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Circulation. 1996;93:1556-1566

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(Circulation. 1996;93:1556-1566.)
© 1996 American Heart Association, Inc.

Articles

Calcium Responsiveness in Regional Myocardial Short-term Hibernation and Stunning in the In Situ Porcine Heart

Inotropic Responses to Postextrasystolic Potentiation and Intracoronary Calcium

Gerd Heusch, MD; Jochen Rose, MD; Andreas Skyschally, PhD; Heiner Post; Rainer Schulz, MD

From the Abteilung für Pathophysiologie, Zentrum für Innere Medizin des Universitätsklinikums Essen, Federal Republic of Germany.

Correspondence to Prof Dr Gerd Heusch, FESC, FACC, Abteilung für Pathophysiologie, Zentrum für Innere Medizin, Universitätsklinikum Essen, Hufelandstraße 55, 45122 Essen, Federal Republic of Germany.


*    Abstract
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 *Abstract
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Background We tested the hypothesis that decreased calcium responsiveness is responsible for the reduction in contractile function in regional hibernating and stunned myocardium in situ.

Methods and Results In 19 anesthetized swine, the left anterior descending coronary artery flow was reduced to decrease anterior myocardial work index (sonomicrometry) by {approx}60%. During 90 minutes of hypoperfusion, creatine phosphate recovered (as determined by biopsy specimens and bioluminescence) and no necrosis developed (as determined by staining with triphenyl tetrazolium chloride). In 10 swine, changes in the intracellular calcium concentration were induced by systematic variation of the postextrasystolic time interval at a constant prematurity. In 9 additional swine, a graded IC calcium infusion was performed. Under control conditions, anterior myocardial work increased with a fully compensated postextrasystolic time interval from 380±93 (mean±SD) to 523±98 mm Hg·mm. IC calcium infusion increased anterior myocardial work under control conditions from 356±85 to a maximum of 428±93 mm Hg·mm. Although the maximal responses were decreased during postextrasystolic potentiation (222±68 versus 523±98 mm Hg·mm) and calcium infusion (176±32 versus 428±93 mm Hg·mm) after 90 minutes of ischemia, the relationships between increases in anterior myocardial work and, respectively, postextrasystolic time interval and IC calcium were not different. The same was true after 30 minutes of reperfusion.

Conclusions Both regional hibernating myocardium and stunned myocardium in situ are characterized by a decrease in overall myocardial calcium responsiveness; however, there appears to be no significant myocardial desensitization to calcium.


Key Words: stunning, myocardial • calcium • ischemia • contractility


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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When a severe reduction of coronary blood flow persists for >20 minutes, myocardial necroses begin to develop and contractile function eventually is irreversibly lost.1 When myocardial ischemia is less severe, the myocardium can remain viable. Contractile function of such myocardium is reduced but recovers at the occurrence of reperfusion. This situation of chronic contractile dysfunction that is reversible on reperfusion has been termed myocardial hibernation.2 The recovery of contractile function on reperfusion, however, may require substantial time, ie, the myocardium is stunned.3 The metabolic status of hibernating myocardium improves over the first few hours as myocardial lactate production is attenuated4 5 and CP, after an initial reduction, returns toward control values.5 6 7 8 The hibernating myocardium can respond to an inotropic stimulation with increased contractile function, however, at the expense of a renewed worsening of the metabolic status.5

The alterations in excitation-contraction coupling that ultimately underlie the reduction in contractile function in hibernating myocardium are not clear in detail. Alterations in sarcoplasmic reticulum function during short-term global ischemia have been observed.9 Alterations at the level of the myofibrils are particularly unclear, largely because of the methodological difficulties in analyzing the intracellular calcium concentration and contractile function simultaneously in a physiological setting. Isolated, buffer-perfused heart preparations that permit the measurement of intracellular free calcium by use of aequorin-fluorescence or nuclear magnetic resonance spectroscopy are characterized by low contractile performance,10 11 12 13 14 15 16 17 even at unphysiologically high extracellular calcium concentrations.11 12 13 14 On the other hand, in vivo preparations that are more physiological do not permit the measurement of intracellular calcium concentrations and rely on the ability of biophysical (eg, postextrasystolic potentiation18 ) or pharmacological maneuvers (eg, IC calcium, catecholamines5 19 ) to alter the intracellular calcium concentration or to modify the calcium sensitivity of the myofibrils (eg, calcium sensitizers).20 21 Marban,22 therefore, emphasized the importance of a synopsis based on both types of preparations: more insightful but more artificial and more indirect but more physiological models.

