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Experimental Biology and Medicine 227:520-528 (2002)
© 2002 Society for Experimental Biology and Medicine

ORIGINAL ARTICLE

Influence of Ischemic Preconditioning on Intracellular Sodium, pH, and Cellular Energy Status in Isolated Perfused Heart

Andriy Babsky*,1, Shahryar Hekmatyar*, Suzanne Wehrli{dagger},, Nicolai Doliba{dagger},, Mary Osbakken{dagger},,§ and Navin Bansal*,1

* Department of Radiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104;
{dagger} Children's Hospital, Philadelphia, Pennsylvania 19104;
{dagger} Department of Biochemistry/Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104; and
§ Aventis, Bridgewater, New Jersey 08807


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Conclusion
 References
 
The possible relationships between intracellular Na+ (Nai+), bioenergetic status and intracellular pH (pHi) in the mechanism for ischemic preconditioning were studied using 23Na and 31P magnetic resonance spectroscopy in isolated Langendorff perfused rat heart. The ischemic preconditioning (three 5-min ischemic episodes followed by two 5-min and one 10-min period of reperfusion) prior to prolonged ischemia (20 min stop-flow) resulted in a decrease in ischemic acidosis and faster and complete recovery of cardiac function (ventricular developed pressure and heart rate) after 30 min of reperfusion. The response of Nai during ischemia in the preconditioned hearts was characterized by an increase in Nai+ at the end of preconditioning and an accelerated decrease during the first few minutes of reperfusion. During post-ischemic reperfusion, bioenergetic parameters (PCr/Pi and ßATP/Pi ratios) were partly recovered without any significant difference between control and preconditioned hearts. The reduced acidosis during prolonged ischemia and the accelerated decrease in Nai+ during reperfusion in the preconditioned hearts suggest activation of Na+/H+ exchanger and other ion transport systems during preconditioning, which may protect the heart from intracellular acidosis during prolonged ischemia, and result in better recovery of mechanical function (LVDP and heart rate) during post-ischemic reperfusion.

Key Words: perfused heart • preconditioning • sodium • NMR


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Conclusion
 References
 
Several theories have been proposed to explain the effect of brief periods of ischemia (ischemic preconditioning) to reduce necrosis in the heart during subsequent prolonged ischemia. The roles of adenosine, bradykinin, opioids, catecholamines, prostaglandins, free radicals, stress proteins, protein kinase C, sarcolemmal and mitochondrial KATP channels, glycolytic rate, etc. have been discussed as potential mechanisms by which preconditioning protects ischemic heart (see reviews 1–3). The possible role of ionic alterations (Na+, Ca2+, H+) for ischemic preconditioning was first described by Steenbergen et al. (4). Others have demonstrated that Ca2+ overload and ischemic acidosis are important intracellular alterations, which could lead to damage in ischemic cardiomyocytes (5,6). Na+ is involved in regulating both Ca2+ and H+ concentrations in the cell through Na+/H+, Na+/Ca2+, Na+-K+-2Cl- and Cl-/HCO3- ion transport mechanisms. Furthermore, Na+ is an important regulator of bioenergetic processes in healthy and diseased cardiomyocytes (7).

There is still controversy concerning the time and magnitude of changes in intracellular sodium (Nai+), which occur during preconditioning, post-preconditioning ischemia, and post-ischemia recovery (8–10). Steenbergen et al. (4) have demonstrated that preconditioning attenuated the increase Nai+ during ischemia, and that there was no difference in the rate of Nai+ recovery during reperfusion. Ramasamy et al. (11) have demonstrated that preconditioning did not reduce Nai+ accumulation during ischemia, but the decline in Nai+ during reperfusion was significantly greater in the preconditioned hearts compared to the control. Na+ transport systems (Na+/K+-ATPase, Na+/H+ and Na+/Ca2+ exchangers and Na+-K+-2Cl- co-transporter) could play a key role in preconditioning, most likely by limiting intracellular acidosis and protecting intracellular Ca2+ overload.

The goal of our investigation was to examine the relationships between the changes in Nai, cellular energy status, and pH during ischemic preconditioning, prolonged ischemia, and post-ischemia recovery during reperfusion in the Langendorff perfused rat heart. We employed 23Na nuclear magnetic resonance (NMR) spectroscopy and the paramagnetic shift reagent (SR) thulium(III) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phosphonate) (TmDOTP5-) for measurement of Nai+ and 31P MRS for measurement of phosphocreatine (PCr), adenosine triphosphate (ATP), inorganic phosphate (Pi), and intracellular pHi.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Conclusion
 References
 
Isolated Perfused Heart.
Male Sprague-Dawley rats (220–270 g, n = 10) were obtained from the Charles River Co. The study was reviewed and approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania and conforms with the PHS Guide for the Care and Use of Laboratory Animals and the U.S. Interagency Research Animal Committee Principles for the Utilization and Care of Research Animals. The rats were anesthetized by an intraperitoneal injection of sodium Nembutal (100 mg/kg). Hearts (1.26 ± 0.07 g) were quickly removed and perfused via the aorta using the Langendorff method (100 mmHg at 37°C) with modified Krebs-Henseleit buffer: NaCl 118 mM, KCl 4.7 mM, MgSO4 1.2 mM, CaCl2 2.0 mM, Na2•EDTA 0.5 mM, NaHCO3 25 mM, glucose 11 mM. The buffer was equilibrated with 95% O2/5% CO2.

