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Am J Physiol Heart Circ Physiol 281: H2645-H2653, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 6, H2645-H2653, December 2001

Postischemic mechanoenergetic inefficiency is related to contractile dysfunction and not altered metabolism

Christian Korvald1, Odd Petter Elvenes1, Ebrahim Aghajani1, Eivind S. P. Myhre2, and Truls Myrmel1

1 Department of Thoracic and Cardiovascular Surgery, University Hospital Tromsø, N-9038 Tromsø; and 2 Department of Medical Physiology, Institute of Medical Biology, University in Tromsø, N-9038 Tromsø, Norway


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mechanoenergetic inefficiency in postischemic nonnecrotic myocardium may partly be explained by an increased fatty acid (FA) oxidation rate. In the present study, left ventricular (LV) postischemic energy transfer was characterized in 10 intact anesthetized pigs. The LV was stunned by 11 brief left main coronary artery occlusions/reperfusions (20-min accumulated ischemia). Seven pigs served as time controls. The relationship between myocardial oxygen consumption (MVO2) and LV pressure-volume area (PVA) was assessed. [14C]glucose and [3H]oleate markers were used to discriminate between glucose and FA consumption. In stunned hearts, severe postischemic dysfunction was observed, and contractile efficiency was reduced (increased MVO2-PVA slope, P = 0.001). Unloaded (nonmechanical) MVO2 was not affected by ischemia. We observed only a small transient increase in FA preference and conclude that the contribution from increased FA utilization to postischemic mechanoenergetic inefficiency is insignificant. Disrupted postischemic chemical-to-mechanical energy transfer in vivo is, therefore, related to inefficient energy utilization in the contractile apparatus.

stunning; pig; ventricular function; energy metabolism; contractile efficiency


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN THE "STUNNED" MYOCARDIUM (4), there is a marked uncoupling between myocardial oxygen consumption (MVO2) and mechanical energy output (6, 9, 16, 19, 22, 26, 32, 33, 40). In theory, several possible mechanisms can explain this mechanoenergetic mismatch, including mitochondrial dysfunction (6), heterogeneous microcirculation (19, 33), dyssynchronized contraction (16, 33), altered excitation-contraction (EC) coupling (16, 26, 32), and/or an increased energy demand for contractile work (9, 32). However, in acute ischemia, an increase in arterial levels of fatty acids (FA) can occur (39), and a shift toward increased utilization of FA in stunned myocardium has been identified (6, 22, 23, 28). We have previously shown that an increase in circulating FA has a profound effect on the relationship between MVO2 and left ventricular (LV) total mechanical energy (pressure-volume area, PVA) (14). The relative increase in MVO2 with high levels of FA is due to a nonmechanical energy demand, i.e., increased unloaded MVO2 (the y-intercept of the MVO2-PVA relationship). Therefore, a FA preference in the stunned heart might explain part of the altered postischemic mechanoenergetic efficiency. This possibility has not yet been addressed in a model relating postischemic MVO2 to total mechanical energy (8, 20, 30).

The aim of the present study was, therefore, to assess whether a postischemic FA preference significantly contributes to mechanoenergetic inefficiency in the stunned myocardium. Postischemic LV dysfunction was induced in an intact pig model (14, 15) with concomitant analysis of global LV mechanical function and MVO2. The mechanoenergetic efficiency was analyzed using the MVO2-PVA relationship, as developed by Suga (35). This model enables a quantitative analysis of the relative contribution of mechanical and nonmechanical energy consuming processes to total LV energy consumption (MVO2). Myocardial oxidation rates of both glucose and FA were analyzed to assess any preference for FA or glucose in reperfused hearts, and their possible influence on efficiency.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. The experimental protocol was approved by the local steering committee of the Norwegian Experimental Animal Board and was conducted in compliance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Revised 1985). Twenty-three castrated male Norwegian landrace pigs (Sus scrofa domesticus) were used. Pigs were habituated to the animal facilities over 5-7 days and fasted overnight with free access to water before experiments. Intramuscular ketamine (20 mg/kg) and atropine (1 mg) were used as premedication. Anesthesia was induced with boluses of pentobarbital-Na and fentanyl, and maintained by continuous intravenous infusions of pentobarbital-Na, fentanyl, and midazolam, as earlier reported (14, 15).

