224 Original research
Impaired myocardium energetics associated with the risk for new-onset atrial fibrillation after isolated coronary artery bypass graft surgery Dian-Min Suna,c,*, Xin Yuana,b,*, Hua Weid,*, Shen-Jun Zhue, Peng Zhanga,b, Shi-Ju Zhanga,b, Hong-Guang Fana,b, Yan Lia,b, Zhe Zhenga,b and Xiao-Cheng Liua,c Background New-onset postoperative atrial fibrillation (POAF) is one of the most common complications occurring in 10–40% of patients after coronary artery bypass graft (CABG) surgery. Recent studies suggest that dysmetabolism may contribute to the pathogenesis of atrial fibrillation; however, the putative mechanism in patients undergoing CABG surgery is unknown. Peroxisome proliferator-activated receptor c coactivator-1a (PGC-1a) has been demonstrated as a master regulator of myocardial energy metabolism, and glucose transporter 3 (GLUT3) has both a higher affinity for glucose and a much greater transport capacity compared with GLUT1, GLUT2, and GLUT4. We sought to evaluate the role of energy metabolism, especially the glucose metabolism, on patients after isolated CABG surgery. Methods and results Right atrial appendages were obtained from 79 patients who were in normal sinus rhythm and undergoing isolated CABG; those who exhibited new-onset POAF (n = 22) or remained in sinus rhythm (n = 57) were prospectively matched on the basis of preoperative, intraoperative, and postoperative characteristics. POAF was assessed by electrocardiogram and must have required the initiation of antiarrhythmic therapy or anticoagulation. Local PGC-1a and GLUT3 concentrations were quantified by enzyme-linked immunosorbent assay in tissue homogenates. The comparison of mRNA expression was tested by
Introduction Postoperative atrial fibrillation (POAF) is one of the most common arrhythmias after cardiac surgery, and occurs mostly within 3–5 days after operation [1–3]. In this setting, most POAF is self-limiting but prone to lead to tachycardia, heart failure, and cerebrovascular emboli. Even uncomplicated POAF requires extra medical and nursing time and prolongs the hospital stay [1–7]. Although many studies have correlated some risk factors with POAF (age, mechanical damage, length of ischemic period, lack of b-blockade, obesity, etc.), the mechanisms underlying its pathophysiology remain unclear. Myofibril has complex and tightly regulated high-energy phosphate production and utilization capabilities [8–10]. Altered myofibrillar energetics may contribute to atrial c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0954-6928
quantitative real-time PCR. PGC-1a and GLUT3 levels and the related protein mRNA expression were significantly reduced in POAF patients compared with controls (P < 0.05). This selective reduction in PGC-1a was associated with the presence of diabetes mellitus (P < 0.05). Conclusion Patients who have low PGC-1a and GLUT3 levels are at increased risk for new-onset POAF. The myofibrillar energetic impairment may be important in the pathogenesis of atrial fibrillation. Coron Artery Dis c 2014 Wolters Kluwer Health | Lippincott 25:224–229 Williams & Wilkins. Coronary Artery Disease 2014, 25:224–229 Keywords: coronary artery bypass graft, energy metabolism, postoperative atrial fibrillation a Peking Union Medical College, Chinese Academy of Medical Science, bFuwai Hospital, Peking Union Medical College, Chinese Academy of Medical Science, Beijing, cTEDA International Cardiovascular Hospital, Tianjin, China, dCardiac Signaling Center of USC, MUSC & Clemson University, Charleston, South Carolina and eMetrowest Medical Center, Framingham, Massachusetts, USA
Correspondence to Xiao-Cheng Liu, MD, TEDA International Cardiovascular Hospital, No. 61, 3rd Ave, TEDA, Tianjin 300457, China Tel: + 86 22 65208030; fax: + 86 22 65208001; e-mail: [email protected] *Dian-Min Sun, Xin Yuan, and Hua Wei are co-first authors. Received 26 September 2013 Revised 5 December 2013 Accepted 12 December 2013
contractile dysfunction [11]. Glycolytic enzymes undergo compensatory upregulation during persistent atrial fibrillation (AF) [9], and discordant metabolic alterations are evident in individuals susceptible to POAF [12]. Numerous studies demonstrate that when the heart is subjected to ischemia, dramatically enhanced fatty acid (FA) oxidation and markedly suppressed glucose oxidation contribute to the cardiac dysfunction and reperfusion injury. However, the mechanism of regulation of mitochondrial oxidative metabolism and the capacity of glucose uptake in human myocardium during ischemia/ reperfusion are unknown. Recent studies have demonstrated that peroxisome proliferator-activated receptor g coactivator-1a (PGC-1a) serves as a master regulator of mitochondrial oxidative metabolism that coordinates the capacity of each step required for ATP synthesis [13]. DOI: 10.1097/MCA.0000000000000081
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Impaired myocardium energetics Sun et al. 225
Activation of the PGC-1a regulatory cascade increases cardiac mitochondrial oxidative capacity in the heart. Glucose transporter 3 (GLUT3) was the third glucose transporter to be discovered, first reported in human myocardium in 1999 [14], and is preferentially expressed in cells with high-energy demand but in lower proportion as compared with GLUT4 and GLUT1. Little is known about the regulation of heart GLUT3. This carrier is the most efficient among the different GLUTs, as it has the lowest Km [15]. In the present study, we tested the hypothesis that both the maladaptive energy metabolism and diminished capacity of glucose uptake are associated with the risk for new-onset atrial fibrillation after isolated coronary artery bypass graft (CABG) surgery.
Methods Patients
All patients were diagnosed as coronary artery disease requiring CABG and as angiographically defined multivessel coronary stenosis, with at least 70% stenosis. Atrial appendages were obtained as surgical specimens from patients undergoing cardiac surgery using the procedures approved by the Institutional Review Board of the Fu Wai Hospital. The study was approved by the local ethics committees and all patients gave informed consent. Right atrial appendages were obtained from 79 patients who were undergoing routine cardiac bypass graft surgery (mean age, 59.88±8.62 years). Tissue was harvested during cannulation of the right atrium for bypass, and Clinical and demographic characteristics of POAF patients, SR patients, and controls
Table 1
Male sex (%) Age (years) BMI Hypertension [n (%)] Diabetes mellitus [n (%)] Hyperlipidemia [n (%)] Total cholesterol (mmol/l) Previous MI [n (%)] Left atrial diameter NYHA class III or IV (%) LVEF at enrollment (%) ACEI/ARB [n (%)] b-Blockers [n (%)] Statins [n (%)] CCB [n (%)] CPB (min) ACC (min) Number of grafts ICU (h)
SR (n = 57)
POAF (n = 22)
P value
73 58.64±8.97 26.08±2.79 59.1 41.3 40.9 4.4 17 (29.8) 50 8.6 60.37 25 (43.9) 55 (96.5) 23 (40.4) 32 (56.1) 61±18 30±11 3±1 53
76 63.04±6.46 26.33±3.49 61.4 47.6 43.8 4.2 5 (22.7) 49 14.2 58.27 9 (40.9) 21 (95.5) 8 (36.4) 16 (72.7) 62±25 30±8 3±1 59
0.738 0.038 0.113 0.853 0.815 0.813 0.487 0.531 0.736 0.995 0.749 0.813 0.830 0.805 0.179 0.72 0.99 0.93 0.327
Postoperative AF was observed in 22 patients (27.8%) with a peak incidence of AF occurring on the second postoperative day. In-hospital postoperative treatment with b-blockers was initiated in a similar percentage of patients from each group (i.e. in 96.5% of SR patients and in 95.5% of patients who developed AF, P = 0.83). ACC, aortic clamp time; ACEI, angiotensin-converting enzyme inhibitor; AF, atrial fibrillation; ARB, angiotensin II receptor blocker; CCB, calcium-channel blocker; CPB, cardiopulmonary bypass time; LVEF, left ventricular ejection fraction; MI, myocardial infarction; NYHA, New York Heart Association; POAF, postoperative atrial fibrillation; SR, sinus rhythm.
