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(Circulation Research. 1996;79:237-246.)
© 1996 American Heart Association, Inc.

Articles

Intracellular Ca²⁺, Intercellular Electrical Coupling, and Mechanical Activity in Ischemic Rabbit Papillary Muscle

Effects of Preconditioning and Metabolic Blockade

Lukas R.C. Dekker, Jan W.T. Fiolet, Ed VanBavel, Ruben Coronel, Tobias Opthof, Jos A.E. Spaan, Michiel J. Janse

the Department of Experimental Cardiology (L.R.C.D., J.W.T.F., R.C., T.O., M.J.J.) and the Department of Medical Physics and Informatics (E.V., J.A.E.S.), Academic Medical Center, Amsterdam, The Netherlands.

Correspondence to L.R.C. Dekker, Department of Experimental Cardiology, Academic Medical Center, M-0-54, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.

	Abstract

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During myocardial ischemia, electrical uncoupling and contracture herald irreversible damage. In the present study, we tested the hypothesis that an increase of intracellular Ca²⁺ is an important factor initiating these events. Therefore, we simultaneously determined tissue resistance, mechanical activity, pH_o, and intracellular Ca²⁺ (with the fluorescent indicator indo 1, Molecular Probes, Inc) in arterially perfused rabbit papillary muscles. Sustained ischemia was induced in three experimental groups: (1) control, (2) preparations preconditioned with two 5-minute periods of ischemia followed by reperfusion, and (3) preparations pretreated with 1 mmol/L iodoacetate to block anaerobic metabolism and minimize acidification during ischemia. In a fourth experimental group, intracellular Ca²⁺ was increased under nonischemic conditions by perfusing with 0.1 mmol/L ionomycin and 0.1 µmol/L gramicidin. Ca²⁺ transients and contractions rapidly disappeared after the induction of ischemia. In the control group, diastolic Ca²⁺ began to rise after 12.6±1.3 minutes of ischemia; uncoupling, after 14.5±1.2 minutes of ischemia; and contracture, after 12.6±1.5 minutes of ischemia (mean±SEM). Preconditioning significantly postponed Ca²⁺ rise, uncoupling, and contracture (21.5±4.0, 24.0±4.1, and 23.0±5.3 minutes of ischemia, respectively). Pretreatment with iodoacetate significantly advanced these events (1.9±0.7, 3.6±0.9, and 1.9±0.2 minutes of ischemia, respectively). In all groups, the onset of uncoupling always followed the start of Ca²⁺ rise, whereas the start of contracture was not different from the rise in Ca²⁺. Perfusion with ionomycin and gramicidin permitted estimation of a threshold [Ca²⁺] for electrical uncoupling of 685±85 nmol/L. In conclusion, the rise in intracellular Ca²⁺ is the main trigger for cellular uncoupling during ischemia. Contracture is closely associated with the increase of intracellular Ca²⁺ during ischemia.

Key Words: ischemia • intracellular Ca²⁺ • cellular coupling resistance • contracture • preconditioning

	Introduction

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Cellular electrical uncoupling, evident as a sudden increase of gap junctional resistance, occurs after 10 to 15 minutes of myocardial ischemia.¹ It coincides with the onset of contracture and with the secondary phase of the rise in extracellular K⁺ and is considered a sign of irreversible ischemic cell damage.² Preconditioning the myocardium with one or more short periods of ischemia postpones the onset of irreversible ischemic damage³ and ischemia-induced cellular uncoupling.⁴

Many modulators of gap junctional resistance have been identified. Elevated intracellular Ca²⁺,^{5 6 7 8} intracellular protons,^{6 9} long-chain acylcarnitines,^{10 11} and decreased ATP content¹² raise intercellular coupling resistance. High extracellular [Ca²⁺] advances the onset of electrical uncoupling and contracture during ischemia, whereas pretreatment with Ca²⁺ entry blocker verapamil postpones these events.^{2 13} However, the mechanism underlying ischemia-induced uncoupling and contracture has not yet been clarified.

The aim of the present study was to test the hypothesis that an increase of [Ca²⁺]_i is an important factor initiating uncoupling and contracture during ischemia. Therefore, we quantified the relationship between [Ca²⁺]_i, cellular uncoupling, and contracture during sustained ischemia and during sustained ischemia preceded by either ischemic preconditioning or pretreatment with iodoacetate. Iodoacetate blocks glycolysis and thus impairs anaerobic metabolism and acidification during ischemia.¹⁴ [Ca²⁺]_i, tissue resistance (R_t), and mechanical activity were simultaneously measured in arterially perfused rabbit papillary muscles. In this model, changes in intercellular coupling resistance are directly reflected by changes in R_t.¹ [Ca²⁺]_i was assessed by means of the fluorescent indicator indo 1. In an additional experimental group, [Ca²⁺]_i was increased under nonischemic conditions by perfusion with a Ca²⁺ ionophore (ionomycin) and a Na⁺-K⁺ exchanger (gramicidin) to further analyze the relation between Ca²⁺ and uncoupling.

The present results suggest that the increase of [Ca²⁺]_i is the main stimulus for cellular uncoupling during ischemia in normal, ischemically preconditioned, and metabolically inhibited myocardium. Contracture is closely associated with the rise in [Ca²⁺]_i during ischemia. Under nonischemic conditions, the estimated threshold [Ca²⁺] for uncoupling is 685±85 nmol/L. During ischemia-induced acidification, higher [Ca²⁺] levels are required for cellular uncoupling.

