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J Thorac Cardiovasc Surg 2005;130:1151
© 2005 The American Association for Thoracic Surgery

Cardiopulmonary Support and Physiology

Cardioplegia prevents ischemia-induced transcriptional alterations of cytoprotective genes in rat hearts: A DNA microarray study

^a Division of Cardiothoracic Surgery, Case Western Reserve University, University Hospitals of Cleveland, Cleveland, Ohio
^b Department of Pediatrics, Vanderbilt University, Nashville, Tenn
^c Cardiology Section, Medical Service, Louis Stokes Department of Veterans Affairs Hospital and Case Western Reserve University, Cleveland, Ohio
^d Division of Cardiothoracic Surgery, Louis Stokes Department of Veterans Affairs Hospital, Case Western Reserve University, University Hospitals of Cleveland, Cleveland, Ohio.

Received for publication March 3, 2005; revisions received May 25, 2005; accepted for publication June 8, 2005.

^_* Address for reprints: Brian L. Cmolik, MD, Cardiothoracic Surgery, University Hospitals of Cleveland, 11100 Euclid Ave, Cleveland, OH 44106-5011 (Email: blc3{at}case.edu).

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
BACKGROUND: Energy conservation and calcium homeostasis contribute to myocardial protection provided by hyperkalemic cardioplegia during ischemia. Complimenting these established mechanisms of protection, previous work suggested that activation of cytoprotective signaling pathways also contributes to reduced injury with cardioplegia. We proposed that cardioplegia would recruit cytoprotective pathways and investigated the transcriptional response of the heart after cardioplegia-protected ischemia compared with that after ischemia alone.

METHODS: Isolated perfused rat hearts underwent 40 minutes of global ischemia alone or with St Thomas cardioplegia, followed by 120 minutes of reperfusion. The expression profiles of isolated RNA were determined by using Affymetrix microarrays and assessed by comparing cardioplegia-protected hearts and hearts undergoing unprotected ischemia with time-matched control hearts. The content of selected proteins was assessed by means of immunoblotting.

RESULTS: Cardioplegia preserved the expression of multiple genes involved in carbohydrate and fatty acid metabolism, glycolysis, and electron transport compared with ischemia alone. The expression of the sodium-calcium exchanger and ryanodine receptor was preserved in line with the ability of cardioplegia to decrease calcium overload. The expression of multiple cytoprotective molecules, including protein-tyrosine kinase, calcineurin B, p38 mitogen-activated protein kinase, voltage-dependent anion channel, protein kinase C , heat shock protein 70, and manganese superoxide dismutase all showed decreased expression in ischemia but were preserved to near nonischemic levels by cardioplegia.

CONCLUSION: Cardioplegia during ischemia maintained an expression profile similar to that seen in nonischemic hearts for genes involved in energy conservation, calcium homeostasis, and cytoprotective pathways, whereas ischemia alone did not. Exposing the transcriptional differences in cytoprotective genes during untreated and cardioplegia-treated ischemia provides valuable insight into an additional mechanism of cardioprotection induced by cardioplegia.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cardioplegic arrest is used to provide myocardial protection in the majority of the 686,000 patients who undergo cardiac surgical procedures in the United States each year. ¹ The arrested heart, although advantageous to the surgeon, remains susceptible to ischemic injury. Hyperkalemic cardioplegia (CP) is used to maintain diastolic arrest and provide myocardial protection by attenuating the progression of ischemic injury. Despite modifications to the formulation and route of administration, suboptimal myocardial protection remains a problem. The protective effect of CP is thought to be largely the result of conservation of cellular energy stores and preservation of calcium homeostasis. In addition to these accepted mechanisms of myocardial protection, previous work from our laboratory demonstrated that the myocardial protection provided by CP is substantially attenuated if intracellular signaling pathways involving protein kinase C (PKC) or tyrosine kinase are inhibited. ² Activation of these signaling kinases leads to cytoprotection and has been implicated in the robust cardioprotective response of ischemic preconditioning (IPC). This evolving appreciation of the key contribution of the activation of intracellular signaling pathways to myocardial protection ³ raises the question of whether similar mechanisms might contribute to the cardioprotection provided by CP. To identify potential key contributing mechanisms involved in the cardioprotection of CP, we used DNA microarray technology to profile the gene expression pattern of rat hearts subjected to ischemia and reperfusion alone compared with that seen in hearts subjected to CP-protected ischemia and reperfusion. The goal of this study was to evaluate transcriptional changes in genes related to previously known mechanisms of CP and to assess for evidence of the novel involvement of cytoprotective pathways.

