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Am J Physiol Heart Circ Physiol 288: H2484-H2490, 2005. First published January 6, 2005; doi:10.1152/ajpheart.00848.2004
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Direct action of T3 on phosphorylation potential in the sheep heart in vivo

Michael A. Portman,1,2 Kun Qian,1 Julia Krueger,1 and Xue-Han Ning1,2

1Division of Cardiology, Department of Pediatrics, University of Washington School of Medicine, and 2Children's Hospital and Regional Medical Center, Seattle, Washington

Submitted 19 August 2004 ; accepted in final form 20 December 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Thyroid acting through ligand binding to nuclear receptors modifies myocardial respiratory kinetics and oxidative phosphorylation in the heart. Direct nongenomic action of thyroid hormone on high-energy phosphate concentrations and respiratory kinetics has never been proven in vivo but might be responsible for observed changes in oxygen utilization efficiency immediately after triiodothyronine (T3) administration. We tested the hypothesis that T3 directly and rapidly modifies myocardial high-energy phosphate concentrations and phosphorylation potential in vivo. Anesthetized sheep (age 28–40 days) thyroidectomized shortly after birth (Thy) and euthyroid age-matched controls (Con) underwent median sternotomy and received T3 infusion (0.8 µg/kg), followed by epinephrine infusion to increase myocardial oxygen consumption (MO2). 31P magnetic resonance spectra were monitored via a surface coil over the left ventricle. T3 increased phosphocreatine (PCr)/ATP and decreased ADP in Thy animals without causing a change in MO2. T3 produced no changes in high-energy phosphates in Con animals. T3 did not modify the PCr/ATP or ADP response to epinephrine and elevation in MO2 in either group. Cardiac mitochondria isolated from Thy and Con animals showed no change in respiratory rate or ADP/ATP exchange efficiency after T3 incubation. T3 infusion in a hypothyroid state decreases ADP concentration, thereby altering the equilibrium between phosphorylation potential and myocardial respiratory rate. These T3-induced effects are not due to changes in ADP/ATP exchange efficiency through action at the adenine nucleotide translocator but may be due to T3 mediation of substrate utilization, confirmed in other models.

adenine nucleotide translocator; myocardial energy metabolism; substrate oxidation

THYROID HORMONE REGULATES myocardial metabolism at the transcriptional level through binding to nuclear receptors. Ligand-dependent binding of these receptors to thyroid receptor elements controls transcription of various target genes involved in contractile and metabolic processes (5, 16). Although these nuclear receptor-mediated processes have been investigated to some extent, thyroid hormone as its active component, triiodothyronine (T3), also regulates cellular processes through direct binding to membranes or enzymes (2).

The direct or nongenomic actions of T3 on cardiac energy metabolism and high-energy phosphate kinetics remain somewhat obscure. In previous studies, we (20) demonstrated that T3 directly regulates myocardial substrate oxidation in isolated, perfused hearts. The relationship between substrate oxidation and phosphorylation potential has been established in several cardiac models (23, 43). An apparent equilibrium exists between mitochondrial NADH/NAD and cytosolic phosphorylation potential. Accordingly, we considered that T3 could also elevate phosphorylation potential in vivo. Prior studies in sheep in vivo implicate the adenine nucleotide translocator (ANT), which facilitates ADP/ATP exchange across the mitochondrial membrane, as a major influence on the dynamic response of phosphorylation potential to changes in cardiac work and myocardial respiratory rate (33, 39). Furthermore, thyroid hormone regulates the ANT through transcriptional and posttranscriptional mechanisms during maturation in sheep. The specific role for direct nongenomic regulation of mitochondrial ADP/ATP exchange by T3 in the heart has not been determined. In this study, we tested the hypothesis that T3 rapidly modifies myocardial high-energy phosphate concentrations and phosphorylation potential in vivo. We also sought to determine whether deficiency in immediate or nongenomic T3 action plays a role in maintaining ADP-dependent respiratory control in thyroidectomized sheep. We tested these hypotheses in mature sheep exhibiting normal ANT accumulation within the mitochondrial membrane, as well as in thyroid-deficient sheep. Studies used dynamic 31P magnetic resonance spectroscopy techniques in the sheep heart in vivo.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Sheep were thyroidectomized within 24–48 h after birth at the Washington State University School of Veterinary Medicine with a longitudinal subcricoid incision (3, 12). Prior studies demonstrated efficacy of this procedure in reducing T3 and thyroxine (T4) to below detectable levels within 7 days (33). After recovery, lambs were returned to ewes. Sheep were transported to the University of Washington, allowing at least 48-h recovery from stress of transport before performance of experimental procedures. All procedures were approved by each university's respective institutional animal care and use committee and were in compliance with National Institutes of Health guidelines.