	Materials and Methods

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Preparation
The technique to isolate the arterially perfused right ventricular papillary muscle of the rabbit has been described in detail previously.^{1 15} New Zealand White rabbits (3 to 3.5 kg, of either sex) were anesthetized with sodium pentobarbital (75 mg/kg IV) and heparinized (1000 U IV). After sternotomy, the heart was taken out and rapidly submerged in ice-cold Tyrode's solution (for composition, see below). After removal of the atria and the left ventricular free wall, the left side of the interventricular septum was secured to a perspex plate. Within 4 minutes after excision of the heart, the septal artery was cannulated, and perfusion was started. The right ventricular free wall was carefully removed, and the preparation was positioned in an organ chamber. Selected papillary muscles had an average length of 4.2±0.3 mm, a diameter of 1.1±0.1 mm, and a single insertion of the tendon. The papillary muscle was connected to a force transducer (Sensonor AE801) by a ligature around the tendon and was horizontally positioned (Fig 1). The resting length of the muscle was stretched to 120% of slack length. A fine silver stimulating wire was tied around the muscle tendon. A large Ag/AgCl electrode on the perspex plate served as ground. Myocardial temperature was 37°C. The preparation was surrounded by a water-saturated gas mixture of 95% O₂/5% CO₂. Temperature and PO₂ of the atmosphere in the organ chamber were continuously monitored.

View larger version (38K): [in this window] [in a new window]   Figure 1. Schematic representation of an arterially perfused papillary muscle in the organ chamber. The preparation is fixed to a perspex plate, and the tendon of the papillary muscle is attached to a force transducer. Oxygen tension and temperature of the water-saturated surrounding atmosphere (dotted area) are constantly monitored. The objective of the fluorescence microscope is heated to prevent condensation of water on its lens. Subthreshold and excitatory current pulses are injected at the muscle apex through a 100-µm silver wire and are measured by a current meter (Amp). E1 and E2 represent extracellular electrodes.

Perfusion
During an initial equilibration period of 30 minutes and after indo 1 loading (see below), the heart was perfused with Tyrode's solution containing (mmol/L) Na⁺ 155.5, K⁺ 4.7, Ca²⁺ 1.45, Mg²⁺ 0.6, Cl^- 136.5, HCO₃^- 27.0, HPO₄^2- 0.4, probenecid 0.1, and glucose 10, along with 10 U/L insulin and 1.0% FCS. Probenecid is an anion transport blocker and has been shown to prevent loss of tetracarboxylate fluorescence indicators.^{16 17} The perfusate was gassed with a mixture of 95% O₂/5% CO₂ to yield a pH of 7.4. A flow rate of 1.0 to 1.2 mL/min per gram was maintained by a constant pressure perfusion system (35 to 45 mm Hg). Ischemia was induced by stopping perfusion and at the same time replacing the 95% O₂/5% CO₂ gas mixture with 95% N₂/5% CO₂. During ischemia, oxygen tension in the organ chamber was <3 mm Hg. Since the light absorption spectrum of hemoglobin is dependent on oxygen tension, erythrocytes were omitted from the perfusate in order to prevent errors in fluorescence measurements after the induction of ischemia.

Fluorescence Measurements
The intracellular fluorescent Ca²⁺ probe indo 1 (Molecular Probes, Inc) was loaded as the membrane-permeable acetoxymethyl ester (indo 1-AM) via the vasculature. Inside the cells, indo 1-AM is deesterified by cytosolic esterases into the Ca²⁺-sensitive form, which does not leak out of the cell easily.¹⁸ Adequate loading was achieved by recirculating 30 mL Tyrode's solution containing 5 µmol/L indo 1-AM (initially dissolved in dimethyl sulfoxide containing 6% [wt/vol] pluronic F-127), 5% FCS, and 1 mmol/L probenecid for 25 to 35 minutes at 30°C. Residual indo 1-AM was washed out by perfusing with control Tyrode's solution for 30 minutes at 37°C. The loading procedure increased fluorescence of the heart by a factor of 8 to 10 compared with fluorescence measured before loading (autofluorescence).

Excitation light from a xenon-arc lamp (75 W) was filtered by a 340-nm interference filter and focused on the surface of the papillary muscle using appropriate UV optics. Illumination via a 10x objective (numerical aperture, 0.50; Fluar) was confined to a circular area with a diameter of 1.3 mm. Emitted light was collected by the same objective and divided by a 455-nm dichroic mirror. Light bundles were then filtered at 405 nm and 495 nm and measured simultaneously by two separate photomultiplier tubes. Photomultiplier currents were converted to voltages and digitized and analyzed with a personal computer. After subtracting the autofluorescence for both emission wavelengths (F_auto,405 and F_auto,495, respectively), the ratio (R) of the 405-nm signal and the 495-nm signal was calculated as follows:

(E1)

In 11 muscles not subjected to sustained ischemia, fluorescence signals were calibrated in order to calculate [Ca²⁺]_i. R_max (R at saturating [Ca²⁺]_i) was determined by perfusing with 95% N₂/5% CO₂–bubbled Tyrode's solution to which a Ca²⁺ ionophore (0.1 mmol/L ionomycin) and a Na⁺-K⁺ exchanger (0.1 mmol/L gramicidin) had been added. To determine R_min (R at depleted [Ca²⁺]_i) a 100% N₂–bubbled Ca²⁺-free HEPES-buffered solution was used containing (mmol/L) EGTA 20, ionomycin 0.1, and gramicidin 0.1. [Ca²⁺]_i (nmol/L) was calculated according to the equation defined by Grynkiewicz et al¹⁸ :

