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.

Nonlinear magnetic field gradients can reduce SAR in flow-driven arterial spin labeling measurements.

This work describes how custom-built gradient coils, designed to generate magnetic fields with amplitudes that vary nonlinearly with position, can be used to reduce the potential for unsafe tissue heating during flow-driven arterial spin labeling processes. A model was developed to allow detailed analysis of the adiabatic excitation process used for flow-driven arterial water stimulation with elimination of tissue signal (FAWSETS) an arterial spin labeling method developed specifically for use in skeletal muscle. The model predicted that, by adjusting the amplitude of the gradient field, the specific absorption rate could be reduced by more than a factor of 6 while still achieving effective labeling. Flow phantom measurements and in vivo measurements from exercising rat hind limb confirmed the accuracy of the model's predictions. The modeling tools were also applied to the more widely used continuous arterial spin labeling (CASL) method and predicted that specially shaped gradients could allow similar reductions in SAR.

Gradient-enhanced FAWSETS perfusion measurements.

This work describes the use of custom-built gradients to enhance skeletal muscle perfusion measurements acquired with a previously described arterial spin labeling technique known as FAWSETS (flow-driven arterial water stimulation with elimination of tissue signal). Custom-built gradients provide active control of the static magnetic field gradient on which FAWSETS relies for labeling. This allows selective, 180 degrees modulations of the phase of the perfusion component of the signal. Phase cycling can then be implemented to eliminate all extraneous components leaving a signal that exclusively reflects capillary-level perfusion. Gradient-enhancement substantially reduces acquisition time and eliminates the need to acquire an ischemic signal to quantify perfusion. This removes critical obstacles to application of FAWSETS in organs other than skeletal muscle and makes the measurements more desirable for clinical environments. The basic physical principles of gradient-enhancement are demonstrated in flow phantom experiments and in vivo utility is demonstrated in rat hind limb during stimulated exercise.

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.

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.

Time-courses of perfusion and phosphocreatine in rat leg during low-level exercise and recovery.

PURPOSE: To develop a noninvasive protocol for measuring local perfusion and metabolic demand in muscle tissue with sufficient sensitivity and time resolution to monitor kinetics at the onset of low-level exercise and during recovery. MATERIALS AND METHODS: Capillary-level perfusion, the critical factor that determines oxygen and substrate delivery to active muscle, was measured by an arterial spin labeling (ASL) technique optimized for skeletal muscle. Phosphocreatine (PCr) kinetics, which signal the flux of oxidative phosphorylation, were measured by (31)P MR spectroscopy. Perfusion and PCr measurements were made in parallel studies before, during, and after three different intensities of low-level, stimulated exercise in rat hind limb. RESULTS: The data reveal close coupling between the perfusion response and PCr changes. The onset and recovery time constants for PCr changes were independent of contractile force over the range of forces studied. Perfusion time constants during both onset of exercise and recovery tended to increase with contractile force. CONCLUSION: These results demonstrate that the protocol implemented can be useful for probing the mechanisms that control skeletal muscle blood flow, the physiological limits to muscle performance, and the causes for the attenuated exercise-induced hyperemia observed in disease states.

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.

Molecular mechanisms of cross-talk between thyroid hormone and peroxisome proliferator activated receptors: focus on the heart.

Thyroid hormone receptors (TR) and peroxisome proliferator activated receptors (PPAR) regulate cardiac metabolism. Numerous studies have examined TR and PPAR function since PPAR was first discovered in the early 1990s, however few have evaluated TR and PPAR interactions. Although ligands for these members of the nuclear steroid receptor family are under evaluation for treatment of congestive heart failure and various metabolic diseases, their interactions have not been investigated in detail in heart. These interactions are remarkably complicated. Nevertheless, their identification and elucidation is extremely important for further development of specific drugs. We review here the fundamental ways TRs and PPARs are regulated and how their cross-talk patterns mediate transcription of their target genes.

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.

FAWSETS perfusion measurements in exercising skeletal muscle.

