RESUMO
The regulation of mitochondrial DNA (mtDNA) expression is crucial for mitochondrial biogenesis during development and differentiation. We have disrupted the mouse gene for mitochondrial transcription factor A (Tfam; formerly known as m-mtTFA) by gene targetting of loxP-sites followed by cre-mediated excision in vivo. Heterozygous knockout mice exhibit reduced mtDNA copy number and respiratory chain deficiency in heart. Homozygous knockout embryos exhibit a severe mtDNA depletion with abolished oxidative phosphorylation. Mutant embryos proceed through implantation and gastrulation, but die prior to embryonic day (E)10.5. Thus, Tfam is the first mammalian protein demonstrated to regulate mtDNA copy number in vivo and is essential for mitochondrial biogenesis and embryonic development.
Assuntos
DNA Mitocondrial , Proteínas de Ligação a DNA/genética , Morte Fetal/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Mitocondriais , Proteínas Nucleares , Fatores de Transcrição/genética , Proteínas Virais , Animais , Proteínas de Ligação a DNA/metabolismo , Implantação do Embrião , Feminino , Retardo do Crescimento Fetal/genética , Dosagem de Genes , Coração/embriologia , Heterozigoto , Proteínas de Grupo de Alta Mobilidade , Integrases/genética , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mutação , Fosforilação , Fatores de Transcrição/metabolismoRESUMO
Mutations of mitochondrial DNA (mtDNA) cause several well-recognized human genetic syndromes with deficient oxidative phosphorylation and may also have a role in ageing and acquired diseases of old age. We report here that hallmarks of mtDNA mutation disorders can be reproduced in the mouse using a conditional mutation strategy to manipulate the expression of the gene encoding mitochondrial transcription factor A (Tfam, previously named mtTFA), which regulates transcription and replication of mtDNA. Using a loxP-flanked Tfam allele (TfamloxP) in combination with a cre-recombinase transgene under control of the muscle creatinine kinase promoter, we have disrupted Tfam in heart and muscle. Mutant animals develop a mosaic cardiac-specific progressive respiratory chain deficiency, dilated cardiomyopathy, atrioventricular heart conduction blocks and die at 2-4 weeks of age. This animal model reproduces biochemical, morphological and physiological features of the dilated cardiomyopathy of Kearns-Sayre syndrome. Furthermore, our findings provide genetic evidence that the respiratory chain is critical for normal heart function.
Assuntos
Cardiomiopatia Dilatada/genética , DNA Mitocondrial , Proteínas de Ligação a DNA , Regulação da Expressão Gênica , Bloqueio Cardíaco/genética , Coração/fisiopatologia , Proteínas de Grupo de Alta Mobilidade , Proteínas Mitocondriais , Proteínas Nucleares , Transativadores , Fatores de Transcrição/biossíntese , Proteínas Virais , Proteínas de Xenopus , Animais , Cardiomiopatia Dilatada/fisiopatologia , Creatina Quinase/genética , Modelos Animais de Doenças , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Bloqueio Cardíaco/fisiopatologia , Humanos , Integrases/genética , Masculino , Camundongos , Camundongos Transgênicos , Músculo Esquelético , Miocárdio , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fatores de Transcrição/genéticaRESUMO
We recently described a mouse model that reproduces important pathophysiological features of mitochondrial DNA (mtDNA) mutation diseases. The gene for mouse mitochondrial transcription factor A, Tfam (also called mtTFA), a nucleus-encoded key regulator of mtDNA expression, was targeted with loxP sites (Tfam(loxP)) and disrupted in vivo by transgenic expression of cre-recombinase from the muscle creatinine kinase (Ckmm) promoter. This promoter is active from embryonic day 13, and the knockouts had normal respiratory chain function in the heart at birth and developed mitochondrial cardiomyopathy postnatally. In this paper, we describe a heart-knockout strain obtained by mating Tfam(loxP) mice to animals expressing cre-recombinase from the alpha-myosin heavy chain (Myhca) promoter. This promoter is active from embryonic day 8, and the knockouts had onset of mitochondrial cardiomyopathy during embryogenesis. The age of onset of cardiac respiratory chain dysfunction can thus be controlled by temporal regulation of cre-recombinase expression. Further characterization demonstrated that approximately 75% of the knockouts died in the neonatal period, whereas, surprisingly, approximately 25% survived for several months before dying from dilated cardiomyopathy with atrioventricular heart conduction blocks. Modifying gene(s) affect the life span of the knockouts, because approximately 95% of the knockout offspring from an intercross of the longer-living knockouts survived the neonatal period. Thus, the tissue-specific knockouts we describe here not only reproduce important pathophysiological features of mitochondrial cardiomyopathy but also provide a powerful system by which to identify modifying genes of potential therapeutic value.