(E2)

where K_d, the indo 1 dissociation constant for Ca²⁺, is 250 nmol/L. F_495,min and F_495,max are the background corrected emission intensities at 495 nm of indo 1 in Ca²⁺-depleted and Ca²⁺-saturated preparations, respectively. Fig 2 shows an example of fluorescence signals corrected for autofluorescence and a single transient of the calculated [Ca²⁺]_i. Experimental protocols comprised a 60-minute period of ischemia, after which reperfusion and calibration were not feasible. In these instances, we used the ratio as an indicator of the [Ca²⁺]_i.

View larger version (21K): [in this window] [in a new window]   Figure 2. Top, Original recordings of fluorescence signals at 405 and 495 nm, corrected for autofluorescence (F405 and F495). Bottom, Calculated [Ca2+]i transient and force signal (dashed line) in a perfused papillary muscle under control conditions. Excitatory pulse (S) is applied at the muscle apex at t=40 milliseconds.

To exclude the possibility that residual unhydrolyzed indo 1-AM remained in the cells after 30 minutes of washout, five preparations were perfused with Tyrode's solution to which 10 mmol/L MnCl₂ and 0.1 mmol/L ionomycin had been added. Mn²⁺ only quenches deesterified indo 1, which is the Ca²⁺-sensitive form. Fluorescence after the quenching procedure includes fluorescence from unhydrolyzed indo 1-AM and autofluorescence. In four hearts not loaded with indo 1, fluorescence signals were recorded during 90 minutes of perfusion and 50 minutes of ischemia (n=2) or iodoacetate infusion and subsequent ischemia (n=2) in order to evaluate changes in autofluorescence. The degree of possible mitochondrial indo 1 loading was assessed in three papillary muscles. Administration of 100 µmol/L MnCl₂ (in the absence of ionomycin) for 60 minutes selectively quenches cytosolic indo 1. Residual fluorescence is a measure of the amount of indo 1 compartmentalized in the mitochondria.¹⁹ In four indo 1–loaded hearts, the contribution of endothelial fluorescence to total fluorescence was evaluated by administration of bradykinin (10 µmol/L).²⁰ Bradykinin selectively increases cytosolic Ca²⁺ in endothelium.

Measurement of R_t
Measurement of R_t is based on the method first described by Weidmann²¹ and later applied to the perfused rabbit papillary muscle by Kleber and colleagues.^{1 15} The cylindrical shape of the papillary muscle and the homogeneous distribution of resistance along the longitudinal axis permit cable analysis in this preparation during conditions of normal perfusion and ischemia. According to cable theory, longitudinal R_t consists of intracellular (r_i) and extracellular (r_o) longitudinal resistances in parallel, where r_i is the series resistance of the intracellular space and the gap junctions and r_o is the resistance of the extracellular space. During ischemia, the onset of cellular uncoupling can be appreciated as a sudden increase of R_t that is caused by an increase of r_i.¹ This increase of R_t was used to assess the onset of uncoupling. We defined the onset of uncoupling as the moment after the induction of ischemia at which R_t rises at least 10% above its baseline level and subsequently continues to rise.

A 7-ms subthreshold current pulse was applied at the apex of the papillary muscle, and the voltage drop between two extracellular electrodes was measured (Fig 1). Extracellular Ag/AgCl electrodes were connected to the surface of the muscle via salt bridges consisting of fine silk threads led through thin polyethylene tubes, placed on either side of the area of tissue used for fluorescence measurements. Longitudinal electrical resistance (r_t, in /cm) was calculated as follows:

(E3)

where V_o is the voltage drop between the extracellular electrodes, x is the distance between the two extracellular electrodes, and I is the strength of the subthreshold current pulse.

Normalizing for diameters of different preparations' R_t (in ·cm) is calculated as follows:

(E4)

where A is the area of transverse section between the extracellular electrodes (cm²).

Interelectrode distance and muscle diameter were measured through a micrometer in the eyepiece of the fluorescence microscope.

An excitatory current pulse (twice threshold and 1-millisecond duration) was delivered 20 milliseconds after the start of the subthreshold pulse (basic cycle length, 450 milliseconds) to activate the apical end of the muscle. A differential electrogram was measured between the two extracellular electrodes.

Experimental Groups
Four experimental groups were studied. In group 1 (n=6), sustained ischemia was induced after 30 minutes of control perfusion. In group 2 (n=6), sustained ischemia was preceded by the following preconditioning protocol: 5 minutes of ischemia, 15 minutes of reperfusion, 5 minutes of ischemia, and 5 minutes of reperfusion. In group 3 (n=4), 1 mmol/L iodoacetate (blocker of glycolysis) was administered 2 minutes before sustained ischemia. This procedure inhibits anaerobic metabolism and thus production of lactate during the subsequent ischemic episode. Sustained ischemia in group 3 was induced after 30 minutes of control perfusion. In group 4 (n=6), [Ca²⁺]_i was increased by adding 0.1 mmol/L ionomycin and 0.1 µmol/L gramicidin to the perfusate. Perfusion pressures were increased up to 100 mm Hg to maintain adequate flow. In three experiments from this group, mechanical activation was prevented by administering 10 mmol/L 2,3-butanedione monoxime (BDM, an inhibitor of excitation contraction coupling).

