Effect of Aminophylline on High-energy Phosphate Metabolism and Fatigue in the Diaphragm

Our preparation has been described previously (Figure 1). [7]In brief, piglets (n = 44; 6-10 weeks old) weighing 10-16 kg were anesthetized with pentobarbital intravenously (20-mg/kg loading dose followed by 10-15-mg *symbol* kg sup -1 *symbol* h sup -1 continuous infusion). The animals' lungs were ventilated by tracheostomy with a Harvard animal ventilator to maintain normocapnia and arterial Oxygen²tension > 100 mmHg.

Venous catheters were placed in the left and right internal jugular veins for maintenance fluid, continuous pentobarbital and aminophylline administration. Arterial catheters were placed in the left carotid artery for blood sampling, in the left ventricle via the right femoral artery for microsphere injection, and in the abdominal aorta via the left femoral artery for microsphere withdrawal.

After a right thoracotomy, helical stainless steel stimulating electrodes were positioned around each phrenic nerve and then connected to a nerve stimulator (S8, Grass Medical Instruments, Quincy, MA) for phrenic nerve pacing. A midline laparotomy was performed to place an elliptical (5 x 2 cm) MRS surface coil, which had been tuned to both proton (200 MHz) and phosphorus (80.5 MHz) resonance frequencies, on the abdominal side of the right hemidiaphragm. The surface coil was shielded from surrounding intercostal and abdominal wall muscle with wet gauze padding. Air was evacuated from the chest and the incisions were closed in layers.

Esophageal pressure (Pes) was measured with an air-filled balloon-tipped catheter positioned through an esophagotomy in the neck. A similar catheter was inserted into the abdomen by laparotomy to determine abdominal pressure (Pab). A needle-tipped catheter was introduced into the endotracheal tube lumen to measure airway pressure. Diaphragmatic force output was given by the transdiaphragmatic pressure (Pdi), which was taken as Pab - Pes at end-expiration with the airway occluded. Diaphragmatic fatigue was defined as a decrease in Pdi by > 20% compared with the initial value during pacing. Constant transpulmonary pressure (taken as airway pressure - Pes) was used as an index of constant lung volume during the experiment.

Arterial blood samples for pH, CO²tension, and Oxygen²tension were measured with a blood gas analyzer (ABL3, Radiometer America, Cleveland, OH). Hemoglobin, Oxygen²saturation, and Oxygen²content were analyzed with a co-oximeter (IL 282 CO-oximeter, Instrumentation Laboratory, Lexington, MA). Plasma aminophylline concentrations were analyzed by fluorescent polarization immunoassay technique (TDX system, Abbott Laboratories, Dallas, TX) with a sensitivity of 0.4 mg/l and < 1% cross reactivity.

Approximately 1.3 x 10⁶microspheres (15 plus/minus 0.5 micro meter in diameter) (du Pont-New England Nuclear Products, Boston, MA) were injected over 30 s into the left ventricle for each diaphragmatic blood flow (Qdi) measurement. [8]Withdrawal of the reference blood sample from the abdominal aorta using a withdrawal pump (Harvard Apparatus Co., Millis, MA) began 20 s before the injection and lasted until 5 ml blood had been withdrawn. The withdrawal rate was 2.47 ml/min.

At the conclusion of the experiment the animal was killed with an overdose of Sodium pentobarbital and KCl. The diaphragm was sectioned, weighed, placed in 15-ml Poly Q vials, and counted in a multichannel analyzer (Minaxi model 5330, Packard Instrument Co., Downer's Grove, IL) with a 7.6-cm through-hole NaI crystal. Qdi was calculated as Qdi = C^Dx (Q^R/C^R) x 100, where C^D= counts/g diaphragm; Q^R= reference blood flow (2.47 ml/min); and C^R= total counts in the reference sample.

