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Cardiovascular Research 1999 44(1):101-112; doi:10.1016/S0008-6363(99)00179-0
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Intracellular Ca2+ and delay of ischemia-induced electrical uncoupling in preconditioned rabbit ventricular myocardium{star}

Lukas R.C. Dekker*, Ruben Coronel, Ed VanBavel, Jos A.E. Spaan and Tobias Opthof

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

* Corresponding author. Tel.: +31-20-566-3266; fax:+31-20-697-5458 L.R.Dekker{at}AMC.UVA.NL

Received 30 December 1998; accepted 11 May 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 
Objective: Short periods of ischemia and reperfusion alter myocardial Ca2+ handling and temporarily induce a mild increase of [Ca2+]i. We hypothesized that these alterations are involved in the cardioprotective mechanism of ischemic preconditioning, possibly via a Ca2+-dependent activation of protein kinase C (PKC). Methods and Results: In arterially perfused rabbit papillary muscles, we determined Ca2+ transients (indo 1) and indicators of the onset of irreversible ischemic damage, including [Ca2+]i rise, electrical uncoupling and contracture. We tested three protocols of ischemic preconditioning (1–3). In addition, the effects of infusion of staurosporine, a blocker of PKC (4), or glibenclamide, a blocker of K+ATP channels (5) were analyzed. Furthermore, pretreatment with phorbol 12-myrisate 13-acetate (PMA), an activator of PKC (6), or cyclopiazonic acid (CPA), an inhibitor of the SR Ca2+ pump (7) was tested. During periods of reperfusion in the preconditioning protocols, the duration of the Ca2+ transient and the diastolic Ca2+ level temporarily increased. Only if sustained ischemia was induced during these changes of the transients, cardioprotection was present. Similar alterations of the Ca2+ transient concurring with cardioprotection were induced by pretreatment with PMA as well as CPA. Staurosporine and glibenclamide antagonized the reperfusion-induced changes of the Ca2+ transients as well as cardioprotection. If reperfusion was extended until the Ca2+ transient had normalized, cardioprotection was also absent. Under all conditions tested, the diastolic Ca2+ elevation or the Ca2+ transient prolongation prior to sustained ischemia correlated with the postponement of ischemic injury. Conclusions: A pre-ischemic mild increase of [Ca2+]i presents a common effector of preconditioning. Our data suggest that activation of PKC or opening of K+ATP channels may initiate the pathway leading to an alteration of Ca2+ metabolism and a protected status of the myocardium.

KEYWORDS Experimental; Heart; Pathophysiology; Preconditioning; Calcium; Ischemia; Reperfusion


   1 Introduction
Top
 Abstract
 1 Introduction
2 Materials and methods
 3 Results
 4 Discussion
 References
 
Ischemic preconditioning activates protective mechanisms postponing the onset of irreversible myocardial damage during subsequent sustained ischemia [1]. The precise mechanism of ischemic preconditioning remains poorly understood. Although various mediators of preconditioning seem to converge in activation of protein kinase C (PKC) [2,3], the role of PKC remains controversial [4]. Some studies have shown that PKC activation is not mandatory for cardioprotection [5,6]. Miyawaki et al. have recently shown that short repetitive periods of Ca2+ depletion and repletion, called ‘Ca2+-preconditioning’, protect the myocardium during subsequent sustained ischemia to an extent that is comparable to ischemic preconditioning [7]. Similarly, Meldrum et al. have shown that pre-ischemic infusion of ryanodine is cardioprotective during ischemia in rat hearts [8]. Since no actual measurements of [Ca2+]i were performed in any of these studies, it could only be inferred that a pre-ischemic mild rise of [Ca2+]i induces cardioprotection mediated via Ca2+-dependent activation of PKC [7–9].

Glibenclamide, which closes the ATP-sensitive potassium channel (K+ATP channel), abolishes the protective effects of preconditioning [10]. The protective action of openers of these channels corroborates the involvement of K+ATP channels in preconditioning [11]. However, the mechanism is undetermined, since several studies have shown that action potential shortening or opening of the sarcolemmal K+ATP channels is not a prerequisite for cardioprotection by K+ATP-channel openers [12,13]. Interestingly, an extensive study by Armstrong et al. has indicated that K+ATP-channel opening may act as an initiator of preconditioning via a PKC-dependent pathway [14].

