Cardiomyopathy and abnormal mitochondrial function.

The diagnosis of cardiomyopathy is mainly based on clinical and morphological criteria. While metabolic, viral, chemical, genetic, and immunological factors are often proposed as causes of cardiomyopathy, little is known about the role of the respiratory chain and other biochemical mitochondrial defects in this group of diseases. Research on the biochemistry and molecular biology of cardiomyopathies offers an opportunity for a better understanding of the pathogenesis and for finding specific therapy.

Abnormal cardiac and skeletal muscle mitochondrial function in pacing-induced cardiac failure.

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.

Specific mitochondrial DNA deletions in idiopathic dilated cardiomyopathy.

OBJECTIVE: Structural changes in human mitochondrial DNA (mtDNA) have been implicated in a number of clinical conditions with dysfunctions in oxidative phosphorylation called OX-PHOS diseases, some of which have cardiac involvement. The objective of this study was to assess the frequency and extent of specific mitochondrial DNA deletions in idiopathic dilated cardiomyopathy. METHODS: DNA extracted from tissue derived from the left ventricle of 41 patients with idiopathic dilated cardiomyopathy and 17 controls was amplified by polymerase chain reaction using specific primers to assess the incidence and proportion of 5-kb and 7.4-kb deletions in mitochondrial DNA. RESULTS: In reactions using primers to detect the 5-kb deletion, an amplified product of 593 bp was found in low abundance relative to undeleted mitochondrial DNA but with high frequency in a number of controls and patients. A second deletion of 7.4 kb in size was also frequently present in controls and patients. In contrast to previous reports, these deletions were found to be present in both controls and in cardiomyopathic patients, 18 years and younger, including several infants. The 7.4-kb deletion was prominently increased in both frequency and in its proportion relative to undeleted mitochondrial DNA in patients 40 years and older with idiopathic dilated cardiomyopathy. CONCLUSIONS: At variance with current literature our study reports a significant presence of both 5 and 7.4-kb deletions in the young and a higher frequency and quantity of the 7.4-kb deletion in the older cardiomyopathic patients in comparison with controls. The increased accumulation of the 7.4-kb deletion as both a function of aging and cardiomyopathy is suggestive that this specific mitochondrial DNA deletion arises more likely as an effect of heart dysfunction rather than as a primary cause of cardiomyopathy.

Mitochondrial DNA defects in cardiomyopathy.

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.

Is age a contributory factor of mitochondrial bioenergetic decline and DNA defects in idiopathic dilated cardiomyopathy?

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.

Isolation and characterization of maltose non utilizing (mnu) mutants mapping outside the MAL1 locus in Saccharomyces cerevisiae.

The MAL1 locus of Saccharomyces cerevisiae comprises three genes necessary for maltose utilization. They include regulatory, maltose transport and maltase genes designated MAL1R, MAL1T and MAL1S respectively. Using a MAL1 strain transformed with an episomal, multicopy plasmid carrying the MAL2 locus, five recessive and one dominant mutant unable to grow on maltose, but still retaining a functional MAL1 locus were isolated. All the mutants could use glycerol, ethanol, raffinose and sucrose as a sole carbon source; expression of the maltase and maltose permease genes was severely and coordinately reduced. Only the dominant mutant failed to accumulate the MAL1R mRNA.

Kearns-Sayre syndrome with a novel mitochondrial DNA deletion.

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.

Mitochondrial biogenesis defects and neuromuscular disorders.

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.

Skeletal muscle mitochondrial defects in nonspecific neurologic disorders.

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.

Mitochondrial cardiomyopathy: molecular and biochemical analysis.

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.

Mitochondrial cardiomyopathy.

An adolescent with mitochondrial cardiomyopathy is described. Skeletal and cardiac biopsies revealed abnormal mitochondria, with biochemical analysis showing cytochrome c oxidase deficiency.

Genetic mapping and biochemical analysis of mutants in the maltose regulatory gene of the MAL1 locus of Saccharomyces cerevisiae.

The MAL1 locus of Saccharomyces cerevisiae comprises three genes necessary for maltose utilization: a regulatory (MALR), a maltose transport (MALT) and a maltase gene (MALS). A fine structure genetic map of the MAL1R gene was constructed and the order of mutations was confirmed by plasmid-mediated chromosomal recombination. The mutations cluster non-randomly within the 5' half of the gene, where the putative DNA binding domain of the encoded protein is located. Only mutations mal1R-22 and MAL1R-72 map in the 3' terminal half of the gene; these mutations cause a different pattern of transcriptional regulation of plasmid-borne MAL6T genes. Experiments supporting a direct involvement of the MALR-encoded protein in carbon catabolite repression of MAL gene expression are reported.