Experimental setup. The experimental model employs an open chest preparation, described in detail earlier (14, 15), with the following modifications. Coronary flow was measured by transit-time probes (Medi-Stim AS, Oslo, Norway) on the main stems of the right coronary, the left anterior descending (LAD) and the circumflex (Cx) arteries. To create coronary flow perturbations, a rubber band was looped around the stem of the left main coronary artery (LCA). When LCA occlusion was performed, the band was tightened sufficiently to demonstrate zero flow in the LAD and Cx flow signals. The great cardiac vein was catheterized in a retrograde fashion; from the cranial caval vein via the coronary sinus, to ensure sampling of LV venous blood. The pulmonary artery was catheterized directly for measurement of mean pulmonary arterial pressure (MPAP) and injection of 4 ml 10% saline boluses (see below).

Central venous pressure was held constant at 4 ± 1 mmHg by infusion of 0.9% NaCl enriched with glucose (1.25 g/l). Heparin was administered intravenously twice, 2,500 international units during venous catheterization and 5,000 international units after allocation of the pig to an experimental group (see Experimental protocol). Urine was drained via a cystostomy. To assess myocardial oxidative metabolism, oleate and glucose isotopes were infused. This was performed as described earlier (14). [3H]oleate (NET-289; [9,10(n)-3H]oleic acid) and [14C]glucose (NEC-042X; D-[U-14C]glucose) (NEN, Research Products, Du Pont) were used.

LV pressure-volume relations were assessed using the combined conductance-pressure catheter technique (1, 34), and volumes were adjusted for parallel conductance (Vp, non-LV cavity derived conductance) assessed by the hypertonic (10%) saline technique at each time point (Fig. 1) (1, 15, 34). The gain correction factor (1) relating conductance-derived stroke volume (SV) to an independent measure of SV (transit-time ultrasonic flow probe) was close to 1.0 (range 0.9-1.1) throughout the present study, and was not applied in volume calculations. Beat-to-beat LV conductance and pressure signals were sampled, digitized, and stored at 250 Hz during 10- to 12-s runs (Leycom Sigma 5 conditioner and Conduct PC software, both from CD Leycom). Other hemodynamic parameters were continuously sampled, digitized and stored at 0.25 Hz (LabView 3.1.1, National Instruments; Austin, TX).


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  		Fig. 1.   The experimental protocol. Hatched area, ischemia/reperfusion protocol (see text). Thirty and 90 min indicate time after ischemia-reperfusion or sham when assessment of mechanical and metabolic variables were started. Text boxes: the periods of myocardial oxygen consumption (MVO2) and pressure-volume area (PVA) registrations lasted ~10 min. NIC, nonischemic time controls. n, no. of pigs.

Ischemia-reperfusion protocol. Stunning was induced by a series of LCA occlusions, affecting ~80% of the LV including the septum (see RESULTS). A large ischemic area was chosen to assess global LV mechanical and metabolic dysfunction. The two initial occlusions lasted 1 min to adapt the LV myocardium to later occlusions, and thus minimize ischemia-induced arrhythmias (38). Subsequently a sequence of nine 2-min LCA occlusions were performed, giving a total ischemic time of 20 min. Reperfusion times between occlusions were 60-120 s, steered by the time needed to reestablish preocclusion mean arterial pressure (MAP) levels. Multiple brief ischemic episodes is a clinically relevant problem (2), and myocardial stunning induced by this technique has earlier been validated to give reversible ischemic injury and cumulative severe stunning in pigs (36). Preconditioning is negated in this model after the third or fourth occlusion (3). However, the series of occlusions induce a "late" preconditioning against stunning for up to 48 h (36).

