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BACKGROUND: Previous studies have shown that marked changes in myocardial mitochondrial structure and function occur in human cardiac failure. To further understand the cellular events and to clarify their role in the pathology of cardiac failure, we have examined mitochondrial enzymatic function and peptide content, and mitochondrial DNA (mtDNA) integrity in a canine model of pacing-induced cardiac failure. METHODS: Myocardium and skeletal muscle tissues were evaluated for levels of respiratory complex I-V and citrate synthase activities, large-scale mtDNA deletions as well as peptide content of specific mitochondrial enzyme subunits. Levels of circulating and cardiac tumor necrosis factor-alpha (TNF-alpha), and of total aldehyde content in left ventricle were also assessed. RESULTS: Specific activity levels of complex III and V were significantly lower in both myocardial and skeletal muscle tissues of paced animals compared to controls. In contrast, activity levels of complex I, II, IV and citrate synthase were unchanged, as was the peptide content of specific mitochondrial enzyme subunits. Large-scale mtDNA deletions were found to be more likely present in myocardial tissue of paced as compared to control animals, albeit at a relatively low proportion of mtDNA molecules (<0.01% of wild-type). In addition, the reduction in complex III and V activities was correlated with elevated plasma and cardiac TNF-alpha levels. Significant increases in left ventricle aldehyde levels were also found. CONCLUSIONS: Our data show reductions in specific mitochondrial respiratory enzyme activities in pacing-induced heart failure which is not likely due to overall decreases in mitochondrial number, or necrosis. Our findings suggest a role for mitochondrial dysfunction in the pathogenesis of cardiac failure and may indicate a commonality in the signaling for pacing-induced mitochondrial dysfunction in myocardial and skeletal muscle. Increased levels of TNF-alpha and oxidative stress appear to play a contributory role.

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Defects in myocardial bioenergetics have been reported in patients with cardiomyopathy but their molecular basis and role in pathophysiology remain unclear. We sought to establish a molecular basis for cardiac mitochondrial respiratory enzyme abnormalities frequently present (75%) in a group of 16 children (including 2 neonates) with end-stage cardiomyopathy. Decreased specific activity levels were found in complexes I, III, IV and V but not in II, the only complex that is entirely nuclear encoded. Sequence analysis of cardiac mtDNA revealed 4 patients harbouring heteroplasmic mtDNA mutations in cytb, tRNAArg, and ND5 at highly conserved positions. These mutations were present neither in controls nor in patients without enzymatic defect. In addition, 4 patients exhibited marked reduction in cardiac mtDNA levels. The basis for respiratory enzyme abnormalities can be explained in a subset of our patients as a result of either pathogenic mtDNA mutation or depletion. Patients harbouring both DNA and enzymatic defects fulfil rigorous criteria defining mitochondrial cardiomyopathy.

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Previous studies in our laboratory demonstrated significant changes in bovine heart mitochondrial bioenergetics during fetal growth and development. To further understand mitochondrial biogenesis in early human development, the activity and subunit content levels of specific mitochondrial enzymes in fetal and neonatal heart were determined. Comparing early gestation (EG, 45-65 day) later gestation (LG, 85-110 day) and neonate (birth-1 month), specific activity of citrate synthase (CS), a Krebs cycle enzyme showed a 2 fold increase from EG to LG and a 2 fold increase from LG to neonate. Specific activities of complex IV and complex V increased similarly 1.8-2 fold from EG to LG. However during the later fetal period from LG to neonate, complex IV activity increased only 1.3 fold and complex V showed no significant increase. Peptide content of COX-II subunit increased 2 fold from EG to LG and by 3.5 fold from LG to neonate. Levels of COX-IV and ATP synthase alpha subunits were undetectable in EG hearts, clearly detectable in LG heart and 3 fold increased from LG to neonate. Unexpectedly, mitochondrial transcription factor A (mt-TFA) levels were not significantly different during these developmental stages. Mitochondrial DNA (mtDNA) levels increased 1.8 fold from EG to LG, and 3.8 fold increase from EG to neonate and correlated with CS activity levels. In conclusion, these data indicate coordinated regulation of some nuclear-encoded (COX-IV and CS activity) and mitochondrial components (COX-II and mtDNA), and strongly suggest that mitochondrial content increases particularly during the early fetal cardiac development and reveal a distinct pattern of regulation for mt-TFA.

