Triiodothyronine increases myocardial function and pyruvate entry into the citric acid cycle after reperfusion in a model of infant cardiopulmonary bypass.

Triiodothyronine (T3) supplementation improves clinical outcomes in infants after cardiac surgery using cardiopulmonary bypass by unknown mechanisms. We utilized a translational model of infant cardiopulmonary bypass to test the hypothesis that T3 modulates pyruvate entry into the citric acid cycle (CAC), thereby providing the energy support for improved cardiac function after ischemia-reperfusion (I/R). Neonatal piglets received intracoronary [2-(13)Carbon((13)C)]pyruvate for 40 min (8 mM) during control aerobic conditions (control) or immediately after reperfusion (I/R) from global hypothermic ischemia. A third group (I/R-Tr) received T3 (1.2 µg/kg) during reperfusion. We assessed absolute CAC intermediate levels and flux parameters into the CAC through oxidative pyruvate decarboxylation (PDC) and anaplerotic carboxylation (PC) using [2-(13)C]pyruvate and isotopomer analysis by gas and liquid chromatography-mass spectrometry and (13)C-nuclear magnetic resonance spectroscopy. When compared with I/R, T3 (group I/R-Tr) increased cardiac power and oxygen consumption after I/R while elevating flux of both PDC and PC (∼4-fold). Although neither I/R nor I/R-Tr modified absolute CAC levels, T3 inhibited I/R-induced reductions in their molar percent enrichment. Furthermore, (13)C-labeling of CAC intermediates suggests that T3 may decrease entry of unlabeled carbons at the level of oxaloacetate through anaplerosis or exchange reaction with asparate. T3 markedly enhances PC and PDC fluxes, thereby providing potential substrate for elevated cardiac function after reperfusion. This T3-induced increase in pyruvate fluxes occurs with preservation of the CAC intermediate pool. Our labeling data raise the possibility that T3 reduces reliance on amino acids for anaplerosis after reperfusion.

EPAS1 and EGLN1 associations with high altitude sickness in Han and Tibetan Chinese at the Qinghai-Tibetan Plateau.

High altitude sickness (HAS) occurs among humans visiting or inhabiting high altitude environments. Genetic differences in the EPAS1 and EGLN1 genes have been found between lowland (Han) and highland (Tibetan) Chinese. Three SNPs within EPAS1 and EGLN1 were evaluated in Han and Tibetan patients with acute mountain sickness (AMS) and chronic mountain sickness (CMS). We compared 85 patients with AMS to 79 Han unaffected with mountain sickness (MS) as well as 45 CMS patients to 34 unaffected Tibetan subjects. The three SNPs studied were EPAS1 [ch2: 46441523 (hg18], EGLN1 (rs480902) and (rs516651). Direct sequencing was used to identify individual genotypes for the three SNPs. Age was found to be significantly associated with the EPAS1 SNP in the CMS patients while heart rate (HR) and oxygen saturation level of hemoglobin (SaO(2)) were found to be significantly associated with the EGLN1 (rs480902) SNP in the Han patients with AMS. The individuals with CMS were found to diverge significantly for the EPAS1 SNP compared to their Tibetan control group as measured by genetic distance (0.123) indicating positive selection of the EPAS-G allele with age and illness. The EGLN1 (rs480902) SNP had a significant correlation with hematocrit (HCT), HR and SaO(2) in AMS patients. AMS and CMS were found to be significantly associated with the EPAS1 and EGLN1 SNPs compared to their Han and Tibetan control groups, respectively, indicating these nucleotide alterations have a physiological effect for the development of high altitude sickness.

Aging impairs myocardial fatty acid and ketone oxidation and modifies cardiac functional and metabolic responses to insulin in mice.

