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Cardiovascular Research 1998 38(3):676-684; doi:10.1016/S0008-6363(98)00064-9
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Cyclosporine A limits myocardial infarct size even when administered after onset of ischemia

Christof Weinbrenner1, Guang S Liu, James M Downey and Michael V Cohen*

Departments of Physiology and Medicine, MSB 3050, University of South Alabama, College of Medicine, Mobile, AL 36688-0002, USA

* Corresponding author. Tel.: +1 (334) 460-6812; fax: +1 (334) 460-6464.

Received 27 November 1997; accepted 3 February 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 
Objective: The role of the immunosuppressant cyclosporine A as a preconditioning-mimetic in the rabbit heart was examined. Methods: Cyclosporine A, a potent protein 2B or calcium/calmodulin-dependent phosphatase (PP) inhibitor, was administered to isolated rabbit hearts starting either 15 min prior to or 10 or 20 min after the onset of a 30 min period of regional ischemia and continuing until the onset of reperfusion. The effect of pretreatment with a second PP2B antagonist, FK-506, was also examined. In an additional protocol L-NAME was perfused for 50 min starting 5 min before the 45-min infusion of cyclosporine A. After 2 h of reperfusion infarct size was measured with triphenyltetrazolium chloride. In a second study left ventricular biopsies of isolated rabbit hearts were obtained to measure the effect of cyclosporine A on dephosphorylation of Formula phosphorylase kinase by calcium/calmodulin-dependent phosphatases. Results: Pretreatment with cyclosporine A resulted in only 10.0% infarction of the risk zone, significantly less than that in untreated control hearts (28.7%, p<0.001) but comparable to the extent of infarction in ischemically preconditioned hearts (10.0%, p<0.001 vs. control). Equivalent protection was also observed in hearts with treatment delayed for 10 min following the onset of ischemia (10.4% infarction, p<0.001 vs. control). However, protection waned when cyclosporine A was administered only during the last 10 min of the 30-min ischemic period (25.5% infarction, p=n.s. vs. control). Pretreatment with FK-506 also resulted in myocardial salvage (10.4% infarction, p<0.001 vs. control). When hearts were exposed to a co-infusion of L-NAME and cyclosporine A, protection was still evident (18.1% infarction, p<0.05 vs. L-NAME), although not as robust as that seen with the PP2B blocker alone. In hearts pretreated with cyclosporine A dephosphorylation of Formula phosphorylase kinase by calcium/calmodulin-dependent phosphatases was inhibited by 67%. Conclusions: Cyclosporine A and FK-506, potent PP2B inhibitors, can protect the ischemic rabbit heart, and at least cyclosporine A continues to be effective when infusion is delayed until after the onset of ischemia. The mechanism of this protection may be related to inhibition of phosphatases and prolongation of the phosphorylation state of ischemic cells.

KEYWORDS Ischemic preconditioning; Phosphorylation; Protein phosphatases; Cyclosporine A; FK-506; L-NAME; PP2B; Infarct size; Nitric oxide


   1 Introduction
Top
 Abstract
 1 Introduction
2 Materials and methods
 3 Results
 4 Discussion
 References
 
Preconditioning the heart with a brief episode of ischemia protects it against ischemic injury from a subsequent ischemic insult [1]. While many receptor systems trigger this protection [2–10], the actual mechanism of the salutary effect remains elusive. Activation of kinases including mitogen-activated protein kinase as well as protein kinase C (PKC) has been proposed to be a key step in this protection, at least in rat, rabbit and human hearts [11–15]. Phosphorylation of an as yet unidentified protein would then protect the heart from infarction [16, 17]. While proteins are phosphorylated by kinases, they are dephosphorylated by phosphatases. Thus inhibition of phosphatases should have an effect similar to activation of kinases. Armstrong and Ganote [18]have shown that inhibition of protein phosphatases (PPs) by okadaic acid or calyculin A attenuated the ischemia-induced increase in osmotic fragility seen in isolated, ischemic rat cardiomyocytes. More recently this group demonstrated that ischemic rabbit cardiomyocytes were similarly protected by calyculin A, and the effect was equivalent to that seen with ischemic preconditioning [19]. However, because very high concentrations of okadaic acid (10 µmol/l) and calyculin A (0.2 and 1 µmol/l) were used, non-selective inhibition of many of the cell's protein phosphatases would have occurred. In an attempt to determine which of the phosphatases might be critical to this protection, we selected for study fostriecin, a PP inhibitor which is highly selective for PP2A [20]. In both isolated rabbit hearts [21]and isolated, ischemic rabbit and porcine cardiomyocytes [22]this selective phosphatase inhibitor had a striking protective effect, and the salvage of ischemic tissue in rabbit hearts could be realized even when fostriecin was administered after ischemia had begun [21]. These observations suggested that potentiation of phosphorylation of some protein which is a natural substrate for PP2A can be very protective to the heart.

We then wondered if inhibition of another phosphatase could also confer protection. Cyclosporine A (CsA) and FK-506, 2 widely used immunosuppressive agents [23], inhibit T-cell activation through inhibition of the calcium/calmodulin-dependent PP2B, also known as calcineurin [24, 25], by forming complexes with the cytoplasmic binding proteins cyclophilin and FK-binding protein (FKBP), respectively [26]. Indeed Griffiths and colleagues found that CsA could preserve post-ischemic contractile function [27, 28], but they ascribed the protection to inhibition of a Ca2+-dependent pore in heart mitochondria [27–31]. Massoudy et al. [32]also noted that CsA treatment preserved post-ischemic function, but their results suggested a nitric oxide-dependent mechanism mediated by endothelin. Further suggestive evidence of a protective effect of ischemic myocardium by PP2B inhibition has been demonstrated in dogs with phenothiazines, inhibitors of calmodulin [33].

The present study was designed to test whether CsA could also protect against infarction. Furthermore, we examined whether CsA, like fostriecin, would still be protective if administered after the onset of ischemia. As a third step, we attempted to determine whether CsA's protection is a direct result of phosphatase inhibition. We measured CsA's ability to inhibit calcium/calmodulin-dependent protein phosphatase activity in left ventricular tissue from intact hearts. Additionally we tested whether another PP2B inhibitor, FK-506, which has no effect on mitochondrial pores could also protect myocardium. Finally, we examined whether a nitric oxide synthase inhibitor could affect CsA's protective effect.


