Mutations with mtDNA have the potential to cause cardiomyopathy and sudden death, as seen in certain rare, inherited syndromes (Kearn-Sayres and others). An accumulation of mutated forms of mtDNA relative to functional, intact mitochondrial genomes also has been implicated in cardiac contractile dysfunction associated with much more common disorders, including: advanced age, idiopathic dilated cardiomyopathy, hypertrophic cardiomyopathy, chronic ischemic heart disease, and drug toxicity (AZT or doxorubicin). In these latter conditions, however, direct evidence for a causal relationship between defects in mtDNA and cardiac dysfunction has been lacking. Up to now, experimental manipulation of mtDNA in animals has not been possible. In this application, the applicant proposes a set of experiments to determine the relationship between DNA copy number, respiratory function, and contractile performance in the intact heart. First, the investigator will complete the molecular cloning of the mouse mitochondrial DNA polymerase (mtPOL) and a factor that terminates mtDNA replication (mtTER). He hypothesizes that altering expression of these proteins in vivo will have one of two sequelae: changes in the copy number of normal mtDNA genomes or increased frequency of mtDNA deletions. To test this hypothesis, the applicant will manipulate expression of these genes in cultured skeletal myotubes (using indelible overexpression and antisense oligonucleotides) and assess the effects on mtDNA copy number and the abundance of deleted forms of mtDNA. Subsequently, he expects to modulate mtDNA copy number and generate deleted forms of mtDNA in the intact heart of transgenic mice using cardiac-specific knock-out of the mtPOL gene and conventional knock-out of an essential mtTER subunit gene, respectively. In addition, the investigator proposes to generate myogenic cell lines and transgenic mice with physiologically significant point mutations in mtDNA by forced expression of a mutated variant of mtPOL defective in 3'-5' exonuclease (proofreading) function. Heart rate and left ventricular dP/dtmax will be measured in these animals, and cardiac respiratory function will be assessed in isolated hearts using 31P NMR spectroscopy under normoxic and ischemic conditions. The applicant hopes these experiments will determine, quantitatively, the severity of contractile and metabolic dysfunction resulting from a given reduction in mtDNA copy number, or from a given degree of heteroplasmy for defined deletions or point mutations in mtDNA.
