Improved Protection of the Hypertrophied Left Ventricle by Histidine-Containing Cardioplegia
Background Myocardial hypertrophy has been shown to lead to increased susceptibility to ischemia with accelerated loss of high-energy nucleotides, greater accumulation of H+ and lactate, and earlier onset of contracture.
Methods and Results To determine whether promoting anaerobic glycolysis during ischemia by buffering H+ results in improved preservation of the hypertrophied heart, we studied the effect of a histidine-containing solution (HBS) on recovery of contractile function and energetic state. Hypertrophied rabbit hearts (aortic banding at 10 days) were subjected to 40 minutes of 37°C ischemia and reperfusion in an isolated Langendorff model. This group was compared with groups receiving St Thomas solution and high-potassium Krebs buffer solution (KCl). Although both phosphocreatine (PCr) and ATP were lower in hypertrophied hearts by end-ischemia compared with nonhypertrophied age-matched controls, there was significantly higher PCr, ATP, and intracellular pH in the HBS group compared with the St Thomas and KCl groups. Recovery of left ventricular developed pressure was best in the HBS group (91% of preischemic values) as was end-diastolic pressure after 30 minutes of reperfusion. Lactate production was also significantly greater in the HBS group, suggesting augmentation of anaerobic glycolysis.
Conclusions We concluded that administration of histidine-containing cardioplegia promotes anaerobic glycolysis and improves recovery of high-energy phosphates and contractile function in hypertrophied myocardium.
Myocardial hypertrophy is an established risk factor in congenital cardiac surgery.1 During experimental ischemia, hypertrophied hearts exhibit accelerated loss of high-energy nucleotides, greater accumulation of tissue lactate, and earlier onset of contracture.2 Physiological alterations known to occur in pressure overload hypertrophy include increased dependence on glycolysis for energy production, particularly in the endocardium, and altered calcium regulation for excitation-contraction coupling.3 4 However, the mechanism responsible for the decreased tolerance to ischemia in hypertrophied hearts is not well understood. One explanation may be that a higher rate of glycolytic flux during ischemia in hypertrophied myocardium leads to more rapid accumulation of glycolytic end products, including H+ and lactate. The accelerated fall in cyto- solic pH (pHi) and/or lactate accumulation may lead to earlier inhibition of glycolysis and ATP production during ischemia and greater intracellular accumulation of sodium and calcium via the Na+/H+ and Na+/Ca2+ exchangers. Although some controversy exists as to the source of the protons during ischemia, intracellular H+ buffering can be achieved by various agents with a resultant delay in the loss of ATP content during ischemia. We have found that a cardioplegia solution containing histidine to buffer pH exerts a profound protective effect on the normal adult and neonatal heart. The improved protection during ischemia is associated with a sustained intracellular alkalosis and increased glycolytic flux due to disinhibition of glycolytic enzymes.5 6 Because hypertrophied myocardium has been shown to be more glycolysis dependent, particularly in the endocardium, we hypothesized that H+ buffering may provide improved protection during ischemia. The purpose of this study was to determine the efficacy of a histidine-buffered cardioplegia solution formulated to promote glycolysis as compared with Krebs buffer with high potassium and a nonbuffered St Thomas solution in hypertrophied rabbit hearts subjected to a moderate to severe ischemic insult.
Hypertrophied Heart Model
Neonatal New Zealand White rabbits (7 to 10 days old) were used to create the model of left ventricular hypertrophy. After anesthesia was induced with ketamine hydrochloride (15 mg/kg IM), a left thoracotomy was performed under spontaneous respiration. A 2-0 silk suture was placed around the descending aorta just distal to the ligamentum arteriosum. Care was taken to make the banding snug without causing stenosis of the descending aorta. After the chest was closed, air was evacuated from the left thoracic cavity by aspiration with a catheter connected to a syringe, and the animals were allowed to recover. They were returned to their mother and allowed to grow in a normal manner. Thus, as the animal grew, an aortic coarctation gradually developed.
Isolated Perfused Heart Model
After 6 to 8 weeks, the animals were euthanitized with an overdose of ketamine (150 mg/kg IV) and the hearts removed and perfused by use of Langendorff’s method with Krebs buffer. The circumference of the banded portion of the descending aorta was measured and compared with an adjacent proximal nonbanded area. In each case, the banded area of aorta was confirmed to be stenotic (<50% of proximal aorta). At the end of the experiments, the atria and right ventricle were removed and left ventricular and body weight were determined. The ratio of left ventricular weight to body weight was compared with that of hearts from age-matched (6 to 8 weeks old) control (ie, nonhypertrophied) rabbits (see Table 1⇓).
