Clinical outcomes associated with proton pump inhibitor use among clopidogrel-treated patients within CYP2C19 genotype groups following acute myocardial infarction.

We examined clinical outcomes with proton pump inhibitors (PPI) use within CYP2C19 genotype groups during clopidogrel treatment following acute myocardial infarction (AMI). 2062 patients were genotyped for CYP2C19*2 and *17 variants in TRIUMPH. 12 month clinical outcomes were analyzed among patients discharged on clopidogrel within CYP2C19*2 carrier, CYP2C19*17 carrier, and CYP2C19*1 homozygote genotype groups. PPI use was not associated with a difference in mortality. Among clopidogrel-treated Caucasians following AMI, PPI use was associated with a significantly higher rate of cardiac rehospitalization (HR 1.62, 95% CI 1.19-2.19; P=0.002) compared with no PPI use. PPI users who were carriers of the CYP2C19*17 variant experienced significantly higher rates of cardiac rehospitalization (HR 2.05, 95% CI 1.26-3.33; P=0.003), carriers of the CYP2C19*2 variant had a trend toward increased 1-year cardiac rehospitalization (HR 1.69, 95% CI 0.95-2.99; P=0.07), while no significant differences were observed among CYP2C19*1 homozygotes. These results indicate that the risks associated with PPI use among clopidogrel-treated Caucasian post-MI patients are impacted by CYP2C19 genotype, and suggest knowledge of genotype may be useful for personalizing PPI use among patients following AMI to reduce rehospitalization.

Evidence for a novel cardiac-enriched retinoid X receptor partner.

Recent studies indicate that retinoid-mediated pathways play a pivotal role in cardiac morphogenesis and function. To identify proteins that serve as interacting partners of the retinoid X receptor alpha (RXRalpha) in heart, DNA-protein binding studies were performed with an RXR-responsive element (NRRE-1) derived from the medium chain acyl-CoA dehydrogenase gene promoter and nuclear protein extracts prepared from adult rat heart. NRRE-1 is a pleiotropic RXR-responsive element comprised of three potential recognition sites for class II members of the nuclear receptor superfamily. Gel mobility shift assays performed with an NRRE-1 probe in the absence or presence of bacterially overproduced RXRalpha and nuclear protein extracts prepared from adult rat heart, liver, or brain identified a cardiac-specific, RXR-dependent DNA-protein interaction. The NRRE-1-RXR.cardiac-enriched RXR-interacting protein (CERIP) complex exhibited a distinct mobility compared with NRRE-1-RXR.peroxisome proliferator-activated receptor, NRRE-1-RXR.retinoic acid receptor, or NRRE-1-RXR.thyroid receptor complexes. Mutational analysis demonstrated that two of the three potential binding half-sites of NRRE-1 (an everted repeat separated by an 8-base pair spacer) are required for the NRRE-1-RXR. CERIP interaction. Gel mobility shift assays demonstrated that CERIP interacted with RXRalpha and RXRgamma but not with RXRbeta, indicating a receptor subtypespecific binding preference and suggesting an RXR AB region-dependent interaction. The RXR.CERIP complex did not form on NRRE-1 when a mutant GST-RXRalpha fusion protein lacking the NH(2)-terminal AB region (but containing the receptor dimerization domain) of RXRalpha was added in place of the full-length RXRalpha, confirming a role for the AB region in the RXR. CERIP interaction. DNA-protein cross-linking studies demonstrated that CERIP is a DNA-binding protein of approximately 110 kDa. These results provide evidence for the existence of a cardiac-enriched DNA-binding protein that interacts with RXRalpha via the AB region and suggest a mechanism whereby cardiac retinoid signaling is controlled in an RXR subtype-specific manner.

Transcriptional control of a nuclear gene encoding a mitochondrial fatty acid oxidation enzyme in transgenic mice: role for nuclear receptors in cardiac and brown adipose expression.

