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Volume 53 (7): 845-850, 2005
Copyright ©The Histochemical Society, Inc.
Cardiomyocytes of Chronically Ischemic Pig Hearts Express the MDR-1 Gene-encoded P-glycoprotein
Department of Clinical Biochemistry, Faculty of Pharmacy and Biochemistry, Buenos Aires University, Buenos Aires, Argentina (AJL); Department of Pathology, Italian Hospital, Buenos Aires, Argentina (HJGR); Departments of Physiology (GLVJ,AJC) and Pathology (LAC,PMCM,GGY,RPL), Favaloro University, Buenos Aires, Argentina; Scientific Investigation Commission of Buenos Aires Province, La Plata, Argentina (PMCM); and Nuclear Medicine Division, Institute of Cardiology and Cardiovascular Surgery, Favaloro Foundation, Buenos Aires, Argentina (AM)
Correspondence to: Rubén P. Laguens, Favaloro University, Solís 453, 1078 Buenos Aires, Argentina. E-mail: rlaguens{at}ffavaloro.org
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The multidrug-resistant (MDR)-1 gene-encoded P-glycoprotein (Pgp-170) is not normally present in the cardiomyocyte. Given that in other tissues Pgp-170 is not found under normoxic conditions but is expressed during hypoxia, we searched for Pgp-170 in chronically ischemic porcine cardiomyocytes. Pgp-170 was detected and localized via immunohistochemistry in ischemic and nonischemic cardiomyocytes of eight adult pigs 8 weeks after placement of an Ameroid constrictor at the origin of the left circumflex artery (Cx). Regional myocardial ischemia in the Cx bed was documented with nuclear perfusion scans. Pgp-170 mass was quantified using Western blot analysis. In all pigs, Pgp-170 was consistently present in the sarcolemma and T invaginations of the cardiomyocytes of the ischemic zone. Pgp-170 expression decreased toward the border of the ischemic zone and was negative in nonischemic regions as well as in the myocardium of sham-operated animals. Western blot analysis yielded significantly higher Pgp-170 mass in ischemic than in nonischemic areas. We conclude that Pgp-170 is consistently expressed in the cardiomyocytes of chronically ischemic porcine myocardium. Its role in the ischemic heart as well as in conditions such as myocardial hibernation, stunning, and preconditioning may have potentially relevant clinical implications and merits further investigation.
(J Histochem Cytochem 53:845–850, 2005)
Key Words: MDR • P-glycoprotein • ATP-binding cassette • transporters • myocardial ischemia • hypoxia • cardiomyocyte • pig
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Introduction |
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THE MULTIDRUG-RESISTANT (MDR)-1 gene-encoded P-glycoprotein (Pgp-170) is an ATP-dependent cationic efflux pump localized in the plasma membrane (Juliano and Ling 1976
; Blackmore et al. 2001). Pgp-170 is capable of extruding a variety of hydrophobic substances from the cell (Beck 1987; Gottesman and Pastan, 1988; Litman et al. 2001), and has been shown to confer multidrug resistance in cancer (Pastan and Gottesman 1987; Endicott and Ling 1989; Stein et al. 2004). In mammals, Pgp-170 is normally present in the epithelium of the intestine and some excretory organs (e.g., kidney proximal tubes), and in the capillaries of the blood–brain barrier (Thiebaut et al. 1987; Cordon-Cardo et al. 1990).
In the normal heart, Pgp-170 is absent (Cordon-Cardo et al. 1990) or is only expressed in endothelial cells of capillaries and arterioles, but not in cardiomyocytes (Meissner et al. 2002). In myocardial ischemia, few data regarding Pgp-170 expression are available. Given that in other organs (e.g., the brain) Pgp-170 is not detected normally (Cordon-Cardo et al. 1990) but is highly expressed in hypoxic brain injury (Ramos et al. 2004), we hypothesized that the ischemic cardiomyocytes should express Pgp-170 and searched for the presence of Pgp-170 in the hearts of pigs submitted to chronic myocardial ischemia achieved by Ameroid-induced occlusion of a coronary artery. We found consistent expression of Pgp-170 in the cardiomyocyte sarcolemma of ischemic myocardium and confirmed its absence in the cardiomyocytes of normoperfused myocardium.
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Materials and Methods |
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Animal Model
Chronic myocardial ischemia was induced in eight adult Landrace pigs weighing 27 ± 2 kg. Under general anesthesia (premedication: acepromazine maleate 0.3 mg/kg; induction: sodium thiopental 20 mg/kg; maintenance: 3% enflurane in pure oxygen), an Ameroid occluder was positioned at the origin of the left circumflex coronary artery (Cx) by a sterile thoracotomy at the 4th intercostal space. Eight weeks later, the animals were killed with an overdose of intravenous sodium thiopental followed by a bolus injection of potassium chloride. All animals belonged to the placebo group of a protocol designed to study myocardial angiogenesis (Crottogini et al. 2003
) and myogenesis (Laguens et al. 2004). Two sham-operated pigs were used as controls.
