The human mitochondrial myopathies and encephalomyopathies comprise clinically, morphologically, and biochemically diverse disorders that are now beginning to be described genetically. Since 1988, specific mitochondrial DNA (mtDNA) mutations have been found to result in disease. This group of diseases generally affects differentiated (post-mitotic) tissues, such as muscle and certain neural tissues most severely. Our goal is to analyze in muscle cell cultures the phenotypic and molecular genetic consequences of mutations of mtDNA that result in myopathies and encephalomyopathies. This analysis will take advantage of a cell culture system that was developed to analyze mtDNA mutations in post-mitotic cells. The system selectively eliminates the endogenous mitochondrial complement of cells and repopulates the cells with exogenous mitochondria that contain mtDNA, including mutated mtDNA. Specific mtDNA mutations can be introduced into myoblasts that can differentiate into multinucleated myotubes and can be innervated with rat spinal cord neurons to form long-lived, remarkably mature, and functionally active myofibers. Because defined proportions of a specific mutation can be introduced into these cultures, it is feasible to study systematically and under controlled conditions the phenotypic consequences of mtDNA mutations and determine their molecular genetic causes.
The specific aims of this proposal are to investigate the mechanisms by which mtDNA rearrangements impede protein synthesis and respiratory chain activity in aneural and innervated muscle cultures. In addition, the interactions of mitochondria in the nuclear domains of individual myoblasts in mature myotubes and the genetic complementation of mitochondria possessing non-overlapping deletions of mtDNA will be investigated. If the pathogenetic mechanisms of specific defects can be elucidated, it will facilitate the development of a rational therapy for human patients. In this in vitro system, the exact metabolic requirements for muscle cells with impaired respiratory chain function can be determined. In addition, the effects of different growth conditions or treatments on the mutated and wild-type mtDNAs can be examined. If the mutated genome can be preferentially damaged or inhibited in its replication, it may be possible in the future to devise treatments for these currently incurable, and often fatal diseases.
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