Experimental protocol. Figure 1 outlines the experimental protocol. All pigs were treated and prepared identically until completion of baseline registrations, which included assessment of two Vp's, two transient vena cava occlusions (VCO, caval balloon), blood sampling (14), and assessment of MVO2 and PVA at uninfluenced (or control) preload. Using the caval balloon catheter, we then assessed 3-4 additional sets of MVO2 and PVA points at different steps of steady-state reduced preloads, steered by MAP (lower level at 45-50 mmHg). Cardiac venous O2 saturation and coronary flow measurements were performed at each step. Pigs were then allocated to the two groups by drawing lots; a stunned group (receiving repetitive LCA occlusions) or a nonischemic time-control group (NIC) (Fig. 1). Pressure-volume relations were recorded during the ischemia-reperfusion protocol at the end of the 6th and 11th occlusions and corresponding reperfusions. Subsequent measurements were performed at identical time points in the two groups (Fig. 1) and were performed as described for baseline. At the end of each experiment, in vivo staining of the LCA perfusion area was performed (0.5% Evans blue), and the heart was simultaneously arrested (KCl). Energetic indexes and coronary blood flow were normalized to 100 g by the wet weights of the LV and stained myocardium.

Chemical analysis. Analyzation of blood hemoglobin, blood oxygen content, plasma-FA, -glucose, and -lactate, and also details of trapping technique and analysis of isotope degradation products (3H2O and 14CO2) have been described earlier (14). The specific activity of substrates were not different between groups at any time; glucose: 3.5 ± 1.2, 4.1 ± 1.5, and 4.6 ± 1.4 disintegrations per min (dpm)/nmol, and FA: 87 ± 55, 99 ± 50, and 133 ± 72 dpm/nmol (n = 17, baseline, 30 and 90 min after ischemia/sham, respectively). Release of myocardial troponin-T to the great cardiac vein was analyzed in serum using the ELISA technique with standards and controls from Boehringer-Mannheim (Germany).

Calculations. MVO2 was assessed as ml O2 · min-1 · 100 g LV-1 from LV blood flow (ml · min-1 · 100 g-1) and arterial to cardiac venous O2-content difference. When calculating LV mechanoenergetics, MVO2 was converted to joules (1 ml O2 = 20.2 J) and corrected for heart rate (HR). Stroke work (SW, mmHg · ml) was assessed from the area of the pressure volume loop, and the PVA was calculated as the sum of SW and end-systolic potential energy (mmHg · ml). Both SW and PVA were converted to J · beat-1 · 100 g-1 by the constant 1.33 × 10-4 J · mmHg-1 · ml-1 (35). The linear end-systolic pressure-volume relationship was assessed (ESPVR) using consecutive beats during VCO to determine the size of the potential energy. The 4-5 preload-varied, steady-state MVO2 and PVA points assessed at each sampling period were used to obtain a MVO2-PVA relationship by linear regression. In this relationship the slope defines MVO2 used for PVA generation (excess MVO2), and the y-axis intercept MVO2 for nonmechanical purposes (unloaded MVO2). Oxidation and uptake rates of FA and glucose are given as µmol · min-1 · 100 g-1. Lactate was assessed as uptake only. For calculation details and equations see our earlier works (14, 15).

LV contractile performance was assessed from the linear beat-to-beat SW to end-diastolic volume (Ved) relationship during VCO runs [SW = Mw(Ved - Vw) where Mw is slope and Vw is the x-intercept] (11). The Mw is widely used as an index for LV contractility, provided and unchanged Vw (10). To address uncertainties in predicting contractile performance by the Mw when there is a concomitant horizontal shift in the x-axis intercept of the relationship, a maximum recruitable SW (SWmax) was calculated. This was performed according to the above formula, but with the maximum Ved within each experiment as a constant instead of a time-varying Ved (10). The time-constant of isovolumic relaxation (tau ) was calculated according to Mirsky (25). The end-diastolic pressure-volume relationship (EDPVR) was assessed from consecutive beats during VCO, and fitted by an exponential equation Ped = alpha  · e(beta · Ved), where Ped is end-diastolic pressure. End-diastolic stiffness was evaluated by the slope coefficient (beta , dimensionless) of the exponential EDPVR.