specimens were immediately snap-frozen and stored in liquid nitrogen. Table 1 describes detailed clinical characteristics. Protein quantification with ELISA
Expression of PGC-1a and GLUT3 by myofibrils in each surgical frozen sample was respectively assayed using the Human PGC-1a enzyme-linked immunosorbent assay (ELISA) Kit (Uscn Life, Double Lake, Missouri City, Texas, USA) and the Human GLUT3 ELISA Kit (Uscn Life) according to the protocol of the manufacturer. Collect the samples and bring all reagents and samples to room temperature before use. Centrifuge the sample again after thawing before the assay. Refer to the Assay Layout Sheet to determine the number of wells to be used and put any remaining wells and the desiccant back into the pouch and seal the Ziploc; store the unused wells at 41C. Add 100 ml of standard and sample per well. Cover with the adhesive strip provided. Incubate for 2 h at 371C. A plate layout is provided to record the standards and samples assayed. Remove the liquid from each well and add 100 ml of Biotin antibody (1 ) to each well. Cover with a new adhesive strip. Incubate for 1 h at 371C. Aspirate each well and wash, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (200 ml) using a squirt bottle, multichannel pipette, manifold dispenser, or autowasher, and let it stand for 2 min; complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. Add 100 ml of HRP-avidin (1 ) to each well. Cover the microtiter plate with a new adhesive strip. Incubate for 1 h at 371C. Repeat the wash process five times and add 90 ml of TMB substrate to each well. Incubate for 15–30 min at 371C. Add 50 ml of Stop Solution to each well and gently tap the plate to ensure thorough mixing. Determine the optical density of each well within 5 min, using a microplate reader set at 450 nm. If wavelength correction is available, set at 540 nm. Subtract the readings at 540 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. At the end, the protein values were normalized to the total protein content in each surgical atrial fragment. The standard points and samples were determined in duplicate. Quantitative real-time PCR
The mRNA was isolated from the samples using the mRNA Isolation Kit (Sigma Aldrich, St Louis, Missouri, USA) and then reverse transcribed using M-MLV reverse transcriptase and oligo(dT)18 primer. Real-time PCR was performed using SYBR Green I (Perkin Elmer Life Sciences, Boston, Massachusetts, USA) in an ABI Prism 7300 thermocycler (Applied Biosystems, Foster City, California, USA). The thermal profile for PCR was 951C for 10 min, followed by 40 cycles at 951C for 15 s and at
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226 Coronary Artery Disease 2014, Vol 25 No 3
Continuous variables are expressed as mean±SD. Protein levels are described by median (Q1–Q3). The differences between patients who developed AF and those who maintained sinus rhythm (SR) during the postoperative period were analyzed with analysis of variance and twosample t-test for continuous variables or the w2-test for categorical variables. A P value less than 0.05 was considered significant. Statistical comparisons of protein between the groups were performed with Wilcoxon’s rank-sum test. The correlation analysis of protein levels with quantitative measurements was performed with the Spearman correlation coefficient (rs). Binary regression analysis was used to determine the correlations between the results. Statistical significance was designated as a probability of less than 0.05. For all analyses, commercially available statistical package software was used (SPSS, version 13.0; SPSS Inc., Chicago, Illinois, USA).
Results Clinical characteristics of the POAF patients and controls are presented in Table 1. POAF was observed in 22 individuals (27.8%) with a peak incidence of AF occurring on the second postoperative day. No significant differences between the groups were found with respect to treatment, cardiovascular risk factors and comorbidity, duration of CPB or aortic cross-clamp, and number of grafted vessels (Table 1). However, patients who developed POAF were apparently older and they also had lower PGC-1a and GLUT3 levels compared with those who Fig. 1
GLUT3 (mg/μl)
Statistical analysis
Fig. 2
∗
14 000.000 12 000.000 10 000.000 8000.000 6000.000 4000.000 2000.000 .000
POAF
SR
GLUT3 protein levels in patients with POAF and SR. The samples of the patients with DM and with POAF and SR were collected and centrifuged after thawing before the assay. The protein expression of GLUT3 was measured with Human GLUT3 ELISA Kit (Uscn Life). Data are presented as mean±SD in triplicate from three independent experiments. *P < 0.05 vs. the control. DM, diabetes mellitus; GLUT3, glucose transporter 3; POAF, postoperative atrial fibrillation; SR, sinus rhythm.
Fig. 3
∗
30 mRNA PGC-1α/β-actin
601C for 1 min. To obtain a relative quantitation, the results were normalized to those obtained for the corresponding actin mRNAs. Each sample was run in duplicate.
25 20 15 10 5 0
POAF
SR
PGC-1a mRNA expression in patients with POAF and SR. The samples of the patients with POAF and SR were collected and centrifuged after thawing before the assay. The mRNAs were isolated using the mRNA Isolation Kit (Sigma Aldrich) and then reverse transcription and real-time PCR were performed. Data are presented as mean±SD in triplicate from three independent experiments. *P < 0.05 vs. the control. PGC1a, peroxisome proliferator-activated receptor g coactivator-1a; POAF, postoperative atrial fibrillation; SR, sinus rhythm.
∗ 3.5
Fig. 4
2.5 2 1.5 1 0.5 0
POAF
SR
PGC-1a protein levels in patients with POAF and SR. The samples from the patients with POAF and SR were collected and centrifuged after thawing before the assay. The protein expression of PGC-1a was measured with Human PGC-1a ELISA Kit (Uscn Life). Data are presented as mean±SD in triplicate from three independent experiments. *P < 0.05 vs. the control. PGC-1a, peroxisome proliferator-activated receptor g coactivator-1a; POAF, postoperative atrial fibrillation; SR, sinus rhythm.
mRNA GLUT3/β-actin
PGC-1α (mg/μl)
3 3
∗
2
1
0
POAF
SR
GLUT3 mRNA expression in patients with POAF and SR. The samples of the patients with POAF and SR were collected and centrifuged after thawing before the assay. The mRNAs were isolated using the mRNA Isolation Kit (Sigma Aldrich) and then reverse transcription and real-time PCR were performed. Data are presented as mean±SD in triplicate from three independent experiments. *P < 0.05 vs. the control. GLUT3, glucose transporter 3; POAF, postoperative atrial fibrillation; SR, sinus rhythm.
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Impaired myocardium energetics Sun et al.
Table 2
Multivariable predictors of postoperative atrial fibrillation
Age (years) BMI Diabetes (no/yes) Total cholesterol PGC-1a
OR
95% CI
P value
1.077 1.783 0.648 0.318 0.407
0.996–1.164 0.941–1.385 0.188–1.971 0.516–1.386 0.191–0.850
0.064 0.182 0.421 0.573 0.017
Binary logistic regression identified PGC-1a (P = 0.017) as an independent predictor for the development of postoperative AF among the 79 patients. AF, atrial fibrillation; CI, confidence interval; OR, odds ratio; PGC-1a, peroxisome proliferator-activated receptor g coactivator-1a.
remained in SR (Figs 1 and 2). The comparison of mRNA expression was similar to protein expression between the two groups (Figs 3 and 4). Atrial PGC-1a levels were also lower in patients with diabetes mellitus (DM) (n = 34) compared with those without DM (n = 45) (P = 0.015). Specifically, the PGC-1a-related mRNA expression was obviously lower in the DM group. The GLUT3 level was not significantly different between the DM group and the non-DM group; the same was true for GLUT3-related mRNA expression. In-hospital postoperative treatment with b-blockers was initiated in a similar percentage of patients from each group (i.e. in 96.5% of SR patients and in 95.5% of patients who developed AF, P = 0.83) (Table 1). There was no difference in the postoperative use of angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers or in the time of initiation of treatment after surgery (Table 1). Binary logistic regression identified PGC-1a (P = 0.017) as an independent predictor for the development of POAF among the 79 patients. Age was no longer associated with an increased risk for POAF after the addition of PGC-1a to the model. Other predictors for POAF that were included in the regression model but did not reach a level of statistical significance were age, history of DM, BMI, and total cholesterol (Table 2), according to the Hosmer and Lemeshow Goodness-of-Fit test for the final model (P = 0.736, d.f. = 8). Characterization of all patients according to the quartiles of atrial PGC-1a confirmed a powerful association between these measurements and the occurrence of POAF (P < 0.001), whereas the correlation between POAF and DM is uncertain, although the PGC-1a levels are obviously lower in patients with POAF and DM compared with those without DM.
