and muscle contracture. DMD patients are, however, associated with high incidence of anesthesia-related complications including severe hyperkalemia, rhabdomyolysis, malignant hyperthermia, arrhythmia and cardiac arrest. The detailed mechanisms leading to these complications or the therapeutics to prevent these complications are not established yet. It has been widely accepted that autophagy/mitophagy functions as a cytoprotection mechanism defending cells from many forms of stresses. Other lines of evidence have established that general anesthesia causes mitochondrial uncoupling. Previously, we have documented that general anesthesia-induced skeletal muscle autophagy was upregulated. It has not been studied, however, whether myofibers from DMD subjects who have defective autophagy/mitophagy functions can cope with such uncoupling stress imposed by general anesthesia. In this study, we analyzed details of autophagy/mitophagy dysfunction in DMD under anesthesia.

Methods: We anesthetized wild type (WT) and mdx mice (an animal model of DMD) with or without GFP-LC3 expression by isoflurane (1.25% inhalation) or intraperitoneal injection of pentobarbital (65mg/kg). At 150 minutes after induction of anesthesia, tibialis anterior muscles were harvested and blood samples were collected. Plasma levels of potassium, sodium, calcium and creatine kinase (CK) were determined by biochemical analyses. Western Blotting was performed according to the standard procedure using the harvested tibialis anterior muscle samples. In vivo confocal microscopy was performed using the sternomastoid muscle of live WT and mdx mice expressing the autophagy marker transgene, tf-LC3, with or without rapamycin. Fluorescent spectrum of signals from tf-LC3 enables distinction between premature and mature autophagosomes, as green (GFP) and red (RFP) or red (RFP) only labeled vesicles, respectively.

Results: Both isoflurane or pentobarbital anesthesia induced autophagy in WT mice in a time dependent fashion. Western blot analysis showed that the expression of LC3II, an autophagy marker protein increased in both mdx and WT. When autophagy turnover was blocked by E64+PepstatinA (E+A), however, only wild type showed further accumulation of LC3II, suggesting that mdx mice are defective in the maturation step. In vivo microscopy revealed that under the stress imposition, by a local rapamycin injection, WT mice showed both mature and premature autophagosomes (matured autophagosome was 32.14%), but mdx mice only showed premature autophagosomes (matured autophagosome was 0.02%), suggesting that the induction of autophagosome occurred in both WT and mdx mice but the maturation process is defective in mdx mice. Mdx mice with defective autophagy functions showed elevated levels of plasma potassium as compare to control mice at 150 minutes after anesthesia with 1.25% isoflurane. (4.9+/-0.4 vs. 16.1+/-2.3 for GFP-LC3 mdx and GFP-LC3 WT control mice, respectively. Ave+/-SE. Experiments were stopped at moribound when heart rate was <60bpm.) At higher dose of isoflurane with 2%, mdx mice showed higher mortality rate as compared to wild type at 3 hours (75% vs. 0% for GFP-LC3 mdx and GFP-LC3 WT control mice, respectively, N=4).

Conclusions: In this study, in vivo microscopy, and Western Blotting showed the induction of skeletal muscle autophagy after anesthesia in both WT and mdx mice. However, the maturation of skeletal muscle autophagy was severely defective in mdx mice leading to anesthesia-induced hyperkalemia. The skeletal muscle autophagy is likely important in anesthesia- induced complications.

Copyright © 2014 American Society of Anesthesiologists