Statistics. Data are presented as means ± SD, unless otherwise mentioned. For all variables, change from baseline was calculated at 30 and 90 min, and groups were then compared for differences in two-way repeated-measures analysis of variance design (GLM procedure, RANOVA). The P values reported are between group differences in change from baseline irrespective of time, unless otherwise mentioned. Pearsons correlation coefficient was used to investigate the association between changes in selected variables. Where appropriate, nonrepeated variables were compared by paired t-test, independent samples t-test, or Mann-Whitney U test. P values were adjusted for multiple comparisons where appropriate (Bonferroni). Computer software was used for statistical analysis (SPSS 10.0). Significance level was set to P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Of the 23 pigs used, 17 were entered for final analysis; 7 in the NIC group and 10 in the stunned group. Exclusions were based on the following: excessive surgical bleeding (one pig), pulmonary stenosis and sepsis of unknown origin (one pig), severe chronic aortic regurgitation (one pig), and ventricular fibrillation after the second or third LCA occlusion (three pigs). Pig body weights were 26.9 ± 2.1 and 26.2 ± 1.0 kg (stunned vs. control, P = 0.42), and LV weights were 3.18 ± 0.14 and 3.13 ± 0.34 g/kg pig body wt, respectively (septum included, P = 0.68). The LCA perfused 81.5 ± 2.0% vs. 81.1 ± 2.7% of the LV (stunned vs. control, P = 0.72). Gross postmortem examination of sliced hearts revealed no area of suspect myocardial infarct (discoloration or induration). Postischemic troponin-T levels (ng/ml) in the great cardiac vein gave no sign of developing myocardial necrosis, median (95% confidence); stunned group: 0.00 (0.00-0.12), 0.01 (0.00-0.12), and 0.00 (0.00-0.09); control group: 0.00 (0.00-0.12), 0.00 (0.00-0.01), and 0.00 (0.00-0.03) at baseline, 30, and 90 min after ischemia/sham, respectively (P > 0.2, between groups at time points, Mann-Whitney U-test). Parallel volume (Vp), determined by the hypertonic saline technique, was 53 ± 14 and 57 ± 15 ml at baseline (stunning and control, respectively), and increased by 12 ± 11 and 16 ± 12 ml in the stunned group versus 2 ± 3 and 2 ± 5 ml in the control group (30 and 90 min, respectively, P = 0.01).

Ischemia-reperfusion protocol. Coronary flow and MAP during LCA occlusions and reperfusions are presented in Fig. 2. The responses presented in Fig. 2 were similar for all 10 animals in the stunned group. As shown, MAP decreased progressively during occlusions, but were never below 30 mmHg. Summarized effects of LCA occlusions and reperfusions are presented in Fig. 3. The LV response to occlusion of the LCA was an increase in HR and Ped, acute dilation (increased Ved), and severely depressed external mechanical energy output (SW down to 13% of baseline). Both Ped and Ved were increased, not only during occlusions, but also during reperfusions (measured at the 6th and 11th reperfusions). SW was significantly decreased during the 11th reperfusion compared with both baseline and the 6th reperfusion.


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  		Fig. 2.   Effect of repeated left main coronary arterial (LCA) occlusions on LCA flow and mean arterial pressure (MAP) in a single experiment. Recorded at 0.25 Hz.



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  		Fig. 3.   Heart rate (HR, beats/min), end-diastolic volume (Ved, ml) and pressure (Ped, mmHg), and stroke work (SW, mmHg · ml) during the ischemia-reperfusion protocol, determined at the end of the 6th and 11th occlusions and reperfusions. The 11th reperfusion values were determined ~90-120 s after occlusive release. Bars are mean and error bars are SD. The 6th occlusion and reperfusion (n = 9 pigs), the 11th occlusion (n = 7 pigs), the 11th reperfusion (n = 8 pigs). Baseline values appear in Tables 1 and 2. Conductance offset volume (Vp) obtained at baseline was used when Ved was assessed. *P < 0.05 vs. baseline; dagger P < 0.01 vs. baseline; Dagger P < 0.05, 6th vs. 11th reperfusion (paired t-tests).

