84 Mitochondrial gene shifting and aging: Resistance is not futile

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of controls, using oligonucleotide microarrays resulted in the detection of a number of differentially expressed genes. Dystrophin (4.1- to 1.1-fold), SOD2 (1.1- to 1.6-fold) and importin 7 (1.9to 3.6-fold) are significantly down-regulated, while peroxin 19 (1.1to 1.5-fold) is significantly up-regulated in both A3243G and A3260G patients. Interestingly, aldolase B is differentially expressed gene between the A3243G and A3260G group. The A3243G group displays a marginal decrease in the level of this enzyme (0.9 to 0.6), while the A3260G group displays a marked increase in its expression (11.5to 13.0-fold). Such differential gene expression profiles may prove to be a beneficial guide for the diagnosis of specific mitochondrial DNA mutations. This research was supported by CIHR and Warren Lammert and Kathy Corkins. doi:10.1016/j.mito.2007.08.085

82 Clinical case histories suggest intracellular mitochondrial up regulation reverses genomic instability Mike Nagel, Micheal Nichols *, Cardiovascular Specialist, Past President of the California Chapter of the American College of Cardiology Tempus Clinic Hypothesis: Clinical case histories suggest that intracellular mitochondrial up regulation reverses genomic instability either by orchestrating nuclear gene expression for wild-type or demand driven natural selecting for the most efficient mitochondrial turnover. Energy demand up regulation with selective mitochondria turnover clearly improves VO2 Max that can only be explained by increased intracellular mitochondrial efficiency or density, or both. Clinical case results from mitochondrial/hormonal transformation therapeutic protocols based on an extrapolated understanding emerging scientific knowledge of mitochondrial animal and fungus studies, as well as both molecular and cellular mitochondrial research has reversal a spectrum of chronic mitochondrial diseases. Although further study is needed to prove the origins of the mitochondrial improvement, the correlation of VO2 Max and disease reversal is strong. Looking at cases having VO2 Max gains of most over 25%, and analyzing both body mass and composition data, would suggest significant gains in intracellular mitochondrial output. Case 1: Non-alcoholic Fatty Liver Disease Reversal. Case 2: Lipid Profile Reversal (while weaning from drugs). Case 3: Coronary Artery Disease: Coronary Calcium Reversal. Case 4: Osteoporosis with CAD: Improved Bone Mineral Density with simultaneous Coronary Calcium Reversal. Case 5: Osteoporosis: Improved Bone Mineral Density. Case 6: Diabetes Type 2: Medical Marker Reversal (while weaning from prescribed drugs). Case 7: Insulin resistance: Medical Marker Reversal (without drugs). Case 8: Post Operative Cancer recovery. Case 9: Post Chemotherapy Cancer recovery. Case 10: Obesity. Conclusion: Although energy demand up regulation causes whole body mitochondrial output metrics to improve, clinical case histories suggest that new tissue cell growth is not sufficient to explain total oxygenation results. Therefore intracellular mitochondriogenesis must result in increased mitochondrial density, up regulated efficiency or both. doi:10.1016/j.mito.2007.08.086

83 Increased risk of type 2 diabetes in patients with mitochondrial cytopathy and the relationship with obesity Heathcliff D’Sa a,*, Sandeep Raha a,d, Murray Potter c, Mark a,b,d Tarnopolsky a Department of Pediatrics, McMaster University, Hamilton, Ont., L8N 3Z5; b Department of Medicine, McMaster University, Hamilton, Ont., L8N 3Z5; c Department of Molecular and Laboratory Medicine, McMaster University, Hamilton, Ont., L8N 3Z5; d Department of Medical Sciences, McMaster University, Hamilton, Ont., L8N 3Z5 Recent studies have established that in lean offspring of parents with type 2 diabetes there is a reduction in mitochondrial function. Other studies have shown that in obese individuals that have a predisposition towards diabetes, skeletal muscle mitochondria are smaller in size, and have reduced oxidative capacity. To test the hypothesis that impairments in mitochondrial function are associated with obesity and diabetes, subjects with primary mitochondrial diseases (MITO) (44 ± 10 yrs) were compared with obese (41 ± 7 yrs) and lean (39 ± 6 yrs) individuals on the basis of various tests of metabolic and mitochondrial function. Subjects with normal fasting glucose completed an oral glucose tolerance test (OGTT) to measure glucose utilization, and resting blood samples were collected for analysis of markers of inflammation and obesity, along with muscle biopsy analysis for measurement of respiratory chain enzyme activity. In spite of normal fasting blood glucose, MITO subjects had elevated twohour plasma glucose values (p < 0.05). Among MITO subjects, 7% showed impaired glucose tolerance and 43% had type 2 diabetes. No significant differences were found in the inflammatory markers TNF-a and IL-6. Leptin concentrations were increased in the obese group (p < 0.05), while adiponectin levels showed no differences between group. Cytochrome c oxidase (COX) activity was lower in the MITO group and citrate synthase (CS) activity was higher in the MITO vs. obese group (p < 0.05). COX/CS ratio was significantly lower in the MITO group (p < 0.05) and beta-oxidation (SCHAD activity) was significantly higher in the MITO vs. lean group (p < 0.05). The metabolic pattern found in obese individuals who have a predisposition to diabetes was not found in patients who have mitochondrial disease, indicating that the development of diabetes in the MITO group is a consequence of their primary mitochondrial dysfunction. This research was supported by CIHR, The Hamilton Health Sciences Foundation, and Warren Lammert and Kathy Corkins. doi:10.1016/j.mito.2007.08.087