Arterial spin labeling (ASL) techniques are now recognized as valid tools for providing accurate measurements of cerebral and cardiac perfusion. The labeling process used with most ASL techniques creates two problems, magnetization transfer (MT) effects and arterial transit time effects, that require compensation. The compensation process limits time resolution and hinders absolute quantification. MT effects are particularly problematic in skeletal muscle because they are large and change rapidly during exercise. The protocol presented here was developed specifically for quantification of perfusion in exercising skeletal muscle. The ASL technique that was implemented, FAWSETS, eliminates MT effects and arterial transit times. Localized, single-voxel perfusion measurements were acquired from rat hind limbs at rest, during ischemia and during three different levels of stimulated exercise. The results demonstrate sufficient sensitivity to determine the time constants for perfusion changes at onset of, and during recovery from, exercise and to distinguish the differences in the amplitude of the perfusion response to different levels of exercise. Additional measurements were conducted to demonstrate insensitivity to MT effects. The exercise protocol is easily adaptable to phosphorous magnetic resonance measurements, allowing the possibility to acquire local measurements of perfusion and metabolism from the same tissue in future experiments.

Validation and advantages of FAWSETS perfusion measurements in skeletal muscle.

This work discusses the strengths, limitations and validity of a novel arterial spin labeling technique when used specifically to measure perfusion in limb skeletal muscle. The technique, flow-driven arterial water stimulation with elimination of tissue signal (FAWSETS), offers several advantages over existing arterial spin labeling techniques. The primary goal of this study was to determine the perfusion signal response to changes in net hind limb flow that were independently verifiable. The range of perfusate flow was relevant to skeletal muscle during mild to moderate exercise. Localized, single voxel measurements were acquired from a 5 mm-thick slice in the isolated perfused rat hind limb at variable net flow rates. The results show that the perfusion signal is linearly proportional to net hind limb flow with a correlation coefficient of 0.974 (p = 0.0013). FAWSETS is especially well suited for studies of skeletal muscle perfusion, where it eliminates the need to compensate for magnetization transfer and arterial transit time effects. A conceptual discussion of the basic principles underlying these advantages is presented.

Hypothermia preserves myocardial function and mitochondrial protein gene expression during hypoxia.

Hypothermia before and/or during no-flow ischemia promotes cardiac functional recovery and maintains mRNA expression for stress proteins and mitochondrial membrane proteins (MMP) during reperfusion. Adaptation and protection may occur through cold-induced change in anaerobic metabolism. Accordingly, the principal objective of this study was to test the hypothesis that hypothermia preserves myocardial function during hypoxia and reoxygenation. Hypoxic conditions in these experiments were created by reducing O2 concentration in perfusate, thereby maintaining or elevating coronary flow (CF). Isolated Langendorff-perfused rabbit hearts were subjected to perfusate (Po2 = 38 mmHg) with glucose (11.5 mM) and perfusion pressure (90 mmHg). The control (C) group was at 37 degrees C for 30 min before and 45 min during hypoxia, whereas the hypothermia (H) group was at 29.5 degrees C for 30 min before and 45 min during hypoxia. Reoxygenation occurred at 37 degrees C for 45 min for both groups. CF increased during hypoxia. The H group markedly improved functional recovery during reoxygenation, including left ventricular developed pressure (DP), the product of DP and heart rate, dP/dtmax, and O2 consumption (MVo2) (P < 0.05 vs. control). MVo2 decreased during hypothermia. Lactate and CO2 gradients across the coronary bed were the same in C and H groups during hypoxia, implying similar anaerobic metabolic rates. Hypothermia preserved MMP betaF1-ATPase mRNA levels but did not alter adenine nucleotide translocator-1 or heat shock protein-70 mRNA levels. In conclusion, hypothermia preserves cardiac function after hypoxia in the hypoxic high-CF model. Thus hypothermic protection does not occur exclusively through cold-induced alterations in anaerobic metabolism.