HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 285/3/H1055    most recent 00865.2002v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (6) Citing Articles via Google Scholar Google Scholar Articles by Elmoselhi, A. B. Articles by Dhalla, N. S. Search for Related Content PubMed PubMed Citation Articles by Elmoselhi, A. B. Articles by Dhalla, N. S.

Preconditioning attenuates ischemia-reperfusion-induced remodeling of Na⁺-K⁺-ATPase in hearts

Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, and Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R2H 2A6, Canada

Submitted 2 October 2002 ; accepted in final form 15 May 2003

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
The aim of this study was to determine whether changes in protein content and/or gene expression of Na⁺-K⁺-ATPase subunits underlie its decreased enzyme activity during ischemia and reperfusion. We measured protein and mRNA subunit levels in isolated rat hearts subjected to 30 min of ischemia and 30 min of reperfusion (I/R). The effect of ischemic preconditioning (IP), induced by three cycles of ischemia and reperfusion (10 min each), was also assessed on the molecular changes in Na⁺-K⁺-ATPase subunit composition due to I/R. I/R reduced the protein levels of the ₂-, ₃-, ₁-, and ₂-isoforms by 71%, 85%, 27%, and 65%, respectively, whereas the ₁-isoform was decreased by <15%. A similar reduction in mRNA levels also occurred for the isoforms of Na⁺-K⁺-ATPase. IP attenuated the reduction in protein levels of Na⁺-K⁺-ATPase ₂-, ₃-, and ₂-isoforms induced by I/R, without affecting the ₁- and ₁-isoforms. Furthermore, IP prevented the reduction in mRNA levels of Na⁺-K⁺-ATPase ₂-, ₃-, and ₁-isoforms following I/R. Similar alterations in protein contents and mRNA levels for the Na⁺/Ca²⁺ exchanger were seen due to I/R as well as IP. These findings indicate that remodeling of Na⁺-K⁺-ATPase may occur because of I/R injury, and this may partly explain the reduction in enzyme activity in ischemic heart disease. Furthermore, IP may produce beneficial effects by attenuating the remodeling of Na⁺-K⁺-ATPase and changes in Na⁺/Ca²⁺ exchanger in hearts after I/R.

ischemic preconditioning; myocardium; Na⁺-K⁺-ATPase gene expression; Na⁺/Ca²⁺ exchanger

Brief periods of ischemia are known to render the heart more resistant to the effects of longer periods of ischemia (25), and this phenomenon is termed ischemic preconditioning (IP). The protection offered by IP appears in two phases: one occurs soon after I/R (classic or early IP), whereas the second becomes evident after 24 h (delayed or second window of protection) (23). IP-induced protection of the heart is manifest as a reduction in infarct size, prevention of ventricular arrhythmias, and attenuation of abnormalities in cardiac performance following I/R (4, 13). Although the exact mechanism of IP is unknown, several pathways are implicated in IP such as activation of adenosine receptors, G protein-coupled receptors, protein kinase C, and ATP-sensitive K⁺ (K_ATP) channels, as well as synthesis and upregulation of various proteins such as heat shock protein 72, MnSOD, inducible nitric oxide synthase, and Bcl2 (21, 23, 31, 40, 42). In a recent study, Nawada et al. (26) demonstrated that IP protects the rabbit heart against the I/R-induced reduction in Na⁺-K⁺-ATPase activity. However, the mechanism of this protection is unclear, because these authors only measured ATPase activity. In the present study, we assessed the changes in Na⁺-K⁺-ATPase subunits induced by I/R and determined whether IP protects against these changes. The changes in different isoforms were measured at both the protein and mRNA levels. The effects of I/R and IP on the Na⁺/Ca²⁺ exchanger in isolated hearts were also assessed to determine whether changes in exchanger protein content and mRNA levels were similar to those seen for Na⁺-K⁺-ATPase.

	METHODS

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Isolated heart function. Experiments were conducted in accordance with the guide to the care and use of experimental animals issued by the Canadian Council in Animal Care. Male Sprague-Dawley rats (250–350 g) were anesthetized with a mixture of ketamine (60 mg/kg) and xylazine (10 mg/kg). The heart was quickly removed and mounted on a Langendorff apparatus, where it was perfused with Krebs-Henseleit medium containing (in mM) 120 NaCl, 25 NaHCO₃, 11 glucose, 4.7 KCl, 1.2 H₂PO₄, 1.2 MgSO₄, and 1.25 CaCl₂ at a constant rate of 10 ml/min (37°C). The perfusion solution was gassed with 95% O₂-5% CO₂ resulting in pH 7.4. The heart was kept in a humidified chamber maintained at 37°C. The atrioventricular node was crushed, and the heart was electrically stimulated at a rate of 300 beats/min by using a Phipps and Bird stimulator (Richmond, VA). A water-filled balloon was inserted into the left ventricle and connected to a pressure transducer to measure contractile parameters. Left ventricular (LV) developed pressure (LVDP) was measured as the systolic minus the diastolic pressure. The LV end-diastolic pressure (LVEDP) was adjusted to 10 mmHg at the beginning of the experiment, and the LV pressure was differentiated to estimate the rate of ventricular pressure development (+dP/dt) and the rate of ventricular pressure decay (–dP/dt). Data were acquired using Acqknowledge 3.03 software for Windows (Biopac System).