A latex balloon (size 4, Kent Scientific Corp., Litchfield, CT) filled with the perfusate buffer containing 10 mM TmDOTP5- and 108 mM methylphosphonic acid (MPA) was inserted through the mitral valve and secured in the left ventricle. The balloon was connected to a pressure transducer (Ohmeda Medical Devices, Madison, WI) and a physiological monitor (Columbia Instruments, Columbus, OH) for monitoring the heart rate and left ventricular developed pressure (LVDP, systolic minus diastolic pressure). Heart rate and LVDP were used as indices of mechanical function. The coronary flow rate was measured and quantitated by collecting the coronary artery effluent in a calibrated graduated cylinder.

Magnetic Resonance Spectroscopy.
A Bruker 9.4-Tesla magnet interfaced to a state-of-the-art Bruker AVANCE 400 DMX console was used. Isolated perfused beating hearts were placed in a 20-mm NMR tube for acquisition of 23Na and 31P spectra. 23Na NMR spectra were obtained using a 34-µsec excitation pulse (90° flip angle) followed by acquisition of 3,072 data points over a spectral width of 10 kHz. One hundred free induction decays (FIDs) were averaged over 25 sec using 0.2-sec repetition time. The Nai+ and extracellular sodium (Nae+) resonances were discriminated with the paramagnetic SR TmDOTP5- (3.6 mM) added to the perfusate buffer. 31P NMR spectra were acquired using a 40-µsec excitation pulse (60° flip angle) followed by acquisition of 16k data points over a spectral width of 16 kHz. One hundred twenty-eight FIDs were averaged over 3 min using 1.5-sec repetition time.

Experimental Protocol.
As shown in Figure 1Go, two groups of hearts, one with preconditioning (PC group) and the other without preconditioning (CON group) were examined. After 15 min of baseline perfusion, preconditioning was produced over 35 min. For preconditioning, two 5-min ischemic episodes were each followed by 5 min of reperfusion. Another 5-min ischemic episode was then followed by 10 min of reperfusion. The control hearts were perfused normally during the 35 min preconditioning period. Both the CON (n = 6) and PC (n = 4) hearts were subjected to 20-min stop-flow ischemia followed by 30 min of reperfusion.



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  		Figure 1. Schematic representation of the experimental protocols. DMMP (dimethyl methylphosphonate), PPA (phenylphosphonic acid), and SR (shift reagent) were added in perfusion buffer. I–VII are the steps of the experimental protocol described in the Methods.

 
In the beginning of perfusion two 31P spectra were obtained to determine the cellular energy status of the isolated hearts (Fig. 1Go, step I). After addition of dimethyl methylphosphonate (DMMP) and phenylphosphonic acid (PPA), which were used for measurement of intracellular volume (12), usually two 31P and two 23Na spectra were collected (step II). The SR, TmDOTP5- (3.6 mM) was added to the perfusate to separate Nai+ and Nae+ signals. Additional CaCl2 (1.3–1.6 mM) was added to the perfusate to bring the free [Ca2+] in physiological range. 23Na spectra were continually acquired during the SR equilibration period, which took 20–25 min (step III). After a steady-state separation between the Nai+ and Nae+ resonances was achieved, one 31P spectrum was collected to determine the effects of SR on cellular bioenergetics and pH, and two 23Na spectra were obtained to determine the initial Nai+ level (baseline period, 15 min, step IV). During preconditioning, six 23Na spectra were collected in PC hearts: one for each 5-min ischemic episode and one for each reperfusion episode (3 ischemia/reperfusion episodes) (preconditioning period, 35 min, step V). During the third reperfusion episode in the preconditioning period, one 31P spectrum was obtained to determine the effect of preconditioning on cellular energetics. In the CON group, the same number of 23Na and 31P spectra were collected during the 35-min regular perfusion (step V). During 20-min ischemia (step VI), four 23Na spectra followed by one 31P spectrum were obtained. Similarly, during the 30-min reperfusion (step VII), six 23Na spectra followed by one 31P spectrum were collected. The coronary flow rate was measured at the end of the initial stabilization period, SR equilibration period, preconditioning, and reperfusion.

Data Analysis and Statistics.
NMR data were transferred to a personal computer and processed using NMR Utility Transform Software (Acorn NMR, Fremont, CA) program for Windows 95/NT. Line broadenings of 25 Hz for 31P and 10 Hz for 23Na were applied, and the resulting FID was Fourier transformed. The resonance areas in 31P and 23Na spectra were determined by integration. Intracellular pH (pHi) was calculated using the following relation (13):

Volumes in the beating, perfused hearts were determined, using ratios of DMMP, MPA, and PPA resonance areas in 31P MRS spectra (12). The intracellular volume was calculated from the difference between the total water volume and extracellular volume.