	Materials and Methods

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All experiments were approved by the Case Western Reserve University Institutional Animal Care and Use Committee and conformed to the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources, National Research Council and published by the National Academy Press (revised 1996).

Isolated Rat Heart Preparation
The isolated rat heart preparation was used as previously described. ² Male Sprague-Dawley rats (320-360 g) were anesthetized (heparin, 1000 units/kg administered intraperitoneally; pentobarbital, 100 mg/kg administered intraperitoneally). Hearts were excised, mounted on the Langendorff apparatus, and perfused with Krebs-Henseleit (KH) buffer delivered at a perfusion pressure of 65 mm Hg. After collection of baseline data during the equilibration period, the hearts were randomized into one of the 3 study groups. Time-matched control hearts (n = 7) were perfused for 180 minutes. Hearts in the ischemic group (n = 7) were equilibrated for 20 minutes and then underwent 40 minutes of normothermic global ischemia, followed by 120 minutes of reperfusion with KH buffer. Hearts in the CP group (n = 7) were equilibrated for 17 minutes, perfused with St Thomas' Cardioplegia II (NaCl, 110.0 mmol/L; KCl, 16.0 mmol/L; CaCl₂, 1.20 mmol/L; MgCl₂*6H₂O, 16.0 mmol/L; and NaHCO₃, 10.0 mmol/L; pH of 7.8 ± 0.2, T = 4°C) for 3 minutes, and underwent 40 minutes of normothermic global ischemia, followed by 120 minutes of reperfusion with KH buffer. After 180 minutes, left ventricular (LV) tissue was excised and immediately frozen in liquid nitrogen. Three hearts from each group were used for RNA preparation, and 4 hearts were used independently for protein quantitation.

Microarray Analysis
Samples of total RNA were processed by the Gene Expression Array Core Facility at Case Western Reserve University.

RNA preparation
Total RNA was isolated from LV tissue by using Trizol Reagent (Invitrogen). RNA preparations from each group (ischemic, CP, and control) were pooled (n = 3), and their quality and quantity (0.79, 0.91, and 1.10 µg RNA/mg LV tissue) were assessed with spectrophotometry (260/280) and gel electrophoresis. Samples were cleaned and eluted by using a column from a Qiagen RNeasy kit (part no. 74106) precipitated with ammonium acetate and ethanol centrifuged, washed, and resuspended.

Synthesis of cDNA
The Affymetrix protocol for cDNA synthesis was used. The reaction was primed by annealing an oligo-dT primer coupled to a T₇ RNA polymerase promoter to the RNA sample. RNA was reverse transcribed with Superscript II reverse transcriptase. Second-strand synthesis was carried out immediately in the presence of Escherichia coli DNA pol I, RNAse H, and DNA ligase; incubated in the presence of T₄ DNA pol; and terminated with ethylenediamine tetraacetic acid (EDTA). The sample was cleaned (Qiagen cDNA clean-up column).

Synthesis of biotin-labeled complementary RNA
Complementary RNA was generated in an in vitro transcription reaction by using a BioArray High Yield ENZO kit (Affymetrix). Samples were mixed, centrifuged, and returned to the incubator every 40 minutes. In vitro transcription samples were cleaned (Qiagen RNA clean-up columns) and eluted. The quality was confirmed spectrophotometrically (260/280).

Fragmentation and hybridization to test array
Samples were fragmented (40 mmol/L Tris acetate [pH 8.1], 100 mmol/L potassium acetate, and 30 mmol/L magnesium acetate at 94°C for 35 minutes) and added to hybridization buffer (final concentrations: 100 mmol/L morpholinoethanesulfonic acid (MES); 1 mol/L [Na⁺]; 20 mmol/L EDTA; 0.01% Tween 20; 0.1 mg/mL Herring sperm DNA; and 0.5 mg/mL acetylated bovine serum albumin) to improve hybridization to the oligonucleotide array. Eukaryotic hybridization controls (BioB, BioC, BioD, and cre; final concentrations of 1.5, 5, 25, and 100 pmol/L, respectively) and control oligonucleotide (50 pmol/L) were added to the cocktail. The hybridization cocktail was denatured (99°C for 5 minutes), transferred to the test array, and incubated (42°C for 16 hours), and the hybridization cocktail was removed. The array was washed, stained, and scanned for background fluorescence and expression levels of control oligonucleotides by using Affymetrix Microarray Suite software.