Aging presumably initiates shifts in substrate oxidation mediated in part by changes in insulin sensitivity. Similar shifts occur with cardiac hypertrophy and may contribute to contractile dysfunction. We tested the hypothesis that aging modifies substrate utilization and alters insulin sensitivity in mouse heart when provided multiple substrates. In vivo cardiac function was measured with microtipped pressure transducers in the left ventricle from control (4-6 mo) and aged (22-24 mo) mice. Cardiac function was also measured in isolated working hearts along with substrate and anaplerotic fractional contributions to the citric acid cycle (CAC) by using perfusate containing (13)C-labeled free fatty acids (FFA), acetoacetate, lactate, and unlabeled glucose. Stroke volume and cardiac output were diminished in aged mice in vivo, but pressure development was preserved. Systolic and diastolic functions were maintained in aged isolated hearts. Insulin prompted an increase in systolic function in aged hearts, resulting in an increase in cardiac efficiency. FFA and ketone flux were present but were markedly impaired in aged hearts. These changes in myocardial substrate utilization corresponded to alterations in circulating lipids, thyroid hormone, and reductions in protein expression for peroxisome proliferator-activated receptor (PPAR)alpha and pyruvate dehydrogenase kinase (PDK)4. Insulin further suppressed FFA oxidation in the aged. Insulin stimulation of anaplerosis in control hearts was absent in the aged. The aged heart shows metabolic plasticity by accessing multiple substrates to maintain function. However, fatty acid oxidation capacity is limited. Impaired insulin-stimulated anaplerosis may contribute to elevated cardiac efficiency, but may also limit response to acute stress through depletion of CAC intermediates.

Rapid progressive central cooling to 29 degrees C by extracorporeal circuit preserves cardiac function and hemodynamics in immature swine.

The International Liaison Committee on Resuscitation (ILCOR) consensus statement includes recommendations and guidelines for therapeutic hypothermia in infants and children. The information supporting these recommendations is sparse, and reveals a need for target temperature and cooling mode data in age-appropriate animal models. Accordingly, we determined cardiac function and hemodynamic indices in immature piglets (<28 days) undergoing graded and rapid central cooling from 36 to 20 degrees C over 20 min by directing cardiac output through an extracorporeal circuit. Functional parameters were recorded continuously using aortic flow probes and left ventricular (LV) pressure capacitance catheters. Stroke volume and work increased during temperature reduction, peaking at 29 degrees C, while systemic vascular resistance did not change. Although, heart rate decreased steadily, cardiac output, power, and LV dP/dt(max) was maintained until 29 degrees C. All function parameters decreased below 29 degrees C, implying a critical threshold had been exceeded at lower temperatures. These data show that the temperature range (30+/-1) degrees C maintains cardiac function and that this target should be further evaluated as a target for therapeutic hypothermia.

SNPs, linkage disequilibrium, and chronic mountain sickness in Tibetan Chinese.

Chronic mountain sickness (CMS) is estimated at 1.2% in Tibetans living at the Qinghai-Tibetan Plateau. Eighteen single-nucleotide polymorphisms (SNPs) from nine nuclear genes that have an association with CMS in Tibetans have been analyzed by using pairwise linkage disequilibrium (LD). The SNPs included are the angiotensin-converting enzyme (rs4340), the angiotensinogen (rs699), and the angiotensin II type 1 receptor (AGTR1) (rs5186) from the renin-angiotensin system. A low-density lipoprotein apolipoprotein B (rs693) SNP was also included. From the hypoxia-inducible factor oxygen signaling pathway, the endothetal Per-Arnt-Sim domain protein 1 (EPAS1) and the egl nine homolog 1 (ENGL1) (rs480902) SNPs were included in the study. SNPs from the vascular endothelial growth factor (VEGF) signaling pathway included are the v-akt murine thymoma viral oncogene homolog 3 (rs4590656 and rs2291409), the endothelial cell nitric oxide synthase 3 (rs1007311 and rs1799983), and the (VEGFA) (rs699947, rs34357231, rs79469752, rs13207351, rs28357093, rs1570360, rs2010963, and rs3025039). An increase in LD occurred in 40 pairwise comparisons, whereas a decrease in LD was found in 55 pairwise comparisons between the controls and CMS patients. These changes were found to occur within and between signaling pathways, which suggests that there is an interaction between SNP alleles from different areas of the genome that affect CMS.

Thyroid hormone receptor isoforms localize to cardiac mitochondrial matrix with potential for binding to receptor elements on mtDNA.

