Molecular remodeling of ion channels, exchangers and pumps in atrial and ventricular myocytes in ischemic cardiomyopathy (original) (raw)

• Journal List
• HHS Author Manuscripts
• PMC2891309

Channels (Austin). Author manuscript; available in PMC 2011 Mar 18.

Published in final edited form as:

PMCID: PMC2891309

NIHMSID: NIHMS170352

Naomi Gronich

1 National Institute on Aging; National Institutes of health; Baltimore, MD USA;

Azad Kumar

1 National Institute on Aging; National Institutes of health; Baltimore, MD USA;

Yuwei Zhang

1 National Institute on Aging; National Institutes of health; Baltimore, MD USA;

Igor R. Efimov

2 Washington University in Saint Louis; St. Louis, MO USA

Nikolai M. Soldatov

1 National Institute on Aging; National Institutes of health; Baltimore, MD USA;

1 National Institute on Aging; National Institutes of health; Baltimore, MD USA;

2 Washington University in Saint Louis; St. Louis, MO USA

*Correspondence to: Nikolai M. Soldatov; National Institute on Aging; NIH; 251 Bayview Blvd., Suite 100; Baltimore, MD 21224, USA; vog.hin.ain.crg@Nvotadlos

†Both authors have contributed equally in this study

‡Current address: Internal Medicine and Department of Community Medicine and Epidemiology, Carmel Medical Center, 7 Michal St., Haifa 34362, Israel.

§Current address: Cell Biology and Gene Expression Unit, Laboratory of Neurogenetics, NIA, NIH, Builiding 35, Room 1A1006, 35, Convent Drive, Bethesda, MD 20892, USA.

¶Current address: Surgery Branch, NCI, NIH, Building 10, 3-5848, Bethesda, MD 20892, USA.

Abstract

Existing molecular knowledge base of cardiovascular diseases is rudimentary because of lack of specific attribution to cell type and function. The aim of this study was to investigate cell-specific molecular remodeling in human atrial and ventricular myocytes associated with ischemic cardiomyopathy. Our strategy combines two technological innovations, laser-capture microdissection of identified cardiac cells in selected anatomical regions of the heart and splice microarray of a narrow catalog of the functionally most important genes regulating ion homeostasis. We focused on expression of a principal family of genes coding for ion channels, exchangers and pumps (CE&P genes) that are involved in electrical, mechanical and signaling functions of the heart and constitute the most utilized drug targets. We found that (1) CE&P genes remodel in a cell-specific manner: ischemic cardiomyopathy affected 63 CE&P genes in ventricular myocytes and 12 essentially different genes in atrial myocytes. (2) Only few of the identified CE&P genes were previously linked to human cardiac disfunctions. (3) The ischemia-affected CE&P genes include nuclear chloride channels, adrenoceptors, cyclic nucleotide-gated channels, auxiliary subunits of Na+, K+ and Ca2+ channels, and cell-surface CE&Ps. (4) In both atrial and ventricular myocytes ischemic cardiomyopathy reduced expression of CACNG7 and induced overexpression of FXYD1, the gene crucial for Na+ and K+ homeostasis. Thus, our cell-specific molecular profiling defined new landmarks for correct molecular modeling of ischemic cardiomyopathy and development of underlying targeted therapies.

Keywords: ischemic cardiomyopathy, molecular profiling, atrial myocytes, ventricular myocytes, ion channels, phospholemman, Na+, K+-ATPase, CLIC

Introduction

Cardiovascular disease is the leading cause of human morbidity and mortality. Ischemic cardiomyopathy1 (ICM) affects approximately 1 out of 100 people in the United States, most often middle-aged to elderly men. ICM is the most common form of cardiomyopathy leading to dilation of the cardiac chambers and congestive heart failure. In spite of great efforts, molecular markers and targets of ICM are not currently well understood.2 The tissue-specific pathogenesis pathways in the human heart reportedly include approximately 20 genes3 coding for structural proteins and those associated with Ca2+ homeostasis and energy metabolism. However, some of the implicated genes may be secondary to ICM because intrinsic noise of investigative platforms precludes from detecting low-signal cell-type-specific critical targets at the whole-tissue level. Different cell types remodel in the development and disease following their unique program. Moreover, this remodeling is anatomically heterogeneous. To overcome these problems and characterize ICM-induced molecular remodeling in a cell-type and anatomical region-specific fashion, we combined two technological innovations, laser-capture microdissection of cells of interest and microarray optimized for detection of alternative RNA splicing events in a narrow catalog of genes coding for ion channels, exchangers and pumps (CE&P genes), which comprise the most acclaimed target classes for top-selling prescription drugs. Because CE&P molecules are involved in electrical, signaling and mechanical functions of the heart, their remodeling in ICM may result in decreased cardiac contractile functions.4 It is likely that pathophysiological conditions of ICM affect regulation of expression of CE&P splice variants.5 Thus, molecular profiling of disease-induced CE&P remodeling may narrow the search for crucial players and enable better understanding of underlying disease mechanisms and effective ICM prevention and treatment. We focused our research on CE&P molecular remodeling in atrial and ventricular cardiomyocytes because of their ultimate role in cardiac contraction targeted by ICM. This research strategy addressed the complexity of cardiovascular system and tissue heterogeneity complicated by differential gene expression and splice variation. Our findings associate ICM with altered expression of CACNG7 and FXYD1 in atrial and ventricular myocytes and show that these cells remodel in ICM following their unique programs involving different subsets of CE&P genes.

