Human Heart Mitochondria Research Articles

Mitochondrial Telomerase Reverse Transcriptase Protects From Myocardial Ischemia/Reperfusion Injury by Improving Complex I Composition and Function.

The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), has protective functions in the cardiovascular system. TERT is not only present in the nucleus but also in mitochondria. However, it is unclear whether nuclear or mitochondrial TERT is responsible for the observed protection, and the appropriate tools are missing to dissect this. We generated new mouse models containing TERT exclusively in the mitochondria (mitoTERT mice) or the nucleus (nucTERT mice) to finally distinguish between the functions of nuclear and mitochondrial TERT. Outcome after ischemia/reperfusion, mitochondrial respiration in the heart, and cellular functions of cardiomyocytes, fibroblasts, and endothelial cells, as well, were determined. All mice were phenotypically normal. Although respiration was reduced in cardiac mitochondria from TERT-deficient and nucTERT mice, it was increased in mitoTERT animals. The latter also had smaller infarcts than wild-type mice, whereas nucTERT animals had larger infarcts. The decrease in ejection fraction after 1, 2, and 4 weeks of reperfusion was attenuated in mitoTERT mice. Scar size was also reduced and vascularization increased. Mitochondrial TERT protected a cardiomyocyte cell line from apoptosis. Myofibroblast differentiation, which depends on complex I activity, was abrogated in TERT-deficient and nucTERT cardiac fibroblasts and completely restored in mitoTERT cells. In endothelial cells, mitochondrial TERT enhanced migratory capacity and activation of endothelial nitric oxide synthase. Mechanistically, mitochondrial TERT improved the ratio between complex I matrix arm and membrane subunits, explaining the enhanced complex I activity. In human right atrial appendages, TERT was localized in mitochondria and there increased by remote ischemic preconditioning. The telomerase activator TA-65 evoked a similar effect in endothelial cells, thereby increasing their migratory capacity, and enhanced myofibroblast differentiation. Mitochondrial, but not nuclear TERT, is critical for mitochondrial respiration and during ischemia/reperfusion injury. Mitochondrial TERT improves complex I subunit composition. TERT is present in human heart mitochondria, and remote ischemic preconditioning increases its level in those organelles. TA-65 has comparable effects ex vivo and improves the migratory capacity of endothelial cells and myofibroblast differentiation. We conclude that mitochondrial TERT is responsible for cardioprotection, and its increase could serve as a therapeutic strategy.

DNA Replication in Human Mitochondria.

DNA replication in human mitochondria has been studied for several decades; however, its mechanism still remains unclear. During the last 15 years, many new experimental data on the mitochondrial replication have appeared, although extremely contradictory. Two asynchronous (strand displacement and RITOLS) and one synchronous (strand-coupled) replication models have been proposed. In the asynchronous models, replication from the origin in the H-chain starts earlier, so that the replication of the two chains ends at different times. The synchronous model is more traditionaland implies two replication forks with leading and lagging strands initiated at the same origin. For each of the three models, both confirming and contradicting experimental data exist. Most likely, there is no single model of mitochondrial replication. It is possible that the unique mitochondrial replication machinery that has originated as a results of endosymbiosis has an unexpected variety of replication strategies to maintain the mitochondrial genome. An unusual combination of enzymes of different origin (phage, bacterial, eukaryotic) and unique features of the mitochondrial genome (existance of heavy and light chains, insertions of ribonucleotides, a variety of origins) can allow replication through different mechanisms. In human mitochondria, asynchronous replication seems to dominate; however, synchronous replication is also possible under certain conditions. In the human heart mitochondria, circular mitochondrial DNA (mtDNA) molecules can rearrange in a network of rapidly replicating linear genomes, thereby suggesting possible existence of a wide range of replication mechanisms in the mitochondria. The review describes the main stages of mtDNA replication and enzymes involved in this process, as well as discusses the prospects of mitochondrial replication studies.