Surgical preparation. At 28–40 days, thyroidectomized lambs and comparable-aged controls were sedated (intramuscular ketamine and xylazine), intubated, and ventilated with room air and oxygen, followed by an intravenous dose of {alpha}-chloralose (40 mg/kg). A femoral arterial cannula was placed for monitoring systemic blood pressure and sampling blood. A median sternotomy was performed, and sheep were separated into one of two protocols.

In vivo studies. Pacing electrodes were sutured to the right atrial appendage. Coronary sinus flow was measured via an extracorporeal shunt between the coronary sinus and the superior vena cava, fashioned by cannulating both jugular veins with heparin-flushed Tygon tubing as previously described (35). Ligation of the hemiazygous vein directed coronary venous flow into the shunt located within the proximal portion of the coronary sinus. A cannulating ultrasonic transit-time probe within the shunt provided for digital display and continuous hard copy tracing. A T connector allowed blood sampling for O2 content. A 2-cm-diameter round NMR surface coil was sutured to the pericardium overlying the left ventricle. The lamb, wrapped in a heating blanket to maintain body core temperature at ~38°C, was placed inside a Lucite cradle and transferred into a spectrometer 26-cm clear bore.

NMR measurements. The NMR surface coil was tuned to 81 MHz and matched to 50 {Omega}. NMR data were collected with a General Electric (Fremont, CA) spectrometer operating at 4.7 T and using resident software. Shimming on the 1H free induction decay at 200 MHz and acquisition of 31P spectra were performed as previously described with cardiac gating (38). The interpulse delay was ~2 s, and the pulse width was optimized for the phosphocreatine (PCr) signal. The cardiac pacemaker triggered spectrometer acquisition. Therefore, atrial pacing rates occurred at the harmonic of 30 beats/s just above the intrinsic sinus node rate. Spectra were acquired with a simple one-pulse sequence, 5,000-Hz sweep width, and 2,048 data points. Thirty-two spectra were collected into data acquisition blocks, which were summed for 8-min periods. Spectra were analyzed with a least-squares fitting program and integration. Fully relaxed spectra were obtained before experimental protocols and used for saturation correction. Intracellular pH was determined from the chemical shift difference Pi – PCr (36).

Protocol. Cardiac pacing was necessary for shimming and gating the magnetic resonance pulse sequence. Throughout the protocol the heart rate was maintained by atrial pacing just above the intrinsic rate, increasing if intrinsic rate rose to compete with the pacing rate. Competition was detected by irregularity in the recorded pulse wave. After completion of an 8-min baseline acquisition period, T3 (0.8 µg/kg as liothyronine) or a comparable volume of saline was infused intravenously over 2 min. After 20 min, an 8-min acquisition period was repeated. Epinephrine infusion was then initiated at 1 µg·kg–1·min–1 and slowly increased over 8 min to reach an approximate two- to threefold increase in myocardial oxygen consumption (MO2). The epinephrine dose was then slowly titrated to maintain a steady coronary sinus flow rate for 8 min during data acquisition. Arterial and coronary venous blood were sampled during the steady-state coronary flow periods at baseline, after T3 or saline infusion, and during epinephrine-stimulated increases in MO2.

Oxygen content was determined with dissolved oxygen calculated from the PO2 and oxyhemoglobin data obtained from a hemoximeter. MO2 was calculated from the coronary arteriovenous difference times coronary sinus flow rate.

Mitochondrial isolation. Mitochondria were isolated from groups of control and thyroidectomized sheep separate from the in vivo infusion protocol. After the median sternotomy, hearts were excised and immediately put into ice-cold 0.9% sodium chloride. After washout, the blood, fat, and connective tissue were removed, and 15–18 g of myocardium from the free left ventricular wall were taken for mitochondria isolation. Mitochondria were isolated from the myocardium as described previously by digestion of the tissue with Nagase (5 mg/g tissue) and differential centrifugation (40).