Thyroid hormone (T(3)) rapidly promotes both nuclear and mitochondrial DNA transcription in cardiomyocytes, suggesting that T3 directly binds and activates mitochondrial genes. We showed for the first time mitochondrial localization for multiple TRalpha isoforms in heart, including truncated versions. Additionally, we demonstrated novel mitochondrial localization for versions of TRalpha(2), the dominant negative isoform lacking a functional ligand-binding domain. We also confirmed by electromobility shift assays, that TRalpha(2) in mitochondrial extracts binds to thyroid receptor response elements present in the 12S rRNA (DRO) and D-loop region (DR2) of mitochondrial DNA. Thus, TRalpha isoforms may directly regulate T(3) responses at mtDNA in the heart.

Mild hypothermic cross adaptation resists hypoxic injury in hearts: a brief review.

Severe cardiac hypoxia is responsible for significant morbidity and mortality in an emergency setting. Most cardiac hypoxia relates to ischemia and surgical events. Although the ischemic mortality rate and the risks of cardiac surgery have significantly decreased in past decades, myocardial protection still plays a major role in survival of hypoxic injury. Cross adaptation as a physiological regulation for homeostasis can resist injury caused by harmful environmental effects and diseases, including hypothermic adaptation. Treatment with hypothermia has been used for fifty years as a protective mechanism to avoid hypoxic injury. Since cold temperatures can cause damage, it is important to gather physiological data to distinguish protective from injurious temperatures. Although results of temperature trials in clinical practice vary, a critical temperature to resist hypoxic/ischemic injury in heart was found to be around 30 degrees C, suggesting a hypothermia protective threshold. Pretreatment with mild hypothermia can resist subsequent hypoxia/ischemia, implying involvement of cross adaptation in protection. Safeguard hypothermia can directly reduce the build up of harmful metabolites and energy demand in hypoxic tissues, as well as preserve mitochondrial membrane specific proteins beta subunit of F1-ATPase and adenine nucleotide translocase isoform 1. Mechanisms of preservation include inactivation of the p53 related pathways, representing anti-apoptosis, and modification of the mRNA level of succinodehydrogenease, indicating a beneficial effect on the aerobic pathway. Stress proteins are also induced. Resultant cellular adaptations serve to maintain myocardial integrity and improve functional recovery during reoxygenation or reperfusion.

Optimal protective hypothermia in arrested mammalian hearts.

Many therapeutic hypothermia recommendations have been reported, but the information supporting them is sparse, and reveals a need for the data of target therapeutic hypothermia (TTH) from well-controlled experiments. The core temperature ≤35°C is considered as hypothermia, and 29°C is a cooling injury threshold in pig heart in vivo. Thus, an optimal protective hypothermia (OPH) should be in the range 29-35°C. This study was conducted with a pig cardiopulmonary bypass preparation to decrease the core temperature to 29-35°C range at 20 minutes before and 60 minutes during heart arrest. The left ventricular (LV) developed pressure, maximum of the first derivative of LV (dP/dtmax), cardiac power, heart rate, cardiac output, and myocardial velocity (Vmax) were recorded continuously via an LV pressure catheter and an aortic flow probe. At 20 minutes of off-pump during reperfusion after 60 minutes arrest, 17 hypothermic hearts showed that the recovery of Vmax and dP/dtmax established sigmoid curves that consisted of two plateaus: a good recovery plateau at 29-30.5°C, the function recovered to baseline level (BL) (Vmax=118.4%±3.9% of BL, LV dP/dtmax=120.7%±3.1% of BL, n=6); another poor recovery plateau at 34-35°C (Vmax=60.2%±2.8% of BL, LV dP/dtmax=28.0%±5.9% of BL, p<0.05, n=6; ), which are similar to the four normothermia arrest (37°C) hearts (Vmax=55.9%±4.8% of BL, LV dP/dtmax=24.5%±2.1% of BL, n=4). The 32-32.5°C arrest hearts showed moderate recovery (n=5). A point of inflection (around 30.5-31°C) existed at the edge of a good recovery plateau followed by a steep slope. The point presented an OPH that should be the TTH. The results are concordant with data in the mammalian hearts, suggesting that the TTH should be initiated to cool core temperature at 31°C.

