Adenine Nucleotide Translocase Isoforms Research Articles

The Role of Adenine Nucleotide Translocase in the Assembly of Respiratory Supercomplexes in Cardiac Cells.

Individual electron transport chain complexes have been shown to assemble into the supramolecular structures known as the respiratory chain supercomplexes (RCS). Several studies reported an associative link between RCS disintegration and human diseases, although the physiological role, structural integrity, and mechanisms of RCS formation remain unknown. Our previous studies suggested that the adenine nucleotide translocase (ANT), the most abundant protein of the inner mitochondrial membrane, can be involved in RCS assembly. In this study, we sought to elucidate whether ANT knockdown (KD) affects RCS formation in H9c2 cardiomyoblasts. Results showed that genetic silencing of ANT1, the main ANT isoform in cardiac cells, stimulated proliferation of H9c2 cardiomyoblasts with no effect on cell viability. ANT1 KD reduced the ΔΨm but increased total cellular ATP levels and stimulated the production of total, but not mitochondrial, reactive oxygen species. Importantly, downregulation of ANT1 had no significant effects on the enzymatic activity of individual ETC complexes I–IV; however, RCS disintegration was stimulated in ANT1 KD cells as evidenced by reduced levels of respirasome, the main RCS. The effects of ANT1 KD to induce RCS disassembly was not associated with acetylation of the exchanger. In conclusion, our study demonstrates that ANT is involved in RCS assembly.

Human adenine nucleotide translocases physically and functionally interact with respirasomes.

Members of the adenine nucleotide translocase (ANT) family exchange ADP for ATP across the mitochondrial inner membrane, an activity that is essential for oxidative phosphorylation (OXPHOS). Mutations in or dysregulation of ANTs is associated with progressive external ophthalmoplegia, cardiomyopathy, nonsyndromic intellectual disability, apoptosis, and the Warburg effect. Binding partners of human ANTs have not been systematically identified. The absence of such information has prevented a detailed molecular understanding of the assorted ANT-associated diseases, including insight into their disparate phenotypic manifestations. To fill this void, in this study, we define the interactomes of two human ANT isoforms. Analogous to its yeast counterpart, human ANTs associate with heterologous partner proteins, including the respiratory supercomplex (RSC) and other solute carriers. The evolutionarily conserved ANT-RSC association is particularly noteworthy because the composition, and thereby organization, of RSCs in yeast and human is different. Surprisingly, absence of the major ANT isoform only modestly impairs OXPHOS in HEK293 cells, indicating that the low levels of other isoforms provide functional redundancy. In contrast, pharmacological inhibition of OXPHOS expression and function inhibits ANT-dependent ADP/ATP exchange. Thus ANTs and the OXPHOS machinery physically interact and functionally cooperate to enhance ANT transport capacity and mitochondrial respiration.

Human Adenine Nucleotide Translocase (ANT) Modulators Identified by High-Throughput Screening of Transgenic Yeast

Transport of ADP and ATP across mitochondria is one of the primary points of regulation to maintain cellular energy homeostasis. This process is mainly mediated by adenine nucleotide translocase (ANT) located on the mitochondrial inner membrane. There are four human ANT isoforms, each having a unique tissue-specific expression pattern and biological function, highlighting their potential as drug targets for diverse clinical indications, including male contraception and cancer. In this study, we present a novel yeast-based high-throughput screening (HTS) strategy to identify compounds inhibiting the function of ANT. Yeast strains generated by deletion of endogenous proteins with ANT activity followed by insertion of individual human ANT isoforms are sensitive to cell-permeable ANT inhibitors, which reduce proliferation. Screening hits identified in the yeast proliferation assay were characterized in ADP/ATP exchange assays employing recombinant ANT isoforms expressed in isolated yeast mitochondria and Lactococcus lactis as well as by oxygen consumption rate in mammalian cells. Using this approach, closantel and CD437 were identified as broad-spectrum ANT inhibitors, whereas leelamine was found to be a modulator of ANT function. This yeast “knock-out/knock-in” screening strategy is applicable to a broad range of essential molecular targets that are required for yeast survival.

