Remedying muscle disorders by targeting muscle apoptosis and autophagy pathways

Abstract

The aim of this thesis was to investigate whether skeletal muscle disorders can be remedied by targeting different molecular cell death pathways. The first muscle disorder being studied in this thesis was related to the impairment of insulin signaling in skeletal muscle, which is also commonly referred to as skeletal muscle insulin resistance, in which muscle cells do not respond adequately to normal physiological insulin concentration and is a key feature of type 2 diabetes. The current thesis showed that 1) insulin signaling in diabetic skeletal muscle was impaired as evidenced by the dysfunctional Akt-PI3k-PDK1 signaling pathway, 2) mTOR signaling was activated by the phosphorylation of PRAS40 at Thr246 and 3) autophagy was inactivated in skeletal muscle under diabetic condition. Our results demonstrated that unacylated ghrelin (UnAG), a natural peptide hormone, restored the impaired insulin signaling in skeletal muscle of diabetic mice (db/db mice). Intriguingly, UnAG was shown to normalize the suppressed autophagic signaling and flux in diabetic muscle. Autophagy is regarded as an evolutionarily conserved intracellular mechanism that degrades and recycles malfunctioning organelles and long-lived proteins. This thesis is the first to propose a cross-talk between insulin signaling pathway and autophagy in skeletal muscle. By targeting autophagic pathway, skeletal muscle insulin resistance can probably be improved. Besides pharmacological activation of autophagy, the current thesis tested the hypothesis that whether long-term habitual exercise could also alter autophagic signaling in skeletal muscle. Interestingly, basal autophagy could be enhanced by long-term habitual exercise. Analyses of autophagic protein markers (e.g., LC3, LC3-II/LC3-I, p62, Atgs and Beclin-1) and the mitochondrial biogenesis factor, PGC-1a, confirmed that the enhancement of basal autophagy and mitochondrial biogenesis were not accompanied by the increased expression of autophagic proteins. Furthermore, this thesis is the first to demonstrate an association between autophagy and muscle fiber-type shifting, which prompted us to speculate that muscle fiber-type shifting might be a result of a series of mitochondrial degradation and biogenesis. Through recycling of mitochondria, the metabolic properties of skeletal muscle might be reformulated. In addition to skeletal muscle autophagy, exercise-induced cardiac muscle autophagy was also studied. Consistently, basal autophagy was enhanced in cardiac muscle by long-term habitual exercise. These findings have important implications in the prevention and treatment of diabetes and skeletal muscle insulin resistance. The second muscle disorder being studied in this thesis was pressure ulcer, which is caused by sustained pressure and shear force being applied on a particular part of the body. Patients with diabetes are more vulnerable to the development of pressure ulcers as high blood sugar causes injures to blood vessels rendering them unable to deliver a sufficient amount of blood to the skeletal muscle cells. Before identifying a therapeutic target to treat ulceration in diabetic patients, the underlying mechanism of pressure ulcer needs to be elucidated. Pressure ulcer, also known as bed sore, can happen to anyone with limited mobility. It has been previously demonstrated that pharmacological inhibition of caspase is effective in relieving muscle damage induced by mechanical compression speculating that pressure ulcer is mediated by intrinsic apoptotic pathway. Therefore, novel BaxBak double-deficient (DKO) mice were generated to test the hypothesis that animals with combined loss of pro-apoptotic genes bax and bak are resistant to the development of pressure ulcer (or pressure-induced injury). Immunobloting data, TUNEL-staining results and Cell Death ELISA-indicating DNA fragmentation results have consistently shown that skeletal muscle apoptosis can be inhibited in our DKO mice. Apart from preventing the development of pressure ulcer in DKO mice, this thesis is the first to 1) evidently prove that pressure-induced inflammation and secondary necrosis are mediated by muscle apoptosis and 2) determine the role of autophagy in pressure ulcer by examining autophagy inhibition in wild type animals, bak single-deficient animals (SKO) and DKO animals. Conclusively, the current thesis has demonstrated that UnAG normalizes impaired insulin signaling and re-activated suppressed autophagic signaling in diabetic muscle. Besides pharmacological treatment, long-term habitual exercise, as a non-pharmacological treatment, has also been shown to increase basal autophagy in both skeletal muscle and cardiac muscle. Last but not least, this thesis has proved that apoptosis mediates the development of pressure ulcer. Animals with combined loss of Bax and Bak have been shown to be resistant to the development of pressure ulcer. Collectively, these significant findings may provide some hints on remedying muscle disorders.