All data are presented as the mean ± SE. Statistical analyses of the data were performed by two-way analysis of variance (ANOVA) (Statistica/w 5.1 program). Post hoc comparisons among the experimental groups were then performed using least-significant differences (LSD) test. A P value <= 0.05 was used to denote a statistical significance.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Conclusion
 References
 
Cardiac Function.
Table IGo shows the heart rate, LVDP, and coronary flow rate before and after the addition of SR and during preconditioning, ischemia, and reperfusion for the CON and PC hearts. During each 5-min preconditioning ischemic episode, the LVDP decreased to approximately 3–4 mmHg. However, heart rate and LVDP returned back to baseline values during each preconditioning reperfusion episode. At the end of ischemic preconditioning, heart rate, LVDP and coronary flow were not significantly different from the values before preconditioning. Significant differences were found between CON and PC hearts during reperfusion after the 20-min ischemia. After 5 min of reperfusion, LVDP was recovered to 62 ± 16% compared to the baseline value in the PC hearts. In contrast, it recovered to only 31 ± 16% in the CON hearts (P <= 0.05 CON vs. PC). After 30 min of reperfusion the difference in LVDP was even larger and more significant (PC, 82 ± 13% recovery vs. CON, 38 ± 16% recovery, P <= 0.01) (Fig. 2Go). Two-way ANOVA showed a significant effect of preconditioning on LVDP between CON and PC hearts (F(1, 24) = 11.69, P < 0.002) during post-ischemic reperfusion. Repeated-measure ANOVA showed changes in LVDP values (F(2, 24) = 9.50, P < 0.0004) with time in both the groups and effect of preconditioning with time (F(2, 24) = 3.00, P < 0.069). In addition, the recovery of heart rate was also better in PC hearts. After 30 min of reperfusion, the heart rate was recovered to 229 ± 12 beats/min in PC hearts and only to 189 ± 31 beats/min in CON hearts (Table IGo).


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  		Table I. Myocardial Function in Isolated Perfused Heart*
 


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  		Figure 2. Relative changes in left ventricular developed pressure (LVDP) in control (CON) and preconditioned (PC) hearts during ischemia (20 min of stop-flow) and reperfusion (30 min). Baseline LVDP is normalized to 100%. The PC hearts showed 82 ± 16% recovery of LVDP after 30 min of reperfusion compared to only 39 ± 13% in CON hearts; n = 6 for CON and n = 5 for PC hearts. Values are reported as Mean ± SE. Significance: *P < 0.05, **P < 0.01 (CON vs. PC hearts).

 
23Na MRS of Perfused Heart.
Figure 3Go shows 23Na spectra from an isolated, perfused heart before and after addition of 3.6 mM TmDOTP5- to the perfusate. The chemical shift of tissue Na+ before addition of the SR was set to 0 ppm, while the signal from Na+ in the reference bulb containing 10 mM TmDOTP5- was shifted to 10.5 ppm. Ten minutes after addition of SR, one shifted peak became visible at ~2.7 ppm (spectrum 2). It is likely that this shifted peak was from Na+ in the perfusate and the unshifted peak contained contributions from both intracellular and interstitial sodium in the heart. A few minutes later, a second shifted peak appeared at ~1.5 ppm (spectrum 3). This shifted peak may largely be from interstitial Na+ in the heart. The two shifted 23Na peaks from the perfusate and interstitial Na+ converged after 16 min (spectrum 4). Only a single shifted Nae+ peak was visible at about 20 min, when a steady state was reached (spectra 5 and 6). This is in contrast with a recent report in which multiple Nae+ peaks were observed even after 75 min of perfusion with the same SR but Ca2+ free perfusate (14). In our experiments 3.5 mM TmDOTP5- shifted the Nae+ signal sufficiently to obtain adequate resolution for reliable measurement of changes in Nai+ signal intensity.



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  		Figure 3. Stacked-plot of typical 23Na spectra from an isolated perfused rat heart after addition of the sodium shift reagent (SR), TmDOTP5- (3.6 mM). Nai, intracellular sodium; Nat, total sodium; Nae, extracellular sodium; Naref, signal from reference capillary containing 10 mM TmDOTP5-. Time after TmDOTP5- addition is indicated on the right. Addition of the SR initially produced two shifted 23Na resonance from perfusate and interstitial sodium (spectrum 3). However, the two shifted Nai+ signals coalesced into a single sharp resonance after 20–25 min (spectra 5 and 6).

 
Figure 4Go shows relative changes in Nai+ signal intensity for the PC and CON hearts during preconditioning, ischemia, and reperfusion. The three ischemia/reperfusion episodes during preconditioning progressively increased Nai signal intensity. During the third ischemic episode of preconditioning, the Nai signal intensity in PC hearts (132 ± 8.4%) was 33.0% higher (P < 0.01) compared to CON hearts (98.8 ± 4.0%). Nai+ signal intensity increased from 126 ± 12.9% to 174 ± 8.6% during the 20 min of ischemia in PC hearts and from 108 ± 4.0 to 151 ± 22.2% in CON hearts. The increase in Nai+ signal intensity relative to the pre-ischemic value was not significantly different in the two groups. During the later periods of reperfusion Nai signal intensity decreased to similar levels in both the experimental groups. However, as shown in Figure 5Go, during the first few minutes of reperfusion, the decrease in Nai+ was faster in the PC hearts compared to CON hearts. Nai+ decreased by 31.3 ± 8.3% (from 174 ± 8.6% to 143 ± 12.2%) during the initial 3-min of reperfusion in the PC hearts, but it changed by only 3.75 ± 19.7% (from 151 ± 22.2% to 148 ± 13.3%) in the CON hearts.



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  		Figure 4. Relative changes in intracellular sodium (Nai+) resonance area as a function of time in control (CON, n = 6) and preconditioned (PC, n = 4) hearts. Nai baseline is normalized to 100. The Nai+ level in the PC hearts was increased by approximately 33% at the end of preconditioning. On reperfusion after prolonged ischemia, the decrease in Nai+ was faster compared to the CON hearts. Significance: *P < 0.01 (PC vs. CON), #P < 0.05 (PC group vs. end of ISC), &P < 0.01 (vs. pre-ischemic level for each group).