Discussion Main findings
Recent studies confirmed that AF denotes a hypermetabolic state [9,11,12,16], but how the metabolism influences the persistence or onset of AF is unclear. This study on a cohort of patients undergoing isolated CABG surgery used the emerging techniques, such as ELISA and quantitative real-time PCR, to provide novel insights into the regulation of myocardial energy metabolism. This was a prospective study to analyze the role of PGC-1a
227
and GLUT3 in myocardium tissue, and demonstrates that, inter alia, a discordant regulation of energy metabolites and the glycometabolic disorder precede the new-onset POAF after CABG surgery. We also found that the reduction in PGC-1a level and the level of transcript expression was associated with the presence of DM; however, the underlying mechanism is unclear. PGC-1a and energy metabolism in myocardium
PGC-1a, initially identified as a PPARg coactivator, has been proven to serve coactivator functions for both nuclear receptors and other classes of transcription factors. Recently, PGC-1a has been demonstrated as the master regulator of mitochondrial oxidative metabolism [13]. Despite a relative abundance of information on the effects of PGC-1a on the isolated myocardium or rodent model systems, the regulation and physiological roles of PGC-1a in human heart, to our knowledge, are unknown. In cardiac myocytes in culture, PGC-1a increases mitochondrial number, upregulates the expression of mitochondrial enzymes, and increases the rates of FA oxidation and coupled respiration [17,18]. PGC-1a transcriptionally regulates the genes involved in the cellular uptake and mitochondrial oxidation of FAs through direct coactivation of PPARs and estrogenrelated receptors [13,19]. Those genes encoding enzymes are involved at multiple steps of these metabolic pathways, such as uptake, esterification, mitochondrial transport, and oxidation [20]. FA and glucose oxidation are the main ATP-generating pathways in human heart. AcetylCoA derived from FA and glucose oxidation is further oxidized in the TCA cycle to generate NADH and FADH2, which enter the electron transport/oxidative phosphorylation pathway and drive ATP synthesis. The glucose uptake/oxidation and electron transport/oxidative phosphorylation pathways are also regulated by PGC-1a through other transcription factors, including MEF2 [21], NRF-1 [22], and cytochrome c [23]. Our study, which applied the ELISA technique directly to human cardiac tissue, first described significant changes in PGC1a levels in fibrillating atrial tissue and then suggested a molecular mechanism that seems to contribute to POAF. GLUT3 and myocardial glucose uptake in reperfusion heart
Glucose and lactate are the major fuels for a healthy heart. The capacity of the cell to absorb glucose relies on the fraction of the glucose transporters that reside in the plasma membrane. Three isoforms from the glucose transporter family have been identified in the myocardium: GLUT1, GLUT4, and GLUT3 [24]. Although GLUT4 is predominant in the heart, GLUT3 has both a higher affinity for glucose and at least a five-fold greater transport capacity than GLUT1 and GLUT4 [25]. Thus, we selected GLUT3 as the biomarker of glucose uptake in this study instead of GLUTs in the myocardium. The consequence of the high rates of FA oxidation during
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228 Coronary Artery Disease 2014, Vol 25 No 3
reperfusion is a decrease in glucose oxidation that in turn contributes to a decrease in pH during ischemia, and a delayed recovery of pH following ischemia [26]. As the intracelluar H + level rises, the H + /Na + exchange and Na + /Ca2 + exchange activities are increased, resulting in Ca2 + overload, electrical instability, mechanical dysfunction, and mitochondrial dysfunction [27]. In our study, we first found that the GLUT3 level and protein-related mRNA expression were both lower in patients of the POAF group compared with those who remained in SR, with the difference being statistically significant. This suggested that the impaired capacity of glucose uptake can abate the efficiency of glucose oxidation in the reperfusion heart and can induce the electrical instability to a certain extent.
that a discordant regulation of energy metabolites precedes the onset of arrhythmia after surgery. They also found that the lower the ratio of glycolytic end products to the end products of lipid metabolism in human atrial tissue, the earlier the onset of arrhythmia in CABG surgery patients. In our study, we found that the reduction in PGC-1a level was significantly associated with new-onset POAF and DM, and the comparison of mRNA expression was similar to protein expression. We know that DM is a highly prevalent risk factor in the cardiac surgical population. Thus, energy metabolic profiles may help to further classify those persons who are at high risk of developing POAF in the elderly patients undergoing CABG surgery. Limitations
PGC-1a and diabetes mellitus in the heart
PGC-1a regulates PPARa and other transcription factors in the heart to couple the metabolic needs to the expression of genes involved in the control of energy metabolism. The expression and activity of PGC-1a and its coactivator PPARs can be dynamically regulated in metabolic diseases. In diabetes, the heart is exposed to a hyperglycemic and hyperlipidemic environment. The elevation of plasma nonesterified FA levels in diabetes results in the activation of PPARa [28,29]. Young et al. [30] showed that progression of diabetes is associated with a marked decrease in the expression of PPARa within the rat heart. Failure to adequately control the intracellular glucose levels has also been implicated in the development of insulin resistance and in the generation of reactive oxygen species in various tissues [31,32]. Glucose may downregulate the expression of FAmetabolizing genes through PPARa repression, leading to accelerated lipid deposition within the cardiomyocyte and resulting in cardiac dysfunction. Roduit et al. [33] have shown that, in the islet cells, glucose exposure decreases the expression of PPARa and several PPARaregulated genes involved in FA metabolism. Recently, a series of studies on humans have demonstrated an inverse correlation of the muscle PGC-1a levels and mitochondrial activity with insulin resistance and diabetes [34–36]. Metabolic alterations and postoperative atrial fibrillation
Recent demonstration in an animal model that glycolytic inhibition predisposes to AF also confirmed the assumption that metabolic alterations might facilitate the onset of arrhythmia [20]. Some other studies also revealed the relationship between energy metabolism and POAF in patients. Kim et al. [37] found that the atrial NADPH oxidase activity is independently associated with an increased risk for POAF, suggesting that this oxidase system may be a key mediator of atrial oxidative stress leading to the development of AF after cardiac surgery. In the study by Mayr et al. [12], they found the metabolic adaptation processes in persistent AF and demonstrated
Some potential limitations may have influenced our results. All patients included in our study underwent cardiac surgery because of coronary artery disease. Thus, no comment can be made about the possible impact of energy metabolism changes in other patient populations. Only right atrial samples were examined; therefore, our present findings may not be representative of other parts of the atria. Although our present study provides new evidence of mechanistic insight regarding energy metabolism impairment for a relatively small sample capacity, we did not find significant association between POAF and metabolic syndrome, which is in disagreement with other studies [38–40].
Acknowledgements This study was supported by the Chinese National Natural Science Foundation (81270302). The authors thank Professor Guowei He from NanKai University for helpful comments. They also thank Dr Zahner from Peking Union Medical College for her genuine suggestions. Finally, all authors thank Dr Tang Yue, Dr Wei Wang, Dr Li-Qing Wang, and Dr Han-Song Sun from Fu Wai Hospital, Peking Union Medical College, and Chinese Academy of Medical Science for helping us to obtain right atrial appendage tissues. Conflicts of interest
There are no conflicts of interest.