Changes in the expression of the human adenine nucleotide translocase isoforms condition cellular metabolic/proliferative status.

Human cells express four mitochondrial adenine nucleotide translocase (hANT) isoforms that are tissue-specific and developmentally regulated. hANT1 is mainly expressed in terminally differentiated muscle cells; hANT2 is growth-regulated and is upregulated in highly glycolytic and proliferative cells; and hANT3 is considered to be ubiquitous and non-specifically regulated. Here, we studied how the expression of hANT isoforms is regulated by proliferation and in response to metabolic stimuli, and examined the metabolic consequences of their silencing and overexpression. In HeLa and HepG2 cells, expression of hANT3 was upregulated by shifting metabolism towards oxidation or by slowed growth associated with contact inhibition or growth-factor deprivation, indicating that hANT3 expression is highly regulated. Under these conditions, changes in hANT2 mRNA expression were not observed in either HeLa or HepG2 cells, whereas in SGBS preadipocytes (which, unlike HeLa and HepG2 cells, are growth-arrest-sensitive cells), hANT2 mRNA levels decreased. Additionally, overexpression of hANT2 promoted cell growth and glycolysis, whereas silencing of hANT3 decreased cellular ATP levels, limited cell growth and induced a stress-like response. Thus, cancer cells require both hANT2 and hANT3, depending on their proliferation status: hANT2 when proliferation rates are high, and hANT3 when proliferation slows.

Expression of adenine nucleotide translocase (ANT) isoform genes is controlled by PGC-1α through different transcription factors.

Adenine nucleotide translocase (ANT) isoforms are mitochondrial proteins encoded by nuclear DNA that catalyze the exchange of ATP generated in the mitochondria for ADP produced in the cytosol. The aim of this study was to determine the role of the transcriptional coactivator PGC-1α (peroxisome proliferator-activated receptor-γ [PPAR-γ] coactivator 1α), a master regulator of mitochondrial oxidative metabolism, in the regulation of the expression of ANT isoform genes and to identify the transcription factors involved. We found that PGC-1α overexpression induced the expression of all ANT human and mouse isoforms but to different degrees. The transcription factor ERRα was involved in PGC-1α-induced expression of all human ANT isoforms (hANT1-3) in HeLa cells as well as in the regulation of mouse isoforms (mANT1-2) in C2C12 myotubes and 3T3-L1 adipocytes, even though ANT isoforms have important physiological differences and are regulated in a tissue-specific manner. In addition to ERRα, PPARδ and mTOR pathways were involved in the induction of mANT1-2 by PGC-1α in C2C12 myotubes, while PPARγ was involved in PGC-1α-regulation of mANT1-2 in 3T3-L1 adipocytes. Furthermore, the regulation of mANT genes by PGC-1α was also observed in vivo in knockout mouse models lacking PGC-1α. In summary, our results show that the regulation of genes encoding ANT isoforms is controlled by PGC-1α through different transcription factors depending on cell type.

ANT-VDAC1 interaction is direct and depends on ANT isoform conformation in vitro

The voltage-dependent anion channel (VDAC) and the adenine nucleotide translocase (ANT) have central roles in mitochondrial functions such as nucleotides transport and cell death. The interaction between VDAC, an outer mitochondrial membrane protein and ANT, an inner membrane protein, was studied in isolated mitochondria and in vitro. Both proteins were isolated from various mitochondrial sources and reconstituted in vitro using a biomimetic system composed of recombinant human VDAC isoform 1 (rhVDAC1) immobilized on a surface plasmon resonance (SPR) sensor chip surface. Two enriched-preparations of (H)ANT (ANT from heart, mainly ANT1) and (L)ANT (ANT from liver, mainly ANT2) isoforms interacted differently with rhVDAC1. Moreover, the pharmacological ANT inhibitors atractyloside and bongkrekic acid modulated this interaction. Thus, ANT-VDAC interaction depends both on ANT isoform identity and on the conformation of ANT.