Mitochondrial respiratory rates. Substrate oxidation rates and ADP-to-O ratios (ADP/O) were determined polarographically described by Chance et al. (4). Mitochondria mixed with K2HPO4 (6 mM) and substrates (glutamate and malate, each 5.5 mM) were added (final volume 120 µl) to an oxygen chamber. State 3 respiration after addition of a known ADP quantity and subsequent state 4 respiration were measured with a Clark electrode calibrated with standard techniques. Uncoupled respiratory rates were measured after addition of 250 nM FCCP to state 4 mitochondria. Mitochondrial respiratory rates were normalized to mitochondrial protein and cytochrome a (Cyt a) contents. Mitochondrial Cyt a content was determined with the technique of Williams (52) and modifications described more recently (1, 26). Isolated mitochondria were solubilized in a 2% solution of Triton X-100 and placed in the sample cuvette of an Aminco spectrometer. Potassium cyanide (2 mm) was added to the cuvette to achieve full reduction of the cytochromes. The Cyt a content was measured by using the difference between absorbencies measured at 605- and 630-nm wavelengths with an extinction coefficient of 12.5. Optical densities were corrected for any difference between these wavelengths in the unreduced spectrum. Respiratory control index was calculated as the ratio of state 3 to state 4 respiratory rates. As described above, mitochondria from euthyroid and hypothyroid sheep were incubated with either T3 or control vehicles during the measurement to test the direct effect of T3 in vitro.

ANT efficiency. ANT function was assessed by modification of the classic "back-exchange" and atractyloside-stop method described by Klingenberg et al. (19). Efficiency of ANT was measured as the exchange of extramitochondrial ATP against intramitochondrial ADP. Under the substrate conditions defined above, the freshly prepared mitochondria (1 mg) were loaded with 5 µl of [14C] ADP (0.02 mCi/ml) by incubation for 30 min. Loaded mitochondria were collected and washed twice to remove remaining extramitochondrial radioactivity. The mitochondria were resuspended and incubated with either 1 µl of T3 (10 mg/ml) or an equal volume of vehicles for 10 min. The outflow of ATP was initiated with the addition of 25 µl of unlabeled ADP. The reaction was terminated by adding 50 µl of carboxyatractyloside (CAT, 1 mM) after incubation for precisely 60 s. The mitochondria were sedimented, and the supernatant and mitochondrial pellet radioactivity (ex) (counts per minute, cpm) were measured separately. The results were corrected for the independent efflux of ADP (con), which occurred in the presence of CAT. The back-exchange was calculated with the modified equation:

Statistical analyses. The reported values are means ± SE. Data were evaluated with single-factor analysis of variance across groups with the Statview 4.5 (FPV) Program (Abacus Concepts, Berkeley, CA). When significant F values were obtained, individual group means were tested for differences with an unpaired t-test. For in vivo study comparisons, baseline parameters were also compared with parameters after T3 infusion with paired t-tests. The effect of T3 incubation in isolated mitochondria was evaluated with paired and unpaired t-tests. The criterion for significance was P < 0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Hemodynamics and MO2 in vivo. Thyroidectomy in sheep resulted in total T3 levels below the detectable range (<30 ng/dl). T3 supplementation increased levels to 354 ± 49 ng/dl at the end of infusion, whereas controls starting at 151 ± 23 ng/dl increased to 401 ± 72 ng/dl at the end of T3 infusion. Hemodynamic data and oxygen consumption for four groups of sheep are included in Table 1: control with T3 supplementation or saline vehicle and thyroidectomy with T3 supplementation or saline vehicle. Heart rate and pressure-rate product were significantly lower in thyroidectomized sheep. T3 infusion yielded no significant change in any of the hemodynamic and cardiac function indexes or in coronary blood flow or MO2 before epinephrine. Epinephrine infusion elevated several parameters and caused a greater than twofold increase in MO2 (>3-fold in some groups). The protocol was designed to increase epinephrine to reach comparable relative increases in coronary flow and MO2 for each group. Accordingly, no differences in coronary flow or MO2 occurred among the groups. Although comparable MO2 levels were achieved among the four groups, some differences in functional parameters occurred during epinephrine infusion: pressure-rate product was higher in thyroidectomized sheep receiving T3 than in those receiving saline, and heart rate was lower in control sheep receiving T3 than in those with saline.