Whereas intracellular calcium concentration is increased consistently during no-flow ischemia,10 11 15 16 17 23 24 25 26 27 the few existing data during low-flow ischemia indicate a decreased12 28 29 30 or unchanged intracellular calcium concentration.31 Maximal developed isovolumic pressure decreases rapidly and calcium sensitivity is decreased during no-flow ischemia.25 Data during low-flow ischemia are lacking. In an isolated buffer-perfused ferret-heart model of short-term hibernation, characterized by a perfusion pressure of 60 mm Hg without concomitant metabolic evidence of ischemia, the reduction in intracellular calcium concentration was sufficient to explain the decrease in contractile function, whereas maximal calcium-activated isovolumic pressure was unaltered.12 Data from more physiological in situ preparations on excitation-contraction coupling in short-term hibernating myocardium are not available. We therefore investigated the calcium responsiveness in regional hibernating myocardium in situ.

In addition, as part of the protocol, the calcium responsiveness in stunned myocardium was reexamined, since changes in calcium responsiveness in stunned myocardium remain controversial. Whereas maximal calcium-activated force was reduced in buffer-perfused hearts,14 15 32 33 maximal calcium-activated segment shortening was not different from control in an in situ canine-heart preparation.19 Calcium sensitivity was reported to be decreased14 32 33 34 or unaltered.15


*    Methods
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 *Methods
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The experimental protocols used in the present study were approved by the bioethical committee of the district of Düsseldorf, and they adhere to the guiding principles of the American Physiological Society.

Experimental Model
Nineteen Göttinger miniswine (weight, 20 to 40 kg) of either sex were initially sedated with ketamine hydrochloride (1 g IM) and then anesthetized with thiopental (Trapanal 500 mg IV). Through a midline cervical incision, the trachea was intubated for connection to a respirator (Dräger). Anesthesia was then maintained with enflurane (1% to 1.5%) with an oxygen/nitrous oxide mixture (40%:60%). Arterial blood gases were monitored frequently in the initial stages of the preparation until stable and then periodically throughout the study (Radiometer). Rectal temperature was monitored and body temperature was kept above 37°C by the use of a heated surgical table and drapes.

Through the cervical incision, both common carotid arteries and internal jugular veins were isolated. The arteries were cannulated with polyethylene catheters, one for the measurement of arterial pressure and the other to supply blood to the extracorporeal circuit. The jugular veins were cannulated for volume replacement with warmed 0.9% NaCl and for the return of blood to the animal from the coronary venous line (see below).

A left lateral thoracotomy was performed in the fourth intercostal space and the pericardium opened. A micromanometer (P7, Konigsberg Instruments) was placed in the LV through the apex together with a saline-filled polyethylene catheter (used to calibrate the micromanometer in situ). Ultrasonic dimension gauges were implanted in the LV myocardium to measure the thickness of the anterior and posterior (control) walls (System 6, Triton Technologies, Inc).

The proximal LAD was dissected over a distance of 1.5 cm, ligated, and cannulated, and the distal LAD was perfused from an extracorporeal circuit. Before coronary cannulation, the pigs were anticoagulated with 20 000 IU sodium heparin; additional doses of 10 000 IU were given at hourly intervals. The system included a roller pump, windkessel, and two side ports, one for the injection of radiolabeled microspheres, the other for dobutamine or calcium infusion.5 35 Coronary arterial pressure was measured from the sidearm of a polyethylene T-connector (Cole-Parmer) used as the catheter tip with an external transducer (Bell and Howell type 4-327I). The large epicardial vein parallel to the LAD was dissected and cannulated. Coronary venous blood was drained to an unpressurized reservoir and then returned to a jugular vein through use of a second roller pump. Heart rate was controlled throughout the study by left atrial pacing (Hugo Sachs Elektronik type 215/T). The completed preparation was allowed to stabilize for at least 30 minutes before control measurements were made. The flow-constant perfusion pump was adjusted so that the minimum coronary arterial pressure was not <70 mm Hg under control conditions to avoid initial hypoperfusion. Therefore, mean coronary arterial pressure exceeded LVPP.

Regional Myocardial Function
End diastole was defined as the point when LV 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 LV dP/dt.36 Systolic wall thickening was calculated as end-systolic wall thickness minus end-diastolic wall thickness, divided by the end-diastolic wall thickness.

Because the regional calcium and dobutamine infusion changed the contraction pattern of the stimulated area in some swine such that an augmented early systolic thickening was followed by late systolic thinning, the regional myocardial work performed by the LAD-perfused myocardium was calculated in addition to systolic wall thickening. Regional myocardial work was determined as the sum of the instantaneous LV pressure–wall thickness product over the time of the cardiac cycle, with the equation:


where ed=end diastole, n=actual cardiac cycle, m=sampling point within cardiac cycle n at a sampling frequency of 5 ms, LVPn,m=instantaneous LV pressure within cardiac cycle n and at sampling point m, LVPn,min=minimum LV pressure within cardiac cycle n, and WTh=wall thickness. The maximal work value during systole is reported as WI.5 35

Ischemia decreased the baseline value of WI. Therefore, to analyze responses of WI to postextrasystolic potentiation or IC calcium infusion (see below) during control conditions and during ischemia and reperfusion, increases in WI were expressed as a fraction of the maximal increase during the respective intervention, as reported previously by other investigators.19 25

Regional Myocardial Blood Flow
Radiolabeled microspheres (15-µm diameter, 141Ce, 114In, 51Cr, 113Sn, 103Ru, 95Nb, or 46Sc; NEN/DuPont Co) were injected into the coronary perfusion circuit (1 to 2x105 suspended in 1 mL saline) to determine the regional myocardial blood flow and its distribution throughout the LAD perfusion bed. This procedure for the determination of blood flow has been validated extensively.35 Blood flow to the tissue at the site of the ultrasonic crystals is reported, and this piece of tissue was divided into transmural thirds of approximately equal thickness.