Measurement of pH_o
In three preparations from group 1 and in three preparations from group 3, pH_o was measured. The pH electrode consisted of a 100-µm silver wire locally covered with a H⁺-sensitive membrane. This membrane contained (by weight) 23% polyvinyl chloride, 76% dioctylsebacicacid, and 1% H⁺ ionophore cocktail A (Fluka). The potential difference between this extracellular H⁺–sensitive electrode and an extracellular reference electrode was converted to changes in pH_o after in vitro calibration.

Data Acquisition and Analysis of Data
Signals from the extracellular electrodes were DC-amplified by high input impedance amplifiers. Electrograms, current signals, and output of the photomultipliers and of the force transducer were digitized, stored, and analyzed on a personal computer. Sampling rate was 4 kHz. The recording interval was 1 minute in groups 1 and 2 and 0.5 minutes in group 3. Data are expressed as mean±1 SEM. Statistical analysis was performed by the paired Student's t test or by the Mann-Whitney U test, as appropriate.

Similar to the definition of the onset of uncoupling (see above), we defined the onset of the increase of [Ca²⁺]_i and the start of contracture in each individual experiment as the moment after the induction of ischemia at which the ratio or resting tension increased at least 10% above baseline and continued to increase.

	Results

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Control Experiments for Indo 1 Fluorescence Measurements
Background fluorescence measured before indo 1 loading constituted at most 12.5% of the final diastolic fluorescence levels after indo 1 loading at both emission wavelengths. Fluorescence after quenching indo 1 in five preparations was not different from background fluorescence, indicating the absence of significant intracellular unhydrolyzed indo 1-AM. In four muscles not loaded with indo 1, background fluorescence levels were stable during 90 minutes of control perfusion. After the induction of ischemia, background fluorescence levels maximally increased by 25±5% at 405 nm and by 70±6% at 495 nm during the first minute and were stable during the remainder of the ischemic episode. This implies that during diastole autofluorescence at 405 nm increased from 12.5% to 15.5% of final fluorescence levels, whereas autofluorescence at 495 nm increased from 12.5% to 21% after the induction of ischemia. Likewise, during systole relative autofluorescence increased from 9.5% to 12.0% at 405 nm and from 15.5% to 23.5% at 495 nm. Since during the entire protocol preischemic background fluorescence levels are subtracted (see Equation 1), this causes overestimation of fluorescence levels at both wavelengths as soon as ischemia is induced. These disproportionate changes decrease the diastolic and systolic ratio by 5% and 8%, respectively. The larger effect of NAD(P)H fluorescence on the systolic ratio compared with the diastolic ratio rapidly diminishes as the systolic ratio approaches the diastolic ratio during the first minutes of ischemia.

During indo 1 loading, developed force decreased. After indo 1 washout, peak tension was only 8±1% lower compared with preloading conditions, whereas time to peak tension and time to 50% relaxation were similar. Treatment with 100 µmol/L MnCl₂ progressively quenched fluorescence intensities. After 40 minutes of infusion, fluorescence levels at 405 and 495 nm had decreased by 90±5% and 88±3%, respectively, and remained constant during the remainder of the 60-minute period. MnCl₂ infusion did not significantly affect the contraction signals.¹⁹ Endothelial indo 1 loading is another potential artifact in fluorescence measurements. We assumed limited indo 1 loading in the endothelium, since bradykinin infusion increased diastolic and systolic ratio levels by only 9±2% and 7±2%, respectively, in four experiments. Administration of bradykinin increased flow by 10±3%.

In 11 indo 1–loaded muscles subjected to the calibration procedure, mean diastolic [Ca²⁺]_i was 160±13 nmol/L, and mean peak systolic [Ca²⁺]_i was 832±33 nmol/L. Fig 2 shows a simultaneous registration of the Ca²⁺ transient and the contraction signal under control conditions.

Sustained Ischemia
Fig 3 shows simultaneous recordings of the indo 1 ratio, the developed tension, and the bipolar electrogram of a typical experiment from group 1 during control perfusion and after 2 and 20 minutes of ischemia. After 2 minutes of ischemia, the systolic ratio and the developed tension had decreased substantially, whereas the diastolic ratio and resting tension were unchanged. The subthreshold voltage drop between the two extracellular electrodes (V_o) had increased by 20% after 2 minutes of ischemia, corresponding to a rise in r_o shortly after the induction of ischemia.¹ After 20 minutes of ischemia, R_t had increased to 230% of control, and local electrical activation following the stimulus was absent. The changes at 20 minutes of ischemia indicate inexcitability and uncoupling of the myocytes, high intracellular Ca²⁺, and contracture.