 


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  		Figure 5. Relative changes in normalized intracellular sodium (Nai+) resonance area at the beginning of ischemia and reperfusion in control (CON) and preconditioned (PC) hearts. Areas are derived from data shown in Figure 4Go. The Nai+ increase during initial 6 min of ischemia was similar in CON and PC hearts. However, PC hearts showed a faster initial recovery of Nai+ during reperfusion compared to the CON hearts.

 
Two-way ANOVA showed significant differences in Nai+ between CON and PC hearts during preconditioning (F(1, 48) = 6.15, P < 0.0167) and ischemic periods (F(1, 32) = 8.46, P < 0.0065) but not during post-ischemic reperfusion (F(1, 43) = 0.025, P < 0.875).

31P MRS of Perfused Heart.
Cellular energetics.
Figure 6Go shows PCr/Pi and ßATP/Pi for the PC and CON hearts during initial perfusion, and at the end of preconditioning, ischemia, and post-ischemic reperfusion. At the beginning of perfusion (baseline) the cellular energy status of the PC and CON hearts was similar. The PCr/Pi was 3.39 ± 0.11 in PC hearts and 3.23 ± 0.17 in CON hearts, and ßATP/Pi was 2.95 ± 0.2 in PC hearts and 2.96 ± 0.24 in CON hearts. Addition of PPA, DMMP, and TmDOTP5- to the perfusate did not produce significant changes in the cellular energy status in either group (data are not presented). After preconditioning, in the PC hearts, PCr/Pi and ßATP/Pi were 1.79 ± 0.75 and 1.33 ± 0.44, respectively, and, in the CON hearts, 2.68 ± 0.54 and 2.07 ± 0.83, respectively. Preconditioning produced a significant decrease in ATP/Pi in the PC hearts compared to the baseline value. However, there was no significant difference in the cellular energy status between the PC and CON hearts before prolonged ischemia. At the end of ischemia PCr/Pi and ßATP/Pi levels were reduced to almost zero in both the groups. At the end of reperfusion PCr/Pi and ßATP/Pi ratios were partly recovered without any significant difference between the PC and CON hearts. PCr/Pi and ATP/Pi were 1.2 ± 0.3 and 0.4 ± 0.1, respectively, in PC hearts and 1.0 ± 0.4 and 0.31 ± 0.05, respectively, in CON hearts. Two-way ANOVA did not show significant differences between CON and PC hearts with respect to PCr/Pi (F(1, 28) = 0.316, P < 0.579) and ßATP/Pi (F(1, 28) = 0.656, P < 0.425).



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  		Figure 6. Changes in PCr/Pi (A) and ATP/Pi (B) ratios in control (CON) and preconditioned (PC) hearts (mean ± SE, n = 6 (CON), n = 4(PC)). After preconditioning, ATP/Pi were reduced compared to the baseline value in the PC heart. At the end of ischemia, PCr/Pi and ATP/Pi were reduced to almost zero in both CON and PC groups. At the end of post-ischemic reperfusion, PCr/Pi and ATP/Pi were partly recovered without any significant difference between the two groups. Significance: (i) PCr/Pi data, *P < 0.01 (ischemia vs. baseline), #P < 0.05 (reperfusion-PC vs. ischemia-PC); (ii) ATP/Pi data, *P < 0.05 (preconditioning vs. baseline), **P < 0.01 (ischemia vs. baseline), #P < 0.01 (reperfusion-PC vs. ischemia-PC).

 
Intracellular pH.
Figure 7Go shows pHi (calculated from 31P spectra) for the PC and CON hearts during initial perfusion, and at the end of preconditioning, ischemia, and post-ischemic reperfusion. At the beginning of perfusion, pHi of the PC (7.06 ± 0.05) and CON (7.09 ± 0.06) hearts was similar. At the end of preconditioning, pHi fell to 6.75 ± 0.15 in the PC hearts (P < 0.05). At the same time point, pHi did not change significantly in the CON hearts (6.97 ± 0.10). The end-ischemic pHi was decreased compared to the baseline values in both the groups (P < 0.001). However, compared to the pre-ischemic levels, ischemia did not change pHi significantly in the PC group and it decreased pHi by 0.59 ± 0.08 unit (P < 0.001) in CON group. At the end of 20 min of ischemia, pHi was significantly acidic in the CON hearts (6.38 ± 0.27) compared to the PC hearts (6.65 ± 0.12) (P < 0.05). Reperfusion returned pHi to approximately the same levels in PC (6.98 ± 0.19) and CON (6.90 ± 0.15) hearts. Two-way ANOVA showed significant differences between CON and PC hearts with respect to pHi in different experimental periods of perfusion (F(3, 33) = 4.19, P < 0.013).



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  		Figure 7. Changes in intracellular pH in control (CON) and preconditioned (PC) rat hearts. At the end of preconditioning, pHi was significantly reduced compared to baseline. Compared to pre-ischemic levels, ischemia decreased pHi by 0.59 ± 0.08 unit in CON hearts but it did not change pHi significantly in the PC hearts. Reperfusion returned pHi to approximately the same levels in PC and CON hearts. Significance: *P < 0.05 (PC vs. CON (baseline 2)), **P < 0.05 (CON (ischemia) vs. PC (ischemia); #P < 0.001 (CON: ischemia vs. baseline2), ##P < 0.01 (CON: reperfusion vs. ischemia); &P < 0.05 (PC: reperfusion vs. ischemia).