Regional Left Ventricular Perfusion
At the end of the study, regional myocardial ischemia was documented with single photon emission computed tomography (SPECT) using 99mTc-sestamibi at rest and under pharmacological stress (dobutamine) on two consecutive days (day 1: stress; day 2: rest) using an ADAC Vertex Dual Detector Camera System (Milpitas, CA).
For the stress study, dobutamine in saline solution was infused intravenously in increasing doses (5, 10, 20, 30, and 40 µg/kg/min) under electrocardiogram monitoring. The infusion lasted until the heart rate had increased at least 50% above rest values (or until it had increased above 200 bpm). At that time point, 99mTc-sestamibi was injected. The corresponding SPECT images were acquired 1 to 2 hr later.
In each experimental condition (stress and rest), the regional perfusion value of ischemic (Cx bed) and nonischemic (left anterior descending or right coronary artery bed) zones was expressed as a percentage of the maximally uptaking (perfused) segment of the individual circumferential count profiles (polar plots). The difference between the perfusion value at stress and at rest was calculated both in the ischemic and nonischemic territories. Within the Cx bed, those segments showing an ischemic pattern (lower perfusion value at stress than at rest) were considered for analysis; those showing a behavior consistent with necrosis (fixed perfusion defect) were not included in this analysis or in the histological study.
Histology and Immunohistochemistry
The heart was cut transversally at a plane equidistant to the apex and the mitral annulus. A 5-mm-thick slice, cut from the distal end of the upper half, was fixed flat in 10% buffered formaldehyde. After 48 hr of fixation, the slice was divided into eight pieces corresponding to the interventricular septum and the posterior, lateral, and anterior left ventricular wall (2 pieces each). All fragments were embedded in paraffin. Four-µm-thick tissue sections were routinely stained with hematoxylin-eosin, PAS and Masson's trichrome. For the immunohistochemical study, antigen retrieval was performed by incubating the hydrated sections in 10 mM sodium citrate buffer (pH 6.0) in a microwave oven for 5 min. After incubating sections for 1 hr at room temperature with two specific monoclonal antibodies against Pgp-170 (clone C494, Signet Laboratories, Dedham, MA; clone MDR-88, Biogenex, San Ramon, CA) diluted 1:100, antibody binding was visualized with a commercial biotin-streptavidin-peroxidase kit, with EAC as the chromogen (Biogenex). Clone C494 (Signet) antibody detects an epitope present only in the MDR-1 isoform of the P-glycoprotein and cross-reacts with piruvate carboxilase, a mitochondrial enzyme. Unequivocal plasma membrane patterns of immunostaining represent true P-glycoprotein expression. Clone MDR-88 (Biogenex) is a monoclonal antibody against a recombinant P-glycoprotein containing four tandem repeats of the amino acid sequence 1092–1252. Positive controls of the reaction were murine brain and kidney tissue sections.
Western Blot Analysis
Frozen myocardium samples from the ischemic (but not infarcted) left ventricular postero-lateral wall and from the anterior wall (nonischemic zone) of four pigs were sliced into small pieces and thawed in lysis buffer containing 10 mM KCl, 1.5 mM MgCl2, and 10 mM TrisCl (pH 7.4) in 0.5% (wt/vol) SDS supplemented with leupeptin (2 µg/ml), aprotinin (2 µg/ml), and E64 (1 µg/ml). DNA was sheared by sonication. Temperature was maintained at 4C throughout all procedures. Protein concentrations were determined using the micromethod of Bradford (Bio-Rad, CA). Samples containing 100 µg of protein were fractioned by SDS in 7% PAGE and then transferred to Hybond P membrane (Amersham Pharmacia Biotech; Amersham Place, England, UK) via electroblotting. The filters were incubated with 3% (wt/vol) nonfat milk for 1 hr at room temperature and then hybridized overnight at 4C in the same buffer containing 0.5 µg/ml of the monoclonal antibody C494 or anti-ß actin (Santa Cruz Biotechnology; Santa Cruz, CA) as internal protein loading controls. The filters were subsequently incubated for 1 hr with 1/1000 horseradish peroxidase-conjugated goat anti-mouse IgG (Dako: Carpinteria, CA). Detection was made with ECL reagent (Amersham Pharmacia Biotech) according to the manufacturer's instructions. The blots were subjected to autoluminography for 20 min with Kodak Biomax ML films (Rochester, NY). The autoradiography films were scanned, and densitometry was performed using Gel-Pro Analyzer software (version 3.1; Media Cybernetics, Silver Spring, MA). Tissues with high expression of Pgp-170 (liver, colon, and kidney from rat and sheep) were used as positive controls.