View larger version (13K): [in this window] [in a new window]   Figure 3. Simultaneous recordings from a papillary muscle of group 1 during control perfusion (left panels) and after 2 and 20 minutes of ischemia (middle and right panels). Fluorescence measurements (indo 1 ratio) are localized to a small area on the papillary muscle, whereas the force signal (tension) represents the lumped mechanical activity of the entire preparation. The bipolar extracellular electrogram (E1-E2) consists of three components: (1) a negative subthreshold voltage drop (Vo), from which tissue resistance (Rt) is calculated, followed by a compensatory positive pulse and (2) the stimulus artifact from an excitatory current pulse (S) followed by (3) local electrical activation (absent in right panel). After 2 minutes of ischemia (middle panels), Ca2+ transient and developed tension have declined and Vo has increased by 20%. After 20 minutes of ischemia (right panels), Ca2+ transients and contractions have disappeared, and diastolic Ca2+ and resting tension are high. Cellular uncoupling is indicated by 130% increase of the Vo. Note that the time scale for the electrograms is different for the indo 1 ratio and tension.

Fig 4 shows the diastolic and systolic indo 1 ratios, R_t, and the resting and developed tensions of the same experiment as shown in Fig 3. After 4 minutes of ischemia, fluorescent transients and contractile activity had disappeared. Diastolic ratio and resting tension started to increase after 10 minutes of ischemia. The onset of uncoupling occurred at 12 minutes of ischemia. Fig 5 shows the time course of averaged values of R_t and diastolic ratio during ischemia from all experiments of group 1. Because of interexperimental variability in fluorescence levels and time to uncoupling, we expressed diastolic ratio as a percentage of preischemic levels and defined a relative time scale using the moment of the onset of uncoupling as t=0 in individual experiments. In all preparations of group 1, the increase in fluorescence ratio preceded the onset of uncoupling.

View larger version (14K): [in this window] [in a new window]   Figure 4. Plots showing diastolic and peak systolic fluorescence indo 1 ratio (top panel), tissue resistance (Rt, middle panel), and resting and developed tensions (bottom panel) during ischemia in the same experiment as shown in Fig 3. Values at t=0 are preischemic control values. Systolic Ca2+ levels and developed tension rapidly decline after the induction of ischemia. Small initial increase of Rt is caused by a rise in the extracellular longitudinal resistance. Diastolic Ca2+ and resting tension start to increase at 10 minutes of ischemia, followed by an abrupt increase of Rt at 12 minutes of ischemia, indicating the onset of uncoupling.

View larger version (18K): [in this window] [in a new window]   Figure 5. Average tissue resistance (Rt) and changes in ratio of control preparations (group 1, n=6) during ischemia plotted on a relative time scale. Moment at which uncoupling started was assigned t=0 for each experiment. See text for further details.

On average, the ratio started to increase after 12.6±1.3 minutes, the onset of uncoupling was at 14.5±1.2 minutes, and contracture started at 12.6±1.5 minutes after the induction of ischemia (Table).

View this table: [in this window] [in a new window]   Table 1. Moments of Indo 1 Ratio Increase, Uncoupling,and Contracture After Induction of Ischemia

Sustained Ischemia Preceded by Ischemic Preconditioning
Fig 6 shows a typical example of an experiment from the preconditioning group. After induction of sustained ischemia, fluorescent transients and contractile performance declined rapidly. After an initial stable phase, the diastolic ratio and the resting tension began to increase at 23 minutes of ischemia; uncoupling began at 25 minutes of ischemia. In all ischemically preconditioned muscles exposed to sustained ischemia, the rise in Ca²⁺ preceded the onset of uncoupling. On average, the increase of the ratio started at 21.5±4.0 minutes; uncoupling, at 24.0±4.1 minutes; and contracture, at 23.0±5.3 minutes of ischemia (Table).

View larger version (15K): [in this window] [in a new window]   Figure 6. Effect of ischemic preconditioning on peak systolic and diastolic indo 1 ratio (top panel), tissue resistance (Rt, middle panel), and resting and developed tensions (bottom panel) during ischemia in a papillary muscle from group 2. Values at t=0 indicate values after preconditioning just before sustained ischemia. Systolic Ca2+ and developed tension rapidly decrease after induction of ischemia. Diastolic Ca2+ and resting tension start to increase after 23 minutes of ischemia. The rise in Rt after 25 minutes of ischemia marks the onset of uncoupling.

Sustained Ischemia Preceded by Metabolic Inhibition
Fig 7 shows a characteristic experiment from group 3. Pretreatment with 1 mmol/L iodoacetate 2 minutes before ischemia did not change the measured parameters during control perfusion. Ratio, R_t, and resting tension increased shortly after the induction of ischemia in metabolically inhibited muscles. In group 3, the ratio started to increase at 1.9±1.5 minutes; R_t, at 3.6±1.9 minutes of ischemia; and contracture, at 1.9±0.7 minutes of ischemia (Table).

View larger version (14K): [in this window] [in a new window]   Figure 7. Effect of pretreatment with 1 mmol/L iodoacetate on peak systolic and diastolic fluorescence ratio (top panel), tissue resistance (Rt, middle panel), and resting and developed tensions (bottom panel) during ischemia in an individual experiment. Values at t=0 indicate preischemic values after pretreatment with iodoacetate. Decreasing systolic Ca2+ and developed tension merge into the terminal increase of diastolic Ca2+ and resting tension at 1.0 minute of ischemia. The increase of Rt at 3 minutes of ischemia indicates the onset of uncoupling.