 
Cellular volume.
The intra- and extracellular volumes in the beating, perfused hearts were 0.36 ± 0.07 and 6.46 ± 1.3 ml, respectively, and did not change significantly during preconditioning, ischemia, and reperfusion.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Conclusion
 References
 
Cardiac Function and Intracellular pH.
The results reported here show that the hearts that were subjected to ischemic preconditioning have a smaller decrease in pHi during prolonged ischemia and better recovery of mechanical function (LVDP and heart rate) during reperfusion after prolonged ischemia compared to hearts without preconditioning. These data support the well-known effect that short ischemic episodes protect the heart from ischemic/reperfusion injury and reduce infarct size (15–17).

LVDP recovery during reperfusion after ischemia is determined by many factors, including pH (18). In our experiments there was an initial rapid recovery of LVDP during post-ischemic reperfusion in PC hearts (reaching 64% of the pre-ischemic level within 5 min and 82% within 30 min of reperfusion). A better recovery of post-ischemic LVDP could be explained by improved pH homeostasis most likely via Na+-coupled net H+ efflux (Na+-HCO3- co-influx and Na+/H+ exchange) (19) along with contribution from lactate and CO2 efflux (20). In non-preconditioned hearts LVDP did not return back to the pre-ischemic level even after 30 min of reperfusion following prolonged ischemia (only 39% compare to 100% pre-ischemic value), despite the recovery of intracellular pH.

The LVDP in our perfused heart preparations was significantly less than the normal values for the isolated perfused rat heart (Table IGo). This was perhaps due to the combined effects of the anesthesia and the sodium SR. All SRs, including TmDOTP5-, are known to complex with extracellular Ca2+ and decrease the free Ca2+ concentration. To avoid this problem we added extra 1.3–1.6 mM CaCl2 to the perfusion medium, but that did not circumvent the problem. Despite this problem, the other mechanical functions of the heart (heart rate, 249–277 beats/min, and coronary flow, 8.8–8.9 ml/min) during baseline perfusion were within physiological range in our experiments. Furthermore, these mechanical functions (in preconditioned hearts) and the intracellular pH (in both the experimental groups) were recovered after post-ischemic reperfusion.

Na+ Accumulation During Preconditioning and Ischemia/Reperfusion.
In our experiments, preconditioning resulted in an increase in Nai+ during the last (third) ischemic episode. Intracellular sodium during preconditioning has also been measured in other studies (4,9,11). A study by Tosaki et al. (9) using destructive chemical analysis and another study by Steenbergen et al. (4) using NMR did not show significant changes of Nai+ during preconditioning. This may be due to (i) large errors associated with the measurement of Nai+ by atomic absorption method (9), (ii) use of calcium chelator 5F-BAPTA in the perfusion media (for measurement of free intracellular Ca2+), which can lead to decreased developed pressure throughout the experiment, and/or (iii) presence of high concentration of NH4+ (20 mM) as a counter-cation with TmDOTP5- (4). Our results are similar to the data reported by Ramasamy et al. (11), who used DyTTHA3- as a Na+ SR and showed a significant increase in Nai+ at the last (fourth) ischemic episode during preconditioning compared to CON hearts. The preconditioning-induced increase in Nai+ may be due to the activation of Na+/H+ exchanger.

To establish that preconditioning activates Na+/H+ exchanger, the ratios of changes in Nai+ to changes in pHi ({Delta}Nai/{Delta}pHi) during preconditioning, prolonged ischemia, and post-ischemic reperfusion periods were calculated. In CON hearts, {Delta}Na/{Delta}pH values were -88, -75, and -52 during preconditioning, ischemia, and reperfusion periods, respectively, and were largely unchanged. In PC hearts, {Delta}Na/{Delta}pH was -75 during the preconditioning period, which is similar to the value for CON group. However, during prolonged ischemia the value increased to -482 in preconditioned hearts. During reperfusion it reduced to -209 but was still higher than the control hearts. The preconditioning induced increase in {Delta}Na/{Delta}pH was due to an increase in Na+ influx (Fig. 3Go) and a relatively small change in pHi (Fig. 6Go). These data support our hypothesis that preconditioning activated the Na+/H+ exchanger in the isolated perfused heart.

In our experiments, both PC and CON hearts showed a rapid increase in Nai during prolonged ischemia. Compared to the baseline period (i.e., before preconditioning) the relative increase in Nai+ at the end of ischemia was 50% for the CON hearts versus 75% for the PC hearts (Fig. 4Go). However, the relative increase in Nai+ compared to the values before prolonged ischemia was similar in the two groups (+43.3% for the CON group and +47.9% for PC group). Ramasamy et al. (11) have shown similar results using a different preconditioning protocol (four 5-min ischemia and 5-min reperfusion episodes) and Dy(TTHA)3- as SR. Preconditioning-induced increase in Na+/H+ exchanger activity did not lead to a significant difference in the change in Nai+ level between the two experimental groups during prolonged ischemia because of a dramatic decrease in cellular energy metabolites (Fig. 6Go) resulting in a decrease in Na+/K+-ATPase activity, which is most important in maintaining the transmembrane Na+ gradient (21). In contrast to our data and the data reported by Ramasamy et al. (11), Steenbergen et al. (4) and Imahashi et al. (10) observed an attenuated increase in Nai+ during ischemia in PC hearts compared to CON hearts. Tani et al. (8) have suggested that the increase in Nai+ during ischemia may be age related. They reported an attenuated increase in Nai+ during ischemia in preconditioned hearts from 12-week-old rats but not from 50- and 100-week-old animals. We used 9- to 10-week-old rats and observed identical relative increase in Nai+ during prolonged ischemia in PC and CON hearts and at the same time observed cardioprotective effect of preconditioning on mechanical function.

