Functional diversity among cardiolipin binding sites on the mitochondrial ADP/ATP carrier.

Lipid-protein interactions play a multitude of essential roles in membrane homeostasis. Mitochondrial membranes have a unique lipid-protein environment that ensures bioenergetic efficiency. Cardiolipin (CL), the signature mitochondrial lipid, plays multiple roles in promoting oxidative phosphorylation (OXPHOS). In the inner mitochondrial membrane, the ADP/ATP carrier (AAC in yeast; adenine nucleotide translocator, ANT in mammals) exchanges ADP and ATP, enabling OXPHOS. AAC/ANT contains three tightly bound CLs, and these interactions are evolutionarily conserved. Here, we investigated the role of these buried CLs in AAC/ANT using a combination of biochemical approaches, native mass spectrometry, and molecular dynamics simulations. We introduced negatively charged mutations into each CL-binding site of yeast Aac2 and established experimentally that the mutations disrupted the CL interactions. While all mutations destabilized Aac2 tertiary structure, transport activity was impaired in a binding site-specific manner. Additionally, we determined that a disease-associated missense mutation in one CL-binding site in human ANT1 compromised its structure and transport activity, resulting in OXPHOS defects. Our findings highlight the conserved significance of CL in AAC/ANT structure and function, directly tied to specific lipid-protein interactions.

Kearns-Sayre syndrome with rare imaging finding of SLC25A4 Mutation.

Kearns-Sayre Syndrome (KSS) is a subtype of chronic progressive external ophthalmoplegia (CPEO). In this case, A 21-year-old man diagnosed with KSS, and presented with chronic progressive blepharoptosis (ptosis) and external ophthalmoplegia, diffuse depigmentation of the retinal pigment epithelium, and cerebellar ataxia, with a cerebrospinal fluid protein of 254 mg/dL, was reported. Genetic screening revealed a novel mutated gene in SLC25A4 in the patient as well as in his mother: NM_001151:c.170G>C in exon 2. Its imaging finding is a characteristic progressive atrophy of the right cerebellar hemisphere. In conclusion, we found a case of KSS with a novel mutated gene in SLC25A4: NM_001151:c.170G>C in exon 2 as the pathogenic mechanism, and found that KSS can be caused only when the proportion of mutations in the SLC25A4 gene reach a certain degree, and the patient with KSS showed a unique cranial imaging feature of unilateral progressive cerebellar atrophy.

A Yeast-Based Screening Unravels Potential Therapeutic Molecules for Mitochondrial Diseases Associated with Dominant ANT1 Mutations.

Mitochondrial diseases result from inherited or spontaneous mutations in mitochondrial or nuclear DNA, leading to an impairment of the oxidative phosphorylation responsible for the synthesis of ATP. To date, there are no effective pharmacological therapies for these pathologies. We performed a yeast-based screening to search for therapeutic drugs to be used for treating mitochondrial diseases associated with dominant mutations in the nuclear ANT1 gene, which encodes for the mitochondrial ADP/ATP carrier. Dominant ANT1 mutations are involved in several degenerative mitochondrial pathologies characterized by the presence of multiple deletions or depletion of mitochondrial DNA in tissues of affected patients. Thanks to the presence in yeast of the AAC2 gene, orthologue of human ANT1, a yeast mutant strain carrying the M114P substitution equivalent to adPEO-associated L98P mutation was created. Five molecules were identified for their ability to suppress the defective respiratory growth phenotype of the haploid aac2M114P. Furthermore, these molecules rescued the mtDNA mutability in the heteroallelic AAC2/aac2M114P strain, which mimics the human heterozygous condition of adPEO patients. The drugs were effective in reducing mtDNA instability also in the heteroallelic strain carrying the R96H mutation equivalent to the more severe de novo dominant missense mutation R80H, suggesting a general therapeutic effect on diseases associated with dominant ANT1 mutations.

Mitochondrial energy deficiency leads to hyperproliferation of skeletal muscle mitochondria and enhanced insulin sensitivity.