Control hearts were perfused for 120 min. For the I/R group, hearts were perfused for 60 min and then subjected to 30 min of global ischemia followed by reperfusion for 30 min. IP hearts were subjected to three 10-min cycles of ischemia and reperfusion. For the IP + I/R group, hearts were subjected to three 10-min cycles of I/R, followed by 30 min of global ischemia and 30 min of reperfusion. At the end of each experiment, the heart was immediately frozen by being clamped, immersed in liquid nitrogen, and then stored at –70°C before use.

Isolation of cardiac sarcolemmal preparations. Ventricular tissue of three hearts were pooled, and purified sarcolemmal membrane fraction was isolated according to the method of Pitts (30) as modified by Kaneko et al. (16). The final pellet was suspended in 0.25 M sucrose-10 mM histidine (pH 7.2), quickly frozen, and stored at –70°C. Marker enzyme activities (10, 16, 29) revealed a 16- to 18-fold purification of the membrane with respect to Na⁺-K⁺-ATPase activity in the heart homogenate and minimal cross contamination with other subcellular organelles such as mitochondria, sarcoplasmic reticulum, and myofibrils.

Measurement of ATPase enzyme activities and Na⁺-dependent Ca²⁺ uptake. Na⁺-K⁺-ATPase activity was measured using a method described previously with some modifications (10). Briefly, the sarcolemma membrane (40 µg) was incubated for 5 min at 37°C with the following (in mM): 1.0 EGTA (Tris) (pH 7.4), 5 NaN₃, 6 MgCl₂, 100 NaCl, and 10 KCl. An ATP-regenerating system was added to the incubation medium to maintain the ATP concentration; this consisted of 2.5 mM phosphoenolpyruvate and 10 IU/ml pyruvate kinase. The reaction was started by adding 0.025 ml of 80 ml Tris-ATP (pH 7.4) and terminated after 5 min with 0.5 ml cold 12% trichloroacetic acid. The liberated phosphate was estimated by the method of Taussky and Shorr (38). Different concentrations of Mg-ATP were used in some experiments, and the amounts of Mg²⁺ and ATP required to achieve the final concentration of Mg-ATP in the incubation medium were determined according to the "SPECS" FORTRAN program developed by Fabiato (11). Na⁺-K⁺-ATPase activity was calculated as the difference between activities with and without Na⁺ plus K⁺.Mg²⁺-ATPase activity was determined as the difference between the activities with and without Mg²⁺ in the absence of Na⁺ and K⁺ in the incubation medium. The Na⁺-dependent Ca²⁺ uptake was measured as described previously (9). Briefly, sarcolemmal vesicles from various groups (7.5 µg protein/tube) were preloaded with NaCl-MOPS buffer at 37°C for 30 min; it was then rapidly diluted (50 times) with Ca²⁺ uptake medium containing 140 mM KCl, 20 mM MOPS, 0.4 µM valinomycin, 0.3 µCi ⁴⁵Ca²⁺, and 20 µM Ca²⁺ concentration in pH 7.4. After 2 s, the reaction was stopped by the addition of 0.03 ml ice-cold stopping buffer containing (in mM) 140 KCl, 1 LaCl₃, and 20 MOPS at pH 7.4. Samples of the total reaction mixture were filtered through 0.45-µm Millipore filters and washed twice with 2.5 ml ice-cold washing solution containing (in mM) 140 KCl, 0.1 LaCl₃, and 20 MOPS in pH 7.4. The radioactivity of the filters was measured using a Beckman LS 1701 counter. Na⁺-dependent Ca²⁺ uptake activity was corrected by subtraction of the nonspecific Ca²⁺ uptake values.

RNA isolation and Northern blot analysis. Total cellular RNA was extracted from the ventricular tissue of six hearts from each group by using guanidinium thiocyanate methods (7). Samples normalized to 20 µg of the total RNA were denatured with formaldehyde and run on a 1% agarose-formaldehyde gel. The fractionated mRNA transcripts were transferred to a charged nylon filter (Nytran Maximum Strength Plus, Scheicher and Schuell; Keene, NH) for 24 h. The membrane was then cross-linked with ultraviolet light (UV Stratalinker 2400, Stratagene). The blots were prehybridized at 42°C overnight in an Innova 4080 incubator (New Brunswick Scientific; Edison, NJ) oscillating at 65 rpm. Labeled probes were added to the prehybridization solution and left overnight under the same conditions. Specific isoform cDNA probes derived from rat brain sequences were used to identify specific isoforms as follows: 0.332-kb cDNA fragment (89–421 nt) for ₁-isoform; 0.381-kb cDNA fragment (121–502 nt) for ₂-isoform; 0.278-kb cDNA fragment (53–331 nt) for ₃-isoform; and 0.271-kb cDNA fragment (813–1,184 nt) for ₁-isoform (American Type Culture Collection; Rockville, MD), and 1.0-kb cDNA fragment of the dog heart for the Na⁺/Ca²⁺ exchanger (courtesy of Dr. K. D. Philipson; Los Angeles, CA). The cDNA fragments were radiolabeled with -[³²P]dCTP (NEN Life Sciences Products; Boston, MA) with a Random Primers DNA Labeling Kit (GIBCO). For 18S rRNA measurement, we used a 25-mer synthetic oligonucleotide complementary to the rat 18S rRNA sequence (1,046–1,070 nt) (5). Synthetic oligonucleotides were 5'-end labeled using T4 polynucleotide kinase and [-³²P]ATP (GIBCO). The hybridized blots were exposed to X-ray film (Kodak-X-OMAT), and radiolabeled mRNA bands were scanned using an image densitometer (model GS-670, Bio-Rad; Mississauga, ON, Canada) and quantified with Image Analysis software. The densitometric value for the band was divided by the corresponding 18S rRNA value to obtain the value normalized for loading.