The Adenine Nucleotide Translocase 2, a Mitochondrial Target for Anticancer Biotherapy

Apoptosis or programmed cell death is one of the most important signaling pathways, which controls the cell fate and is frequently impaired in cancer cells. The major consequences of apoptosis inhibition are the accumulation of mutated cells and their enhanced resistance to chemotherapeutic agents. More generally, intrinsic or acquired apoptosis resistance may favor tumor growth and dissemination of mutated cells, and this resistance can be responsible of treatment failure. Mitochondria are central organelles in the signaling pathway of apoptosis and have been proposed as favorite candidates for anticancer biotherapy because they accommodate potential biological targets. Indeed, although cancer cells are highly glycolytic and become energetically independent of oxidative phosphorylation. Mitochondrial proteins involved in the so-called mitochondrial membrane permeabilization (MMP), such as the adenine nucleotide translocase (ANT) can be instrumental to elicit cancer cell death. Thus, multiple pharmacological and molecular studies revealed ANT could be a promising therapeutic target for the following reasons: (i) ANT is a bi-functional protein, it mediates the vital exchange of cytosolic ADP and mitochondrial ATP and participates to MMP via its capacity to become a lethal pore in the mitochondrial inner membrane; (ii) both ANT functions are under the control of the (anti)-oncogenes from the Bax/Bcl-2 family, (iii) several chemotherapeutic agents directly modulate the pore-forming activity of ANT and (iv) ANT2 isoform, which is anti-apoptotic, can be overexpressed in human cancers and its invalidation sensitize cells to apoptosis. In this review, we will introduce the knowledge of the role of ANT in MMP, illustrate the modulation of ANT by several strategies and propose the possibility to target preferentially the ANT2 isoform for induction of cancer cell apoptosis.

Functional expression of human adenine nucleotide translocase 4 in Saccharomyces cerevisiae.

The adenine nucleotide translocase (ANT) mediates the exchange of ADP and ATP across the inner mitochondrial membrane. The human genome encodes multiple ANT isoforms that are expressed in a tissue-specific manner. Recently a novel germ cell-specific member of the ANT family, ANT4 (SLC25A31) was identified. Although it is known that targeted depletion of ANT4 in mice resulted in male infertility, the functional biochemical differences between ANT4 and other somatic ANT isoforms remain undetermined. To gain insight into ANT4, we expressed human ANT4 (hANT4) in yeast mitochondria. Unlike the somatic ANT proteins, expression of hANT4 failed to complement an AAC-deficient yeast strain for growth on media requiring mitochondrial respiration. Moreover, overexpression of hANT4 from a multi-copy plasmid interfered with optimal yeast growth. However, mutation of specific amino acids of hANT4 improved yeast mitochondrial expression and supported growth of the AAC-deficient yeast on non-fermentable carbon sources. The mutations affected amino acids predicted to interact with phospholipids, suggesting the importance of lipid interactions for function of this protein. Each mutant hANT4 and the somatic hANTs exhibited similar ADP/ATP exchange kinetics. These data define common and distinct biochemical characteristics of ANT4 in comparison to ANT1, 2 and 3 providing a basis for study of its unique adaptation to germ cells.