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  		Table 1. Hemodynamic parameters and myocardial oxygen consumption

 
High-energy phosphates in vivo. Representative 31P magnetic resonance spectra from a single thyroidectomized sheep experiment are illustrated in Fig. 1. Difference spectra exhibit the increase in PCr with T3 infusion, with relative stability of ATP. Spectra at peak oxygen consumption achieved through epinephrine show the decrease in PCr and the corresponding increase in Pi. ADP values were calculated from the creatine kinase reaction equation using baseline ATP and total creatine values obtained from previous studies in thyroidectomized and control sheep (33). For these calculations, we assumed that total creatine did not change through the protocol. 31P spectra indicated that no significant change in ATP or intracellular pH occurred during these protocols. T3 infusion significantly increased steady-state PCr/ATP ratio and decreased calculated free ADP in the thyroidectomized group only (Figs. 2 and 3). Low signal-to-noise ratio precluded accurate measurement of Pi, although a qualitative decrease was noted on spectra. Epinephrine infusion in conjunction with increased MO2 yielded no significant changes in PCr/ATP or calculated ADP for the control groups. However, PCr/ATP decreased and calculated free ADP increased in the thyroidectomized groups. T3 did not obviate the increase in ADP at peak oxygen consumption in the thyroidectomized group.



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  		Fig. 1. In vivo cardiac 31P spectra from a thyroidectomized sheep. Peaks are labeled as phosphocreatine (PCr), intracellular Pi, and 3 ATP peaks: {gamma}, {alpha}, and {beta}. A: baseline spectra demonstrate the principal phosphate peaks. C: spectra obtained after triiodothyronine (T3) infusion. B: difference spectra (C – A) show that PCr/ATP is greater after T3 infusion. D: spectra obtained during peak myocardial oxygen consumption (MO2) induced by epinephrine. Pi is visible because blood from mature sheep, unlike other species, does not contain high concentrations of 2,3-diphosphoglycerate, which overlap with Pi in the spectrum (29). Pi increases during a corresponding decrease in PCr compared with A and C.

 


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  		Fig. 2. PCr/ATP for each step in the protocol: baseline (Base), T3 or saline vehicle infusion (I), and epinephrine infusion (Epi; peak MO2). Data for 4 sheep groups are shown: control with T3 or saline vehicle and thyroidectomy (Thy) with T3 or saline vehicle; n values for each group are reported in Table 1. PCr/ATP increases with T3 infusion in the thyroidectomy group and decreases with epinephrine infusion in both thyroidectomy groups.

 


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  		Fig. 3. Calculated free cytosolic ADP concentration ([ADP]) for each step in the protocol with the format in Fig. 2. ADP changes inversely to PCr/ATP changes in Fig. 2 with a decrease after T3 in the thyroidectomy group and an increase in both thyroidectomy groups with epinephrine infusion. [ADP] is constant throughout the protocol in the control groups.

 
Isolated mitochondria. Respiratory rates normalized to mitochondrial Cyt a (cytochrome oxidase) content and ANT exchange efficiencies are noted in Table 2. Mitochondria were included for evaluation if the respiratory control index (RCI) exceeded 6.0. There were no significant differences between state 3 and uncoupled state with FCCP, indicating that mitochondria were maximally respiring in state 3. Additionally, no significant change in respiratory rate per unit Cyt a, RCI, or ADP/O occurred with T3 incubation. There were no differences in ADP/ATP exchange efficiency among the groups. Furthermore, T3 did not alter ANT exchange efficiency in cardiac mitochondria from either thyroidectomized or euthyroid sheep.


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  		Table 2. Mitochondrial respiratory rates and ADP exchange efficiency in control and thyroidectomized sheep

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Thyroid hormone exerts profound effects on cardiovascular physiology (16). However, the vast majority of thyroid hormone research, particularly in the heart, has focused on genomic or nuclear receptor-mediated actions. Only a few investigators have considered the importance of immediate nongenomic and non-nuclear receptor responses to T3 in the heart (20, 51). The scientific literature regarding T3 influence on high-energy phosphate metabolism generally includes studies involving several days of T3 injections to euthyroid or occasionally to hypothyroid animals to establish physiological or metabolic responses (14, 41, 47). These types of investigations tend to ignore any contribution through nongenomic action. The cardiac surgical literature includes several studies that investigated immediate T3 action after clinical cardiopulmonary bypass in humans (17, 34) or after conditions emulating cardiac surgery in animal models (18, 31). Cardiopulmonary bypass through various mechanisms reduces circulating T4 and T3 levels. T3 supplementation in these scenarios rapidly modifies cardiac physiology and energy metabolism. Two research groups have noted that T3 supplementation elevates the contractile work-to-oxygen consumption ratio after hypothermic ischemic circulatory arrest in baboons (30) or after cross-perfusion in dogs. (18) However, these studies did not specifically evaluate direct T3 action without complicating influences of hypothermia and ischemia. The current study offers a more directed evaluation of cardiac energy metabolism by examining the relationship between MO2 and phosphorylation potential. No prior study has evaluated acute effects of T3 supplementation on these relationships in hypothyroid or euthyroid models either in perfused heart or in vivo.