Western blot analysis. The relative protein contents of Na⁺-K⁺-ATPase isoforms and Na⁺/Ca²⁺ exchanger were determined as previously described (17, 18). Sarcolemmal membranes (20 µg of total protein/lane) were separated on a 10–12% SDS-PAGE gel and electroblotted to polyvinylidene difluoride membranes (Boehringer Mannheim). The Na⁺-K⁺-ATPase isoforms and Na⁺/Ca²⁺ exchanger were detected using the following primary antibodies: monoclonal anti-₁ mouse IgG (0.05 µg/ml); polyclonal anti-₂ rabbit IgG (1: 1,000); polyclonal anti-₃ rabbit IgG (1 µg/ml); monoclonal anti-₁ mouse IgG (0.8 µg/ml); polyclonal anti-₂ rabbit IgG (1:1,000) (Upstate Biotechnology; Lake Placid, NY); and monoclonal anti-Na⁺-Ca²⁺ mouse IgG (1:2,000) (Research Diagnostic; Flanders, NJ). Secondary antibodies consisted of biotinylated anti-mouse IgG (1:3,000) for ₁, ₁, and Na⁺/Ca²⁺ exchanger (Amersham Life Science) and biotinylated anti-rabbit IgG (1:3,000) for ₂, ₃, and ₂ (Upstate Biotechnology). Membranes were incubated for 1 h with strepdavidin-conjugated horseradish peroxidase (1:5,000) and then processed for chemiluminescence (ECL Kit) on hyperfilm-ECL (Amersham Life Science). An imaging densitometer (model GS-670, Bio-Rad; Hercules, CA) was used to scan the bands, which were quantified using the Image Analysis Software (version 1.3). A purified microsomal preparation from a rat brain (Upstate Biotechnology) was used as a positive control to identify different Na⁺-K⁺-ATPase isoforms. No standards are commercially available for any of the Na⁺-K⁺-ATPase isoforms; thus we could not measure absolute levels of any subunit. To calculate the distribution of Na⁺-K⁺-ATPase isoforms, we determined the percentage of each subunit with respect to the total protein content for the ₁-, ₂-, ₃-, ₁-, and ₂-isoforms in each group.

Statistical analysis. Values are reported as means ± SE unless otherwise indicated. Statistical analysis among groups was performed using one-way ANOVA and confirmed by two-way Student's t-test. P < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Contractile performance, ATPase enzyme activities, and Na⁺-dependent Ca²⁺ uptake. Cardiac contractile function was evaluated by measuring LVDP, LVEDP, +dP/dt, and –dP/dt in isolated hearts from various groups (Table 1). There was no significant change in LVDP between the control and IP hearts. However, LVDP was decreased by 77% in the I/R hearts compared with controls (P < 0.05). This decrease was prevented by IP before the I/R (IP + IR) (P < 0.05). The I/R hearts showed a significant increase (50-fold) in LVEDP, which was prevented by IP (IP + IR) (P < 0.05). A significant depression (80%) of both +dP/dt and –dP/dt occurred in the I/R hearts, but this was completely prevented in the IP + I/R hearts (P < 0.05). Table 1 also shows the results of Na⁺-K⁺-ATPase, Mg²⁺-ATPase activities, and Na⁺-dependent Ca²⁺ uptake in the various groups. Na⁺-K⁺-ATPase activity fell by 27% in the I/R hearts compared with controls; this reduction was prevented in the IP + I/R hearts. The reduction of Mg²⁺-ATPase in the I/R hearts was even more severe (54%) compared with controls but was also completely prevented in the IP + I/R hearts. Na⁺-dependent Ca²⁺ uptake did not change in IP hearts but was decreased by 33% in the I/R hearts versus control (P < 0.05). The I/R-induced reduction was completely prevented in the IP + IR hearts.

Effect of I/R and IP on protein expression. The protein levels of Na⁺-K⁺-ATPase isoforms in the various groups (n = 6 for each group) were measured using Western blot analysis and are shown in Fig. 1. The bands of the Na⁺-K⁺-ATPase ₁-, ₂-, and ₃-isoforms are located at 110 kDa, with ₁ being the most prominent isoforms. The ₁- and ₂-bands are located at 55 and 45 kDa, respectively. A ₃-band was also detected at 40 kDa, but it was too faint to accurately assess any change among the various groups. There was also a nonspecific band that always appeared between 66 and 90 kDa, which may be due to a cross-reaction with secondary antibodies.

View larger version (58K): [in this window] [in a new window]   Fig. 1. Effects of ischemia-reperfusion (I/R) and ischemic preconditioning (IP) on protein levels of different Na+-K+-ATPase isoforms. Autoradiograms of Western blots for different isoforms in the treatment groups are shown. C, control; IP + I/R, ischemic preconditioning preceding I/R. A–E: analysis of various isoforms (n = 4–6). Each protein sample is a collection of three hearts. Protein samples were solubilized by incubation at 37°C for 30 min before separation on SDS-polyacrylamide gel, transferred to membranes, and incubated with corresponding antibodies. Bands for 1, 2, and 3 were located at 110 kDa, whereas 1 and 2 were located at 55 and 45 kDa, respectively. *P < 0.05 vs. control; #P < 0.05 vs. ischemia.