Adenine nucleotide translocase family: four isoforms for apoptosis modulation in cancer

Mitochondria have important functions in mammalian cells as the energy powerhouse and integrators of the mitochondrial pathway of apoptosis. The adenine nucleotide translocase (ANT) is a family of proteins involved in cell death pathways that perform distinctly opposite functions to regulate cell fate decisions. On the one hand, ANT catalyzes the adenosine triphosphate export from the mitochondrial matrix to the intermembrane space with the concomitant import of ADP from the intermembrane space to the matrix. On the other hand, during periods of stress, ANT could function as a lethal pore and trigger the process of mitochondrial membrane permeabilization, which leads irreversibly to cell death. In human, ANT is encoded by four homologous genes, whose expression is not only tissue specific, but also varies according to the pathophysiological state of the cell. Recent evidence revealed a differential role of the ANT isoforms in apoptosis and a deregulation of their expression in cancer. In this review, we introduce the current knowledge of ANT in apoptosis and cancer cells and propose a novel classification of ANT isoforms.

Adenine nucleotide translocase: a component of the phylogenetically conserved cell death machinery

Lethal mitochondrial membrane permeabilization has been depicted as the result of two fundamentally distinct processes, namely primary mitochondrial outer membrane permeabilization (MOMP) versus permeability transition (PT) ignited at the level of the mitochondrial inner membrane. MOMP and PT have been connected to apoptosis and necrosis, respectively. Moreover, it has been thought that MOMP was mediated by pro-apoptotic multidomain proteins of the Bcl-2 family (Bax and Bak), which would operate near-to-independently from the permeability transition pore complex (PTPC) composed by voltage-dependent anion channel (VDAC), adenine nucleotide translocase (ANT) and cyclophilin D. A recent paper in Molecular and Cellular Biology now reveals the obligate contribution of one particular ANT isoform to the execution of developmental and homeostatic cell death in Caenorhabditis elegans. The physical and functional interaction between CED-9, the sole multidomain Bcl-2 protein of C. elegans, and ANT emphasizes the existence of an intricate, phylogenetically conserved crosstalk between Bcl-2 family proteins and constituents of the PTPC. In this issue of Cell Death and Differentiation, Malorni et al. further corroborate this notion by showing that type 2 transglutaminase (TG2) is essential for the correct assembly/function of ANT1, and that, at least in some experimental settings, TG2 might be required to enable and/or stabilize the pro-apoptotic association of Bax with ANT1.

Altered expression of the adenine nucleotide translocase isoforms and decreased ATP synthase activity in skeletal muscle mitochondria in heart failure

Exercise intolerance is a component of heart failure (HF) syndrome. We aimed to identify the defects in skeletal muscle mitochondria which may contribute to the development of peripheral myopathy. Subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria were isolated from gastrocnemius muscle of control dogs ( N = 5) and dogs with pacing-induced HF ( N = 5). The measurement of integrated mitochondrial function (oxidative phosphorylation) and of individual activities of mitochondrial electron transport chain (ETC) complexes was complemented with the assessment of the amount and activity of the components of the phosphorylation apparatus. Both populations of skeletal muscle mitochondria isolated from HF have significantly decreased ADP-stimulated (state 3) respiratory rates with complex I, II and III substrates. The decrease in respiratory rates of skeletal muscle SSM are neither relieved upon collapsing the mitochondrial potential with an uncoupler nor increased in the presence of maximal ADP concentrations showing a defect in the ETC, which needs further investigation. In contrast, respiratory rates of skeletal muscle IFM from HF were relieved with the uncoupler and partially improved in the presence of maximal ADP concentrations. In these IFM, alterations in the phosphorylation apparatus were detected with a decreased amount of ANT isoform 2 and increased amount of isoform 1. The IFM dysfunction may be explained by this shift in ANT isoforms. In conclusion, pacing-induced HF causes a decrease in the oxidative phosphorylation of skeletal muscle mitochondria due to defects in the ETC and phosphorylation apparatus.