Numerous studies have demonstrated that short periods of ischemia and reperfusion temporarily induce a mild increase of [Ca2+]i possibly by decreasing Ca2+ uptake or increasing Ca2+ efflux from the sarcoplasmic reticulum (SR) [15–17]. In the present study we estimated [Ca2+]i and analyzed the relationship between the changes of [Ca2+]i and the extent of cardioprotection under different experimental conditions. First, we used three different preconditioning protocols, in which one or two 5-min periods of ischemia were followed by 5 or 15 min of reperfusion. Secondly, we further examined the interrelations between [Ca2+]i, PKC and cardioprotection by activating PKC with phorbol 12-myrisate 13-acetate (PMA) in control myocardium and by inhibiting PKC in ischemically preconditioned myocardium with staurosporin [18]. Thirdly, we analyzed the alterations of Ca2+ transients in preparations, in which the protective effect of preconditioning is blocked by glibenclamide [10]. Finally, we tested whether administration of cyclopiazonic acid (CPA), which selectively inhibits sarcoplasmic reticulum (SR) Ca2+-ATPase [19,20], induces cardioprotection similar to Ca2+-preconditioning or ryanodine infusion [7,8]. Indo-1 fluorescence ratios and the extent of protection against ischemic damage were determined in the arterially perfused papillary muscle of the rabbit. In this rabbit model the onset of ischemic damage and, thus, the effect of cardioprotective interventions, are determined by three markers of ischemic damage, including the start of the terminal Ca2+ rise, electrical uncoupling and contracture [21–23].

The present study shows that during the periods of reperfusion in the preconditioning protocol the duration of the Ca2+ transient and the diastolic Ca2+ level temporarily increase. The extent of the changes of the Ca2+ transient at the start of sustained ischemia correlates with the degree of cardioprotection during sustained ischemia. The reperfusion-induced alterations of the Ca2+ transient as well as the delay of ischemic injury are qualitatively equaled by PKC activation. PKC blockade antagonizes the reperfusion-induced changes of the Ca2+ transient and cardioprotection in ischemically preconditioned myocardium. Likewise, K+ATP-channel blockade antagonizes the reperfusion-induced alterations of the Ca2+ transient as well as cardioprotection. Alternatively, inhibiting SR Ca2+ uptake by perfusing with CPA increases [Ca2+]i and induces a cardioprotective effect during subsequent sustained ischemia. It is suggested that an alteration of myocardial Ca2+ handling resulting in a mild increase of [Ca2+]i is a common effector in the complex mechanism of cardioprotection induced by preconditioning.


   2 Materials and methods
Top
 Abstract
 1 Introduction
 2 Materials and methods
3 Results
 4 Discussion
 References
 
2.1 Preparation
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). The technique to simultaneously measure [Ca2+]i, specific tissue resistance (Rt) and mechanical activity from the isolated arterially perfused papillary muscle of the right ventricle of the rabbit has been described in detail previously [22]. Briefly, New Zealand White rabbits (3–3.5 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 Tyrode’s solution at 4°C (for composition see below). The atria and the left ventricular free wall were removed and the left side of the interventricular septum was secured to a silicone plate. Within 4-min after excision of the heart the septal artery was cannulated and perfusion was started. After resection of the right ventricular free wall the preparation was positioned in an organ chamber. The papillary muscle was horizontally connected to a force transducer (Sensonor AE801, Norway) by a ligature around the tendon (Fig. 1). Selected papillary muscles had an average length of 4.0±0.1 mm, a diameter of 1.1±0.1 mm and a single insertion of the tendon. The resting length of the muscle was slightly stretched by about 15% of slack length. A fine silver stimulating wire was tied around the muscle apex. A large Ag/AgCl electrode on the perspex plate served as ground. The preparation was surrounded by a water saturated gas mixture of 95% O2+5% CO2. Myocardial temperature was maintained at 37°C by means of the thermostated organ chamber.


Figure 1
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  		Fig. 1 Left panel: Schematic representation of the preparation. Subthreshold and excitatory current pulses are injected at the muscle apex and measured by a current meter (Amp). E1 and E2 represent the extracellular electrodes. The objective of the fluorescent microscope is heated to prevent condensation of water on its lens. Oxygen tension and temperature of the water-saturated surrounding atmosphere (dotted area) are continuously monitored. Right panel: Simultaneous registrations of the electrogram, fluorescent ratio and mechanical activity during control conditions. The bipolar extracellular electrogram (E1–E2) consists of three components: (1) a negative subthreshold voltage drop (Vo), from which Rt is calculated (followed by a compensatory positive pulse) and (2) the excitatory stimulus artifact (S) followed by (3) local electrical activation. Tension (mN) represents the lumped mechanical activity of the entire preparation. Note different time scales.

 
2.2 Perfusion
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, HPO42– 0.4, probenecid 0.1, glucose 10, insulin 10 U/l and fetal calf serum (FCS) 2.0%. Probenecid is an anion transport blocker and has been shown to prevent loss of tetracarboxylate fluorescent indicators [24,25]. The perfusate was gassed with a mixture of 95% O2 and 5% CO2 to yield a pH of 7.4. A flow-rate of 1.1–1.4 ml/min/g (Transonic T206 flowmeter) was maintained by a constant pressure perfusion system (35–45 mmHg). Ischemia was induced by stopping flow and at the same time replacing the 95% O2 and 5% CO2 gas mixture in the surrounding atmosphere by 95% N2 and 5% CO2. During ischemia oxygen tension in the organ chamber was less than 3 mmHg.

