Earlier studies, mainly in rodents, have shown that diabetic

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1 Downregulation of Myocardial Myocyte Enhancer Factor 2C and Myocyte Enhancer Factor 2C Regulated Gene Expression in Diabetic Patients With Nonischemic Heart Failure Peter Razeghi, MD; Martin E. Young, DPhil; Tonya C. Cockrill, MS; O. Howard Frazier, MD; Heinrich Taegtmeyer, MD, DPhil Background In animal studies, diabetes has been shown to induce changes in gene expression of key regulators in cardiac energy metabolism and calcium homeostasis. In the present study, we tested the hypothesis that metabolic gene expression in nonischemic failing hearts of diabetic patients differs from that in nonischemic failing hearts of nondiabetic patients. Methods and Results Left ventricular tissue was obtained from nonfailing hearts (n 6) and from nonischemic failing hearts of patients with or without type 2 diabetes. Myocardial transcript levels of key regulators in energy substrate metabolism (glucose transporter 1, glucose transporter 4, pyruvate dehydrogenase kinase 4, peroxisome proliferator activated receptor, muscle carnitine palmitoyl transferase-1, medium-chain acyl-coa dehydrogenase, and uncoupling protein 3), calcium homeostasis (sarcoplasmic reticulum Ca 2 -ATPase [SERCA2a], phospholamban, and cardiac ryanodine receptor), and contractile function (myosin heavy chain ) were measured using real-time quantitative reverse transcription polymerase chain reaction. In addition, we measured myocyte enhancer factor 2C (MEF2C) and SERCA2a protein levels. Only MEF2C regulated transcripts (glucose transporter 4, SERCA2a, and myosin heavy chain ) were lower in the diabetic group compared with the nondiabetic group. MEF2C protein content was also decreased. Conclusion MEF2C and MEF2C-regulated genes are decreased in the failing hearts of diabetic patients. This transcriptional mechanism may contribute to the contractile dysfunction in heart failure patients with diabetes. (Circulation. 2002;106: ) Key Words: diabetes mellitus cardiomyopathy metabolism polymerase chain reaction Earlier studies, mainly in rodents, have shown that diabetic cardiomyopathy is associated with impairment in numerous cell functions, including energy substrate metabolism, calcium homeostasis, and cell signaling. 1 At the same time, we have observed profound changes in the gene expression profile of hearts from insulin-deficient rats. 2 These observations have led us to propose that the diabetic human myocardium may exhibit abnormalities in metabolic gene expression. Methods Patients Clinical data were collected retrospectively. Nonfailing heart tissue (left ventricular apex) was obtained (up to 12 hours after death) from 6 male donors who died in motor vehicle accidents (age 35 8 years, no significant medical history). Left ventricular tissue (apex) of the heart failure patients was obtained during the implantation of a left ventricular assist device. All tissue samples were immediately frozen in liquid nitrogen. Informed consent was obtained from each patient or the next of kin, and the protocol was approved by the respective Institutional Review Boards. Gene Expression RNA was extracted by standard methods 3 and analyzed by reverse transcription followed by real-time quantitative polymerase chain reaction for the transcripts of interest. The methodology of quantitative polymerase chain reaction has been previously described in detail. 