The protein level of the Na⁺-K⁺-ATPase ₁-isoform decreased to a lesser extent (<15%) in the I/R hearts relative to other isoforms, and this decrease was not prevented by IP. The Na⁺-K⁺-ATPase ₂-, ₃-, ₁-, and ₂-protein levels, on the other hand, were decreased in the I/R hearts by 71%, 85%, 27%, and 65%, respectively, compared with control hearts. However, only the changes in protein levels of ₂, ₃, and ₂ were attenuated in the IP + I/R group (by 53, 49, and 39%, respectively; P < 0.05). When the data for each isoform were analyzed in terms of the total Na⁺-K⁺-ATPase protein content (Table 2), there was upregulation of the ₁-isoform following I/R by 137% compared with control. ₁ was unchanged, whereas ₂, ₃, and ₂ were downregulated by 46%, 20%, and 56%, respectively. Thus a change of the relative percentage of various isoforms occurred following I/R. However, IP preceding I/R prevented these changes, whereas IP alone did not have much effect. The bands of the Na⁺/Ca²⁺ exchanger are located at 120, 160, and 70 kDa (29, 30). In Fig. 2A, the density of the 120-kDa band was compared among various groups. This band alone showed a decrease of 72% in the I/R group, and this reduction was almost completely prevented in the IP + I/R group (P < 0.05). A similar pattern was seen when we compared other bands of the Na⁺/Ca²⁺ exchanger.

View larger version (48K): [in this window] [in a new window]   Fig. 2. Effects of I/R and IP on Na+/Ca2+ exchanger (NCE) at the protein level (A) and mRNA level (B). A: Western blot analysis for various groups; n = 4–6. Each protein sample is a collection of three hearts. Numbers to the top left are molecular mass markers (kDa). B: Northern blot analysis for various groups; n = 6. mRNA band was located at 7 kb. *P < 0.05 vs. control; #P < 0.05 vs. ischemia.

Effect of I/R and IP on gene expression. We measured the steady-state level of mRNA of Na⁺-K⁺-ATPase isoforms in various groups (n = 6 for each group) (Fig. 3). Northern blot analysis showed that I/R reduced the level of Na⁺-K⁺-ATPase ₂, ₃, and ₁ mRNA by 55, 67, and 38% compared with controls, respectively (P < 0.05). The band for the ₁-isoform was reduced to a lesser extent (15%) compared with other isoforms, and this reduction was unaffected by IP. The ₁ mRNA level increased in the IP group, but the reason for this increase is not clear. IP preceding I/R (IP + IR) attenuated the reduction of mRNA levels of the ₂-, ₃-, and ₁-isoforms to 20, 30, and 32%, respectively. This pattern was also in agreement with the protein levels of the various isoforms except that the protein level of ₁ was not significantly affected by IP. The mRNA level of the Na⁺/Ca²⁺ exchanger decreased by 43% in the I/R group, and this reduction was completely prevented by IP (P < 0.05) (Fig. 2B).

View larger version (54K): [in this window] [in a new window]   Fig. 3. Effects of I/R and IP on mRNA levels for different Na+-K+-ATPase isoforms. Autoradiograms of Northern blots for the different isoforms in the treatment groups are shown. A–D: Northern blot analysis of the various isoforms (n = 6). RNA was isolated from heart tissues of the different groups and assayed for mRNA with respective probes; mRNA 18S was used as an internal standard to account for differences in nucleic acid loading or transfer. mRNA sizes (invariant band) were as follows: 1, 3.7 kb; 2, 3.4 kb and 5.3 kb; 3, 3.7 kb; and 1, 2.35 and 2.7 kb. *P < 0.05 vs. control; #P < 0.05 vs. ischemia.

The assays for mRNA and protein levels of Na⁺-K⁺-ATPase subunits used in this study are not quantitative. Thus differential changes in the levels of subunits could potentially be due to an underestimation of the changes in some of the subunits as a consequence of oversaturation of the density of these bands. However, assays for each of the isoforms in the control, IP, I/R, and IP + I/R groups were performed under identical conditions and argues against this being the case. Furthermore, the band for any one isoform should not be compared with other bands because the density of the band depends on the quality of the antibody (or molecular probe) and the exposure time. Thus caution needs to be exercised when interpreting these data. Nonetheless, the relation between protein content and optical density was linear for the Western blots (Fig. 4). It should be noted that the purified rat brain microsomal Na⁺-K⁺-ATPase preparation has at least 10 times higher activity than the heart sarcolemmal preparation. Thus the amount of sarcolemmal protein used in this study should be well within the linear range of the Western blot assay. The Northern blots in Fig. 5 also indicate that the amount of mRNA used was within the linear range. Although it is not readily apparent in Figs. 1 and 2, the data in Table 2 clearly indicate that both the ₂- and ₃-isoform levels in the control and IP hearts are expressed at markedly lower levels than the ₁-isoform. This is consistent with previous observations by our group and others (22, 43, 44).

View larger version (33K): [in this window] [in a new window]   Fig. 4. Relation between protein content and optical density (OD) for the 1-, 2-, 3-, 1-, 2-, and 3-Na+-K+-ATPase isoforms obtained using purified rat brain microsomal Na+-K+-ATPase. Regression squared (r2) values (n = 4) as well as representative Western blots are shown for each isoform.