Expression of the gene encoding medium-chain acyl coenzyme A dehydrogenase (MCAD), a nuclearly encoded mitochondrial fatty acid beta-oxidation enzyme, is regulated in parallel with fatty acid oxidation rates among tissues and during development. We have shown previously that the human MCAD gene promoter contains a pleiotropic element (nuclear receptor response element [NRRE-1]) that confers transcriptional activation or repression by members of the nuclear receptor superfamily. Mice transgenic for human MCAD gene promoter fragments fused to a chloramphenicol acetyltransferase gene reporter were produced and characterized to evaluate the role of NRRE-1 and other promoter elements in the transcriptional control of the MCAD gene in vivo. Expression of the full-length MCAD promoter-chloramphenicol acetyltransferase transgene (MCADCAT.371) paralleled the known tissue-specific differences in mitochondrial beta-oxidation rates and MCAD expression. MCADCAT.371 transcripts were abundant in heart tissue and brown adipose tissue, tissues with high-level MCAD expression. During perinatal cardiac developmental stages, expression of the MCADCAT.371 transgene paralleled mouse MCAD mRNA levels. In contrast, expression of a mutant MCADCAT transgene, which lacked NRRE-1 (MCADCATdeltaNRRE-1), was not enriched in heart or brown adipose tissue and did not exhibit appropriate postnatal induction in the developing heart. Transient-transfection studies with MCAD promoter-luciferase constructs containing normal or mutant NRRE-1 sequences demonstrated that the nuclear receptor binding sequences within NRRE-1 are necessary for high-level transcriptional activity in primary rat cardiocytes. Electrophoretic mobility shift assays demonstrated that NRRE-1 was bound by several cardiac and brown adipose nuclear proteins and that these interactions required the NRRE-1 receptor binding hexamer sequences. Antibody supershift studies identified the orphan nuclear receptor COUP-TF as one of the endogenous cardiac proteins which bound NRRE-1. These results dictate an important role for nuclear receptors in the transcriptional control of a nuclear gene encoding a mitochondrial fatty acid oxidation enzyme and identify a gene regulatory pathway involved in cardiac energy metabolism.

Activation of a novel metabolic gene regulatory pathway by chronic stimulation of skeletal muscle.

To determine whether expression of a nuclear gene encoding a mitochondrial fatty acid oxidation enzyme is regulated in parallel with skeletal muscle fibre-type-specific energy substrate preference, expression of the gene encoding medium-chain acyl-CoA dehydrogenase (MCAD) was delineated in canine latissimus dorsi muscle subjected to chronic motor nerve stimulation. In predominantly fast-twitch canine latissimus dorsi muscle, MCAD mRNA levels were regulated by chronic stimulation in a biphasic pattern. During the 1st wk of stimulation, steady-state MCAD mRNA levels decreased to 50% of unstimulated levels. MCAD mRNA levels began to increase during the 3rd wk of stimulation to reach a level 3.0-fold higher than levels in unstimulated contralateral control muscle by day 70. Immunodetectable MCAD mRNA levels throughout the stimulation period. The temporal pattern and magnitude of MCAD mRNA accumulation in response to muscle stimulation was distinct from that of mRNAs encoding other enzymes known to be regulated by this stimulus, including glyceraldehyde phosphate dehydrogenase, citrate synthase, and sarcoplasmic reticulum Ca-ATPase, but paralleled the protein levels of the peroxisome proliferator-activated receptor (PPAR), an orphan member of the nuclear hormone receptor superfamily known to regulate genes encoding fatty acid oxidation enzymes in liver. The skeletal muscle expression pattern of PPAR was also similar to that of MCAD in unstimulated rat skeletal muscles with distinct fiber-type compositions. These results demonstrate that a nuclear gene encoding a mitochondrial beta-oxidation enzyme is dynamically regulated in a pattern that parallels skeletal muscle fiber-type-specific energy substrate utilization and implicate an orphan nuclear receptor transcription factor as a candidate transducer of this response.

Structure and chromosomal location of the mouse medium-chain acyl-CoA dehydrogenase-encoding gene and its promoter.

Medium-chain acyl-coenzyme A dehydrogenase (MCAD; mouse gene Acadm; human gene ACADM) catalyzes the initial step of fatty acid beta-oxidation in mitochondria. Inherited MCAD deficiency is an autosomal recessive disorder that occurs at high frequency in humans and is associated with considerable morbidity and mortality. We have cloned and characterized mouse Acadm which spans approximately 25 kb and contains 12 exons. The promoter region does not contain TATA or CAAT boxes and is G + C-rich (60%) within 200 bp of the cap site. A CpG island extends from 5' of the transcription start point into intron 1. The 5' regulatory region and a portion of intron 1 contain several Sp1 consensus sites and three regions containing hexamer DNA sequences that match the binding consensus for steroid/thyroid nuclear receptors. These putative nuclear receptor response elements (NRRE) share DNA sequence homology and electrophoretic mobility shift characteristics with known NRRE in the human ACADM promoter [Carter et al., J. Biol. Chem. 268 (1993) 13805-13810]. We have mapped mouse Acadm to the distal end of chromosome 3. Sequences previously localized to chromosome 8 are shown to be a pseudogene, and an additional pseudogene was identified on chromosome 11.