Hypothermic protection of the ischemic heart via alterations in apoptotic pathways as assessed by gene array analysis.

Hypothermia improves resistance to ischemia in the cardioplegia-arrested heart. This adaptive process produces changes in specific signaling pathways for mitochondrial proteins and heat-shock response. To further test for hypothermic modulation of other signaling pathways such as apoptosis, we used various molecular techniques, including cDNA arrays. Isolated rabbit hearts were perfused and exposed to ischemic cardioplegic arrest for 2 h at 34 degrees C [ischemic group (I); n = 13] or at 30 degrees C before and during ischemia [hypothermic group (H); n = 12]. Developed pressure, the maximum first derivative of left ventricular pressure, oxygen consumption, and pressure-rate product (P < 0.05) recovery were superior in H compared with in I during reperfusion. mRNA expression for the mitochondrial proteins, adenine translocase and the beta-subunit of F1-ATPase, was preserved by hypothermia. cDNA arrays revealed that ischemia altered expression of 13 genes. Hypothermia modified this response to ischemia for eight genes, six related to apoptosis. A marked, near fivefold increase in transformation-related protein 53 in I was virtually abrogated in H. Hypothermia also increased expression for the anti-apoptotic Bcl-2 homologue Bcl-x relative to I but decreased expression for the proapoptotic Bcl-2 homologue bak. These data imply that hypothermia modifies signaling pathways for apoptosis and suggest possible mechanisms for hypothermia-induced myocardial protection.

VEGFA SNPs and transcriptional factor binding sites associated with high altitude sickness in Han and Tibetan Chinese at the Qinghai-Tibetan Plateau.

Mountain sickness (MS) occurs among humans visiting or inhabiting high altitude environments. We conducted genetic analyses of seven single nucleotide polymorphisms (SNPs) in the promoter region of VEGFA gene for lowland (Han) and highland (Tibetan) Chinese. The seven SNPs were evaluated in Han and Tibetan patients with acute (A) and chronic (C) MS. We compared 64 patients with AMS with 64 Han unaffected with MS, as well as 48 CMS patients with 32 unaffected Tibetans. The SNPs studied are rs699947, rs34357231, rs79469752, rs13207351, rs28357093, rs1570360, and rs2010963 which are found in the promoter ranging from -2,578 to -634 bp from the transcriptional start site (TSS), respectively. Direct sequencing was used to identify individual genotypes for these SNPs. Arterial oxygen saturation of hemoglobin (SaO2) was found to be significantly associated with the rs699947, rs34357231, rs13207351, and rs1570360 SNPs in Han patients with AMS, while the rs2010963 SNP was found to approach significance in the AMS study group, but found to be significantly associated in the normal Tibetan study group. The Han and Tibetan control groups were found to diverge significantly for the rs28357093 and rs2010963 SNPs, as measured by genetic distances of 0.073 and 0.054, respectively. All the SNPs are found in transcriptional factor binding sites (TFBS), and their possible role in gene regulation was evaluated with regard to MS. MS was found to be significantly associated with these SNPs compared with their Han and Tibetan control groups, indicating that these nucleotide substitutions result in TFBS changes which apparently have a physiological effect on the development of high altitude sickness.

The adaptor-related protein complex 2, alpha 2 subunit (AP2α2) gene is a peroxisome proliferator-activated receptor cardiac target gene.