View larger version (31K): [in this window] [in a new window]   Fig. 5. Relation between total RNA and OD for the 1-, 2-, 3-, and 1-Na+-K+-ATPase isoforms. Regression squared (r2) values (n = 4) as well as representative Northern blots (insets) are shown for each isoform.

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
The present study demonstrates that I/R reduces myocardial contractile function and Na⁺-K⁺-ATPase activity in the isolated rat heart. I/R induced a large decrease in Na⁺-K⁺-ATPase ₂- and ₃-protein levels and a lesser reduction in ₁-protein levels. Also, ₂-protein levels were reduced to a greater extent than ₁-protein levels. A similar pattern was seen at the mRNA level. A change in the percentage of various isoforms relative to the total subunits also occurred following I/R for both protein and mRNA levels. Thus differential changes in Na⁺-K⁺-ATPase subunits seem to occur following I/R, and this change in the molecular structure of the enzyme may represent remodeling of the Na⁺-K⁺-ATPase. Our differential remodeling agrees with the literature with respect to expression of various Na⁺-K⁺-ATPase isoforms in the myocardium during development and in different pathological conditions. The nonsignificant change in ₁ expression and upregulation of protein and mRNA following I/R correlates well with its role as the ubiquitous maintenance isoform in the rat heart. Cardiac hypertrophy is also associated with no change in ₁-protein and mRNA expression (6). Alternatively, ₂- and ₃-isoforms, the ouabain-sensitive isoforms, were significantly reduced after I/R. A similar reduction of ₂-isoform levels was also reported in cardiac hypertrophy (6) and hypokalemia (1). ₁ is the predominant -subunit of the heart (35). The ₁-protein level was decreased following I/R in the present study but to a lesser extent than the ₂-protein levels. Similarly, ₁ mRNA levels are unchanged in cardiac hypertrophy (6). Thus ₁ may share a comparable role to ₁ in terms of "housekeeping" of the heart. In agreement with the differential remodeling in our results, a study on rat cardiac hypertrophy showed that the -isoforms are independently regulated at the pretranslational level, and Na⁺-K⁺-ATPase expression is controlled by distinct regulatory mechanisms (3, 6). However, extensive studies need to be carried out to fully understand the differential changes in Na⁺-K⁺-ATPase isoforms in the I/R hearts.

The reason for the alteration of various isoforms in our study is still speculative, but their sensitivity to I/R-induced environmental changes such as oxidative stress, Ca²⁺ overload, and proteolysis, as well as their distinct structure, may play a key role. Oxidative stress is involved in I/R damage (19, 33). Previous studies reported that ₂- and ₃-isoforms have a greater sensitivity to H₂O₂ and hydroxyl radical than ₁-isoforms (14, 41), and these differences in sensitivities of various Na⁺-K⁺-ATPase isoforms are attributed to distinct structural features (14). These results are consistent with our findings of a greater reduction of ₂- and ₃-isoform levels compared with ₁ following I/R. Na⁺-K⁺-ATPase ₂- and ₃-isoforms mRNA in the rat heart are expressed at higher levels in the conduction system and junctional complexes, whereas ₁ is distributed ubiquitously in both atrial and ventricular tissues (43). Thus the ₂- and ₃-isoforms may play a distinct role in impulse transmission (44). In this perspective, we can hypothesize areas of the heart containing abundant ₂- and ₃-isoforms may be more susceptible to damage by I/R than other areas of the myocardium. Furthermore, it is known that oxidative stress during I/R is implicated in altering cellular protein structure and function in the heart (14, 39). Specifically, the importance of protein and nonprotein sulfhydryl groups in affecting Na⁺-K⁺-ATPase function was recently demonstrated by depleting the protein sulfhydryl oxidation and glutathione, which led to depressed Na⁺-K⁺-ATPase activity (12). The distribution of protein sulfhydryl groups and reduced glutathione in the various - and -isoforms is not clear. However, several reports suggest higher numbers of free sulfhydryl groups for ₂- and ₂-isoforms than other isoforms (34, 36, 37). Thus a differential remodeling in the protein content of the different isoforms following I/R may reflect their relative distribution of protein sulfhydryl groups and/or reduced glutathione.

Human heart failure is also associated with a reduction of Na⁺-K⁺-ATPase activity and ₁-, ₃-, and ₁-protein levels, whereas ₂- and Na⁺/Ca²⁺ exchanger protein levels did not change (32). This led to the suggestion that the failing heart may have an enhanced sensitivity to cardiac glycosides. Our data showed a different pattern of alterations of Na⁺-K⁺-ATPase and Na⁺/Ca²⁺ exchanger following I/R. It is unclear why our findings in the rat differ from those in human hearts, but this may reflect differences in the etiology of the insults or species differences. In any case, there appears to be import structural and functional differences among Na⁺-K⁺-ATPase isoforms and the Na⁺/Ca²⁺ exchanger during various myocardial insults. Further investigation is needed to evaluate the change in sensitivity of Na⁺-K⁺-ATPase isoforms to cardiac glycosides, especially after ischemia or I/R injury.