Regional Myocardial Metabolism
Oxygen content was measured with use of anaerobically sampled blood drawn simultaneously from the cannulated coronary vein and an artery (Cavitron/LexO2-Con-k, Dr B.G. Schlag). 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 by use of enzymatic dehydrogenation and subsequent photometry of NADH,37 and lactate consumption was calculated by multiplying the arterial-coronary venous difference by the transmural blood flow at the crystal site.

Transmural myocardial biopsy specimens ({approx}10 mg each) were taken with a modified dental drill from the LAD perfusion bed for analysis of ATP and CP content. Care was taken to ensure that the biopsy samples originated from within the LAD perfusion territory (with epicardial arteries used as landmarks) but distal to the site of the ultrasonic dimension gauges and blood-flow measurements. Samples that required >1 to 2 seconds for acquisition were not used for this analysis. The analytical procedures (bioluminescence) have been described in detail previously.5

Morphology
At the end of each study, the heart was removed and sectioned from base to apex into five transverse slices in a plane parallel to the atrioventricular groove. The tissue slices were immersed in a 0.09 mol/L sodium phosphate buffer (pH 7.4) containing 1.0% triphenyl tetrazolium chloride (TTC, Sigma) and 8% dextran (mol wt, 77 800) for 20 minutes at 37°C to verify the absence of infarcted tissue.

Experimental Protocols
In both groups of swine, a 90-minute ischemic period was followed by 120 minutes of reperfusion. During ischemia, coronary inflow was decreased to reduce WI by {approx}60%; this degree of ischemia has been previously shown to be compatible with the development of myocardial hibernation.5 38 39 Under control conditions and at 5 and 85 minutes of ischemia, pairs of arterial and coronary venous blood samples were simultaneously withdrawn. During the blood sampling, microspheres were injected into the LAD perfusion system for measurement of regional myocardial blood flow, and hemodynamic and regional dimension data were recorded. Coronary arterial pressure was continuously monitored during the microsphere injection to ensure that it was unaffected by the injection. Immediately after the microsphere injection, myocardial biopsy specimens were taken. A set of measurements was obtained within 2 to 3 minutes.

Group 1 (n=10): Postextrasystolic Potentiation
The prematurity of the electrically induced extrasystole that resulted in maximal postextrasystolic potentiation of WI was determined under control conditions and then kept constant. Extrasystoles with constant prematurity were then followed by three different postextrasystolic time intervals: a fully compensated interval, an interval as long as the regular beat-to-beat interval, and an abbreviated interval (set to 65% of the regular beat-to-beat interval). Sets of measurements, including postextrasystolic potentiation with the three different postextrasystolic time intervals, were performed under control conditions and at 5 and 85 minutes of ischemia. After 85 minutes of ischemia, dobutamine (2.5±1 µg/min) was infused into the coronary perfusion system. The infusion rate was set to increase LV dP/dtmax by 30%. After 2 to 3 minutes of the dobutamine infusion, a further set of measurements was performed. Thereafter, the myocardium was reperfused and a final set of measurements was obtained at 30 minutes of reperfusion.

Group 2 (n=9): IC Calcium Infusion
Under control conditions, CaCl2 was infused into the perfusion system with a syringe pump, starting at an infusion rate of 10 mL/h, with stepwise increases up to 240 mL/h. These calcium infusion rates were normalized for the actual LAD inflow and finally expressed in micrograms of added CaCl2 per milliliter of blood. The recruitment of maximal calcium-activated work was verified by the lack of further increases of WI despite further increased calcium infusion rates. Since IC calcium infusion increased contractile function, it was also expected to deteriorate the metabolic status of the myocardium during ischemia, as previously shown for dobutamine.39 Therefore, to not interfere with the development of hibernating myocardium, IC calcium infusion early during ischemia was avoided. After 85 minutes of ischemia, the three doses of IC calcium that resulted in an {approx}10% increase, half-maximal increase, and maximal increase in WI during control conditions were repeated. The infusion rates were corrected for the reduced coronary inflow during hypoperfusion to obtain identical calcium doses (in micrograms of added CaCl2 per milliliter of blood) as during control conditions. These doses are referred to as Ca1 (56.6±20.3 µg/mL), Ca2 (164.9±42.8 µg/mL), and Ca3 (407.5±117.9 µg/mL). With the maximal functional response to added calcium, the last microsphere measurement was performed. Thereafter, the myocardium was reperfused and at 30 minutes of reperfusion, the stepwise calcium infusion was repeated. Calcium infusion rates were once more corrected for the increased coronary inflow during reperfusion.