   2 Materials and methods
Top
 Abstract
 1 Introduction
 2 Materials and methods
3 Results
 4 Discussion
 References
 
All procedures were in conformance 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 1985).

2.1 Isolated heart model
New Zealand White rabbits of either sex were anesthetized with intravenous sodium pentobarbital (30 mg/kg) administered into a marginal ear vein. The trachea was intubated through a cervical incision. The rabbits were ventilated with 100% oxygen with a positive pressure respirator (MD Industries, Mobile, AL) at a rate of 30–35 breaths per minute and tidal volume of approximately 15 ml.

The chest was opened and the heart exposed. The heart was excised and quickly mounted on a Langendorff apparatus by the aortic root in 60 s or less. The heart was perfused at constant pressure (100 cm H2O) with a Krebs–Henseleit buffer containing in mmol/l NaCl 118.5, KCl 4.7, MgSO4 1.2, CaCl2 2.5, NaHCO3 24.8, KH2PO4 1.2, and glucose 10. The perfusate was gassed with 95% O2/5% CO2 and the perfusate temperature was maintained at 38°C. A saline-filled latex balloon connected by a catheter to a pressure transducer was inserted into the left ventricle. The balloon volume was adjusted to provide a left ventricular end-diastolic pressure of 5–10 mmHg. All hearts experienced an initial 20-min stabilization period.

2.2 Measurement of infarct and risk zones
A 2-0 silk suture on a curved taper needle was passed around a prominent branch of the left coronary artery, and the ends were pulled through a small vinyl tube to form a snare. The coronary branch was occluded by tightening the snare, which was then fixed by clamping the tube with a small hemostat. Reperfusion was achieved by releasing the snare and was confirmed by enhanced coronary flow and developed pressure. Infarction was induced by 30 min of regional ischemia, which was then followed by 2 h of reperfusion.

At the end of the experiment the coronary artery was reoccluded, and 1–10 µm zinc cadmium sulfide fluorescent particles (Duke Scientific, Palo Alto, CA) were infused into the perfusate to demarcate the risk zone as the tissue without fluorescence. The heart was then removed from the apparatus, weighed, frozen, and cut into 2-mm thick slices. The slices were incubated in 1% triphenyltetrazolium chloride (TTC) in pH 7.4 phosphate buffer for 15 min at 37°C. The areas of infarct (TTC negative tissue) and risk zone (nonfluorescent under ultraviolet light) were determined by planimetry. Infarct and risk zone volumes were then calculated by multiplying each area by the slice thickness and summing the products. Infarct size was expressed as a percentage of the risk zone.

2.3 Experimental protocols: infarct size studies
Five groups with 6 to 7 hearts in each group were studied. Group 1 rabbits served as controls and received vehicle (0.05% ethanol) for 45 min starting 15 min prior to the 30-min period of regional ischemia followed by 2 h of reperfusion (Fig. 1). Group 2 was ischemically preconditioned (PC) with 5 min of global ischemia followed by 10 min of reperfusion before the onset of 30 min of regional ischemia. In the third group (Pre-CsA 750) 750 nmol/l cyclosporine A was infused over 45 min, starting 15 min prior to onset of ischemia and continuing through the ischemic period. In group 4 (Post-10-CsA), 750 nmol/l CsA was infused over 20 min, beginning 10 min after the onset of ischemia and continuing until the beginning of reperfusion. In group 5 (Post-20-CsA), 750 nmol/l CsA was infused for only the last 10 min of the 30 min of regional ischemia.


Figure 1
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  		Fig. 1 Experimental protocol. See text for details.

 
In a second protocol 3 additional groups of hearts (6 to 7 hearts in each group) were investigated. Group 1 (Pre-CsA 100) received 100 nmol/l CsA for 45 min beginning 15 min before the 30-min coronary occlusion. In group 2 (Pre-FK 7.5) 7.5 nmol/l and in group 3 (Pre-FK 150) 150 nmol/l FK-506 were infused for similar 45-min periods.

In a third protocol (5 to 6 hearts in each group) hearts of the first group were perfused with 100 µmol/l N{omega}-nitro-L-arginine methyl ester (Pre-L-NAME) for 50 min starting 20 min before the 30 min of regional ischemia and continuing to the end of the ischemic period. A second group received 750 nmol/l CsA as detailed in the Pre-CsA 750 protocol in addition to L-NAME (Pre-CsA-L-NAME). Finally, the third group was ischemically preconditioned as in PC and also received an L-NAME infusion (PC-L-NAME).

2.4 Experimental protocols: protein phosphatase measurements in left ventricular biopsies
Isolated rabbit hearts (n=3) were stabilized for 20 min and subsequently perfused with 100 nmol/l CsA for 15 min followed by a 15-min perfusion period with 750 nmol/l CsA. Three transmural biopsies (35–99 mg each) were obtained from the left ventricular free wall of each heart with a motor-driven biopsy tool and immediately frozen in liquid nitrogen. Tissue near a major coronary artery was avoided. The first biopsy was taken after the 20-min equilibration period, the second after low-dose perfusion with CsA, and the third after high dose perfusion. A similar protocol was employed with FK-506 infusions at 7.5 and 150 nmol/l.

2.4.1 Tissue preparation
Biopsies were weighed and homogenized with a Mini-Beadbeater (Biospec Products, Bartlesville, OK) for 10 s in 500 µl buffer chilled to 4°C and containing Tris-HCl 50 mmol/l (pH 7.4), EDTA 1 mmol/l, EGTA 4 mmol/l, PMSF 1 mmol/l, and benzamidine 1 mmol/l. Subsequently the samples were centrifuged at 1000xg for 5 min and the supernatant was centrifuged again at 15 000xg for 30 s. The pellet was discarded and the protein content of the supernatant was determined using the DC protein assay (BioRad) with bovine serum albumin as a standard. Calcium/calmodulin-dependent protein phosphatase activity of the supernatant was determined as described below.