Very interestingly, our data show that in the beginning of post-ischemic reperfusion, the rate of Nai recovery was faster in the PC hearts compared to the CON hearts (Fig. 5Go). This is in contrast with Steenbergen et al. (4) who did not find any difference in the rate of post-ischemic Nai+ recovery. On the other hand, Tosaki et al. (9) demonstrated unchanged Nai+ levels during post-ischemic reperfusion in both control and preconditioned hearts. Our data are consistent with Ramasamy et al. (11), who reported an accelerated recovery of Nai+ during reperfusion in preconditioned hearts. We have shown that most of the decrease in Nai+ occurred during the initial 10 min of post-ischemia reperfusion and that the changes in Nai+ during the later 20 min were similar in PC and CON groups. Nai+ remained approximately 20% higher compared to baseline after 30 min of post-ischemic reperfusion in both CON and PC groups. Thus, the most important and significant response of Nai+ in the PC hearts was an increase in Nai+ at the end of preconditioning and a rapid decrease Nai+ during the first few minutes of post-ischemic reperfusion compared to the CON hearts. These data suggest that changes in ion transport processes involving Na+ are altered in the PC hearts and may protect the heart from ischemic damage.

Our data show that ischemia decreased pHi by approximately 0.6 unit compared to the pre-ischemic value in the control hearts but did not produce a significant change in the PC hearts. Ischemic preconditioning may stimulate Na+/H+ exchanger, which could potentially reduce intracellular acidosis during prolonged ischemia. Activation of Na+/H+ exchanger in this case could increase acid extrusion during ischemia and support faster recovery of mechanical function in PC hearts during post-ischemic reperfusion. In PC hearts the {Delta}Nai/{Delta}pHi value, which represents Na+/H+ exchange activity, was decreased during post-ischemic reperfusion compared to ischemia, but it was still higher than the value during the baseline period. It has been shown by others that short repetitive intracellular acidification (a likely preconditioning stimulus) activates proton efflux via Na+/H+ exchanger (23). Activation of Na+/H+ exchanger may be related to activation of protein kinase C during preconditioning. Liu et al. (24) have shown that protein kinase C-dependent phosphorylation is necessary for the protective effects of preconditioning. In addition, Wallert et al. (25) have shown that the cardiac Na+/H+ exchanger is stimulated by activation of protein kinase C and that stimulation of the Na+/H+ exchanger normalized pHi after an acid load. Thus, preconditioning may activate protein kinase C, which in turn activates the Na+/H+ exchanger. Another possibility is that preconditioning or repeated intracellular acidification increases the number of sites or copies of the exchanger in the sarcolemma (23).

Contrary to our hypothesis that activation of Na+/H+ exchange during preconditioning could be beneficial for reducing intracellular acidosis, recently, the use of Na+/H+ exchanger antagonists has been suggested as a mechanism to prevent ischemic damage to the myocardium (16,26,27). Use of Na+/H+ exchange inhibitors in human clinical trials, however, have shown disappointing results; the reasons for this are not clear (28,29). Our results support the viewpoint that use of Na+/H+ exchange inhibitor would likely be detrimental in patients with ischemic heart disease and possibly explain the poor efficacy or no efficacy of such inhibitors in preventing ischemic damage.

The pHi at the end of ischemia was higher in our experiments compared to what has been reported in some previous publications (30–32). However, other publications have reported similar end-ischemic intracellular pH values as we observed. For example, Steenbergen et al. reported a pHi of 6.3 in control hearts and 6.5 in preconditioned hearts after 30 min of ischemia (4). The exact reason for the difference in end-ischemic pHi in various studies is not clear. It does not appear to be related to the different buffer systems (NaHCO3/HCO3- vs. NaH2PO4/H2PO4-) or substrate(s) (glucose only vs. glucose plus pyruvate) in the perfusion medium used in the previous studies. Another possibility is that the different studies used different equations for calculating the pH from 31P NMR data. We recalculated pHi using different equations that have been used previously for the heart but that did not change our pHi values significantly.

In addition to Na+/H+ exchanger, functional coupling between Na+-K+-2Cl- co-transporter and Cl-/HCO3- exchanger may be involved in controlling the pH during ischemia in PC hearts. Our data show that Nai+ is increased during preconditioning. This increase may result from an increase in the activity of Na+-K+-2Cl- co-transporter in addition to other ion transport mechanisms. If this is true, then intracellular Cl- should also increase during preconditioning. The increased Cl- during preconditioning may facilitate transport of HCO3- into the cells via Cl-/HCO3- exchanger, thus buffering the cell (11,20) during prolonged ischemia.

We have shown in the present study that Nai+ is increased at the end of preconditioning and further increased during prolonged ischemia in PC hearts compared to CON. One traditional view is that the increase in Nai+ in cardiomyocytes can stimulate the Na+/Ca2+ exchanger, leading to Ca2+ overloading in the myocardium, and increase susceptibility to ischemia/reperfusion injury (33). However, we observed better recovery of mechanical function (LVDP and heart rate) and less ischemic acidification in PC hearts despite the higher Nai+ level. Consistent with our data, a previous study by Tosaki et al. (9) has shown that ischemic preconditioning causes less accumulation of Ca2+ by cells during post-ischemic reperfusion and better recovery of cardiac function during reperfusion. One explanation for this phenomenon is that during ischemia, the Na+/Ca2+ exchanger may work in reverse direction, i.e., transport Ca2+ out of the cells and Na+ into the cells (34).