Statistical Analysis
Perfusion and optical density data for ischemic and nonischemic zones were compared using Student's t-tests for unpaired data. Results are expressed as the mean ± SEM. Significance was set at p<0.05.
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SPECT Perfusion Scans
Representative stress and rest polar plots are shown in Figure 1A. It can be seen that the resting perfusion defect increases its magnitude during stress. Figure 1B shows the perfusion values at stress and rest. At stress, perfusion was 60.2 ± 6.5% in the ischemic zone and 96.6 ± 5.8% in the nonischemic zone (p<0.001). Likewise, at rest, ischemic zone perfusion was lower than nonischemic zone perfusion (65.6 ± 7.9 vs 95.3 ± 6.4; p<0.001). Figure 1C shows the stress–rest perfusion difference in both zones (ischemic: –5.3 ± 2.6%; nonischemic: 1.3 ± 2.3%, p<0.001). Note the clear-cut ischemic pattern displayed by the segments of the Cx bed considered for analysis.
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Immunohistochemistry
In the left Cx artery bed, the cardiomyocytes presented a positive reaction located at the sarcolemma level (Figures 2A and 2B). Staining was positive at the myocyte lateral walls. According to the plane of section and cell orientation, the reaction was also positive in the T invaginations of the lateral sarcolemma (Figure 2C). The pattern of staining was similar with both monoclonal antibodies. The proportion of Pgp-170–positive cardiomyocytes varied according to the area of the free left ventricular wall examined. In the lateral wall, which was shown by the SPECT study to be the maximally ischemic zone, all the myocytes were positive for Pgp-170, independently of their localization within the ventricular wall thickness. The number of positive cardiomyocytes decreased at the border zone between the lateral and the inferior wall. In this area, clusters of positive myocytes appeared intermingled among negative cells, and in distant areas there were no more Pgp-170–positive myocytes (Figure 2D). In control tissue sections of the kidney and brain, an intense positive reaction was observed in the cortical tubes and in the capillary endothelium, respectively (Figure 3). However, in the hearts of sham-operated animals, a search of Pgp-170 rendered consistently negative results, as occurred in the nonischemic areas (septum and right ventricular free wall) of pigs with myocardial ischemia.
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Western Blot
Western blot analysis revealed that Pgp-170 was significantly increased in the ischemic areas [25.7 ± 5.4 optical density units (ODU)] with respect to nonischemic areas (9.4 ± 2.3 ODU; p<0.002). Figure 4 shows the positive staining band with a molecular weight of 170 kDa.
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Discussion |
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Our results indicate that, as occurs in brain parenchyma (Ramos et al. 2004
), chronic ischemia induces the expression of Pgp-170 in the cardiomyocyte, a cell that does not express this ATP-binding cassette transporter under normal conditions. In agreement with previous studies reporting that Pgp-170 is not detected in normal cardiomyocytes (Cordon-Cardo et al. 1990; Smit et al. 1994), we did not find its expression in the myocardium of sham-operated animals and in normoperfused areas of our study hearts.
On account that stem cells or resident cardiomyoblasts may express Pgp-170 (Urbanek et al. 2003), the possibility that cells with positive Pgp-170 plasmalemma stain could belong to those series should be considered. However, the large size of positive cells and the presence of cross striations, features not present either in stem cells or in cardiomyoblasts, indicate that they were adult cardiomyocytes.
Our results differ from the recent observation (Meissner et al. 2002) that in explanted hearts of patients with end-stage cardiac failure of diverse etiologies, Pgp-170 is expressed in the endothelium of coronary capillaries and arterioles but not in cardiomyocytes. Even in the patients in whom the etiology of heart failure was ischemia, the level of Pgp-170 expression did not vary with respect to non-failing hearts. However, that report did not establish whether the hearts were ischemic at the time of explantation, nor did it establish whether patients were under pharmacological treatment with drugs that are known to be substrates of Pgp-170 or inhibitors of Pgp-170 expression, a condition that may have influenced the results. In our study, all animals had regional myocardial ischemia, as documented by the 99mTc-sestamibi scans of their respective protocols. With regard to drugs that may have influenced our results, a reference should be made to sodium thiopental, which was administered immediately before sacrifice. Barbiturates have been reported to upregulate MDR-1 expression in cell cultures (Schuetz et al. 1996). However, in rats treated with phenobarbital over 11 days, no significant P-glycoprotein increases were seen in the brain (Seegers et al. 2002). It is thus unlikely that in our model the single sodium thiopental dose given a few seconds before death could have upregulated MDR-1 expression, especially considering that the sham-operated animals also received sodium thiopental and did not show Pgp-170 in their cardiomyocytes, and that no Pgp-170 was detected in cardiomyocytes belonging to the normoperfused areas of the experimental animals.