A peroxisome proliferator-actived receptor (PPAR) response element (RE) in the promoter region of the adaptor-related protein complex 2, alpha 2 subunit (AP2α2) of mouse heart has been identified. The steroid hormone nuclear PPARs and the retinoid X receptors (RXRs) are important transcriptional factors that regulate gene expression, cell differentiation and lipid metabolism. They form homo- (RXR) and hetero- (PPAR-RXR) dimers that bind DNA at various REs. The AP2α2 gene is part of complex and process that transports lipids and proteins from the plasma membrane to the endosomal system. A PPAR activator (Wy14643) and DMSO (vehicle) was introduced into control and δ337T thyroid hormone receptor (TRß1) transgenic mice. Heart tissue was extracted and AP2α2 gene expression was compared using Affymetrix expression arrays and qRT PCR among four groups [control, control with Wy14643, δ337T TRß1 and δ337T TRß1 with Wy14643]. The gene expression of AP2α2 in the Wy14643 control and transgenic mouse groups was significantly up regulated over the vehicle mouse groups in both the array (p < 0.01) and qRT PCR (p < 0.01) studies. Duplex oligo DNAs containing the PPAR/RXR motif (AGGTCA/TCCAGT) from the AP2α2 promoter were used in EMSA to verify binding of the PPAR and RXR receptors to their REs. pGL4.0 [Luc] constructs of the AP2α2 promoter with and without the PPAR/RXR motifs were co-transfected with mouse PPARα, ß or γ1 into HepG2 cells and used in lucerifase assays to verify gene activation. In conclusion our study revealed that PPARα regulates the mouse cardiac AP2α2 gene in both the control and transgenic mouse.

AKT3, ANGPTL4, eNOS3, and VEGFA associations with high altitude sickness in Han and Tibetan Chinese at the Qinghai-Tibetan Plateau.

Mountain sickness (MS) occurs among humans visiting or inhabiting high altitude environments. We conducted genetic analyses of the AKT3, ANGPTL4, eNOS3 and VEGFA genes in lowland (Han) and highland (Tibetan) Chinese. Ten single nucleotide polymorphisms (SNPs) were evaluated in Han and Tibetan patients with acute (A) and chronic (C) MS. We compared 74 patients with AMS to 79 Han unaffected with MS, as well as 48 CMS patients to 31 unaffected Tibetans. The ten SNPs studied are AKT3 (rs4590656, rs2291409), ANGPTL4 (rs1044250), eNOS3 (rs1007311, rs1799983) and VEGFA (rs79469752, rs13207351, rs28357093, rs1570360, rs3025039). Direct sequencing was used to identify individual genotypes for these SNPs. Hemoglobin (Hb), hematocrit (Hct), and red blood cell count (RBC) were found to be significantly associated with the AKT3 SNP (rs4590656), Hb was found to be associated with the eNOS3 SNP (rs1007311), and RBC was found to be significantly associated with the VEGFA SNP (rs1570360) in Tibetan patients with CMS. CMS patients were found to diverge significantly for both eNOS3 SNPs as measured by genetic distance (0.042, 0.047) and for the VEGFA SNP (rs28357093) with a genetic distance of 0.078 compared to their Tibetan control group. Heart rate (HR) was found to be significantly associated with the eNOS3 SNP (rs1799983) and arterial oxygen saturation of hemoglobin (SaO2) was found to be significantly associated with the VEGFA SNPs (rs13207351, rs1570360) in Han patients with AMS. The Han and Tibetan control groups were found to diverge significantly for the ANGPTL4 SNP and VEGFA SNP (rs28357093), as measured by genetic distances of 0.049 and 0.073, respectively. Seven of the SNPs from non-coding regions are found in the transcriptional factor response elements and their possible role in gene regulation was evaluated with regard to MS. AMS and CMS were found to be significantly associated with the four genes compared to their Han and Tibetan control groups, respectively, indicating that these nucleotide alterations have a physiological effect for the development of high altitude sickness.

Genetic associations with mountain sickness in Han and Tibetan residents at the Qinghai-Tibetan Plateau.