To determine the effect of ischemia on cardiac function and high-energy phosphates, hearts were perfused by use of Langendorff’s method, and left ventricular function, high-energy phosphate, and glycolytic metabolites were measured. Details of this preparation have been reported previously.5 Briefly, anesthesia was induced with ketamine hydrochloride (150 mg/kg IV) and systemic anticoagulation was attained with heparin (500 U/kg IV). The hearts were rapidly excised and immediately placed in a 4°C perfusate bath, and the aorta was cannulated for coronary artery perfusion with a polyethylene cannula. Hearts were perfused at 80 mm Hg pressure by Krebs-Ringer solution bubbled with a gas mixture of 95% O2 and 5% CO2 at 37°C. All hearts were perfused for at least 30 minutes before ischemia to allow for stabilization. The hypertrophied hearts were then divided into three groups (Table 2⇓). Group 1 received Krebs buffer to which KCl was added to raise K+ to 20 mmol/L (KCl group). Group 2 received St Thomas solution (St Thomas group) and group 3 received a high-potassium, low-sodium formulation containing histidine to buffer H+ (HBS group). Thirty milliliters of cardioplegia was administered over 2 minutes at the onset of ischemia. The hearts were then subjected to 40 minutes of ischemia at 37°C followed by 30 minutes of reperfusion. These three groups were compared with an age-matched (6 to 8 weeks old) nonhypertrophied control group of hearts receiving Krebs buffer plus KCl (Krebs+KCl).
Cardiac Function Measurements
Cardiac function was assessed by an intracavitary balloon inserted into the left ventricle and connected to a catheter-tipped micromanometer (Millar Instruments Co). The balloon was filled with saline solution from a calibrated syringe to permit incremental volume changes with simultaneous pressure measurements. Peak developed pressure was measured at a preischemic diastolic pressure of 5 to 7 mm Hg and the same balloon volume was used to measure peak developed pressure after 30 minutes of reperfusion. Measurements of function were made at the end of the 30-minute equilibration period and after 30 minutes of reperfusion.
31P NMR Spectroscopy
In a separate set of experiments, age-matched control hearts (n=5) and three groups of hypertrophied hearts (n=5 per group) were studied with 31P NMR spectroscopy. High-energy phosphates were measured in perfused hearts in a 40-cm horizontal-bore 4.7 T Bruker BioSpec NMR spectrometer (Bruker Instrument, Inc) operating at a 31P frequency of 81 MHz. The perfusion chamber was surrounded by a 3.1-cm diameter, five-turn solenoid coil tuned to 81 MHz for 31P spectroscopy. Ninety-degree radiofrequency pulses were applied with a recycle time of 1 second during a period of 2 minutes and 40 seconds, for a total of 148 acquisitions per spectrum. A bulb containing dimethylene phosphonic acid (DMPA) as an internal 31P standard was placed within the right ventricle, and the right atrium was sutured closed. The areas under the beta-ATP, phosphocreatine (PCr), and DMPA peaks were determined by integration after baseline correction with the polynomial curve-fitting routine supplied by Bruker Instruments, Inc. All peaks were normalized to the DMPA peaks. Intracellular pH was determined by the following equation:
where a is the chemical shift difference in parts per million between Pi and PCr. Concentration measurements of each of the phosphorus-containing metabolites were obtained by integration of the areas under the individual peaks. The experimental protocol was the same as described above. Developed pressure was monitored continuously during these experiments.
To measure total lactate released from the heart and tissue lactate content from endocardium and epicardium, another 12 hypertrophied hearts were used and divided into three groups. Lactate released into the coronary effluent from the 30 mL flush of cardioplegia solution and the initial 1 minute of reperfusion plus total tissue lactate at end-ischemia were determined by enzymatic assay (Lactate-glucose analyzer, 2300 stat, YSI) and expressed as micromoles of total lactate produced per gram wet weight of heart tissue.
All animals received humane care in compliance with the principles of laboratory animal care formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources and published by the NIH (NIH publication no. 86-23, revised 1985). This protocol was reviewed and approved by the animal care committee at Children’s Hospital of Pittsburgh.
For comparison of multiple data points between two groups, ANOVA for repeated measures was used with the Bonferroni correction. All data are expressed as mean±SEM; a value of P<.05 was considered statistically significant.