Although we did not investigate the molecular mechanisms involved in MDR-1 gene expression, it may be hypothesized that it was induced by transcription factors activated by cell ischemia, such as hypoxia-inducible factor 1 (HIF-1), a transcription factor mediating mechanisms of cell protection against ischemia (Zaman et al. 1999; Bergeron et al. 2000) that is expressed in the ischemic myocardium (Martin et al. 1998; Stroka et al. 2001; Cai et al. 2003). In support of this assumption, it has recently been shown that the MDR-1 gene is activated by HIF-1 (Comerford et al. 2002). In addition, it has been reported that there is no Pgp-170 overexpression in myocardial damage not mediated by hypoxia (e.g., tachycardia-induced heart failure) (Sims et al. 2004).
Given that the MDR-1 gene-encoded Pgp-170, acting as a cationic efflux pump, confers multidrug resistance in cancer cells, it is tempting to speculate that Pgp-170 may be a molecule involved in extruding from the cell the toxic products derived from hypoxia. In addition, because it is known that Pgp-170 overexpression protects against cell death induced by Fas ligand and tumor necrosis factor (TNF) (Johnstone et al. 1999), even independently from ATPase activity and hence of pumping ability (Tainton et al. 2004), the possibility that expression of PgP-170 may confer a protection against TNF-induced heart damage should be considered, on account that this cytokine plays a role in heart failure (Bozkurt et al. 1998). However, because we did not intend to assess the processes involved in the effect of Pgp-170, the preceding considerations are strictly speculative.
Study Limitations
Some drawbacks of this study should be noted. First, the study was retrospective, using tissue samples from animals originally prepared for other protocols. Second, we did not use molecular techniques such as RT-PCR, which would have allowed detecting transcripts of the MDR-1 gene both in the ischemic and nonischemic tissues. However, it should be noted that our intention was only to assess for the presence or absence of Pgp-170 in the ischemic cardiomyocytes; in this regard, the immunostaining used permitted us to not only fulfill this objective but also to reveal the cytological localization of Pgp-170. Besides, Western blot analysis confirmed that the mass of Pgp-170 was significantly greater in the ischemic myocardium. An additional flaw is the lack of direct measurement of myocardial blood flow using the radioactive or color-coded microsphere technique. Although the use of this method would have allowed precise quantification of tissue flow, it must be noted that all animals had undergone 99mTc-sestamibi SPECT scans for their respective original protocols. These scans documented myocardial hypoperfusion in the zones considered ischemic in the present study and normoperfusion in those considered normal.
Clinical Implications
Our observation may have potentially relevant clinical implications. If the presence of Pgp-170 represents a cell defense mechanism against stress insults that play a role in myocardial dysfunction and/or cardiomyocyte death, it would be important to investigate if Pgp-170 plays a role in ischemia-reperfusion injury, a condition in which extrusion of potentially harmful compounds (e.g., products derived from free radicals) would be of benefit. In addition, it would be reasonable to hypothesize that Pgp-170 may be involved in other cell protection processes such as myocardial preconditioning (Kloner and Jennings 2001) and postconditioning (Zhao et al. 2003). If this were the case, it could be speculated that pharmacological induction of MDR-1 expression may be of therapeutic value in coronary heart disease and in situations such as cardiac surgery or transplant organ preservation, in which protection against cell hypoxia is mandatory.
Conclusion
In the present study, we demonstrate expression of the MDR-1 gene-encoded Pgp-170 in the cardiomyocytes of chronically ischemic porcine myocardium and confirm the absence of expression in the cardiomyocytes of normoperfused myocardium. The mechanisms involved, as well as the role of this cationic efflux pump in conditions such as myocardial hybernation, stunning, and preconditioning, require further investigation.
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Acknowledgments |
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This study was supported by grants from the René G. Favaloro University Foundation.
We thank veterinarians María Inés Besansón, Pedro Iguain, and Marta Tealdo for assisting in anesthesia, and animal house personnel Juan Ocampo, Osvaldo Sosa, and Juan Carlos Mansilla for dedicated care of the animals. The technical help of Julio Martínez and Fabián Gauna is gratefully acknowledged.
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Footnotes |
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Received for publication October 4, 2004; accepted January 19, 2005
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