MRS data were obtained in 24 animals in a 40-cm-bore, 4.7-T magnet (CSI, General Electric, Freemont, CA). Noise was filtered from the MRS signal by attaching a filter to the stimulating electrodes, which were grounded on the wall of the magnet at the bore aperture. Homogeneity of the magnet field was maximized by adjusting the shim coil current at the start of the experiment. Signal to noise ratio was further improved by gating the signal acquisition to changes in airway pressure, which varied primarily in response to mechanical ventilation. Free induction decays for phosphorus were acquired after 80-W radiofrequency pulses of 85-micro second duration. The interval between successive radiofrequency pulses was determined by airway pressure changes and was usually 3.0 s. A single spectrum was produced by averaging free induction decays over 2 min. Fourier transform of the free induction decay was performed after exponential weighting with a line width of 40 Hz.

Areas of beta-adenosine triphosphate (beta-ATP), PCr, and Pi were computed by a least-squares best-fit technique. To adjust for relaxation effects, the PCr and Pi areas were corrected relative to beta-ATP by factors of 1.3 and 1.1, respectively. The areas of each phosphate peak were expressed as fractions of the total phosphate area to adjust for changes in signal intensity. Based on studies by Chance et al. [9]and Gutierrez et al. [10]we assumed that Pi/PCr greater or equal to 1 reflected a state of inadequate oxidative metabolism in the muscle. Intracellular pH (pHi) was determined by the chemical shift of the Pi. [11].

Because measurement of CAP amplitude interferes with the MRS signal, a separate group of 20 animals was used to evaluate CAP of the diaphragmatic EMG during pacing with and without aminophylline. The same surgical procedures and protocol were used for this group except for placement of a bipolar stainless steel electrode in the abdominal side of the left hemidiaphragm to measure CAP. A reference electrode was placed in the adjacent rectus abdominis muscle. A copper plate applied to the back of the animal served as the ground electrode.

The CAP electrodes were connected to a signal averager (Quantum 84, Cadwell Laboratories, Kennewick, WA). CAP and Pdi were measured at the onset of pacing and then every 5 min. The CAP amplitude data at each 5 min time point represented the average of 24 individual compound action potentials recorded within an 800-ms interval. Bandpass filters of the signal averager were set at 100 and 10,000 Hz. The CAP amplitude was taken as the distance between the trough of the initial downward deflection and the peak of the subsequent upward deflection.

To assess the effects of aminophylline on diaphragmatic force output, activation, and high-energy phosphate metabolism, the animals (n = 44) were divided into four groups: (1) saline control (n = 11), (2) aminophylline 10 mg/kg (A10; n = 11), aminophylline 20 mg/kg (A20; n = 11), and (4) aminophylline 40 mg/kg (A40; n = 11). The aminophylline loading doses of 10, 20, and 40 mg/kg intravenously were followed by continuous infusions of aminophylline of 1, 2, and 4 mg *symbol* kg sup -1 *symbol* h sup -1, respectively. A 60-min recovery was allowed after completion of the surgical procedures. Thereafter, MRS data were acquired every 5 min during four phases of the experiment: (1) control with the diaphragm at rest before drug administration (10 min); (2) diaphragm rest during saline or aminophylline loading (10-20 min); (3) diaphragm pacing and saline or aminophylline continuous infusion (25 min); and (4) recovery (no pacing, 10 min). Diaphragm pacing required stimulation of both phrenic nerves in the chest with supramaximal voltage, stimulation frequency 30 Hz, duty cycle 0.33, and 10 trains/min. These parameters were chosen to mimic a normal respiratory pattern with 10 breaths/min, an inspiratory to expiratory time ratio of 1:2 (duty cycle 0.33), and a phrenic nerve firing rate in the physiologic range (stimulation frequency 30 Hz). [12]The animals' lungs were also mechanically ventilated throughout the experiment to suppress all spontaneous respiratory effort and maintain adequate oxygenation and CO²elimination.