Hybridization of fragmented samples to species microarray
Sample hybridization cocktails were thawed (45°C) and centrifuged. The Affymetrix chip arrays (RG-U34A, part no. 510338), containing probe sets for more than 7000 known genes, were equilibrated to room temperature. Sample cocktails were introduced into the chamber of the preconditioned chips and incubated (45°C for 16 hours). The chips were washed, stained, and scanned with an Agilent Gene Array scanner 2000 driven by Affymetrix MicroArray Suite 5.0. Genes with unknown products or duplicate entries were omitted. Functional or biologic classification was determined from the Affymetrix database, the PubMed database, or both.

Statistical analysis of species microarray
Scanned images were analyzed with Affymetrix MAS 5.0 software. For complete description of the statistical algorithms, refer to the Statistical Algorithm Description Document available from Affymetrix. The following tunable parameters were used: 1 = 0.04 and 2 = 0.06 for single-array analysis, and = 0.015, 1L = 0.0025, 1H = 0.0025, 2L = 0.003, and 2H = 0.003 for comparison analysis. Statistical significance is determined by query of 3 parameters: detection, fold change, and change. To be regarded as increased in the treated sample compared with the control, detection was "P" (present) or "M" (marginal), fold change was 2 or more, and change was "I" (increased). For decreases, detection in the sample was "P" or "M," fold change was –2 or less, and change was D (decreased). The change algorithm uses the Wilcoxon signed-rank test with corresponding cutoffs of (P < .0025 vs control).

Western Analysis
Frozen ventricular tissue was placed in buffer (Tris-HCl, 50 mmol/L [pH 7.4]; sodium orthovanadate, 0.1 mmol/L; sodium fluoride, 50 mmol/L; sucrose, 150 mmol/L; phenylmethylsulfonyl fluoride, 1 mmol/L; EDTA, 5 mmol/L; ethyleneglycol-bis-(ß-aminoethylether)-N,N,N',N'tetraacetic acid, 2 mmol/L; 0.2% Triton X-100, Sigma protease inhibitor cocktail 1:200 [producing final concentrations of 4-(2-aminoethyl)benzenesulfonylfluoride HCl (AEBSF), 500 µmol/L; Aprotinin, 0.4 µmol/L; Leupeptin, 10 µmol/L; Bestatin, 18 µmol/L; Pepstatin, 7.5 µmol/L; E-64, 7 µmol/L]) and homogenized (Polytron, 2 bursts of 30 seconds, speed 5). The lysate was mixed on ice for 60 minutes. The solubilized proteins were centrifuged at 360g for 10 minutes, and the resulting supernatant was assayed for total protein concentration by using the Bradford method. Total protein (15 µg) was separated by means of sodium dodecylsulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane. Nonspecific binding was blocked with 5% nonfat milk, and the membrane was incubated with primary antibody (anti-p38, mouse monoclonal sc-7972, Santa Cruz Biotechnology Inc; anti–protein-tyrosine kinase [anti-JAK2], rabbit polyclonal, Upstate Biotechnology; anti–sodium-calcium exchanger, rabbit polyclonal, Swant). The membrane was washed and incubated with the appropriate horseradish peroxidase–linked secondary antibody, washed, and then incubated with a chemiluminescent agent (Luminol, Santa Cruz Biotechnology). Photographic film was exposed to the chemiluminescence, and the bands were quantified by means of densitometry (Scion Image, Scion Corp) as normalized to a control lysate.

Statistical Analysis of Left Ventricular Developed Pressure Recovery, Lactate Dehydrogenase Release, and Protein Content
Data are expressed as means ± SEM. One-way analysis of variance with the Tukey post-hoc test were performed on left ventricular developed pressure (LVDP) recovery, lactate dehydrogenase (LDH) release, and protein content.

	Results

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LVDP and LDH
Time-matched control hearts performed as expected for this model, with preserved LVDP after 180 minutes. Ischemic hearts showed decreased recovery of function compared with control hearts. CP treatment before ischemia markedly protected against the decrease in LVDP (Figure 1). Ischemic hearts showed a significant increase in LDH release compared with that seen in nonischemic control hearts, as well as compared with hearts undergoing CP-treated ischemia (Figure 1). Values for LVDP and LDH release are comparable with those obtained previously in this model. ²

View larger version (12K): [in this window] [in a new window]   Figure 1. Recovery of function (top) at 180 minutes and LDH release during minutes 60 through 90. LVDP, Left ventricular developed pressure; LDH, lactate dehydrogenase. *P < .001 versus control or cardioplegia groups.