The human medium chain Acyl-CoA dehydrogenase gene promoter consists of a complex arrangement of nuclear receptor response elements and Sp1 binding sites.

Expression of the gene encoding the mitochondrial fatty acid. beta-oxidation enzyme, medium-chain acyl-CoA dehydrogenase (MCAD), is regulated among tissues during development and in response to alterations in substrate availability. To identify and characterize cis-acting MCAD gene promoter regulatory elements and corresponding transcription factors, DNA-protein binding studies and mammalian cell transfection analyses were performed with hjman MCAD gene promoter fragments. DNA:protein binding studies with nuclear protein extracts prepared from hepatoma G2 cells, 3T3 fibroblasts, or Y-1 adrenal tumor cells identified three sequences (nuclear receptor response element 1 or NRRE-1, NRRE-2, and NRRE-3) that bind orphan members of the steroid/thyroid nuclear receptor superfamily including chicken ovalbumin upstream promoter transcription factor and steroidogenic factor 1. Sp1 binding sites (A-C) were identified in close proximity to each of the NRREs. NRRE-3 conferred cell line-specific transcriptional repression by interacting with chicken ovalbumin upstream promoter transcription factor or activation via steroidogenic factor 1. In contrast, the Sp1 binding site A behaved as a transcriptional activator in all cell lines examined. We propose that multiple nuclear receptor transcription factors interact with MCAD gene promoter elements to differentially regulate transcription among a variety of cell types.

Impaired left atrial function after heart transplantation: disparate contribution of donor and recipient atrial components studied on-line with quantitative echocardiography.

BACKGROUND: Because of a lack of noninvasive techniques, left atrial function after orthotopic heart transplantation has not been well characterized. METHODS: Global left atrial performance and the relative contributions of donor and recipient atrial components were assessed with transthoracic echocardiography with on-line automated border detection in 20 patients with normal left ventricular systolic function 1 to 6 years (mean 3.5 +/- 0.3 years [standard error]) after heart transplantation. RESULTS: The mean left atrial area at ventricular end-systole was 22.9 +/- 1.5 cm2, the mean left atrial emptying fraction ([left atrial area at ventricular end-systole--left atrial area at ventricular end-diastole]/left atrial area at ventricular end-systole) was 29.7% +/- 2.6%, and the fractional area change caused by active contraction was 27.8% +/- 3.1%. Compared with controls, patients had larger atria, depressed emptying, and reduced fractional active contraction. Although the recipient to donor area ratio was 3:2, the proportion of atrial emptying (change in area from mid-to-late ventricular diastole divided by the total left atrial change during ventricular diastole) contributed by the recipient component was greatly diminished when compared with that of the donor component (1.4% +/- 3.5% versus 31% +/- 2.7%) (p = 0.0001). CONCLUSIONS: Despite being anatomically smaller, the functional contractile contribution of the donor component dominated atrial emptying. Thus, after heart transplantation, global left atrial function is depressed, predominantly because of dysfunction of the recipient atrial component.

The peroxisome proliferator-activated receptor regulates mitochondrial fatty acid oxidative enzyme gene expression.

Medium-chain acyl-CoA dehydrogenase (MCAD) catalyzes a pivotal reaction in mitochondrial fatty acid (FA) beta-oxidation. To examine the potential role of FAs and their metabolites in the regulation of MCAD gene expression, we measured MCAD mRNA levels in animals fed inhibitors of mitochondrial long-chain FA import. Administration of carnitine palmitoyltransferase I inhibitors to mice or rats resulted in tissue-limited increases in steady-state MCAD mRNA levels. HepG2 cell cotransfection experiments with MCAD promoter reporter plasmids demonstrated that this was a transcriptional effect mediated by the peroxisome proliferator-activated receptor (PPAR). The activity mapped to a nuclear receptor response element that functioned in a heterologous promoter context and specifically bound immunoreactive PPAR in rat hepatic nuclear extracts, confirming an in vivo interaction. PPAR-mediated transactions of this promoter and element were also induced by exogenously added FA and fibric acid derivatives. Induction of PPAR transactivation by perturbation of this discrete metabolic step is unusual and indicates that intracellular FA metabolites that accumulate during such inhibition can regulate MCAD expression and are likely candidates for PPAR ligand(s). These results dictate an expanded role for the PPAR in the regulation of FA metabolism.