2.3 Fluorescence measurements
Adequate loading with indo 1 (Molecular Probes Inc., Eugene, OR, USA) was achieved by recirculating 30 ml Tyrode’s solution containing 5 µmol/l indo 1 AM (initially dissolved in dimethyl sulfoxide containing 6% w/v pluronic F-127, with final concentrations 0.5% v/v and 0.05% w/v, respectively), 5% v/v FCS and 1 mmol/l probenecid, for 25–35 min at 30°C. After a 30-min period of washout at 37°C, fluorescence of the heart had increased by a factor 8–10 compared to fluorescence measured before loading (autofluorescence).

A circular area on the surface of the papillary muscle with a diameter of 1.3 mm was illuminated by 340 nm excitation light from a xenon-arc lamp (75 W) via a 10x objective (NA 0.50, Fluar, Zeiss). Emitted light was measured simultaneously by two photomultiplier tubes (Hamamatsu R1166) at 405 nm and 495 nm. Following I/V conversion (±100 nA/V) the ratio (R) of the 405-nm and 495-nm signals, after subtracting the autofluorescence at both wavelengths, was used as an indicator of [Ca2+]i [26]. Calibration of fluorescence signals and calculation of actual [Ca2+]i was not feasible, since at the conclusion of the experiments the myocardium was irreversibly damaged by sustained ischemia. In a previous study we calculated actual [Ca2+]i in the arterially perfused rabbit papillary muscle according to the formula defined by Grynkiewicz [22,26]. Under normal conditions diastolic and systolic [Ca2+]i were 160 and 830 nmol/l, respectively.

Three indo 1-loaded preparations were treated for 30 min with the excitation–contraction uncoupler L 2,3 butanedione monoxime (BDM; 10 mmol/l) in order to assess the contribution of motion artifacts in the observed Ca2+ transients. Fig. 2 shows a typical example. BDM treatment eliminating mechanical activity did not affect the diastolic level or duration of the Ca2+ transient; it reduced Ca2+-transient amplitude by 11±1%.


Figure 2
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  		Fig. 2 Typical example of the effects of 10 mmol/l L 2,3 butanedione monoxime (BDM) on Ca2+ transient and contraction signal.

 
We evaluated the effects of NAD(P)H fluorescence during repeated cycles of 5-min periods of ischemia and 15-min periods of reperfusion in four hearts not loaded with indo 1. During the first minute of ischemia autofluorescence levels increased 28±6% at 405 nm and 63±5% at 495 nm and were stable during the remainder of the ischemic episode. This implies that during ischemia diastolic autofluorescence at 405 nm increased from 12.5% to 16% of final fluorescence levels whereas autofluorescence at 495 nm increased from 12.5% to 20%. Due to these disproportionate changes the ratio was underestimated by about 5% after the induction of ischemia. Elevated background fluorescence returned to control values within the first minute of reperfusion. Infusion of CPA, staurosporine or glibenclamide did not affect autofluorescence levels as assessed in three hearts not loaded with indo 1.

Duration of the Ca2+-dependent fluorescent transient was determined by measuring the interval (in ms) between the moment of 10% rise and the moment of 90% decay relative to the maximal range between diastolic and peak systolic ratio levels.

2.4 Measurement of tissue resistance
The cylindrical shape of the papillary muscle and the homogeneous distribution of resistance along the longitudinal axis permit cable analysis during conditions of normal perfusion, ischemia and reperfusion [21]. In this model longitudinal tissue resistance (rt) consists of the intracellular (ri) and extracellular (ro) longitudinal resistances in parallel, where ri is the series resistance of the intracellular space and the gap junctions.

Two extracellular Ag/AgCl electrodes were placed on either side of the area of tissue used for fluorescence measurements. Prior to the excitatory pulse a 7-ms subthreshold current pulse was applied at the apex of the papillary muscle. From the subthreshold voltage drop (Vo in the differential electrogram in Fig. 1) and the distance between two extracellular electrodes longitudinal electrical resistance (rt) was calculated [21]. To correct for differences in muscle dimensions, rt was multiplied by the surface area of transverse section between the extracellular electrodes, resulting in the total specific resistance, Rt. During ischemia the onset of cellular uncoupling can be appreciated as a sudden increase of Rt that is caused by an increase in ri [21].

An excitatory current pulse (S; twice threshold and 1-ms duration) was given 20 ms after the start of the subthreshold pulse (basic cycle length 400–450 ms) to activate the apical end of the muscle. A differential electrogram of the propagating activation front was measured between the two extracellular electrodes. Longitudinal conduction velocity (cm/s) was calculated from the conduction time between the two extracellular electrodes (time between the steepest deflection and inflection of the differential electrogram) and the interelectrode distance. Fig. 1 shows a typical example of the electrogram, fluorescence ratio and contraction signal measured simultaneously under normal conditions.