At the end of each study, the digital reading of the roller pump was calibrated by timed collection of arterial blood in a graduated cylinder.

Data Analysis and Statistics
Hemodynamic data were recorded on an 8-channel recorder (Gould MK 200A) and stored directly on the hard disk of an AT-type computer. Hemodynamic and functional parameters were digitized and recorded over a 20-second period during each microsphere injection ({approx}33 consecutive beats over at least two complete respiratory cycles) with CORDAT II software (Triton Technologies, Inc).40 Hemodynamic parameters reported are heart rate, LVEDP and LVPP, LV dP/dtmax, and mean LAD pressure. Regional wall function parameters include AWED, AWT, and AWI,35 and PWT. Metabolic parameters include the myocardial contents of ATP and CP and the consumption of oxygen and lactate (positive values indicate myocardial uptake).

In group 1, {approx}33 consecutive beats were averaged for hemodynamic and regional wall function parameters and compared with single beats with an abbreviated, regular, or fully compensated postextrasystolic time interval. In group 2, {approx}33 consecutive beats were averaged for the calculation of hemodynamic and regional wall function parameters.

Statistical analyses were performed by use of SYSTAT software. In both groups of swine, changes in regional myocardial blood flow and metabolism over the time course of the experiment were evaluated by one-way ANOVA.

In addition, in group 1, changes in systemic hemodynamics and regional myocardial function during postextrasystolic potentiation were assessed by two-way ANOVA, accounting for the different time points throughout the experiment and the three postextrasystolic time intervals. In group 2, changes in systemic hemodynamics and regional myocardial function during increasing calcium infusion rates were assessed by two-way ANOVA, accounting for the different time points throughout the experiment and the three different calcium doses (minor, half-maximal, and maximal increase in myocardial work). When significant differences were detected, individual mean values were compared by use of post hoc tests.

Linear regression analyses between the postextrasystolic time interval (expressed on a logarithmic scale, group 1) and all doses of added calcium (expressed on a logarithmic scale,19 group 2) and the response in the work index (expressed as a fraction of the maximal response19 25 ) were performed in groups 1 and 2. Regression lines were compared by ANCOVA.

All data are reported as mean±SD, and a value of P<.05 was accepted as indicating a significant difference.


*    Results
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 *Results
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In both groups of swine, there was no myocardial necrosis after 90 minutes of ischemia and 2 hours of reperfusion.

Group 1: Postextrasystolic Potentiation
Data regarding systemic hemodynamics, regional myocardial function, blood flow, and metabolism are summarized in Tables 1Down and 2Down. An original tracing from one experiment in group 1 is given in Fig 1Down for a fully compensated postextrasystolic time interval, and the effects of postextrasystolic potentiation on AWI are displayed in Fig 2Down.


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  		Table 1. Systemic Hemodynamics and Regional Myocardial Function Under Control Conditions, at 5 and 85 Minutes of Ischemia, at 90 Minutes of Ischemia and IC Dobutamine Infusion, and at 30 Minutes of Reperfusion Under Resting Conditions and During Postextrasystolic Potentiation


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  		Table 2. Regional Myocardial Blood Flow and Metabolism Under Control Conditions, at 5 and 85 Minutes of Ischemia, and at 90 Minutes of Ischemia and IC Dobutamine Infusion



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  		Figure 1. Original tracing from one experiment in group 1. Displayed are a regular beat, an extrasystole, and a postextrasystolic beat after a fully compensated postextrasystolic time interval. The end-diastolic wall thickness in the postextrasystolic beat is decreased and systolic wall thickening is enhanced in both walls under control conditions. AWT is then decreased by 60% for 90 minutes by decreasing inflow into the cannulated LAD. Despite a decreased baseline wall thickening in the anterior wall, the postextrasystolic beat is characterized by an enhanced wall thickening in both walls compared with the respective baseline beat at 5 and 85 minutes of ischemia and 30 minutes of reperfusion. Also, the decrease in end-diastolic wall thickness of the postextrasystolic beat is comparable to that during control conditions.



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  		Figure 2. AWI during resting conditions (REST) and at an abbreviated (ABBR), regular (REG), and fully compensated (COMP) postextrasystolic time interval after extrasystoles with a constant prematurity. Under control conditions, AWI increased at a regular and fully compensated postextrasystolic time interval and decreased at an abbreviated postextrasystolic time interval. At 5 and 85 minutes of ischemia and at 30 minutes of reperfusion, AWI was significantly reduced compared with control conditions. AWI again increased at regular and fully compensated postextrasystolic time intervals. The maximal responses, however, were significantly reduced compared with those under control conditions. *P<.05 vs respective control; {ddagger}P<.05 vs respective REST.

