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Life without mitochondrial DNA : studies of transgenic mice
Mitochondrial DNA (mtDNA) is a closed circular DNA genome that resides in the mitochondrial network. Mutations of mtDNA cause spontaneous and hereditary disorders known as mitochondrial diseases. Mitochondrial transcription factor A (Tfam) is a key factor for transcription of mtDNA in vitro. We disrupted the mouse Tfam gene by using the cre-loxP recombination system to study the in vivo roles of Tfam. This thesis focuses on the analyses of germline knockout mice and the characterization of tissue-specific knockout mice with cardiomyopathy due to the disruption of oxidative phosphorylation (OXPHOS) in heart. We also sought to dissect the pathogenesis of mitochondrial disease by studying the role of apoptosis in these animal models.
Germline Tfam knockout mice (Tfam-/Tfam-) displayed embryonic lethality. The homozygous mutant embryos showed growth retardation at embryonic day (E)8.5 with absence of heart and optic discs and delayed neural development. The knockouts had abnormal mitochondrial ultrastructure and enzyme histochemical studies demonstrated the lack of OXPHOS. Molecular biological studies showed the absence of Tfam protein and mtDNA. We thus conclude that has a critical role in maintaining mitochondrial function and OXPHOS in vivo (Paper I).
We crossed Tfam1oxP/Tfam1oxP mice to muscle creatinine kinase (Ckmm)-cre transgenic mice to selectively disrupt Tfam in heart and skeletal muscle. Tissue-specific knockout mice (Tfam1oxP/Tfam1oxP, +/Ckmm-cre developed dilated cardiomyopathy with atrioventricular heart conduction blocks in the postnatal period and died at the age of 2-4 weeks. These animals showed reduced mtRNA and mtDNA levels in the heart and skeletal muscle, reduced levels of the mtDNA-encoded ATP8 protein, impaired respiratory chain function and morphologically abnormal mitochondria in the heart, whereas the respiratory chain function was normal in skeletal muscle. We have thus generated an animal model that closely reproduces pathophysiological features of mitochondrial cardiomyopathy (Paper II).
In order to study the role of OXPHOS in embryonic heart development, we produced a novel heart-specific knockout strain by mating Tfam1oxP mice to [alpha]-myosin heavy chain (Myhca)-cre transgenics that express cre-recombinase in the heart early in embryogenesis. As expected, the knockouts developed mitochondrial cardiomyopathy during embryogenesis and 75% of the knockouts succumbed in the neonatal period. Surprisingly, 25% survived for several months before developing mitochondrial cardiomyopathy. When we mated the long-living knockouts to each other, 95% of the resulting offspring survived the neonatal period. Our data suggest that modifying gene(s) affect the life span of these knockout animals. Our data are compatible with the hypothesis that the modifying genes do not affect the cre-loxP recombination efficiency, but rather act to stabilize mitochondrial protein levels (Paper III).
Cell loss is observed in human mitochondrial disease and also in animal models of these diseases. We addressed the question if cells with deficient OXPHOS can undergo apoptosis in vivo. We demonstrated apoptosis in the hearts of Tfam1oxP/Tfam1oxP, +/Ckmm-cre animals by TUNEL staining and DNA ladder gel assays and in E9.5 day Tfam-/Tfam-embryos by TUNEL staining and immunostaining for cleaved caspase 3. We further demonstrated that a human cell line lacking mtDNA ([rho]0 cells) undergo apoptosis after stimulation by various inducers. We conclude that cells lacking OXPHOS are more prone to undergo apoptosis in vivo. This study provides an insight into the mechanism of cell loss found in human mitochondrial disorders (Paper IV).
List of scientific papers
I. Larsson NG, Wang J, Wilhelmsson H, Oldfors A, Rustin P, Lewandoski M, Barsh GS, Clayton DA (1998). "Mitochondrial transcription factor A is necessary for mtDNA maintenance and embryogenesis in mice. " Nat Genet 18(3): 231-6
https://pubmed.ncbi.nlm.nih.gov/9500544
II. Wang J, Wilhelmsson H, Graff C, Li H, Oldfors A, Rustin P, Bruning JC, Kahn CR, Clayton DA, Barsh GS, Thoren P, Larsson NG (1999). "Dilated cardiomyopathy and atrioventricular conduction blocks induced by heart-specific inactivation of mitochondrial DNA gene expression. " Nat Genet 21(1): 133-7
https://pubmed.ncbi.nlm.nih.gov/9916807
III. Li H, Wang J, Wilhelmsson H, Hansson A, Thoren P, Duffy J, Rustin P, Larsson NG (2000). "Genetic modification of survival in tissue-specific knockout mice with mitochondrial cardiomyopathy. " Proc Natl Acad Sci U S A 97(7): 3467-72
https://pubmed.ncbi.nlm.nih.gov/10737799
IV. Wang J, Silva JP, Gustafsson C, Rustin P, Larsson NG (2000). "Increased apoptosis in vivo in cells lacking mitochondrial DNA gene expression." (Manuscript)
History
Defence date
2000-12-01Department
- Department of Molecular Medicine and Surgery
Publication year
2000Thesis type
- Doctoral thesis
ISBN-10
91-628-4491-1Number of supporting papers
4Language
- eng