Cellular responses to respiratory chain dysfunction

Mitochondria are network-like organelles present in most mammalian cells. They contain the respiratory chain (RC) that produces the majority of the energy needed in the cell, in the form of ATP. RC deficiency can arise due to mutations in the mitochondrial DNA (mtDNA) or in nuclear genes encoding mitochondrial proteins. A number of distinct genetic mitochondrial disease syndromes are known and impairment of RC function has also been implicated in the pathophysiology of several age-associated diseases, such as neurodegeneration, diabetes mellitus II and heart failure, as well as in aging. The pathology of mitochondrial diseases has been studied in numerous patients and it is often presumed that decreased ATP production and increased reactive oxygen species (ROS) formation are critical links between the RC dysfunction and the observed pathology. There are currently no efficient treatments available for mitochondrial diseases and development of novel therapies will require in depth understanding of key pathophysiological events. We have therefore created and characterized mouse models with RC dysfunction. The general aim of this thesis was to investigate how cells respond to RC dysfunction in mitochondrial disease and aging, with focus on RC deficiency occurring in the mouse heart. We selectively disrupted the nuclear gene encoding mitochondrial transcription factor A (TFAM) in heart and skeletal muscle. The TFAM protein is imported into mitochondria where it is essential for maintenance and transcription of mtDNA. Critical components of t he RC are encoded by mtDNA and loss of TFAM therefore results in a severe RC deficiency. The tissue-specific Tfam knockout mice developed severe dilated cardiomyopathy and had a maximal life span of ~12 weeks. The progressive nature of the RC deficiency allowed us to follow temporal changes in gene expression at different stages of mitochondrial cardiomyopathy. Microarray analyses revealed substantial changes in the expression of nuclear genes in the RC deficient mouse heart. We observed a coordinated reduction of transcripts encoding enzymes involved in fatty acid degradation in parallel with an increase of some transcripts encoding glycolytic enzymes at a very early disease stage. This metabolic switch in cardiac energy metabolism is unlikely to be beneficial for the RCdeficient heart and may instead accelerate the pathology. Cardiac excitation-contraction (E-C) coupling was altered in Tfam knockout cardiomyocytes. Transcripts encoding proteins involved in E-C coupling were reduced in RC deficient hearts and smaller and faster Ca2+ transients and reduced Ca2+ storage capacity of the sarcoplasmic reticulum was found in isolated Tfam knockout cardiomyocytes. Interestingly, beta-adrenergic stimulation induced an irregular Ca2+ release pattern that predisposed to severe arrhythmias. These findings may be important when considering treatment strategies for patients with mitochondrial cardiomyopathy. The mtDNA mutator mouse expresses an altered form of the mitochondrial DNA polymerase with impaired proofreading capacity. These knockin mice have elevated levels of somatic point mutations of mtDNA, which cause a progressive RC dysfunction, premature aging symptoms and reduced lifespan. We found no or minor changes of ROS production, expression of ROS scavenging enzymes and oxidative damage in cell lines and tissues from mtDNA mutator mice. These findings contradict the previous dogma that mutations of mtDNA. will lead not only to impaired RC enzyme activities but also to a high production of ROS, which, in turn, further will damage the mtDNA and proteins in the mitochondria and thereby elicit a vicious circle. In summary, RC dysfunction causes distinct changes in Ca2+ handling as well as a metabolic switch in respiratory chain deficient mouse hearts. In addition, our results do not support the hypothesis that increased ROS production and oxidative damage are critical for creating the premature aging phenotypes associated with increased levels of somatic mtDNA mutations