Diabetes is associated with impaired glucose metabolism in the presence of excess insulin. Glucose and fatty acids provide reducing equivalents to mitochondria to generate energy, and studies have reported mitochondrial dysfunction in type II diabetes patients. If mitochondrial dysfunction can cause diabetes, then we hypothesized that increased mitochondrial metabolism should render animals resistant to diabetes. This was confirmed in mice in which the heart-muscle-brain adenine nucleotide translocator isoform 1 (ANT1) was inactivated. ANT1-deficient animals are insulin-hypersensitive, glucose-tolerant, and resistant to high fat diet (HFD)-induced toxicity. In ANT1-deficient skeletal muscle, mitochondrial gene expression is induced in association with the hyperproliferation of mitochondria. The ANT1-deficient muscle mitochondria produce excess reactive oxygen species (ROS) and are partially uncoupled. Hence, the muscle respiration under nonphosphorylating conditions is increased. Muscle transcriptome analysis revealed the induction of mitochondrial biogenesis, down-regulation of diabetes-related genes, and increased expression of the genes encoding the myokines FGF21 and GDF15. However, FGF21 was not elevated in serum, and FGF21 and UCP1 mRNAs were not induced in liver or brown adipose tissue (BAT). Hence, increased oxidation of dietary-reducing equivalents by elevated muscle mitochondrial respiration appears to be the mechanism by which ANT1-deficient mice prevent diabetes, demonstrating that the rate of mitochondrial oxidation of calories is important in the etiology of metabolic disease.

Drosophila melanogaster Mitochondrial Carriers: Similarities and Differences with the Human Carriers.

Mitochondrial carriers are a family of structurally related proteins responsible for the exchange of metabolites, cofactors and nucleotides between the cytoplasm and mitochondrial matrix. The in silico analysis of the Drosophila melanogaster genome has highlighted the presence of 48 genes encoding putative mitochondrial carriers, but only 20 have been functionally characterized. Despite most Drosophila mitochondrial carrier genes having human homologs and sharing with them 50% or higher sequence identity, D. melanogaster genes display peculiar differences from their human counterparts: (1) in the fruit fly, many genes encode more transcript isoforms or are duplicated, resulting in the presence of numerous subfamilies in the genome; (2) the expression of the energy-producing genes in D. melanogaster is coordinated from a motif known as Nuclear Respiratory Gene (NRG), a palindromic 8-bp sequence; (3) fruit-fly duplicated genes encoding mitochondrial carriers show a testis-biased expression pattern, probably in order to keep a duplicate copy in the genome. Here, we review the main features, biological activities and role in the metabolism of the D. melanogaster mitochondrial carriers characterized to date, highlighting similarities and differences with their human counterparts. Such knowledge is very important for obtaining an integrated view of mitochondrial function in D. melanogaster metabolism.

Recurrent De Novo Dominant Mutations in SLC25A4 Cause Severe Early-Onset Mitochondrial Disease and Loss of Mitochondrial DNA Copy Number.

Mutations in SLC25A4 encoding the mitochondrial ADP/ATP carrier AAC1 are well-recognized causes of mitochondrial disease. Several heterozygous SLC25A4 mutations cause adult-onset autosomal-dominant progressive external ophthalmoplegia associated with multiple mitochondrial DNA deletions, whereas recessive SLC25A4 mutations cause childhood-onset mitochondrial myopathy and cardiomyopathy. Here, we describe the identification by whole-exome sequencing of seven probands harboring dominant, de novo SLC25A4 mutations. All affected individuals presented at birth, were ventilator dependent and, where tested, revealed severe combined mitochondrial respiratory chain deficiencies associated with a marked loss of mitochondrial DNA copy number in skeletal muscle. Strikingly, an identical c.239G>A (p.Arg80His) mutation was present in four of the seven subjects, and the other three case subjects harbored the same c.703C>G (p.Arg235Gly) mutation. Analysis of skeletal muscle revealed a marked decrease of AAC1 protein levels and loss of respiratory chain complexes containing mitochondrial DNA-encoded subunits. We show that both recombinant AAC1 mutant proteins are severely impaired in ADP/ATP transport, affecting most likely the substrate binding and mechanics of the carrier, respectively. This highly reduced capacity for transport probably affects mitochondrial DNA maintenance and in turn respiration, causing a severe energy crisis. The confirmation of the pathogenicity of these de novo SLC25A4 mutations highlights a third distinct clinical phenotype associated with mutation of this gene and demonstrates that early-onset mitochondrial disease can be caused by recurrent de novo mutations, which has significant implications for the application and analysis of whole-exome sequencing data in mitochondrial disease.

Ant1 mutant mice bridge the mitochondrial and serotonergic dysfunctions in bipolar disorder.