BACKGROUND: Acute (AMS) and chronic (CMS) mountain sicknesses are illnesses that occur among humans visiting or inhabiting high-altitude environments, respectively. Some individuals are genetically less fit than others when stressed by an extreme high-altitude environment. Seven blood physiological parameters and five genetic polymorphisms were studied in Han patients with AMS and Tibetan patients with CMS. METHODS: We compared 98 AMS patients with 60 Han controls as well as 50 CMS patients with 36 Tibetan controls. The genetic loci studied are ACE I/D (rs4340), AGT M235T (rs699), AGTR1 A1166C (rs5186), GNB3 A(-350)G (rs2071057) and APOB A/G (rs693). RESULTS: All physiological parameters (RBC, HCT, Hb, SaO(2), HR, and BPs/d) studied significantly changed in the CMS patients while SaO(2) and HR changed in the AMS Han patients compared to their controls. The ACE D and AGT 235M alleles were found to be significantly associated with AMS and CMS, respectively, while a significantly high incidence of the G-protein (GNB3) (-350)A allele was found in the AMS patients. ACE (I/D) was significantly associated with HR in CMS patients while the AGT M235T was significantly associated with SaO(2) and BPs/d in AMS patients. APOB A/G was significantly associated with BPs/d in AMS and HR in CMS patients. CONCLUSION: AMS and CMS share very similar genetic results for the ACE I/D and AGT M235T polymorphisms indicating that these mutations have an effect on both illnesses.

Cardiac PPARalpha Protein Expression is Constant as Alternate Nuclear Receptors and PGC-1 Coordinately Increase During the Postnatal Metabolic Transition.

Gene expression data obtained in mouse heart indicate that increased expression for the nuclear receptor, peroxisomal proliferator activated receptor alpha (PPARalpha), prompts the postnatal transition from predominantly carbohydrate to fatty acid oxidation preference. However, no phenotypic or proteomic data are available to confirm downstream signaling and metabolic transition in mice. We studied the hypothesis that shifts in nuclear receptor expression trigger the newborn metabolic switch in a newborn sheep. This species is well characterized with regards to developmental changes in substrate oxidative metabolism. Heart tissues from fetal (130 days gestation), newborn

Superior cardiac function via anaplerotic pyruvate in the immature swine heart after cardiopulmonary bypass and reperfusion.

Pyruvate produces inotropic responses in the adult reperfused heart. Pyruvate oxidation and anaplerotic entry into the tricarboxylic acid (TCA) cycle via carboxylation are linked to the stimulation of contractile function. The goals of this study were to determine if these metabolic pathways operate and are maintained in the developing myocardium after reperfusion. Immature male swine (age: 10-18 days) were subjected to cardiopulmonary bypass (CPB). Intracoronary infusion of [2-(13)C]pyruvate (to achieve an estimated final concentration of 8 mM) was given for 35 min, starting either during weaning (group I) and after its discontinuation (group II) or without (control) CPB. Hemodynamic data were collected. 13C NMR spectroscopy was used to determine the fraction of pyruvate entering the TCA cycle via pyruvate carboxylation (PC) to total TCA cycle entry (PC plus decarboxlyation via pyruvate dehydrogenase). Liquid chromatography-mass spectrometry was used to determine total glutamate enrichment. Pyruvate infusion starting during the weaning of mechanical circulatory support improved maximum dP/dt (P<0.05) but waiting to start the infusion until after the discontinuation of CPB did not. Glutamate fractional enrichment was confirmed by liquid chromatography-mass spectroscopy as adequate (>5%) to provide signal to noise in the NMR experiment in all groups. The ratio of pyruvate carboxylase to total pyruvate entry into the TCA cycle did not differ between groups (group I: 20+/-4%, group II: 23+/-7%, and control: 27+/-7%). These data show that robust PC operates in the neonatal pig heart and is maintained during reperfusion under conditions that emulate CPB and reperfusion in human infants.

Cardioselective dominant-negative thyroid hormone receptor (Delta337T) modulates myocardial metabolism and contractile efficiency.