Steenbergen et al. (4) have shown that there is a good correlation between tissue ATP levels and Ca2+ concentration during ischemia. Lundmark et al. (23) have shown that preconditioning or repetitive acidosis attenuates the decrease of ATP during prolonged ischemia in isolated perfused rat hearts. Thus, the higher ATP level during ischemia in preconditioned hearts may help in maintaining the cellular Ca2+ level. A higher ATP level during ischemia may also be responsible for the faster recovery of Nai+ during the first few minutes of reperfusion in the preconditioned hearts. In our experiments the cellular ATP and PCr levels in the PC and CON hearts were similar during the preconditioning, ischemia, and reperfusion periods. Perhaps we did not observe better bioenergetics in the PC hearts compared to the CON hearts because we collected 31P NMR spectra only at the end of each experimental period, as our main goal was to monitor the changes in Nai+ continuously.


   Conclusion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
References
 
Ischemic preconditioning resulted in decreased cellular acidosis during prolonged ischemia and faster and more complete recovery of cardiac function upon reperfusion. The response of Nai+ in the PC hearts was an increase in Nai+ at the end of preconditioning and an accelerated decrease during the first few minutes of post-ischemic reperfusion. These data suggest that PC stimulated preliminary activation of ion transport processes involving Na+ may protect the heart from intracellular acidosis during prolonged ischemia, promote rapid recovery of pHi during reperfusion, and thus result in better recovery of mechanical function (LVDP and heart rate) during post-ischemic reperfusion.


   Footnotes
 
This work was supported in part by grant R01-HL54574 (to N.B.) from the National Institutes of Health.

1 To whom requests for reprints and correspondence should be addressed at 207 Anatomy-Chemistry Building, University of Pennsylvania, 37th & Hamilton Walk, Philadelphia, PA 19104. E-mail: andriy10{at}yahoo.com; bansal{at}rad.upenn.edu Back