4 The nucleotide sequences for probes as well as forward and reverse primers have been previously published 5 and are shown in the Table. The transcript for the constitutive gene product -actin was used as a reporter gene for data normalization. Internal RNA standards were prepared using the T7 RNA polymerase method (Ambion). Because nonfailing human heart tissue was obtained up to 12 hours after death, we measured transcript levels of glucose transporter 4 (GLUT4) immediately after death and 12 hours thereafter in rat heart. There was no significant difference GLUT4 expression between the 2 time points ( versus GLUT4 mrna/ -actin mrna). Received April 2, 2002; revision received May 28, 2002; accepted June 5, From the Division of Cardiology, University of Texas Houston Medical School (P.R., M.E.Y., T.C.C., H.T.), and St Luke s Episcopal Hospital and Texas Heart Institute (O.H.F., H.T.), Houston, Tex. Guest editor for this article was Gerd Hasenfuss, MD, Georg-August-Universitat Gottingen, Gottingen, Germany. Correspondence to Heinrich Taegtmeyer, MD, DPhil, Department of Internal Medicine, Division of Cardiology, University of Texas Houston Medical School, 6431 Fannin, MSB 1.246, Houston, TX Heinrich.Taegtmeyer@uth.tmc.edu 2002 American Heart Association, Inc. Circulation is available at DOI: /01.CIR DC 407

2 408 Circulation July 23, 2002 Transcript Assays Gene Primer/Probe Sequence Genbank Accession # ctri Forward (72 90) 5 -CAACTACCGCGCTTATGCC-3 NM Reverse ( ) 5 -CAGCTCTTGCTTTGCAATCTG-3 Probe ( ) 5 -FAM-AGCAGAGTCTTCAGCTGCAATTTTCTCGA-TAMRA-3 PPAR Forward ( ) 5 -TCCCTTTTTGTGGCTGCTATC-3 NM Reverse ( ) 5 -GTTGCTCTGCAGGTGGAGTC-3 Probe ( ) 5 -FAM-CTGTGGAGATCGTCCTGGCCTTCTAAAC-TAMRA-3 SERCA2a Forward ( ) 5 -CATCTTCCAGATCACACCGC Reverse ( ) 5 -CCAGTATTGCAGGTTCCAGG-3 Probe ( ) 5 -FAM-TCCTTGCCCGTGATTCTCATGGATG-TAMRA-3 Plb Forward ( ) 5 -CCCACGTAAACACCCGTAAGA-3 AH Reverse ( ) 5 -AAAGCTGGCAGCCAAATATGA-3 Probe ( ) 5 -FAM-TGATCACAGCTGCCAAGGCTACCTAAAAGA-TAMRA-3 RyR2 Forward ( ) 5 -GACTTGGACAGAGTTCGCACA-3 NM_ Reverse ( ) 5 -CCCGCACATACCATATAGCAG-3 Probe ( ) 5 -FAM-TGCATGAAAGCATCAAACGCAGCA-TAMRA-3 MEF2A Forward ( ) 5 -GAAACCAGATCTTCGAGTTGTCATC-3 U49020 Reverse ( ) 5 -TGGCTTGAGAACTACTGATCCTTTG-3 Probe ( ) 5 -FAM-CCACTATCGGAGGAAGAGGAATTGGAGTTGA-TAMRA-3 -actin Forward ( ) 5 -CCCTGGCACCCAGCAC-3 BC Reverse ( ) 5 -GCCGATCCACACGGAGTAC-3 Probe ( ) 5 -FAM-ATCAAGATCATTGCTCCTCCTGAGCGC-TAMRA-3 ctri indicates cardiac troponin I; PPAR, peroxisome proliferator-activated receptor ; SERCA2a, sarcoplasmic reticulum Ca 2 -ATPase; Plb, phospholamban; RyR2, cardiac ryanodine receptor; and MEF2A, myocyte enhancer factor 2A. Protein Expression Proteins from 8 nonischemic failing hearts (4 nondiabetic and 4 diabetic patients) were isolated as previously described. 6 Briefly, samples were homogenized in extraction buffer and centrifuged (30 minutes at g) and the supernatant was isolated. Proteins were fractionated by 6% PAGE and transferred to a nitrocellulose membrane. Antibodies were purchased from Santa Cruz Biotechnology (sc-313, sc-2030) and Affinity Bioreagents (MA3-919). The primary antibody against myocyte enhancer factor 2 (MEF2) binds to MEF2A and MEF2C. The different isoforms were identified by their different molecular weight of 70 and 60 kda, respectively. Statistical Analysis All data are presented as mean SEM. Differences between the groups for normally distributed gene expression were calculated by 1-way ANOVA followed by the Bonferroni test. Nonnormally distributed data were analyzed by using the Kruskal-Wallis test followed by the Bonferroni test. A P value 0.05 was considered as significant. Results Clinical Data of Heart Failure Patients Clinical data of 8 (male/female: 4/4) nonischemic heart failure patients without diabetes and 7 (male/female: 6/1) nonischemic heart failure patients with type 2 diabetes of more than 5 years duration were retrospectively analyzed. There were no significant differences in age ( versus years), left ventricular diastolic diameter ( versus cm), or cardiac index ( versus L/min per m 2 ) between nondiabetic and diabetic heart failure patients. Left ventricular ejection fraction was less than 20% in all patients. Fasting serum glucose levels and body