We used 31P magnetic resonance spectroscopy to define the relationship between MO2 and phosphorylation potential in euthyroid and hypothyroid juvenile sheep. Myocardial oxidative phosphorylation and mitochondrial membrane transport systems efficiently supply ATP for use by energy-consuming processes in the cardiomyocyte. Several investigators using various species have demonstrated that the ATP production rate generally matches the overall rate of ATP hydrolysis in the mature heart in vivo if carbon substrate and oxygen supplies are ample (10, 13, 28, 39, 53). The cellular energy state can be defined by the phosphorylation potential ([ATP]/[ADP][Pi]). Energy available for contractile processes and defined as the free energy of ATP hydrolysis ({Delta}GATP) directly relates to phosphorylation potential as:

(1)
where {Delta}G is the standard free energy change (concentrations at 1 M), R is the gas constant, and T is the absolute temperature in Kelvins.

As noted, some investigators have evaluated the immediate effect of T3 administration on efficiency of energy use for contractile work after ischemic cardiac arrest. DiPierro and coauthors (6) showed that T3 administration in euthyroid sheep in vivo increased cardiac contractile work without raising MO2. The improved oxygen utilization efficiency observed in most of these studies has generally been attributed to changes in ventriculo-arterial coupling and systemic arterial vasodilation. Elevation in {Delta}GATP could also contribute to this phenomenon but has not been previously considered as a T3-mediated cause for change in oxygen utilization efficiency.

Our study shows that T3 elevates PCr/ATP without concomitant change in MO2. PCr provides the major reservoir of high-energy phosphates within the cytosol. In magnetic resonance spectroscopy studies, PCr/ATP ratio is considered directly proportional to phosphorylation potential. The creatine kinase equilibrium reaction describes the relationship between PCr and ADP.

(2)
in following:

(3)
where Cr is creatine Keq = 1.66 x 10–9, as reported by Lawson and Veech (24).

The data obtained in this study show a significant and rapid decrease in [ADP] by T3 in the hypothyroid hearts. According to the relationships defined in these equations, and with the reasonable assumption that the total creatine pool does not change during these protocols, T3 significantly increases myocardial {Delta}GATP, thereby improving efficiency of ATP utilization. Furthermore, the increase in {Delta}GATP is amplified by relative decrease in Pi. The T3-induced alteration in phosphorylation potential and {Delta}GATP occurs only in the hypothyroid condition, possibly indicating the influence of the baseline energetic state and/or the buffering capacity of the intrinsic circulating T3 pool in the control euthyroid sheep heart. Conceivably, T3 binding sites in the control hearts are already saturated or near saturated in the presence of normal circulating levels. Alternatively, in the hypothyroid state, the operative yet undefined T3 binding sites may be upregulated or exhibit greater sensitivity in a manner similar to the documented enhancement of nuclear thyroid receptors by prolonged thyroid deficiency (11, 22).

The mechanisms through which T3 effects change in [ADP] and phosphorylation potential are likely multifold. We previously documented that observed reductions in phosphorylation potential during elevations in cardiac work state in the neonatal sheep in vivo did not occur in mature counterparts. [ADP] increases and phosphorylation potential declines with moderate MO2 elevation in the neonatal heart (37, 39). The relationship between MO2 and [ADP] emulates a respiratory control pattern consistent with first-order Michaelis-Menten kinetics. Transition to the mature and less ADP-dependent type respiratory control occurs in the first month of development and parallels accumulation of ANT in the mitochondrial membrane (39). These findings in a sheep model in vivo correspond to results from studies performed in isolated mitochondria from other species (44, 45). These studies together confirm that [ADP], phosphorylation potential, and {Delta}GATP depend in part on ANT function. The ADP/ATP exchange rate must increase in concert with an increase in ATP hydrolysis at ATPase sites to maintain the steady-state [ADP]. Under dynamic conditions such as epinephrine or dobutamine stimulation, a diminution in ANT functional capacity, whether resulting from immaturity or heart failure, produces elevated cytosolic ADP with reductions in phosphorylation potential and {Delta}GATP (Eq. 1; Refs. 28, 29, 39).