References 1
2 3
4
Aranki SF, Shaw DP, Adams DH, Rizzo RJ, Couper GS, VanderVliet M, et al. Predictors of atrial fibrillation after coronary artery surgery: current trends and impact on hospital resources. Circulation 1996; 94:390–397. Creswell LL, Schuessler RB, Rosenbloom M, Cox JL. Hazards of postoperative atrial arrhythmias. Ann Thorac Surg 1993; 56:539–549. Mathew JP, Parks R, Savino JS, Friedman AS, Koch C, Mangano DT, et al. Atrial fibrillation following coronary artery bypass graft surgery: predictors, outcomes, and resource utilization. MultiCenter Study of Perioperative Ischemia Research Group. JAMA 1996; 276:300–306. Taylor GJ, Malik SA, Colliver JA, Dove JT, Moses HW, Mikell FL, et al. Usefulness of atrial fibrillation as a predictor of stroke after isolated coronary artery bypass grafting. Am J Cardiol 1987; 60:905–907.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Impaired myocardium energetics Sun et al. 229
5
6
7 8
9
10 11
12
13 14
15
16
17
18
19
20
21
22
Borzak S, Tisdale JE, Amin NB, Goldberg AD, Frank D, Padhi ID, et al. Atrial fibrillation after bypass surgery: does the arrhythmia or the characteristics of the patients prolong hospital stay? Chest 1998; 113:1489–1491. Kim MH, Deeb GM, Morady F, Bruckman D, Hallock LR, Smith KA, et al. Effect of postoperative atrial fibrillation on length of stay after cardiac surgery (the Postoperative Atrial Fibrillation in Cardiac Surgery Study [PACS(2)]). Am J Cardiol 2001; 87:881–885. Ommen SR, Odell JA, Stanton MS. Atrial arrhythmias after cardiothoracic surgery. N Engl J Med 1997; 336:1429–1434. Vega RB, Huss JM, Kelly DP. The coactivator PGC-1 cooperates with peroxisome proliferator-activated receptor a in transcriptional control of nuclear genes encoding mitochondrial fatty acid oxidation enzymes. Mol Cell Biol 2000; 20:1868–1876. Barth AS, Merk S, Arnoldi E, Zwermann L, Kloos P, Gebauer M, et al. Reprogramming of the human atrial transcriptome in permanent atrial fibrillation: expression of a ventricular-like genomic signature. Circ Res 2005; 96:1022–1029. Wagoner LE, Walsh RA. The cellular pathophysiology of progression to heart failure. Curr Opin Cardiol 1996; 11:237–244. Mihm MJ, Yu F, Carnes CA, Reiser PJ, McCarthy PM, Van Wagoner DR, et al. Impaired myofibrillar energetics and oxidative injury during human atrial fibrillation. Circulation 2001; 104:174. Mayr M, Yusuf S, Weir G, Chung YL, Mayr U, Yin X, et al. Combined metabolomic and proteomic analysis of human atrial fibrillation. J Am Coll Cardiol 2008; 51:585–594. Huss JM, Kelly DP. Mitochondrial energy metabolism in heart failure: a question of balance. J Clin Invest 2005; 115:547–555. Grover-McCay M, Walsh SA, Thompson SA. Glucose transporter 3 (GLUT3) protein is present in human myocardium. Biochim Biophys Acta 1999; 1416:145–154. Colville CA, Seatter MJ, Jess TJ, Gould GW, Thomas HM. Kinetic analysis of the liver-type (GLUT2) and brain-type (GLUT3) glucose transporters in Xenopus oocytes: substrate specificities and effects of transport inhibitors. Biochem J 1993; 290:701–706. Thijssen VL, Ausma J, Borgers M. Structural remodelling during chronic atrial fibrillation: act of programmed cell survival. Cardiovasc Res 2001; 52:14–24. Lehman JJ, Barger PM, Kovacs A, Saffitz JE, Medeiros DM, Kelly DP. Peroxisome proliferator-activated receptor gamma coactivator-1 promotes cardiac mitochondrial biogenesis. J Clin Invest 2000; 106:847–856. Huss JM, Pine´da Torra I, Staels B, Gigue`re V, Kelly DP. ERRa directs PPAR a signaling in the transcriptional control of energy metabolism in cardiac and skeletal muscle. Mol Cell Biol 2004; 24:9079–9091. Schreiber SN, Knutti D, Brogli K, Uhlmann T, Kralli A. The transcriptional coactivator PGC-1 regulates the expression and activity of the orphan nuclear receptor estrogen-related receptor a (ERRa). J Biol Chem 2003; 278:9013–9018. Ono N, Hayashi H, Kawase A, Lin SF, Li H, Weiss JN, et al. Spontaneous atrial fibrillation initiated by triggered activity near the pulmonary veins in aged rats subjected to glycolytic inhibition. Am J Physiol Heart Circ Physiol 2007; 292:639–648. Michael LF, Wu Z, Cheatham RB, Puigserver P, Adelmant G, Lehman JJ, et al. Restoration of insulin-sensitive glucose transporter (GLUT4) gene expression in muscle cells by the transcriptional coactivator PGC-1. Proc Natl Acad Sci USA 2001; 98:3820–3825. Wu Z, Puigserver P, Andersson U, Zhang C, Adelmant G, Mootha V, et al. Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell 1999; 98:115–124.
23
24
25
26
27
28
29
30
31
32
33
34 35
36
37
38
39
40
Short KR, Vittone JL, Bigelow ML, Proctor DN, Rizza RA, Coenen-Schimke JM, et al. Impact of aerobic exercise training on age-related changes in insulin sensitivity and muscle oxidative capacity. Diabetes 2003; 52: 1888–1896. Gavete ML, Agote M, Martin MA, Alvarez C, Escriva F. Effects of chronic undernutrition on glucose uptake and glucose transporter proteins in rat heart. Endocrinology 2002; 143:4295–4303. Simpson IA, Dwyer D, Malide D, Moley KH, Travis A, Vannucci SJ. The facilitative glucose transporter GLUT3: 20 years of distinction. Am J Physiol Endocrinol Metab 2008; 295:E242–E253. Liu Q, Docherty JC, Rendell JC, Clanachan AS, Lopaschuk GD. High levels of fatty acids delay the recovery of intracellular pH and cardiac efficiency in post-ischemic hearts by inhibiting glucose oxidation. J Am Coll Cardiol 2002; 39:718–725. Karmazyn M. The role of the myocardial sodium–hydrogen exchanger in mediating ischemic and reperfusion injury. From amiloride to cariporide. Ann N Y Acad Sci 1999; 874:326–334. Gulick T, Cresci S, Caira T, Moore DD, Kelly DP. The peroxisome proliferatoractivated receptor regulates mitochondrial fatty acid oxidative enzyme gene expression. Proc Natl Acad Sci USA 1994; 91:11012–11016. Brandt JM, Djouadi F, Kelly DP. Fatty acids activate transcription of the muscle carnitine palmitoyltransferase I gene in cardiac myocytes via the peroxisome proliferator-activated receptor alpha. J Biol Chem 1998; 273:23786–23792. Young ME, Patil S, Ying J, Depre C, Ahuja HS, Shipley GL, et al. Uncoupling protein 3 transcription is regulated by peroxisome proliferator-activated receptor (alpha) in the adult rodent heart. FASEB J 2001; 15:833–845. Rossetti L, Smith D, Shulman GI, Papachristou D, DeFronzo RA. Correction of hyperglycemia with phlorizin normalizes tissue sensitivity to insulin in diabetic rats. J Clin Invest 1987; 79:1510–1515. Rosen P, Du X, Tschope D. Role of oxygen derived radicals for vascular dysfunction in the diabetic heart: prevention by alpha-tocopherol? Mol Cell Biochem 1998; 188:103–111. Roduit R, Morin J, Masse´ F, Segall L, Roche E, Newgard CB, et al. Glucose down-regulates the expression of the peroxisome proliferator-activated receptor-alpha gene in the pancreatic beta-cell. J Biol Chem 2000; 275:35799–35806. Attie AD, Kendziorski CM. PGC-1a at the crossroads of type 2 diabetes. Nat Genet 2003; 34:244–245. Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar J, et al. PGC-1a-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet 2003; 34:267–273. Patti ME, Butte AJ, Crunkhorn S, Cusi K, Berria R, Kashyap S, et al. Coordinated reduction of genes of oxidative metabolism in humans with insulin resistance and diabetes: potential role of PGC1 and NRF1. Proc Natl Acad Sci USA 2003; 100:8466–8471. Kim YM, Kattach H, Ratnatunga C, Pillai R, Channon KM, Casadei B. Association of atrial nicotinamide adenine dinucleotide phosphate oxidase activity with the development of atrial fibrillation after cardiac surgery. J Am Coll Cardiol 2008; 51:68–74. Wang TJ, Parise H, Levy D, D’Agostino RB Sr, Wolf PA, Vasan RS, Benjamin EJ. Obesity and the risk of new-onset atrial fibrillation. JAMA 2004; 292:2471–2477. Dublin S, French B, Glazer NL, Wiggins KL, Lumley T, Psaty BM, et al. Risk of new-onset atrial fibrillation in relation to body mass index. Arch Intern Med 2006; 166:2322–2328. Zacharias A, Schwann TA. Obesity and risk of new-onset atrial fibrillation after cardiac surgery. Circulation 2005; 112:3247–3255.