2.4.2 Preparation of phosphoprotein substrate
Formula labeled phosphorylase kinase was prepared by phosphorylation of 200 U phosphorylase kinase with 150 µg of the catalytic subunit of cyclic-AMP-dependent protein kinase according to the method of Stewart et al. [34]with slight modifications [35]. The reaction was carried out for 1 min at 30°C in 250 µl (final volume) of buffer containing 2 mmol/l magnesium acetate, 6 mmol/l sodium glycerophosphate, 0.25 mmol/l EDTA, and 0.15 mmol/l Formula ATP. The reaction was terminated by addition of 12.5 µl each of 1 mol/l sodium fluoride and 200 mmol/l EDTA, and the reaction mixture was subsequently passed through a column of Sephadex G50 superfine (25x1 cm) equilibrated in 50 mmol/l Tris-HCl pH 7.0 and 0.3% (v/v) 2-mercaptoethanol.

2.4.3 Determination of protein phosphatase activity
Tissue protein phosphatase activity was determined in duplicate by quantification of the remaining Formula phosphate in phosphorylase kinase following incubation with the samples. Calcium/calmodulin-dependent phosphatase activity was assayed in aliquots of homogenate in buffer (final volume 40 µl) containing 50 mmol/l Tris-HCl (pH 7.0), 15 mmol/l 2-mercaptoethanol, 0.0033% Brij-35, 0.2 mg/ml BSA, Formula labeled phosphorylase kinase, and 25 U calmodulin according to Stewart and Cohen [36]and Ganapathi and Lee [35]. The reaction was carried out either with the addition of 2.5 mmol/l calcium chloride (to measure calcium/calmodulin-dependent and independent phosphatase activity) or without calcium but instead with 4 mmol/l EGTA to chelate calcium ions and 150 µmol/l trifluoperazine to inhibit calmodulin (to measure calcium/calmodulin-independent phosphatase activity). The reaction was initiated by addition of homogenate (15 µg) and was conducted at 30°C for 10 min. The reaction was stopped by the addition of SDS-sample buffer, and the reaction mixture was separated on a 6% polyacrylamide gel according to Laemmli [37]. After staining with Coomassie brilliant blue R-250 the gels were dried and subjected to autoradiography. The Formula phosphate content in the isolated {alpha}-subunit of phosphorylase kinase was quantified by scanning the band using a computer scanner and calculating the density with Sigmagel software (Jandel Scientific). The calcium/calmodulin-dependent phosphatase activity was calculated as the difference between total activity and the calcium/calmodulin-independent activity. Finally the calcium/calmodulin-dependent phosphatase activity for each of the two CsA concentrations was normalized to the activity without CsA. A similar procedure was used for determination of FK-506 activity. Since all biopsies from one heart were processed at the same time and run on the same gel and because the protein content of each sample was equal, it was not necessary to correct for differences of protein content or exposure times.

2.5 Chemicals
L-NAME, phosphorylase kinase, the catalytic subunit of cyclic-AMP-dependent protein kinase (peak II), calmodulin, and trifluoperazine were purchased from Sigma Chemicals (St. Louis, MO, USA). CsA was obtained from Calbiochem and was diluted in ethanol to a stock solution of 4.5 mmol/l from which it was further diluted with Krebs buffer. FK-506, also known as Tacrolimus (Prograf®), was obtained from Fujisawa (Deerfield, IL, USA). It had been dissolved by the company in polyoxyl 60 hydrogenated castor oil (6.1 mmol/l). A stock solution (1 mmol/l) was prepared in ethanol prior to the final dilution in Krebs buffer.

2.6 Statistics
Values are presented as means±SEM. One-way analysis of variance with repeated measures and Tukey–Kramer post hoc tests were used to test for differences within groups, and without repeated measures to test for differences in infarct size (Instat, Graphpad Software, San Diego, CA). A p value <0.05 was considered significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
4 Discussion
 References
 
3.1 Hemodynamics
Baseline hemodynamic parameters were comparable in the eleven groups of hearts (Table 1). In the 3 L-NAME groups heart rate and developed pressure decreased slightly after drug treatment. However, coronary flow was dramatically reduced before and during coronary occlusion with little recovery with reperfusion. In the PC, Pre-CsA 750, and Pre-FK 150 groups there were small but significant falls in developed pressure after the intervention (p<0.05).


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  		Table 1 Hemodynamic data

 
3.2 Infarct size studies
There were no differences in body weight, heart weight or risk zone sizes among the groups (Table 2). Infarct sizes normalized as a percentage of the ischemic (risk) zone are shown in Fig. 2Fig. 3Fig. 4. Hearts pretreated with 750 nmol/l CsA had only 10.0±2.9% infarction as compared to 28.7±3.3% in control hearts (p<0.001) (Fig. 2). This level of protection is comparable to that seen with ischemic preconditioning (10.0±1.6%; p<0.001 vs. control). When CsA was given 10 min after the onset of occlusion, protection was still present with 10.4±2.0% infarction (p<0.001 vs. control). However, protection waned when CsA was administered for only the last 10 min of the 30-min ischemic period (25.5±3.9%). Pretreatment with low dose (100 nmol/l) CsA was nearly as protective as the higher concentration of CsA (11.9±1.9%, p<0.005 vs. control) (Fig. 3). Although the low concentration of FK-506 (7.5 nmol/l) had little effect on infarct size (24.5±3.5%, p=n.s. vs. control), 150 nmol/l FK-506 was as protective as CsA (10.4±2.8%, p<0.001 vs. control and FK 7.5).


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  		Table 2 Infarct size data

 

Figure 2
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  		Fig. 2 Infarct size normalized as a percentage of the ischemic (risk) zone in isolated perfused rabbit hearts. Control hearts (n=7) experienced only 30 min of regional ischemia. Hearts were ischemically preconditioned with 5 min of global ischemia followed by 10 min of reperfusion before the 30 min of regional ischemia (PC; n=7). Cyclosporine A (CsA), 750 nmol/l, was either administered for 15 min prior to and during the regional ischemia (Pre-CsA 750; n=6) or added to the buffer 10 (Post-10-CsA; n=7) or 20 (Post-20-CsA; n=7) min after the onset of the 30-min ischemic period. Pretreatment with CsA as well as ischemic preconditioning resulted in a significant reduction in infarction compared to control hearts (**p<0.001). Late CsA treatment beginning 10 min after the onset of regional ischemia still significantly reduced infarct size. However, protection was no longer evident when CsA was given in only the last 10 min of ischemia.

