Cardiovascular Research

Cardiovascular Research 1998 39(2):318-326; doi:10.1016/S0008-6363(98)00086-8
© 1998 by European Society of Cardiology

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Copyright © 1998, European Society of Cardiology

Inorganic phosphate content and free energy change of ATP hydrolysis in regional short-term hibernating myocardium

Claus Martin, Rainer Schulz, Jochen Rose and Gerd Heusch^*

Abteilung für Pathophysiologie, Zentrum für Innere Medizin des Universitätsklinikums, Essen, 45122 Essen, Federal Republic of Germany

^* Corresponding author. Tel.: +49 (201) 723 4480; Fax: +49 (201) 723 4481; E-mail: gerd.heusch@uni-essen.de

Received 14 November 1997; accepted 25 February 1998

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: Short-term myocardial hibernation is characterized by an adaptation of contractile function to the reduced blood flow, the recovery of creatine phosphate content and lactate balance back towards normal, whereas ATP content remains reduced at a constant level. We examined the hypothesis that, despite the absence of ATP recovery, the short-term hibernating myocardium regains an energetic balance. Methods: An enzymatic method was modified for the measurement of inorganic phosphate (P_i) in transmural myocardial drill biopsies (about 5 mg). In 12 anaesthetized swine, moderate ischemia was induced by reduction of coronary inflow into the cannulated left anterior descending coronary artery to decrease regional myocardial function (sonomicrometry) by 50%. Results: The development of short-term hibernation was verified by the recovery of creatine phosphate content, the persistence of inotropic reserve in response to dobutamine and the absence of necrosis (triphenyl tetrazolium chloride). At 5-min ischemia, P_i was increased from 3.6±0.3 (SD) to 8.1±1.1 µmol/g_{wet wt} (p<0.05). The free energy of ATP hydrolysis (G_ATP) was decreased from –57.8±0.8 to –52.2±1.4 kJ/mol (p<0.05). The relationships between function and P_i (r=–0.81) and G_ATP (r=–0.83), respectively, during control and at 5-min ischemia became invalid at 90-min ischemia, as myocardial blood flow and function remained reduced at a constant level, but P_i decreased back to 4.9±0.9 µmol/g (p<0.05 vs. control and 5-min ischemia), and G_ATP fully recovered back to –57.2±1.3 kJ/mol (p<0.05 vs. 5-min ischemia). Conclusions: In short-term hibernating myocardium, myocardial inorganic phosphate content recovers partially and the free energy change of ATP hydrolysis returns to control values. Contractile function remains reduced by mechanisms other than an energetic deficit.

KEYWORDS Short-term hibernation; Swine; Inorganic phosphate; Free energy of ATP hydrolysis

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Short-term myocardial hibernation is characterized by perfusion–contraction matching, lack of necrosis, and the recovery of some metabolic signs of ischemia despite ongoing hypoperfusion and persistent reduction in contractile function. Following an initial and transient decrease, the myocardial creatine phosphate content (CP) recovers back to preischemic values within 60- to 90-min ischemia, and lactate production is attenuated [1–5]. The content of ATP tends to decrease in transmural [2, 4]and is decreased more markedly in subendocardial biopsies [1, 6]; ATP content remains reduced at a constant level throughout ischemia. In contrast to the recovery of lactate balance and CP content, the persistent reduction in myocardial ATP appears inconsistent with the idea that a regulatory reduction in contractile function makes the short-term hibernating myocardium regain its energetic balance. However, it is not the ATP content but the free energy change of ATP hydrolysis that determines the energetic state of the myocardium. Recovery of the myocardial ATP-phosphorylation potential has been previously observed in a hibernation model subjecting the isolated working, saline-perfused guinea pig heart to global low-flow ischemia. In this model, the entire heart was freeze-clamped for the measurement of high-energy phosphates and calculation of the phosphorylation potential [7]. We have now established a method for biopsy-based sequential determinations of G_ATP from regionally ischemic myocardium in situ and applied this technique to an established pig model of short-term hibernation [2, 8, 9]. Since P_i [10–12]and G_ATP [13–15]are considered as potentially causal factors in the reduction of myocardial contractile work, we tested whether or not this also holds true in short-term hibernating myocardium in situ. Our results confirm the complete recovery of G_ATP at the end of a 90-min short-term hibernation protocol and support the idea of a restoration of the energetic balance in short-term myocardial hibernation [16, 17].

