Stimulation of the left ventricle through the coronary sinus with a newly developed 'over the wire' lead system--early experiences with lead handling and positioning.

AIMS: This report describes the initial clinical results with a newly designed guiding catheter and an 'over the wire' pacing lead based on angiolasty technology to stimulate the left ventricle using the transvenous route via the coronary sinus (OTW-CV lead). METHODS AND RESULTS: In 75% of the 15 patients (6 males, 9 females, mean age of 53 +/- 9 years) with congestive heart failure, access to coronary sinus required less than 2 min; in one patient. the attempt failed. Mean R wave amplitudes plus or minus the standard deviation, measured at apical, mid-ventricular and basal positions the anterior (11.4 +/- 9.2, 10.8 +/- 6.2, 9.3 +/- 6.3 mV) and lateral or posterior veins (10.1 +/- 10.7, 8.6 +/- 6.4, 7.7 +/- 4.3 mV) showed a trend favouring the apex without statistical significance. Pacing impedance, measured at the same sites and vein tributaries, ranged from 670 +/- 191 to 915 +/- 145 ohms. Pacing thresholds measured at apical and mid ventricular sites were significantly lower than at the base in the anterior vein 2.5 +/- 2.8 and 2.8 +/- 1.8 vs 5.6 +/- 2.7 V at 0.5 ms, P<0.001). Thresholds in the lateral/posterior veins showed a similar trend but did not reach statistical significance (3.0 +/- 1.7, 3.6 +/- 1.4 +/- 1.8 V at 0.5 ms). In patients, in whom thresholds were determined in more than one vein, the 'best' mean threshold was 1.6 +/- 0.7 V. CONCLUSION: The new 'over the wire' lead and guiding catheter system allows uncomplicated access to the coronary sinus and the depth of the coronary vein tributaries. Left ventricular sensing and pacing thresholds are acceptable for chronic use in implanted cardiac rhythm management systems.

Tom22 is a multifunctional organizer of the mitochondrial preprotein translocase.

Mitochondrial preproteins are imported by a multisubunit translocase of the outer membrane (TOM), including receptor proteins and a general import pore. The central receptor Tom22 binds preproteins through both its cytosolic domain and its intermembrane space domain and is stably associated with the channel protein Tom40 (refs 11-13). Here we report the unexpected observation that a yeast strain can survive without Tom22, although it is strongly reduced in growth and the import of mitochondrial proteins. Tom22 is a multifunctional protein that is required for the higher-level organization of the TOM machinery. In the absence of Tom22, the translocase dissociates into core complexes, representing the basic import units, but lacks a tight control of channel gating. The single membrane anchor of Tom22 is required for a stable interaction between the core complexes, whereas its cytosolic domain serves as docking point for the peripheral receptors Tom20 and Tom70. Thus a preprotein translocase can combine receptor functions with distinct organizing roles in a multidomain protein.

The J-related segment of tim44 is essential for cell viability: a mutant Tim44 remains in the mitochondrial import site, but inefficiently recruits mtHsp70 and impairs protein translocation.

Tim44 is a protein of the mitochondrial inner membrane and serves as an adaptor protein for mtHsp70 that drives the import of preproteins in an ATP-dependent manner. In this study we have modified the interaction of Tim44 with mtHsp70 and characterized the consequences for protein translocation. By deletion of an 18-residue segment of Tim44 with limited similarity to J-proteins, the binding of Tim44 to mtHsp70 was weakened. We found that in the yeast Saccharomyces cerevisiae the deletion of this segment is lethal. To investigate the role of the 18-residue segment, we expressed Tim44Delta18 in addition to the endogenous wild-type Tim44. Tim44Delta18 is correctly targeted to mitochondria and assembles in the inner membrane import site. The coexpression of Tim44Delta18 together with wild-type Tim44, however, does not stimulate protein import, but reduces its efficiency. In particular, the promotion of unfolding of preproteins during translocation is inhibited. mtHsp70 is still able to bind to Tim44Delta18 in an ATP-regulated manner, but the efficiency of interaction is reduced. These results suggest that the J-related segment of Tim44 is needed for productive interaction with mtHsp70. The efficient cooperation of mtHsp70 with Tim44 facilitates the translocation of loosely folded preproteins and plays a crucial role in the import of preproteins which contain a tightly folded domain.