Results

Laser capture microdissection is an established method for isolation with confidence of histochemically identified cells from heterogeneous cell populations allowing the rational design of comprehensive splice variant profiling of CE&P remodeling in cardiomyocytes. Results of microarray analysis are presented in Table 3 and summarized in Table 4. We found that ICM caused remodeling of 12 genes in atrial myocytes. Increased expression in relation to healthy hearts was found for the FXYD1 gene coding for protein regulator of Na+,K+-ATPase phospholemman (2.3-fold) and intracellular chloride channels CLIC1 and CLIC4 (both 1.6-fold). The other nine identified genes were significantly down-regulated including FXYD7, the inward rectifier Kir2.4 channel, the acetylcholine receptor α10 subunit, the Ca2+ channel Cavγ subunits, the AMPA-selective glutamate receptor 3, the Kv4.3 K+ channel involved in initial phase of repolarization and setting the plateau voltage of the action potential, the polyunsaturated fatty acids-activated and mechano-sensitive K2P4.1 channel, the Na+ channel Navβ1 subunit. In ventricular myocytes we identified 63 genes affected by ICM. An increase (2.5-fold) was found only for FXYD1. The other 62 CE&P genes were down-regulated 1.5–3.5 fold. Some of them belong to the same functional groups that are affected by ICM in atrial myocytes (Table 4) including FXYD3, three A-type K+ channels, two inward rectifiers, three tandem-pore-domain K+ channels, three Cavγ subunits, six acetylcholine receptor subunits, and the AMPA1 glutamate receptor. In addition, we identified three delayed rectifiers, the Ca2+-activated KCa3.1 channel, seven modifiers and β-subunits of K+ channels, six voltage-gated Ca2+ channels, two ENaC and trp channels, cyclic nucleotide-gated channels HCN2 and CNGA3, three connexins, NMDA and glycine receptors, GABA receptor subunits, and three adrenoceptors.

Table 3

Top-list ANOVA gene-score annotation for CE&P genes altered by ICM in atrial and ventricular myocytes as compared to healthy subjects

Table 4

CE&P genes altered by ICM in atrial and ventricular myocytes

To find out whether the splice microarray results reported in Table 3 are disease-type specific, we supplemented the same splice microarray of ICM ventricular myocytes with one additional sample prepared from left ventricle of a 41-years old dilated cardiomyopathy male donor. The top-list ANOVA gene score annotation for combined altered CE&P genes in ventricular myocytes (Table 3) was reduced from 63 to just 4 genes (FXYD1 (Gfold = +2.4), HCN2 (−1.5), GLRA1 (−1.8) and GJC1 (−2.3)). This result suggests that dilated and ischemic cardiomyopathy may affect different CE&P subsets and only four indicated genes may be common among the ones affected by these diseases, overexpression of FXYD1 being the most obvious characteristic feature.

Discussion

Our study is based on the precept that to characterize the ICM-induced molecular remodeling it is essential to investigate it at the level of cells and molecules responsible for altered cardiac function. To achieve this goal, we developed the investigative approach combining laser capture microdissection of identified cardiac cells from tissue biopsies with splice microarray of custom-narrowed sets of CE&P genes that play ultimate role in cardiac contraction targeted by ICM. Our approach for the first time enabled direct comparison of altered gene expression caused by ICM in atrial and ventricular myocytes and identified new essential players.

First of all, we found that atrial and ventricular myocytes remodel following their unique programs where ICM affects different CE&P genes. Although some of these genes were already reported to be expressed in the heart4,79 (see also references in Tables 3 and ​4), only few of them were previously linked to known cardiac pathologies.21,39,64,80–84 These include (1) α1B, α2C and β1-adrenoceptors whose down-regulation in cardiomyopathy was linked to pathological remodeling in failing ventricular myocardium.42,51,85 (2). KCND2. In diabetic ventricle, a switch from Kv4.2 to Kv1.4 may underlie the slower kinetics of the Ito K+ current.40 (3). KCND3. Down- regulation of cardiac Kv4.3 and minK channels may be associated with arrhythmia and atrial fibrillation.14,61,62,86 It was reported that congestive heart failure and hypertrophy decrease Kv4.3 expression in terminally failing human hearts.87 In line with this finding, KCND3 gene transfer abrogates the hypertrophic response to aortic stenosis.88 (4) HCN2. Transfer of this gene was also tested as gene therapy for cardiac arrhythmias in experimental animals with positive results.89 However, the existing knowledge base remains rudimentary in the absence of attribution to certain cardiac cell types and functions.

We found that ICM does not alter expression of Cav1.2 and Nav5 channel isoforms and Na+,K+-ATPase but rather affects some of their accessory subunits. The most profound change in both atrial and ventricular myocytes was overexpression of phospholemman. Binding of phospholemman to Na+,K+-ATPase induces a decrease in the affinity of α1–β1 and α2–β1 isozymes to external K+ and approximately 2-fold decrease in the affinity to internal Na+.90 Inhibition of Na+/Ca2+ exchanger by phospholemman91 may add to the disbalance of Na+, K+ and Ca2+ gradients across the plasma membrane and contribute to hypertrophy of ICM muscle cells due to overexpression of FXYD1. Another unexpected finding that may have profound functional consequences is underexpression of Navβ1. Although the functional role of this single- membrane-spanning-repeat protein in the heart remains uncertain, it co-localizes with the Nav1.5 pore-forming α1 subunit in the T-tubule system and intercalating discs levels in cardiomyocytes17 and modulates Nav1.5 channels in the heart by increasing the Na+ current density.92 Confirming its crucial role in the heart, SCN1B knock-out caused prolongation of QT and RR intervals93 and development of cardiomyopathy.94 Post-transcriptional gene silencing of Navβ1 reduced mRNA and protein levels of Nav1.5, KChIP2 mRNA and Kv4.3 resulting in markedly decreased Na+ and Ito currents.95 Thus, underexpression of Navβ1 may lead to a suppression of Ito, action potential prolongation and increased susceptibility of the heart to ventricular arrhythmia.