General hemodynamic data. Hemodynamic data from the reperfusion period are presented in Table 1. There was a markedly greater decrease (relative to baseline) in MAP, LV developed pressure, and stroke volume after ischemia compared with time controls. Although both HR (P < 0.001) and MPAP (P < 0.001) were increased with time in the stunned group, significant differences were not detected compared with the control group. Neither LV blood flow nor MVO2 were affected by time or intervention.

                              
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  		Table 1.   Hemodynamic variables

LV performance and energetics. End-systolic volume was 27 ± 3 in the stunned compared with 23 ± 3 ml in the control hearts at baseline, and did not change significantly between groups. Variables of LV systolic function are presented in Fig. 4. Unexpectedly, the Mw (slope of SW-Ved relation) was not different between the two groups. However, when controlling for a leftward shift of the SW-Ved relationship (increased Vw) by the calculation of SWmax, preload recruitable SW decreased to 72% of baseline at 30 min after ischemia in the stunned hearts. This may be illustrated also by Fig. 5, showing restricted pressure-volume loop areas at 30 and 90 min after ischemia (reduced SW). This together with a reduced maximum rate of systolic pressure generation (dP/dtmax) demonstrates impaired systolic function after repetitive ischemic insults.


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  		Fig. 4.   Left ventricular (LV) systolic function. Mw and Vw, slopes and y-intercept of the SW-Ved relationship; dP/dtmax, maximum first derivative of LV pressure per time unit; SWmax, see Calculations. P, probability for a between-groups difference (RANOVA). Bars are mean and error bars are SD. NIC, nonischemic time controls.



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  		Fig. 5.   Representative traces of control (unifluenced preload) LV pressure-volume relations before and after ischemia in one experiment within the stunned group. Recorded at 250 Hz. The area of one loop constitutes SW.

Parameters of LV diastolic function are presented in Fig. 6. The dP/dtmin became less negative value in the stunned compared with the control hearts. However, when isovolumic relaxation was expressed as the time constant tau  no between-group difference was observed (P = 0.12). In end diastole, there was an increase in both LV pressure (Ped) and stiffness (beta ), along with a reduced filling volume (Ved) in stunned hearts. Together, the observed changes in systolic function, Vw, Ved, and LV end-diastolic stiffness, indicates a restriction of the "preload-range" within which SW may be generated (Ved - Vw). This range was calculated to 46 ± 6 ml at baseline. After ischemia, it was reduced by 35%, compared with only 6% in the time-control group (P < 0.001).


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  		Fig. 6.   LV diastolic function. dP/dtmin, peak negative first derivative of LV pressure per time unit; Ved, end-diastolic volume; beta , end-diastolic stiffness (see Calculations); Ped, end-diastolic pressure; P, probability for a between-groups difference (RANOVA). NIC, nonischemic time controls. Bars are mean and error bars are SD.

The slopes and y-axis intercepts of the MVO2-PVA relationships are expressed in Table 2. These values were comparable between groups at baseline, and the regression lines had an excellent linearity (r2). In every animal within in the stunned group, a higher slope value of the MVO2-PVA relationship was found at both 30 and 90 min after ischemia. This gave a mean increase in slope of 54 and 50% at 30 and 90 min in the stunned group, respectively. Unloaded MVO2 was unchanged over time in both groups. According to Suga (35), the inverse of the MVO2-PVA slope is an expression of contractile efficiency. In the stunned hearts, LV contractile efficiency was 44.2 ± 9.5% at baseline versus 29.7 ± 7.2 and 29.8 ± 6.4% at 30 and 90 min after ischemia. Thus the decrease in mechanoenergetic efficiency after intervention in the stunned group was related to a relative increase in MVO2 for generation of PVA (excess MVO2).