Pdi was determined at 5-min intervals in all animals. Pi, PCr, ATP, and pHi were measured in 24 animals: 6 animals in each of the saline, A10, A20, and A40 groups. Simultaneous Qdi determination with the microsphere technique was obtained in 20 animals (5 in each of the four groups). Because there were only six isotopes available, microsphere injection took place at control; during saline or aminophylline loading; at 1, 5, and 25 min of pacing; and during recovery. Diaphragmatic activation as measured by CAP amplitude was determined every 5 min during pacing in 20 animals (n = 5 in each of the four groups).

The data were analyzed by analysis of variance to determine differences between groups and within a single group over time. Critical differences between the mean values were assessed with Scheffe's F test or the paired t test with Bonferroni's correction. P < 0.05 was considered significant. Results are expressed as mean plus/minus SE.

Arterial blood gases and pHa (Table 1), transpulmonary pressure, hemoglobin concentration, and mean arterial pressure did not vary as a function of pacing or aminophylline dose. After the aminophylline loading dose, heart rate increased in the A40 group compared with the saline group (P < 0.05, Table 2).

Plasma aminophylline concentrations varied in a dose-dependent manner (Figure 2). At the onset of pacing, the plasma aminophylline concentration was 12.2 plus/minus 0.7 mg/l in the A10 group, 21.9 plus/minus 2.4 mg/l in the A20 group (P < 0.05 compared with A10 group), and 44.9 plus/minus 3.6 mg/l in the A40 group (P < 0.05 compared with A10 and A20 groups). Aminophylline concentrations decreased gradually over time in all groups despite a continuous infusion of aminophylline.

(Figure 3and Figure 4) demonstrate the effects of aminophylline on diaphragmatic force output and activation. The original tracings of Pab, Pes, and CAP show preservation of Pab and Pes (and hence Pdi) despite decreasing CAP amplitude when the animal is treated with aminophylline 40 mg/kg (Figure 3(A)) in contrast to the decrease in Pab, Pes, and CAP amplitude during pacing in a saline control animal (Figure 3(B)). Mean data confirm that aminophylline did not affect CAP amplitude during pacing (Figure 4(A)). CAP amplitude was reduced in all groups by 30% after 25 min of pacing. Conversely, aminophylline had a dose-dependent effect on diaphragmatic force output (Figure 4(B)). Pdi was significantly greater in the A40 group than in the saline group during pacing (P < 0.01) such that fatigue (> 20% decrease in Pdi) was prevented in the A40 animals for the first 20 min of pacing, whereas fatigue occurred in the saline group within the first 5 min of pacing. When the diaphragm was allowed to rest for 10 min after pacing, Pdi returned to baseline in the A40 group (103 plus/minus 16%), whereas Pdi remained depressed in the saline group (72 plus/minus 6%) (P < 0.05 compared with the initial value). The A10 and A20 groups exhibited Pdi levels intermediate between these two extremes at each time point consistent with a dose-dependent effect of aminophylline.

(Figure 5(A)) displays the effects of aminophylline on Pi/PCr during pacing. The normal resting Pi/PCr ratio of 0.2-0.3 did not change during saline or aminophylline infusion. Immediately after the onset of pacing, Pi/PCr increased to 1.01 plus/minus 0.09 in the saline group (P < 0.05 compared with rest and with A40 group during pacing). Subsequently, Pi/PCr decreased progressively to 0.61 plus/minus 0.06 by the end of pacing and returned to a resting control value of 0.30 plus/minus 0.03 after a 10-min recovery. In contrast, Pi/PCr did not differ significantly from the resting levels throughout pacing with aminophylline 40 mg/kg. In the A10 and A20 groups, Pi/PCr was intermediate between the saline and A40 levels during pacing such that Pi/PCr increased to 0.56 plus/minus 0.12 (A10) and 0.49 plus/minus 0.14 (A20) immediately after the onset of pacing (P < 0.05 compared with resting value and with saline value at the onset of pacing). Pi/PCr plateaued at these levels for the next 15 min of pacing in contrast to the sharp increase and subsequent decrease in Pi/PCr in the saline group.