Other new potential targets for drug discovery are _CLIC_s.8,35 Members of the p64 family, CLIC proteins localize to the cell nucleus and exhibit both nuclear and plasma-membrane chloride channel activity, but their functions are not well defined. CLIC2, which shares sequence similarity with CLIC1, modulates cardiac ryanodine receptors and inhibits Ca2+ release from the sarcoplasmic reticulum.96 Thus, ICM-induced remodeling of _CLIC_s in cardiomyocytes may affect membrane potential, intracellular pH and cell volume.

The four identified Cavγ calcium channel subunits downregulated in ICM show a broad spectrum of modulating activities that may have a role in cardiac myocytes. The γ7, which is homologous to γ5, regulates stability of certain mRNAs97 and, along with γ2 and γ8, controls trafficking and gating of AMPA receptors.98,99 It remains to be studied whether remodeling of AMPA receptors in ICM is associated with Cavγs. Acetylcholine,72 AMPA and NMDA receptors73, also downregulated in ICM, are present in cardiac neuromuscular junctions and intercalating disk, but little is known about their non-neuronal expression and roles in the heart.

Our study is the first step in molecular characterization of ICM with organ- and cell-specific annotation of altered expression of CE&P genes. Our study does not determine whether upregulation or downregulation of the identified CE&P genes are the primary drivers of ICM or reflect pathophysiological response to the disease. However, it provides an objective context within which it would be easier to find therapeutic targets among the elucidated markers of ICM. Future extension of this study may clarify links between CE&P genes expression and drug therapy, duration of disease, age, gender and race as potential factors in ICM and define key aspects of the principal gene network in relation to the development of ICM at the cellular and specific intercellular levels. In conjunction with protein and immunohistochemical analyses this may yield a more robust approach to better understanding mechanisms and pathophysiology of ICM - a critical need in the clinical utilization of this field.

Methods

Human cardiac tissue samples

Healthy (Table 1) and ICM hearts (Table 2) of anonymous donors were obtained from the Cardiac Transplantation Center at the Washington University in St. Louis, MO. Healthy hearts were excluded from transplantation after exceeding 6 h-limit of allograft ischemic time. Showing no evidence of hemorrhagic stroke, they retained normal structure and function evidenced by bimodal biophotonic imaging and optical mapping of the atrioventricular junction.100 There was no delay in collecting donor’s heart tissue. Decision on suitability for transplantation was made prior to harvest of organs, our team members were notified in advance (at least 2 h before cross clamping) and were present during organ harvest. Heart was cardioplegically arrested, harvested and transferred to us immediately after removal from the chest. Tissue samples (0.5–2 g) were dissected from the area outside of scar burden (if any) of left ventricle and left atrial appendages under cold cardiopledic arrest conditions within approximately 30–40 min after removal of the heart from donor’s chest. Tissue was washed in 4°C saline, immersed in Tissue-Tek OCT Compound (Electron Microscopy Sciences, Hatfield, PA), flash frozen in liquid nitrogen and stored at −80°C until use. Although availability of donors suitable for this study was limited, the number of selected human hearts (Tables 1 and ​2) is in full compliance with the NCBI-recommended MIAME guidelines to microarray101 and enabled us to analyze all individual donor’s samples, grouped by cell type, simultaneously on one microarray slide, thus excluding possible slide-to-slide variations.

Table 1

Characteristics of the healthy heart donors

Table 2

Characteristics of the ICM heart patients

Laser-capture microdissection and isolation of mRNA

Serial cryostat sections (7–8 μm thick) were cut from the frozen tissue samples using Minotome Plus microtome cryostat. Before laser-capture microdissection, cardiomyocytes in the sections were quickly (10 s) stained with Eosin Y (Sigma-Aldrich, St. Louis, MO) according to standard procedure. Given the notably large size of cardiac muscle cells relative to other cells in the tissue, this method of identification is sufficient to distinguish stained cardiomyocytes from other cells under microscope. Laser-capture microdissection was performed with PixCell II system (Arcturus, Mountain View, CA) using a 7.5-μm laser spot as described earlier.5 The excised cardiomyocytes were picked up from the slide surface and captured on LCM Caps. To confirm the quality of cell isolation, the sectional images taken before and after microdissection were thoroughly inspected to exclude contamination with non-muscle cells, and only then the captured samples from serial sections were joined together. High-quality cellular RNA was recovered from the collected cells using PicoPure™ RNA isolation kit (Arcturus) and treated with RNase-free DNase (Qiagen, Valencia, CA). Quality of RNA was tested right before the labeling for microarray by measuring the OD and 28S/18S RNA ratios. On average, the OD ratio at 260/280 nm and 260/230 nm was > 1.8 and the ratio of 28S/18S in the RNA samples selected for experiments was ≥ 1.6. Individual samples of total RNA were optionally amplified without 3′-bias using the TransPlex™ Whole Transcriptome Amplification kit (Rubicon Genomics, Ann Arbor, MI). (The non-bias 3′-amplification was confirmed by the automated RT-PCR with capillary electrophoretic quantification of amplicons executed on a commercial human cardiac total RNA samples (Sigma) with primers to CACNA1C and CACNB2, calcium channel genes characterized by complex alternative splicing). The amplified products were purified using QIAquick PCR purification kit (Qiagen).

cDNA labeling and microarray

The PCR products were fragmented with DNase I, denatured and end-labeled with Cy-3 fluorescent dye. The individual donor’s samples, grouped according to cell type, were analyzed simultaneously on Human Ion Channel Splice Arrays 8-pack 4×44K slides (ExonHit Therapeutics, Gaithersburg, MD) manufactured on the Ion Channel Splice Array sv1.1 platform representing 287 human CE&P, including 248 alternatively spliced ones in total 1655 splicing events and supplemented with additional capabilities to recognize connexins and ryanodine receptors.

