The objective of this proposal is to elucidate the molecular mechanism of the pathogenesis of mitochondrial myopathy and encephalomyopathy. We propose to employ both in vitro and in vivo approaches to identify the metabolic disorders in skeletal muscle of patients and to combine the techniques of biochemistry and biophysics together with clinical investigation. The following approaches will be used: (I). To identify the metabolic disorder(s) in skeletal muscle of patients by evaluating the biochemical and biophysical properties of preparations derived from skeletal muscle biopsies. A. To characterize the isolated mitochondrial preparations with respect to the respiratory and phosphorylating properties, the various partial reactions of the respiratory chain, the ATPase activities, the respiratory chain carrier contents, fatty acid and ketoacid oxidative metabolism and the functional aspects of the mitochondrial membrane; B. to characterize the crude tissue extracts of small biopsy specimens with respect to the respiratory and phosphorylating properties, and various partial reactions of the respiratory chain and those associated with the TCA cycle intermediates; and C. to identify site(s) of structural alterations in biopsy specimens and the preparations derived therefrom by means of ultrastructural examination in parallel with the biochemical studies. (II). To elucidate the molecular mechanism of the therapeutic effects of glucocorticoids in patients. A. To evaluate the in vivo muscle energy metabolism before and after the glucocorticoid treatment by means of 31P-NMR spectroscopy; B. To examine the phospholipase A2 and its inhibitor protein activities in patients' plasma before and after glucocorticoid treatment for possible induction of lipomodulin biosynthesis. C. To elucidate the action of glucocorticoid on mitochondrial metabolism or rats: i. to examine the in vitro effects of glucocorticoids on energy metabolism of isolated mitochondrial preparations of skeletal muscle, liver and heart of rat; and ii. to examine the in vivo effects of glucocorticoids in rats by studying the oxidative metabolism of mitochondrial preparations derived therefrom, and by examing the phospholipase A2 activities of the rat plasma before and after drug treatment. Analysis of the biochemical studies, the 31P-NMR studies and the clinical studies will lend assistance in searching for possible links between the pathological states and the metabolic disorders, in identifying the cause of disease, and ultimately in formulating the proper therapeutic treatments.