                              
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  		Table 2.   MVO2-PVA relationships before and after ischemia

Substrate metabolism. At baseline the arterial levels of FA were 390 ± 155 versus 340 ± 115 µmol/l, and glucose levels were 5.7 ± 1.4 versus 5.7 ± 1.5 mmol/l (stunning vs. control). Neither changed significantly between groups over time. Arterial lactate was 1.0 ± 0.2 versus 1.3 ± 0.3 mmol/l at baseline, remained stable in the stunned group, but fell with time in the control group (P < 0.001). LV oxidation rates of FA and glucose, together with uptake rates of FA, glucose, and lactate are shown in Table 3. The net effect after 30 and 90 min of reperfusion was an increased uptake rate of glucose in the stunned group. Glucose oxidation rate was significantly changed over time in the stunned group only, with a reduced oxidation rate at 30 min compared with baseline (P = 0.045). Additionally, an interaction between time and groups was found for FA oxidation (P = 0.031). This may indicate some minor shifts in substrate metabolism after ischemia. For calculation of ATP production from glucose and FA oxidation rates, we applied the following stoichiometrics: a mean of 105 mol ATP per mole FA oxidized and 32 mol ATP per mole glucose oxidized (27). The total ATP production was 668 ± 238 and 530 ± 151 µmol · min-1 · 100 g-1 at baseline (stunned vs. control) and did not change significantly with time between groups. FA preference of the myocardium was calculated as the fraction of FA derived ATP per total ATP. This fraction was 0.58 ± 0.21 versus 0.63 ± 0.13 at baseline (stunned vs. controls), increased in the stunned hearts to 0.75 ± 16 at 30 min, but returned to baseline values at 90 min (P = 0.027 between groups). Correlation analysis between delta values on increased FA preference at 30 min and the corresponding decrease in contractile efficiency did not demonstrate any association (P > 0.2).

                              
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  		Table 3.   Oxidation and uptake rates of left ventricular substrates


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To our knowledge, the present work is the first to integrate detailed global LV mechanoenergetic function and oxidative metabolism after reversible ischemia in an intact (in vivo) preparation. In this model, repetitive episodes of brief ischemia induced a mechanoenergetic inefficient LV described by a steeper slope and an unchanged y-intercept of the MVO2-PVA relationship. Even though oxidation rate of glucose and calculated total ATP production were quite balanced after ischemia, a small preference toward an increased utilization of FA within 30 min of reperfusion was demonstrated. However, no association between postischemic mechanoenergetic inefficiency and FA preference was found, and the inefficiency persisted when oxidative metabolism was normalized 90 min after reperfusion. Thus a relatively increased postischemic MVO2 may not be explained by a shift in myocardial substrate preference, but rather by an inefficient coupling between chemical energy production and the contractile apparatus (contractile inefficiency).

Postischemic myocardial energy production. A metabolic hallmark of stunned myocardium is its ability to resume oxidative metabolism (40). In no-flow ischemic myocardium, metabolism is strictly anaerobic, and within 1 to 2 min of reperfusion, oxidative phosphorylation again becomes the major source of ATP production (40). An increased preference for utilization of FA intermediates, accumulated during ischemia, has been shown in reperfused myocardium (6, 22, 23, 28). In contrast to normal myocardium, the FA preference may continue despite high arterial lactate concentrations (40). An increased utilization of FA may lead to an increase in oxygen consumption and thus contribute to postischemic mechanoenergetic inefficiency. The increase in MVO2 is explained primarily by phosphate/oxygen (P/O) ratio differences between myocardial substrates, with a P/O ratio of 2.8 for pure FA oxidation in contrast to 3.2 for glucose (27). Also, during high intracellular levels of FAs an energy-consuming futile cycling of FAs (triglyceride cycle) has been proposed (6, 27).

A postischemic FA preference was first demonstrated in pigs by Liedtke et al. (22), showing an 89% increase in 14CO2 production from [14C]palmitate. However, glucose oxidation rate or mechanoenergetic relationships were not assessed simultaneously in this study. Later work has suggested a metabolic inefficiency in stunning, due to a decreased mitochondrial capacity and increased FA oxidation rate combined with reduced stroke volume (6). A metabolic defect has been suggested also in postischemic rat hearts, based on reductions in efficiency ratios (23). It should be noted, however, that both MVO2 and tricarboxylic acid cycle activity was little affected in the latter study, and that the findings were more likely due to impaired cardiac work. In addition, palmitate has been found to reduce mechanical function in reperfused rabbit hearts (13), and reduced transport rates of metabolites across the mitochondrial membranes have been demonstrated (21). Collectively, these studies advocate inefficiency at the level of intermediary metabolism. On the other hand, because creatine phosphate content is often increased (20), mitochondrial function may be minimally affected (29), response to inotropy is normal (12), and pharmacological restoration of myocardial ATP content do not influence recovery of contractile function (20), a crucial role of intermediary metabolism in postischemic inefficiency is controversial. Furthermore, in some of the previous studies (6, 23), judgment of the metabolic inefficiency ratios were difficult, because they did not separate total MVO2 into mechanical and nonmechanical MVO2. In the present study, however, a clear relative increase in PVA-related MVO2 was seen (contractile inefficiency). Combined with an unaffected nonmechanical MVO2, and a FA preference not related to any shifts in efficiency, an important metabolic defect in stunning was not supported.