Microarray data analysis

The statistically significant differential expression patterns between ICM and healthy atrial and ventricular cell samples was analyzed using long- to short-form ratio statistics and expression level statistics to identify genes and splice events affected by ICM. All analyses were performed at ExonHit using Partek Genomics Suite. Principal Component Analysis was carried out to illustrate the level of spread between samples and experimental groups. A two-way ANOVA model was used to perform statistical tests on the probe set level intensities, to compare ICM vs. healthy cells. A Source of Variation plot was generated from this data to find the relative level of difference contributed by each factor. A cutoff level was determined to generate “top hit lists” of probe sets that indicate the most statistically significant differences between the sample groupings. The raw and transformed data sets are submitted to Gene Expression Omnibus, accession # GSE17294 and GSE17530. The method accounted for specific splice variants shown in the results, but did not evaluate the ICM-induced changes in alternative splicing. Low variability between the individual RNA samples in meaningful (p < 0.01) probesets, estimated as average of individual probesets standard deviations normalized to the respective mean values (0.08 ± 0.04, mean ± st.dev), suggests that differences in donor’s age and gender in our study have not notably affected the results of microarray. Additional details are presented in Supplementary Methods.

Supplementary Material

Acknowledgments

The authors thank Kevin Becker and William H. Wood (Gene Expression and Genomics Unit, NIA) for development of splice microarray slides, Heather Jordan and Weiyin Zhou (ExonHit Therapeutics) for help with splice microarray analysis, and Cardiac Transplantation Unit of the Washington University in St. Louis, MO for help with cardiac tissues. This work was supported in part by the Intramural Research Program of the National Institutes of Health, National Institute on Aging (Z01 AG000294-08 to N.M.S.) and by the National Heart, Lung and Blood Institute grant (RO1 HL085369 to I.R.E.).

Abbreviations

ICM

ischemic cardiomyopathy

CE&P

channels, exchangers and pumps

References

1. Felker GM, Shaw LK, O’Connor CM. A standardized definition of ischemic cardiomyopathy for use in clinical research. J Am Coll Cardiol. 2002;39:210–18. [PubMed] [Google Scholar]

2. Kittleson MM, Minhas KM, Irizarry RA, Ye SQ, Edness G, Breton E, et al. Gene expression analysis of ischemic and nonischemic cardiomyopathy: Shared and distinct genes in the development of heart failure. Physiol Genomics. 2005;21:299–307. [PubMed] [Google Scholar]

3. Sanoudou D, Vafiadaki E, Arvanitis DA, Kranias E, Kontrogianni-Konstantopoulos A. Array lessons from the heart: focus on the genome and transcriptome of cardiomyopathies. Physiol Genomics. 2005;21:131–43. [PubMed] [Google Scholar]

4. Mangoni ME, Nargeot J. Genesis and regulation of the heart automaticity. Physiol Rev. 2008;88:919–82. [PubMed] [Google Scholar]

5. Tiwari S, Zhang Y, Heller J, Abernethy DR, Soldatov NM. Atherosclerosis-related molecular alteration of the human Cav1.2 calcium channel α1C subunit. Proc Natl Acad Sci USA. 2006;103:17024–29. [PMC free article] [PubMed] [Google Scholar]

6. Bibert S, Roy S, Schaer D, Horisberger J-D, Geering K. Phosphorylation of phospholemman (FXYD1) by protein kinases A and C modulates distinct Na,K-ATPase isozymes. J Biol Chem. 2008;283:476–86. [PubMed] [Google Scholar]

7. Fuller W, Eaton P, Bell JR, Shattock MJ. Ischemia-induced phosphorylation of phospholemman directly activates rat cardiac Na/K-ATPase. FASEB J. 2004;18:197–99. [PubMed] [Google Scholar]

8. Berryman M, Bretscher A. Identification of a novel member of the chloride intracellular channel gene family (CLIC5) that associates with the actin cytoskeleton of placental microvilli. Mol Biol Cell. 2000;11:1509–21. [PMC free article] [PubMed] [Google Scholar]

9. Harrell MD, Harbi S, Hoffman JF, Zavadil J, Coetzee WA. Large-scale analysis of ion channel gene expression in the mouse heart during perinatal development. Physiol Genomics. 2007;28:273–83. [PubMed] [Google Scholar]

10. Liu GX, Derst C, Schlichthörl G, Heinen S, Seebohm G, Brüggemann A, et al. Comparison of cloned Kir2 channels with native inward rectifier K+ channels from guinea-pig cardiomyocytes. J Physiol. 2001;532:115–26. [PMC free article] [PubMed] [Google Scholar]

11. Sgard F, Charpantier E, Bertrand S, Walker N, Caput D, Graham D, et al. A novel human nicotinic receptor subunit, α10, that confers functionality to the α9-subunit. Molecular Pharmacology. 2002;61:150–59. [PubMed] [Google Scholar]

12. Chu P-J, Robertson HM, Best PM. Calcium channel γsubunits provide insights into the evolution of this gene family. Gene. 2001;280:37–48. [PubMed] [Google Scholar]

13. Gill SS, Pulidoa OM, Muellera RW, McGuire PF. Molecular and immunochemical characterization of the ionotropic glutamate receptors in the rat heart. Brain Res Bull. 1998;46:429–34. [PubMed] [Google Scholar]

14. Brundel BJJM, Van Gelder IC, Henning RH, Tieleman RG, Tuinenburg AE, Wietses M, et al. Ion channel remodeling is related to intraoperative atrial effective refractory periods in patients with paroxysmal and persistent atrial fibrillation. Circulation. 2001;103:684–90. [PubMed] [Google Scholar]

15. Ozaita A, Vega-Saenz de Miera E. Cloning of two transcripts, HKT4.1a and HKT4.1b, from the human two-pore K+ channel gene KCNK4. Chromosomal localization, tissue distribution and functional expression. Mol Brain Res. 2002;102:18–27. [PubMed] [Google Scholar]