Regulation of MAL gene expression in yeast: gene dosage effects.

Both the MAL1 and MAL6 loci in Saccharomyces strains have been shown by functional and structural studies to comprise a cluster of at least three genes necessary for maltose utilization. They include regulatory, maltose transport and maltase genes designated MALR, MALT and MALS, respectively. Subclones of each gene derived from the MAL6 locus were inserted into the multicopy shuttle plasmid YEp13, introduced into MAL1 and mal1 strains and the effects of altered gene dosage of each gene, or a combination of them, on MAL gene expression investigated. MAL1 strains transformed with a plasmid carrying the MAL6S gene showed coordinate four to five fold increases in both maltase enzyme activity and its mRNA, whereas no increase in maltose transport activity or of MALT mRNA was observed when MAL6T was present on multicopy plasmids. The presence of the MAL6R gene on a multicopy plasmid led to greatly increased transcription of both inducible and constitutive mRNAs with homology to the regulatory gene; it also gave rise to two fold increases in both induced maltase mRNA levels and enzyme activity, but only in the presence of maltose. However, it had no apparent effect on the accumulation of MALT mRNA. Finally, the induction kinetics of plasmid-borne and chromosomal MALS and MALT gene expression were examined under conditions of altered gene dosage of the MAL6 regulatory and structural genes. The results of these experiments indicate that MALR encodes a trans-acting positive activator that requires maltose for induction of MALS and MALT transcription even when the regulatory gene is present on a multicopy plasmid. Maltose transport can be a rate-limiting factor in MAL gene expression, at least in the early stages of induction. The regulation of the MALS and MALT genes, whose activities are coordinately induced in MAL1 strains by maltose, may in fact exhibit some important differences.

Human mitochondrial function during cardiac growth and development.

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.

Mitochondrial DNA of the blowfly Phormia regina: restriction analysis and gene localization.

A study of an invertebrate mitochondrial genome, that of the blowfly Phormia regina, has been initiated to compare its structural and functional relatedness to other metazoan mitochondrial genomes. A restriction map of mitochondrial DNA (mtDNA) isolated from sucrose gradient-purified mitochondria has been established using a combination of single and double restriction endonuclease digestions and hybridizations with isolated mtDNA fragments, revealing a genome size of 17.5 kilobases (kb). A number of mitochondrial genes including those encoding the 12 S and 16 S ribosomal RNA, the cytochrome c oxidase I subunit (COI) and an unidentified open reading frame (URF2) have been located on the Phormia mtDNA by Southern blot analysis using as probes both isolated mtDNA fragments and oligonucleotides derived from the sequences of previously characterized genes from rat and Drosophila yakuba mtDNAs. These data indicate that for those regions examined, the mitochondrial genome organization of blowfly mtDNA is the same as that of Drosophila yakuba, the order being COI-URF2-12 S-16 S. These data also report the presence of an A + T-rich region, located as a 2.5-kb region between the URF2 and the 12 S rRNA genes, and its amplification by the polymerase chain reaction is described.

Mitochondrial gene expression in rat heart and liver during growth and development.

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.

Cloning and molecular analysis of the human citrate synthase gene.

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.

Presence of a mitochondrial DNA deletion in fetal and adult bovine cardiac tissue.

A deletion of about 5.3 kilobases has been detected in the mitochondrial DNA of bovine cardiac tissue. This deletion appears to be somatic in origin given its sporadic presence in the various heart compartments examined. Cardiac tissue derived from developmental stages including fetal, early and older adult animals harbored this mutation with increased levels (100-1000 fold) found in older adults. The deleted region of the mitochondrial genome maps to relatively the same area (deleting ATPase6, COXIII, ND2, ND4 and a portion of ND5) as the common 5 kb deletion reported in humans, but its presence in fetal tissue, as well as its decreased age dependence distinguish it relative to the reported human deletion.

Mitochondrial dysfunction after fetal alcohol exposure.

Specific mitochondrial enzyme activities and mRNA levels were measured in the heart, brain, and liver tissues of a group of 1-day-old neonatal rats whose mothers were alcohol-fed during pregnancy and compared with a control group. The results show a significant decrease in mitochondrial ATP synthase activity in both the brain and liver, as well as a decrease in complex III activity in the liver of rats exposed to alcohol. Other mitochondrial enzymes activities (e.g., citrate synthase, cytochrome c oxidase, and complex I), as well as specific mitochondrial transcript levels, were not significantly affected. Heart mitochondrial enzyme activities were not significantly affected. These data reveal that a tissue-specific response occurs after fetal exposure to alcohol and may explain some of the cellular events occurring in fetal alcohol syndrome resulting in abnormal growth and neurological development.