In hypertrophied hearts, there was significantly higher developed pressure preischemia than in the age-matched control hearts, reflecting the degree of left ventricular hypertrophy. After 40 minutes of 37°C ischemia and 30 minutes of reperfusion, developed pressure decreased from 71±9 to 58±14 mm Hg (82% of preischemic value) in the nonhypertrophied control hearts receiving Krebs+KCl cardioplegia solution. In hypertrophied hearts, developed pressure decreased from 94±9 to 41±12 mm Hg (44% of preischemic value) in the KCl group, from 81±12 to 49±9 mm Hg (60% of preischemic value) in the St Thomas group, and from 89±12 to 81±12 mm Hg (91% of preischemic value) in the HBS group (see Fig 1⇓).
Similarly, end-diastolic pressure (EDP) measured at the same balloon volume used before ischemia was significantly elevated after reperfusion in the St Thomas group (from 6.0±1 mm Hg at preischemia to 19±6.8 mm Hg at the end of reperfusion) and in the KCl groups (from 6.0±0.2 to 47±15 mm Hg). In the group receiving the histidine-containing cardioplegia, however, the rise in EDP after reperfusion was significantly less (from 6.2±0.1 mm Hg preischemia to 9.5±2.0 mm Hg postischemia), similar to that seen in the age-matched control group (from 5.6±0.5 to 9.3±1.3 mm Hg) (see Fig 1⇑).
Total lactate released into coronary effluent during cardioplegia administration and during the first minute of reperfusion was significantly higher in the HBS group (109±13 μmol/g wet weight) compared with 37±7 and 77±1 μmol/g wet weight in the St Thomas and KCl groups, respectively (see Fig 2⇓). There were no significant differences in tissue lactate concentrations at end-ischemia between the groups, nor was there a difference between epicardium and endocardium with respect to tissue lactate levels. The greater total lactate production in the HBS group suggests an increase in glycolytic flux during ischemia.
There was rapid and significant decline in intracellular pH in the St Thomas (from 7.11±0.02 to 6.57±0.04 pH units), Krebs+KCl (from 7.12±0.04 to 6.25±0.09 pH units) and nonhypertrophied control groups (from 7.12±0.03 to 6.41±0.07 pH units) during ischemia (see Fig 3⇓). In the HBS group, however, intracellular pH was significantly higher (6.75±0.04 pH units) at the end of ischemia. There was rapid recovery of pHi by 5 minutes of reperfusion in all groups except the Krebs+KCl group, where pHi normalized more slowly.
At the end of the 40-minute ischemic period, PCr was best preserved in the nonhypertrophied control hearts (26±2% of preischemic values). Among the hypertrophied heart groups, however, PCr was significantly better preserved in the HBS group (19±2% of preischemic values compared with 9±2% and 2.5±2.5% of preischemic value in St Thomas and KCl groups, respectively) (Fig 4⇓). With reperfusion, there was complete recovery of PCr and nearly complete recovery of ATP in the HBS group, significantly better than the St Thomas and Krebs+KCl hearts (Fig 5⇓). In the KCl group, PCr never returned to preischemic levels by the end of 30 minutes of reperfusion (45±21% of preischemic values) and ATP levels did not recover either, indicating irreversible injury in this group of hearts.
This study confirms previous work by other investigators that pressure overload hypertrophy leads to increased susceptibility to ischemia.2 We have demonstrated that hypertrophy leads to a more rapid loss of high-energy phosphates during ischemia, earlier onset of irreversible injury, and impaired postischemic cardiac function (control versus hypertrophy KCl groups). Furthermore, by delaying the decline in intracellular pH during ischemia by buffering protons with histidine, we were able to retard the fall of high-energy phosphates and improve recovery of contractile function. The improved preservation of high-energy phosphates was associated with greater total lactate production during ischemia, indicative of increased glycolytic flux.