16. Dixon JE, Shi W, Wang H-S, McDonald C, Yu H, Wymore RS, et al. Role of the Kv4.3 K+ channel in ventricular muscle: A molecular correlate for the transient outward current. Circ Res. 1996;79:659–68. [PubMed] [Google Scholar]

17. Domínguez JN, de la Rosa Á, Navarro F, Franco D, Aránega AE. Tissue distribution and subcellular localization of the cardiac sodium channel during mouse heart development. Cardiovasc Res. 2008;78:45–52. [PubMed] [Google Scholar]

18. Makita N, Bennett PB, Jr, George AL., Jr Voltage-gated Na+ channel β1 subunit mRNA expressed in adult human skeletal muscle, heart, and brain is encoded by a single gene. J Biol Chem. 1994;269:7571–79. [PubMed] [Google Scholar]

19. Mitchell JW, Larsen JK, Best PM. Identification of the calcium channel α1E (Cav2.3) isoform expressed in atrial myocytes. Biochim Biophys Acta - Gene Str Expr. 2002;1577:17–26. [PubMed] [Google Scholar]

20. Weiergräber M, Henry M, Hescheler J, Schneider T. Ablation of the Cav2.3 containing E- type voltage-gated calcium channel results in cardiac arrhythmia and altered autonomic control within the murine cardiovascular system. Heart Rhythm. 2005;2:S35–S36. [PubMed] [Google Scholar]

21. Ackerman MJ. Cardiac channelopathies: it’s in the genes. Nat Med. 2004;10:463–64. [PubMed] [Google Scholar]

22. Choi G, Kopplin LJ, Tester DJ, Will ML, Haglund CM, Ackerman MJ. Spectrum and frequency of cardiac channel defects in swimming-triggered arrhythmia syndromes. Circulation. 2004;110:2119–24. [PubMed] [Google Scholar]

23. Czirják G, Tóth ZE, Enyedi P. Characterization of the heteromeric potassium channel formed by Kv2.1 and the retinal subunit Kv8.2 in Xenopus oocytes. J Neurophysiol. 2007;98:1213–22. [PubMed] [Google Scholar]

24. Patel AJ, Maingret F, Magnone V, Fosset M, Lazdunski M, Honore E. TWIK-2, an inactivating 2P domain K+ channel. J Biol Chem. 2000;275:28722–30. [PubMed] [Google Scholar]

25. Yajima T, Nagashima H, Tsutsumi-Sakai R, Hagiwara N, Hosoda S, Quertermous T, et al. Functional activity of the CFTR Cl− channel in human myocardium. Heart Vessels. 1997;12:255–61. [PubMed] [Google Scholar]

26. Abramowitz J, Birnbaumer L. Physiology and pathophysiology of canonical transient receptor potential channels. FASEB J. 2009;23:297–328. [PMC free article] [PubMed] [Google Scholar]

27. Nakayama H, Wilkin BJ, Bodi I, Molkentin JD. Calcineurin-dependent cardiomyopathy is activated by TRPC in the adult mouse heart. FASEB J. 2006;20:1660–70. [PMC free article] [PubMed] [Google Scholar]

28. Gaborit N, Le Bouter S, Szuts V, Varro A, Escande D, Nattel S, et al. Regional and tissue specific transcript signatures of ion channel genes in the non-diseased human heart. J Physiol. 2007;582:675–93. [PMC free article] [PubMed] [Google Scholar]

29. Mangoni ME, Couette B, Bourinet E, Platzer J, Reimer D, Striessnig J, et al. Functional role of L-type Cav1.3 Ca2+ channels in cardiac pacemaker activity. Proc Natl Acad Sci U S A. 2003;100:5543–48. [PMC free article] [PubMed] [Google Scholar]

30. Xiao J, Yang B, Lin H, Lu Y, Luo X, Wang Z. Novel approaches for gene-specific interference via manipulating actions of microRNAs: Examination on the pacemaker channel genes HCN2 and HCN4. J Cell Physiol. 2007;212:285–92. [PubMed] [Google Scholar]

31. Kang D, Mariash E, Kim D. Functional expression of TRESK-2, a new member of the tandem-pore K+ channel family. J Biol Chem. 2004;279:28063–70. [PubMed] [Google Scholar]

32. O’Driscoll KE, Hatton WJ, Burkin HR, Leblanc N, Britton FC. Expression, localization, and functional properties of Bestrophin 3 channel isolated from mouse heart. Am J Physiol Cell Physiol. 2008;295:C1610–24. [PMC free article] [PubMed] [Google Scholar]

33. Fonfria E, Murdock PR, Cusdin FS, Benham CD, Kelsell RE, McNulty S. Tissue distribution profiles of the human TRPM cation channel family. J Recep Sign Transduct. 2006;26:159–78. [PubMed] [Google Scholar]

34. Kong B, Liu Y-l, Lü X-d. Microarray-bioinformatics analysis of altered genomic expression profiles between human fetal and infant myocardium. Chinese Med J. 2008;121:1257–64. [PubMed] [Google Scholar]

35. Qian Z, Okuhara D, Abe MK, Rosner MR. Molecular cloning and characterization of a mitogen-activated protein kinase-associated intracellular chloride channel. J Biol Chem. 1999;274:1621–27. [PubMed] [Google Scholar]

36. Chiang C-S, Huang C-H, Chieng H, Chang Y-T, Chang D, Chen J, Jr, et al. The Cav3.2 T-type Ca2+ channel is required for pressure overload-induced cardiac hypertrophy in mice. Circ Res. 2009;104:522–30. [PubMed] [Google Scholar]

37. Cribbs LL, Lee JH, Yang J, Satin J, Zhang Y, Daud A, et al. Cloning and characterization of α1H from human heart, a member of the T-type Ca2+ channel gene family. Circ Res. 1998;83:103–09. [PubMed] [Google Scholar]