IP prevents many defects induced by I/R (4, 39). In the present study, IP alone did not alter myocardial contractile function, Na⁺-K⁺-ATPase activity, or protein mRNA levels compared with controls. IP preceding I/R, however, protected against myocardial dysfunction and the I/R-induced decrease in Na⁺-K⁺-ATPase activity. Our results agree with a recent finding that IP protects Na⁺-K⁺-ATPase activity in rabbit hearts subjected to 20 min of sustained ischemia in vivo (26). At the protein level, we found that IP preceding I/R prevented the reduction of Na⁺-K⁺-ATPase ₂-, ₃-, and ₂-isoforms but not the decrease in ₁-isoform induced by I/R. A similar pattern was also seen at the mRNA levels, except that the decrease in the ₁-isoform levels was completely prevented by IP. Prevention of the ₁-isoform reduction at the gene, but not the protein level, during the short duration of this study suggests that this protection may occur only at the transcriptional level and that the ₁-isoform may play more of a role in the second window of IP than in the acute classic phase of IP. From the available data, it is difficult to predict whether Na⁺-K⁺-ATPase isoforms in the I/R and IP + I/R hearts are regulated independently or if there is cross talk between the subunits.

We also measured the Na⁺/Ca²⁺ exchanger protein and mRNA levels to assess the specificity of the effects of I/R and IP on Na⁺-K⁺-ATPase isoforms in our study. I/R significantly reduced the protein and mRNA levels of the Na⁺/Ca²⁺ exchanger. The reduction of Na⁺/Ca²⁺ exchanger protein and mRNA levels correlate with the decrease in Na⁺/Ca²⁺ exchanger activity seen here and previously (3, 9). Moreover, our results show that IP prevents the I/R-induced decrease in Na⁺/Ca²⁺ exchanger at both the protein and mRNA levels. In agreement with our data, Nawada et al. (26) reported that IP preserved the Na⁺/Ca²⁺ exchanger activity and that Na⁺/Ca²⁺ exchanger activity was actually increased in ischemic versus nonischemic areas of preconditioned, but not control, rabbit hearts. Thus protection of Na⁺/Ca²⁺ exchanger activity may be due to prevention of the alterations in protein content and mRNA levels induced by I/R.

Prevention of the I/R-induced remodeling of Na⁺-K⁺-ATPase isoforms by IP is probably a functional modification and consequently a mediator of IP, rather than the final end effector. IP prevents the loss of Na⁺-K⁺-ATPase activity during the sustained ischemia, and this may underlie its protective effects in the myocardium. IP hearts would be better able to handle the intracellular [Na⁺] and intracellular [Ca²⁺] overload that occurs during I/R because Na⁺-K⁺-ATPase activity is essentially preserved in these hearts. In this regard, it is emphasized that the present study reports two major and novel findings. First, I/R rapidly induces differential changes of Na⁺-K⁺-ATPase isoforms at both the protein and mRNA level. This remodeling of Na⁺-K⁺-ATPase may be responsible, at least in part, for the decline in enzyme activity following I/R and suggests distinct roles and differential sensitivity of individual isoforms to I/R. Second, IP prevents the changes in Na⁺-K⁺-ATPase isoforms induced by I/R at both the protein and mRNA levels. These changes are consistent with the IP-induced protection of myocardial contractile dysfunction and Na⁺-K⁺-ATPase activity. Inhibition of Na⁺-K⁺-ATPase activity is known to reduce the infarct limiting size of IP (26), but our results do not provide any information regarding the cause and effect of IP-mediated protection on Na⁺-K⁺-ATPase subunits or enzyme activity. Indeed, our data indicate that IP has no effect on Na⁺-K⁺-ATPase activity or isoform content. Thus it is unlikely that the IP-mediated protection against the I/R induced remodeling of Na⁺-K⁺-ATPase is due to a direct effect of IP.

	DISCLOSURES

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
This work was supported by a grant from the Canadian Institute of Health Research (CIHR) (CIHR Group in Experimental Cardiology) and a grant from the CIHR Institute of Circulatory and Respiratory Health on Gene Environment Interaction in Heart Failure. A. B. Elmoselhi was a postdoctoral fellow supported by AstraPharma/Heart and Stroke Foundation of Canada/CIHR; P. Ostadal was a postdoctoral fellow supported by the St. Boniface General Hospital Research Foundation; N. S. Dhalla holds the CIHR/Pharmaceutical Research & Development Chair in Cardiovascular Research supported by Merck Frost Canada.

	FOOTNOTES

 

Address for reprint requests and other correspondence: N. S. Dhalla, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Ave., Winnipeg, Manitoba, Canada R2H 2A6 (E-mail: cvso{at}sbrc.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 