Although mitochondrial and serotonergic dysfunctions have been implicated in the etiology of bipolar disorder (BD), the relationship between these unrelated pathways has not been elucidated. A family of BD and chronic progressive external ophthalmoplegia (CPEO) caused by a mutation of the mitochondrial adenine nucleotide translocator 1 (ANT1, SLC25A4) implicated that ANT1 mutations confer a risk of BD. Here, we sequenced ANT1 in 324 probands of NIMH bipolar disorder pedigrees and identified two BD patients carrying heterozygous loss-of-function mutations. Behavioral analysis of brain specific Ant1 heterozygous conditional knockout (cKO) mice using lntelliCage showed a selective diminution in delay discounting. Delay discounting is the choice of smaller but immediate reward than larger but delayed reward and an index of impulsivity. Diminution of delay discounting suggests an increase in serotonergic activity. This finding was replicated by a 5-choice serial reaction time test. An anatomical screen showed accumulation of COX (cytochrome c oxidase) negative cells in dorsal raphe. Dorsal raphe neurons in the heterozygous cKO showed hyperexcitability, along with enhanced serotonin turnover in the nucleus accumbens and upregulation of Maob in dorsal raphe. These findings altogether suggest that mitochondrial dysfunction as the genetic risk of BD may cause vulnerability to BD by altering serotonergic neurotransmission.

Molecular analysis of inherited cardiomyopathy using next generation semiconductor sequencing technologies.

BACKGROUND: Cardiomyopathies are the most common clinical and genetic heterogeneity cardiac diseases, and genetic contribution in particular plays a major role in patients with primary cardiomyopathies. The aim of this study is to investigate cases of inherited cardiomyopathy (IC) for potential disease-causing mutations in 64 genes reported to be associated with IC. METHODS: A total of 110 independent cases or families diagnosed with various primary cardiomyopathies, including hypertrophic cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, left ventricular non-compaction, and undefined cardiomyopathy, were collected after informed consent. A custom designed panel, including 64 genes, was screened using next generation sequencing on the Ion Torrent PGM platform. The best candidate disease-causing variants were verified by Sanger sequencing. RESULTS: A total of 78 variants in 73 patients were identified. After excluding the variants predicted to be benign and VUS, 26 pathogenic or likely pathogenic variants were verified in 26 probands (23.6%), including a homozygous variant in the SLC25A4 gene. Of these variants, 15 have been reported in the Human Gene Mutation Database or ClinVar database, while 11 are novel. The majority of variants were observed in the MYH7 (8/26) and MYBPC3 (6/26) gene. Titin (TTN) truncating mutations account for 13% in our dilated cardiomyopathy cases (3/23). CONCLUSIONS: This study provides an overview of the genetic aberrations in this cohort of Chinese IC patients and demonstrates the power of next generation sequencing in IC. Genetic results can provide precise clinical diagnosis and guidance regarding medical care for some individuals.

Movement disorders in genetically confirmed mitochondrial disease and the putative role of the cerebellum.

BACKGROUND: Mitochondrial disease can present as a movement disorder. Data on this entity's epidemiology, genetics, and underlying pathophysiology, however, is scarce. OBJECTIVE: The objective of this study was to describe the clinical, genetic, and volumetric imaging data from patients with mitochondrial disease who presented with movement disorders. METHODS: In this retrospective analysis of all genetically confirmed mitochondrial disease cases from three centers (n = 50), the prevalence and clinical presentation of video-documented movement disorders was assessed. Voxel-based morphometry from high-resolution MRI was employed to compare cerebral and cerebellar gray matter volume between mitochondrial disease patients with and without movement disorders and healthy controls. RESULTS: Of the 50 (30%) patients with genetically confirmed mitochondrial disease, 15 presented with hypokinesia (parkinsonism 3/15), hyperkinesia (dystonia 5/15, myoclonus 3/15, chorea 2/15), and ataxia (3/15). In 3 patients, mitochondrial disease presented as adult-onset isolated dystonia. In comparison to healthy controls and mitochondrial disease patients without movement disorders, patients with hypo- and hyperkinetic movement disorders had significantly more cerebellar atrophy and an atrophy pattern predominantly involving cerebellar lobules VI and VII. CONCLUSION: This series provides clinical, genetic, volumetric imaging, and histologic data that indicate major involvement of the cerebellum in mitochondrial disease when it presents with hyper- and hypokinetic movement disorders. As a working hypothesis addressing the particular vulnerability of the cerebellum to energy deficiency, this adds substantially to the pathophysiological understanding of movement disorders in mitochondrial disease. Furthermore, it provides evidence that mitochondrial disease can present as adult-onset isolated dystonia. © 2017 International Parkinson and Movement Disorder Society.