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The experimental protocols used in this study were approved by the local authorities of the district of Düsseldorf, and they conform with the Guide for the Care and Use of Laboratory Animals published by the US National Research Council (National Academy Press, Washington, DC, 1996).

2.1 Experimental model
The experimental model has been described in detail previously [2, 8, 9]. In brief, in 12 enflurane/nitrous oxide-anaesthetized Göttinger miniswine, a left-lateral thoracotomy was performed and a micromanometer (P7, Konigsberg Instr, Pasadena, Calif) was placed in the left ventricle through the apex. Ultrasonic dimension gauges were implanted in the left-ventricular myocardium to measure the thickness of the anterior and the posterior (control) walls (System 6, Triton Technologies, San Diego, Calif). The proximal left anterior descending coronary artery (LAD) was cannulated and perfused from an extracorporeal circuit at constant flow. LAD perfusion pressure was measured from the sidearm of the extracorporeal circuit. The large epicardial vein parallel to the LAD was dissected and cannulated to sample coronary-venous blood. Heart rate was controlled throughout the study by left-atrial pacing (type 215/T, Hugo Sachs Elektronik, Hugstetten, FRG).

2.2 Regional myocardial function
End-diastole was defined as the point when left-ventricular dP/dt started its rapid upstroke after crossing the zero-line. Regional end-systole was defined as the point of maximal wall thickness within 20 ms before peak negative left-ventricular dP/dt [18]. Systolic wall thickening was calculated as end-systolic wall thickness minus end-diastolic wall thickness divided by the end-diastolic wall thickness. In addition, a regional myocardial work index was determined as the sum of the instantaneous left-ventricular pressure–wall thickness product over the time of the cardiac cycle, using the equation:

[ed=end-diastole, n=actual cardiac cycle, m=sampling point within cardiac cycle n at a sampling interval of 5 ms, LVP_n,m=instantaneous left-ventricular pressure within cardiac cycle n and at sampling point m, LVP_min=minimum left-ventricular pressure, WTh=wall thickness]. The maximal work index value during systole is reported as WI [9].

2.3 Regional myocardial blood flow and metabolism
Radiolabelled microspheres (NEN, Du Pont, Boston, Mass) were injected into the coronary perfusion circuit to determine the regional myocardial blood flow and its distribution throughout the LAD perfusion bed [19]. Oxygen content was measured using anaerobically sampled blood drawn simultaneously from the cannulated coronary vein and an artery, and oxygen consumption of the anterior myocardial wall (MO₂) was calculated by multiplying the arterial–coronary venous oxygen difference by the transmural blood flow at the crystal site. Lactate was measured in simultaneously drawn coronary-venous and arterial blood samples [20], and lactate consumption was calculated by multiplying the arterial–coronary venous difference by transmural blood flow at the crystal site.

2.4 Inorganic phosphate, high energy phosphates and G_ATP
Transmural drill biopsies (about 5 mg weight) were taken from the LAD perfusion bed. A 1.5-mm-diameter bit was used in conjunction with a modified dental drill. Negative pressure within the drill bit facilitated the taking of the sample, whereas reversal to positive pressure was used to rapidly expel the sample into a stainless-steel mortar cooled with liquid nitrogen. The sample was then immediately ‘freeze clamped’ using a steel pestle also cooled with liquid nitrogen. The time from tissue sampling to clamping was 1–2 s; samples requiring longer time for acquisition were not used for this analysis. Holes in the myocardium resulting from the biopsies were closed using a shallow purse-string suture [2]. The tissue samples were stored in liquid nitrogen until the subsequent analysis.

2.4.1 Chemicals
Inosine, phosphate-free nucleoside phosphorylase (NP), phosphate-free xanthine oxidase (XO), creatine kinase, pyruvate kinase, lactate dehydrogenase and glutamate pyruvate transaminase were obtained from Boehringer Mannheim (Mannheim, FRG). 2,6-Dichloroindophenol (DCIP) was from Sigma Chemie (Deisenhofen, FRG). All other chemicals were of analytical grade.