Genes with increased expression in the ischemic group but not in the CP-treated group represent products that display a diverse array of functions (Table E1 ) and include some potentially related to cardioprotection. Among the genes exhibiting decreased expression after ischemia but that were not diminished with CP protection (Tables 1 and E2 ) are 29 genes involved in carbohydrate metabolism, fatty acid metabolism, glycolysis, electron transport, or other cellular processes involved in energy production. Five genes involved in calcium regulation or calcium binding, including the sodium-calcium exchanger (NCx) and ryanodine receptor, had decreased expression after ischemia unless protected with CP. Genes for proteins involved in cytoprotection, including JAK2, calcineurin B, p38 mitogen-activated protein kinase (MAPK), voltage-dependent anion channel, PKC-binding protein enigma, PKC-, heat shock protein 70–related gene and precursor, and mitochondrial manganese superoxide dismutase, also did not maintain baseline levels of their transcripts after ischemia unless first protected with CP.

View larger version (28K): [in this window] [in a new window]   Figure 2. Myocardial abundance of p38 mitogen-activated protein kinase (p38, top) and protein-tyrosine kinase (JAK2, bottom) in time-matched control (open bar), ischemic (hatched bar), or cardioplegia-treated (filled bar) hearts after 2 hours of reperfusion or equivalent time for control hearts (n = 4). Units are relative to a common, nonischemic heart value set at 1.00. Western blotting shows representative hearts (n = 2) for each group. *P < .05 compared with control or cardioplegia-treated hearts.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The Langendorff model chosen, using 40 minutes of normothermic global ischemia, eliminates the confounding effects of hypothermic ischemia and was shown to provide good resolution between treatments in functional end points (Figure 1). ² Microarray analysis in the 3 groups revealed differences corroborating the known protective mechanisms of CP, namely energy conservation and calcium homeostasis. The transcriptional data also support the preservation of a nonischemic phenotype by cardioplegia, a new observation.

It is important to recall that although collectively ischemia is damaging to cardiac function, the changes in the expression of individual genes is not necessarily detrimental. Likewise, although CP protection improves function, each of the changes in individual gene expression levels is not necessarily beneficial. The stimulus of ischemia or CP might evoke a combination of responses activating adaptive or protective mechanisms, as well as disrupting normal cell functions. A comparison of gene expression levels between ischemia and CP-protected ischemia therefore might provide clues as to the mechanism of cardioprotection, as well as its limitations. Additionally, gene expression studies are not definitive but are rather preliminary studies to focus further investigation.

The greatest disruption to the normal transcription process occurred in the untreated ischemic group, with 133 genes demonstrating a 2-fold or greater decrease in expression. CP treatment limited the decreased transcript content to merely 28 genes. The ability of CP treatment to maintain expression levels nearer to control for genes otherwise altered by ischemia suggests that CP, in general, preserves a nonischemic transcriptional profile and is likely to maintain a nonischemic phenotype. The protein content data shown in Figure 2 support this notion.

The ability of CP treatment to maintain transcription levels of genes involved in carbohydrate metabolism, fatty acid metabolism, glycolysis, electron transport, and calcium homeostasis suggests mechanisms of CP involving the relative conservation of energy and prevention of cellular calcium overload. The NCx and ryanodine receptor are important regulators of Ca²⁺ homeostasis. Increased expression of NCx and ryanodine receptor has been demonstrated as an adaptive response to low-flow ischemia. ⁴ Although we were unable to demonstrate differences in the protein content for NCx, here we show increased gene expression in CP-protected ischemia compared with that in ischemia alone for these important regulators of calcium homeostasis.

Assessing the changes that occur to gene expression by cardioplegia protection but not by ischemia alone (Table 2) arguably would appear most pertinent to the mechanism of cardioplegia. Surprisingly, this category contained relatively few genes. Furthermore, the magnitude of change was small, with just one gene exhibiting greater than a 3-fold increase and one with a greater than 3-fold decrease. This finding refutes our hypothesis that cardioplegia would activate numerous cytoprotective pathways but rather suggests that the cardioprotection of cardioplegia might be less a factor of transcriptional changes induced by the treatment and more a function of minimizing ischemia-induced alterations in transcription.