1. Azuma KK, Hensley CB, Putnam DS, and McDonough AA. Hypokalemia decreases Na⁺-K⁺-ATPase ₂- but not ₁-isoform abundance in heart, muscle, and brain. Am J Physiol Cell Physiol 260: C958–C964, 1991.[Abstract/Free Full Text]
2. Beller GA, Conroy J, and Smith TW. Ischemia-induced alterations in myocardial (Na⁺+K⁺)-ATPase and cardiac glycoside binding. J Clin Invest 57: 341–350, 1976.[ISI][Medline]
3. Book CB, Moore RL, Semanchik A, and Ng YC. Cardiac hypertrophy alters expression of Na⁺,K⁺-ATPase subunit isoforms at mRNA and protein levels in rat myocardium. J Mol Cell Cardiol 26: 591–600, 1994.[ISI][Medline]
4. Cave AC. Preconditioning induced protection against post-ischaemic contractile dysfunction: characteristics and mechanisms. J Mol Cell Cardiol 27: 969–979, 1995.[ISI][Medline]
5. Chan YL, Gutell R, Noller HF, and Wool IG. The nucleotide sequence of a rat 18 S ribosomal ribonucleic acid gene and a proposal for the secondary structure of 18 S ribosomal ribonucleic acid. J Biol Chem 259: 224–230, 1984.[Abstract/Free Full Text]
6. Charlemagne D, Orlowski J, Oliviero P, Rannou F, Sainte BC, Swynghedauw B, and Lane LK. Alteration of Na,K-ATPase subunit mRNA and protein levels in hypertrophied rat heart. J Biol Chem 269: 1541–1547, 1994.[Abstract/Free Full Text]
7. Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159, 1987.[ISI][Medline]
8. Dhalla NS, Temsah RM, and Netticadan T. Role of oxidative stress in cardiovascular diseases. J Hypertens 18: 655–673, 2000.[ISI][Medline]
9. Dixon IM, Eyolfson DA, and Dhalla NS. Sarcolemmal Na⁺-Ca²⁺ exchange activity in hearts subjected to hypoxia reoxygenation. Am J Physiol Heart Circ Physiol 253: H1026–H1034, 1987.[Abstract/Free Full Text]
10. Dixon IM, Hata T, and Dhalla NS. Sarcolemmal calcium transport in congestive heart failure due to myocardial infarction in rats. Am J Physiol Cell Physiol 262: C664–C671, 1992.[Abstract/Free Full Text]
11. Fabiato A. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol 157: 378–417, 1988.[ISI][Medline]
12. Haddock PS, Shattock MJ, and Hearse DJ. Modulation of cardiac Na⁺-K⁺ pump current: role of protein and nonprotein sulfhydryl redox status. Am J Physiol Heart Circ Physiol 269: H297–H307, 1995.[Abstract/Free Full Text]
13. Hagar JM, Hale SL, and Kloner RA. Effect of preconditioning ischemia on reperfusion arrhythmias after coronary artery occlusion and reperfusion in the rat. Circ Res 68: 61–68, 1991.[Abstract/Free Full Text]
14. Huang W, Lai CC, Wang Y, Askari A, Klevay LM, Askari A, and Chiu TH. Altered expressions of cardiac Na/K-ATPase isoforms in copper deficient rats. Cardiovasc Res 29: 563–568, 1995.[ISI][Medline]
15. James PF, Grupp IL, Grupp G, Woo AL, Askew GR, Croyle ML, Walsh RA, and Lingrel JB. Identification of a specific role for the Na,K-ATPase ₂ isoform as a regulator of calcium in the heart. Mol Cell 3: 555–563, 1999.[ISI][Medline]
16. Kaneko M, Elimban V, and Dhalla NS. Mechanism for depression of heart sarcolemmal Ca²⁺ pump by oxygen free radicals. Am J Physiol Heart Circ Physiol 257: H804–H811, 1989.[Abstract/Free Full Text]
17. Kato K, Lukas A, Chapman DC, and Dhalla NS. Changes in the expression of cardiac Na⁺-K⁺-ATPase subunits in the UM-X7.1 cardiomyopathic hamster. Life Sci 67: 1175–1183, 2000.[ISI][Medline]
18. Kato K, Lukas A, Chapman DC, Rupp H, and Dhalla NS. Differential effects of etomoxir treatment on cardiac Na⁺-K⁺-ATPase subunits in diabetic rats. Mol Cell Biochem 232: 57–62, 2002.[ISI][Medline]
19. Kim MS and Akera T. O₂ free radicals: cause of ischemia-reperfusion injury to cardiac Na⁺-K⁺-ATPase. Am J Physiol Heart Circ Physiol 252: H252–H257, 1987.[Abstract/Free Full Text]
20. Kim DH, Akera T, and Kennedy RH. Ischemia-induced enhancement of digitalis sensitivity in isolated guinea-pig heart. J Pharmacol Exp Ther 226: 335–342, 1982.
21. Kuzuya T, Hoshida S, Yamashita N, Fuji H, Oe H, Hori M, Kamada T, and Tada M. Delayed effects of sublethal ischemia on the acquisition of tolerance to ischemia. Circ Res 72: 1293–1299, 1993.[Abstract/Free Full Text]
22. Lucchesi PA and Sweadner KJ. Postnatal changes in Na,K-ATPase isoform expression in rat cardiac ventricle. Conservation of biphasic ouabain affinity. J Biol Chem 266: 9327–9331, 1991.[Abstract/Free Full Text]
23. Marber MS, Latchman DS, Walker JM, and Yellon DM. Cardiac stress protein elevation 24 h after brief ischemia or heat stress is associated with resistance to myocardial infarction. Circulation 88: 1264–1272, 1993.[Abstract/Free Full Text]
24. McDonough AA, Geering K, and Farley RA. The sodium pump needs its beta subunit. FASEB J 4: 1598–1605, 1990.[Abstract]