Chemosensitization by Knockdown of Adenine Nucleotide Translocase-2

Mitochondrial membrane permeabilization (MMP) is a rate-limiting step of apoptosis, including in anticancer chemotherapy. Adenine nucleotide translocase (ANT) mediates the exchange of ADP and ATP on the inner mitochondrial membrane in healthy cells. In addition, ANT can cooperate with Bax to form a lethal pore during apoptosis. Humans possess four distinct ANT isoforms, encoded by four genes, whose transcription depends on the cell type, developmental stage, cell proliferation, and hormone status. Here, we show that the ANT2 gene is up-regulated in several hormone-dependent cancers. Knockdown of ANT2 by RNA interference induced no major changes in the aspect of the mitochondrial network or cell cycle but provoked minor increase in mitochondrial transmembrane potential and reactive oxygen species level and reduced intracellular ATP concentration without affecting glycolysis. At expression and functional levels, ANT2 depletion was not compensated by other ANT isoforms. Most importantly, ANT2, but not ANT1, silencing facilitated MMP induction by lonidamine, a mitochondrion-targeted antitumor compound already used in clinical studies for breast, ovarian, glioma, and lung cancer as well as prostate adenoma. The combination of ANT2 knockdown with lonidamine induced apoptosis irrespective of the Bcl-2 status. These data identify ANT2 as an endogenous inhibitor of MMP and suggest that its selective inhibition could constitute a promising strategy of chemosensitization.

Overexpression of adenine nucleotide translocase reduces Ca 2+ signal transmission between the ER and mitochondria

The adenine nucleotide translocase (ANT), besides transferring ATP from the mitochondrial matrix to the rest of the cell, has also been proposed to be involved in mitochondrial permeability transition (MPT), and accordingly in mitochondrial Ca 2+ homeostasis. In order to assess the role of ANT in Ca 2+ signal transmission from the endoplasmic reticulum (ER) to mitochondria, we overexpressed the various ANT isoforms and measured the matrix [Ca 2+] ([Ca 2+] m) increases evoked by stimulation with IP 3-dependent agonists. ANT overexpression reduced the amplitude of the [Ca 2+] m peak following Ca 2+ release, an effect that was markedly greater for ANT-1 and ANT-3 isoforms than for ANT-2. Three further observations might explain these findings. First, the effect was partially reversed by treating the cells with cyclosporine A, suggesting the involvement of MPT. Second, the effect was paralleled by alterations of the 3D structure of the mitochondria. Finally, ANT-1 and ANT-3 overexpression also caused a reduction of ER Ca 2+ loading that caused a marginal decrease in the cytosolic Ca 2+ responses. Overall, these data provide evidence for the involvement of ANT-1 and ANT-3 in the induction of MPT and indicate the relevance of this phenomenon in ER–mitochondria Ca 2+ transfer.

Hypothyroidism Reduces Tricarboxylate Carrier Activity and Expression in Rat Liver Mitochondria by Reducing Nuclear Transcription Rate and Splicing Efficiency

The tricarboxylate carrier (TCC), also known as citrate carrier, is an integral protein of the mitochondrial inner membrane. It is an essential component of the shuttle system by which mitochondrial acetyl-CoA, primer for both fatty acid and cholesterol synthesis, is transported into the cytosol, where lipogenesis occurs. The effect of hypothyroidism on the activity and expression of the hepatic mitochondrial TCC was investigated in this study. TCC activity was significantly decreased in hypothyroid rats as compared with euthyroid animals. This hormone deficiency effect was due to a reduction in the amount of carrier protein, which resulted from a proportionate decrease of the specific mRNA. Hypothyroidism did not influence TCC mRNA stability. On the other hand, nuclear run-on assay revealed that the transcriptional rate of TCC mRNA decreased by approximately 40% in the nuclei from hypothyroid versus euthyroid rats. In addition, the ribonuclease protection assay showed that, in the nuclei of hypothyroid rats, the ratio of mature to precursor RNA decreased, indicating that the splicing of TCC RNA is affected. Furthermore, we found that the ratio of polyadenylated/unpolyadenylated TCC RNA as well as the length of the TCC RNA poly(A) tail were similar in both euthyroid and hypothyroid rats. Thus, the rate of formation of the TCC 3'-end is not altered in hypothyroidism. These results suggest that hypothyroidism affects TCC expression at both the transcriptional and post-transcriptional levels.