Relation Between [Ca²⁺]_i, Uncoupling, and Contracture
The Table summarizes the moments of Ca²⁺ rise, uncoupling, and contracture in all experimental groups during ischemia. Compared with control ischemia (group 1), preconditioning and metabolic blockade significantly postpone and advance, respectively, the Ca²⁺ increase, uncoupling, and contracture during ischemia (Mann-Whitney U test, P<.05). In each group, the onset of uncoupling is significantly later than the rise in [Ca²⁺]_i (paired t test, P<.01). The start of contracture is not statistically different from the moment of Ca²⁺ rise after the induction of ischemia. Fig 8 shows the moment of Ca²⁺ rise on the abscissa versus the onset of uncoupling on the ordinate during ischemia for each experiment of groups 1, 2, and 3. All points are above the line of identity, indicating that during ischemia the onset of uncoupling always followed Ca²⁺ rise. The average delay was 2.1±0.2 minutes.

View larger version (19K): [in this window] [in a new window]   Figure 8. The relation between the moment of diastolic Ca2+ rise (on the abscissa) and the moment of the onset of uncoupling (on the ordinate) after the induction of ischemia for all experiments. IAA+ischemia indicates sustained ischemia in preparations pretreated with 1 mmol/L iodoacetate; ischemia, sustained ischemia in control preparations; and PC+ischemia, sustained ischemia in ischemically preconditioned preparations. The oblique line is the line of identity. Uncoupling always follows the increase of intracellular Ca2+. The average interval is 2.1±0.2 minutes.

In order to further analyze the relation between intracellular Ca²⁺ and cellular uncoupling, [Ca²⁺]_i was increased under nonischemic conditions (group 4). In these experiments, calibration of the fluorescence signals and calculation of actual [Ca²⁺] was feasible. Diastolic and systolic [Ca²⁺] levels in this group were 161±26 nmol/L and 835±58 nmol/L, respectively. After administration of ionomycin and gramicidin, Ca²⁺ transients rapidly disappeared, and diastolic Ca²⁺ started to rise. Uncoupling started when intracellular Ca²⁺ exceeded 685±85 nmol/L; this level was reached 0.9±0.3 minutes after the start of Ca²⁺ rise. In the absence of BDM, [Ca²⁺]_i and resting tension increased simultaneously, whereas in the presence of BDM, contracture followed the rise in Ca²⁺ after a delay of 1.5±0.5 minutes, and the rate of rise of contracture was significantly reduced. Attenuation of contracture by infusion of BDM had no effect on increase of [Ca²⁺]_i and cellular uncoupling after infusion with ionomycin and gramicidin.

pH_o During Ischemia in Control Preparations and in Metabolically Inhibited Preparations
In three muscles from group 1, we measured pH_o. After the induction of ischemia, there was a characteristic²² acidification: after 20 minutes of ischemia, pH_o was, on average, 1.4±0.05 pH units lower than during control perfusion.

In three metabolically inhibited preparations from group 3, in which glycolysis was blocked, pH_o maximally decreased 0.15 pH units after the induction of ischemia. Iodoacetate did not change preischemic pH_o.

	Discussion

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In the present study, we have shown that increase of [Ca²⁺]_i, uncoupling, and contracture during ischemia are closely coupled in untreated, preconditioned, and metabolically inhibited myocardium. The onset of uncoupling always follows the increase of [Ca²⁺]_i during ischemia, with an average interval of 2.1±0.2 minutes. The moment of the onset of contracture after the induction of ischemia is not different from the moment of rise in [Ca²⁺]_i. Under nonischemic perfused conditions, estimated threshold [Ca²⁺] for cellular uncoupling is 685±85 nmol/L.

Methodological Considerations
Many factors can artifactually influence fluorescence measurements on the endocardial surface of myocardial tissue. Ca²⁺ buffering was considered low, since contraction signals before and after loading were nearly identical.²³ Fluorescence from endothelial indo 1, quantified by selectively increasing endothelial [Ca²⁺]_i by infusion with bradykinin,²⁰ constituted a minor component of total fluorescence. Significant contribution of fluorescence from unhydrolyzed indo 1-AM was excluded, since fluorescence levels after quenching with 10 mmol/L Mn²⁺ and 0.1 mmol/L ionomycin were not different from background fluorescence levels before loading.²³

In contrast to iontophoretic loading, indo 1-AM loading may cause considerable mitochondrial uptake of indo 1. Since after perfusion with 100 µmol/L MnCl₂, fluorescence intensities at 405 and 495 nm fell by 90% and 88%, we presume that only a small amount of indo 1 is compartmentalized in the mitochondria.¹⁹ Loading conditions applied in the present study (30°C and 5 µmol/L indo 1-AM in the perfusate) suppress mitochondrial loading²³ compared with studies^{19 24} showing significant mitochondrial fluorescence (23°C and 25 µmol/L indo 1-AM). This was confirmed by a study on rabbit hearts showing that after indo 1-AM loading under conditions similar to the conditions used in the present study, fluorescence from isolated mitochondria was only 3.6% of total fluorescence.²⁵ In addition, another report showed that after loading in cultured cells with low concentrations of indo 1-AM (3 µmol/L) at 37°C, indo 1 is hardly subject to compartmentalization.²⁶ Diastolic and systolic [Ca²⁺] levels measured in iontophoretically loaded rat trabeculas²⁷ correspond to [Ca²⁺] measured in the present study.