Of particular focus in this study were changes in genes known to be involved in cytoprotective mechanisms, including IPC. As with other cluster groups, CP-protected ischemia produced expression levels closer to baseline levels than did unprotected ischemia for genes related to cytoprotective pathways (Figure 3). Interestingly, several genes with decreased expression in ischemia but preserved with CP treatment have previously been shown to have roles in the cardioprotective response during ischemia and reperfusion (Tables 1 and E2). These include JAK2, ⁶ calcineurin B, ^7,8 p38 MAPK, ⁹ voltage-dependent anion channel, ³ PKC-binding protein enigma, ¹⁰ PKC-, ¹¹ heat shock protein 70–related gene and precursor, ¹² and manganese superoxide dismutase. ¹³ Although the precise role for each of these gene products is unclear, the evidence implicating them in ischemia or cardioprotective mechanisms makes the alterations observed in our study intriguing. Repressed gene expression of these mediators after ischemia, as we have demonstrated for p38 and JAK2 (Figure 2), might indicate the cell's inability to maintain appropriate content of these peptides and thus contribute to injury. The ability of the CP-protected heart to maintain expression levels much closer to the baseline levels suggests mechanisms of cardioplegia-induced protection that were previously unknown.

View larger version (29K): [in this window] [in a new window]   Figure 3. Cardioplegia preserves expression of genes with cytoprotective function. Open bars represent unprotected ischemia, and filled bars represent cardioplegia-protected ischemia. Data are displayed relative to control values of 1, such that a 2-fold decrease (–2) is equivalent to 0.5. HSP70, 70-kd Heat shock protein; KATP-1, adenosine triphosphate–sensitive potassium channel uKATP-1; MAPK P-1, mitogen-activated protein kinase phosphatase 1; HSP70 precursor, 70-kd heat shock protein precursor; MAP3K8, serine-threonine protein kinase (Map3k8); PKC-e, protein kinase C ; MnSOD, manganese superoxide dismutase; Heme Ox, heme oxygenase; HSP-related (hst70), heat shock protein-related gene hst70; SCS-3, suppressor of cytokine signaling 3; VDAC, voltage-dependent anion channel; p38 MAPK, p38 mitogen-activated protein kinase; JAK2, protein-tyrosine kinase.

Also noteworthy is the disproportionate number of genes involved in the inflammatory response, which displayed decreased expression in ischemia and CP (Table E3). Recognizing the relationship of ischemia to oxidative stress and of the latter to inflammation, we anticipated increased expression for many inflammatory markers in the ischemic group but not in the CP group. Perhaps this represents the extent of dysfunction caused in ischemia and in CP treatment before ischemia or occurs because of the use of the buffer-perfused heart as the model of experimental ischemia. This finding nonetheless suggests that the heightened inflammatory response prevalent after cardiopulmonary bypass (CPB) might not occur as a result of CP but rather is activated by other factors. The unique expression profile in human atrial tissue after CPB and cardioplegic arrest ¹⁴ provides valuable data to support this statement. These investigators noted increased expression of inflammatory genes after CPB. Our observation of predominantly decreased inflammatory genes in the isolated perfused heart suggests that the increases observed in the clinical setting might occur because of one or more of the additional factors associated with the clinical cardiac surgical setting, including perhaps the CPB circuit, hypothermia, surgical trauma, anesthesia, or preexisting ischemia. Additionally, induced genes unique to the clinical study might be reflective of differences between analysis of LV tissue from an isolated rat heart and atrial tissue from CPB surgery or merely limitations of the microarray technique. Future studies will be needed to delineate these differences.

The current study evaluated differences in the gene expression pattern between hearts subjected to ischemia alone or hearts protected with hyperkalemic CP before ischemia. Ischemia altered the pattern of expression to a much greater extent in untreated hearts than in hearts first protected with CP, suggesting that CP-protected ischemia tends to preserve the nonischemic transcriptional phenotype. Many of the genes exhibiting decreased expression caused by ischemia alone are important in energy production and calcium homeostasis, which is consistent with proposed mechanisms of CP-induced myocardial protection. In addition, the cardioprotection provided by CP maintains cytoprotective peptides reminiscent of IPC. The novel concept that cardioplegia can prevent ischemic disruptions to the transcriptional processes of many genes, including those involved in cytoprotection, is worthy of consideration for advancing myocardial protective strategies during cardiac surgery.

	Acknowledgments

 
We appreciate the assistance of Ms Michelle Quicci in preparation of the RNA and Patrick Leahy, PhD, in conducting the microarray analysis. We also thank Christine Moravec, PhD, for helpful discussions and review of the manuscript and Mark Schluchter, PhD, for review of the statistical methods.

	Footnotes

 
Supported by the Jay L. Ankeney Endowment. Dr Lesnefsky was supported by grants 2RO1AG12447 and 1PO15885 from the National Institutes of Health and by the Medical Research Service, Department of Veterans Affairs. Dr Hedayati was an Allen Fellow supported by the Jay L. Ankeney Professorship in Cardiothoracic Surgery, Case Western Reserve University School of Medicine, Cleveland, Ohio.

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