2.5 Experimental groups
Fig. 3 is a diagram of the protocols used. Protocols were started after 30 min of control perfusion following 30 min of indo-1 washout. In the control group (control) sustained ischemia of 45 min was induced after an additional 30 min of control perfusion. Three different preconditioning protocols (PC) preceding sustained ischemia were studied. PC-1: 5 min of ischemia, 15 min of reperfusion, 5 min of ischemia and 5 min of reperfusion. PC-2: 5 min of ischemia, 5 min of reperfusion, 5 min of ischemia and 15 min of reperfusion. PC-3: 5 min of ischemia and 5 min of reperfusion. In the PC+Stau group 0.1 µmol/l staurosporine was added during the second period of reperfusion of the PC-1 protocol. In the Stau group 0.1 µmol/l staurosporine infusion was started 5 min prior to sustained ischemia. In the PC+Glib 15 µmol/l glibenclamide was added to the perfusate during the second period of reperfusion of the PC-1 protocol, whereas in the Glib group 15 µmol/l glibenclamide was infused starting 5 min prior to sustained ischemia. In the PMA-group phorbol 12-myrisate 13-acetate (10 nmol/l) was administered for 5 min followed by 5 min of washout prior to sustained ischemia. We titrated a low dose of PMA, as it has been shown previously that high dosages of PMA have physiologically important effects that are not PKC-dependent [27]. Furthermore, the effect of the present concentration of PMA on flow was negligible. In the CPA group we administered 20 µmol/l cyclopiazonic acid starting 8 min prior to sustained ischemia. This is a specific inhibitor of SR Ca2+-ATPase inducing Ca2+ loss from the SR into the cytosol [19,28]. Sodium-nitroprusside (SNP; 30 µmol/l) was co-infused to prevent vasoconstriction and subsequent hypoperfusion caused by Ca2+ overload in vascular smooth muscle cells. In every experimental group four hearts were studied. In three additional experiments preparations were pretreated with SNP (30 µmol/l) for 8 min prior to sustained ischemia in order to evaluate a possible effect of SNP on the onset of irreversible ischemic damage. We preferred CPA over thapsigargin, since two independent studies have shown that the effects of thapsigargin also include other unknown actions, especially under conditions of ischemia and reperfusion [19,29].


Figure 3
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  		Fig. 3 Experimental design. n=4 in every group. See Section 2.5 for further details.

 
2.6 Data acquisition, analysis of data and statistics
Signals from the extracellular electrodes were DC-amplified by high input impedance amplifiers. Electrograms, current signals, output of the photomultipliers and of the force transducer were digitized, stored and analyzed with a personal computer. Sampling rate was 4 kHz. The recording interval was 1 min. Data are expressed as mean±1 standard error of the mean (SEM). Statistical analysis was performed by the Student’s t-test or by the Mann–Whitney test, as appropriate.

We defined the onset of uncoupling as the moment after the induction of ischemia at which Rt rises at least 10% above its baseline level and subsequently continues to rise. Likewise, the start of the terminal rise in [Ca2+]i and the start of contracture in individual experiments were defined as the moment after the induction of ischemia at which the diastolic ratio or tension increases at least by 10% above baseline and subsequently continues to rise.


   3 Results
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
4 Discussion
 References
 
3.1 The effects of ischemic preconditioning
Fig. 4 shows a typical example of the changes of the Ca2+ transients during the PC-1 protocol. After the induction of ischemia, systolic Ca2+ levels and transient duration rapidly decline, whereas diastolic Ca2+ does not change, as shown previously [22]. In all experimental groups the rate of decline of the systolic ratio during ischemia was equal; transients were absent within 5 min of ischemia. Within a few seconds after reperfusion, Ca2+ transients reappear. During reperfusion the duration of the Ca2+ transient and the diastolic and systolic levels temporarily increase. After 15 min of reperfusion, the transients are similar to the control. The Ca2+ transient after 5 min in the first period of reperfusion is equal to the Ca2+ transient after 5 min in the second period of reperfusion.


Figure 4
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  		Fig. 4 Changes of the Ca2+ transient of a typical experiment from the PC-1 group. Upper panel: Ratio of fluorescent signals (arbitrary units) and developed tension (mN) registered at moments indicated in lower panel. Lower panel: Changes of duration of the transient and systolic and diastolic ratio levels from same experiment as shown in upper panel. C, I and R indicate control conditions, ischemia and reperfusion, respectively. Third ischemic episode is the initial phase of sustained ischemia.

 
Similar reperfusion-induced changes of the Ca2+ transient configuration occur in all experiments of the PC-1,2,3 groups. The Ca2+ transient prolongs during all 20 periods of reperfusion (12 experiments). Elevation of the diastolic and systolic ratio levels during reperfusion is observed in 10 out of 12 preconditioned muscles. In the eight 15-min periods of reperfusion in the PC-1 and PC-2 protocols, the changes of the Ca2+ transient configuration peak between 3 and 5 min (range) and disappear within 11–14 (range) min of reperfusion. On average in the PC-1,2,3 groups, after 5 min of reperfusion following the 5-min period of ischemia (n=20) the transient duration increases to 225±5 ms compared to 173±8 ms (n=12) under baseline conditions, whereas diastolic and systolic levels rise by 22±4% and 11±5% compared to baseline fluorescence levels, respectively (p<0.05, unpaired t-test).