(Figure 5(B)) shows beta-ATP in the presence and absence of aminophylline during rest and pacing. The resting beta-ATP area referenced to the total phosphate area was approximately 0.2 in all groups. Neither pacing nor aminophylline infusion affected beta-ATP subsequently. Similarly, pHi was approximately 7.2 in all groups and remained constant during pacing regardless of aminophylline dose (Figure 5(C)). These changes are evident in Figure 6, which depicts original MRS scans during saline (Figure 6(A)) and aminophylline 40 mg/kg (Figure 6(B)) administration.

Qdi is affected by pacing but not by aminophylline (Figure 7). Once pacing commenced, Qdi increased to 300-410 ml *symbol* min sup -1 *symbol* 100 g sup -1 at 1 and 5 min and then decreased to 200-250 ml *symbol* min sup -1 *symbol* 100 g sup -1 in all groups by the end of pacing.

The major findings of this study are that (1) aminophylline delays the onset of diaphragmatic fatigue despite a reduction in diaphragmatic activation during pacing; (2) aminophylline limits the increase in Pi/PCr that is otherwise observed during fatigue; (3) the effects of aminophylline on Pdi and Pi/PCr cannot be explained by differences in Qdi; and (4) the effects of aminophylline are dose-dependent.

Our data suggest a direct effect of aminophylline on diaphragmatic muscle with improved diaphragmatic force output and delayed onset of fatigue during pacing despite decreasing CAP amplitude (Figure 4). This result is consistent with previous findings in animals [13]and humans [14]in which aminophylline had no effect on diaphragmatic EMG but increased Pdi. Our findings are not consistent with reports that suggest aminophylline acts primarily through increased activation either at the brainstem or neuromuscular junction. The data supporting a central stimulatory effect of aminophylline come primarily from neonates in whom aminophylline is used to treat apnea of prematurity. These studies have documented a reduction in the frequency of central apnea, but have not measured diaphragmatic EMG. [15]Hence, it is possible that the central effects of aminophylline lie in the regular initiation of discharges from the respiratory center rather than in an increase in amplitude of those discharges. Similarly, aminophylline has been reported to antagonize neuromuscular blockade with pancuronium, raising the possibility that aminophylline may act by increasing cyclic adenosine monophosphate at the motor endplate, which in turn increases acetylcholine release. [16]This mechanism has been called into question by the finding that neuromuscular blockade produced by vecuronium is not antagonized by aminophylline. [17]Furthermore, the observed improvement in strength after aminophylline administration during neuromuscular blockade with pancuronium also may be explained by increased myoplasmic Calcium²+ concentration resulting from the aminophylline (which would leave diaphragmatic EMG unchanged) rather than increased neurotransmitter release (which should increase diaphragmatic EMG). Thus our data and others', [18]in which diaphragmatic activation was measured directly, show that the primary action of aminophylline is within the muscle rather than along the activation pathway with the exception of stabilizing respiratory center discharge in patients with central apnea.

Although others' work [18]supports our result of improved diaphragmatic force output with aminophylline, some investigators have noted no effect [6]or even decreased force output. [19]These differing results reflect the complexity of the interaction between aminophylline and respiratory muscle, including factors such as aminophylline dose, developmental stage, muscle fiber type composition, and type of diaphragmatic contractions (paced vs. spontaneous breathing). The particular importance of a large aminophylline dose is confirmed by our data.

In a dose-dependent manner, aminophylline prevents the increase in Pi/PCr ratio while preserving force output of the diaphragm during pacing. Others [20]have shown that contractile failure of cardiac and limb muscle as a result of inadequate energy supply relative to demand is accompanied by an increase in Pi/PCr to greater or equal to 1. We have demonstrated previously the same phenomenon in the diaphragm. [7]Marsh et al. [21]used MRS to demonstrate that theophylline administration improves forearm contractile force in association with improved aerobic metabolism. The current report extends these observations on the link between energy metabolism and force output by demonstrating that force output can be improved with administration of aminophylline, which also prevents the increase in Pi/PCr in the diaphragm.