In a prior study, sheep thyroidectomy on day 1 after birth delayed normal postnatal mitochondrial accumulation of ANT, thereby reducing total ANT exchange capacity and resulting in persistence of newborn-type respiratory kinetics at 4 wk of age (33). The immature respiratory control mode was demonstrated in that study and confirmed in the current study in both thyroidectomy groups by the decrease in PCr/ATP and the increase in ADP, which accompany epinephrine-stimulated elevation in oxygen consumption. The previous study also showed that early thyroidectomy, although reducing overall myocardial ANT protein content, did not affect qualitative function at individual ANT exchanger sites. Additionally, thyroidectomy did not alter distribution among the three isoforms. Therefore, the persistence of ADP-dependent respiratory kinetics in thyroidectomized sheep appeared to result from reduced total ANT exchange capacity controlled at the transcriptional level, not from modifications at individual exchange sites.

Our prior work (33) in the sheep heart evaluated T3 mediation of transcriptional pathways, which influence ADP/ATP exchange and high-energy phosphate kinetics, without considering an additional contribution through nongenomic mechanisms. However, deficiency in direct T3 stimulation of ADP/ATP exchange could explain in part the increase in ADP associated with epinephrine infusion in the thyroidectomized sheep. This consideration stems from reports from several research groups identifying specific T3-binding activity in the inner mitochondrial membrane in association with rapid T3 enhancement of ADP/ATP exchange efficiency (27, 42, 48, 49). Sterling et al. (50) observed stimulation of respiratory rate in liver mitochondria isolated 30 min after T3 injection into a rat in the presence of protein synthesis inhibitors. Mowbray and Hardy (27) demonstrated that reduced ADP/ATP exchange efficiency in liver mitochondria isolated under hypothyroid conditions can be normalized to euthyroid levels within 15 min of T3 exposure provided either in vivo or in vitro. The precise mechanism for immediate T3 action on liver mitochondria ANT remains controversial, but some studies implicate T3 allosteric modification of an ADP-ribosyl transferase, which covalently modifies ANT, as opposed to direct T3 binding to ANT (27). Importantly, T3 did not accelerate ANT exchange in similarly prepared testes or spleen mitochondria, implying organ specificity due to differences in isoform distribution (50). Previous investigators did not evaluate mitochondria from cardiac or skeletal muscle, prompting our detailed analyses performed both in vivo and in vitro.

To determine whether T3 similarly induced a rapid effect on respiratory kinetics in sheep heart exhibiting an immature respiratory mode due to deficiency in ANT content, we provided T3 supplementation to thyroidectomized sheep before the epinephrine challenge. We found that T3 administration did not alter the dynamic PCr/ATP or ADP response to increasing oxygen consumption in thyroidectomized sheep or in euthyroid control sheep. Therefore, reduction in nongenomic action by T3 did not cause the persistence of the ADP-dependent mode of respiratory control in thyroidectomized sheep in vivo.

To confirm that our study results did not deviate from prior studies conducted with liver mitochondria because of differences in experimental format, we then also evaluated mitochondria in vitro with similar methodology (49). T3 produced no change in ADP/ATP exchange efficiency with the classic back-exchange methods with atractyloside, a specific ANT exchange inhibitor. Thus we concluded that T3 direct stimulation of ANT exchange did not occur in heart and was not responsible for the ADP increase caused by T3 administration. These findings support the organ specificity theory suggested by Sterling's work, as heart expresses predominantly ANT isoform 1 in contrast to dominant expression of ANT-2 in liver (7).

In summary, we present here the first analysis of rapid or nongenomic T3 action on high-energy phosphate kinetics in the heart in vivo. We demonstrated direct and rapid T3-mediated reduction of [ADP], and thus elevation in phosphorylation potential, in hypothyroid sheep heart in vivo without change in MO2. However, T3 does not immediately modify ANT efficiency in the hypothyroid heart, shown in isolated mitochondria studies in vitro and further supported by experiments in vivo defining the relation between high-energy phosphates and MO2. The latter finding supports our previously published data in hypothyroid sheep implicating the delay in ANT accumulation after birth as the primary cause for delayed maturation in respiratory control.