 

Figure 3
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  		Fig. 3 Infarct size normalized as a percentage of the ischemic (risk) zone in isolated perfused rabbit hearts. Pre-CsA 100 hearts (n=7) received 100 nmol/l CsA for 45 min starting 15 min prior to the regional ischemia. The Pre-FK 7.5 (n=6) and Pre-FK 150 (n=7) groups received FK-506 for a similar duration at concentrations of 7.5 nmol/l and 150 nmol/l, respectively. For comparison the control and Pre-CsA 750 data from Fig. 2 are shown again. Pretreatment with 100 nmol/l CsA was nearly as protective as the high concentration of CsA (*p<0.005 vs. control). The low concentration of FK-506 (7.5 nmol/l) showed no protection, while 150 nmol/l FK-506 was as protective as CsA (**p<0.001 vs. control).

 

Figure 4
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  		Fig. 4 Infarct size normalized as a percentage of the ischemic (risk) zone in isolated perfused rabbit hearts. L-NAME-treated hearts (n=5) received 100 µmol/l L-NAME for 50 min starting 20 min prior to the 30-min period of regional ischemia. Pre-CsA-L-NAME hearts (n=6) were perfused with 750 nmol/l CsA for 45 min beginning 15 min prior to ischemia in addition to L-NAME as above. The PC-L-NAME group (n=5) was ischemically preconditioned with a single cycle of 5-min occlusion/10-min reperfusion in addition to receiving L-NAME as above. For comparison the control, PC, and Pre-CsA 750 data from Fig. 2 are shown again. L-NAME itself had no influence on infarct size and did not affect the protection of PC. CsA continued to be protective despite the presence of L-NAME ({ddagger}p<0.05 vs. L-NAME). Although L-NAME tended to increase infarct size in the CsA-treated hearts, this trend was not significant. **p<0.001 vs. control.

 
Fig. 4 presents the data for L-NAME infusions. L-NAME itself had no influence on infarct size (30.6±2.4%), and it had no effect on the protection mediated by ischemic preconditioning (9.6±2.5%, p<0.001 vs. Pre-L-NAME). On the other hand, infarction averaged 18.1±2.7% of the risk zone in hearts exposed to both L-NAME and CsA. These infarcts were significantly smaller than those in animals treated with only L-NAME (p<0.05). Infarctions tended to be larger than in hearts treated with only CsA, but this difference was not significant.

3.3 Protein phosphatase measurements
Baseline hemodynamic data after the equilibration period were comparable in the hearts from which biopsies were obtained. Following perfusion with 100 nmol/l CsA dephosphorylation of Formula labeled phosphorylase kinase was reduced to 37±8% of control, while 750 nmol/l CsA decreased dephosphorylation to 33±7% of the baseline level (p<0.01 vs. control). The lower dose of FK-506 had a small effect (69% of control), whereas 150 nmol/l decreased dephosphorylation to 19% of the baseline level.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
References
 
The present study demonstrates that CsA, a potent inhibitor of calcium/calmodulin-dependent protein phosphatase (PP2B), was highly protective against infarction in the isolated rabbit heart. This protection was equivalent to that seen with ischemic preconditioning. It is important to note that CsA was still protective even when given after the onset of ischemia, which is different from what we have found with other preconditioning mimetics such as adenosine [38]. Another PP2B inhibitor, FK-506, was also as protective as CsA when given as pretreatment.

PP2B, a member of the large family of serine/threonine phosphatases, is well characterized and was first purified from bovine heart [39–41]. Still, relatively little is known about the physiological role of PP2B in the heart. Caroni et al. [42]noted that a calcium/calmodulin-dependent protein phosphatase deactivated the Na+/Ca2+ exchanger in heart sarcolemma, and Zimmermann et al. [43]showed an increase in the phosphorylation state, especially of myosin light chain and troponin I, following CsA treatment in guinea pig cardiomyocytes.

CsA and FK-506 are both potent inhibitors of PP2B in different tissues [24, 25, 43–45]. These agents are well known for their immunosuppressive properties. Normally activation of PP2B by increasing cellular calcium leads to dephosphorylation of the cytosolic subunit of the transcription factor NF-ATc which then migrates to the nucleus where, after combining with nuclear factors, it binds to and activates the interleukin-2 promoter gene [46]. Increased production of interleukin-2 and T-cell proliferation account for rejection of foreign tissue. This process can be interrupted by CsA which binds with a cytosolic binding protein, cyclophilin [25, 26, 47]. The drug–protein complex then binds to and inhibits PP2B, thus effectively blocking interleukin-2 production and T-cell proliferation [46, 47]. FK-506 binds to its own binding protein (FKBP), and this complex similarly inhibits PP2B [25, 26, 47]with subsequent suppression of interleukin-2 transcription and T-cell proliferation [46, 47].

Our protein phosphatase assays in myocardial tissue indeed demonstrate that CsA is an effective PP2B inhibitor in rabbit heart. It is recognized that the assay system we used may not be totally specific for PP2B and may possibly be influenced by other unidentified protein phosphatases in the homogenate which might recognize phosphorylase kinase or inhibitor 1 as a substrate. We assayed serial samples from each heart on the same gel with the same protein loading with and without the calcium chelator EGTA and the calmodulin antagonist trifluoperazine. This allowed us to measure the calcium/calmodulin-dependent phosphatase activity. Because we did not purify phosphorylase kinase or inhibitor-1, the substrates for the phosphatase assay, it was necessary to separate the substrates with polyacrylamide gel electrophoresis. It was assumed (but not tested) that the bands identified as substrate were not contaminated with other phosphatase substrates. The results obtained with our method are supported by the data of Zimmermann et al. [43]who showed that CsA enhanced the phosphorylation state of cardiomyocytes in a concentration-dependent manner.