Heart failure–induced activation of phospholipase iPLA2γ generates hydroxyeicosatetraenoic acids opening the mitochondrial permeability transition pore

Congestive heart failure typically arises from cardiac myocyte necrosis/apoptosis, associated with the pathological opening of the mitochondrial permeability transition pore (mPTP). mPTP opening decreases the mitochondrial membrane potential leading to the activation of Ca2+-independent phospholipase A2γ (iPLA2γ) and the production of downstream toxic metabolites. However, the array of enzymatic mediators and the exact chemical mechanisms responsible for modulating myocardial mPTP opening remain unclear. Herein, we demonstrate that human heart failure activates specific myocardial mitochondrial phospholipases that increase Ca2+-dependent production of toxic hydroxyeicosatetraenoic acids (HETEs) and attenuate the activity of phospholipases that promote the synthesis of protective epoxyeicosatrienoic acids (EETs). Mechanistically, HETEs activated the Ca2+-induced opening of the mPTP in failing human myocardium, and the highly selective pharmacological blockade of either iPLA2γ or lipoxygenases attenuated mPTP opening in failing hearts. In contrast, pharmacological inhibition of cytochrome P450 epoxygenases opened the myocardial mPTP in human heart mitochondria. Remarkably, the major mitochondrial phospholipase responsible for Ca2+-activated release of arachidonic acid (AA) in mitochondria from non-failing hearts was calcium-dependent phospholipase A2ζ (cPLA2ζ) identified by sequential column chromatographies and activity-based protein profiling. In contrast, iPLA2γ predominated in failing human myocardium. Stable isotope kinetics revealed that in non-failing human hearts, cPLA2ζ metabolically channels arachidonic acid into EETs, whereas in failing hearts, increased iPLA2γ activity channels AA into toxic HETEs. These results mechanistically identify the sequelae of pathological remodeling of human mitochondrial phospholipases in failing myocardium. This remodeling metabolically channels AA into toxic HETEs promoting mPTP opening, which induces necrosis/apoptosis leading to further progression of heart failure.

Cytochrome c Effect on Respiration of Heart Mitochondria: Influence of Various Factors

The effect of exogenous cytochrome c on respiration rate of the rat and human heart mitochondria was assessed in situ, using permeabilized fibers. It was (i) much more pronounced in State 2 and 4 than in State 3 with all the respiratory substrates (pyruvate+malate, succinate, palmitoyl-CoA+carnitine and octanoyl-L-carnitine), (ii) different with different substrates, (iii) much higher after ischemia in both metabolic states, particularly in the case of succinate oxidation compared to pyruvate+malate, (iv) the highest in State 4 with succinate as a substrate. Similar results were obtained with the isolated rat and rabbit heart mitochondria. The differences in the degree of stimulation of mitochondrial respiration by cytochrome c and, thus, sensitivity of cytochrome c test in evaluation of the intactness/injury of outer mitochondrial membrane are probably determined by the differences in the cytochrome c role in the control of mitochondrial respiration in the above-described conditions.

Location of the Coenzyme Binding Site in the Porcine Mitochondrial NADP-dependent Isocitrate Dehydrogenase

The structure of crystalline porcine mitochondrial NADP-dependent isocitrate dehydrogenase (IDH) has been determined in complex with Mn2+-isocitrate. Based on structural alignment between this porcine enzyme and seven determined crystal structures of complexes of NADP with bacterial IDHs, Arg83, Thr311, and Asn328 were chosen as targets for site-directed mutagenesis of porcine IDH. The circular dichroism spectra of purified wild-type and mutant enzymes are similar. The mutant enzymes exhibit little change in Km for isocitrate or Mn2+, showing that these residues are not involved in substrate binding. In contrast, the Arg83 mutants, Asn328 mutants, and T311A exhibit 3-20-fold increase in the Km(NADP). We propose that Arg83 enhances NADP affinity by hydrogen bonding with the 3'-OH of the nicotinamide ribose, whereas Asn328 hydrogen bonds with N1 of adenine. The pH dependence of Vmax for Arg83 and Asn328 mutants is similar to that of wild-type enzyme, but for all the Thr311 mutants, pK(es) is increased from 5.2 in the wild type to approximately 6.0. We have previously attributed the pH dependence of Vmax to the deprotonation of the metal-bound hydroxyl of isocitrate in the enzyme-substrate complex, prior to the transfer of a hydride from isocitrate to NADP's nicotinamide moiety. Thr311 interacts with the nicotinamide ribose and is the closest of the target amino acids to the nicotinamide ring. Distortion of the nicotinamide by Thr311 mutation will likely be transmitted to Mn2+-isocitrate resulting in an altered pK(es). Because porcine and human mitochondrial NADP-IDH have 95% sequence identity, these results should be applicable to the human enzyme.

