AMP Deaminase 1 Transcriptional Regulation and Knockdown Mouse Skeletal Muscle Function
Open AccessThe group of idiopathic inflammatory myopathies, collectively known as myositis, have traditionally been diagnosed and treated as autoimmune diseases. They involve cytotoxic T cells and antibodies attacking host tissue, usually respond to immunosuppressive treatment, and are associated with both environmental exposures as well as inherited alleles of immune genes like MHC Class I. Patients with myositis may experience muscle weakness, fatigue, or pain and often have systemic pathologies of the skin, lungs, or gastrointestinal tract. The standard therapy is administration of glucocorticoids and immunosuppressive drugs, but there currently are no treatments specifically for myositis. Although the current drugs can eliminate inflammatory cells within the muscle, most patients do not fully regain strength. Many recent studies have shown that there is no correlation between the amount of muscle inflammatory infiltration and weakness. Therefore, we sought other explanations for refractory muscle weakness in myositis. We and others have observed an acquired deficiency of the muscle-specific, metabolic enzyme AMP deaminase 1 (AMPD1) in both patients and a mouse model of myositis. AMPD1 catalyzes the rate-limiting step of the purine nucleotide cycle, and a deficiency in this enzyme has been hypothesized to cause muscle weakness, fatigue, or cramping. Our hypothesis was that an acquired AMPD1 deficiency is at least partially responsible for muscle weakness in myositis.In this dissertation, our three aims were to identify drugs that could increase the amount of AMPD1 in muscle, uncover mechanisms of how an AMPD1 deficiency could be acquired in myositis, and create a genetically engineered mouse in which Ampd1 is turned off to assess the gene’s effect on muscle strength and fatigue. We used the cutting-edge technique of quantitative high throughput screening to test over 4,000 compounds at more than 7 concentrations each and found that microtubule inhibitor drugs such as podophyllotoxin can increase the expression of AMPD1 in cell culture. To investigate how an AMPD1 deficiency could be acquired, we subjected both cultured muscle cells as well as mice to factors associated with myositis pathology and found that TLR7 stimulation, ER stress, and MHC Class I over-expression can decrease Ampd1 expression. Finally, we created a conditional, skeletal muscle-specific, Ampd1 knockdown (KD) mouse and performed standardized and rigorous protocols to measure muscle strength, fatigue, and recovery from fatigue. Compared to normal wild-type mice, the KD mice were not weak and did not fatigue quicker, but unexpectedly, their isolated extensor digitorum longus muscles had an improved recovery from ex vivo fatigue protocols.Although Ampd1 KD did not cause weakness nor fatigue by our measures in mice, a deficiency might still cause these symptoms in otherwise normal humans, in the presence of a disease, or in the context of other genes. Our drug screening efforts have identified the first known compounds to increase the expression of AMPD1, namely the microtubule inhibiting drugs like podophyllotoxin. These drugs were very potent and a custom synthetic library of aza-podophyllotoxin analogues is currently being explored for an improved therapeutic profile. Our studies on the regulation of Ampd1 expression have provided insight to up-stream causes of AMPD1 deficiency occurring in myositis. We found that MHC class I over-expression is likely the cause of ER stress as well as Ampd1 down-regulation. The finding that Ampd1 KD muscle recovers more force after resting from fatigue requires further research and may be related to altered metabolite levels or improved blood flow observed by others’ in humans deficient for AMPD1. Although an acquired AMPD1 deficiency may not be the cause of muscle weakness in myositis, we have discovered some of the mechanisms that control its transcriptional regulation and found that it may play a role in muscle recovery after fatigue.
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