Systemic Hemodynamics
Under control conditions, postextrasystolic potentiation at an abbreviated postextrasystolic time interval reduced LVPP (P=NS) and LV dP/dtmax (P<.05), whereas LVEDP (P<.05) was increased. At a regular and fully compensated postextrasystolic time interval, LVEDP (P=NS), LVPP (P=NS), and LV dP/dtmax (P<.05) were increased (Table 1Up). With the reduction in coronary inflow, LVPP (P=NS) and LV dP/dtmax (P<.05) were decreased, with no further significant changes when ischemia was prolonged to 85 minutes (Table 1Up). Infusion of dobutamine after 85 minutes of ischemia significantly increased LV dP/dtmax. After 30 minutes of reperfusion, LVPP and LV dP/dtmax tended to be reduced further. The responses to postextrasystolic potentiation at 5 and 85 minutes of ischemia and at 30 minutes of reperfusion were comparable to the responses under control conditions (Table 1Up).

Regional Myocardial Function
Under control conditions, postextrasystolic potentiation at an abbreviated postextrasystolic time interval increased the AWED (P<.05) but reduced AWT and AWI (both P<.05). Also, PWT was decreased (P<.05). At a regular and fully compensated postextrasystolic time interval, AWED was decreased (P=NS) and AWT, AWI (Fig 2Up), and PWT were increased. At 5 minutes of ischemia, AWED tended to be reduced (P=NS), and AWT and AWI were significantly decreased to 17.3±6.9% and 133±51 mm Hg·mm, respectively, whereas PWT remained unchanged. Again, postextrasystolic potentiation at an abbreviated postextrasystolic time interval increased AWED (P<.05) and reduced AWT, AWI, and PWT (all P<.05). At a regular and fully compensated postextrasystolic time interval, AWED was decreased (P=NS), whereas AWT and AWI were increased. However, the absolute values of AWT and AWI always remained below their respective values during control conditions (P<.05, Fig 2Up). In contrast, the increase in PWT during postextrasystolic potentiation at a regular and fully compensated postextrasystolic time interval was comparable to that observed during control conditions. With prolongation of ischemia to 85 minutes, neither the absolute values of AWT, AWI (Fig 2Up), or PWT nor their responses to postextrasystolic potentiation were changed. With IC dobutamine infusion, the contraction pattern of the anterior wall changed in that the maximal wall thickness was reached early during systole followed by a late systolic wall thinning. During dobutamine infusion, AWI was increased (P<.05), whereas AWT and PWT remained unchanged. With reperfusion, AWT and AWI remained depressed. Postextrasystolic potentiation again induced similar responses such as seen during 5 and 85 minutes of ischemia (Fig 2Up).

Coronary Arterial Pressure, Regional Myocardial Blood Flow, and Metabolism
With the reduction in coronary inflow, coronary arterial pressure was significantly reduced to 51±7 mm Hg. Regional myocardial blood flow was reduced, particularly in the subendocardium (Table 2Up). Myocardial CP and ATP content as well as MO2 were decreased, and myocardial lactate consumption under control conditions was reversed to net lactate production. When ischemia was prolonged to 85 minutes, coronary arterial pressure, regional myocardial blood flow, MO2, and the myocardial ATP content remained unchanged, whereas the myocardial CP content recovered toward its control value, and lactate production was attenuated. With infusion of dobutamine at unchanged coronary arterial pressure, regional myocardial blood flow, and MO2, myocardial CP content again decreased and myocardial lactate production again increased, whereas myocardial ATP content decreased further.

Group 2: IC Calcium Infusion
Data regarding systemic hemodynamics, regional myocardial function, blood flow, and metabolism are summarized in Tables 3Down and 4Down. The effects of IC calcium infusion on AWI are displayed in Fig 3Down.


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  		Table 3. Systemic Hemodynamics and Regional Myocardial Function Under Control Conditions, at 85 Minutes of Ischemia, and at 30 Minutes of Reperfusion Under Resting Conditions and in Response to IC Infusion of Three Different Calcium Doses (Ca1, Ca2, Ca3)


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  		Table 4. Regional Myocardial Blood Flow and Metabolism Under Control Conditions, at 5 and 85 Minutes of Ischemia, and at 90 Minutes of Ischemia and IC Calcium Infusion



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  		Figure 3. AWI during resting conditions (REST) and in response to IC infusion of three different calcium doses (Ca1, Ca2, Ca3). Under control conditions, the stepwise IC calcium infusion increased AWI. At 85 minutes of ischemia and at 30 minutes of reperfusion, AWI was significantly reduced compared with control conditions. AWI was again increased during the IC calcium infusion. The maximal responses, however, were significantly reduced compared with those under control conditions. *P<.05 vs respective control; {ddagger}P<.05 vs respective REST.