Supramolecular Assemblies of Human Frataxin are Formed via Subunit–Subunit Interactions Mediated by a Non-conserved Amino-terminal Region

The mitochondrial protein frataxin is emerging as a novel mechanism to promote iron metabolism while also providing anti-oxidant protection. Recombinant frataxin proteins from different species are able to form large molecular assemblies that store Fe(III) as a stable mineral in vitro. Furthermore, monomeric and assembled forms of frataxin donate Fe(II) to the Fe-S cluster scaffold protein IscU, [3Fe-4S]1+ aconitase, and ferrochelatase in vitro. However, little is known about the speciation of frataxin in vivo, and the physiologically relevant form(s) of the protein remains undefined. Here, we report that human heart mitochondria contain frataxin species of increasing negative surface charge and molecular mass, ranging from monomer to polymers of >1 MDa. Moreover, we show that the main partner protein of frataxin, IscU, binds in a stable manner to frataxin oligomers. These results suggest that assembly is a physiologic property of frataxin. Biochemical analyses further reveal that, unlike the prokaryotic and yeast frataxin homologues, which require iron-protein interactions for assembly, human frataxin uses stable subunit-subunit interactions involving a non-conserved amino-terminal region. We propose that human frataxin is a modular protein that depends on self-assembly to accomplish its diverse functions.

The high-resolution structure of the NADP(H)-binding component (dIII) of proton-translocating transhydrogenase from human heart mitochondria

Background: Transhydrogenase, located in the inner membranes of animal mitochondria and the cytoplasmic membranes of bacteria, couples the transfer of reducing equivalents between NAD(H) and NADP(H) to proton pumping. The protein comprises three subunits termed dI, dII and dIII. The dII component spans the membrane. The dI component, which contains the binding site for NAD +/NADH, and the dIII component, which has the binding site for NADP +/NADPH, protrude from the membrane. Proton pumping is probably coupled to changes in the binding affinities of dIII for NADP + and NADPH. Results: The first X-ray structure of the NADP(H)-binding component, dIII, of human heart transhydrogenase is described here at 2.0 Å resolution. It comprises a single domain resembling the classical Rossmann fold, but NADP + binds to dIII with a reversed orientation. The first βαβαβ motif of dIII contains a Gly-X-Gly-X-X-Ala/Val ‘fingerprint’, but it has a different function to that in the classical Rossmann structure. The nicotinamide ring of NADP + is located on a ridge where it is exposed to interaction with NADH on the dI subunit. Two distinctive features of the dIII structure are helix D/loop D, which projects from the β sheet, and loop E, which forms a ‘lid’ over the bound NADP +. Conclusions: Helix D/loop D interacts with the bound nucleotide and loop E, and probably interacts with the membrane-spanning dII. Changes in ionisation and conformation in helix D/loop D, resulting from proton translocation through dII, are thought to be responsible for the changes in affinity of dIII for NADP + and NADPH that drive the reaction.

Evidence for the stabilization of NADPH relative to NADP + on the dIII components of proton-translocating transhydrogenases from Homo sapiens and from Rhodospirillum rubrum by measurement of tryptophan fluorescence

A unique Trp residue in the recombinant dIII component of transhydrogenase from human heart mitochondria ( hsdIII), and an equivalent Trp engineered into the dIII component of Rhodospirillum rubrum transhydrogenase ( rrdIII.D155W), are more fluorescent when NADP + is bound to the proteins, than when NADPH is bound. We have used this to determine the occupancy of the binding site during transhydrogenation reactions catalysed by mixtures of recombinant dI from the R. rubrum enzyme and either hsdIII or rrdIII.D155W. The standard redox potential of NADP +/NADPH bound to the dIII proteins is some 60–70 mV higher than that in free solution. This results in favoured reduction of NADP + by NADH at the catalytic site, and supports the view that changes in affinity at the nucleotide-binding site of dIII are central to the mechanism by which transhydrogenase is coupled to proton translocation across the membrane.

Mitochondrial endonuclease activity in the rat varies markedly among tissues in relation to the rate of tissue metabolism.

Rat heart mitochondria contain a potent endonuclease activity that closely resembles the endonuclease of bovine and human heart mitochondria, and shows a striking preference for an evolutionarily conserved sequence that resides just upstream from the heavy (H)-strand origin of DNA replication (Ori H), (Low, R.L. et al. (1988) Nucleic Acids Res. 16, 6427-6425). This study reports that while the site-directed endonuclease is evident in the mt fractions of several rat organs, the levels of activity among them varies in an unexpected and marked fashion. There is nearly 200-times more of this endonuclease activity per mg of mt protein in the heart than in the liver (or spleen). Levels intermediate to those in heart and liver are found in the kidney and brain. The large variations in endonuclease activity do not correlate with reported rates of mtDNA turnover among tissues and are in contrast to the much smaller variations in levels of mtDNA and DNA polymerase-gamma activity. However, there may be some relationship between the amount of the endonuclease and the rate of oxidative phosphorylation.