Dominant-negative thyroid hormone receptors (TRs) show elevated expression relative to ligand-binding TRs during cardiac hypertrophy. We tested the hypothesis that overexpression of a dominant-negative TR alters cardiac metabolism and contractile efficiency (CE). We used mice expressing the cardioselective dominant-negative TRbeta(1) mutation Delta337T. Isolated working Delta337T hearts and nontransgenic control (Con) hearts were perfused with (13)C-labeled free fatty acids (FFA), acetoacetate (ACAC), lactate, and glucose at physiological concentrations for 30 min. (13)C NMR spectroscopy and isotopomer analyses were used to determine substrate flux and fractional contributions (Fc) of acetyl-CoA to the citric acid cycle (CAC). Delta337T hearts exhibited rate depression but higher developed pressure and CE, defined as work per oxygen consumption (MVo(2)). Unlabeled substrate Fc from endogenous sources was higher in Delta337T, but ACAC Fc was lower. Fluxes through CAC, lactate, ACAC, and FFA were reduced in Delta337T. CE and Fc differences were reversed by pacing Delta337T to Con rates, accompanied by an increase in FFA Fc. Delta337T hearts lacked the ability to increase MVo(2). Decreases in protein expression for glucose transporter-4 and hexokinase-2 and increases in pyruvate dehydrogenase kinase-2 and -4 suggest that these hearts are unable to increase carbohydrate oxidation in response to stress. These data show that Delta337T alters the metabolic phenotype in murine heart by reducing substrate flux for multiple pathways. Some of these changes are heart rate dependent, indicating that the substrate shift may represent an accommodation to altered contractile protein kinetics, which can be disrupted by pacing stress.

The dominant negative thyroid hormone receptor beta-mutant {Delta}337T alters PPAR{alpha} signaling in heart.

PPARalpha and TR independently regulate cardiac metabolism. Although ligands for both these receptors are currently under evaluation for treatment of congestive heart failure, their interactions or signaling cooperation have not been investigated in heart. We tested the hypothesis that cardiac TRs interact with PPARalpha regulation of target genes and used mice exhibiting a cardioselective Delta337T TRbeta1 mutation (MUT) to reveal cross-talk between these nuclear receptors. This dominant negative transgene potently inhibits DNA binding for both wild-type (WT) TRalpha and TRbeta. We used UCP3 and MTE-1 as principal reporters and analyzed gene expression from hearts of transgenic (MUT) and nontransgenic (WT) littermates 6 h after receiving either specific PPARalpha ligand (WY-14643) or vehicle. Interactions were determined through qRT-PCR analyses, and the extent of these interactions across multiple genes was determined using expression arrays. In the basal state, we detected no differences between groups for protein content for UCP3, PPARalpha, TRalpha2, RXRbeta, or PGC-1alpha. However, protein content for TRalpha1 and the PPARalpha heterodimeric partner RXRalpha was diminished in MUT, whereas PPARbeta increased. We demonstrated cross-talk between PPAR and TR for multiple genes, including the reporters UCP3 and MTE1. WY-14643 induced a twofold increase in UCP3 gene expression that was totally abrogated in MUT. We demonstrated variable cross-talk patterns, indicating that multiple mechanisms operate according to individual target genes. The non-ligand-binding TRbeta1 mutation alters expression for multiple nuclear receptors, providing a novel mechanism for interaction that has not been previously demonstrated. These results indicate that therapeutic response to PPARalpha ligands may be determined by thyroid hormone state and TR function.

Moderate hypothermia (30 degrees C) maintains myocardial integrity and modifies response of cell survival proteins after reperfusion.

Hypothermia preserves myocardial function, promotes signaling for cell survival, and inhibits apoptotic pathways during 45-min reperfusion. We tested the hypothesis that signaling at the transcriptional level is followed by corresponding proteomic response and maintenance of structural integrity after 3-h reperfusion. Isolated hearts were Langendorff perfused and exposed to mild (I group; n = 6, 34 degrees C) or moderate (H group; n = 6, 30 degrees C) hypothermia during 120-min total ischemia with cardioplegic arrest and 180-min 37 degrees C reperfusion. Moderate hypothermia suppressed anaerobic metabolism during ischemia and significantly diminished left ventricular end-diastolic pressure at the end of ischemia from 52.7 +/- 3.3 (I group) to 1.8 +/- 0.9 (H group) mmHg. Unlike the I group, which showed poor cardiac function and high left ventricular pressure, the H group showed preservation of myocardial function, coronary flow, and oxygen consumption. Compared with normal control hearts without ischemia (n = 5), histological staining in the I group showed marked disarray and fragmentation of collagen network (score 4-5), while the H group showed preserved collagen integrity (score 0-1). The apoptosis-linked tumor suppressor protein p53 was expressed throughout the I group only (score 4-5). The H group produced elevated expression for hypoxia-inducible factor 1alpha and heme oxygenase 1, but minimally affected vascular endothelial growth factor expression. The H group also elevated expression for survival proteins peroxisomal proliferator-activated receptor-beta and Akt-1. These results show in a constant left ventricular volume model that moderate hypothermia (30 degrees C) decreases myocardial energy utilization during ischemia and subsequently promotes expression of proteins involved in cell survival, while inhibiting induction of p53 protein. These data also show that 34 degrees C proffers less protection and loss of myocardial integrity.