Motion artifact was canceled using the ratio of the two emission light signals. Calculated diastolic and systolic [Ca²⁺] levels during control perfusion were in agreement with other reports, in which indo 1, aequorin, or fura 2 was used to measure [Ca²⁺]_i in myocardial tissue.^{27 28 29} The K_d of indo 1 used in the present study is an in vitro value that is commonly extrapolated to intracellular circumstances.^{18 30 31} However, recently, some controversy has arisen on the assumption that the K_d is not different intracellularly.^{30 31 32}

No experimental data are available on the rate of rise of Ca²⁺ transients in intact perfused rabbit myocardium at 37°C. However, when comparing the Ca²⁺ transient as shown in Fig 2 to transients measured at lower temperatures^{33 34} or in other species^{19 23 35} it might be argued that the rising limb of the present Ca²⁺ transients is relatively slow. This could be explained by the fact that fluorescence is measured from a rather large area and that an apparent slowing of the Ca²⁺ transient occurs because of propagation of the activation front. To experimentally test this hypothesis, we replaced the gaseous atmosphere of the preparation with Tyrode's solution at 37°C in three experiments. Under these conditions, we measured Ca²⁺ transients and compared stimulation at the apex of the papillary muscle to homogeneous field stimulation of the entire preparation. Instantaneous activation after field stimulation increased the rate of rise of the Ca²⁺ transient by 34% compared with propagating activation after apical stimulation. Ca²⁺ transients during field stimulation were similar to transients measured in isolated myocytes.^{19 36 37} Therefore, we conclude that because of the relatively large area of fluorescence measurements, the rising limb of the Ca²⁺ transient is affected by propagation.

The changes in ratio, due to background fluorescence increasing shortly after the induction of ischemia [mainly NAD(P)H fluorescence], were small. The intracellular presence of indo 1 did not modify the ischemic processes ultimately leading to irreversible damage, since the times to onset of uncoupling and contracture during ischemia in the present study were not different from values measured in preparations not loaded with indo 1.^{1 4} The absence of erythrocytes did not change the time course of uncoupling or the effect of preconditioning during sustained ischemia.⁴

Effects of Sustained Ischemia in Untreated, Preconditioned, and Metabolically Inhibited Preparations
After the induction of sustained ischemia in control muscles (group 1), diastolic Ca²⁺ levels were stable, whereas systolic Ca²⁺ levels and developed tension rapidly declined. The effect of NAD(P)H fluorescence causing an underestimation of maximally 8% of the systolic ratio during ischemia is small compared with the decline of the systolic ratio during the first minutes of ischemia. This rapid decline of the systolic ratio is in contrast to other studies showing that during the first minutes of ischemia diastolic and systolic Ca²⁺ levels increased while contractile force rapidly declined.^{25 29 33} The mechanism underlying the elevation of the Ca²⁺ transients during the first minutes of ischemia is controversial.^{25 29 33} This early increase of [Ca²⁺]_i during ischemia was not observed in intact ischemic hearts by nuclear magnetic resonance imaging.^{38 39} As stated previously by Lorell et al,²⁰ since in the former studies^{25 29 33} the degree of indo 1 loading in the endothelium was not assessed, elevation of endothelial Ca²⁺ during the first minutes of ischemia could explain this discrepancy. Furthermore, distinct methodological differences between the above-mentioned studies and the present study could further contribute to the disparity between experimental data.

In the present study, diastolic Ca²⁺ levels in the control group were stable until 12.6±1.3 minutes of ischemia. At that moment, [Ca²⁺]_i started to increase and continued to rise. Measurements of [Ca²⁺]_i with nuclear magnetic resonance imaging during sustained ischemia also demonstrate an abrupt Ca²⁺ increase after an initial stable period of 10 to 15 minutes of ischemia.^{40 41}

Ischemic preconditioning significantly postponed the rise in [Ca²⁺]_i during sustained ischemia. After an initial stable phase, the diastolic Ca²⁺ level started to increase at 21.5±4.0 minutes of sustained ischemia. Nuclear magnetic resonance measurements in ischemic rat hearts have also shown that ischemic preconditioning delays the ischemia-induced increase of [Ca²⁺]_i.^{42 43}

The onset of uncoupling in ischemically preconditioned muscles was postponed to 24.0±4.1 minutes of ischemia compared with 14.5±1.2 minutes in untreated muscles. The effect of preconditioning on the onset of uncoupling is in close agreement with previous data from our group obtained in the same model.⁴

Reports in the literature on the effect of preconditioning on the start of contracture are conflicting. Studies in both rat and rabbit hearts,^{44 45} including the present study, show that the start of contracture during sustained ischemia was postponed in preconditioned hearts compared with control hearts. Opposite results were obtained in studies involving the recovery of postischemic contractile function in rat hearts; these studies showed that preconditioning advanced ischemia-induced contracture during sustained ischemia.^{46 47} This discrepancy is surprising since preconditioning delays the development of necrosis,³ whereas contracture is strongly indicative of irreversible ischemic damage and necrosis.

Increases of [Ca²⁺]_i, uncoupling, and contracture during ischemia were significantly advanced after metabolic blockade. Pretreatment with iodoacetate blocks anaerobic metabolism and causes a rapid depletion of ATP during ischemia.¹⁴ The absence of lactate production minimizes acidification during ischemia. This was indirectly demonstrated by a relatively small decrease of pH_o during ischemia after metabolic inhibition. Metabolic inhibition significantly reduced ischemic acidification compared with no treatment. Changes in pH_o in the untreated group correspond to previous data.^{22 48}

Relations Between [Ca²⁺]_i, Uncoupling, and Contracture
The close association between uncoupling and contracture during ischemia corresponds to previous reports.^{2 11 13} However, data on the relation between [Ca²⁺]_i and contracture are conflicting. Reports on contracture starting before as well as after the rise in [Ca²⁺]_i have been published previously.^{39 40 49} We did not observe a significant time difference between the increase of [Ca²⁺]_i and the start of contracture during ischemia.