Systemic Hemodynamics
With increasing IC calcium doses (Ca1, Ca2, and Ca3), LVPP tended to be elevated and LV dP/dtmax was increased in a stepwise fashion (P<.05 at Ca3) (Table 3Up). After 85 minutes of ischemia and at 30 minutes of reperfusion, baseline values of LVPP (P=NS) and LV dP/dtmax (P<.05) were reduced. Increasing IC calcium doses, however, induced hemodynamic responses comparable to those under control conditions.

Regional Myocardial Function
Under control conditions with increasing IC calcium doses, AWED remained stable, whereas AWT (P=NS) and AWI (P<.05, Fig 3Up) were increased at the highest IC calcium dosage. At 85 minutes of ischemia, baseline AWT and AWI were significantly reduced to 18.3±8.2% and 132±38 mm Hg·mm, respectively. There was no further change at 30 minutes of reperfusion. Again, during both ischemia and reperfusion at the highest IC calcium dose, AWI was increased (P<.05 at Ca3, Fig 3Up). However, at any given calcium dose, AWI was reduced during ischemia and reperfusion compared with control conditions. Systolic thickening of the posterior myocardium remained unchanged during the entire protocol.

Coronary Arterial Pressure, Regional Myocardial Blood Flow, and Metabolism
Coronary arterial pressure, regional myocardial blood flow, and metabolism under control conditions and at 5 and 85 minutes of ischemia were comparable to those observed in group 1. With the IC calcium infusion at unchanged coronary arterial pressure, regional myocardial blood flow, and MO2, myocardial CP content was again decreased and myocardial lactate production was again increased, whereas myocardial ATP content was decreased further.

AWI–Postextrasystolic Time Interval and AWI-Added Calcium Relationships
The relationships between increases in AWI (expressed as a fraction of the maximal increase in AWI) and the postextrasystolic time interval were not different under control conditions, at 5 and 85 minutes of ischemia, and at 30 minutes of reperfusion (Fig 4Down). Also, the relationships between AWI and added calcium were superimposable under control conditions, at 85 minutes of ischemia (Fig 5ADown), and at 30 minutes of reperfusion (Fig 5BDown).



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  		Figure 4. Relation between the duration of the postextrasystolic time interval (expressed on a logarithmic scale) and the increase in regional myocardial work (expressed as a fraction of the maximal increase). The close relation between postextrasystolic time interval and fractional increase in regional myocardial work under control conditions remains unaltered at 5 and 85 minutes of ischemia as well as at 30 minutes of reperfusion (P=NS by ANCOVA).




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  		Figure 5. A, Relation between the added calcium chloride (expressed on a logarithmic scale) and the increase in regional myocardial work (expressed as a fraction of the maximal increase). The relations obtained under control conditions and at 85 minutes of ischemia are superimposable (P=NS by ANCOVA). B, Relation between the added calcium chloride (expressed on a logarithmic scale) and the increase in regional myocardial work (expressed as a fraction of the maximal increase). The relations obtained under control conditions and at 30 minutes of reperfusion are superimposable (P=NS by ANCOVA).


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The major finding of the present study is that both regional short-term hibernating myocardium and stunned myocardium are characterized by a decrease in maximal calcium-activated work without apparent alterations in calcium sensitivity in the in situ porcine heart.

Critique of Methods
Strengths and Limitations of the Experimental Preparation
The present model of regional myocardial hibernation with its strengths and limitations has been discussed in detail previously.5 38 39 The degree of flow reduction, the decrease in regional myocardial oxygen consumption, and the contractile dysfunction of the present study were comparable to former studies (Tables 1Up and 3Up).5 38

In the in situ heart preparation, neither intracellular calcium concentrations nor maximal calcium-activated force can be measured; however, both can be determined in isolated myocytes, papillary muscles, or buffer-perfused heart preparations. Given the advantage of direct access to the measurement of intracellular calcium concentrations, myofilament calcium sensitivity, and maximal calcium-activated force after pharmacological elimination of the sarcoplasmic reticulum and tetanization, such preparations, however, suffer from unphysiological external conditions, ie, lack of blood perfusion, lack of autoregulation,12 lack of physiological contraction mode (isovolumic rather than ejecting), and decreased strength of contraction,10 12 13 14 15 16 17 28 even despite elevated extracellular calcium concentrations.11 12 13 14 In blood-perfused hearts with an intact autoregulation and a physiological contraction mode, responses of contractile function to IC calcium or to manipulation of the intracellular calcium concentration with varying coupling intervals during postextrasystolic potentiation must serve as surrogates for intracellular calcium concentration, myofilament calcium sensitivity, and maximal calcium-activated force. Supporting our approach, postextrasystolic potentiation or increasing concentrations of extracellular calcium have been directly demonstrated in buffer-perfused hearts to enhance the intracellular calcium concentration and to result in proportionate increases in contractile function.25 41 However, we cannot exclude that IC calcium infusion and postextrasystolic potentiation recruit different amounts of activator calcium under control conditions, in hibernating myocardium, and in stunned myocardium. In the present study, therefore, only overall myocardial calcium responsiveness can be studied under defined pathophysiological conditions such as regional myocardial ischemia and reperfusion.