Duration and peak of the contraction force signals during reperfusion parallel the changes of the Ca2+ transients (data not shown). However, we never observe higher resting tensions, despite the fact that diastolic Ca2+ levels are elevated. During reactive hyperemia in the 15-min episodes of reperfusion, flow maximally increases by 41±14% and returns to control after 7.1±0.4 min (n=8). Conduction velocity is 55±2 cm/s during control conditions and immediately returns to control value after the start of reperfusion (n=12).

In the PC-1 group, ischemia-induced Ca2+ rise, uncoupling and contracture are significantly postponed compared to the control group (Table 1). However, in the PC-2 group, in which the total duration of preconditioning ischemia and reperfusion is equal to PC-1, cardioprotection is absent (Table 1). Although in the PC-3 group only a single 5-min period of ischemia precedes sustained ischemia, the onset of ischemic damage is significantly postponed and not different from PC-1 (Table 1).


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  		Table 1 Ca2+ transient duration (ms) and diastolic Ca2+ level (relative to baseline conditions) measured just prior to the induction of sustained ischemia, and the time to increase of [Ca2+]i, onset of uncoupling and start of contracture after the induction of sustained ischemia (min) for all experimental groupsa

 
3.2 Effects of staurosporine
The relation between [Ca2+]i and PKC-activation in ischemically preconditioned myocardium is analyzed by administration of 0.1 µmol/l staurosporine during the second period of reperfusion. Preconditioning periods of ischemia are, therefore, not modified by PKC blockade and not different between experimental groups. Furthermore, it is possible to compare the effects of PKC blockade on the Ca2+ transients during the second period of reperfusion with the first period of reperfusion.

Changes of the Ca2+ transient during the first, drug-free 15-min period of reperfusion in the PC+Stau group are similar to the PC-1,2 groups: after 5 min of reperfusion transient duration increases to 268±24 ms compared to 186±15.7 ms under baseline conditions (n=4; p<0.05, paired t-test), whereas mean diastolic level increases by 21±4.5% relative to baseline (n=4; p<0.05, paired t-test). As staurosporine is washed in from the start of the second reperfusion, the effects of staurosporine become apparent only after the first minute of reperfusion. The initial increase of the duration and the diastolic level of the Ca2+ transient reverses into a gradual decrease after 1 min of reperfusion with staurosporine. On average, at the start of sustained ischemia, after 5 min of reperfusion with staurosporine, duration and diastolic and systolic levels of the Ca2+ transient are not different from baseline values (Table 1).

In the PC+Stau group the moments of the Ca2+ rise, uncoupling and contracture during sustained ischemia are not different from the control group (Table 1). Staurosporine infusion for 5 min in four non-preconditioned hearts does not affect the Ca2+ transients, the contraction signals and Rt. Also, changes during subsequent sustained ischemia are not different from the control (Table 1). Staurosporine does not affect flow or the degree of reactive hyperemia (mean peak flow increase 36±15%).

3.3 Effects of glibenclamide
As K+ATP channel opening has been implicated in the initiation of preconditioning [14], we tested whether blocking preconditioning with glibenclamide [10] also affects the reperfusion-induced changes of the Ca2+ transient. Fig. 5 shows a typical example of an experiment in the PC+Glib group. During the 15 min of drug-free reperfusion in the PC+Glib group, the Ca2+ transients show changes similar to the PC-1,2 groups: on average, after 5 min of reperfusion the Ca2+ transient duration is 251±19 ms compared to 165±17.2 ms under baseline conditions (n=4; p<0.05, paired t-test), whereas mean diastolic level increases by 18±5.0% relative to baseline (n=4; p<0.05, paired t-test).


Figure 5
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  		Fig. 5 Changes of the duration and diastolic and systolic levels of the Ca2+ transient during the PC+Glib protocol. I indicates ischemia. Third ischemic episode is the initial phase of sustained ischemia.

 
After an initial wash-in period of maximally 1 min, glibenclamide treatment markedly attenuates the changes of the Ca2+ transients and contraction signals during the second period of reperfusion (Fig. 5). In this group the diastolic and systolic levels and the duration of the Ca2+ transient at the moment of induction of sustained ischemia are not different from baseline values. Also, treatment with glibenclamide completely blocks the protective effect of ischemic preconditioning (Table 1). In the Glib and PC+Glib groups the Ca2+ transient disappears within the first 5 min of ischemia, as in the other experimental groups.

In the Glib group perfusion with glibenclamide decreases flow by 20%. This does not affect the Ca2+ transients, Rt and contraction signals during perfused conditions as well as the start of the markers of irreversible damage during subsequent ischemia. Also, in the PC+Glib group reactive hyperemia constitutes a maximal flow increase of 35±15% compared to preischemic flow.