The increase of [Ca²⁺]_i preceded the onset of uncoupling during ischemia, with an average interval of 2.1±0.2 minutes (Fig 8). The relation between [Ca²⁺]_i and uncoupling was maintained under ischemic conditions with reduced acidification (group 3).

Furthermore, we could also demonstrate this relation in the absence of ischemia by perfusing with ionomycin and gramicidin. In these experiments, a threshold [Ca²⁺] for cellular uncoupling of 685±85 nmol/L was found. This threshold concentration is consistent with previous reports.^{6 50 51} Since the threshold [Ca²⁺] is within the range of a normal transient, one might wonder if gap junction conductance decreases during a normal Ca²⁺ transient. The process of uncoupling is probably too slow to produce a sizable decrease of gap junction conduction during a Ca²⁺ transient under normal conditions.⁶

We can only speculate about at which [Ca²⁺]_i level the myocytes start to uncouple under ischemic conditions, because it is impossible to calibrate indo 1 signals in irreversibly damaged ischemic myocardium. Moreover, since K_d of indo 1 increases at low pH, [Ca²⁺] levels are underestimated when preischemic K_d is used to calculate [Ca²⁺]_i in acidified circumstances.⁵² At the moment of onset of uncoupling, the "diastolic" ratio in groups 1 and 2 was about the same as the peak systolic ratio during control perfusion, whereas in group 4 the indo 1 ratio at the moment of uncoupling was always lower than preischemic systolic values. Because at the moment of uncoupling the ratio as well as K_d are higher in groups 1 and 2 than in group 4, threshold [Ca²⁺] for uncoupling during ischemia in groups 1 and 2 is higher than that found in group 4 (685 nmol/L). Although many possible modulating factors change during ischemia, this discrepancy may at least partly be explained by the fact that at decreasing pH levels, higher [Ca²⁺] levels are required to obtain the same closing effect, as shown by Noma and Tsuboi.⁶

The interval between the start of Ca²⁺ increase and the moment of uncoupling is significantly smaller in group 4 (0.9±0.3 minutes) compared with the other groups (2.1±0.2 minutes). This is probably due to the fact that during ischemia the rate of rise of the indo 1 ratio was slower compared with group 4, requiring more time to reach the threshold [Ca²⁺] for cellular uncoupling.

In the present study, uncoupling occurred under conditions of small or absent acidification (groups 3 and 4). Although pH plays a modulating role in the interaction between Ca²⁺ and gap junctions,⁶ intracellular acidosis is not the main stimulus for cellular uncoupling, as already shown in isolated Purkinje fibers.⁵³ After 15 minutes of ischemia, pH_i in the rabbit papillary muscle is 6.5.²² Another study clearly demonstrated that at this pH (and at unchanged [Ca²⁺]_i), gap junctional conductance is still unaltered.⁶ The observation that uncoupling evolves similarly in ischemia and hypoxia (with only minor acidosis)⁵⁴ further corroborates the fact that pH is a subordinate trigger for cellular uncoupling during ischemia.²

Although in ischemic preparations contracture and uncoupling appear about simultaneously after the induction of ischemia, the physical disruption of gap junctions due to contracture is not a probable cause of cellular uncoupling. Attenuation of contracture by BDM had no effect on Ca²⁺ increase⁵⁵ and uncoupling or on their interrelation. Furthermore, physical disruption can be excluded in isolated cell pairs showing Ca²⁺-induced uncoupling.⁸

Study Limitations
We cannot be definite about the moments of uncoupling and Ca²⁺ rise on a cellular level. Due to a spatial and temporal heterogeneity of uncoupling on a cellular level, R_t might have started to increase when a considerable number of cells were already uncoupled. Moreover, cells that were not superficial could have contributed to an increase in R_t, whereas their Ca²⁺ rise was not registered by our epifluorescence microscope.

In this model, we have not explicitly measured other possible factors of uncoupling, such as pH_i or ATP.^{6 9 12} Therefore, we cannot completely exclude their potential contribution to the process of uncoupling during ischemia. The effects of pH (see above) and our measurements of pH_o cancel an important role of pH. The fact that ATP levels are very similar from 15 minutes of ischemia in preconditioned and control tissue,^{42 56} whereas the rise in Ca²⁺ and the onset of uncoupling are postponed after preconditioning, suggests that ATP is not the prominent trigger for ischemia-induced uncoupling.

In summary, Ca²⁺ is the primary trigger for cellular uncoupling during ischemia in normal, preconditioned, and metabolically inhibited myocardium. Contracture is closely associated with the increase of intracellular Ca²⁺ during ischemia. Under nonischemic conditions, threshold [Ca²⁺] for cellular uncoupling is estimated at 685±85 nmol/L. Ischemia-induced acidification augments the [Ca²⁺] required for electrical cellular uncoupling.

Received November 20, 1995; accepted May 7, 1996.

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