Regional Myocardial Function
Depending on the duration of the postextrasystolic time interval, preload and afterload of the postextrasystolic beat were altered. Thus, apart from alterations in excitation-contraction coupling and inotropy, changes in loading conditions, in particular preload recruitment, probably contributed to the increased regional myocardial function during postextrasystolic potentiation. Therefore, the increases in regional myocardial function during postextrasystolic beats were substantially more pronounced than those during the highest calcium infusion rate and during dobutamine infusion, because calcium and dobutamine infusion did not increase preload, as reflected by the unchanged AWED and LVEDP (Table 3Up). Afterload tended to be increased during postextrasystolic potentiation at a regular and fully compensated postextrasystolic time interval and during calcium and dobutamine infusion, as reflected by the slightly increased LVPP (Tables 1Up and 3Up). However, the recruitment of preload during postextrasystolic potentiation and the increase in afterload during postextrasystolic potentiation and IC calcium infusion were comparable in intact, ischemic, and reperfused myocardium (Tables 1Up and 3Up), thus permitting an intraindividual comparison of regional myocardial inotropic responses during the experimental protocol.

To better evaluate the influence of prolonged ischemia and stunning on the inotropic responses to postextrasystolic potentiation and IC calcium infusion, the apparent calcium sensitivity of normoperfused, hibernating, and stunned myocardium was assessed by plotting the response in regional myocardial work expressed as a fraction of the maximal response during each intervention against the postextrasystolic time interval or the added IC calcium dose.19 The calcium dose was calculated in micrograms per milliliter of blood to account for changes in blood flow during ischemia and reperfusion. The curves under control conditions, ischemia, and reperfusion were virtually superimposable, although during ischemia and reperfusion, there was a tendency for a small rightward shift, ie, the negative log(added calcium) for the half-maximal increase in fractional work was decreased by 2.8% (hibernation) and 0.8% (stunning), respectively.

Hibernating Myocardium
Contractile dysfunction during moderate ischemia represents more than a reflection of the severity of ischemia and impending infarction. The energy-sparing effect from the loss of regional contractile function serves as an important mechanism through which the myocardium adjusts to an ischemic period.42 43 Severe ischemia with total coronary occlusion causes cell death within {approx}20 minutes in the inner wall, with progressive necrosis across the wall over time, the so-called wave front of infarction.1 More moderate ischemia, such as in the present study, is characterized by a less intense decrease in regional contractile function (persistent systolic work). Such moderate ischemia can be maintained for prolonged periods without development of irreversible damage.5 38 44 45 46 47

Whereas during such moderate ischemia, myocardial creatine phosphate content initially decreases during the first few minutes of ischemia but then returns to near-control values, the ATP content remains constant at a slightly reduced level despite ongoing hypoperfusion.5 6 7 38 Finally, the ischemia-induced net lactate production becomes attenuated.4 5 38 Although basal contractile function is depressed, the hypoperfused myocardium retains its responsiveness to an inotropic challenge, supporting the notion that the reduction in contractile function is not secondary or not entirely secondary to an energetic deficit. When, after 85 minutes of ischemia, dobutamine is infused selectively into the ischemic region, contractile function increases, although regional blood flow remains unchanged or slightly decreases further5 38 (Table 2Up). In the present study, regional myocardial function in the hibernating myocardium also increased during IC calcium infusion (Table 3Up). The extent of functional improvement during IC dobutamine and calcium infusion was comparable and was associated with worsening of metabolic parameters by both interventions, as myocardial CP content again decreased and lactate production increased (Tables 2Up and 4Up).

Overall myocardial responsiveness to extracellular calcium comprises three aspects: the intracellular free calcium concentration during each cardiac cycle, ie, the calcium transient; the calcium sensitivity of the myofilaments; and the maximal calcium-activated force.32 Studies that used isolated buffer-perfused hearts with low-flow ischemia reported unchanged12 28 31 or decreased12 29 30 end-diastolic and unchanged31 or decreased12 28 29 30 peak systolic intracellular calcium concentrations, ie, the calcium transient during low-flow ischemia was either unchanged31 or slightly decreased.12 28 29 30 If we assume an unchanged calcium transient, the unchanged relationships between AWI and postextrasystolic time interval (Fig 4Up) or added calcium (Fig 5AUp) indicate a largely unchanged calcium sensitivity in hibernating myocardium. If we assume a decreased calcium transient, which has actually been reported,12 these unchanged relationships even indicate an increase in calcium sensitivity in hibernating myocardium. The decrease in contractile function in hibernating myocardium is therefore most likely explained by a decrease in the maximal calcium-activated work (Figs 2Up and 3Up).