mass index did not differ between the 2 groups (109 4 versus versus mg/dl, P 0.10, and 25 2 versus 26 1 kg/m 2, P 0.61, respectively). All patients received diuretics, angiotensin-converting enzyme inhibitors, lowdose -blockers, and positive inotropic agents. Five diabetic patients were treated with sulfonylureas, metformin, and insulin, and 2 only with insulin. Gene Expression Data Myocyte-Specific Marker We assayed for transcript levels of the myocyte-specific marker cardiac troponin I to exclude the possibility of differences in gene expression between the 3 groups being caused by differences in the myocyte/nonmyocyte ratio. There was no significant difference in troponin I transcript levels between the groups (nonfailing: ; failing: ; failing diabetic: troponin 1 mrna/ -actin mrna). Regulators of Energy Substrate Metabolism Transcript level of GLUT1 did not significantly differ between the groups (Figure 1a). In contrast, gene expression of GLUT4 was significantly decreased in failing hearts of diabetic patients when compared with nonfailing and failing hearts of nondiabetics (Figure 1b). Transcript levels of pyruvate dehydrogenase kinase 4 were increased in failing

3 Razeghi et al Myocyte Enhancer Factor 2C and Heart Failure 409 Figure 1. Transcript levels of genes regulating energy metabolism, calcium homeostasis, and contractile function. GLUT4, SERCA2a, and MHC expression are significantly decreased in nonischemic heart failure patients with diabetes. NF indicates nonfailing; HF, nonischemic heart failure patients; and D, diabetics. *P 0.05 vs nonfailing; P 0.05 vs failing. hearts of diabetics when compared with nonfailing hearts (Figure 1c). Gene expressions of the transcription factor peroxisome proliferator activated receptor (PPAR ) and 3 PPAR regulated genes (muscle carnitine palmitoyl transferase-1, medium-chain acyl-coa dehydrogenase, and uncoupling protein 3) are shown in Figure 1d through 1g. PPAR, mediumchain acyl-coa dehydrogenase, and uncoupling protein 3 expressions were decreased in both failing groups when compared with the nonfailing group, but did not differ between nondiabetic and diabetic heart failure patients. Regulators of Calcium Metabolism Figure 1h through 1j shows transcript levels of genes regulating calcium homeostasis. Gene expression of sarcoplasmic reticulum Ca 2 -ATPase (SERCA2a) was significantly depressed in the diabetes group with failing hearts when compared with the nonfailing and failing groups of nondiabetic patients (Figure 1h). Marker of Contractile Function Transcript levels of myosin heavy chain (MHC) were significantly decreased in both heart failure groups when compared with the nonfailing groups. Heart failure patients with diabetes showed an even greater decrease when compared with heart failure patients without diabetes (Figure 1k). MEF2 and SERCA2a Protein Expression Previous studies have shown that MEF2 is involved in the transcriptional regulation of GLUT4, SERCA2a, and MHC. 7 9

4 410 Circulation July 23, 2002 Figure 2. MEF2C protein content. a, 2 representative western blots of each group. b, a significant decrease in MEF2C protein content is seen between the 2 groups. HF indicates nonischemic heart failure patients; D, diabetes. *P 0.05; n 4 for each group. Therefore, we assayed protein content of MEF2A and MEF2C in 4 heart failure patients with diabetes and 4 without diabetes. MEF2C protein was significantly decreased in the heart failure group with diabetes (Figure 2). In contrast, neither MEF2A transcript nor protein levels were significantly different between the 2 groups ( versus MEF2A mrna/ -actin mrna, protein: versus arbitrary units). In addition, SERCA2a protein levels showed a decrease of marginal significance ( versus 36 9 arbitrary units, P 0.07). Discussion We have shown that transcript levels of GLUT4, SERCA2a, and