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Impaired myocardium energetics associated with the risk for new-onset atrial fibrillation after isolated coronary artery bypass graft surgery Dian-Min Suna,c,*, Xin Yuana,b,*, Hua Weid,*, Shen-Jun Zhue, Peng Zhanga,b, Shi-Ju Zhanga,b, Hong-Guang Fana,b, Yan Lia,b, Zhe Zhenga,b and Xiao-Cheng Liua,c Background New-onset postoperative atrial fibrillation (POAF) is one of the most common complications occurring in 10–40% of patients after coronary artery bypass graft (CABG) surgery. Recent studies suggest that dysmetabolism may contribute to the pathogenesis of atrial fibrillation; however, the putative mechanism in patients undergoing CABG surgery is unknown. Peroxisome proliferator-activated receptor c coactivator-1a (PGC-1a) has been demonstrated as a master regulator of myocardial energy metabolism, and glucose transporter 3 (GLUT3) has both a higher affinity for glucose and a much greater transport capacity compared with GLUT1, GLUT2, and GLUT4. We sought to evaluate the role of energy metabolism, especially the glucose metabolism, on patients after isolated CABG surgery. Methods and results Right atrial appendages were obtained from 79 patients who were in normal sinus rhythm and undergoing isolated CABG; those who exhibited new-onset POAF (n = 22) or remained in sinus rhythm (n = 57) were prospectively matched on the basis of preoperative, intraoperative, and postoperative characteristics. POAF was assessed by electrocardiogram and must have required the initiation of antiarrhythmic therapy or anticoagulation. Local PGC-1a and GLUT3 concentrations were quantified by enzyme-linked immunosorbent assay in tissue homogenates. The comparison of mRNA expression was tested by
Introduction Postoperative atrial fibrillation (POAF) is one of the most common arrhythmias after cardiac surgery, and occurs mostly within 3–5 days after operation [1–3]. In this setting, most POAF is self-limiting but prone to lead to tachycardia, heart failure, and cerebrovascular emboli. Even uncomplicated POAF requires extra medical and nursing time and prolongs the hospital stay [1–7]. Although many studies have correlated some risk factors with POAF (age, mechanical damage, length of ischemic period, lack of b-blockade, obesity, etc.), the mechanisms underlying its pathophysiology remain unclear. Myofibril has complex and tightly regulated high-energy phosphate production and utilization capabilities [8–10]. Altered myofibrillar energetics may contribute to atrial c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0954-6928
quantitative real-time PCR. PGC-1a and GLUT3 levels and the related protein mRNA expression were significantly reduced in POAF patients compared with controls (P < 0.05). This selective reduction in PGC-1a was associated with the presence of diabetes mellitus (P < 0.05). Conclusion Patients who have low PGC-1a and GLUT3 levels are at increased risk for new-onset POAF. The myofibrillar energetic impairment may be important in the pathogenesis of atrial fibrillation. Coron Artery Dis c 2014 Wolters Kluwer Health | Lippincott 25:224–229 Williams & Wilkins. Coronary Artery Disease 2014, 25:224–229 Keywords: coronary artery bypass graft, energy metabolism, postoperative atrial fibrillation a Peking Union Medical College, Chinese Academy of Medical Science, bFuwai Hospital, Peking Union Medical College, Chinese Academy of Medical Science, Beijing, cTEDA International Cardiovascular Hospital, Tianjin, China, dCardiac Signaling Center of USC, MUSC & Clemson University, Charleston, South Carolina and eMetrowest Medical Center, Framingham, Massachusetts, USA
Correspondence to Xiao-Cheng Liu, MD, TEDA International Cardiovascular Hospital, No. 61, 3rd Ave, TEDA, Tianjin 300457, China Tel: + 86 22 65208030; fax: + 86 22 65208001; e-mail: [email protected] *Dian-Min Sun, Xin Yuan, and Hua Wei are co-first authors. Received 26 September 2013 Revised 5 December 2013 Accepted 12 December 2013
contractile dysfunction [11]. Glycolytic enzymes undergo compensatory upregulation during persistent atrial fibrillation (AF) [9], and discordant metabolic alterations are evident in individuals susceptible to POAF [12]. Numerous studies demonstrate that when the heart is subjected to ischemia, dramatically enhanced fatty acid (FA) oxidation and markedly suppressed glucose oxidation contribute to the cardiac dysfunction and reperfusion injury. However, the mechanism of regulation of mitochondrial oxidative metabolism and the capacity of glucose uptake in human myocardium during ischemia/ reperfusion are unknown. Recent studies have demonstrated that peroxisome proliferator-activated receptor g coactivator-1a (PGC-1a) serves as a master regulator of mitochondrial oxidative metabolism that coordinates the capacity of each step required for ATP synthesis [13]. DOI: 10.1097/MCA.0000000000000081
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Impaired myocardium energetics Sun et al. 225
Activation of the PGC-1a regulatory cascade increases cardiac mitochondrial oxidative capacity in the heart. Glucose transporter 3 (GLUT3) was the third glucose transporter to be discovered, first reported in human myocardium in 1999 [14], and is preferentially expressed in cells with high-energy demand but in lower proportion as compared with GLUT4 and GLUT1. Little is known about the regulation of heart GLUT3. This carrier is the most efficient among the different GLUTs, as it has the lowest Km [15]. In the present study, we tested the hypothesis that both the maladaptive energy metabolism and diminished capacity of glucose uptake are associated with the risk for new-onset atrial fibrillation after isolated coronary artery bypass graft (CABG) surgery.
Methods Patients
All patients were diagnosed as coronary artery disease requiring CABG and as angiographically defined multivessel coronary stenosis, with at least 70% stenosis. Atrial appendages were obtained as surgical specimens from patients undergoing cardiac surgery using the procedures approved by the Institutional Review Board of the Fu Wai Hospital. The study was approved by the local ethics committees and all patients gave informed consent. Right atrial appendages were obtained from 79 patients who were undergoing routine cardiac bypass graft surgery (mean age, 59.88±8.62 years). Tissue was harvested during cannulation of the right atrium for bypass, and Clinical and demographic characteristics of POAF patients, SR patients, and controls
Table 1
Male sex (%) Age (years) BMI Hypertension [n (%)] Diabetes mellitus [n (%)] Hyperlipidemia [n (%)] Total cholesterol (mmol/l) Previous MI [n (%)] Left atrial diameter NYHA class III or IV (%) LVEF at enrollment (%) ACEI/ARB [n (%)] b-Blockers [n (%)] Statins [n (%)] CCB [n (%)] CPB (min) ACC (min) Number of grafts ICU (h)
SR (n = 57)
POAF (n = 22)
P value
73 58.64±8.97 26.08±2.79 59.1 41.3 40.9 4.4 17 (29.8) 50 8.6 60.37 25 (43.9) 55 (96.5) 23 (40.4) 32 (56.1) 61±18 30±11 3±1 53
76 63.04±6.46 26.33±3.49 61.4 47.6 43.8 4.2 5 (22.7) 49 14.2 58.27 9 (40.9) 21 (95.5) 8 (36.4) 16 (72.7) 62±25 30±8 3±1 59
0.738 0.038 0.113 0.853 0.815 0.813 0.487 0.531 0.736 0.995 0.749 0.813 0.830 0.805 0.179 0.72 0.99 0.93 0.327
Postoperative AF was observed in 22 patients (27.8%) with a peak incidence of AF occurring on the second postoperative day. In-hospital postoperative treatment with b-blockers was initiated in a similar percentage of patients from each group (i.e. in 96.5% of SR patients and in 95.5% of patients who developed AF, P = 0.83). ACC, aortic clamp time; ACEI, angiotensin-converting enzyme inhibitor; AF, atrial fibrillation; ARB, angiotensin II receptor blocker; CCB, calcium-channel blocker; CPB, cardiopulmonary bypass time; LVEF, left ventricular ejection fraction; MI, myocardial infarction; NYHA, New York Heart Association; POAF, postoperative atrial fibrillation; SR, sinus rhythm.