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We describe a 17-year-old boy with a clinical neurologic picture consistent with Kearns-Sayre syndrome. His manifestations included progressive external ophthalmoplegia, bilateral ptosis, retinitis pigmentosa, and muscle weakness. He was found to harbor an abundant novel deletion in skeletal muscle mitochondrial DNA. Biochemical analysis of the patient's biopsied skeletal muscle showed that the specific activities of all four respiratory complexes with mitochondrial DNA-encoded subunits were markedly reduced in contrast to normal activity levels of entirely nuclear DNA-encoded enzyme activities (eg, complex II and citrate synthase). Ultrastructural analysis also indicated the presence of strikingly abnormal mitochondria with both unusual cristae and frequent paracrystalline inclusions. The great amount of the deleted mitochondrial DNA in this patient's muscle, as well as the concomitant reduction in specific respiratory complex activity, suggests that the mitochondrial DNA deletion plays a role in the pathogenesis of this neurologic disease.

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A variety of mitochondrial DNA (mtDNA) defects, ranging from point mutations and large-scale deletions to severe reduction in the overall quantity of mtDNA (mtDNA depletion), may be associated with neuromuscular disorders. The nuclear genome, which encodes most of the proteins involved in mitochondrial biogenesis (regulation of maintenance, replication, and transcription of mtDNA), appears to be implicated in many of the mtDNA defects. In this review, we describe some of the mtDNA defects discovered by our laboratory and others in patients with neurologic disorders and analyze their potential relationship with the pathways and mechanisms involved in mitochondrial biogenesis.

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BACKGROUND: Previous studies have shown that mitochondrial DNA (mtDNA) mutations are often present in patients with myocardial dysfunction. We sought to assess the prevalence and significance of heart mtDNA sequence changes in patients with idiopathic dilated cardiomyopathy (DCM). METHODS AND RESULTS: DNA sequence of all the transfer ribonucleic acid (tRNA), ribosomal RNA (rRNA), and structural genes in cardiac mtDNA of 28 patients with DCM was determined and compared with a control group that had no evidence of heart disease. An increased number of point mutations were found in DCM cardiac mtDNA when compared with controls. Both novel and previously reported mutations were found in mitochondrial tRNA and structural genes. One of these mutations was heteroplasmic and resulted in changing a highly conserved nucleotide in tRNAArg. Novel, heteroplasmic mtDNA mutations (n = 4) specifying changes in moderate to highly conserved amino acid residues were found in COII, COIII, ND5, and cytb. These novel mtDNA mutations were found only in patients with severe reduction in mitochondrial enzyme activities. CONCLUSIONS: Our results indicate that a high incidence of mtDNA nucleotide sequence changes in both tRNA and structural genes are present in DCM. Five heteroplasmic mutations were detected that both changed evolutionarily conserved residues (which may impair the function of proteins or tRNAs) and were associated with specific enzymatic defects. These mutations could play an important role in the pathogenesis of cardiomyopathy.

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A group of 25 children (5 months to 20 years of age) presenting with intractable seizures, developmental delay, and severe hypotonia, who did not fall into the known categories of mitochondrial encephalomyopathies, underwent muscle biopsy for evaluation of mitochondrial function and were compared with age-matched control subjects. Biopsied skeletal muscle was analyzed for six mitochondrial enzyme-specific activities, mitochondrial DNA point mutations and deletions, and mitochondrial DNA levels. The data reveal a high incidence of specific mitochondrial enzyme activity defects. Reduced activity levels were evident in complex I (11 patients), III (24 patients), IV (nine patients), and V (10 patients). Two patients also exhibited pronounced reduction in mitochondrial DNA levels (80% reduction compared with control subjects). Two patients manifested increased levels of 5-kb and 7.4-kb mitochondrial DNA deletions. Pathogenic mutations previously described in association with mitochondrial encephalomyopathies were not evident. The data suggest that mitochondrial dysfunction, including extensive defects in specific enzyme activities, may be frequently present in children with seizures, developmental delay, and hypotonia that do not fall within the known mitochondrial encephalomyopathies. These mitochondrial deficiencies can be primarily ascertained by biochemical analysis and are rarely accompanied by mitochondrial ultrastructural changes. The molecular basis of these defects, their role in these disorders, and potential treatment warrant further study.