Transvenous biventricular pacing for heart failure: can the obstacles be overcome?

Despite increasing evidence of hemodynamic benefit and long-term improvement in clinical status of congestive heart failure (CHF) patients with left ventricular and biventricular pacing, the risks and technical limitations of placing a permanent left ventricular pacing lead have prevented widespread clinical adoption of this therapy. Results of this and other recent investigations suggest it is necessary to target specific sites on the left ventricle to maximize hemodynamic benefit. However, limitations and variations of coronary vein anatomy, as well as patient safety, lead dislodgement, pacing thresholds, lead handling, and ease-of-use issues, present technical challenges for current transvenous permanent pacing lead designs. However, a new transvenous lead system based on an over-the-wire design appears to solve many of these problems and has proved feasible in acute clinical studies.

Separation of structural and dynamic functions of the mitochondrial translocase: Tim44 is crucial for the inner membrane import sites in translocation of tightly folded domains, but not of loosely folded preproteins.

The essential gene TIM44 encodes a subunit of the inner mitochondrial membrane preprotein translocase that forms a complex with the matrix heat-shock protein Hsp70. The specific role of Tim44 in protein import has not yet been defined because of the lack of means to block its function. Here we report on a Saccharomyces cerevisiae mutant allele of TIM44 that allows selective and efficient inactivation of Tim44 in organello. Surprisingly, the mutant mitochondria are still able to import preproteins. The import rate is only reduced by approximately 30% compared with wild-type as long as the preproteins do not carry stably folded domains. Moreover, the number of import sites is not reduced. However, the mutant mitochondria are strongly impaired in pulling folded domains of preproteins close to the outer membrane and in promoting their unfolding. Our results demonstrate that Tim44 is not an essential structural component of the import channel, but is crucial for import of folded domains. We suggest that the concerted action of Tim44 and mtHsp70 drives unfolding of preproteins and accelerates translocation of loosely folded preproteins. While mtHsp70 is essential for import of both tightly and loosly folded preproteins, Tim44 plays a more specialized role in translocation of tightly folded domains.

The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested preproteins in the absence of matrix Hsp70-Tim44.

Preprotein import into mitochondria is mediated by translocases located in the outer and inner membranes (Tom and Tim) and a matrix Hsp70-Tim44 driving system. By blue native electrophoresis, we identify an approximately 90K complex with assembled Tim23 and Tim17 as the core of the inner membrane import site for presequence-containing preproteins. Preproteins spanning the two membranes link virtually all Tim core complexes with one in four Tom complexes in a stable 600K supercomplex. Neither mtHsp70 nor Tim44 are present in stoichiometric amounts in the 600K complex. Preproteins in transit stabilize the Tim core complex, preventing an exchange of subunits. Our studies define a central role for the Tim core complexes in mitochondrial protein import; they are not passive diffusion channels, but can stably interact with preproteins and determine the number of translocation contact sites. We propose the hypothesis that mtHsp70 functions in protein import not only by direct interaction with preproteins, but also by exerting a regulatory effect on the Tim channel.

Multiple interactions of components mediating preprotein translocation across the inner mitochondrial membrane.

The protein transport machinery of the inner mitochondrial membrane contains three essential Tim proteins. Tim17 and Tim23 are thought to build a preprotein translocation channel, while Tim44 transiently interacts with the matrix heat shock protein Hsp70 to form an ATP-driven import motor. For this report we characterized the biogenesis and interactions of Tim proteins. (i) Import of the precursor of Tim44 into the inner membrane requires mtHsp70, whereas import and inner membrane integration of the precursors of Tim17 and Tim23 are independent of functional mtHsp70. (ii) Tim17 efficiently associates with Tim23 and mtHsp70, but only weakly with Tim44. (iii) Depletion of Tim44 does not affect the co-precipitation of Tim17 with antibodies directed against mtHsp70. (iv) Tim23 associates with both Tim44 and Tim17, suggesting the presence of two Tim23 pools in the inner membrane, a Tim44-Tim23-containing sub-complex and a Tim23-Tim17-containing sub-complex. (v) The association of mtHsp70 with the Tim23-Tim17 sub-complex is ATP sensitive and can be distinguished from the mtHsp70-Tim44 interaction by the differential influence of an amino acid substitution in mtHsp70. (vi) Genetic evidence, suppression of the protein import defect of a tim17 yeast mutant by overexpression of mtHsp70 and synthetic lethality of conditional mutants in the genes of Tim17 and mtHsp70, supports a functional interaction of mtHsp70 with Tim17. We conclude that the protein transport machinery of the mitochondrial inner membrane consists of dynamically interacting sub-complexes, each of which transiently binds mtHsp70.