3.4 The effects of pretreatment with phorbol 12-myrisate 13-acetate
As shown previously [27], a low dose of PMA increases the duration as well as the diastolic and systolic level of the Ca2+ transient (Fig. 6). In two experiments the transient continues to change during the 5-min period of washout (like in Fig. 6), whereas in the other two experiments in the PMA group, the transient reaches a new steady state at the end of PMA-infusion, which does not change during subsequent washout. On average, in this group transient duration increases from 163±16 ms under control conditions to 213±12 ms at 5 min of washout following 5 min of PMA treatment (p<0.05; paired t-test). Also, diastolic and systolic ratios at the moment of induction of sustained ischemia are 107±1% (p<0.05; paired t-test) and 104±2.1% (not significant) of preischemic control values, respectively. In the PMA group the markers of ischemic damage, the onset of Ca2+ rise, uncoupling and contracture, are significantly postponed compared to control hearts (Table 1). The differences in changes of the Ca2+ transient and degree of protection between the PC-1 or PC-3 groups and the PMA group do not reach statistical significance.


Figure 6
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  		Fig. 6 Changes of the duration and diastolic and systolic levels of the Ca2+ transient during the PMA protocol. The black bar indicates the initial phase of sustained ischemia (I).

 
3.5 The effects of pretreatment with cyclopiazonic acid
Fig. 7 shows a typical example of the effect of CPA/SNP treatment on the configuration of the Ca2+ transient. A new steady state is reached after 8 min of infusion. CPA/SNP infusion significantly increases the Ca2+ transient duration from 180±9 ms under baseline conditions to 231±8 ms (p<0.05, paired t-test) after 8 min of infusion (Table 1). This treatment also increases the diastolic level by 25±4% (p<0.05, paired t-test), whereas it decreases the systolic level by 4±2% (not significant). Despite the fact that diastolic Ca2+ levels are elevated during CPA pretreatment, we never observe higher resting tensions. Duration of the contraction signal increased, whereas systolic tension decreased by 15% (not significant). Myocardial flow is raised by 30±8%. Infusion of SNP without CPA increases flow by 34±7%, but does not affect Ca2+ transients or contraction signals (data not shown).


Figure 7
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  		Fig. 7 Ca2+ transients under control conditions and after 8 min of combined cyclopiazonic acid/nitroprusside infusion of a typical example from the CPA group.

 
In preparations pretreated with CPA/SNP indicators of ischemic damage, Ca2+ rise, uncoupling and contracture, are significantly delayed compared to control hearts, respectively (Table 1). After single SNP infusion changes during sustained ischemia are not different from the control group (data not shown).

3.6 Configuration of the Ca2+ transient and cardioprotection
Transient durations under baseline conditions (i.e. before any intervention) are not different between groups (173±5 ms; n=40). Baseline fluorescence levels and basic cycle lengths (range 400–450 ms) vary between experiments. Therefore, changes of the Ca2+ transient of individual experiments at the moment of induction of sustained ischemia are expressed relative to baseline measurements 40 min prior to sustained ischemia. Table 1 summarizes the changes of the diastolic ratio level and of the duration of the Ca2+ transient just prior to the moment of induction of sustained ischemia as well as the time to the start of terminal Ca2+ rise, uncoupling and contracture during subsequent sustained ischemia. Since the reperfusion-induced change of the systolic ratio level at the moment of induction of sustained ischemia is not significant between experimental groups, it is excluded from further analysis.

Fig. 8 shows the relations between the duration of the Ca2+ transient (upper panel) or the diastolic Ca2+ level (lower panel) at the moment of induction of sustained ischemia and the delay of the onset of electrical uncoupling. The extent of prolongation of the Ca2+ transient and the elevation of the diastolic Ca2+ level just prior to induction of sustained ischemia significantly correlate with the delay of the moment of uncoupling. The preischemic changes of the diastolic Ca2+ level and the Ca2+ transient duration also significantly, positively correlate with the other two indicators of the onset of ischemic injury, i.e. the moment of Ca2+ rise (r=0.80 and r=0.79, respectively) and the start of contracture (r=0.82 and r=0.83, respectively).


Figure 8
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  		Fig. 8 Relation between the relative Ca2+ transient prolongation (upper panel) or the relative elevation of the diastolic Ca2+ level (lower panel) measured just prior to sustained ischemia (abscissa) and the moment of onset of electrical uncoupling after induction of sustained ischemia (ordinate) for all groups except the control, Stau and Glib groups. The inset shows the changes of Rt during sustained ischemia from typical examples of the PC-1 (closed squares) and PC-2 groups (open squares). Arrows indicate moments of uncoupling.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
References
 
The present study shows that the temporary increase of the Ca2+ transient duration and of the diastolic Ca2+ level during reperfusion after 5 min of ischemia correlate with the postponement of ischemic injury in preconditioned myocardium. Staurosporine and glibenclamide antagonize the reperfusion-induced changes of the Ca2+ transient and block the protective effect of preconditioning. In preparations pretreated with PMA preischemic changes of the Ca2+ transient and delay of ischemic damage are qualitatively similar to the ischemically preconditioned preparations, in which the onset of ischemic damage is delayed. Increasing the diastolic Ca2+ level and the transient duration by blocking SR Ca2+-uptake with CPA also postpones ischemic injury.