Cardiac growth secondary to hemodynamic overload has been extensively studied in several animal species including humans.3 Some of the more important changes that relate to tolerance to ischemia include: altered Ca regulation for excitation contraction coupling, with greater dependence on transsarcolemmal calcium flux3 7 ; a shift in substrate utilization, with increased dependence on glycolysis4 ; an increased number of mitochondria; and a switch to fetal isoforms of myosin with slower myosin ATPase activity. The change in substrate preference to glucose has been demonstrated by increased incorporation of 14C-glucose into glycogen stores and increased rate of uptake of 14C-deoxyglucose.4 There is also a decrease in fatty acid utilization, with lower myocardial carnitine and coenzyme A content seen in pressure overload hearts.4 Both of these changes would lead to accelerated glycolysis in pressure overload hearts, which may explain the accelerated lactate accumulation and decreased intracellular pH seen during ischemia. In previous studies, we have demonstrated that intracellular H+ buffering can be achieved by various buffers, with a resultant delay in the loss of ATP content during ischemia. In particular, histidine buffering in cardioplegia exerts a profound protective effect on the heart in association with a sustained intracellular alkalosis.5 6 Histidine is a potent proton buffer at physiological pH (pKa=6.8 at 25°C) and is the amino acid most responsible for the intrinsic intracellular buffering capacity. Its ability to enter the myocardial cell and maintain a high buffering capacity makes it an ideal agent to maintain intracellular pH at physiological range during ischemia.8 9 Administration of a high concentration of histidine through the coronary vessels also may be beneficial, because of its ability to facilitate proton removal from the cytosol by anionic carriers such as lactate. Thus, histidine facilitates removal of both H+ and lactate from the cytosol, which in turn prevents inhibition of glycolysis by the accumulation of these end products. Other beneficial effects of histidine may be related to its ability to bind calcium, which has been shown to increase in the cytosol during ischemia; there is also evidence that histidine can scavenge singlet oxygen, which may be produced during ischemia/reperfusion.10 Because we did not directly measure either of these properties of histidine in the present study, we can only presume that at least some of the beneficial effects of histidine in this model were due to these effects.
In the oxygenated perfused heart, glycolysis is not thought to be a significant source of high-energy compounds because the tri-carboxyl acid cycle in mitochondria is so much more efficient in ATP production.11 Indeed, the preferred substrate in the blood-perfused heart is fatty acids, with glucose accounting for <20% of oxygen consumption in the working heart.12 Nevertheless, there is a growing body of evidence that suggests that glycolysis, particularly during early reperfusion, may have an important role in supplying ATP for the membrane ion pumps and thus may serve an important role in maintaining intracellular ionic homeostasis.13 During ischemia, anaerobic glycolysis is the only potential source of ATP, since oxidative phosphorylation is rapidly inhibited by lack of oxygen and accumulation of NADH in the mitochondria. In ischemic hearts, inhibition of glycolytic flux has been temporally associated with the onset of contracture, and chemical inhibition of glycolysis has been associated with an earlier onset of contracture.14 Glycolysis may also play a crucial role in generating ATP for sarcolemmal ion pump function, including sodium and calcium homeostasis during early reperfusion.15 When glycolysis is inhibited during early reperfusion, the myocardium may be unable to compensate for increased Ca2+ entry resulting from Na+/Ca2+ exchange. This may lead to a larger increase in cytosolic Ca2+ and mitochondrial Ca2+ overload with uncoupling of oxidative phosphorylation.16 Because accumulation of protons and lactate during ischemia may delay flux through glycolytic enzymes during early reperfusion, buffering protons during ischemia may also contribute to the faster recovery of high-energy phosphates seen in the present experiments.
Some of the limitations of this study are that we did not directly test the effects of histidine on recovery of contractile function or lactate production in this model. However, we have shown that in the nonhypertrophied myocardium, addition of histidine to the cardioplegia significantly augments anaerobic glycolysis and ATP preservation during ischemia.5 6 Also, we chose to use the crystalloid-perfused rabbit heart preparation to permit measurements of high-energy phosphates by 31P NMR spectroscopy. Although an ejecting blood–perfused model in which left atrial filling pressure is varied is a closer model of the in vivo heart, the isovolumic heart preparation used in these experiments is stable for the duration of the study period and does provide a direct measurement of changes in systolic function and diastolic compliance.
The present study confirms the observation that pressure overload hypertrophy leads to increased susceptibility to ischemia and impaired postischemic cardiac function. A high concentration of exogenous histidine is able to buffer protons and remove lactate to promote anaerobic glycolysis for ATP production during ischemia in association with improved recovery of contractile function.
This work was supported in part by American Heart Association Grant no. 94002280, NIH grant HL-46207, and NIH center grant 1P41-RR03631. We greatly appreciate MaryAnn Butowicz for her technical assistance and Glenda Johnson for her assistance with manuscript preparation.
Presented in part at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1995, and published in abstract form (Circulation. 1994;90[pt 2]:I-422).
- Copyright © 1995 by American Heart Association
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