Stunned myocardium demonstrates a net increase in the uptake of glucose (2, 7), primarily used for restoration of depleted glycogen stores (27). An increased glycolytic rate has also been observed, concomitantly with a reduced rate of glucose oxidation (23, 24). The present study is in line with these observations, most likely explained by a relative inhibition of pyruvate dehydrogenase due to a somewhat increased rate of beta -oxidation, and thus reduced glycolytic entry to the TCA cycle (27).

Postischemic mechanoenergetic "oxygen waste": altered Ca2+-transport or myofilament activation? Use of the MVO2-PVA frame for discussing LV mechanoenergetics, enabled us to demonstrate that the relatively increased MVO2 in the reperfused LV is related to generation of mechanical energy (contractile processes). Thus the level of "oxygen waste" in stunning is dependent on the size of the PVA. Although this establishes an inefficient use of ATP for mechanical energy generation, the MVO2-PVA model does not indicate the site of lesion in the transition process from ATP to mechanical energy.

An increased energy use for Ca2+ handling (EC coupling) may potentially explain mechanoenergetic inefficiency in reperfused myocardium (17, 18). This would imply an increase in unloaded MVO2 (35), but no studies have demonstrated this in stunned hearts (26, 31, 32). However, an unaltered, unloaded MVO2 could, in fact, imply a relative increase when the contractility is reduced (26, 31, 32, 35). In our study, the small increase in FA utilization might have contributed to uphold nonmechanical MVO2 in the early phase of reperfusion, but the impact of basal and EC coupling metabolisms on postischemic MVO2 seems minimal.

Alternatively, an increased demand, or inefficient use, of ATP by the myosin ATPase for myofilamental activation may also be causative (20). To our knowledge, no studies have actually investigated the function of the myosin ATPase in stunned myocardium. However, ample evidence suggests that the ischemic insult, and subsequent reperfusion, damage the myofilaments and reduce their Ca2+ sensitivity by the effects of free oxygen radicals and Ca2+ overload (3). Thus a link between a reduced Ca2+ sensitivity of myofilaments, and an inefficient use of ATP by the myosin ATPase, is a possibility. Our data demonstrate a marked and isolated reduction in contractile efficiency with preserved myocardial metabolism, making an inefficiency at the level of the myosin ATPase reasonable.

This explanation is also supported by earlier reports based on isolated rabbit (32) and in situ ovine hearts (9). However, results obtained in isolated heart models have limitations, because the interaction between organ and organism is bypassed. Also, the FA-free Krebs-Hensleit buffer with bovine erythrocyte suspension used by Schipke et al. (32), makes the heart solely dependent on glucose metabolism, creating a setup different from the balanced in vivo perfusion used in the present study. Furthermore, the observation by Furukawa et al. (9), using 30 min of global ischemia, were largely inconclusive, due to the use of cardiopulmonary bypass (CPB), the lack of any control group, and only a fair correlation between MVO2 and PVA (see also Methodological considerations).