2.4.2 Analyses
The powdered tissue samples (Mikro Dismembrator, B. Braun Melsungen, Melsungen, FRG) were extracted with 0.3 mol/l perchloric acid and neutralized with 0.9 mol/l KOH, 170 mmol/l K₂SO₄, 60 mmol/l Tris. Aliquots were used for the measurements of P_i, high-energy phosphates and creatine (Cr).

For the measurement of P_i, almost all conventional colorimetric and enzymatic methods have inherent disadvantages, such as low sensitivity and interference from other compounds, decomposition of labile organic phosphates or contamination of chemicals and enzyme preparations with P_i [21–25]. We modified an enzymatic assay based on nucleoside phosphorylase–xanthine oxidase-coupled reactions [26, 27]. P_i is transferred to inosine with phosphorolytic cleavage. The formed hypoxanthine is oxidized by XO, transferring hydrogen to DCIP which is reduced to its colourless dihydroderivative. Each molecule of P_i is equivalent to two molecules of reduced dye, thus extending the lower detection limit of the assay. DCIP (0.16 mmol/l) and inosine (5 mmol/l) were dissolved in 0.2 mol/l Tris/HCl buffer (pH 7.2). A volume of 820 µl of this solution was placed in a cuvette with a 10-mm optical pathlength tempered to 37°C. A volume of 10 µl each of NP and XO, respectively, dissolved in the above Tris/HCl buffer were added to a final concentration of 0.2 units/ml. Then, 60 µl of the sample were added and the absorbance at 606 nm was recorded, until the reaction endpoint was reached (8452 A Diode Array Spectrophotometer, Hewlett Packard GmbH, Waldbronn, FRG). External, identically treated P_i-standards were used for calibration. P_i samples ranging from 0.1 to 12 nmol had a linear response in the decrease of UV absorbance. The lower detection limit of about 10 µmol/l sample concentration is equivalent to a tissue content of about 0.6 µmol/g_{wet wt} P_i in a 5-mg tissue sample.

High-energy phosphates were measured using HPLC on an anion-exchanger column (Protein Pak DEAE 5 PW, Millipore-Waters GmbH, Eschborn, FRG). The elution buffer consisted of 3 mmol/l Tris/H₂SO₄ (pH 9.0) with a linear gradient from 6 to 290 mmol/l K₂SO₄ within the first 7 min of the run; the flow was 1.0 ml/min. UV absorption was monitored at 214 nm. The retention times were 8, 10, 14, 16 min for CP, AMP, ADP, ATP, respectively.

Creatine was determined by the enzymatic method of Bernt et al. [28].

G_ATP was calculated with the use of the mass equilibrium constant of the creatine kinase reaction using the equation:

[Cr], [P_i], [CP] refer to the cytosolic free concentrations, G_ATP^o' is the standard free energy change of ATP hydrolysis (–30.5 kJ/mol) [29], R the universal gas constant, T the absolute temperature and K_CK the equilibrium constant of the creatine kinase reaction (1.66·10⁹) [30]. The cytosolic free concentrations were calculated from the tissue contents according to Giesen and Kammermeier [31]. The cytosolic pH of 7.2 during normoperfusion, of 6.9 at 5 min and of 7.0 at 90 min of hypoperfusion was derived from a NMR study in open-chest pigs with a comparable reduction in regional subendocardial blood flow [32](from 0.96±0.16 (SEM) to 0.30±0.06 in that study vs. from 0.79±0.26 (SD) to 0.29±0.12 ml/min/g in the present study).

2.5 Morphology
At the end of each study, following 120-min reperfusion, the absence of infarcted tissue was verified by the triphenyl tetrazolium chloride technique as previously described in our model [9].

2.6 Experimental protocol
Coronary inflow was decreased for 90 min to reduce the regional myocardial work index by approximately 50%; this degree of ischemia has previously been shown to be compatible with the development of short-term myocardial hibernation [2, 8, 33]. Sets of measurements were performed under control conditions, at 5- and 90-min ischemia, and in 4 animals additionally after 5 min of dobutamine infusion (2.5 µg/min) into the LAD perfusion circuit under control conditions and at 90-min ischemia. These measurements included the simultaneous withdrawal of pairs of arterial and coronary-venous blood samples. During the blood sampling, microspheres were injected into the LAD perfusion system for the measurement of regional myocardial blood flow, and systemic hemodynamic and regional dimension data were recorded. Immediately after the microspheres injection, myocardial biopsies were taken.