25. Murry CE, Jennings RB, and Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74: 1124–1136, 1986.[Abstract/Free Full Text]
26. Nawada R, Murakami T, Iwase T, Nagai K, Morita Y, Kouchi I, Akao M, and Sasayama S. Inhibition of sarcolemmal Na⁺,K⁺-ATPase activity reduces the infarct size-limiting effect of preconditioning in rabbit hearts. Circulation 96: 599–604, 1997.[Abstract/Free Full Text]
27. Nicoll DA, Longoni S, and Philipson KD. Molecular cloning and functional expression of the cardiac sarcolemmal Na⁺-Ca²⁺ exchanger. Science 250: 562–565, 1990.[Abstract/Free Full Text]
28. Philipson KD, Longoni S, and Ward R. Purification of the cardiac Na⁺-Ca²⁺ exchange protein. Biochim Biophys Acta 945: 298–306, 1988.[Medline]
29. Pierce GN and Dhalla NS. Sarcolemmal Na⁺-K⁺-ATPase activity in diabetic rat heart. Am J Physiol Cell Physiol 245: C241–C247, 1983.[Abstract/Free Full Text]
30. Pitts BJ. Stoichiometry of sodium-calcium exchange in cardiac sarcolemmal vesicles. Coupling to the sodium pump. J Biol Chem 254: 6232–6235, 1979.[Abstract/Free Full Text]
31. Schultz JE, Hsu AK, and Gross GJ. Morphine mimics the cardioprotective effect of ischemic preconditioning via a glibenclamide-sensitive mechanism in the rat heart. Circ Res 78: 1100–1104, 1996.[Abstract/Free Full Text]
32. Schwinger RH, Wang J, Frank K, Muller-Ehmsen J, Brixius K, McDonough AA, and Erdmann E. Reduced sodium pump alpha1, alpha3, and beta1-isoform protein levels and Na⁺,K⁺-ATPase activity but unchanged Na⁺-Ca²⁺ exchanger protein levels in human heart failure. Circulation 99: 2105–2112, 1999.[Abstract/Free Full Text]
33. Shao Q, Matsubara T, Bhatt SK, and Dhalla NS. Inhibition of cardiac sarcolemma Na⁺-K⁺-ATPase by oxyradical generating systems. Mol Cell Biochem 147: 139–144, 1995.[ISI][Medline]
34. Shoot BM, De Pont JJ, and Bonting SL. Studies on (Na⁺+K⁺)-activated-ATPase. XLII Evidence for two classes of essential sulfhydryl groups. Biochim Biophys Acta 522: 602–613, 1978.[Medline]
35. Shyjan AW, Gottardi C, and Levenson R. The Na,K-ATPase beta 2 subunit is expressed in rat brain and copurifies with Na,K-ATPase activity. J Biol Chem 265: 5166–5169, 1990.[Abstract/Free Full Text]
36. Sweadner KJ. Two molecular forms of (Na⁺+K⁺)-stimulated-ATPase in brain. Separation and difference in affinity for strophanthidin. J Biol Chem 254: 6060–6067, 1979.[Abstract/Free Full Text]
37. Sweadner KJ. Isozymes of the Na⁺/K⁺-ATPase. Biochim Biophys Acta 988: 185–220, 1989.[Medline]
38. Taussky HH and Schorr E. A microcalorimetric method for determination of inorganic phosphorus. J Biol Chem 202: 678–685, 1953.
39. Temsah RM, Netticadan T, Chapman D, Takeda S, Mochizuki S, and Dhalla NS. Alterations in sarcoplasmic reticulum function and gene expression in ischemic-reperfused rat heart. Am J Physiol Heart Circ Physiol 277: H584–H594, 1999.[Abstract/Free Full Text]
40. Thornton JD, Liu GS, and Downey JM. Pretreatment with pertussis toxin blocks the protective effects of preconditioning: evidence for a G-protein mechanism. J Mol Cell Cardiol 25: 311–320, 1993.[ISI][Medline]
41. Xie Z, Jack-Hays M, Wang Y, Periyasamy SM, Blanco G, Huang WH, and Askari A. Different oxidant sensitivities of the alpha 1 and alpha 2 isoforms of Na⁺/K⁺-ATPase expressed in baculovirus-infected insect cells. Biochem Biophys Res Commun 207: 155–159, 1995.[ISI][Medline]
42. Yellon DM, Baxter GF, Garcia-Dorado D, Heusch G, and Sumeray MS. Ischaemic preconditioning: present position and future directions. Cardiovasc Res 37: 21–33, 1998.[Abstract/Free Full Text]
43. Zahler R, Brines M, Kashgarian M, Benz EJ, and Gilmore-Hebert M. The cardiac conduction system in the rat expresses the 2 and 3 isoforms of the Na⁺,K⁺-ATPase. Proc Natl Acad Sci USA 89: 99–103, 1992.[Abstract/Free Full Text]
44. Zahler R, Sun W, Ardito T, and Kashgarian M. Na-K-ATPase -isoform expression in heart and vascular endothelia: cellular and developmental regulation. Am J Physiol Cell Physiol 270: C361–C371, 1996.[Abstract/Free Full Text]

This article has been cited by other articles:

				S. V. Pierre, C. Yang, Z. Yuan, J. Seminerio, C. Mouas, K. D. Garlid, P. Dos-Santos, and Z. Xie Ouabain triggers preconditioning through activation of the Na+,K+-ATPase signaling cascade in rat hearts Cardiovasc Res, February 1, 2007; 73(3): 488 - 496. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 285/3/H1055    most recent 00865.2002v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (6) Citing Articles via Google Scholar Google Scholar Articles by Elmoselhi, A. B. Articles by Dhalla, N. S. Search for Related Content PubMed PubMed Citation Articles by Elmoselhi, A. B. Articles by Dhalla, N. S.