Postischemic LV function. In the hearts subjected to ischemic insults, a reduced LV mechanical function was observed over the 90 min of reperfusion, with no concomitant deficit in myocardial perfusion. This complies with the criteria of stunning (3). We did not assess the reversibility and duration of this condition. However, normalized function has been shown after ~5 h in a similar model (36). The mechanical dysfunction was primarily characterized by reduced pressure generation (dP/dtmax and LVDP) and a reduced mechanical energy generation. In the present model the LV dilated during ischemia and immediate reperfusion, but a reduced Ved, combined with a "diastolic creep" of Vw (10), was observed over the following 90 min of reperfusion (decreased "preload range"). Combined with the increase in end-diastolic stiffness, this indicates a moderate development of contracture with reduced filling as one explanation for reduced contractile performance. This resulted in a marked decrease in SWmax, a more sensitive index than the SW-Ved relation for characterization of postischemic contractile function (10). Because the dP/dtmin is relatively load dependent (25), and the more load-independent, time-constant of isovolumic relaxation (tau ) was unchanged (37), the postischemic dysfunction observed seems less related to alterations in the active LV relaxational properties.

Methodological considerations. Our model induces a nearly global LV postischemic dysfunction (including septum). The right coronary artery supplies the posterior part of the septum and the tip of the apical region in pigs (our own observations), and these regions were not stunned (<20% of the LV). Thus minor inhomogeneity of LV contraction could be expected in this model, but should not impair the accuracy of volume measurements. Furthermore, by not inducing right ventricular ischemia, a confounding factor for assessment of postischemic LV dysfunction was omitted (5). Another reason for choosing an LCA occlusion protocol, was observations during pilot studies where a model of global ischemia and CPB assist was tested. After three experiments with stunning and four controls, we experienced a too unpredictable hemodynamic status after CPB, and mechanoenergetic differences between stunned and control hearts were not obvious. This suggested a significant negative mechanoenergetic effect from CPB alone, an observation leading us to abandon the use of CPB.

Ideally, when LV mechanoenergetics are described, direct assessment of MVO2 in a totally unloaded beating LV (EC coupling + basal metabolism), and MVO2 during cardiac arrest (basal metabolism only), should be assessed to distinguish between energy used for EC coupling and basal metabolism (35). Our in vivo model did not permit this. Thus the unloaded MVO2 estimates are sums of MVO2 for EC coupling and basal metabolism (35), and have some degree of uncertainty due to extrapolation of the MVO2-PVA relationship to the y-axis. However, an estimate of slope does not suffer from the same uncertainty, and therefore, does not influence our observation of a contractile inefficiency.

We employed anesthetized animals. This may have hidden a stress-induced increase in FA levels, as it occurs in conscious animals and humans during ischemia (39). Infusion of FA emulsion was not employed to mimic this, because we were mainly interested in purely ischemia-induced shifts in myocardial metabolism. However, gross postischemic increases in arterial FA concentrations and myocardial FA oxidation rates, may in clinical situations have an additional negative effect on the total efficiency ratio (14).

In conclusion, experimental observations do not automatically apply to clinical settings. However, patients undergoing cardiac surgery, coronary angioplasty, or reperfusion during acute coronary syndromes, are prone to develop postischemic dysfunction (2). Our results give a detailed description of the pathophysiology of postischemic mechanoenergetics and oxidative metabolism in a setting of global LV dysfunction. No obvious link between the increased FA preference and the decreased contractile efficiency was established, and the study therefore does not indicate correctable metabolic alterations in postischemic myocardium. The main observation in the study was a profound contractile inefficiency, pointing to a defect in the chemical-to-mechanical energy-coupling in stunned hearts, possibly at the level of the myosin ATPase.


    ACKNOWLEDGEMENTS

The staff at the Surgical Research Laboratory, Institute of Clinical Medicine, University of Tromsø, is greatly acknowledged for technical assistance.


    FOOTNOTES

The present work was supported in parts by grants from the Norwegian Council on Cardiovascular Diseases, the Norwegian Research Council, and the Norwegian Odd Fellow Association.

Parts of the present work were presented on August 28, 2000, at the 22nd Congress of the ESC, Amsterdam, the Netherlands (Eur Heart J 21, Suppl: 253, 2000).

Address for reprint requests and other correspondence: Christian Korvald, Dept. of Thoracic and Cardiovascular Surgery, Univ. Hospital Tromsø, N-9038 Tromsø, Norway (E-mail: christian.korvald{at}rito.no).

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

Received 16 May 2001; accepted in final form 4 September 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 281(6):H2645-H2653
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