Intracellular pH was approximately 7.2 in all groups during diaphragm pacing and was not affected by the onset of fatigue or by the administration of aminophylline (Figure 5(C)). We have shown previously that diaphragmatic fatigue induced by phrenic nerve pacing is not associated with significant changes in pHi. [7]Others have also noted that changes in pHi cannot account for muscle fatigue, [22]possibly because lactate production during muscle stimulation is balanced by H+ consumption during PCr hydrolysis. [23].

The few studies on the effects of aminophylline on pHi have provided conflicting results. Connett [24]noted an association between intracellular alkalosis and Calcium²+ release in frog sartorius muscle during aminophylline administration. Conversely, Shee et al. [25]found no effect of aminophylline on pHi in the resting state and a decrease in pHi during stimulated rat diaphragm contractions in the presence of aminophylline. These differing results may reflect different muscle fiber types or difference between in vitro, superfused muscle versus in vivo, perfused muscle. When adult subjects voluntarily increase forearm contraction to the point of exhaustion, intracellular acidosis is noted and is ameliorated by theophylline administration. [21]Intracellular acidosis may be more likely during progressively increasing voluntary contractions to the point of maximum output as opposed to the constant stimulation frequency producing submaximal paced contractions used in this investigation. Alternatively, differences in fiber type composition may play a role. Regardless of the reported effects of aminophylline on pHi, these studies agree with our finding that the improvement in force output in the presence of aminophylline is not mediated by an effect on pHi.

ATP was unaffected by pacing or aminophylline in our study. This observation is consistent with our previous data [7]and that of others. [26]The constancy of ATP during fatiguing contractions reflects the activity of the creatine kinase reaction to buffer changes in ATP at the expense of PCr hydrolysis and Pi production. Extreme hypoxia or prolonged maximal tetanic contractions may overwhelm the creatine kinase buffer and lead to ATP depletion. [27]In general, studies in humans showing ATP depletion during fatigue have used maximal voluntary contraction to exhaustion. [28]Our experimental protocol used constant stimulation intensity to produce submaximal contractions of the diaphragm, and the extreme conditions described above were not used. Finally, it is possible that changes ATP concentration in subcellular compartments are obscured because the 31P MRS measurement of ATP provides the average ATP in the volume of tissue sampled.

We used supramaximal phrenic nerve stimulation to activate the diaphragm to control the stimulus intensity. However, this method leads to recruitment of all diaphragm fibers simultaneously rather than the incremental recruitment observed during spontaneous breathing. If fatigue and the increase in Pi/PCr develop because of early activation of fatigable, glycolytic type IIb fibers in the diaphragm (which might not have been activated during spontaneous breathing), then it is possible that aminophylline improves force output and aerobic metabolism by preferentially affecting type IIb fibers. This possibility could not be tested in our model because the MRS surface coil samples a volume of tissue that is approximately the size of the diameter of the coil and that contains all fiber types.

Qdi increased during pacing, but the increases in Qdi were not affected by aminophylline dose (Figure 7). Thus the improved force output and aerobic energy metabolism in the presence of the large dose of aminophylline cannot be explained by increased Qdi. Our series confirms the results of a previous study on Qdi in the piglet by Mayock et al. [29]The pattern of Qdi changes during muscle contraction may depend on developmental stage, because studies in adult humans and animals have shown increased blood flow during aminophylline administration.

The study design sought to assess the metabolic and mechanical effects of various doses of aminophylline on the diaphragm in vivo. This design limited the ability to control heart rate in the various groups because high dose aminophylline produces tachycardia. However, we do not believe the tachycardia in the A20 and A40 groups affected the results because there was no difference in Qdi between the groups.

Aminophylline has been shown to recruit expiratory muscles in the spontaneously breathing dog. [18]It is unknown whether this effect is also seen during diaphragm pacing. However, it raises the possibility that expiratory muscle recruitment could have led to changes in diaphragm length during pacing that in turn may have affected force output and metabolism. Although we cannot exclude small changes in length, the protocol was designed to minimize this possibility by obtaining measurements during end-expiration with the airway occluded. Constant transpulmonary pressure during the experiment suggests that there were no major changes in lung volume.

We speculate that the known effects of aminophylline on intracellular Calcium²+ (Caⁱ²+) metabolism may explain the relation between force output and metabolism. Aminophylline increases Caⁱ²+ concentration through sarcolemmal Caⁱ²+ channels and by increased Caⁱ²+ release from the sarcoplasmic reticulum. [30]Increased Caⁱ²+ leads to increased crossbridge formation and force output. Aminophylline may also improve relaxation of the diaphragm by enhancing Calcium²+ sequestration into sarcoplasmic reticulum after a contraction. [31]The improved relaxation leads a more optimal diaphragm length and hence increased force output during the next contraction. Conversely, delayed relaxation may reduce contractile force, as has been shown in cardiac muscle, in which relaxation defects have been associated with heart failure, [32]myocardial ischemia, [33]and "stunned" myocardium. [34].

The increase in Caⁱ²+ may also improve aerobic energy metabolism within muscle. [35]Once myoplasmic Calcium²+ concentrations have increased, mitochondrial Calcium²+ uptake is mediated by an electrophoretic uniporter mechanism that is stimulated by increasing myoplasmic Calcium²+ concentration. [36]Conversely, Calcium²+ efflux from mitochondria by the Sodium sup +/Calcium²+ exchanger is inhibited by increased extramitochondrial Calcium²+ concentrations. [37]The resultant increase in intramitochondrial Calcium²+ concentration activates three intramitochondrial dehydrogenases: pyruvate dehydrogenase, [38]isocitrate dehydrogenase, [39]and alpha-ketoglutarate dehydrogenase. [40]The increase in activity of these Calcium²+ sensitive enzymes leads to an increase in turnover of the Krebs cycle, an increase in NADH production, and ultimately an increase in overall oxidative phosphorylation of adenosine diphosphate to ATP. Thus changes in intracellular Calcium²+ concentration balance the consumption of ATP by muscle work with ATP production by oxidative phosphorylation.

A variety of compounds, including epinephrine, phenylephrine, and caffeine, stimulate intramitochondrial dehydrogenases and oxidative metabolism. [41-43]This stimulatory effect is blocked by ruthenium red, an inhibitor of Calcium²+ uptake into mitochondria. [43]The effects of aminophylline on Calcium²+ -sensitive intramitochondrial dehydrogenase activity have not, to our knowledge, been investigated directly. However, our data are consistent with the interpretation that aminophylline raises intracellular Calcium²+ such that the increase in myosin adenosine triphosphatase activity during diaphragmatic work is balanced by increased aerobic metabolism and oxidative phosphorylation, which in turn is reflected in the low Pi/PCr ratio during pacing. The finding that another methylxanthine, caffeine, affects aerobic metabolism in a similar dose-dependent fashion further supports this interpretation. [43]Because Qdi is unchanged with aminophylline, it is likely that the effects of aminophylline on aerobic metabolism are associated with increased Oxygen²extraction and consumption.

In summary, we have demonstrated that aminophylline improves aerobic metabolism and delays the onset of fatigue in the diaphragm in a dose-dependent manner. We speculate that increased Caⁱ²+ concentration resulting from aminophylline provides the link between aerobic metabolism and force output. Even if sufficient improvement in diaphragmatic force output may not be uniformly achievable with nontoxic aminophylline doses, a clearer understanding of the relation between energy metabolism and force output may permit the development of other drugs to increase diaphragmatic force by preserving aerobic metabolism.