specimens were immediately snap-frozen and stored in liquid nitrogen. Table 1 describes detailed clinical characteristics. Protein quantification with ELISA
Expression of PGC-1a and GLUT3 by myofibrils in each surgical frozen sample was respectively assayed using the Human PGC-1a enzyme-linked immunosorbent assay (ELISA) Kit (Uscn Life, Double Lake, Missouri City, Texas, USA) and the Human GLUT3 ELISA Kit (Uscn Life) according to the protocol of the manufacturer. Collect the samples and bring all reagents and samples to room temperature before use. Centrifuge the sample again after thawing before the assay. Refer to the Assay Layout Sheet to determine the number of wells to be used and put any remaining wells and the desiccant back into the pouch and seal the Ziploc; store the unused wells at 41C. Add 100 ml of standard and sample per well. Cover with the adhesive strip provided. Incubate for 2 h at 371C. A plate layout is provided to record the standards and samples assayed. Remove the liquid from each well and add 100 ml of Biotin antibody (1 ) to each well. Cover with a new adhesive strip. Incubate for 1 h at 371C. Aspirate each well and wash, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (200 ml) using a squirt bottle, multichannel pipette, manifold dispenser, or autowasher, and let it stand for 2 min; complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. Add 100 ml of HRP-avidin (1 ) to each well. Cover the microtiter plate with a new adhesive strip. Incubate for 1 h at 371C. Repeat the wash process five times and add 90 ml of TMB substrate to each well. Incubate for 15–30 min at 371C. Add 50 ml of Stop Solution to each well and gently tap the plate to ensure thorough mixing. Determine the optical density of each well within 5 min, using a microplate reader set at 450 nm. If wavelength correction is available, set at 540 nm. Subtract the readings at 540 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. At the end, the protein values were normalized to the total protein content in each surgical atrial fragment. The standard points and samples were determined in duplicate. Quantitative real-time PCR
The mRNA was isolated from the samples using the mRNA Isolation Kit (Sigma Aldrich, St Louis, Missouri, USA) and then reverse transcribed using M-MLV reverse transcriptase and oligo(dT)18 primer. Real-time PCR was performed using SYBR Green I (Perkin Elmer Life Sciences, Boston, Massachusetts, USA) in an ABI Prism 7300 thermocycler (Applied Biosystems, Foster City, California, USA). The thermal profile for PCR was 951C for 10 min, followed by 40 cycles at 951C for 15 s and at
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226 Coronary Artery Disease 2014, Vol 25 No 3
Continuous variables are expressed as mean±SD. Protein levels are described by median (Q1–Q3). The differences between patients who developed AF and those who maintained sinus rhythm (SR) during the postoperative period were analyzed with analysis of variance and twosample t-test for continuous variables or the w2-test for categorical variables. A P value less than 0.05 was considered significant. Statistical comparisons of protein between the groups were performed with Wilcoxon’s rank-sum test. The correlation analysis of protein levels with quantitative measurements was performed with the Spearman correlation coefficient (rs). Binary regression analysis was used to determine the correlations between the results. Statistical significance was designated as a probability of less than 0.05. For all analyses, commercially available statistical package software was used (SPSS, version 13.0; SPSS Inc., Chicago, Illinois, USA).
Results Clinical characteristics of the POAF patients and controls are presented in Table 1. POAF was observed in 22 individuals (27.8%) with a peak incidence of AF occurring on the second postoperative day. No significant differences between the groups were found with respect to treatment, cardiovascular risk factors and comorbidity, duration of CPB or aortic cross-clamp, and number of grafted vessels (Table 1). However, patients who developed POAF were apparently older and they also had lower PGC-1a and GLUT3 levels compared with those who Fig. 1
GLUT3 (mg/μl)
Statistical analysis
Fig. 2
∗
14 000.000 12 000.000 10 000.000 8000.000 6000.000 4000.000 2000.000 .000
POAF
SR
GLUT3 protein levels in patients with POAF and SR. The samples of the patients with DM and with POAF and SR were collected and centrifuged after thawing before the assay. The protein expression of GLUT3 was measured with Human GLUT3 ELISA Kit (Uscn Life). Data are presented as mean±SD in triplicate from three independent experiments. *P < 0.05 vs. the control. DM, diabetes mellitus; GLUT3, glucose transporter 3; POAF, postoperative atrial fibrillation; SR, sinus rhythm.
Fig. 3
∗
30 mRNA PGC-1α/β-actin
601C for 1 min. To obtain a relative quantitation, the results were normalized to those obtained for the corresponding actin mRNAs. Each sample was run in duplicate.
25 20 15 10 5 0
POAF
SR
PGC-1a mRNA expression in patients with POAF and SR. The samples of the patients with POAF and SR were collected and centrifuged after thawing before the assay. The mRNAs were isolated using the mRNA Isolation Kit (Sigma Aldrich) and then reverse transcription and real-time PCR were performed. Data are presented as mean±SD in triplicate from three independent experiments. *P < 0.05 vs. the control. PGC1a, peroxisome proliferator-activated receptor g coactivator-1a; POAF, postoperative atrial fibrillation; SR, sinus rhythm.
∗ 3.5
Fig. 4
2.5 2 1.5 1 0.5 0
POAF
SR
PGC-1a protein levels in patients with POAF and SR. The samples from the patients with POAF and SR were collected and centrifuged after thawing before the assay. The protein expression of PGC-1a was measured with Human PGC-1a ELISA Kit (Uscn Life). Data are presented as mean±SD in triplicate from three independent experiments. *P < 0.05 vs. the control. PGC-1a, peroxisome proliferator-activated receptor g coactivator-1a; POAF, postoperative atrial fibrillation; SR, sinus rhythm.
mRNA GLUT3/β-actin
PGC-1α (mg/μl)
3 3
∗
2
1
0
POAF
SR
GLUT3 mRNA expression in patients with POAF and SR. The samples of the patients with POAF and SR were collected and centrifuged after thawing before the assay. The mRNAs were isolated using the mRNA Isolation Kit (Sigma Aldrich) and then reverse transcription and real-time PCR were performed. Data are presented as mean±SD in triplicate from three independent experiments. *P < 0.05 vs. the control. GLUT3, glucose transporter 3; POAF, postoperative atrial fibrillation; SR, sinus rhythm.
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Impaired myocardium energetics Sun et al.
Table 2
Multivariable predictors of postoperative atrial fibrillation
Age (years) BMI Diabetes (no/yes) Total cholesterol PGC-1a
OR
95% CI
P value
1.077 1.783 0.648 0.318 0.407
0.996–1.164 0.941–1.385 0.188–1.971 0.516–1.386 0.191–0.850
0.064 0.182 0.421 0.573 0.017
Binary logistic regression identified PGC-1a (P = 0.017) as an independent predictor for the development of postoperative AF among the 79 patients. AF, atrial fibrillation; CI, confidence interval; OR, odds ratio; PGC-1a, peroxisome proliferator-activated receptor g coactivator-1a.
remained in SR (Figs 1 and 2). The comparison of mRNA expression was similar to protein expression between the two groups (Figs 3 and 4). Atrial PGC-1a levels were also lower in patients with diabetes mellitus (DM) (n = 34) compared with those without DM (n = 45) (P = 0.015). Specifically, the PGC-1a-related mRNA expression was obviously lower in the DM group. The GLUT3 level was not significantly different between the DM group and the non-DM group; the same was true for GLUT3-related mRNA expression. In-hospital postoperative treatment with b-blockers was initiated in a similar percentage of patients from each group (i.e. in 96.5% of SR patients and in 95.5% of patients who developed AF, P = 0.83) (Table 1). There was no difference in the postoperative use of angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers or in the time of initiation of treatment after surgery (Table 1). Binary logistic regression identified PGC-1a (P = 0.017) as an independent predictor for the development of POAF among the 79 patients. Age was no longer associated with an increased risk for POAF after the addition of PGC-1a to the model. Other predictors for POAF that were included in the regression model but did not reach a level of statistical significance were age, history of DM, BMI, and total cholesterol (Table 2), according to the Hosmer and Lemeshow Goodness-of-Fit test for the final model (P = 0.736, d.f. = 8). Characterization of all patients according to the quartiles of atrial PGC-1a confirmed a powerful association between these measurements and the occurrence of POAF (P < 0.001), whereas the correlation between POAF and DM is uncertain, although the PGC-1a levels are obviously lower in patients with POAF and DM compared with those without DM.