Stunned Myocardium
In most isolated buffer-perfused heart preparations after 10 to 20 minutes of ischemia and 10 to 20 minutes of reperfusion, both end-diastolic and peak systolic intracellular calcium concentrations have returned to control values,10 11 15 27 32 and in only one study,14 peak systolic concentration has remained slightly increased. Consequently, the calcium transient has returned to the normal range10 11 15 27 32 or is slightly increased.14 The maximal calcium-activated force is decreased,14 15 32 and calcium sensitivity remains either unchanged15 or is decreased.14 Also, in skinned myocytes obtained from in situ stunned porcine hearts or in rat ventricular trabeculae, myofilament calcium sensitivity is decreased.33 34 In contrast, with in situ canine and porcine preparations, no changes in the regional inotropic response to epinephrine48 and postextrasystolic potentiation18 48 were found during reperfusion after 15 minutes of ischemia. In particular, IC calcium infusion into a stunned region resulted in an increase in segment shortening that was not different from the value during calcium infusion under control conditions, suggesting an unchanged maximal calcium-activated segment shortening.19

In the present study, baseline contractile function was decreased in reperfused myocardium, whereas the contractile response to postextrasystolic potentiation (expressed as a fraction of the maximal increase, Fig 4Up) and to IC calcium infusion (Fig 5BUp) was unchanged. Because the calcium transient in stunned myocardium (as mentioned above) has returned toward control values, the unchanged relationships between AWI (expressed as a fraction of the maximal increase) and postextrasystolic time interval (Fig 4Up) or added calcium (Fig 5BUp) indicate a largely unchanged calcium sensitivity in stunned myocardium. The decrease in contractile function in stunned myocardium, as in hibernating myocardium, is therefore most likely explained by a decrease in the maximal calcium-activated work (Figs 2Up and 3Up).

Studies from buffer-perfused hearts,14 ventricular trabeculae,33 and skinned fibers34 reported slight but significant decreases in calcium sensitivity in stunned myocardium, ie, -log(Ca2+)i for the half-maximal tension was decreased by 2% to 3%. In the present study, calcium sensitivity also tended to be decreased (-log[added calcium] for the half-maximal increase in fractional work was decreased by 0.8%), but this effect did not reach statistical significance. Therefore, slight decreases in calcium sensitivity in stunned myocardium might not have been detected in the present study in situ. In particular, calcium sensitivity may have decreased if the calcium transient during reperfusion was slightly increased, as reported in only one study.14 Indeed, in a recent study in anesthetized swine, the purported calcium sensitizer EMD 60263 predominantly enhanced regional contractile function of the stunned region.21 Although the study protocol was different with respect to both the duration and severity of ischemia and might thus account for the different findings, the severity of contractile dysfunction was comparable between this and the present study. In particular, however, the calcium sensitizer properties and lack of other inotropic properties of EMD 60263 were not substantiated and were based solely on an unpublished communication by the manufacturer. In contrast, in the present study, we have established dose-response curves of IC calcium and contractile function and found the fractional increase in regional myocardial work during IC calcium infusion in stunned myocardium identical to that under control conditions and at 85 minutes of ischemia. Nevertheless, even if small changes in the calcium sensitivity occurred and were not detected in the present study, these changes are of minor importance compared with the substantial alterations in the maximal calcium-activated work observed in the stunned myocardium in vivo.

Potential Mechanisms Underlying the Depression of Maximal Calcium-Activated Force During Ischemia and Reperfusion
Potential candidate mediators for the decrease in developed force are an accumulation of inorganic phosphate49 50 and a calcium overload.51 52 However, data on inorganic phosphate in hibernating myocardium are lacking, and inorganic phosphate returns to control levels within 20 minutes of reperfusion in stunned myocardium.53 Likewise, the available data indicate no increase in intracellular calcium concentration in hibernating myocardium12 28 29 30 31 and after severe ischemia, a return to normal within 10 to 20 minutes of reperfusion.10 11 15 27 32 Nevertheless, short bouts of increased inorganic phosphate or calcium may induce long-lasting changes in contractile function, a notion supported by the effective attenuation of stunning only by pretreatment with calcium antagonists before ischemia.52


*    Selected Abbreviations and Acronyms
 
AWED = anterior wall end-diastolic thickness
AWI = regional work index of the anterior wall
AWT = anterior systolic wall thickening
CP = creatine phosphate
LAD = left anterior descending coronary artery
LV = left ventricular
LVEDP = left ventricular end-diastolic pressure
LVPP = left ventricular peak pressure
PWT = posterior systolic wall thickening
WI = maximal regional myocardial work value during systole


*    Acknowledgments
 
This study was supported by grants from the German Research Foundation (No. He 1302/8-1 and 8-2). We thank Claus Martin, PhD, for the chemical analyses and Petra Gres, Ursula Prägler, and Anita van de Sand for their technical support.

Received September 6, 1995; revision received October 26, 1995; accepted November 5, 1995.


*    References
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