The preprotein translocase of the inner mitochondrial membrane: evolutionary conservation of targeting and assembly of Tim17.

The preprotein translocase of the inner mitochondrial membrane has only been described in Saccharomyces cerevisiae to date. We report that the essential subunit Tim17 is highly conserved in evolution. The targeting and assembly of yeast Tim17 as well as that of human and Drosophila melanogaster Tim17 were characterized with isolated yeast mitochondria. Targeting signals in the mature protein direct the Tim17 precursors to the receptor Tom70 on the mitochondrial surface. In a membrane potential-dependent step the precursors insert into the inner membrane, adopt a characteristic topology and assemble with Tim23. The mechanisms of targeting and assembly were indistinguishable between the Tim17s from distinct organisms, indicating a high evolutionary conservation.

Mitochondrial protein import: biochemical and genetic evidence for interaction of matrix hsp70 and the inner membrane protein MIM44.

The import of preproteins into mitochondria involves translocation of the polypeptide chains through putative channels in the outer and inner membranes. Preprotein-binding proteins are needed to drive the unidirectional translocation of the precursor polypeptides. Two of these preprotein-binding proteins are the peripheral inner membrane protein MIM44 and the matrix heat shock protein hsp70. We report here that MIM44 is mainly exposed on the matrix side, and a fraction of mt-hsp70 is reversibly bound to the inner membrane. Mt-hsp70 binds to MIM44 in a 1:1 ratio, suggesting that mt-hsp70 is localizing to the membrane via its interaction with MIM44. Formation of the complex requires a functional ATPase domain of mt-hsp70. Addition of Mg-ATP leads to dissociation of the complex. Overexpression of mt-hsp70 rescues the protein import defect of mutants in MIM44; conversely, overexpression of MIM44 rescues protein import defects of mt-hsp70 mutants. In addition, yeast strains with conditional mutations in both MIM44 and mt-hsp70 are barely viable, showing a synthetic growth defect compared to strains carrying single mutations. We propose that MIM44 and mt-hsp70 cooperate in translocation of preproteins. By binding to MIM44, mt-hsp70 is recruited at the protein import sites of the inner membrane, and preproteins arriving at MIM44 may be directly handed over to mt-hsp70.

Identification of the essential yeast protein MIM17, an integral mitochondrial inner membrane protein involved in protein import.

We analyzed four Saccharomyces cerevisiae mutants defective in mitochondrial protein import and found that they are complemented by a novel gene encoding a 17 kDa protein. The protein is integrally located in the mitochondrial inner membrane and is termed MIM17. It shows significant homology to MIM23/Mas6p, a previously identified mitochondrial inner membrane protein required for the import of preproteins. Like MIM23, the precursor of MIM17 is synthesized without a presequence. A deletion of MIM17 is lethal. MIM17 thus joins the small group of mitochondrial proteins that are essential for the viability of yeast. We propose that MIM17 is an essential component of the preprotein import machinery of the mitochondrial inner membrane.

The essential yeast protein MIM44 (encoded by MPI1) is involved in an early step of preprotein translocation across the mitochondrial inner membrane.

The essential yeast gene MPI1 encodes a mitochondrial membrane protein that is possibly involved in protein import into the organelle (A. C. Maarse, J. Blom, L. A. Grivell, and M. Meijer, EMBO J. 11:3619-3628, 1992). For this report, we determined the submitochondrial location of the MPI1 gene product and investigated whether it plays a direct role in the translocation of preproteins. By fractionation of mitochondria, the mature protein of 44 kDa was localized to the mitochondrial inner membrane and therefore termed MIM44. Import of the precursor of MIM44 required a membrane potential across the inner membrane and involved proteolytic processing of the precursor. A preprotein in transit across the mitochondrial membranes was cross-linked to MIM44, whereas preproteins arrested on the mitochondrial surface or fully imported proteins were not cross-linked. When preproteins were arrested at two distinct stages of translocation across the inner membrane, only preproteins at an early stage of translocation could be cross-linked to MIM44. Moreover, solubilized MIM44 was found to interact with in vitro-synthesized preproteins. We conclude that MIM44 is a component of the mitochondrial inner membrane import machinery and interacts with preproteins in an early step of translocation.