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OBJECTIVES: This study sought to examine skeletal muscle of children with cardiomyopathy (CM) for changes in mitochondrial enzyme activities and in mitochondrial DNA (mtDNA). BACKGROUND: Heart mitochondrial enzymatic activity defects have been often found in dilated and hypertrophic CM. The defects primarily involve the activities of the electron transport system and oxidative phosphorylation pathway including respiratory complexes I, III, IV, and V. METHODS: Skeletal muscle biopsies of 8 children with CM were examined for specific mitochondrial enzyme activities, mtDNA copy number and the presence of pathogenic mutations and deletions in mtDNA. RESULTS: A marked deficiency in specific mitochondrial enzyme activities was found in 6 of 8 patients in skeletal muscle as well as in 2 of 3 hearts of those in whom cardiac tissue was available. Specific activity defects were found in complex I (2 cases), complex III (5 cases), complex IV (3 cases), and complex V (4 cases). Complex II and citrate synthase activities were unaffected. None of the previously reported pathogenic mutations associated with CM were detected, nor was there any evidence of mtDNA depletion. The incidence of defective respiratory complex activities in skeletal muscle was similar to the incidence of defective complex activities previously reported in cardiac tissue. CONCLUSIONS: Mitochondrial analysis of skeletal muscle is warranted in the overall clinical evaluation of children with CM, and particularly before consideration for cardiac transplantation.

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While mitochondrial abnormalities are increasingly recognized in cardiac diseases including hypertrophic cardiomyopathy, their presence in idiopathic dilated cardiomyopathy and the role that age plays in their incidence and severity have yet not been assessed. Levels of cardiac respiratory enzyme activities and mitochondrial DNA (mtDNA) were examined in 55 subjects with idiopathic dilated cardiomyopathy divided into 3 age groups. Respiratory enzyme activity levels were significantly lower in 37 patients (67%) compared to age-matched controls and increased activity levels were noted in 9 (16%). Decreased activities were found in complex I (n = 11), III (n = 16), IV (n = 12) and V (n = 13), but not in II, the only respiratory complex entirely nuclear-encoded. No age-specific differences were found in the overall frequency of enzymatic abnormalities. However, older patients had significantly increased multiple enzyme activity defects as well as increases in abundance and frequency of the 7.4 kb deletion. In addition, 3 patients were noted with marked reduction in mtDNA levels. None of the pathogenic mtDNA mutations previously associated with hypertrophic cardiomyopathy were found, nor was there any relationship that could be established between levels of specific mtDNA deletions and enzyme activities. In summary, specific mitochondrial abnormalities are heterogenous and frequent in both adults and children with idiopathic dilated cardiomyopathy. Older patients are more likely to have mtDNA deletions and multiple enzyme activity defects. The molecular basis for these abnormalities remains undefined.

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The nucleotide sequence encoding the citrate synthase (CS) gene was determined from the sequencing of the CS cDNA isolated from a human heart cDNA library. The primary sequence of CS deduced from its nucleotide sequence reveals a highly conserved, albeit slightly larger, protein of 466 amino acids, with 95% homology to its pig homologue. The data also indicate that the human genomic CS gene contains no introns, and confirms the location of the human CS gene on chromosome 12.

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Little information is presently available concerning mitochondrial respiratory and oxidative phosphorylation function in the normal human heart during growth and development. We investigated the levels of specific mitochondrial enzyme activities and content during cardiac growth and development from the early neonatal period (10-20 days) to adulthood (67 years). Biochemical analysis of enzyme specific activities and content and mitochondrial DNA (mtDNA) copy number was performed with left ventricular tissues derived from 30 control individuals. The levels of cytochrome c oxidase (COX) and complex V specific activity, mtDNA copy number and COX subunit II content remained unchanged in contrast to increased citrate synthase (CS) activity and content. The developmental increase in CS activity paralleled increasing CS polypeptide content, but was neither related to overall increases in mitochondrial number nor coordinately regulated with mitochondrial respiratory enzyme activities. Our findings of unchanged levels of cardiac mitochondrial respiratory enzyme activity during the progression from early childhood to older adult contrasts with the age-specific regulation found with CS, a Krebs cycle mitochondrial enzyme.