MHC are depressed in nonischemic heart failure patients with diabetes when compared with nonischemic heart failure patients without diabetes and protein content of MEF2C, a transcription factor regulating GLUT4, SERCA2a, and MHC expression, is also decreased in the diabetic group. Disturbances of Myocardial Energy Metabolism in Diabetes During diabetes, myocardial glucose uptake and subsequent oxidation are impaired. The present study shows that nonischemic heart failure patients with diabetes have decreased myocardial GLUT4, but not GLUT1, transcript levels when compared with nonischemic heart failure patients without diabetes. This distinction between glucose transporter isoform expression may be explained by differences in their transcriptional regulation. A recent study has shown that the transcriptional coactivator proliferator-activated-receptor gamma coactivator-1 mediates increased GLUT4 expression, in large part, by binding to and coactivating MEF2C. 10 This is consistent with our findings of a concomitant decrease in MEF2C protein and GLUT4 gene expression. Genes regulating fatty acid oxidation show no difference in the failing human heart of diabetic and nondiabetic patients. PPAR regulates genes encoding for enzymes, which are involved in fatty acid uptake and oxidation, and has been suggested to be involved in the regulation of cardiac hypertrophy. 11 We have previously shown that myocardial PPAR expression is decreased in a rat model of cardiac hypertrophy and in a model of insulin deficient diabetes. 12 The present study shows that PPAR transcript levels and 2 PPAR regulated gene levels (medium-chain acyl-coa dehydrogenase and uncoupling protein 3) are decreased in both failing groups but do not significantly change in heart failure patients with diabetes when compared with heart failure patients without diabetes. One explanation for this finding is that end-stage heart failure may itself maximally depress PPAR expression. Thus, the downregulation of PPAR expression suggests a transcriptional mechanism for the decrease in PPAR -regulated genes in the failing human heart. Disturbances of Myocardial Calcium Homeostasis and Contractile Function in Diabetes Heart failure in diabetics is characterized by early diastolic dysfunction followed by late systolic dysfunction. 1 In the failing human heart, decreased levels of SERCA2a are associated with impaired cardiac function. 13 The present study shows that diabetes decreases levels of SERCA2a in patients with nonischemic heart failure. These results are consistent with previous studies showing impaired sarcoendoplasmic calcium uptake in the diabetic rat heart. 14 MHC expression is decreased in the failing human heart when compared with the nonfailing human heart. 5 We confirmed the downregulation of MHC in the failing human heart and showed that diabetes augments this depression. A recent report in rat cardiac myocytes showed that MHC isoform expression determines force-velocity relationships, velocity of loaded shortening, and overall power outputgenerating capacity. 15 Thus, together the decrease in SERCA2a and MHC gene expression observed in human failing hearts of diabetics may contribute to cardiac dysfunction. MEF2-Regulated Gene Expression Because MEF2C binds to the GLUT4, SERCA2a, and MHC promoter, we focused our analysis on this transcription factor. Although the present data do not prove that the decrease in MEF2C protein expression causes the downregulation of GLUT4, SERCA2a, and MHC transcript levels, they are consistent with previous animal studies showing a decrease in myocardial MEF2, GLUT4, SERCA2a, and MHC expression with diabetes. 2,7,16 Study Limitations We examined only myocardial tissue of nonischemic heart failure patients because ischemia is known to alter metabolic