This is one of the few studies underlining the importance of changes during reperfusion in the effector mechanism of preconditioning. Our data suggest that a preischemic mild increase of [Ca2+]i is a common effector in this complex mechanism of cardioprotection.

4.1 Ca2+ transients during reperfusion
The prolongation of the Ca2+ transient and the elevation of the diastolic Ca2+ level during reperfusion found in the present study are most likely caused by a temporary reduction of SR function. Various investigators have shown that short periods of ischemia and reperfusion decrease SR Ca2+ uptake or increase SR Ca2+ release causing a SR depletion and a diastolic accumulation of Ca2+ in the cytosol [15–17].

Alternatively, the mild increase of [Ca2+]i during reperfusion could have been caused by an increased Ca2+ influx through the L-type Ca2+ channels or through the Na+/Ca2+ exchanger secondary to intracellular Na+ accumulation [30,31]. However, ischemic preconditioning does not affect the density or kinetics of L-type Ca2+ channels, although the effects on channel availability and open probability are unknown [32]. Since a 5-min period of ischemia is too short to produce a detectable increase of [Na+]i during reperfusion, an increase of [Ca2+]i via the Na+/Ca2+ exchanger is unlikely. Although pH may change the binding kinetics of endogenous Ca2+ ligands, intracellular acidosis is not a probable cause of the prolonged transients during reperfusion, because intracellular pH normalizes within 1 min of reperfusion following a 5-min period of ischemia [33]. The role of indo 1, an exogenous Ca2+ ligand, can be excluded, since reperfusion after 5 min of ischemia in muscles not loaded with indo 1 caused changes in the contraction signals that were identical to those in indo 1 loaded muscles.

Reactive hyperemia, evident during all periods of reperfusion, cannot explain the alterations of the Ca2+ transients and contraction signals. The time courses of the changes in myocardial flow and in the configuration of the Ca2+ transient during reperfusion are different. Also, a 34% increase in myocardial flow during infusion of sodium nitroprusside had no effect on Ca2+ transients or contraction signals [34]. Changes in conduction velocity and in temperature during reperfusion were excluded.

The enlarged contractions observed during reperfusion, also called hypercontractions, are consistent with previous reports in the literature [35,36]. Despite the increase of the diastolic ratio during reperfusion, resting tensions did not rise in the PC 1,2, and 3 groups as well as the CPA and PMA groups. This is in agreement with the S-shaped relation between [Ca2+]i and developed force [37].

4.2 Ca2+ transient and cardioprotection
We found a significant correlation between the increase of the diastolic Ca2+ level or the transient duration just prior to sustained ischemia and three indicators of the onset of irreversible ischemic damage. The cardioprotective significance of such a preischemic mild increase of [Ca2+]i was further analyzed by blocking two alleged initiator pathways of ischemic preconditioning, PKC activation and K+ATP channel opening [2,3,10,11]. Also, we tested the effects of pretreatment with a PKC-activator or a blocker of SR Ca2+ uptake.

Although the involvement of K+ATP channels in the mechanism of preconditioning seems clearly evidenced, its mechanism remains controversial [14,38]. Since action potential shortening is not obligatory for cardioprotection induced by K+ATP-channel openers [12], intracellular sites of action, such as the SR, could play a role instead of the sarcolemmal channels. We speculate that a fraction of the K+ channels, which are present in the SR membrane, is ATP-sensitive [39]. Since these K+ channels ensure electroneutrality across the SR membrane during a Ca2+ transient, changing their conduction state may affect the amount of Ca2+ released by the SR. It may thus be possible that SR K+ATP channel activation after preconditioning facilitates SR Ca2+ release inducing alterations of the Ca2+ transients as described in the present study. Consequently, this effect can be blocked by glibenclamide.

Based on the above observations we, therefore, speculate that the role of the preischemic mild increase of [Ca2+]i is twofold. On the one hand, mild increase of [Ca2+]i could activate PKC via direct Ca2+-dependent activation or via a G protein activated by Ca2+-dependent phospholipase C [4,40]. On the other hand, this mild increase of [Ca2+]i may represent an effector mechanism of cardioprotection. Steenbergen et al. have previously suggested that the degree of SR Ca2+ loading is an important determinant of the rate and extent of the rise in [Ca2+]i and irreversible damage during ischemia [41]. Under ischemic conditions the SR Ca2+-ATPase consumes a substantial portion of total cellular energy [42,43]. A reduction of Ca2+-ATPase activity, transitorily increasing diastolic [Ca2+]i and Ca2+ transient duration, is thermodynamically favorable and may constitute a cardioprotective mechanism. Likewise, inhibiting the SR Ca2+ pump with CPA protects the myocardium during subsequent ischemia. In addition, one of the effects of activated PKC is inhibition of Ca2+ accumulation in the SR [27,37,40]. Concomitantly, in the PMA group preischemic Ca2+ transient duration as well as cytosolic Ca2+ levels are increased concurring with a delay of ischemic injury. Therefore, Ca2+-dependent PKC activation and PKC-mediated transition of Ca2+ from the SR into the cytosol may constitute a positive feedback loop resulting in a thermodynamically favorable and protected status of the myocardium. These speculations are in agreement with two previous papers proposing that an elevation of [Ca2+]i prior to sustained ischemia marks a protected state of the myocardium [7–9]. In these studies, however, [Ca2+]i measurements were not performed

In the present study the protective effect of preconditioning is lost after 15 min of reperfusion. This may seem as a relatively short period compared to other studies, in which the cardioprotective effect persisted for 1 to 2 h of reperfusion using different species and experimental models [1]. A study investigating the effect of the duration of reperfusion on cardioprotection in preconditioned rabbit hearts showed that infarct size reduction started to wane after 30 min of reperfusion [44].