38. Guo W, Jung WE, Marionneau C, Aimond F, Xu H, Yamada KA, et al. Targeted deletion of Kv4.2 eliminates Ito,f and results in electrical and molecular remodeling, with no evidence of ventricular hypertrophy or myocardial dysfunction. Circ Res. 2005;97:1342–50. [PubMed] [Google Scholar]

39. Marionneau C, Brunet S, Flagg TP, Pilgram TK, Demolombe S, Nerbonne JM. Distinct cellular and molecular mechanisms underlie functional remodeling of repolarizing K+ currents with left ventricular hypertrophy. Circ Res. 2008;102:1406–15. [PMC free article] [PubMed] [Google Scholar]

40. Nishiyama A, Ishii DN, Backx PH, Pulford BE, Birks BR, Tamkun MM. Altered K+ channel gene expression in diabetic rat ventricle: isoform switching between Kv4.2 and Kv1.4. Am J Physiol Heart Circ Physiol. 2001;281:H1800–07. [PubMed] [Google Scholar]

41. Kashiwakura Y, Cho HC, Barth AS, Azene E, Marbán E. Gene transfer of a synthetic pacemaker channel into the heart: A novel strategy for biological pacing. Circulation. 2006;114:1682–86. [PubMed] [Google Scholar]

42. Woodcock EA. Roles of α1A- and α1B-adrenoceptors in heart: Insights from studies of genetically modified mice. Clin Exp Pharmacol Physiol. 2007;34:884–88. [PubMed] [Google Scholar]

43. Shuck ME, Bock JH, Benjamin CW, Tsai TD, Lee KS, Slightom JL, et al. Cloning and characterization of multiple forms of the human kidney ROM-K potassium channel. J Biol Chem. 1994;269:24261–70. [PubMed] [Google Scholar]

44. Yeung SY, Pucovsky V, Moffatt JD, Saldanha L, Schwake M, Ohya S, et al. Molecular expression and pharmacological identification of a role for Kv7 channels in murine vascular reactivity. Br J Pharmacol. 2007;151:758–70. [PMC free article] [PubMed] [Google Scholar]

45. Wallner M, Meera P, Toro L. Molecular basis of fast inactivation in voltage and Ca2+-activated K+ channels: A transmembrane β-subunit homolog. Proc Natl Acad Sci USA. 1999;96:4137–42. [PMC free article] [PubMed] [Google Scholar]

46. Brahmajothi MV, Morales MJ, Liu S, Rasmusson RL, Campbell DL, Strauss HC. In situ hybridization reveals extensive diversity of K+ channel mRNA in isolated ferret cardiac myocytes. Circ Res. 1996;78:1083–89. [PubMed] [Google Scholar]

47. Mustapha Z, Pang L, Nattel S. Characterization of the cardiac KCNE1 gene promoter. Cardiovasc Res. 2007;73:82–91. [PubMed] [Google Scholar]

48. Gaborit N, Steenman M, Lamirault G, Le Meur N, Le Bouter S, Lande G, et al. Human atrial ion channel and transporter subunit gene-expression remodeling associated with valvular heart disease and atrial fibrillation. Circulation. 2005;112:471–81. [PubMed] [Google Scholar]

49. Mollnau H, Oelze M, August M, Wendt M, Daiber A, Schulz E, et al. Mechanisms of increased vascular superoxide production in an experimental model of idiopathic dilated cardiomyopathy. Arterioscler Thromb Vasc Biol. 2005;25:2554–59. [PubMed] [Google Scholar]

50. Aggarwal A, Esler MD, Socratous F, Kaye DM. Evidence for functional presynaptic alpha-2 adrenoceptors and their down-regulation in human heart failure. J Am Coll Cardiol. 2001;37:1246–51. [PubMed] [Google Scholar]

51. Brede M, Wiesmann F, Jahns R, Hadamek K, Arnolt C, Neubauer S, et al. Feedback inhibition of catecholamine release by two different α2-adrenoceptor subtypes prevents progression of heart failure. Circulation. 2002;106:2491–96. [PubMed] [Google Scholar]

52. Kardia S, Kelly R, Keddache M, Aronow B, Grabowski G, Hahn H, et al. Multiple interactions between the alpha2C- and beta1-adrenergic receptors influence heart failure survival. BMC Med Genet. 2008;9:93. [PMC free article] [PubMed] [Google Scholar]

53. Kitsios G, Zintzaras E. Genetic variation associated with ischemic heart failure: A HuGE review and meta-analysis. Am J Epidemiol. 2007;166:619–33. [PubMed] [Google Scholar]

54. Bukauskas FF, Kreuzberg MM, Rackauskas M, Bukauskiene A, Bennett MVL, Verselis VK, et al. Properties of mouse connexin 30.2 and human connexin 31.9 hemichannels: Implications for atrioventricular conduction in the heart. Proc Natl Acad Sci U S A. 2006;103:9726–31. [PMC free article] [PubMed] [Google Scholar]

55. Bristow MR, Ginsburg R, Umans V, Fowler M, Minobe W, Rasmussen R, et al. β1- and β2-Adrenergic-receptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective β1-receptor down- regulation in heart failure. Circ Res. 1986;59:297–309. [PubMed] [Google Scholar]

56. Dorn GW, II, Molkentin JD. Manipulating cardiac contractility in heart failure: Data from mice and men. Circulation. 2004;109:150–58. [PubMed] [Google Scholar]

57. Barry DM, Trimmer JS, Merlie JP, Nerbonne JM. Differential expression of voltage-gated K+ channel subunits in adult rat heart: Relation to functional K+ channels? Circ Res. 1995;77:361–69. [PubMed] [Google Scholar]

58. Davies WL, Vandenberg JI, Sayeed RA, Trezise AEO. Cardiac expression of the cystic fibrosis transmembrane conductance regulator involves novel exon 1 usage to produce a unique amino-terminal protein. J Biol Chem. 2004;279:15877–87. [PubMed] [Google Scholar]