Xenotopic expression of alternative electron transport enzymes in animal mitochondria and their impact in health and disease.

The mitochondrial respiratory chain in vertebrates and arthropods is different from that of most other eukaryotes because they lack alternative enzymes that provide electron transfer pathways additional to the oxidative phosphorylation (OXPHOS) system. However, the use of diverse experimental models, such as human cells in culture, Drosophila melanogaster and the mouse, has demonstrated that the transgenic expression of these alternative enzymes can impact positively many phenotypes associated with human mitochondrial and other cellular dysfunction, including those typically presented in complex IV deficiencies, Parkinson's, and Alzheimer's. In addition, these enzymes have recently provided extremely valuable data on how, when, and where reactive oxygen species, considered by many as "by-products" of OXPHOS, can contribute to animal longevity. It has also been shown that the expression of the alternative enzymes is thermogenic in cultured cells, causes reproductive defects in flies, and enhances the deleterious phenotype of some mitochondrial disease models. Therefore, all the reported beneficial effects must be considered with caution, as these enzymes have been proposed to be deployed in putative gene therapies to treat human diseases. Here, we present a brief review of the scientific data accumulated over the past decade that show the benefits and the risks of introducing alternative branches of the electron transport into mammalian and insect mitochondria, and we provide a perspective on the future of this research field.

Mitochondrial functions modulate neuroendocrine, metabolic, inflammatory, and transcriptional responses to acute psychological stress.

The experience of psychological stress triggers neuroendocrine, inflammatory, metabolic, and transcriptional perturbations that ultimately predispose to disease. However, the subcellular determinants of this integrated, multisystemic stress response have not been defined. Central to stress adaptation is cellular energetics, involving mitochondrial energy production and oxidative stress. We therefore hypothesized that abnormal mitochondrial functions would differentially modulate the organism's multisystemic response to psychological stress. By mutating or deleting mitochondrial genes encoded in the mtDNA [NADH dehydrogenase 6 (ND6) and cytochrome c oxidase subunit I (COI)] or nuclear DNA [adenine nucleotide translocator 1 (ANT1) and nicotinamide nucleotide transhydrogenase (NNT)], we selectively impaired mitochondrial respiratory chain function, energy exchange, and mitochondrial redox balance in mice. The resulting impact on physiological reactivity and recovery from restraint stress were then characterized. We show that mitochondrial dysfunctions altered the hypothalamic-pituitary-adrenal axis, sympathetic adrenal-medullary activation and catecholamine levels, the inflammatory cytokine IL-6, circulating metabolites, and hippocampal gene expression responses to stress. Each mitochondrial defect generated a distinct whole-body stress-response signature. These results demonstrate the role of mitochondrial energetics and redox balance as modulators of key pathophysiological perturbations previously linked to disease. This work establishes mitochondria as stress-response modulators, with implications for understanding the mechanisms of stress pathophysiology and mitochondrial diseases.

Yarrowia lipolytica AAL genes are involved in peroxisomal fatty acid activation.

In yeast, ß-oxidation of fatty acids (FAs) essentially takes place in peroxisomes, and FA activation must precede FA oxidation. In Saccharomyces cerevisiae, a single fatty-acyl­CoA-synthetase, ScFaa2p, mediates peroxisomal FA activation. We have previously shown that this reaction also exists in the oleaginous yeast Yarrowia lipolytica; however, the protein involved in this process remains unknown. Here, we found that proteins, named Aal proteins (Acyl/Aryl-CoA-ligases), resembling the 4-coumarate­CoA-ligase-like enzymes found in plants are involved in peroxisomal FA activation in Y. lipolytica; Y. lipolytica has 10 AAL genes, eight of which are upregulated by oleate. All the Aal proteins contain a PTS1-type peroxisomal targeting sequence (A/SKL), suggesting a peroxisomal localization. The function of the Aal proteins was analyzed using the faa1Δant1Δ mutant strain, which demonstrates neither cytoplasmic FA activation (direct result of FAA1 deletion) nor peroxisomal FA activation (indirect result of ANT1 deletion, a gene coding an ATP transporter). This strain is thus highly sensitive to external FA levels and unable to store external FAs in lipid bodies (LBs). Whereas the overexpression of (cytoplasmic) AAL1ΔPTS1 was able to partially complement the growth defect observed in the faa1Δant1Δ mutant on short-, medium- and long-chain FA media, the presence of Aal2p to Aal10p only allowed growth on the short-chain FA medium. Additionally, partial LB formation was observed in the oleate medium for strains overexpressing Aal1ΔPTS1p, Aal4ΔPTS1p, Aal7ΔPTS1p, and Aal8ΔPTS1p. Finally, an analysis of the FA content of cells grown in the oleate medium suggested that Aal4p and Aal6p present substrate specificity for C16:1 and/or C18:0.