5 Razeghi et al Myocyte Enhancer Factor 2C and Heart Failure 411 gene expression and therefore may mask diabetes-induced changes in transcripts of metabolic genes. This may explain why the present study, unlike previous reports, 5,13 could not find any significant decrease for the glucose transporters muscle carnitine palmitoyl transferase-1 and SERCA2a in the nonischemic failing human heart. We cannot exclude the possibility that increasing the number of patients may lead to statistically significant differences between the groups for genes with high variances (eg, phospholamban). Conclusions The downregulation of MEF2C and MEF2C-regulated genes (GLUT4, SERCA2a, and MHC ) in the failing hearts of patients with diabetes suggests a transcriptional mechanism that might contribute to the pathogenesis and contractile dysfunction of heart failure patients with diabetes. 17 Acknowledgment This study was supported in part by grants from the US Public Health Service (RO1-HL/AG and F32HL-67609). References 1. Fein F, Sonnenblick E. Diabetic cardiomyopathy. Cardiovasc Drugs Ther. 1994;8: Depre C, Young ME, Ying J, et al. Streptozotocin-induced changes in cardiac gene expression in the absence of severe contractile dysfunction. J Mol Cell Cardiol. 2000;32: Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162: Depre C, Shipley GL, Chen W, et al. Unloaded heart in vivo replicates fetal gene expression of cardiac hypertrophy. Nat Med. 1998;4: Razeghi P, Young M, Alcorn J, et al. Metabolic gene expression in fetal and failing human heart. Circulation. 2001;104: Gibala M, Young M, Taegtmeyer H. Anaplerosis of the citric acid cycle: role in energy metabolism of heart and skeletal muscle. Acta Physiol Scand. 2000;168: Thai M, Guruswamy S, Cao K, et al. Myocyte enhancer factor 2 (MEF2)- binding site is required for GLUT4 gene expression in transgenic mice. J Biol Chem. 1998;273: Moriscot A, Sayen M, Hartong R, et al. Transcription of the rat sarcoplasmic reticulum Ca2 adenosine triphosphatase gene is increased by 3,5,3 -triiodothyronine receptor isoform-specific interactions with the myocyte-specific enhancer factor-2a. Endocrinology. 1997;138: Lee Y, Nadal-Ginard B, Mahdavi V, et al. Myocyte-specific enhancer factor 2 and thyroid hormone receptor associate and synergistically activate the -cardiac myosin heavy-chain gene. Mol Cell Biol. 1997;17: Michael L, Wu Z, Cheatham R, et al. Restoration of insulin-sensitive glucose transporter (GLUT4) gene expression in muscle cells by the transcriptional coactivator PGC-1. Proc Natl Acad Sci U S A. 2001;98: Jamshidi Y, Montgomery H, Hense H, et al. Peroxisome proliferator activated receptor alpha gene regulates left ventricular growth in response to exercise and hypertension. Circulation. 2002;105: Young ME, Patil S, Ying J, et al. Uncoupling protein 3 transcription is regulated by peroxisome proliferator-activated receptor (alpha) in the adult rodent heart. FASEB J. 2001;15: Hasenfuss G, Reinecke H, Studer R, et al. Relation between myocardial function and expression of sarcoplasmic reticulum Ca2 -ATPase in failing and non-failing human myocardium. Circ Res. 1994;75: Netticadan T, Temsah R, Kent A, et al. Depressed levels of Ca2 -cycling proteins may underlie sarcoplasmic reticulum dysfunction in the diabetic heart. Diabetes. 2001;50: Herron T, Korte F, McDonald K. Loaded shortening and power output in cardiac myocytes are dependent on myosin heavy chain isoform expression. Am J Physiol Heart Circ Physiol. 2001;281:H1217 H Zhong Y, Ahmed S, Grupp I, et al. Altered SR protein expression associated with contractile dysfunction in diabetic rat hearts. Am J Physiol Heart Circ Physiol. 2001;281:H1137 H Stamler J, Vaccaro O, Neaton J, et al. Diabetes, other risk factors, and 12-yr cardiovascular mortality for men screened in the Multiple Risk Factor Intervention Trial. Diabet Care. 1993;16: