Within hibernating myocardium, it is uncertain whether a normal energetic state is present at baseline and whether maintaining that energy state during a catecholamine challenge is dependent on ATP-dependent potassium channel opening. In this study, 16 swine underwent a thoracotomy with placement of an external constrictor on the left anterior descending coronary artery (LAD) (hibernation model). Seven additional swine underwent a sham operation. At 10 wk, the myocardial energetic state in the LAD region was assessed by 31P-NMR spectroscopy, and the ratio of phosphocreatine to ATP (PCr/ATP) was determined at baseline, during glibenclamide treatment (0.5 mg/kg bolus with 50 μg/min iv), and during addition of dobutamine (40 μg·kg−1·min−1 iv). At baseline, transmural blood flow in the LAD and remote region was 0.75 ± 0.11 and 0.88 ± 0.09 ml·min−1·g−1, respectively (P < 0.01), in hibernating hearts and 0.83 ± 0.12 and 0.88 ± 0.15 ml·min−1·g−1, respectively (not significant), in sham-operated hearts. Under basal conditions, PCr/ATP in the LAD region of hibernating and sham pigs was 2.15 ± 0.04 and 2.11 ± 0.05, respectively (not significant). In sham pigs, addition of dobutamine to glibenclamide increased the double product from 10.4 ± 0.8 to 23.9 ± 4.0 mmHg·beats·min−1 × 1,000 (P < 0.05) and decreased transmural PCr/ATP from 2.06 ± 0.06 to 1.69 ± 0.06 (P < 0.05). Dobutamine increased the double product in hibernating pigs in a similar fashion and, despite a 40% lower blood flow response, induced an equivalent decrease in PCr/ATP from 2.04 ± 0.04 to 1.73 ± 0.08 (P < 0.05). In conclusion, we found that, in chronic hibernating swine myocardium with reduced basal blood flow and perfusion reserve, the transmural energetic state, defined by PCr/ATP, is normal during addition of dobutamine, despite inhibition of ATP-dependent potassium channel opening with glibenclamide. These data suggest that important adaptations other than the ATP-dependent potassium channel opening allow hibernating myocardium to operate over a lower range of the oxygen supply-demand relationship to protect against myocardial ischemia.
- myocardial ischemia
- high-energy phosphates
- mitochondrial adaptations
chronic hibernating myocardium has been observed both clinically and experimentally and is characterized by decreased regional blood flow and function at rest in the absence of significant necrosis (15, 18). Metabolically, hibernating myocardial tissue demonstrates increased glucose uptake and glycogen storage at a time that myocardial oxygen consumption is reduced and anaerobic glycolysis is not present (4, 5, 8). In chronically instrumented swine, hibernation evolves after several months of repetitive myocardial ischemia and can be distinguished from chronic stunning by a lower resting blood flow and decreased expression of sarcoplasmic reticulum calcium handling proteins related to contraction (1, 3, 11). Although the mechanisms of protection within hibernating myocardium are unclear, it is conceivable that ischemic preconditioning and hibernation share common signaling pathways that modify the severity of an energy supply-demand imbalance associated with limited perfusion reserve. Within ischemic preconditioned myocardium, ATP-dependent channel opening within the inner membrane of mitochondria may be protective by increasing the matrix volume and preserving the adenine nucleotide pool (2). Within the second window of protection, those channels on the sarcolemma may trigger downstream signaling that leads to cardioprotection (14).
Measurements of the phosphocreatine-to-ATP ratio (PCr/ATP) with 31P-NMR spectroscopy have been useful in predicting the severity of left ventricular (LV) dysfunction in patients with postinfarct remodeled hearts (19) and in identifying those patients with dilated cardiomyopathy who are at increased risk of cardiac death (13). Using 31P-NMR spectroscopy in the present study, we tested whether the transmural energetic state in a swine model of hibernation is normal under basal conditions and during inotropic stimulation. As observed in a swine model of short-term hibernation in which PCr/ATP improves at a time in which function and blood flow remain depressed (7), we postulated that high-energy nucleotides in chronic hibernation remain normal at high work states, despite inhibition of ATP-dependent potassium channel opening.
This study was approved by and performed under the guidance of The Animal Care Committee at the Veterans Affairs Medical Center and University of Minnesota and is in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication 85-23, revised 1996).
Sixteen domestic pigs (8–10 kg) were used for the chronic ischemic pig model. They were sedated with xylazine (2 mg/kg im) and Telazol (4 mg/kg im), ventilated, and anesthetized with isoflurane (1%). The thorax was prepped and draped, and a left thoracotomy was performed in the fifth intercostal space. The left anterior descending coronary artery (LAD) was dissected free, and a C-shaped occluder (3 mm in length and 1.4 mm in internal diameter) was secured around the vessel and gently closed with suture (8, 9). Seven additional pigs (30 kg) also underwent a left thoracotomy with dissection of the LAD without instrumentation. In all animals, the pericardium and chest were closed in layers. Sterile dressings were applied, and a chest tube was placed and removed within 3 days of surgery. Cephazoline (1 g iv) was given before and 12 h after the procedure and repeated daily for 3 days. Pain prophylaxis was provided during the first 3 postoperative days with buprenex (0.5 mg im every 12 h).
Regional Function Measurements
Within 48 h of the final study, a two-dimensional (2D) echocardiogram was done during sedation to assess regional wall thickening. To determine the degree of contractile reserve in this hibernating model, wall thickening was also measured by 2D echocardiogram in six additional sedated swine not used for the terminal studies, at baseline and 5 min after an intravenous infusion of dobutamine (40 μg·kg−1·min−1). In these animals, wall thickening at baseline in the LAD and remote regions was 25 ± 5% and 37 ± 4%, respectively (P < 0.05). End-diastolic wall thickness at baseline in the LAD and remote regions was 9.4 ± 0.3 and 9.3 ± 0.4 mm, respectively (not significant), demonstrating no diastolic thinning. During dobutamine with an increased heart rate to 158 ± 18 beats/min, wall thickening increased slightly but significantly in the LAD region to 33 ± 8% (P < 0.05) but remained less than the increased wall thickening in the remote region, which was 48 ± 7% (P < 0.05). The heart rate increased from 58 ± 1 beats/min at baseline to 158 ± 18 beats/min with dobutamine infusion. These data confirm that functional reserve with dobutamine exists in the chronically ischemic LAD region but to a lesser degree than shown in remote regions.
31P-NMR Spectroscopy Technique
During the terminal study, pigs were sedated with Telazol (4 mg/kg im) and transferred to the Center for Magnetic Resonance Research at the University of Minnesota. Pigs were ventilated and anesthetized with pentobarbital sodium (30 mg/kg bolus followed by 4 mg·kg−1·h−1 infusion); after a midline sternotomy, animals were instrumented with a 31P-MR spectroscopy (MRS) coil on the anterior LV wall. Catheters were placed in the LV apex for administration of microspheres and in the left anterior descending coronary vein for sampling of substrate uptake.
Spatially localized 31P-NMR spectroscopy was performed with the use of the RAPP-ISIS/FSW method. This is the rotating-frame experiment that uses adiabatic plane-rotation pulses for phase modulation (RAPP)-imaging-selected in vivo spectroscopy (ISIS)/Fourier series window (FSW) method. In this application of RAPP-ISIS/FSW, the signal origin was first restricted to a 12 × 12-mm 2D column perpendicular to the LV wall. The signal was later localized into three well-resolved and five partially resolved layers along the column and hence across the LV wall. Localization along the column was based on B1 phase encoding and employed a nine-term FSW. Whole wall spectra were obtained with the ISIS technique, defining a column 12 mm2 perpendicular to the heart wall. The calibration of spectroscopic parameters was facilitated by placing a polyethylene capillary filled with 15 μl of 3 M phosphonoacetic acid into the inner diameter of the surface coil. This phosphonoacetic acid standard was used only for calculating the 90° pulse length of the RAPP-ISIS method. The position of the voxels relative to the coil was set according to the B1 strength at the coil center, which was experimentally determined in each case by measuring the 90° pulse length for the phosphonoacetic acid standard contained in the reference capillary at the coil center. NMR data acquisition was gated to the cardiac and respiratory cycles using the cardiac cycle as the master clock to drive both the respirator and the spectrometer as previously described (19). The surface coil was constructed from a single turn copper wire 25 mm in diameter with each side of the coil leads soldered to a 33-pF capacitor. Complete transmural data sets were obtained in 10-min time blocks using a repetition time of 6–7 s to allow for full relaxation for ATP and Pi and ∼95% relaxation of the PCr resonance. PCr/ATP results were calculated for each transmurally differentiated spectra set, and all resonance intensities were quantified using integration routines provided by SISCO software.
Calculation of free ADP levels.
Myocardial free ADP levels were calculated from the creatine kinase equilibrium expression (where Keq = 1.66 × 109, cytosolic pH = 7.1, and brackets indicate concentration): [ADP] = [ATP][CRfree]/[PCr][H+]Keq (19). PCr and ATP values obtained from the spectra were calibrated from chemically determined ATP levels measured from the tissue biopsy samples following the protocol. Free creatine was calculated by subtracting the PCr values from total creatine measured in the tissue.
1H-NMR spectroscopy technique.
Radiofrequency transmission and signal detection were performed with the dually tuned 28-mm-diameter surface coil. A single-pulse collection sequence with a frequency-selective Gaussian excitation pulse (1 ms) was used to selectively excite the N-δ proton resonance signal of the proximal histidine in deoxymyoglobin (12). This technique provided sufficient water suppression because of the large chemical shift difference between water and deoxymyoglobin (>14 kHz). Adjusting the radiofrequency pulse power with the water signal as a reference optimized the NMR signal. A short repetition time (25 ms) was used because of the short longitudinal relaxation time (T1) of deoxymyoglobin. Each spectrum was acquired in 5 min (10,000 free induction decays). Although the short T1 of deoxymyoglobin and fast acquisition prevent gating to the cardiac cycle, the signal loss due to motion is negligible compared with the inherently broad line width of the deoxymyoglobin peak.
The regulation of oxidative phosphorylation was determined by MRS estimates of transmural PCr/ATP. Systolic blood pressure, heart rate, and rate-pressure double product were determined at each work state. Measurements were determined at baseline, during an intravenous infusion of glibenclamide (0.5 mg/kg bolus followed by 50 μg/min iv), at a dose that blocks the protective effect of ischemic preconditioning in swine (16), and during addition of dobutamine (40 μg·kg−1·min−1 iv) (Fig. 1). Regional myocardial blood flows were determined by standard blood flow techniques, using 1–2 million microspheres (15 μm) labeled with 141Ce, 85Sr, 46Sc, or 95Nb. Arterial-coronary venous lactate, glucose, and free fatty acid concentrations were determined by enzymatic technique, and oxygen saturation levels were calculated from arterial and venous blood-gas measurements. After the NMR study and before death, tissue from the LAD region was extracted with forceps and immediately frozen at −70°C for enzymatic assay of high-energy phosphates (8). The heart was then removed and separated into LAD and non-LAD regions, and the absence of necrosis was confirmed by placing longitudinal sections in freshly prepared triphenyltetrazolium solution. Heart tissue was then fixed for at least 48 h in 10% formalin, and myocardial and reference blood samples were counted in a multichannel analyzer (gamma counter 5000; Packard).
Data are expressed as means and SE. In the hibernating and sham pigs, regional blood flow and function at baseline were compared in the LAD and remote regions by paired Student's t-test. During repeated measurements, statistical differences were tested by ANOVA with post hoc testing using Student's paired t-test and a Bonferroni correction.
Baseline Metabolism in the Hibernating Model
Within 48 h of the terminal study, wall thickening was 27 ± 4% in the LAD region and 39 ± 4% in the remote region from the hibernating pigs (P = 0.01). At the time of the terminal study, regional blood flow by microspheres was 0.75 ± 0.11 ml·min−1·g−1 in the LAD region and 0.88 ± 0.09 ml·min−1·g−1 in the remote region from the hibernating pigs (P < 0.01). No differences in either regional function or blood flow were noted in the sham-operated pig hearts (Fig. 2). Baseline NMR spectra were obtained from the anterior wall of hibernating and sham pigs, and representative examples are shown in Fig. 3. At comparable levels of work during basal conditions, transmural PCr/ATP in hibernating and sham pigs was 2.15 ± 0.04 and 2.11 ± 0.05, respectively (not significant). Regional arterial-venous lactate uptake under basal conditions was 23.6 ± 8.3% in the hibernating pigs and 32.9 ± 8.8% in the sham pigs (Table 1), demonstrating no net differences in lactate production. Similarly, transmural myocardial oxygenation, by in vivo 1H-MRS myoglobin saturation levels, demonstrated no myoglobin desaturation in the hibernating myocardium (data not shown). Together, these metabolic findings demonstrate that hibernating myocardial tissue has adapted favorably so that myocardial ischemia is not observed, despite the presence of reduced wall thickening and depressed regional blood flow at baseline.
Bioenergetics During Increased Work in the Hibernating Model
To determine whether transmural energy is dependent on ATP-dependent potassium channel opening, glibenclamide was infused at a dose that is known to abolish ischemic preconditioning in swine (16). Consistent with its systemic effects on increasing glucose uptake, glibenclamide decreased plasma glucose levels from 8.1 ± 0.7 μmol/ml at baseline to 5.6 ± 0.8 μmol/ml (P < 0.05) in hibernating pigs (Table 1). Glibenclamide tended to decrease transmural PCr/ATP in the hibernating myocardium from 2.15 ± 0.04 to 2.04 ± 0.04 and in the sham-operated hearts from 2.11 ± 0.05 to 2.06 ± 0.06, but those differences did not reach statistical significance. In sham animals, the increase in cardiac work in response to dobutamine was associated with a 2.3-fold increase in transmural myocardial blood flow and PCr/ATP decreased to 1.69 ± 0.06. Although the increase in blood flow in response to dobutamine was 40% lower in LAD tissue of hibernating pigs than that in sham pigs, the decrease in PCr/ATP at the high work state was comparable (1.73 ± 0.08) (Fig. 4), with a similar relationship between increased cardiac work and decreased PCr/ATP in hibernating and sham pig hearts (Fig. 5). Thus the lower basal blood flow and reduced perfusion reserve in response to dobutamine did not result in greater depression of PCr/ATP in the hibernating myocardium. After the stress-inducing protocol was completed, the chemically determined ATP levels were 12.49 ± 0.71 μmol/g dry wt in the hibernating LAD region and 15.46 ± 0.27 μmol/g dry wt in the sham LAD region (P < 0.01). Total creatine and calculated free ADP levels in the hibernating regions were slightly but not significantly different from the sham regions (Table 1).
To address the effects of glibenclamide on the energetic response to dobutamine in sham pigs in the present study, we compared the response of dobutamine from the sham animals in the presence of glibenclamide with the response of dobutamine in historical sham pigs in the absence of glibenclamide (6). The dobutamine infusion increased the rate-pressure product over twofold in both groups of animals, and, as shown in Fig. 6, the magnitude of the effect on transmural PCr/ATP was similar.
Hibernating myocardium is characterized by decreased contractile function associated with a modest but significant decrease in blood flow. Furthermore, the contractile response to inotropic stimulation is blunted in hibernating regions, and this is associated with a subnormal increase in myocardial blood flow. The subnormal response of contractile function could be the result of insufficient energy production due to impaired oxygen delivery (blood flow) in the hibernating region or could represent inotropic hyporesponsiveness of the hibernating myocardium that acts to match contractile function to the limited blood supply. If contractile function is limited by insufficient oxidative ATP production, then free ADP levels will rise. In this situation, the creatine kinase reaction transfers a phosphoryl group from PCr to ADP in an attempt to maintain the ATP level, thereby resulting in a decrease in the PCr/ATP level. Thus examination of the response of PCr/ATP (which bears an inverse relation to cytosolic free ADP) will demonstrate whether the hyporesponsiveness in the hibernating region resulted from insufficient energy production or represents an equilibrium in which the contractile demands for energy are reduced in proportion to the decreased oxygen delivery (blood flow).
The principal finding of this study is that, in a swine model of hibernating myocardium with decreased regional blood flow and function at rest, the energetic state, as determined by PCr/ATP derived by 31P-NMR spectroscopy, was normal. The energetic state remained normal during a catecholamine-induced increase in cardiac work, despite a reduced blood flow response. Furthermore, the equilibrium between energy demands and blood flow was maintained in the presence of ATP-dependent potassium channel (KATP) blockade with glibenclamide. This is of importance because KATP channels are endowed with metabolic sensing capacity and may contribute to downregulation of energy requirements when ATP availability is decreased by causing shortening of the action potential and, therefore, the calcium transient and tension development. The present data support the concept that the decrease in blood flow in hibernating myocardial tissue is decreased in proportion to a reduction in energetic demands (5) and by a mechanism that is independent of ATP-dependent potassium channel opening during an increased work state.
ATP-Dependent Potassium Channels and Hibernation
During baseline conditions, an infusion of glibenclamide at a dose that did not alter either workload or regional perfusion tended to decrease the transmural PCr/ATP. During the increased levels of work with dobutamine, the reductions in PCr/ATP in the hibernating and sham myocardium were equivalent at a time that opening of ATP-dependent potassium channels was inhibited with glibenclamide. These data suggest that preservation of energy in chronic hibernation during a supply-demand mismatch is not dependent on ATP-dependent potassium channel opening. However, the reductions in blood flow at baseline were mild, and the lack of effect with glibenclamide could conceivably have been a result of an insufficient stimulus to maintain KATP channels in the open state. In a swine model of short-term hibernation, with a more moderate reduction in regional blood flow, Shulz et al. (17) have shown that glibenclamide at a similar dose does not inhibit the return of creatine phosphate at a time of sustained reductions in blood flow and function. Those observations are consistent with the present findings and demonstrate that energy preservation in short-term and chronic hibernation is not dependent on opening of ATP-dependent channels during ischemia or during increased work. Opening of ATP-dependent potassium channels on the mitochondrial membrane has been shown to be protective in preconditioned myocardium by preserving the adenine nucleotide pool by a mechanism that involves increased mitochondrial matrix volume (2). Within the second window of protection after ischemic preconditioning, ATP-dependent channel opening on the sarcolemma triggers downstream signaling related to mechanisms involved with cardioprotection (14). Within chronic hibernating myocardium, transcriptional events related to expression of inducible nitric oxide synthase and activation of the mitogen-activated protein kinases are evident (9) and could have conceivably led to a modification of the effects of ATP-dependent potassium channels in a manner different from that during acute sustained reductions in blood flow. In the present study, PCr/ATP results during glibenclamide treatment were comparable in the hibernating and sham myocardium at high workloads, demonstrating that conservation of energy in hibernating tissue with reduced blood flow reserve is not dependent on opening of the ATP-dependent potassium channels.
Hibernation and Protection
Hibernating myocardium has been characterized clinically in patients with severe coronary artery disease and reduced regional blood flow at rest with functional improvement following coronary artery revascularization (15, 18). In the present study, a swine model of hibernation was used by instrumentation of the proximal LAD with a fixed device that does not result in abrupt occlusion of the vessel but rather evolves into a state of chronic supply-demand ischemia (5, 8, 11). Over a period of 10–12 wk, progressive reductions in myocardial blood flow are noted along with a pattern of protein expression associated with the contractile apparatus that differs from chronic myocardial stunning (1, 3). Glucose uptake is increased relative to oxygen consumption in the hibernating myocardial regions during fasted conditions yet evidence of anaerobic glycolysis at rest or during increased myocardial oxygen demand has not been observed (4, 9). The normal energetic state at high cardiac work in the present study demonstrates that adaptations that offset oxidant stress in response to chronic supply-demand mismatch do not necessarily decrease the efficiency of energy production, as defined by transmural PCr/ATP. In tissue extracted after dobutamine stress, transmural ATP levels were slightly but significantly lower in the hibernating vs. sham LAD regions, although total high-energy nucleotides and ADP were not dissimilar. Because the tissue was obtained after the protocol, we cannot exclude the possibility that more rapid hydrolysis of the ATP may have occurred in the hibernating LAD regions. Although under the basal conditions, a stable ATP concentration is maintained in hibernating myocardium, in response to catecholamine stimulation, more ATP loss may occur as a result of previously rebalanced myocardial oxygen delivery and demand under the basal cardiac work state that was disturbed by this stimulation. In the present study, we did not measure ATP in the remote regions after the protocol, which would have allowed us to determine whether the process of hydrolysis of high-energy phosphates is more rapid in tissues from ischemic cardiomyopathy areas after increased work states. We also did not measure regional function during the NMR study and therefore cannot be certain about the stability of regional functional measurements during acquisition of the spectra. Although we have observed that regional wall thickening can be recruited after a brief period of dobutamine treatment in this model, we recognize that temporal differences in the functional response to a catecholamine may differ between 5 and 25 min of persistent stimulation.
The mechanism of preserved energy during a condition of chronically reduced blood flow within hibernating myocardium remains unclear. Our study provides additional support to the notion that hibernating myocardium has undergone important adaptations that improve the relationship between myocardial oxygen consumption and expenditure at increased workload. We have recently shown that mitochondrial protection against anoxia-reoxygenation is observed in this swine model of chronic myocardial ischemia by a mechanism that may involve decreased oxidant damage, possibly by increased expression of uncoupling protein-2 (10). It is possible that intrinsic changes in the mitochondria that reduce oxidant damage within hibernating myocardium preserve energy at high work states. A major unsettled question in cardiovascular physiology however is what factors in the cascade of ATP production, transportation, and utilization control the maximum performance of a normal heart or contribute to the dysfunction of a failing heart. The relationship between steady-state myocardial concentrations of ATP, ADP, Pi, creatine, and PCr and the mitochondrial ATP production capacity remains unclear. This is because in vivo ATP synthetic capacity probably substantially exceeds the expenditure of ATP (as estimated by measurements of myocardial oxygen consumption and assuming a stable P/O) observed in the normal in vivo heart during both open-chest and intact conscious animal studies. Future studies should address the mechanistic basis of the adaptations that lead to the development and maintenance of an energetic steady state in hibernating myocardium. This steady state is characterized by the preservation of cellular integrity. It occurs because there are (“protective”) limitations of myocardial ATP expenditure rates that result from reductions of the capacity to expend ATP in contractile processes. Unfortunately, the latter characteristics of the hibernating state contribute to the progressive heart failure that occurs in patients with advanced coronary artery disease.
Transmural bioenergetics in hibernating myocardium, as determined by PCr/ATP derived by 31P-NMR spectroscopy, is normal at baseline and remains normal during the increased work associated with high-dose dobutamine exposure, despite reduced perfusion reserve and inhibition of ATP-dependent channel opening with glibenclamide. These data support the notion that an adaptive process within hibernating myocardial tissue allows the myocardium to operate over a lower range of the oxygen supply-demand relationship to protect against supply-demand ischemia by a mechanism other than ATP-dependent potassium channel opening.
This work was supported in part by a Department of Veterans Affairs Merit Review Grant (E. O. McFalls), National Heart, Lung, and Blood Institute Grants HL50470 and HL67828 (J. Zhang), and a Thoracic Surgery Foundation for Research and Education Nina Starr Braunwald Award (R. F. Kelly).
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- Copyright © 2007 by the American Physiological Society