Dominance of yeast aac2R96H and aac2R252G mutations, equivalent to pathological mutations in ant1, is due to gain of function.

The mitochondrial ADP/ATP carrier is a nuclear encoded protein, which catalyzes the exchange of ATP generated in mitochondria with ADP produced in the cytosol. In humans, mutations in the major ADP/ATP carrier gene, ANT1, are involved in several degenerative mitochondrial pathologies, leading to instability of mitochondrial DNA. Recessive mutations have been associated with mitochondrial myopathy and cardiomyopathy whereas dominant mutations have been associated with autosomal dominant Progressive External Ophtalmoplegia (adPEO). Recently, two de novo dominant mutations, R80H and R235G, leading to extremely severe symptoms, have been identified. In order to evaluate if the dominance is due to haploinsufficiency or to a gain of function, the two mutations have been introduced in the equivalent positions of the AAC2 gene, the yeast orthologue of human ANT1, and their dominant effect has been studied in heteroallelic strains, containing both one copy of wild type AAC2 and one copy of mutant aac2 allele. Through phenotypic characterization of these yeast models we showed that the OXPHOS phenotypes in the heteroallelic strains were more affected than in the hemiallelic strain indicating that the dominant trait of the two mutations is due to gain of function.

ZNF555 protein binds to transcriptional activator site of 4qA allele and ANT1: potential implication in Facioscapulohumeral dystrophy.

Facioscapulohumeral dystrophy (FSHD) is an epi/genetic satellite disease associated with at least two satellite sequences in 4q35: (i) D4Z4 macrosatellite and (ii) ß-satellite repeats (BSR), a prevalent part of the 4qA allele. Most of the recent FSHD studies have been focused on a DUX4 transcript inside D4Z4 and its tandem contraction in FSHD patients. However, the D4Z4-contraction alone is not pathological, which would also require the 4qA allele. Since little is known about BSR, we investigated the 4qA BSR functional role in the transcriptional control of the FSHD region 4q35. We have shown that an individual BSR possesses enhancer activity leading to activation of the Adenine Nucleotide Translocator 1 gene (ANT1), a major FSHD candidate gene. We have identified ZNF555, a previously uncharacterized protein, as a putative transcriptional factor highly expressed in human primary myoblasts that interacts with the BSR enhancer site and impacts the ANT1 promoter activity in FSHD myoblasts. The discovery of the functional role of the 4qA allele and ZNF555 in the transcriptional control of ANT1 advances our understanding of FSHD pathogenesis and provides potential therapeutic targets.

A Mechanism Study Underlying the Protective Effects of Cyclosporine-A on Lung Ischemia-Reperfusion Injury.

AIM: This study is aimed at validating the hypothesis that administration of cyclosporine-A (CsA) would be protective in lung ischemia-reperfusion (I/R) injury and in exploring the underlying mechanism. METHODS: Rabbits were divided into 4 groups: the control, sham operation, I/R, and I/R with CsA treatment. Flow cytometry was used to measure the mitochondrial membrane potential. Laser scanning confocal microscope was used to analyze mitochondrion permeability transition pore (MPTP). The apoptotic cell was detected by the TUNEL staining. Western blot was performed to analyze the protein expression levels. RESULTS: CsA not only attenuated the histopathologic alterations in lung and mitochondria after I/R injury, but also attenuated I/R injury through increasing MPP and inhibiting MPTP opening. Besides, CsA attenuated I/R injury through suppressing the release of cytochrome-c (CytC), inhibiting cell apoptosis and decreasing the expression levels of cyclophilin-D (Cyp-D), adenine nucleotide translocase 1 (ANT1) and voltage-dependent anion channel 1 (VDAC1). Finally, we found that Cyp-D knockdown inhibits I/R injury-induced MPTP opening and cell apoptosis. CONCLUSION: Our study found that the protective role of CsA on lung I/R injury depends on the inhibition of MPTP and CytC release, suppression of the activation of mitochondrial apoptosis pathway and the expressions of apoptotic-related proteins, as well as the decreased expression levels of ANT1 and VDAC1.