59. Warth JD, Collier ML, Hart P, Geary Y, Gelband CH, Chapman T, et al. CFTR chloride channels in human and simian heart. Cardiovasc Res. 1996;31:615–24. [PubMed] [Google Scholar]

60. Sanguinetti MC, Curran ME, Zou A, Shen J, Specter PS, Atkinson DL, et al. Coassembly of KvLQT1 and minK (IsK) proteins to form cardiac IKS potassium channel. Nature. 1996;384:80–83. [PubMed] [Google Scholar]

61. Restier L, Cheng L, Sanguinetti MC. Mechanisms by which atrial fibrillation-associated mutations in the S1 domain of KCNQ1 slow deactivation of IKs channels. J Physiol. 2008;586:4179–91. [PMC free article] [PubMed] [Google Scholar]

62. Sampson KJ, Terrenoire C, Cervantes DO, Kaba RA, Peters NS, Kass RS. Adrenergic regulation of a key cardiac potassium channel can contribute to atrial fibrillation: evidence from an IKs transgenic mouse. J Physiol. 2008;586:627–37. [PMC free article] [PubMed] [Google Scholar]

63. Vassort G, Talavera K, Alvarez JL. Role of T-type Ca2+ channels in the heart. Cell Calcium. 2006;40:205–20. [PubMed] [Google Scholar]

64. Pitt GS, Dun W, Boyden PA. Remodeled cardiac calcium channels. J Mol Cell Cardiol. 2006;41:373–88. [PubMed] [Google Scholar]

65. Mangoni ME, Traboulsie A, Leoni A-L, Couette B, Marger L, Le Quang K, et al. Bradycardia and slowing of the atrioventricular conduction in mice lacking Cav3.1/α1G T-type calcium channels. Circ Res. 2006;98:1422–30. [PubMed] [Google Scholar]

66. Perez-Reyes E, Cribbs LL, Daud A, Lacerda AE, Barclay J, Williamson MP, et al. Molecular characterization of a neuronal low-voltage-activated T-type calcium channel. Nature. 1998;391:896–900. [PubMed] [Google Scholar]

67. Niwa N, Yasui K, Opthof T, Takemura H, Shimizu A, Horiba M, et al. Cav3.2 subunit underlies the functional T-type Ca2+ channel in murine hearts during the embryonic period. Am J Physiol Heart Circ Physiol. 2004;286:H2257–63. [PubMed] [Google Scholar]

68. Ju Y-K, Chu Y, Chaulet H, Lai D, Gervasio OL, Graham RM, et al. Store-operated Ca2+ influx and expression of TRPC genes in mouse sinoatrial node. Circ Res. 2007;100:1605–14. [PubMed] [Google Scholar]

70. White TW, Srinivas M, Ripps H, Trovato-Salinaro A, Condorelli DF, Bruzzone R. Virtual cloning, functional expression, and gating analysis of human connexin31.9. Am J Physiol Cell Physiol. 2002;283:C960–70. [PubMed] [Google Scholar]

71. Söhl G, Nielsen PA, Eiberger J, Willecke K. Expression profiles of the novel human connexin genes hCx30.2, hCx40.1, and hCx62 differ from their putative mouse orthologues. Cell Commun Adhes. 2003;10:27–36. [PubMed] [Google Scholar]

72. Dvorakova M, Lips KS, Brüggmann D, Slavikova J, Kuncova J, Kummer W. Developmental changes in the expression of nicotinic acetylcholine receptor α-subunits in the rat heart. Cell Tissue Res. 2005;319:201–09. [PubMed] [Google Scholar]

73. Dai S, Hall DD, Hell JW. Supramolecular assemblies and localized regulation of voltage-gated ion channels. Physiol Rev. 2009;89:411–52. [PMC free article] [PubMed] [Google Scholar]

74. Mueller RW, Gill SS, Pulido OM. The monkey (Macaca fascicularis) heart neural structures and conducting system: An immunochemical study of selected neural biomarkers and glutamate receptors. Toxicol Pathol. 2003;31:227–34. [PubMed] [Google Scholar]

75. Perrino C, Rockman HA. Reversal of cardiac remodeling by modulation of adrenergic receptors: a new frontier in heart failure. Current Opinion in Cardiology. 2007;22:443–49. [PubMed] [Google Scholar]

76. Weinberg DH, Trivedi P, Tan CP, Mitra S, Perkins-Barrow A, Borkowski D, et al. Cloning, expression and characterization of human α adrenergic receptors α1a, α1b and α1c. Biochem Biophys Res Commun. 1994;201:1296–304. [PubMed] [Google Scholar]

77. McCloskey DT, Turnbull L, Swigart P, O’Connell TD, Simpson PC, Baker AJ. Abnormal myocardial contraction in α1A- and α1B-adrenoceptor double-knockout mice. J Mol Cell Cardiol. 2003;35:1207–16. [PubMed] [Google Scholar]

78. Rivard K, Trepanier-Boulay V, Rindt H, Fiset C. Electrical remodeling in a transgenic mouse model of α1B-adrenergic receptor overexpression. Am J Physiol Heart Circ Physiol. 2009;296:H704–18. [PubMed] [Google Scholar]

79. Demolombe S, Marionneau C, Le Bouter S, Charpentier F, Escande D. Functional genomics of cardiac ion channel genes. Cardiovasc Res. 2005;67:438–47. [PubMed] [Google Scholar]

80. Borlak J, Thum T. Hallmarks of ion channel gene expression in end-stage heart failure. FASEB J. 2003;17:1592–608. [PubMed] [Google Scholar]

81. Moss AJ, Kass RS. Long QT syndrome: from channels to cardiac arrhythmias. J Clin Invest. 2005;115:2018–24. [PMC free article] [PubMed] [Google Scholar]