2.7 Data analysis and statistics
Hemodynamic parameters reported are left-ventricular end-diastolic and peak pressures, the maximum and minimum of the first derivative of the LV pressure (LV dP/dt_max, LV dP/dt_min), and mean coronary-arterial pressure. Regional wall function is reported as systolic wall thickening and the regional work index of the anterior wall, and—as a control reference—systolic wall thickening of the posterior wall. Metabolic parameters include calculated G_ATP, the myocardial contents of ATP, CP and P_i, and the consumptions of oxygen and lactate (positive values indicate myocardial uptake).

Changes in systemic hemodynamics, regional myocardial function, blood flow and metabolism over the time course of the experiment were evaluated using an analysis of variance for repeated measures, followed by Tukey's t-test for comparison of single mean values. Linear regression analyses between P_i, G_ATP and AWI were performed. All data are reported as mean values±standard deviation, and a p-value less than 0.05 was accepted as indicating a significant difference.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
In all animals, myocardial necrosis after 90-min ischemia and 2-h reperfusion was absent. Data of systemic hemodynamics and regional myocardial function are summarized in Table 1. Data of blood flow and metabolic parameters are summarized in Table 2.

View this table: [in this window] [in a new window]   Table 1 Systemic hemodynamics and regional myocardial function

 

View this table: [in this window] [in a new window]   Table 2 Regional myocardial blood flow and metabolism

 
3.1 Systemic hemodynamics
Heart rate was kept constant by left-atrial pacing throughout the protocol. Infusion of dobutamine increased LV dP/dt_max (p<0.05), while LV dP/dt_min tended to be decreased. With the reduction in coronary inflow, left-ventricular peak pressure and left-ventricular end-diastolic pressure were unchanged. LV dP/dt_max was decreased (p<0.05). There were no further changes in systemic hemodynamics when ischemia was prolonged to 90 min. Infusion of dobutamine at 90-min ischemia increased LV dP/dt_max and decreased LV dP/dt_min (both p<0.05 vs. 90-min ischemia).

3.2 Coronary-arterial pressure, regional myocardial blood flow, and regional myocardial function
During normoperfusion, intracoronary dobutamine increased regional blood flow, systolic wall thickening and the regional work index of the anterior wall (all p<0.05). With the reduction in coronary inflow, coronary-arterial pressure and regional myocardial blood flow were reduced (both p<0.05). At 5-min ischemia, systolic wall thickening and the regional work index of the anterior wall were significantly decreased to 22±8% and 164±56 mmHg·mm, respectively, (both p<0.05), whereas systolic wall thickening of the posterior wall remained unchanged. With prolongation of ischemia to 90 min, regional myocardial blood flow, systolic wall thickening and the work index did not change further. A close correlation existed between the regional work index of the anterior wall and transmural myocardial blood flow during control conditions and throughout ischemia (Fig. 1).

View larger version (24K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Relationship between transmural myocardial blood flow and anterior work index. The symbols represent data from 12 animals each under control conditions and after 5- and 90-minutes moderate ischemia. The regression line (y=373.6x+16.3; n=24; r=0.80) was obtained from data under control conditions and at 5-min ischemia.

 
3.3 Metabolism
With intracoronary dobutamine infusion, myocardial oxygen consumption tended to be increased, P_i content tended to be slightly increased and G_ATP tended to be slightly decreased (all nonsignificant vs. the respective control values). At 5-min ischemia, the myocardial CP content (p<0.05) as well as the myocardial oxygen consumption (p<0.05) were decreased, the myocardial ATP content tended to be decreased (p=0.08), and myocardial lactate consumption was reversed to net lactate production. Myocardial P_i content was increased from 3.6±0.3 to 8.1±1.1 µmol/g_{wet wt} (p<0.05). The P_i content correlated inversely to the regional work index during control conditions and at 5-min ischemia (Fig. 2). The free energy change of ATP hydrolysis (G_ATP), derived from these data, was decreased from –57.8±0.8 to –52.2±1.4 kJ/mol (p<0.05), and G_ATP also correlated to regional myocardial function (Fig. 3).

View larger version (24K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Relationship between myocardial inorganic phosphate content and anterior work index. The symbols represent data from 12 animals each under control conditions and after 5- and 90-min moderate ischemia. The regression line (y=–32.2x+427.1; n=24; r=–0.81) was obtained from data under control conditions and at 5-min ischemia.