ANT1-mediated fatty acid-induced uncoupling as a target for improving myocellular insulin sensitivity.

AIMS/HYPOTHESIS: Dissipating energy via mitochondrial uncoupling has been suggested to contribute to enhanced insulin sensitivity. We hypothesised that skeletal muscle mitochondria of endurance-trained athletes have increased sensitivity for fatty acid (FA)-induced uncoupling, which is driven by the mitochondrial protein adenine nucleotide translocase 1 (ANT1). METHODS: Capacity for FA-induced uncoupling was measured in endurance-trained male athletes (T) and sedentary young men (UT) in an observational study and also in isolated skeletal muscle mitochondria from Zucker diabetic fatty (ZDF) rats and C2C12 myotubes following small interfering RNA (siRNA)-mediated gene silencing of ANT1. Thus, fuelled by glutamate/succinate (fibres) or pyruvate (mitochondria and myotubes) and in the presence of oligomycin to block ATP synthesis, increasing levels of oleate (fibres) or palmitate (mitochondria and myotubes) were automatically titrated while respiration was monitored. Insulin sensitivity was measured by hyperinsulinaemic-euglycaemic clamp in humans and via insulin-stimulated glucose uptake in myotubes. RESULTS: Skeletal muscle from the T group displayed increased sensitivity to FA-induced uncoupling (p = 0.011) compared with muscle from the UT group, and this was associated with elevated insulin sensitivity (p = 0.034). ANT1 expression was increased in T (p = 0.013). Mitochondria from ZDF rats displayed decreased sensitivity for FA-induced uncoupling (p = 0.008). This difference disappeared in the presence of the adenine nucleotide translocator inhibitor carboxyatractyloside. Partial knockdown of ANT1 in C2C12 myotubes decreased sensitivity to the FA-induced uncoupling (p = 0.008) and insulin-stimulated glucose uptake (p = 0.025) compared with controls. CONCLUSIONS/INTERPRETATION: Increased sensitivity to FA-induced uncoupling is associated with enhanced insulin sensitivity and is affected by ANT1 activity in skeletal muscle. FA-induced mitochondrial uncoupling may help to preserve insulin sensitivity in the face of a high supply of FAs. TRIAL REGISTRATION: www.trialregister.nl NTR2002.

Mutation of the mitochondrial carrier SLC25A42 causes a novel form of mitochondrial myopathy in humans.

Myopathies are heterogeneous disorders characterized clinically by weakness and hypotonia, usually in the absence of gross dystrophic changes. Mitochondrial dysfunction is a frequent cause of myopathy. We report a simplex case born to consanguineous parents who presented with muscle weakness, lactic acidosis, and muscle changes suggestive of mitochondrial dysfunction. Combined autozygome and exome analysis revealed a missense variant in the SLC25A42 gene, which encodes an inner mitochondrial membrane protein that imports coenzyme A into the mitochondrial matrix. Zebrafish slc25a42 knockdown morphants display severe muscle disorganization and weakness. Importantly, these features are rescued by normal human SLC25A42 RNA, but not by RNA harboring the patient's variant. Our data support a potentially causal link between SLC25A42 mutation and mitochondrial myopathy in humans.

Severity of cardiomyopathy associated with adenine nucleotide translocator-1 deficiency correlates with mtDNA haplogroup.