84 Mitochondrial gene shifting and aging: Resistance is not futile Adeel Safdar a,*, Sandeep Raha b, Jeremy Paikin b, Steven R. Smith c, Mark A. Tarnopolsky b a Kinesiology, McMaster University, 1200 Main Street West, Hamilton, Canada L8N 3Z5; b Pediatrics and Medicine, McMaster University, 1200 Main Street West, Hamilton, Canada L8N 3Z5; c Endocrinology, Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, LA 70808, USA The role of mitochondrial abnormalities and mitochondrial DNA (mtDNA) deletions in the etiology of skeletal muscle fiber loss with aging has been extensively characterized. In older adults, resistance exercise causes satellite cell activation and skeletal muscle hypertrophy, which is an effective countermeasure to sarcopenia. However, the protective mechanism underlying this effect is unclear. The objectives of this study were (1) to establish the baseline differences in mtDNA deletions and oxidative capacity in mature muscle vs. myoblasts of young vs. older subjects, and (2) to investigate the influence of 6 months of resistance exercise in older subjects on mtDNA deletions, total DNA oxidative damage, and mitochondrial oxidative capacity. We analyzed mature muscle and primary myoblasts derived from the vastus lateralis of young (mean ± SD: 22 ± 2 y, n = 8) and older (70 ± 5 y, n = 8) subjects for mtDNA deletions,

Abstracts / Mitochondrion 7 (2007) 404–433 total DNA 8-hydroxy-2-deoxyguanosine (8-OHdG) content, and cytochrome c oxidase (COX) enzyme activity. The same indices were quantified in the mature muscle of older adults before and after 6 months of resistance training. In myoblasts, there were no mtDNA deletions or differences in COX activity between young and older subjects. In mature muscle, only older subjects showed mtDNA deletions. Older subjects had 8.7-fold higher 8-OHdG (P < 0.001) and 35% lower COX activity (P = 0.003) in mature muscle as compared with the young. Older subjects showed 63% decrease in mtDNA deletions (P = 0.024), a 45% decrease in 8-OHdG (P = 0.018), and a 79% increase in COX activity (P < 0.001) following resistance training. Together, these observations suggest that the improvements in mitochondrial function occurred through resistance exercise-induced recruitment of the satellite cells resulting in ‘‘mitochondrial gene shifting’’ in the skeletal muscle. We propose that the resistance training is a viable therapy to attenuate and/or ‘‘reverse’’ mitochondrial abnormalities associated with sarcopenia. This research was supported by CIHR, and Warren Lammert and Kathy Corkins. doi:10.1016/j.mito.2007.08.088

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Left ventricular hypertrophy (LVH) is one of the most important target organ damages in hypertension. Despite the involvement of multiple factors, the genetic factors including the mitochondrial genomes have been implicated to play an important role in the pathogenesis of LVH. Recently, a systematic and extended mutational screening of mitochondrial genome has been initiated in a large cohort of Chinese clinical population of Geriatric Cardiology Clinic at the Chinese PLA General Hospital, China. Further genetic evaluation suggested that two Chinese families with LVH appeared to be transmitted maternally. Sequence analysis of mitochondrial DNA identified a novel A-G transition at position 4401 (A4401G) at the junction of tRNAMet and tRNAGlu. In fact, this mutation was absent in 272 Chinese control subjects. This mutation appears to affect the processing of precursors in these mitochondrial tRNAs. Functional significance of this mutation was supported that the markedly decreases in the steady-state levels of tRNAMet and tRNAGlu were detected in the cells carrying this mutation. As a result, it may cause the defect in mitochondrial protein synthesis, thereby reducing the rate of respiration. These genetic and biochemical data imply that the novel A4401G mutation is involved in the pathogenesis of left ventricular hypertrophy in these Chinese families. doi:10.1016/j.mito.2007.08.090