4.3 Methodological considerations
It seems likely that in the PC-1 and 3 groups, the alterations of the Ca2+ metabolism underlying the changes of the Ca2+ transients during reperfusion after 5 min of ischemia extend into the period of sustained ischemia. However, detailed analysis of the Ca2+ transients during ischemia is not feasible, because in this study, transients rapidly disappear during early ischemia and, secondly, fluoroscopy during ischemia is hampered by potential artifacts, such as shifts in autofluorescence and changes of pH [22]. These limitations of indo 1 fluorescence do not apply under conditions of reperfusion. The pHi rapidly normalizes during reperfusion after 5 min of ischemia. Also, in the present study we show that autofluorescence returns to preischemic control values within the first minute of reperfusion. Although it can not be completely excluded, it is unlikely that other factors interfered with the Ca2+ measurements during reperfusion. Irreversible changes of indo 1 during preconditioning, such as degradation by oxygen radicals or a transition from the cytosol into the mitochondria, can be excluded, since after 15 min of reperfusion the Ca2+ transient is again similar to its preischemic control.

The alterations of the Ca2+ transient and the contraction signal during SR Ca2+ ATPase inhibition are in agreement with previous publications [19,20,28]. The changes of the systolic ratio levels during reperfusion or CPA treatment were not significantly different from baseline suggesting that systolic ratio elevation is not a prerequisite for cardioprotection. However, since we did not quantify actual [Ca2+]i these changes could have been obscured by the relative inaccuracy of the indo 1 method at higher Ca2+ concentrations (>1 µmol/l). It is unlikely that CPA increased endothelial [Ca2+]i, since such an effect would be expected to induce a parallel shift of the diastolic and systolic ratio without a prolongation of the transient. In this model, endothelial Ca2+ overload induced by bradykinin results in a 10% increase of total fluorescence at most [22]. A minor contribution of endothelial [Ca2+] to the CPA-induced 25% increase of the diastolic ratio cannot be completely excluded.

Staurosporine, as all PKC-inhibitors, suffers from relatively low specificity [4]. Also, PKC-stimulation by PMA does not quantitatively equal the changes of the Ca2+ transient and the degree of cardioprotection induced by cardioprotection. On the one hand, this could be due to the fact that other pathways in parallel to PKC activation play a role in ischemic preconditioning. On the other hand, it could have been caused by the relatively low dose of PMA. However, in a study by Ward et al. it has been convincingly shown that low dosages of PMA have potent PKC-dependent effects, whereas higher dosages of PMA predominantly induce PKC-independent effects [27]. Also, such a low dose does not affect myocardial flow.

In the present and a previous study systolic ratio rapidly declines during the initial phase of ischemia [22]. In contrast, however, others have shown that during the first min of ischemia diastolic and systolic ratio levels increase, whereas the contractile force rapidly decreases [45,46]. The latter observations remain controversial and unexplained. Lorell et al. suggested that in these studies substantial indo 1 loading in the endothelium could play a role [47]. This possibility is excluded in our studies, since in the present model fluorescence from the endothelial cells constitutes only a minor component of total fluorescence [22]. Furthermore, distinct methodological differences could also play a role. Experimental temperature in the present study was 37°C, whereas in other studies 30°C was used. Also, the present model is characterized by stringent ischemic conditions (pO2<5 mmHg). However, ischemic pH changes could be attenuated compared to intact preparations due to less CO2 accumulation in the papillary muscle with a relatively small diameter [48]. Motion artifacts were excluded by the BDM experiments.

Ca2+ rise, electrical uncoupling and contracture are specific markers of the onset of irreversible ischemic damage [21,22]. However, recently some controversy has arisen on the assumption that a postponement of contracture is a good estimate of the effect of preconditioning. Some studies have shown that in rat hearts the onset of contracture during sustained ischemia is advanced after preconditioning [49,50]. This may be a species difference, because in rabbit hearts preconditioning postpones the onset of ischemia-induced contracture [22].

In conclusion, a preischemic mild increase of [Ca2+]i may act as mediator and common end-effector in preconditioning. Our data suggest that both activation of PKC and opening K+ATP channels initiate pathways leading to an alteration of Ca2+ metabolism and a protected status of the myocardium.


   Notes
 
{star} For this paper Dr. K. Sipido (Leuven, Belgium) acted as guest editor. Back


   References
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 

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