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We report the case of a child with severe hypertrophic cardiomyopathy, with decreased activity levels of cardiac mitochondrial respiratory complex I and III, and with a pronounced reduction in cardiac mitochondrial DNA copy number level. Mitochondrial DNA depletion has not been previously reported in hypertrophic cardiomyopathy and it may play a role in its pathogenesis.

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Abnormalities in mitochondrial DNA (mtDNA) including specific deletions and point mutations have been found in an increasing number of cases of both dilated and hypertrophic cardiomyopathy. The role that these mutations may play in contributing to the cardiomyopathic phenotype is discussed in this survey of the recent literature.

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Abnormalities in specific mitochondrial respiratory enzymes and DNA (mtDNA) have been reported in cardiomyopathy. In this study, we report 4 cases of severe hypertrophic cardiomyopathy (HCM) in which specific cardiac mitochondrial enzyme activity defects were found, including complex I (n = 2), complex III (n = 2), complex IV (n = 2) and complex V (n = 1). Other abnormalities were also noted including a marked depletion of mtDNA (n = 1) and decreased content of subunit 2 of cytochrome c oxidase (n = 1). None of the mtDNA point mutations and common deletions previously found in association with cardiomyopathy were detected in these patients. These data indicate that specific respiratory enzyme activity defects are frequently present in HCM. Also, our finding of a marked depletion of mtDNA in 1 patient suggests that cardiac mtDNA depletion, previously unreported in HCM, needs further examination in order to establish whether it plays a primary role in its pathogenesis.

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Abnormalities in cardiac mitochondrial respiratory enzymes and mitochondrial DNA have been found in an increasing number of pediatric cases of both dilated and hypertrophic cardiomyopathy, giving rise to the entity known as mitochondrial cardiomyopathy. Histochemical, biochemical, and molecular findings are described in this review of mitochondrial cardiomyopathy, which should provide assistance in its diagnostic identification.

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The levels of mitochondrial enzyme activities involved in respiration and oxidative phosphorylation and of specific mitochondrial gene transcripts were examined in rat heart and liver tissues during early growth, development, and aging. Increases were shown in cardiac respiratory complex activities I, III, IV, and V and ATPase6 and CoxII transcript levels during the transition from neonate to young adult. This increased mitochondrial gene expression is not associated with a proportionate increase in mitochondrial number. In contrast, no significant changes in liver mitochondrial activities or transcripts were detected during this transition. Marked reductions in the activities of complexes I, III, IV, and V and in ATPase6 and COXII transcripts were demonstrated in older adult as compared with young adult cardiac tissue with no concomitant reduction in cardiac citrate synthase activity and content, and mtDNA copy number. No decline was noted in liver mitochondrial enzyme activity levels and transcripts of old adult rats. These findings suggest that cardiac mitochondrial gene expression is developmentally regulated at a pretranslational level. The pattern of increasing mitochondrial gene expression in the young adult and decreasing gene expression in the aging heart stands in clear contrast to liver mitochondrial gene expression or nuclear-encoded genes such as citrate synthase.

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An infant with Leigh syndrome and associated cardiomyopathy is described. Abnormal activities of mitochondrial respiratory complexes III and V and a change in mtDNA at nt 8993 were detected in heart and skeletal muscle but not in liver.

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The effect of myocardial ischemia on mitochondrial DNA (mtDNA) structure and the presence of specific mtDNA deletions was determined using the model of Ameroid constriction of canine coronary arteries. The incidence of specific deletions was high in both the endocardial and epicardial tissues perfused by the occluded vessel as compared to myocardial tissues perfused by the unconstricted vessels. Our results show that specific mtDNA deletions similar to the 5 kb and 7.4 kb human mtDNA deletions occur following canine myocardial ischemia. However the presence of these deletions did not correlate with specific mitochondrial enzyme defects.

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We report a high incidence of reduced respiratory Complex III activity in heart muscle concomitant with the presence of a specific mutation in cytochrome b (cytb) in patients with ischemic cardiomyopathy. This C-->A mutation at nt 15452 converts the 236th residue of cytb from a leucine to isoleucine, is heteroplasmic and was observed in only 2 of 43 controls. Complex III activity is reduced (> 50%) in 5 of 6 patients with the C-->A15452 mutation suggesting that the cytb mutation is responsible for decreased Complex III activity and may play a role in the pathophysiology of ischemic cardiomyopathy.

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