85 The mitochondrial tRNAIle A4295G mutation is associated with hypertension in two Han Chinese subjects Haiyan Zhu *, Zongbin Li, Yuqi Liu, Rui Chen, Ronghua Li, Shiwen Wang, Min-Xin Guan Institute of Geriatric Cardiology, Chinese PLA General Hospital, Beijing, China Hypertension is one of the most common human sufferings, affecting approximately 1 billion individuals worldwide and 130 million in China. The etiology of hypertension is poorly understood due to the multi-factorial causes. Mutations in mitochondrial DNA (mtDNA) were shown to be one of molecular bases. In particular, the mitochondrial tRNAIle gene appears to be a hot spot of mutations associated with hypertension. To further investigate the role of mitochondrial genome in hypertension, a systematic and extensive screening of mitochondrial tRNAIle gene has been initiated at the Institute of Geriatric Cardiology, Chinese PLA General Hospital and Cincinnati Children’s Hospital Medical Center in a large cohort of Chinese clinical population. As a result, the homoplasmic tRNAIle A4295G mutation, which was previously associated with cardiomyopathy, was identified in two Chinese subjects with essential hypertension. This mutation was absent in 272 Chinese control subjects. Sequence analysis of entire mtDNA revealed that the A4295G mutation is a sole mutation in these mitochondrial genomes. The A4295G mutation is localized at 3 0 end adjacent to the anticodon, at conventional position 37(A37), of tRNAIle. The adenine(A37) at this position of tRNAIle is extraordinarily conserved from bacteria to human mitochondria. The nucleotide at this position of tRNAs is always modified and the modified A37 was shown to contribute to the high fidelity of codon recognition and to the structural formation and stabilization of functional tRNA. Thus, this mutation may lead to a failure in tRNA metabolism, thereby causing a mitochondrial dysfunction essential for the expression of hypertension in these subjects. doi:10.1016/j.mito.2007.08.089

86 The novel mitochondrial DNA A4401G mutation is involved in left ventricular hypertrophy in two Han Chinese pedigrees Shiwen Wang *, Haiyan Zhu, Ronghua Li, Li Yang, Zongbin Li, Yuqi Liu, Rui Chen, Lin Wang, Bin Xu, Yang Li, Min-Xin Guan Institute of Geriatric Cardiology. Chinese PLA General Hospital, Beijing, China

87 Overexpression of mitochondrial Leucyl-tRNA synthetase restore the mitochondrial dysfunctions caused by the MELAS-associated tRNALeu(UUR) A3243G mutation Ronghua Li, Min-Xin Guan * Cincinnati Children’s Hospital Medical Center The A3243G mutation in the tRNALeu(UUR) gene causes mitochondrial encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS) and other disorders including diabetes and deafness. Cytoplasmic hybrids (cybrids) cell studies demonstrated that this mutation results in decreased level and aminoacylation capacity of the tRNALeu(UUR), thereby leading to a decrease in the steady-state levels of affected tRNA. A failure in the tRNALeu(UUR) metabolism is responsible for the reduced rate of mitochondrial protein synthesis and the respiration defects. However, attempts to correct the mitochondrial dysfunctions caused by this mtDNA mutation have so far been unsuccessful. We hypothesized that overexpression of mitochondrial LeuRS in the cybrids carrying the A3243G mutation can correct the defects in mitochondrial tRNA metabolism, consequently increasing the level of mitochondrial translation. For this purpose, human LeuRS cDNA was cloned into a pcDNA3 vector and transfected into 43B cell line carrying nearly homoplasmic A3243G mutation and a cell line derived from same subject but lacking thisd mutation. Resultant stable transfectants expressing the LeuRS cDNA exhibited 50% increase in the level of aminoacylated and steady-state tRNALeu(UUR) as well as other mitochondrial tRNAs, compared with parental cybrids carrying the A3243G mutation. These are likely responsible for increasing of in the rates of mitochondrial protein synthesis and respiration in these resultant stable transfectants expressing the LeuRS cDNA, relative to the parental cybrids carrying the A3243G mutation. This suggests that the overexpression of mitochondrial Leucyl-tRNA synthetase restore the mitochondrial dysfunctions caused by the MELAS-associated tRNALeu(UUR) A3243G mutation.

doi:10.1016/j.mito.2007.08.091