Discussion Main findings
Recent studies confirmed that AF denotes a hypermetabolic state [9,11,12,16], but how the metabolism influences the persistence or onset of AF is unclear. This study on a cohort of patients undergoing isolated CABG surgery used the emerging techniques, such as ELISA and quantitative real-time PCR, to provide novel insights into the regulation of myocardial energy metabolism. This was a prospective study to analyze the role of PGC-1a
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and GLUT3 in myocardium tissue, and demonstrates that, inter alia, a discordant regulation of energy metabolites and the glycometabolic disorder precede the new-onset POAF after CABG surgery. We also found that the reduction in PGC-1a level and the level of transcript expression was associated with the presence of DM; however, the underlying mechanism is unclear. PGC-1a and energy metabolism in myocardium
PGC-1a, initially identified as a PPARg coactivator, has been proven to serve coactivator functions for both nuclear receptors and other classes of transcription factors. Recently, PGC-1a has been demonstrated as the master regulator of mitochondrial oxidative metabolism [13]. Despite a relative abundance of information on the effects of PGC-1a on the isolated myocardium or rodent model systems, the regulation and physiological roles of PGC-1a in human heart, to our knowledge, are unknown. In cardiac myocytes in culture, PGC-1a increases mitochondrial number, upregulates the expression of mitochondrial enzymes, and increases the rates of FA oxidation and coupled respiration [17,18]. PGC-1a transcriptionally regulates the genes involved in the cellular uptake and mitochondrial oxidation of FAs through direct coactivation of PPARs and estrogenrelated receptors [13,19]. Those genes encoding enzymes are involved at multiple steps of these metabolic pathways, such as uptake, esterification, mitochondrial transport, and oxidation [20]. FA and glucose oxidation are the main ATP-generating pathways in human heart. AcetylCoA derived from FA and glucose oxidation is further oxidized in the TCA cycle to generate NADH and FADH2, which enter the electron transport/oxidative phosphorylation pathway and drive ATP synthesis. The glucose uptake/oxidation and electron transport/oxidative phosphorylation pathways are also regulated by PGC-1a through other transcription factors, including MEF2 [21], NRF-1 [22], and cytochrome c [23]. Our study, which applied the ELISA technique directly to human cardiac tissue, first described significant changes in PGC1a levels in fibrillating atrial tissue and then suggested a molecular mechanism that seems to contribute to POAF. GLUT3 and myocardial glucose uptake in reperfusion heart
Glucose and lactate are the major fuels for a healthy heart. The capacity of the cell to absorb glucose relies on the fraction of the glucose transporters that reside in the plasma membrane. Three isoforms from the glucose transporter family have been identified in the myocardium: GLUT1, GLUT4, and GLUT3 [24]. Although GLUT4 is predominant in the heart, GLUT3 has both a higher affinity for glucose and at least a five-fold greater transport capacity than GLUT1 and GLUT4 [25]. Thus, we selected GLUT3 as the biomarker of glucose uptake in this study instead of GLUTs in the myocardium. The consequence of the high rates of FA oxidation during
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228 Coronary Artery Disease 2014, Vol 25 No 3
reperfusion is a decrease in glucose oxidation that in turn contributes to a decrease in pH during ischemia, and a delayed recovery of pH following ischemia [26]. As the intracelluar H + level rises, the H + /Na + exchange and Na + /Ca2 + exchange activities are increased, resulting in Ca2 + overload, electrical instability, mechanical dysfunction, and mitochondrial dysfunction [27]. In our study, we first found that the GLUT3 level and protein-related mRNA expression were both lower in patients of the POAF group compared with those who remained in SR, with the difference being statistically significant. This suggested that the impaired capacity of glucose uptake can abate the efficiency of glucose oxidation in the reperfusion heart and can induce the electrical instability to a certain extent.
that a discordant regulation of energy metabolites precedes the onset of arrhythmia after surgery. They also found that the lower the ratio of glycolytic end products to the end products of lipid metabolism in human atrial tissue, the earlier the onset of arrhythmia in CABG surgery patients. In our study, we found that the reduction in PGC-1a level was significantly associated with new-onset POAF and DM, and the comparison of mRNA expression was similar to protein expression. We know that DM is a highly prevalent risk factor in the cardiac surgical population. Thus, energy metabolic profiles may help to further classify those persons who are at high risk of developing POAF in the elderly patients undergoing CABG surgery. Limitations
PGC-1a and diabetes mellitus in the heart
PGC-1a regulates PPARa and other transcription factors in the heart to couple the metabolic needs to the expression of genes involved in the control of energy metabolism. The expression and activity of PGC-1a and its coactivator PPARs can be dynamically regulated in metabolic diseases. In diabetes, the heart is exposed to a hyperglycemic and hyperlipidemic environment. The elevation of plasma nonesterified FA levels in diabetes results in the activation of PPARa [28,29]. Young et al. [30] showed that progression of diabetes is associated with a marked decrease in the expression of PPARa within the rat heart. Failure to adequately control the intracellular glucose levels has also been implicated in the development of insulin resistance and in the generation of reactive oxygen species in various tissues [31,32]. Glucose may downregulate the expression of FAmetabolizing genes through PPARa repression, leading to accelerated lipid deposition within the cardiomyocyte and resulting in cardiac dysfunction. Roduit et al. [33] have shown that, in the islet cells, glucose exposure decreases the expression of PPARa and several PPARaregulated genes involved in FA metabolism. Recently, a series of studies on humans have demonstrated an inverse correlation of the muscle PGC-1a levels and mitochondrial activity with insulin resistance and diabetes [34–36]. Metabolic alterations and postoperative atrial fibrillation
Recent demonstration in an animal model that glycolytic inhibition predisposes to AF also confirmed the assumption that metabolic alterations might facilitate the onset of arrhythmia [20]. Some other studies also revealed the relationship between energy metabolism and POAF in patients. Kim et al. [37] found that the atrial NADPH oxidase activity is independently associated with an increased risk for POAF, suggesting that this oxidase system may be a key mediator of atrial oxidative stress leading to the development of AF after cardiac surgery. In the study by Mayr et al. [12], they found the metabolic adaptation processes in persistent AF and demonstrated
Some potential limitations may have influenced our results. All patients included in our study underwent cardiac surgery because of coronary artery disease. Thus, no comment can be made about the possible impact of energy metabolism changes in other patient populations. Only right atrial samples were examined; therefore, our present findings may not be representative of other parts of the atria. Although our present study provides new evidence of mechanistic insight regarding energy metabolism impairment for a relatively small sample capacity, we did not find significant association between POAF and metabolic syndrome, which is in disagreement with other studies [38–40].
Acknowledgements This study was supported by the Chinese National Natural Science Foundation (81270302). The authors thank Professor Guowei He from NanKai University for helpful comments. They also thank Dr Zahner from Peking Union Medical College for her genuine suggestions. Finally, all authors thank Dr Tang Yue, Dr Wei Wang, Dr Li-Qing Wang, and Dr Han-Song Sun from Fu Wai Hospital, Peking Union Medical College, and Chinese Academy of Medical Science for helping us to obtain right atrial appendage tissues. Conflicts of interest
There are no conflicts of interest.
References 1
2 3
4
Aranki SF, Shaw DP, Adams DH, Rizzo RJ, Couper GS, VanderVliet M, et al. Predictors of atrial fibrillation after coronary artery surgery: current trends and impact on hospital resources. Circulation 1996; 94:390–397. Creswell LL, Schuessler RB, Rosenbloom M, Cox JL. Hazards of postoperative atrial arrhythmias. Ann Thorac Surg 1993; 56:539–549. Mathew JP, Parks R, Savino JS, Friedman AS, Koch C, Mangano DT, et al. Atrial fibrillation following coronary artery bypass graft surgery: predictors, outcomes, and resource utilization. MultiCenter Study of Perioperative Ischemia Research Group. JAMA 1996; 276:300–306. Taylor GJ, Malik SA, Colliver JA, Dove JT, Moses HW, Mikell FL, et al. Usefulness of atrial fibrillation as a predictor of stroke after isolated coronary artery bypass grafting. Am J Cardiol 1987; 60:905–907.