 

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Relationship between free energy change of ATP hydrolysis and anterior work index. The symbols represent data from 12 animals each under control conditions and after 5- and 90-min moderate ischemia. The regression line (y=–28.5x–1336.6; n=24; r=–0.83) was obtained from data under control conditions and at 5-min ischemia.

 
With prolongation of ischemia to 90 min, regional myocardial blood flow remained unchanged. Net lactate production was no longer present (p<0.05 vs. 5-min ischemia). The myocardial ATP content remained unchanged at its slightly reduced level (p=0.06 vs. control), whereas the myocardial contents of P_i and CP recovered towards their preischemic values: the P_i content decreased back to 4.9±0.9 µmol/g_{wet wt} (p<0.05 vs. control and vs. 5-min ischemia) and the CP content increased back to 8.9±2.2 µmol/g_{wet wt} (ns vs. control, p<0.05 vs. 5-min ischemia). G_ATP recovered to –57.2±1.3 kJ/mol (ns vs. control, p<0.05 vs. 5-min ischemia). Most single data and the means of the data for P_i content vs. function and G_ATP vs. function were outside the 95% confidence intervals of the respective correlations at 90-min ischemia (Figs. 2 and 3).

At 90-min ischemia, intracoronary dobutamine infusion once more increased the P_i content and decreased G_ATP to an extent comparable to that during early ischemia (both nonsignificant vs. 5-min ischemia).

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
In short-term hibernating myocardium, myocardial inorganic phosphate is decreased back towards control values and the free energy change of ATP hydrolysis is restored, despite persistently reduced myocardial blood flow and contractile function.

4.1 Critique of methods
Short-term hibernation was verified by recovery of CP and lactate balance, increased contractile function during inotropic stimulation with dobutamine at the expense of metabolic recovery, and the absence of TTC-detectable necrosis. In TTC positive and thus viable myocardium, no signs of irreversible tissue damage could be demonstrated by light microscopy in a previous set of experiments [2].

G_ATP was calculated from simultaneous measurements of CP, creatine and P_i in single transmural biopsies taken from the LAD bed of anaesthetized swine during normo- and hypoperfusion. Thus, the calculated G_ATP reflects the regional transmural value of the free energy change of ATP hydrolysis. Regional transmural myocardial work was estimated as a work index by multiplying wall thickening by the instantaneous left-ventricular chamber pressure; this product was integrated throughout ventricular systole. Thus, unidimensional excursion against a load during systole was used as an estimate of regional myocardial work. It could obviously not be measured in the same region in which biopsies were taken. Therefore, the relation of G_ATP to the work index assumes comparable degrees of ischemia in both regions. Indeed, analysis of transmural blood flow in adjacent pieces of myocardium within and along different heart slices of the LAD-perfused territory revealed a coefficient of variation below 5%. The mean coefficient of variation in the entire series of experiments averaged 2.36±1.63% (control conditions: 2.90±1.46%, 5-min ischemia: 2.35±2.41%, 90-min ischemia: 2.17±1.02%, dobutamine stimulation: 1.85±1.17%) in a total of 272 pieces with an average weight of 1.41±0.63 g.

G_ATP was calculated from the presumed cytosolic free concentrations of P_i, CP and Cr. In biopsies such as in the present study, only the total cardiac content of these metabolites, but not their myocardial cytosolic free concentrations can be measured. This limitation appears to be less relevant for Cr and CP, since compartmentation and binding of these metabolites are negligible [34]. Their cytosolic concentrations were calculated in proportion to their cytosolic space distribution [31]. The myocardial tissue content of P_i was transferred into a cytosolic concentration by correction for bound P_i and compartmentation of P_i in the extracellular and mitochondrial space, according to Giesen and Kammermeier [31]. These authors also introduced an approximation of cytosolic P_i with reference to the pH gradient across the inner mitochondrial membrane, which is related to myocardial oxygen consumption and derived from an empirical equation [35]. This additional approximation, however, was unnecessary in our calculations for the recovery of G_ATP with prolonged hypoperfusion as myocardial oxygen consumption remained unchanged throughout the ischemic period. The cytosolic H⁺ concentration which also contributes to the calculation of G_ATP could not be measured in our experimental setting. It was therefore derived from a NMR spectroscopy study in open-chest pigs with a comparable reduction in regional myocardial flow [32]. The cytosolic free ADP concentration was calculated by means of the equilibrium constant of the creatine kinase reaction [30].