Conflicting evidence exists regarding the protective mechanism of CsA in the ischemic heart. Griffiths and Halestrap [27, 28]noted in isolated rat hearts that CsA improved post-ischemic left ventricular function. They and others further observed that the opening of a non-specific ion pore in the mitochondrial inner membrane [27–29, 31, 48]occurred in hearts reperfused following ischemia, and formation of that pore was blocked by CsA. A distinct isoform of cyclophilin which functions as a cis–trans-peptidyl–prolyl isomerase is present in the mitochondrial matrix of heart cells, and its interaction with a membrane protein during ischemia is thought to lead to the pore formation. Binding of cyclophilin by CsA inhibits the enzyme's activity, thus blocking mitochondrial pore opening and presumably calcium overload during ischemia [48], the proposed mechanism of protection. In contrast to CsA, FK-506 inhibits neither the mitochondrial cis–trans-peptidyl–prolyl isomerase nor induction of mitochondrial pores [48]. Yet FK-506 was quite protective in rabbit hearts (Fig. 3), thus making mitochondrial pore closure less likely as the mechanism of CsA's protection in the rabbit.

Massoudy et al. [32]studied working and non-working guinea pig hearts and measured recovery of cardiac function and NO release from the hearts during global ischemia and reperfusion. They found that 800, but not 80, nmol/l CsA infused prior to ischemia resulted in significantly improved return of systolic function. NO release in control and CsA-treated hearts was identical before ischemia and returned to preischemic levels within 5 min of reperfusion. The hearts exposed to CsA maintained NO release at this level throughout the 25-min reperfusion period, whereas in control hearts NO release declined after 10 min. The authors concluded that preservation of NO release could be the protective mechanism. This interpretation is somewhat contrary to the observation of Taniguchi et al. [49]in isolated rat hearts. They found NO levels in the coronary effluent following the second and third preconditioning ischemia/reperfusion cycles did not recover to the same extent as seen after the initial ischemic episode, and suggested attenuated NO release was contributing to the protection. The role of NO as a cardioprotective agent is still controversial. Bilinska et al. [50]observed that donors of NO protected against reperfusion arrhythmias in isolated rat hearts. On the other hand, Weselcouch et al. [51]saw no attenuation of function following reperfusion in ischemically preconditioned rat hearts when NO synthesis was inhibited by L-NAME (30 µmol/l). Woolfson et al. [52]found that inhibition of NO synthesis did not block ischemic preconditioning's anti-infarct effect in the in vitro rabbit heart, similar to observations reported here (Fig. 4). Unlike the present findings, however, Woolfson actually found some protection against infarction from the NO blocker itself, suggesting a detrimental role for endogenous NO. In light of the suggestion that NO might contribute to CsA's protective effect, we attempted to attenuate the latter with L-NAME. Administration of L-NAME to hearts treated with CsA tended to increase infarct size over that seen with CsA alone, but the trend was not significant. Therefore, our data do not confirm, but rather tend to negate, a role for NO in CsA's protection.

But even if NO is not clearly involved in the protection of CsA in our rabbit model, our experiments do not exclude a possible role for endothelin. Both CsA [53, 54]and FK-506 [54, 55]cause endothelin to be released from cells in culture, and Massoudy et al. [32]in their guinea pig hearts showed that CsA's ability to preserve post-ischemic function could be aborted if an endothelin receptor blocker were co-administered. Indeed, we have previously demonstrated that exogenously administered endothelin mimics the protection of ischemic preconditioning in the isolated rabbit heart [5]by activating protein kinase C (PKC). Nonetheless it is improbable that the increased release of endothelin mediated by CsA could have resulted in enough PKC stimulation to result in protection. Brunner and colleagues [56]measured an endothelin concentration of approximately 0.05 pmol/l in the coronary effluent of control rat hearts, while CsA increases cellular endothelin release by 40–130% [53, 54]. Yet the endothelin dose required for the triggering of preconditioning in the rabbit heart is 100–200-fold higher [5]. Despite these arguments, however, we cannot completely exclude a role for endothelin in CsA's protection because we did not examine whether CsA could decrease infarction in the presence of specific endothelin receptor antagonists.

Finally, both CsA and FK-506 are also known to block degranulation of mast cells [57]. Linden [58]has suggested that the mechanism of preconditioning is related to the inability of previously activated mast cells to release large quantities of cytotoxic granule contents at the beginning of ischemia. If this hypothesis were correct, it would follow that mast cell stabilizing agents would have a protective effect. However, we have previously provided evidence that mast cells play no role in the protection of ischemic preconditioning, and interference with degranulation at the onset of ischemia had no protective value [59].

Studies from our laboratory [21]as well as Ganote's [18, 19, 22]have clearly demonstrated that inhibitors of protein phosphatases and more specifically PP2A inhibitors can protect whole hearts and isolated ventricular cardiomyocytes. It is not known how PP inhibition actually protects the myocardium, but it seems likely that they promote phosphorylation of one or more kinase substrates in preconditioning's signal transduction pathway. Thus, the effect of inhibiting the phosphatase would be similar to activation of the associated kinase. It is somewhat surprising that a PP2B inhibitor has an effect similar to that of PP2A inhibition because the substrate specificities of the two phosphatases are quite different. If, however, the kinases are arranged in a sequential cascade, then activation at any point in the cascade will result in activation of all down-stream kinases and activation of the final effector.

Coronary collateral flow in a blood-perfused rabbit heart ranges from 1–5% of flow to normal myocardium [60, 61], but is probably higher in the buffer-perfused, isolated heart. Therefore, collateral flow is low, but not absent. A CsA concentration of 100 nmol/l was protective, although the effective minimal concentration was not determined. Therefore, the dose of 750 nmol/l used in the post-ischemia experiments was 7.5-fold greater than the known effective pretreatment concentration, but could actually be many more times the smallest protective perfusate dose. We used this higher concentration to decrease the amount of time required for the meager collaterals to load the ischemic myocardium with an effective dose of CsA.