Identification of MIM23, a putative component of the protein import machinery of the mitochondrial inner membrane.

A screening for yeast mutants impaired in mitochondrial protein import led to the identification of two genes (MPII and MPI2) encoding the essential components MIM44 and MIM17 of the inner membrane import machinery. We analyzed twelve additional mutants obtained in the screening and found two further complementation groups. One group represents mutants of SSC1, the gene encoding mitochondrial hsp70, an essential matrix protein required for protein import across the inner membrane. The second complementation group represents mutants of a new gene (MP13) encoding a 23 kDa integral inner membrane protein (MIM23). MIM23 is synthesized without a presequence, and its import to the inner membrane requires a membrane potential. MIM23 contains a domain homologous to half of MIM17. We speculate that MIM23 is a new member of the protein import machinery of the mitochondrial inner membrane.

Epitope-tagging vectors designed for yeast.

In order to facilitate the process of epitope-tagging of yeast proteins, we have constructed two Saccharomyces cerevisiae-Escherichia coli shuttle vectors that allow fusion of a sequence encoding an epitope of the human c-myc protein at the 3' end of any gene. An example of the use of this technique is presented.

MPI1, an essential gene encoding a mitochondrial membrane protein, is possibly involved in protein import into yeast mitochondria.

To identify components of the mitochondrial protein import pathway in yeast, we have adopted a positive selection procedure for isolating mutants disturbed in protein import. We have cloned and sequenced a gene, termed MPI1, that can rescue the genetic defect of one group of these mutants. MPI1 encodes a hydrophilic 48.8 kDa protein that is essential for cell viability. Mpi1p is a low abundance and constitutively expressed mitochondrial protein. Mpi1p is synthesized with a characteristic mitochondrial targeting sequence at its amino-terminus, which is most probably proteolytically removed during import. It is a membrane protein, oriented with its carboxy-terminus facing the intermembrane space. In cells depleted of Mpi1p activity, import of the precursor proteins that we tested thus far, is arrested. We speculate that the Mpi1 protein is a component of a proteinaceous import channel for translocation of precursor proteins across the mitochondrial inner membrane.

Demarcation of a sequence involved in mediating catabolite repression of the gene for the 11 kDa subunit VIII of ubiquinol-cytochrome c oxidoreductase in Saccharomyces cerevisiae.

A regulatory element has been identified in the promoter region of the gene encoding the 11 kDa subunit VIII of the ubiquinol-cytochrome c oxidoreductase in Saccharomyces cerevisiae. The element, which is approximately 40 bp long and situated 185 bp upstream of the initiator ATG, is essential for induction of gene expression during growth in the presence of non-fermentable carbon sources. This is shown by the regulated synthesis of beta-galactosidase in yeast cells harbouring a CYC1-lacZ fusion gene, in which the CYC1 UAS's had been replaced by a 43 bp subunit VIII gene promoter fragment. In addition two DNA-binding activities, which may represent either separate factors or different forms of a single factor, have been detected. Both factors are abundant and they bind in a mutually exclusive fashion to a DNA sequence just upstream of the regulatory element. Although it is unlikely that these factors are directly involved in the response of the subunit VIII gene to catabolite repression, the position of their binding sites relative to the UAS and to the 3'-terminus of a gene located only 361 bp upstream suggest that they are important in modulating transcriptional activity of this region.

Inactivation of the gene encoding the 11-kDa subunit VIII of the ubiquinol-cytochrome-c oxidoreductase in Saccharomyces cerevisiae.