The inability to measure cytosolic free ADP concentrations is not unique to chemical determinations; its approximation via the CK equilibrium is usually performed in NMR spectroscopy based measurements, too. Both the chemical analysis as well as the NMR technique use further approximations, the one in calculating cytosolic free concentrations from measured tissue contents and assuming cytosolic H⁺ concentrations, the other in transforming peak areas into absolute concentrations of the respective compounds and estimating creatine concentrations. Thus, both methods are complementary.

The course of the intracellular pH values as derived from data in the literature [32]shows an early decrease followed by a slight increase at 90-min ischemia. A corresponding—though less-pronounced course of the coronary-venous pH values was measured in the present study (Table 2). Nevertheless, minor deviations of the assumed from the actual intracellular pH values are not critical for the major results of the present study: a deviation of 0.1 pH units results in a less than 1.5% (mean for all measurements 1.06±0.05%) error in the calculated G_ATP. From the differences in the total amount of creatine (Cr+CP) and phosphates (CP+ATP+P_i) (Table 2), potential errors of about 10% can be assumed for the measurements of the respective tissue contents. The resulting potential error in the calculation of G is, however, considerably lower than the potential errors in the measurements of single tissue contents, as the tissue contents in the calculation appear, after conversion into cytosolic free concentrations, on a logarithmic scale in the formula of G. The potential ‘worst case’-error in the calculated G_ATP (Cr and P_i: +10%; CP: –10%) is below 2% (mean for all measurements: 1.63±0.09%) and thus unlikely to affect the observed changes in G_ATP of about 10%.

The calculation of G_ATP by means of the equilibrium constant of the CK reaction is based on near equilibrium conditions for this reaction in the cytosol. In the course of myocardial hibernation, however, the ratio of CP to ATP content is altered dramatically. Nevertheless, there is good evidence that, despite this altered CP/ATP ratio, the CK reaction still runs at equilibrium: in an isolated rat-heart model of short-term hibernation, the ratio of the pseudo first order rate constants of the foreward and reverse CK reaction remained unchanged throughout control conditions and ischemia, indicating the maintenance of the CK equilibrium [36]. Furthermore, increased energy utilization with pacing [37]or inotropic stimulation [2, 9, 33]after 60 or 90 min of moderate ischemia results in rapid breakdown of CP, as also seen in the present study. Hence, the CP accumulated during short-term hibernation is still available for energy production. Its breakdown during inotropic stimulation at 90-min ischemia is comparable to that at 5-min ischemia (from 8.9±2.2 to 4.3±0.4 vs. from 9.1±0.8 to 3.7±1.4 µmol/g_{wet wt}) indicating an unchanged, high activity of cytosolic CK. Thus, the most plausible explanation for the reduction of ATP with normalized CP results from the equilibrium conditions, where the primary alteration is a loss of total adenine nucleotides, as reported previously [38–41].

Our biopsy technique sampled transmurally since tissue samples became highly distorted by the ‘freeze-clamping’ and could therefore not be further divided. Thus, our technique attempted to freeze tissue as rapidly as possible but sacrificed the possibility of dividing it transmurally. Since swine do not have an extensive collateral circulation, the gradient of subendocardial to subepicardial blood flow is smaller as compared to species with an extensive collateral circulation such as the dog. Thus, despite some transmural perfusion heterogeneity, the metabolic response to ischemia appears to be transmurally more uniform [2, 42], thereby minimizing the underestimation of the metabolic consequences of ischemia due to our examining only transmural samples.

One limitation of all animal experiments that have studied myocardial hibernation is the limited observation period [43]. Myocardial hibernation, as introduced by Rahimtoola [16], is a clinical situation of contractile dysfunction in patients with coronary-artery disease that exists for months and longer but is fully reversible on reperfusion.