Apoptotic and anti-proliferative effects of all- trans retinoic acid. : Adenine nucleotide translocase sensitizes HeLa cells to all- trans retinoic acid

We examined the apoptotic and anti-proliferative effects of all- trans retinoic acid (atRA) in HeLa cells. Our results demonstrated that HeLa cells were more sensitive to the anti-proliferative effects of atRA than to its apoptotic effects. Furthermore, we demonstrated that caspase inhibition attenuates cell death but does not alter the atRA-dependent reduction in cell proliferation, which suggests that atRA-induced apoptosis is independent of the arrest in cell proliferation. To check whether ANT proteins mediated these atRA effects, we transiently transfected cells with expression vectors encoding for individual ANT (adenine nucleotide translocase 1–3). Our results revealed that ANT1 and ANT3 over-expressing HeLa cells increased their atRA sensitivity. Thus, our results not only demonstrate the different functional activities of ANT isoforms, but also contribute to a better understanding of the properties of atRA as an anti-tumoral agent used in cancer therapy.

Ectopic expression of the human adenine nucleotide translocase, isoform 3 (ANT-3). Characterization of ligand binding properties

The adenine nucleotide translocase (ANT) is a key component in maintaining cellular energy homeostasis, and has also been implicated in formation of the mitochondrial permeability transition pore. Human ANT-3 was cloned from a human heart cDNA library and expressed as a histidine-tagged fusion protein in the mitochondria of the Trichoplusia ni. cell line. Over-expression resulted in a concomitant decrease in the endogenous ANT content, allowing for the characterization of binding of known ANT ligands to the human protein. Binding affinities for bongkrekic acid (BKA), ADP, and atractyloside (ATR) were measured in mitochondria from the human ANT-3 expressing cell line, and compared to similar preparations from bovine heart mitochondria by use of a novel radioiodinated derivative of ATR. Binding to ANT-3 by the high affinity inhibitors BKA and ATR, as well as the lower affinity natural ligand ADP, was similar to that measured in bovine heart mitochondria, and to that previously reported for mammalian heart mitochondria. Characterizations such as these of human ANT isoforms may lead to drug development for enhanced mitochondrial function and cellular viability.

Sp1 and chromatin environment are important contributors to the formation of repressive chromatin structures on the transfected human adenine nucleotide translocase-2 promoter

The influence of chromatin on the human adenine nucleotide translocase isoform 2 (ANT2) promoter was investigated in transfected cells treated with the deacetylase inhibitors butyrate and trichostatin A (TSA). Both inhibitors activated the expression of reporter plasmids transfected into HeLa cells, indicating that the promoter was suppressed by hypoacetylated chromatin and activated by hyperacetylation. Inhibitor-dependent activation was traced to the two Sp1-activation elements within the proximal promoter region, indicating that the Sp1 elements are repressed by chromatin structure. Repressive chromatin structures were also formed on the promoter integrated into a stable chromatin environment, as shown by the effects of TSA and butyrate on 14 single-cell-derived NIH3T3 clones bearing the stable integrated ANT2 promoter. Both the basal expression of the luciferase reporter gene and the response to TSA and butyrate varied widely between clones. The range of basal expression (4000-fold) was due partially to variation in the formation of repressive chromatin, since clones with low basal expression were induced by TSA, but those with high basal expression were less effected. These data indicate that chromatin environment surrounding the integrated DNA exerts a strong influence on chromatin-dependent repression of the ANT2 promoter, and that the ability of Sp1 to activate ANT2 expression is compromised in the repressed state.