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
 References
 

  1. Yellon DM, Baxter GF, Garcia-Dorado D, Heusch G, Sumeray MS. Ischaemic preconditioning: present position and future directions. Cardiovasc Res 37:21–33, 1998.[Abstract/Free Full Text]
  2. Cohen MV, Baines CP, Downey JM. Ischemic preconditioning: from adenosine receptor to KATP channel. Annu Rev Physiol 62:79–109, 2000.[Medline]
  3. Schulz R, Cohen MV, Behrends M, Downey JM, Heusch G. Signal transduction of ischemic preconditioning. Cardiovasc Res 52:181–198, 2001.[Free Full Text]
  4. Steenbergen C, Perlman ME, London RE, Murphy E. Mechanism of preconditioning. Ionic alterations. Circ Res 72:112–125, 1993.[Abstract/Free Full Text]
  5. Bouchard JF, Chouinard J, Lamontagne D. Participation of prostaglandin E2 in the endothelial protective effect of ischaemic preconditioning in isolated rat heart. Cardiovasc Res 45(2):418–427, 2000.[Abstract/Free Full Text]
  6. Wang Y, Ashraf M. Activation of {alpha}1-adrenergic receptor during Ca2+ pre-conditioning elicits strong protection against Ca2+ overload injury via protein kinase C signaling pathway. J Mol Cell Cardiol 30(11):2423–2435, 1998.[Medline]
  7. Babsky A, Doliba Nic, Doliba Nat, Savchenko A, Wehrli S, Osbakken M. Na+ effects on mitochondrial respiration and oxidative phosphorylation in diabetic hearts. Exp Biol Med 226(6):543–551, 2001.[Abstract/Free Full Text]
  8. Tani M, Honma Y, Takayama M, Hasegawa H, Shinmura K, Ebihara Y, Tamaki K. Loss of protection by hypoxic preconditioning in aging Fisher 344 rat hearts related to myocardial glycogen content and Na+ imbalance. Cardiovasc Res 41:594–602, 1999.[Abstract/Free Full Text]
  9. Tosaki A, Engelman DT, Engelman RM, Das DK. The evolution of diabetic response to ischemia/reperfusion and preconditioning in isolated working rat hearts. Cardiovasc Res 31:526–536, 1996.[Medline]
  10. Imahashi K, Nishimura T, Yoshioka J, Kusuoka H. Role of intracellular Na+ kinetics in preconditioned rat heart. Circ Res 88(11):176–182, 2001.
  11. Ramasamy R, Hong L, Anderson S, Lundmark J, Schaefer S. Ischemic preconditioning stimulates sodium and proton transport in isolated rat hearts. J Clin Invest 96:1464–1472, 1995.
  12. Clark K, Anderson R, Nedelec J-F, Foster D, Ally A. Intracellular and extracellular spaces and the direct quantification of molar intracellular concentrations of phosphorus metabolites in the isolated rat heart using 31P NMR spectroscopy and phosphate markers. MRM 32:181–188, 1994.
  13. Seshan V, Bansal N. In vivo 31P and 23Na NMR spectroscopy and imaging. In: Bruch MD, Ed. NMR Spectroscopy Techniques (2nd ed). New York: Marcel Dekker, pp557–607, 1996.
  14. Zhao P, Xia ZF, Malloy CR, Sherry AD. TmDOTP5- differentiates two extracellular Na+ signals in hearts perfused with calcium-free buffer. Proc Intl Soc Magn Res Med 7:641, 1999.
  15. Eng S, Maddaford TG, Kardami E, Pierce GN. Protection against myocardial ischemic/reperfusion injury by inhibitors of two separate pathways of Na+ entry. J Mol Cell Cardiol 30:829–835, 1998.[Medline]
  16. Gumina RJ, Buerger E, Eickmeier C, Moore J, Daemmgen J, Gross G. Inhibition of the Na+/H+ exchanger confers greater cardioprotection against 90 minutes of myocardial ischemia than ischemic preconditioning in dogs. Circulation 100:2519–2526, 1999.[Abstract/Free Full Text]
  17. Miura T, Ogawa T, Suzuki K, Goto M, Shimamoto K. Infarct size limitation by a new Na+–H+ exchange inhibitor, Hoe 642: difference from preconditioning in the role of protein kinase C. J Am Coll Cardiol 29(3):693–701, 1997.[Abstract]
  18. Jacobus WE, Pores I, Lucas S, Weisfedt M, Flaherty J. Intracellular acidosis and contractility in the normal and ischemic heart as examined by 31P NMR. J Mol Cell Cardiol 14:13–20, 1982.
  19. Vandenberg JI, Metcalfe J, Grace A. Mechanisms of pHi recovery after global ischemia in the perfused heart. Circ Res 72:993–1003, 1993.[Abstract/Free Full Text]
  20. Grace AA, Kirschelohr HL, Metcalfe JC, Smith GA, Weissberg PI, Cragoe EJ, Vandenberg JI. Regulation of intracellular pH in the perfused heart by external HCO3- and Na+–H+ exchange. Am J Physiol 265:H289–H298, 1993.[Abstract/Free Full Text]
  21. Cross HR, Radda GK, Clarke K. The role of Na+-K+-ATPase activity during low-flow ischemia in preventing myocardial injury: a 31P, 23Na and 87Rb NMR spectroscopic study. Magn Reson Med 34:673–685, 1995[Medline]
  22. Fenton RA, Dickson EW, Meyer TE, Dobson JG. Aging reduces the cardioprotective effect of ischemic preconditioning in the rat heart. J Mol Cell Cardiol 32:1371–1375, 2000.[Medline]
  23. Lundmark JA, Trueblood N, Wang LF, Ramasamy R, Schaefer S. Repetitive acidosis protects the ischemic heart: implications for mechanisms in preconditioned hearts. J Mol Cell Cardiol 31:907–917, 1999.[Medline]
  24. Liu Y, Ytrehus K, Downey J. Evidence that translocation of protein kinase C is key event during ischemic preconditioning of rabbit myocardium. J Mol Cell Cardiol 26:661–668, 1994.[Medline]
  25. Wallert MA, Frohlich O. {alpha}1-Adrenergic stimulation of Na+/H+ exchange in cardiac myocytes. Am J Physiol 263:C1096–C1102, 1992.[Abstract/Free Full Text]
  26. Avkiran M. Protection of the myocardium during ischemia and reperfusion. Na+/H+ exchange inhibition versus ischemic preconditioning. Circulation 100:2469–2472, 1999.[Free Full Text]
  27. Ramasamy R, Schaefer S. Inhibition of Na+–H+ exchanger protects diabetic and non-diabetic hearts from ischemic injury: insight into altered susceptibility of diabetic hearts to ischemic injury. J Mol Cell Cardiol 31:785–797, 1999.[Medline]
  28. Menown IBA, Adgey AAJ. Cardioprotective therapy and sodium–hydrogen inhibition: current concepts and future goals. J Am Coll Cardiol 38:1651–1653, 2001.[Free Full Text]
  29. Avkiran M, Marber MS. Na+–H+ exchange inhibitors for cardioprotective therapy: progress, problems and prospects. J Am Coll Cardiol 39:747–753, 2002.[Abstract/Free Full Text]
  30. Kuprianov VV, Xiang B, Buttler KW, St-Jean M, Deslauriers R. Energy metabolism, intracellular Na+ and contractile function in isolated pig and rat hearts during cardioplegic ischemia and reperfusion: 23Na and 31P NMR studies. Basic Res Cardiol 90:220–233, 1995.[Medline]
  31. Vuorinen K, Yilitalo K, Peuhkurinen K, Raatikainen P, Ala-Rami A, Hassinen IE. Mechanism of ischemic preconditioning in rat myocardium. Circulation 91:2810–2818, 1995.[Abstract/Free Full Text]
  32. Schaefer S, Carr LJ, Prussel E, Ramasamy R. Effect if glycogen depletion on ischemic injury in isolated rat hearts: insights into preconditioning. Am J Physiol 268:H935–H944, 1995.[Abstract/Free Full Text]
  33. Murphy E, Cross H, Steenbergen C. Sodium regulation during ischemia versus reperfusion and its role in injury. Circ Res 84:1469–1470, 1999.[Free Full Text]
  34. Kohomoto O, Barry W. Mechanism of protective effects of Ca2+ channel blockers on energy deprivation contracture in cultured ventricular myocytes. J Pharmacol Exp Ther 248:821–878, 1989.
Received for publication November 21, 2001. Accepted for publication April 4, 2002.




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