Mutations of both nuclear and mitochondrial DNA (mtDNA)-encoded mitochondrial proteins can cause cardiomyopathy associated with mitochondrial dysfunction. Hence, the cardiac phenotype of nuclear DNA mitochondrial mutations might be modulated by mtDNA variation. We studied a 13-generation Mennonite pedigree with autosomal recessive myopathy and cardiomyopathy due to an SLC25A4 frameshift null mutation (c.523delC, p.Q175RfsX38), which codes for the heart-muscle isoform of the adenine nucleotide translocator-1. Ten homozygous null (adenine nucleotide translocator-1(-/-)) patients monitored over a median of 6 years had a phenotype of progressive myocardial thickening, hyperalaninemia, lactic acidosis, exercise intolerance, and persistent adrenergic activation. Electrocardiography and echocardiography with velocity vector imaging revealed abnormal contractile mechanics, myocardial repolarization abnormalities, and impaired left ventricular relaxation. End-stage heart disease was characterized by massive, symmetric, concentric cardiac hypertrophy; widespread cardiomyocyte degeneration; overabundant and structurally abnormal mitochondria; extensive subendocardial interstitial fibrosis; and marked hypertrophy of arteriolar smooth muscle. Substantial variability in the progression and severity of heart disease segregated with maternal lineage, and sequencing of mtDNA from five maternal lineages revealed two major European haplogroups, U and H. Patients with the haplogroup U mtDNAs had more rapid and severe cardiomyopathy than those with haplogroup H.

Characterization of the cardiac succinylome and its role in ischemia-reperfusion injury.

Succinylation refers to modification of lysine residues with succinyl groups donated by succinyl-CoA. Sirtuin5 (Sirt5) is a mitochondrial NAD(+)-dependent deacylase that catalyzes the removal of succinyl groups from proteins. Sirt5 and protein succinylation are conserved across species, suggesting functional importance of the modification. Sirt5 loss impacts liver metabolism but the role of succinylation in the heart has not been explored. We combined affinity enrichment with proteomics and mass spectrometry to analyze total succinylated lysine content of mitochondria isolated from WT and Sirt5(-/-) mouse hearts. We identified 887 succinylated lysine residues in 184 proteins. 44 peptides (5 proteins) occurred uniquely in WT samples, 289 (46 proteins) in Sirt5(-/-) samples, and 554 (133 proteins) were common to both groups. The 46 unique proteins in Sirt5(-/-) heart participate in metabolic processes such as fatty acid ß-oxidation (Eci2) and branched chain amino acid catabolism, and include respiratory chain proteins (Ndufa7, 12, 13, Dhsa). We performed label-free analysis of the peptides common to WT and Sirt5(-/-) hearts. 16 peptides from 9 proteins were significantly increased in Sirt5(-/-) by at least 30%. The adenine nucleotide transporter 1 showed the highest increase in succinylation in Sirt5(-/-) (108.4 fold). The data indicate that succinylation is widespread in the heart and enriched in metabolic pathways. We examined whether the loss of Sirt5 would impact ischemia-reperfusion (I/R) injury and we found an increase in infarct size in Sirt5(-/-) hearts compared to WT littermates (68.5(+)/-1.1% Sirt5(-/-) vs 39.6(+)/(-) 6.8% WT) following 20min of ischemia and 90-min reperfusion. We further demonstrate that I/R injury in Sirt5(-/-) heart is restored to WT levels by pretreatment with dimethyl malonate, a competitive inhibitor of succinate dehydrogenase (SDH), implicating alteration in SDH activity as causative of the injury.

Extracellular ADP prevents neuronal apoptosis via activation of cell antioxidant enzymes and protection of mitochondrial ANT-1.

Apoptosis in neuronal tissue is an efficient mechanism which contributes to both normal cell development and pathological cell death. The present study explores the effects of extracellular ADP on low [K(+)]-induced apoptosis in rat cerebellar granule cells. ADP, released into the extracellular space in brain by multiple mechanisms, can interact with its receptor or be converted, through the actions of ectoenzymes, to adenosine. The findings reported in this paper demonstrate that ADP inhibits the proapoptotic stimulus supposedly via: i) inhibition of ROS production during early stages of apoptosis, an effect mediated by its interaction with cell receptor/s. This conclusion is validated by the increase in SOD and catalase activities as well as by the GSSG/GSH ratio value decrease, in conjunction with the drop of ROS level and the prevention of the ADP protective effect by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a novel functionally selective antagonist of purine receptor; ii) safeguard of the functionality of the mitochondrial adenine nucleotide-1 translocator (ANT-1), which is early impaired during apoptosis. This effect is mediated by its plausible internalization into cell occurring as such or after its hydrolysis, by means of plasma membrane nucleotide metabolizing enzymes, and resynthesis into the cell. Moreover, the findings that ADP also protects ANT-1 from the toxic action of the two Alzheimer's disease peptides, i.e. Aß1-42 and NH2htau, which are known to be produced in apoptotic cerebellar neurons, further corroborate the molecular mechanism of neuroprotection by ADP, herein proposed.