Thornton [38]and others [62, 63]have demonstrated that receptor agonists which mimic preconditioning are not effective when administered after the onset of ischemia. Because treatment prior to acute myocardial infarction is a virtual impossibility in most clinical situations, there has been a great deal of interest in anti-infarct agents which do not require pretreatment. The PP2A inhibitor fostriecin was effective as an infarct-sparing agent in the isolated rabbit heart even when started early in ischemia [21]. The explanation of this cardioprotection is unknown but may result from activation of the signal transduction pathway at a very distal point which would avoid some of the delays associated with upstream components of that pathway. Now CsA, a PP2B antagonist, has been found to exhibit this same property. If these phosphatase inhibitors are indeed acting to promote phosphorylation of some kinase's substrate, then one might imagine that their effect would be lost when the cell's ATP stores have become sufficiently depleted so that kinases can no longer phosphorylate substrate, i.e., after approximately 10 min of ischemia in the rabbit heart. When administration is delayed for 20 min, kinase activity may be so low that blockade of dephosphorylation is meaningless, thus accounting for failure of the Post-20-CsA protocol.

CsA and FK-506 are widely used clinically and their adverse effects are well known [23, 64]. The concentrations of 100 and 750 nmol/l CsA used in this study are close to the effective plasma levels of 100 to 800 ng/ml (depending on the determination method) required in patients after heart transplantation [23]. If CsA or FK-506 could be administered very early to patients presenting with chest pain, then in those patients actually having an acute myocardial infarction the early treatment should result in a powerful anti-infarct effect. Of course, it is unknown whether the hemodynamic side effects would allow such a rapid administration of these agents.

Time for primary review 26 days.


   Acknowledgements
 
Christof Weinbrenner is sponsored by a grant of the Deutsche Forschungsgemeinschaft (We 1955/1-1). This study was supported in part by grants from the National Institutes of Health Heart, Lung, and Blood Institute HL-20648 and HL-50688.


   Notes
 
1 Present address: Department of Cardiology, University of Heidelberg, Bergheimerstr. 58, D-69115 Heidelberg, Germany. Back