82. Bezzina CR, Wilde AAM, Roden DM. The molecular genetics of arrhythmias. Cardiovasc Res. 2005;67:343–46. [PubMed] [Google Scholar]

83. Nattel S, Burstein B, Dobrev D. Atrial remodeling and atrial fibrillation: Mechanisms and implications. Circ Arrhythmia Electrophysiol. 2008;1:62–73. [PubMed] [Google Scholar]

84. Shieh C-C, Coghlan M, Sullivan JP, Gopalakrishnan M. Potassium channels: Molecular defects, diseases, and therapeutic opportunities. Pharmacol Rev. 2000;52:557–94. [PubMed] [Google Scholar]

85. Bristow MR, Raynolds MVA, Port JDA, Rasmussen RA, Ray PEA, Feldman AM. Reduced beta 1 receptor messenger RNA abundance in the failing human heart. J Clin Invest. 1993;92:2737–45. [PMC free article] [PubMed] [Google Scholar]

86. Tsuji Y, Zicha S, Qi X-Y, Kodama I, Nattel S. Potassium channel subunit remodeling in rabbits exposed to long-term bradycardia or tachycardia: Discrete arrhythmogenic consequences related to differential delayed-rectifier changes. Circulation. 2006;113:345–55. [PubMed] [Google Scholar]

87. Zicha S, Xiao L, Stafford S, Cha TJ, Han W, Varro A, et al. Transmural expression of transient outward potassium current subunits in normal and failing canine and human hearts. J Physiol. 2004;561:735–48. [PMC free article] [PubMed] [Google Scholar]

88. Lebeche D, Kaprielian R, del Monte F, Tomaselli G, Gwathmey JK, Schwartz A, et al. In vivo cardiac gene transfer of Kv4.3 abrogates the hypertrophic response in rats after aortic stenosis. Circulation. 2004;110:3435–43. [PubMed] [Google Scholar]

89. Donahue JK. Gene therapy for cardiac arrhythmias: A dream soon to come true? J Cardiovasc Electrophysiol. 2007;18:553–59. [PubMed] [Google Scholar]

90. Crambert G, Füzesi M, Garty H, Karlish S, Geering K. Phospholemman (FXYD1) associates with Na,K-ATPase and regulates its transport properties. Proc Natl Acad Sci U S A. 2002;99:11476–81. [PMC free article] [PubMed] [Google Scholar]

91. Zhang X-Q, Ahlers BA, Tucker AL, Song J, Wang J, Moorman JR, et al. Phospholemman inhibition of the cardiac Na+/Ca2+ exchanger: Role of phosphorylation. J Biol Chem. 2006;281:7784–92. [PMC free article] [PubMed] [Google Scholar]

92. Qu YS, Isom LL, Westenbroek RE, Rogers JC, Tanada TN, Mccormick KA, et al. Modulation of cardiac Na+ channel expression in Xenopus oocytes by β1 subunits. J Biol Chem. 1995;270:25696–701. [PubMed] [Google Scholar]

93. Lopez-Santiago LF, Meadows LS, Ernst SJ, Chen C, Malhotra JD, McEwen DP, et al. Sodium channel Scn1b null mice exhibit prolonged QT and RR intervals. J Mol Cell Cardiol. 2007;43:636–47. [PMC free article] [PubMed] [Google Scholar]

94. Hesse M, Kondo CS, Clark RB, Su L, Allen FL, Geary-Joo CTM, et al. Dilated cardiomyopathy is associated with reduced expression of the cardiac sodium channel Scn5a. Cardiovasc Res. 2007;75:498–509. [PubMed] [Google Scholar]

95. Deschênes I, Armoundas AA, Jones SP, Tomaselli GF. Post-transcriptional gene silencing of KChIP2 and Navβ1 in neonatal rat cardiac myocytes reveals a functional association between Na and Ito currents. J Mol Cell Cardiol. 2008;45:336–46. [PMC free article] [PubMed] [Google Scholar]

96. Dulhunty AF, Pouliquin P, Coggan M, Gage PW, Board PG. A recently identified member of the glutathione transferase structural family modifies cardiac RyR2 substate activity, coupled gating and activation by Ca2+ and ATP. Biochem J. 2005;390:333–43. [PMC free article] [PubMed] [Google Scholar]

97. Ferron L, Davies A, Page KM, Cox DJ, Leroy J, Waithe D, et al. The stargazin-related protein γ7 interacts with the mRNA-binding protein heterogeneous nuclear ribonucleoprotein A2 and regulates the stability of specific mRNAs, including Cav2.2. J Neurosci. 2008;28:10604–17. [PMC free article] [PubMed] [Google Scholar]

98. Tomita S, Chen L, Kawasaki Y, Petralia RS, Wenthold RJ, Nicoll RA, et al. Functional studies and distribution define a family of transmembrane AMPA receptor regulatory proteins. J Cell Biol. 2003;161:805–16. [PMC free article] [PubMed] [Google Scholar]

99. Kato AS, Zhou W, Milstein AD, Knierman MD, Siuda ER, Dotzlaf JE, et al. New transmembrane AMPA receptor regulatory protein isoform, γ-7, differentially regulates AMPA receptors. J Neurosci. 2007;27:4969–77. [PMC free article] [PubMed] [Google Scholar]

100. Hucker WJ, Ripplinger CM, Fleming CP, Fedorov VV, Rollins AM, Efimov IR. Bimodal biophotonic imaging of the structure-function relationship in cardiac tissue. J Biomed Optics. 2008;13:054012. [PMC free article] [PubMed] [Google Scholar]

101. Brazma A, Hingamp P, Quackenbush J, Sherlock G, Spellman P, Stoeckert C, et al. Minimum information about a microarray experiment (MIAME)-toward standards for microarray data. Nat Genet. 2001;29:365–71. [PubMed] [Google Scholar]