Transcription of the adenine nucleotide translocase isoforms in various types of tissues in the rat

Two different isoforms of the adenine nucleotide translocase (ANT1 and ANT2) have been identified in the rat. In order to obtain enhanced knowledge of the ANT isoform expression, we analyzed the transcription pattern of both isoforms and their mRNA levels in various tissues of the rat using the PCR technique. A predominant ANT1 mRNA percentage was recorded in the skeletal muscle, heart and brain, ranging from 81 to 58%. In contrast to these tissues, the percentages of ANT2 were dominant with a range from 59 to 75% in the kidney, lung, spleen and liver. The level of total ANT mRNA varied markedly in the various organs. Tissues with a dominant ANT1 percentage simultaneously showed a high level of total ANT transcription (24–41 attomol/ng total RNA). In comparison to the latter, tissues with a prevalent ANT2 transcription were shown to have an even lower ANT transcription level (2–5 attomol/ng total RNA). The predominance of the ANT1 expression appeared to be restricted to tissues with an inability to regenerate by means of mitotic division, whereas a prevalent ANT2 transcription is found in cell types able to proliferate. The level of total ANT transcription but not the individual ANT isoform expression depends to a great extent on the energy requirements of the tissue.

Alteration of the ADP/ATP translocase isoform pattern improves ATP expenditure in developing rat liver mitochondria

The expression of adenine nucleotide translocase isoforms (AAC) during perinatal development of the rat was studied by measuring mRNA transcript levels of AAC1 and AAC2 genes in liver, heart and brain tissue. In contrast to heart and brain, AAC1 mRNA is not present in adult liver tissue, but is transiently expressed around birth. AAC1 expression in liver did not respond to cold stress (examined with adult rats), therefore a possible involvement of AAC1 in the liver thermogenesis of newborns is excluded. Measurement of the [ 3H]ADP uptake by liver mitochondria revealed that the molecular activity of the AAC protein was significantly higher in mitochondria from 4-day-old neonates compared with adults. We suggest that the transient AAC1 gene expression in the perinatal liver helps to accommodate the mitochondrial ATP supply to the increased cytosolic ATP consumption initiated at birth.

Hypothermia preserves function and signaling for mitochondrial biogenesis during subsequent ischemia.

Hypothermia is known to protect myocardium during ischemia, but its role in induction of a protective stress response before ischemia has not been evaluated. As cold incites stress responses in other tissues, including heat shock protein induction and signaling mitochondrial biogenesis, we postulated that hypothermia in perfused hearts would produce similar phenomena while reducing injury during subsequent ischemia. Studies were performed in isolated perfused rabbit hearts (n = 77): a control group (C) and a hypothermic group (H) subjected to decreasing infusate temperature from 37 to 31 degrees C over 20 min. Subsequent ischemia during cardioplegic arrest at 34 degrees C for 120 min was followed by reperfusion. At 15 min of reperfusion, recovery of left ventricular developed pressure (LVDP), maximum first derivative of left ventricular pressure (LV dP/dtmax), LV -dP/dtmax, and the product of heart rate and LVDP was significantly increased in H (P < 0.01) compared with C hearts. Ischemic contracture started later in H (97.5 +/- 3.6 min) than in C (67.3 +/- 3.3 min) hearts. Myocardial ATP preservation and repletion during ischemia and reperfusion were higher in H than in C hearts. mRNA levels of the nuclear-encoded mitochondrial proteins adenine nucleotide translocase isoform 1 (ANT1) and beta-F1-adenosine-triphosphatase (beta-F1-ATPase) normalized to 28S RNA decreased in C hearts but were preserved in H hearts after reperfusion. Inducible heat shock protein (HSP70-1) mRNA was elevated nearly 4-fold after ischemia in C hearts and 12-fold in H hearts. These data indicate that hypothermia preserves myocardial function and ATP stores during subsequent ischemia and reperfusion. Signaling for mitochondrial biogenesis indexed by ANT1 and beta-F1-ATPase mRNA levels is also preserved during a marked increase in HSP70-1 mRNA.