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

  1. Murry C.E, Jennings R.B, Reimer K.A. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation (1986) 74:1124–1136.[Abstract/Free Full Text]
  2. Liu G.S, Thornton J, Van Winkle D.M, Stanley A.W.H, Olsson R.A, Downey J.M. Protection against infarction afforded by preconditioning is mediated by A1 adenosine receptors in rabbit heart. Circulation (1991) 84:350–356.[Abstract/Free Full Text]
  3. Banerjee A, Locke-Winter C, Rogers K.B, Mitchell M.B, Brew E.C, Cairns C.B, Bensard D.D, Harken A.H. Preconditioning against myocardial dysfunction after ischemia and reperfusion by an {alpha}1-adrenergic mechanism. Circ Res (1993) 73:656–670.[Abstract/Free Full Text]
  4. Tsuchida A, Liu Y, Liu G.S, Cohen M.V, Downey J.M. {alpha}1-Adrenergic agonists precondition rabbit ischemic myocardium independent of adenosine by direct activation of protein kinase C. Circ Res (1994) 75:576–585.[Abstract/Free Full Text]
  5. Wang P, Gallagher K.P, Downey J.M, Cohen M.V. Pretreatment with endothelin-1 mimics ischemic preconditioning against infarction in isolated rabbit heart. J Mol Cell Cardiol (1996) 28:579–588.[CrossRef][Web of Science][Medline]
  6. Liu Y, Tsuchida A, Cohen M.V, Downey J.M. Pretreatment with angiotensin II activates protein kinase C and limits myocardial infarction in isolated rabbit hearts. J Mol Cell Cardiol (1995) 27:883–892.[CrossRef][Web of Science][Medline]
  7. Yao Z, Gross G.J. Acetylcholine mimics ischemic preconditioning via a glibenclamide-sensitive mechanism in dogs. Am J Physiol (1993) 264:H2221–H2225.[Web of Science][Medline]
  8. Thornton J.D, Liu G.S, Downey J.M. Pretreatment with pertussis toxin blocks the protective effects of preconditioning: evidence for a G-protein mechanism. J Mol Cell Cardiol (1993) 25:311–320.[CrossRef][Web of Science][Medline]
  9. Goto M, Liu Y, Yang X.-M, Ardell J.L, Cohen M.V, Downey J.M. Role of bradykinin in protection of ischemic preconditioning in rabbit hearts. Circ Res (1995) 77:611–621.[Abstract/Free Full Text]
  10. Schultz J.E.J, Rose E, Yao Z, Gross G.J. Evidence for the involvement of opioid receptors in ischemic preconditioning in rat hearts. Am J Physiol (1995) 268:H2157–H2161.[Web of Science][Medline]
  11. Ytrehus K, Liu Y, Downey J.M. Preconditioning protects ischemic rabbit heart by protein kinase C activation. Am J Physiol (1994) 266:H1145–H1152.[Web of Science][Medline]
  12. Liu Y, Cohen M.V, Downey J.M. Chelerythrine, a highly selective protein kinase C inhibitor, blocks the antiinfarct effect of ischemic preconditioning in rabbit hearts. Cardiovasc Drugs Ther (1994) 8:881–882.[CrossRef][Web of Science][Medline]
  13. Speechly-Dick M.E, Mocanu M.M, Yellon D.M. Protein kinase C: its role in ischemic preconditioning in the rat. Circ Res (1994) 75:586–590.[Abstract/Free Full Text]
  14. Ikonomidis J.S, Shirai T, Weisel R.D, Derylo B, Rao V, Whiteside C.I, Mickle D.A.G, Li R.-K. Preconditioning cultured human pediatric myocytes requires adenosine and protein kinase C. Am J Physiol (1997) 272:H1220–H1230.[Web of Science][Medline]
  15. Weinbrenner C, Liu G.-S, Cohen M.V, Downey J.M. Phosphorylation of tyrosine 182 of p38 mitogen-activated protein kinase correlates with the protection of preconditioning in the rabbit heart. J Mol Cell Cardiol (1997) 29:2383–2391.[CrossRef][Web of Science][Medline]
  16. Liu Y, Gao W.D, O'Rourke B, Marban E. Synergistic modulation of ATP-sensitive K+ currents by protein kinase C and adenosine: implications for ischemic preconditioning. Circ Res (1996) 78:443–454.[Abstract/Free Full Text]
  17. Yang X.-M, Sato H, Downey J.M, Cohen M.V. Protection of ischemic preconditioning is dependent upon a critical timing sequence of protein kinase C activation. J Mol Cell Cardiol (1997) 29:991–999.[CrossRef][Web of Science][Medline]
  18. Armstrong S.C, Ganote C.E. Effects of the protein phosphatase inhibitors okadaic acid and calyculin A on metabolically inhibited and ischaemic isolated myocytes. J Mol Cell Cardiol (1992) 24:869–884.[CrossRef][Web of Science][Medline]
  19. Armstrong S.C, Hoover D.B, Delacey M.H, Ganote C.E. Translocation of PKC, protein phosphatase inhibition and preconditioning of rabbit cardiomyocytes. J Mol Cell Cardiol (1996) 28:1479–1492.[CrossRef][Web of Science][Medline]
  20. Walsh A.H, Cheng A, Honkanen R.E. Fostriecin, an antitumor antibiotic with inhibitory activity against serine/threonine protein phosphatases types 1 (PP1) and 2A (PP2A), is highly selective for PP2A. FEBS Lett (1997) 416:230–234.[CrossRef][Web of Science][Medline]
  21. Weinbrenner C, Baines CP, Liu G-S, Armstrong SC, Ganote CE, Walsh AH, Honkanen RE, Cohen MV, Downey JM. Fostriecin, an inhibitor of protein phosphatase 2A, limits myocardial infarct size even when administered after onset of ischemia. Circulation 1998; (in press).
  22. Armstrong S.C, Kao R, Gao W, Shivell L.C, Downey J.M, Honkanen R.E, Ganote C.E. Comparison of in vitro preconditioning responses of isolated pig and rabbit cardiomyocytes: effects of a protein phosphatase inhibitor, fostriecin. J Mol Cell Cardiol (1997) 29:3009–3024.[CrossRef][Web of Science][Medline]
  23. Kahan B.D. Cyclosporine. New Engl J Med (1989) 321:1725–1738.[Web of Science][Medline]
  24. Fruman D.A, Klee C.B, Bierer B.E, Burakoff S.J. Calcineurin phosphatase activity in T lymphocytes is inhibited by FK 506 and cyclosporin A. Proc Natl Acad Sci USA (1992) 89:3686–3690.[Abstract/Free Full Text]
  25. Clipstone N.A, Crabtree G.R. Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. Nature (1992) 357:695–697.[CrossRef][Medline]
  26. Liu J, Farmer J.D Jr., Lane W.S, Friedman J, Weissman I, Schreiber S.L. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell (1991) 66:807–815.[CrossRef][Web of Science][Medline]
  27. Griffiths E.J, Halestrap A.P. Protection by cyclosporin A of ischemia/reperfusion-induced damage in isolated rat hearts. J Mol Cell Cardiol (1993) 25:1461–1469.[CrossRef][Web of Science][Medline]
  28. Griffiths E.J, Halestrap A.P. Mitochondrial non-specific pores remain closed during cardiac ischaemia, but open upon reperfusion. Biochem J (1995) 307:93–98.[Web of Science][Medline]
  29. Crompton M, Ellinger H, Costi A. Inhibition by cyclosporin A of a Ca2+-dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress. Biochem J (1988) 255:357–360.[Web of Science][Medline]
  30. Fukuda H, Shima H, Vesonder R.F, Tokuda H, Nishino H, Katoh S, Tamura S, Sugimura T, Nagao M. Inhibition of protein serine/threonine phosphatases by fumonisin B1, a mycotoxin. Biochem Biophys Res Commun (1996) 220:160–165.[CrossRef][Web of Science][Medline]
  31. Duchen M.R, McGuinness O, Brown L.A, Crompton M. On the involvement of a cyclosporin A sensitive mitochondrial pore in myocardial reperfusion injury. Cardiovasc Res (1993) 27:1790–1794.[Free Full Text]