As the rapid elevation in phosphorylation potential during steady-state conditions does not appear to involve direct stimulation of ANT-mediated adenylate exchange, other possible mechanisms for this reduction in [ADP] without concomitant change in MO2 require consideration. Several lines of evidence indirectly support the speculation that T3 modification of substrate oxidation alters the relationship between cytosolic phosphorylation potential and cardiac cellular respiratory rate. From et al. (8) demonstrated in perfused rat hearts that the cytosolic ADP level and phosphorylation potential at a given level of oxygen consumption vary according to the specific substrate undergoing oxidation. The prevailing hypothesis explaining this relationship, supported by some experimental data, states that an equilibrium exists between mitochondrial NADH/NAD and cytosolic NADH/NAD (8). In following cytosolic [ATP]/[ADP], [Pi] operates near equilibrium with [NADH]/[NAD]. Accordingly, oxidation of substrates that induce high reduced mitochondrial nicotinamide (NADHm), such as pyruvate or octanoate, elevate cytosolic phosphorylation potential. In contrast, substrates producing relatively low NADHm, such as glucose (8, 43), effect a decrease. Subsequent studies have confirmed this phenomenon in the heart in vivo (15, 32, 46). Kim et al. (15) showed that phosphorylation potential increased in the canine heart during infusion of ketones in the form of {beta}-hydroxybutyrate, despite constant MO2. Also, Schwartz and coworkers (46) decreased phosphorylation potential during catecholamine infusion by blocking fatty acid oxidation with the carnitine palmitoyl transferase I inhibitor oxfenicine. These studies indicate that mitochondrial NADH redox state regulates cytosolic phosphorylation potential in vivo.

As phosphorylation potential in these multiple experiments performed in perfused heart or in vivo can be modified by substrate switching without concurrent change in myocardial respiratory rate, we postulate that the T3-mediated change in ADP occurs through a similar mechanism. The current study is limited in proving this hypothesis in that we did not explicitly evaluate myocardial substrate oxidation in this model. We previously showed (33) that thyroidectomy did not alter the lactate oxidation contribution to the increase in MO2 caused by epinephrine stimulation in this sheep model. However, we recognized inherent errors in assuming that substrate uptake corresponds to substrate oxidation, particularly when considerable variation occurs in circulating substrate levels. As myocardial substrate uptake, particularly in vivo, does not correspond to substrate oxidation, a thorough investigation would require fairly elaborate tracer techniques using either radioisotopes or 13C labeling (21, 25). Instead, in this study, we measured high-energy phosphates in vivo and did not perform the additional complex experiments necessary to confirm T3 mediation of relative oxidation rates for individual substrates. Nevertheless, we have confirmed our hypothesis suggesting nongenomic T3 mediation of myocardial substrate oxidation in an alternate model. T3 administration immediately increased the free fatty acid fractional contribution of acetyl-CoA to the tricarboxylic acid, while decreasing fractional contribution through lactate in isolated retrograde perfused hearts from euthyroid (20) and thyroidectomized (unpublished data) rats. Thus rat heart data, obtained under more controlled substrate conditions than can be achieved in vivo, support the contention that T3 modification of substrate oxidation yields observed changes in phosphorylation potential in sheep hearts. Confirmation of T3 modification of substrate oxidation in vivo is planned for future experiments using 13C isotopomer techniques (25).

These study findings do not preclude the participation of other mechanisms in T3 regulation of high-energy phosphates. Alternative direct binding targets for T3 or diiodothyronines have been identified. For instance, diiodothyronines specifically bind to and increase activity of cytochrome c oxidase Va (9). Furthermore, we measured hemodynamic indexes and pressure-rate product but did not measure more specific parameters of cardiac work, such as output or ventricular power. Thus it is conceivable that cardiac work efficiency was altered by a T3 effect on afterload as described earlier in a similar sheep model (6). Energy saving may have contributed to increased high-energy phosphate storage, although previously improvement in efficiency produced increased work (6).


   ACKNOWLEDGMENTS
 
This work was supported by National Heart, Lung, and Blood Institute Grant R01-HL-60666 (to M. A. Portman).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. A. Portman, Children's Hospital and Regional Medical Center, 4G-01, Seattle, WA 98105 (E-mail: michael.portman{at}seattlechildrens.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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