4.2 Implications
The P_i content was increased by 125% at 5-min ischemia and was then decreased back towards preischemic values in regional short-term hibernating myocardium. Initially during ischemia, the P_i content correlated inversely to regional function, consistent with the idea that P_i is important in decreasing myocardial contractile force at the onset of ischemia [10–12]. An increase in P_i has been suggested to depress maximal Ca²⁺-activated force in early ischemia [44, 45]. In a previous study from our laboratory [9], short-term hibernating myocardium has been characterized by a decrease in calcium responsiveness without significant desensitization to calcium. However, with the development of short-term hibernating, calcium responsiveness and contractile function, on the one hand, and P_i content, on the other hand, obviously diverge: while calcium responsiveness and contractile function remain reduced at a constant level, the P_i content recovers towards preischemic values, yet remains increased by 36% above its control value (p<0.05). It remains unclear whether or not the remaining increase in P_i content is sufficient to maintain the reduction in contractile function at 90-min ischemia. If so, the initial early ischemic increase in P_i would be overshooting for the observed decrease in contractile function, an idea not consistent with the close relation between P_i and contractile function during early ischemia reported by He et al. [12]and observed in the present study. Alternatively, an initial short bout of substantially increased P_i content could induce more long-lasting effects on the contractile machinery to maintain the reduction of contractile function. Most probably, however, increased P_i content per se is not responsible for the reduced contractile function in hibernating myocardium.

In the concept of myocardial hibernation, the observed recovery of CP and lactate is assumed to indicate an active downregulation of energy demand somewhat below energy supply, thus restoring the energetic balance [1, 37, 46, 47]. However, the recovery of single metabolic parameters can certainly suggest but not prove the restoration of energetic balance. In contrast, G_ATP integrates all single metabolites relevant for the energy available from ATP hydrolysis to various cellular processes, including contractile function.

A close relation between global myocardial function and global G_ATP has been reported from studies in isolated hearts with graded hypoxia or graded hypoperfusion [14, 48, 49]. Recovery of the myocardial phosphorylation potential during ongoing moderate ischemia has been reported by Gao et al. [7]: isolated working, saline-perfused guinea pig hearts were subjected to global low-flow ischemia, and the experiments were terminated by freeze stop either during control or at distinct times of ischemia. At 60-min ischemia, the myocardial phosphorylation potential had completely recovered to preischemic values. In the present study, we have confirmed the recovery of G_ATP during ongoing moderate ischemia and extended the finding of Gao et al. [7]to an in situ model of regional short-term hibernation in pigs with sequential, biopsy-based measurements of high energy phosphates. In this model, neither the initial decrease in G_ATP nor its recovery with the development of short-term hibernation were accompanied by equivalent changes in the myocardial ATP content: the ATP content decreased only slightly and remained at this reduced level during short-term hibernation, consistent with previous studies from our laboratory [2, 8, 9, 33]. The initial slight decrease in myocardial ATP content points to an initial imbalance in ATP demand and supply. However, myocardial mitochondria possess a tremendous reserve capacity for the production of ATP and the maintenance of various energetic steady states depending on demand [50, 51]. When ATP production is reduced by a slow and progressive reduction of blood flow, ATP synthesis and consumption still remain tightly linked: no sustained fall in the CP/ATP ratio and no or minimal lactate production are observed, suggesting an actively regulated reduction of ATP consumption in response to the gradual reduction of ATP production [52]. The results of the present study support the idea of an active downregulation of energy demand since G_ATP recovered while oxidative phosphorylation, as indicated by MO₂, remained stable and anaerobic glycolysis, as indicated by lactate production, was attenuated during ongoing ischemia. There is no evidence for an impaired flux of energy to the contractile machinery through the CK reaction [53, 54]in the present study. On the contrary, the recovery of CP together with its availability for energy production almost exclude transport problems or changes in the overall CK reaction. The recovery of CP demonstrates the availability of ATP for the phosphorylation of creatine although the myocardial ATP content remains unchanged at its reduced level. Loss of adenine nucleotides via the 5' nucleotidase and myokinase reactions is responsible for the persistent reduction of ATP content; moreover, any decrease in cytosolic free ADP content shifts the equilibrium of the CK reaction towards CP formation and increases G_ATP despite an unchanged, reduced ATP content [41].

The present study demonstrates complete restoration of the energetic balance in regionally short-term hibernating myocardium in situ, supporting the regulatory nature of decreased contractile function as a hallmark of the concept of myocardial hibernation.

Time for primary review 25 days

	Acknowledgements

 
This research was supported by the Hans und Gertie Fischer-Stiftung. We thank Petra Gres and Ursula Prägler for their technical support.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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