  32. Massoudy P, Zahler S, Kupatt C, Reder E, Becker B.F, Gerlach E. Cardioprotection by cyclosporine A in experimental ischemia and reperfusion — evidence for a nitric oxide-dependent mechanism mediated by endothelin. J Mol Cell Cardiol (1997) 29:535–544.[CrossRef][Web of Science][Medline]
  33. Gabauer I, Slezak J, Styk J, Ziegelhöffer A. Further assessment of the protective effect of calmodulin inhibitors against reperfusion injury after acute coronary occlusion in the dog. Can J Cardiol (1994) 10:125–132.[Web of Science][Medline]
  34. Stewart A.A, Hemmings B.A, Cohen P, Goris J, Merlevede W. The MgATP-dependent protein phosphatase and protein phosphatase 1 have identical substrate specificities. Eur J Biochem (1981) 115:197–205.[Web of Science][Medline]
  35. Ganapathi M.K, Lee E.Y.C. Dephosphorylation and inactivation of phosphorylase kinase: subunit specificity of rabbit skeletal muscle protein phosphatases. Arch Biochem Biophys (1984) 233:19–31.[CrossRef][Web of Science][Medline]
  36. Stewart A.A, Cohen P. Protein phosphatase-2B from rabbit skeletal muscle: a Ca2+-dependent, calmodulin-stimulated enzyme. Methods Enzymol (1988) 159:409–416.[Web of Science][Medline]
  37. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (1970) 227:680–685.[CrossRef][Medline]
  38. Thornton J.D, Liu G.S, Olsson R.A, Downey J.M. Intravenous pretreatment with A1-selective adenosine analogues protects the heart against infarction. Circulation (1992) 85:659–665.[Abstract/Free Full Text]
  39. Wolf H, Hofmann F. Purification of myosin light chain kinase from bovine cardiac muscle. Proc Natl Acad Sci USA (1980) 77:5852–5855.[Abstract/Free Full Text]
  40. Stewart A.A, Ingebritsen T.S, Manalan A, Klee C.B, Cohen P. Discovery of a Ca2+- and calmodulin-dependent protein phosphatase. Probable identity with calcineurin (CaM-BP80). FEBS Lett (1982) 137:80–84.[CrossRef][Web of Science][Medline]
  41. Stewart A.A, Ingebritsen T.S, Cohen P. The protein phosphatases involved in cellular regulation. 5. Purification and properties of a Ca2+/calmodulin-dependent protein phosphatase (2B) from rabbit skeletal muscle. Eur J Biochem (1983) 132:289–295.[Web of Science][Medline]
  42. Caroni P, Soldati L, Carafoli E. The Na+/Ca2+ exchanger of heart sarcolemma is regulated by a phosphorylation–dephosphorylation process. Soc Gen Physiol Ser (1984) 38:149–160.[Medline]
  43. Zimmermann N, Gams E, Neumann J, Boknik P, Scholz H. Effects of cyclosporine on protein phosphorylation in the heart. Transplant Proc (1994) 26:2875–2876.[Web of Science][Medline]
  44. Groblewski G.E, Wagner A.C.C, Williams J.A. Cyclosporin A inhibits Ca2+/calmodulin-dependent protein phosphatase and secretion in pancreatic acinar cells. J Biol Chem (1994) 269:15111–15117.[Abstract/Free Full Text]
  45. Klee C.B, Krinks M.H, Manalan A.S, Cohen P, Stewart A.A. Isolation and characterization of bovine brain calcineurin: a calmodulin-stimulated protein phosphatase. Methods Enzymol (1983) 102:227–244.[Web of Science][Medline]
  46. Wera S, Hemmings B.A. Serine/threonine protein phosphatases. Biochem J (1995) 311:17–29.[Web of Science][Medline]
  47. Walsh C.T, Zydowsky L.D, McKeon F.D. Cyclosporin A, the cyclophilin class of peptidylprolyl isomerases, and blockade of T cell signal transduction. J Biol Chem (1992) 267:13115–13118.[Abstract/Free Full Text]
  48. Griffiths E.J, Halestrap A.P. Further evidence that cyclosporin A protects mitochondria from calcium overload by inhibiting a matrix peptidyl–prolyl cis–trans isomerase. Implications for the immunosuppressive and toxic effects of cyclosporin. Biochem J (1991) 274:611–614.[Web of Science][Medline]
  49. Taniguchi I, Taniguchi M, Kobayashi M, Onodera T, Seki S, Mochizuki S. Real time measurement of nitric oxide during ischemic preconditioning. J Mol Cell Cardiol (1997) 29:A232. (Abstract).
  50. Bilinska M, Maczewski M, Beresewicz A. Donors of nitric oxide mimic effects of ischaemic preconditioning on reperfusion induced arrhythmias in isolated rat heart. Mol Cell Biochem (1996) 160:265–271.[CrossRef][Medline]
  51. Weselcouch E.O, Baird A.J, Sleph P, Grover G.J. Inhibition of nitric oxide synthesis does not affect ischemic preconditioning in isolated perfused rat hearts. Am J Physiol (1995) 268:H242–H249.[Web of Science][Medline]
  52. Woolfson R.G, Patel V.C, Neild G.H, Yellon D.M. Inhibition of nitric oxide synthesis reduces infarct size by an adenosine-dependent mechanism. Circulation (1995) 91:1545–1551.[Abstract/Free Full Text]
  53. Bunchman T.E, Brookshire C.A. Cyclosporine-induced synthesis of endothelin by cultured human endothelial cells. J Clin Invest (1991) 88:310–314.[Web of Science][Medline]
  54. Takeda Y, Yoneda T, Ito Y, Miyamori I, Takeda R. Stimulation of endothelin mRNA and secretion in human endothelial cells by FK 506. J Cardiovasc Pharmacol (1993) 22:S310–S312. Suppl 8.[Web of Science][Medline]
  55. Goodall T, Kind C.N, Hammond T.G. FK 506-induced endothelin release by cultured rat mesangial cells. J Cardiovasc Pharmacol (1995) 26:S482–S485. Suppl 3.[Web of Science][Medline]
  56. Brunner F, du Toit E.F, Opie L.H. Endothelin release during ischaemia and reperfusion of isolated perfused rat hearts. J Mol Cell Cardiol (1992) 24:1291–1305.[CrossRef][Web of Science][Medline]
  57. Schreiber S.L, Crabtree G.R. The mechanism of action of cyclosporin A and FK506. Immunol Today (1992) 13:136–142.[CrossRef][Web of Science][Medline]
  58. Linden J. Cloned adenosine A3 receptors: pharmacological properties, species differences and receptor functions. Trends Pharmacol Sci (1994) 15:298–306.[CrossRef][Medline]
  59. Wang P, Downey J.M, Cohen M.V. Mast cell degranulation does not contribute to ischemic preconditioning in isolated rabbit hearts. Basic Res Cardiol (1996) 91:458–467.[CrossRef][Web of Science][Medline]
  60. Maxwell M.P, Hearse D.J, Yellon D.M. Species variation in the coronary collateral circulation during regional myocardial ischaemia: a critical determinant of the rate of evolution and extent of myocardial infarction. Cardiovasc Res (1987) 21:737–746.[Abstract/Free Full Text]
  61. Cohen M.V, Yang X.-M, Liu Y, Snell K.S, Downey J.M. A new animal model of controlled coronary artery occlusion in conscious rabbits. Cardiovasc Res (1994) 28:61–65.[Abstract/Free Full Text]
  62. Goto M, Miura T, Iliodoromitis E.K, O'Leary E.L, Ishimoto R, Yellon D.M, Iimura O. Adenosine infusion during early reperfusion failed to limit myocardial infarct size in a collateral deficient species. Cardiovasc Res (1991) 25:943–949.[Abstract/Free Full Text]
  63. Vander Heide R.S, Reimer K.A. Effect of adenosine therapy at reperfusion on myocardial infarct size in dogs. Cardiovasc Res (1996) 31:711–718.[Abstract/Free Full Text]
  64. Fahr A. Cyclosporin clinical pharmacokinetics. Clin Pharmacokinet (1993) 24:472–495.[Web of Science][Medline]

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