The single nuclear gene encoding the 11-kDa subunit VIII of the ubiquinol-cytochrome-c oxidoreductase (complex III) in Saccharomyces cerevisiae has been inactivated by a one-step gene disruption procedure. Inactivation results in a loss of ubiquinol-cytochrome-c oxidoreductase activity (less than 1% wild type) and respiratory deficiency. Cells lacking the 11-kDa protein also display lowered steady-state levels of other complex-III subunits encoded by nuclear genes including the 14-kDa subunit VII and the Rieske Fe-S protein and of the mitochondrially encoded cytochrome b. The steady-state levels of the transcripts from the genes encoding these proteins are however not reduced. The results strongly imply that the 11-kDa protein plays an important role in regulating the synthesis of complex III at the post-transcriptional level, most likely assembly. Separation of chromosomes by pulsed-field gel electrophoresis of DNA of wild-type and of the mutant lacking the 11-kDa-protein gene followed by Southern blot analysis reveals that the latter gene is located on chromosome X rather than on XII as reported by Van Loon et al. [Mol. Gen. Genet. 197 (1984) 219-224].

Nucleotide sequence of the gene encoding the 11-kDa subunit of the ubiquinol-cytochrome-c oxidoreductase in Saccharomyces cerevisiae.

The nucleotide sequence of the gene encoding the 11-kDa subunit VIII of the ubiquinol-cytochrome-c oxidoreductase in Saccharomyces cerevisiae has been determined. The coding sequence has a length of 330 bp and is preceded at a distance of 361 bp by another reading frame, coding for a protein of as yet unknown function. The 11-kDa gene is transcribed independently of the URFx gene and transcription of both is sensitive to catabolite repression. Multiple 5' and 3' termini of transcripts of the gene for the 11-kDa subunit were identified by S1 nuclease protection analysis of DNA X RNA hybrids. The 5' termini map 52 +/- 2 and 60 +/- 2 nucleotides upstream of the initiation codon whereas the 3' termini map 336 +/- 2 and 350 +/- 2 nucleotides downstream of the stop codon. The subunit VIII reading frame encodes a protein with a molecular mass of 12.4 kDa and a polarity of 37.6%. It is predicted to contain a high content of beta-sheet segments, which may be capable of forming a barrel-like structure in a lipid bilayer. A comparison of the sequence with those of the small subunits of the beef heart complex reveals similarity with the 9.5-kDa subunit VII (core-linked protein) characterized by Borchart et al. (1986) FEBS Lett. 200, 81-86. The significance of this is discussed.

Subunit IV of yeast cytochrome c oxidase: cloning and nucleotide sequencing of the gene and partial amino acid sequencing of the mature protein.

The six small subunits (IV-VII, VIIa, VIII) of yeast cytochrome c oxidase are encoded by nuclear genes and imported into the mitochondria. We have isolated the gene for subunit IV from a yeast genomic clone bank and determined its complete nucleotide sequence. We have also isolated subunit IV from purified yeast cytochrome c oxidase and determined most of its amino acid sequence which confirms the positioning of approximately 90% of the amino acid residues. The sequence comparison shows that the coding sequence of the gene lacks introns and that subunit IV is made as a precursor with an amino-terminal extension of 25 residues, five of which are basic and none of them acidic. Precursor processing involves cleavage of a Leu-Gln bond.

Isolation, characterization and regulation of expression of the nuclear genes for the core II and Rieske iron-sulphur proteins of the yeast ubiquinol-cytochrome c reductase.

Cloning and mapping of the yeast nuclear genes for the core II (Mr 40 000) and Rieske iron-sulphur proteins of the mitochondrial ubiquinol-cytochrome c reductase, and comparison with the genomic regions in nuclear DNA from which they are derived, show that the genes are likely to be present in single copies and that they are not closely linked. They have been reintroduced into yeast cells on multi-copy plasmids and, similar to results obtained for the Mr 11 000 subunit [Van Loon et al., EMBO J. 2 (1983) 1765-1770], increase in the dosage of either gene prompts discoordinate synthesis of the encoded protein. Quantitative analysis of transformants carrying extra copies of the gene for the iron-sulphur protein shows that messenger RNA level, rate of synthesis and steady-state concentration of the protein correlate well with each other. This indicates that its level, in contrast to that of the Mr 11 000 subunit, is only determined by the concentration of its messenger RNA. Over-production of these proteins does not interfere with mitochondrial function as judged from growth rates of transformed cells on non-fermentable media. The excess Mr 40 000 protein is imported into the mitochondrion, showing that import of this subunit is not obligatorily coupled to complex assembly.