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5
6
7 8
9
10 11
12
13 14
15
16
17
18
19
20
21
22
Borzak S, Tisdale JE, Amin NB, Goldberg AD, Frank D, Padhi ID, et al. Atrial fibrillation after bypass surgery: does the arrhythmia or the characteristics of the patients prolong hospital stay? Chest 1998; 113:1489–1491. Kim MH, Deeb GM, Morady F, Bruckman D, Hallock LR, Smith KA, et al. Effect of postoperative atrial fibrillation on length of stay after cardiac surgery (the Postoperative Atrial Fibrillation in Cardiac Surgery Study [PACS(2)]). Am J Cardiol 2001; 87:881–885. Ommen SR, Odell JA, Stanton MS. Atrial arrhythmias after cardiothoracic surgery. N Engl J Med 1997; 336:1429–1434. Vega RB, Huss JM, Kelly DP. The coactivator PGC-1 cooperates with peroxisome proliferator-activated receptor a in transcriptional control of nuclear genes encoding mitochondrial fatty acid oxidation enzymes. Mol Cell Biol 2000; 20:1868–1876. Barth AS, Merk S, Arnoldi E, Zwermann L, Kloos P, Gebauer M, et al. Reprogramming of the human atrial transcriptome in permanent atrial fibrillation: expression of a ventricular-like genomic signature. Circ Res 2005; 96:1022–1029. Wagoner LE, Walsh RA. The cellular pathophysiology of progression to heart failure. Curr Opin Cardiol 1996; 11:237–244. Mihm MJ, Yu F, Carnes CA, Reiser PJ, McCarthy PM, Van Wagoner DR, et al. Impaired myofibrillar energetics and oxidative injury during human atrial fibrillation. Circulation 2001; 104:174. Mayr M, Yusuf S, Weir G, Chung YL, Mayr U, Yin X, et al. Combined metabolomic and proteomic analysis of human atrial fibrillation. J Am Coll Cardiol 2008; 51:585–594. Huss JM, Kelly DP. Mitochondrial energy metabolism in heart failure: a question of balance. J Clin Invest 2005; 115:547–555. Grover-McCay M, Walsh SA, Thompson SA. Glucose transporter 3 (GLUT3) protein is present in human myocardium. Biochim Biophys Acta 1999; 1416:145–154. Colville CA, Seatter MJ, Jess TJ, Gould GW, Thomas HM. Kinetic analysis of the liver-type (GLUT2) and brain-type (GLUT3) glucose transporters in Xenopus oocytes: substrate specificities and effects of transport inhibitors. Biochem J 1993; 290:701–706. Thijssen VL, Ausma J, Borgers M. Structural remodelling during chronic atrial fibrillation: act of programmed cell survival. Cardiovasc Res 2001; 52:14–24. Lehman JJ, Barger PM, Kovacs A, Saffitz JE, Medeiros DM, Kelly DP. Peroxisome proliferator-activated receptor gamma coactivator-1 promotes cardiac mitochondrial biogenesis. J Clin Invest 2000; 106:847–856. Huss JM, Pine´da Torra I, Staels B, Gigue`re V, Kelly DP. ERRa directs PPAR a signaling in the transcriptional control of energy metabolism in cardiac and skeletal muscle. Mol Cell Biol 2004; 24:9079–9091. Schreiber SN, Knutti D, Brogli K, Uhlmann T, Kralli A. The transcriptional coactivator PGC-1 regulates the expression and activity of the orphan nuclear receptor estrogen-related receptor a (ERRa). J Biol Chem 2003; 278:9013–9018. Ono N, Hayashi H, Kawase A, Lin SF, Li H, Weiss JN, et al. Spontaneous atrial fibrillation initiated by triggered activity near the pulmonary veins in aged rats subjected to glycolytic inhibition. Am J Physiol Heart Circ Physiol 2007; 292:639–648. Michael LF, Wu Z, Cheatham RB, Puigserver P, Adelmant G, Lehman JJ, et al. Restoration of insulin-sensitive glucose transporter (GLUT4) gene expression in muscle cells by the transcriptional coactivator PGC-1. Proc Natl Acad Sci USA 2001; 98:3820–3825. Wu Z, Puigserver P, Andersson U, Zhang C, Adelmant G, Mootha V, et al. Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell 1999; 98:115–124.
23
24
25
26
27
28
29
30
31
32
33
34 35
36
37
38
39
40
Short KR, Vittone JL, Bigelow ML, Proctor DN, Rizza RA, Coenen-Schimke JM, et al. Impact of aerobic exercise training on age-related changes in insulin sensitivity and muscle oxidative capacity. Diabetes 2003; 52: 1888–1896. Gavete ML, Agote M, Martin MA, Alvarez C, Escriva F. Effects of chronic undernutrition on glucose uptake and glucose transporter proteins in rat heart. Endocrinology 2002; 143:4295–4303. Simpson IA, Dwyer D, Malide D, Moley KH, Travis A, Vannucci SJ. The facilitative glucose transporter GLUT3: 20 years of distinction. Am J Physiol Endocrinol Metab 2008; 295:E242–E253. Liu Q, Docherty JC, Rendell JC, Clanachan AS, Lopaschuk GD. High levels of fatty acids delay the recovery of intracellular pH and cardiac efficiency in post-ischemic hearts by inhibiting glucose oxidation. J Am Coll Cardiol 2002; 39:718–725. Karmazyn M. The role of the myocardial sodium–hydrogen exchanger in mediating ischemic and reperfusion injury. From amiloride to cariporide. Ann N Y Acad Sci 1999; 874:326–334. Gulick T, Cresci S, Caira T, Moore DD, Kelly DP. The peroxisome proliferatoractivated receptor regulates mitochondrial fatty acid oxidative enzyme gene expression. Proc Natl Acad Sci USA 1994; 91:11012–11016. Brandt JM, Djouadi F, Kelly DP. Fatty acids activate transcription of the muscle carnitine palmitoyltransferase I gene in cardiac myocytes via the peroxisome proliferator-activated receptor alpha. J Biol Chem 1998; 273:23786–23792. Young ME, Patil S, Ying J, Depre C, Ahuja HS, Shipley GL, et al. Uncoupling protein 3 transcription is regulated by peroxisome proliferator-activated receptor (alpha) in the adult rodent heart. FASEB J 2001; 15:833–845. Rossetti L, Smith D, Shulman GI, Papachristou D, DeFronzo RA. Correction of hyperglycemia with phlorizin normalizes tissue sensitivity to insulin in diabetic rats. J Clin Invest 1987; 79:1510–1515. Rosen P, Du X, Tschope D. Role of oxygen derived radicals for vascular dysfunction in the diabetic heart: prevention by alpha-tocopherol? Mol Cell Biochem 1998; 188:103–111. Roduit R, Morin J, Masse´ F, Segall L, Roche E, Newgard CB, et al. Glucose down-regulates the expression of the peroxisome proliferator-activated receptor-alpha gene in the pancreatic beta-cell. J Biol Chem 2000; 275:35799–35806. Attie AD, Kendziorski CM. PGC-1a at the crossroads of type 2 diabetes. Nat Genet 2003; 34:244–245. Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar J, et al. PGC-1a-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet 2003; 34:267–273. Patti ME, Butte AJ, Crunkhorn S, Cusi K, Berria R, Kashyap S, et al. Coordinated reduction of genes of oxidative metabolism in humans with insulin resistance and diabetes: potential role of PGC1 and NRF1. Proc Natl Acad Sci USA 2003; 100:8466–8471. Kim YM, Kattach H, Ratnatunga C, Pillai R, Channon KM, Casadei B. Association of atrial nicotinamide adenine dinucleotide phosphate oxidase activity with the development of atrial fibrillation after cardiac surgery. J Am Coll Cardiol 2008; 51:68–74. Wang TJ, Parise H, Levy D, D’Agostino RB Sr, Wolf PA, Vasan RS, Benjamin EJ. Obesity and the risk of new-onset atrial fibrillation. JAMA 2004; 292:2471–2477. Dublin S, French B, Glazer NL, Wiggins KL, Lumley T, Psaty BM, et al. Risk of new-onset atrial fibrillation in relation to body mass index. Arch Intern Med 2006; 166:2322–2328. Zacharias A, Schwann TA. Obesity and risk of new-onset atrial fibrillation after cardiac surgery. Circulation 2005; 112:3247–3255.
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