Short-cycle hypoxia in the intact heart: hypoxia-inducible factor 1alpha signaling and the relationship to injury threshold.

Hypoxia-inducible factor 1alpha (HIF-1alpha) transcriptionally activates multiple genes, which regulate metabolic cardioprotective and cross-adaptive mechanisms. Hypoxia and several other stimuli induce the HIF-1alpha signaling cascade, although little data exist regarding the stress threshold for activation in heart. We tested the hypothesis that relatively mild short-cycle hypoxia, which produces minimal cardiac dysfunction and no sustained or major disruption in energy state, can induce HIF-1alpha activation. We developed a short-cycle hypoxia protocol in isolated perfused rabbit heart to test this hypothesis. By altering cycling conditions, we identified a specific cycle with O(2) content and duration that operated near a threshold for causing functional injury in these rabbit hearts. Mild short-cycle hypoxia for 46 min elevated HIF-1alpha mRNA and protein within 45 min after reoxygenation. Expression also increased for multiple HIF-1alpha target genes, such as VEGF and heme oxygenase 1. After mild hypoxia, VEGF protein accumulation occurred, although HIF-1alpha and VEGF protein accumulation were suppressed after more severe hypoxia, which also caused depletion of ATP and nondiffusible nucleotides. In summary, these results indicate that mild near-threshold hypoxia induces HIF-1alpha cascade, but more severe hypoxia suppresses protein accumulation for this transcription factor and the target genes. Posttranscriptional suppression of these proteins occurs under conditions of altered energy state, exemplified by ATP depletion.

Thyroid hormone controls myocardial substrate metabolism through nuclear receptor-mediated and rapid posttranscriptional mechanisms.

Thyroid hormone regulates metabolism through transcriptional and posttranscriptional mechanisms. The integration of these mechanisms in heart is poorly understood. Therefore, we investigated control of substrate flux into the citric acid cycle (CAC) by thyroid hormone using retrogradely perfused isolated hearts (n = 20) from control (C) and age-matched thyroidectomized rats (T). We determined substrate flux and fractional contributions (Fc) to the CAC by 13C-NMR spectroscopy and isotopomer analyses in hearts perfused with [1,3-(13)C]acetoacetic acid (0.17 mM), L-[3-(13)C]lactic acid (LAC, 1.2 mM), [U-13C]long-chain mixed free fatty acids (FFA, 0.35 mM), and unlabeled glucose. Some T hearts were supplied triiodothyronine (T3, 10 nM; TT) for 60 min. Prolonged hypothyroid state reduced myocardial oxygen consumption, although T3 produced no significant change. Hypothyroidism reduced overall CAC(flux) but selectively altered only FFA(flux) among the individual substrates, though LAC(flux) trended upward. T3 rapidly decreased lactate Fc and flux. 13C labeling of glutamine through glutamate was increased in T with further enhancement in TT. The glutamate-to-glutamine ratio was significantly lower in T and TT. Immunoblots detected a decrease in hypothyroid hearts for muscle carnitine palmitoyltransferase I (CPT I) and a marked increase in pyruvate dehydrogenase kinase (PDK)-2 with no changes in liver CPT I, PDK-4, or hexokinase 2. TT, but not T, displayed elevated glutamine synthetase (GS) expression. These studies showed that T3 regulates cardiac metabolism through integration of several mechanisms, including changes in oxidative enzyme content and rapid modulation of individual substrates fluxes. T3 also moderates forward glutamine flux, possibly by increasing the overall activity of GS.