Analysis of polyadenylated RNA from human heart mitochondria

The content of the mitochondrial poly(A)-RNA fraction of human heart has been analyzed by electrophoresis through agarose slab gels in the presence of methyl mercury hydroxide and staining with ethidium bromide. It is possible to identify all the heavy strand coded mRNAs in the electrophoretic pattern. This pattern is qualitatively similar to that obtained from the mitochondria of cells cultured in vitro (HeLa cells), although differences in the relative amount of some components have been detected.

Antibody-independent activation of C1. II. Evidence for two classes of nonimmune activators of the classical pathway of complement.

Abstract Nonimmune activation of the first component of complement (C1) by cardiolipin (CL) vesicles present specific features which were not demonstrated on immune complexes. CL vesicles which activate C1 in the presence of C1-inhibitor (C1-INH) were found to bind C1s in the absence of C1r, and to induce a specific C1r-independent cleavage of C1q-bound C1s. Therefore, several known natural nonimmune activators were analyzed by comparing their ability to activate C1 in the presence of C1-INH and to mediate a C1r-independent cleavage of C1s. Freshly isolated human heart mitochondria (HHM) activated C1 only in the absence of C1-INH. However, mitoplasts derived from HHM (HHMP) activated C1 regardless of the presence of C1-INH, and induced a specific cleavage of C1q-bound C1s. The same pattern was observed in the case of smooth E. coli and a semi-rough E. coli strain. DNA, known to activate C1 only in the absence of C1-INH, does not induce C1s cleavage in the absence of C1r. Thus, nonimmune activators can be classified into two distinct categories. "Strong" activators, such as CL vesicles, HHMP, or the semi-rough E. coli strain J5 can activate C1 in the presence of C1-INH. By using C1qs2 as a probe, they exhibit a specific, C1r-independent cleavage of C1s. C1s-binding to C1q is a critical factor for the activation process in this group. In the case of "weak" activators, such as E. coli smooth strains, DNA, or HHM, no C1s-binding to activator-bound C1q was detected, and C1r-independent C1s cleavage and C1 activation in the presence of C1-INH were not observed. As in the case of immune complexes, C1r activation appears to play a key role in the C1 activation by "weak" activators.

Palmitate oxidation and cytochromes in human and rat heart

Palmitate oxidation rates were determined in mitochondria and homogenates of human and rat heart muscle. Oxidation rates based on the sum of the production of 14CO 2 and 14C-labeled acid-soluble productsfrom [ 14C]palmitatewere in good agreement with those calculated from the oxygen consumption during palmitate oxidation. With limited amounts of substrate palmitate was completely oxidized to CO 2, but with non-limiting palmitate concentrations acetyl-carnitine and citric acid cycle intermediates accumulated. Palmitate oxidation rates per mg protein were about equal for human and rat heart muscle. Palmitate oxidation rates by homogenates and mitochondria were about equal, when expressed on the basis of cytochrome c oxidase activity for each species, but they differed between rat and man. This difference is caused by the lower activity of cytochrome c oxidase in human heart mitochondria. Difference spectrophotometric measurements showed, however, higher concentrations of cytochrome aa 3 and c + c 1 in human heart mitochondria than in those of the rat.

Incidence of intermembrane alterations in human heart mitochondria: A preliminary ultrastructural study

An alteration consisting of widening of the inner membrane space (approximately 240 A) of the human heart mitochondria was studied and compared in surgical and autopsy material. The presence of these changes in well-preserved surgical material suggests that it may be a morphologic manifestation of an unknown biochemical alteration.

Antibody-Independent Activation of Human C1 after Interaction with Heart Subcellular Membranes

Abstract A 19S, complement-fixing, heat-labile factor reactive with human heart mitochondria and microsomes has been detected in all normal human serum. This serum factor reacted with autologous and homologous heart mitochondria but did not react with mitochondria derived from cardiac tissue from other species. Subsequent characterization has indicated that this heat-labile factor was the first component of human complement. The C1 reacted with the mitochondrial membrane, was activated, and resulted in an apparent antibody-independent activation of the complement system.