Dilated cardiomyopathy (DCM) is characterized by idiopathic dilation and systolic contractile dysfunction of the cardiac chambers. The present work aimed to study the alterations in gene expression of ion channels involved in cardiomyocyte function.
Methods and Results
Microarray profiling using the Affymetrix Human Gene® 1.0 ST array was performed using 17 RNA samples, 12 from DCM patients undergoing cardiac transplantation and 5 control donors (CNT). The analysis focused on 7 cardiac ion channel genes, since this category has not been previously studied in human DCM. SCN2B was upregulated, while KCNJ5, KCNJ8, CLIC2, CLCN3, CACNB2, and CACNA1C were downregulated. The RT-qPCR (21 DCM and 8 CNT samples) validated the gene expression of SCN2B (p < 0.0001), KCNJ5 (p < 0.05), KCNJ8 (p < 0.05), CLIC2 (p < 0.05), and CACNB2 (p < 0.05). Furthermore, we performed an IPA analysis and we found a functional relationship between the different ion channels studied in this work.
Citation: Molina-Navarro MM, Roselló-Lletí E, Ortega A, Tarazón E, Otero M, Martínez-Dolz L, et al. (2013) Differential Gene Expression of Cardiac Ion Channels in Human Dilated Cardiomyopathy. PLoS ONE 8(12): e79792. https://doi.org/10.1371/journal.pone.0079792
Editor: German E. Gonzalez, University of Buenos Aires, Faculty of Medicine. Cardiovascular Pathophysiology Institute., Argentina
Received: March 26, 2013; Accepted: September 25, 2013; Published: December 5, 2013
Copyright: © 2013 Molina-Navarro et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants of the National Institute of Health “Fondo de Investigaciones Sanitarias del Instituto de Salud Carlos III” [RD12/0042/0003; FIS Project PI10/00275]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Dilated cardiomyopathy (DCM) is one of the most frequent diseases that cause heart failure (HF) . DCM is characterized by idiopathic dilation and systolic contractile dysfunction, with an increase in ventricular mass and volume and wall thickness . Ion channel disruptions have been described as contributory to the development of DCM . Nevertheless, there are not studies analyzing the mechanisms involved in cardiac contraction dysfunction at the ion channel gene expression level.
Cardiac muscle contraction produced by the initiation of action potentials (AP) in cardiomyocytes has an important role in the pathogenesis of the disease. Cardiac ion channels are responsible for ion currents that determine and influence the cardiac AP in different parts of the human heart . Furthermore, cardiomyocytes are highly differentiated cells that specialize in excitation-contraction (EC) coupling, and have well-developed mechanical and electrical properties. The sarcomere is the functional unit in the contraction process that spans the area between the Z lines. It is made of three types of filaments: thin (actin), thick (myosin), and elastic (titin or connectin) . Ca2+ ions play an important role through binding directly to sarcomeric proteins allowing the initiation of the myocyte contraction [6,7].
The major ion channels involved in both the depolarization and repolarization of muscle cells are implicated in sodium, potassium, calcium, and chloride ion fluxes [8,9]. A common structure exists in all ion channels, including a transmembrane subunit α that forms the ion-conducting pore, and a variable number of associated subunits that are responsible for the regulation of channel expression and gating [10-12].
Establishing the alterations in gene expression is a proper manner to elucidate the causes or putative treatments of many diseases. We used high-throughput whole-genome microarray as well as the database for annotation, visualization and integrated discovery (DAVID) analysis tool to determine the biological and functional categories of the obtained gene list.
Since low contraction is one of the causes of poor prognosis in patients with DCM, we hypothesized that patients with DCM may show changes in the expression of genes related to cardiac contraction, such as genes encoding ion channels. Therefore, the aim of the study was to evaluate for the first time the differential gene expression of cardiac ion channels in DCM patients compared to control subjects.
The project was approved by the Ethics Committee of Hospital La Fe, Valencia, and all participants gave their written, informed consent. The study was conducted in accordance with the guidelines of the Declaration of Helsinki .
Source of tissue
Experiments were performed with left ventricular (LV) samples from explanted human hearts from patients with DCM undergoing cardiac transplantation. Clinical history, hemodynamic study, ECG, and Doppler echocardiography data were available from all of these patients. Non-ischemic DCM was diagnosed when patients had LV systolic dysfunction (EF <40%) with a dilated non-hypertrophic left ventricle (LVDD >55 mm) on echocardiography. Moreover, patients did not show existence of primary valvular disease and familial DCM. All patients were functionally classified according to the New York Heart Association (NYHA) criteria and they were receiving medical treatment following the guidelines of the European Society of Cardiology .
Non-diseased donor hearts were used as control (CNT) samples. The hearts were initially considered for transplantation, but were subsequently deemed unsuitable for transplantation either because of blood type or size incompatibility. The cause of death was cerebrovascular or motor vehicle accident. All donors had normal LV function and had no history of myocardial disease or active infection at the time of transplantation.
Transmural samples were taken from near the apex of the left ventricle and stored at 4°C for a maximum of 6 h from the time of coronary circulation loss. Samples were stored at -80°C until the RNA and protein extractions were performed.
Of 29 heart samples, 17 were used in the microarray profiling (DCM, n = 12; and CNT n = 5). The 29 total heart samples were used in the validation by RT-qPCR to improve the numerical base with a higher number of patients and control subjects (DCM, n = 21; and CNT, n = 8).
Total RNA isolation
RNA was extracted using a Qiagen RNeasy Fibrous Tissue Mini kit following the manufacturer’s instructions (Qiagen Iberia SL, Spain). The concentration of the obtained RNA was assessed using a NanoDrop 2000 spectrophotometer and the quality was determined using a microfluidic-based platform (2100 Bioanalyzer, Agilent Technologies, Spain SL).
cDNA synthesis was carried out using the WT Expression Sense Target Protocol (Ambion, Life Technologies, Carlsbad, CA, USA), and genome-wide gene expression was determined using Affymetrix Human Gene® 1.0 ST arrays (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instructions. Array hybridization, washing, and scanning were performed using the Gene Chip Scanner 7G System platform (Affymetrix, Santa Clara, CA, USA). The GeneChip® Command Console software was used for initial image processing. Affymetrix Expression ConsoleTM software provided quality control and a probe set summarization to attain gene-level signal data (Affymetrix, Santa Clara, CA, USA). The Partek® Genomics SuiteTM (Partek Inc., Saint Louis, MO, USA) software was used for background correction, normalization, probe summarization and statistical comparison (ANOVA) of expression profiles between the pathological group and the control group using the RMA algorithm. Genes were considered significantly different with a p-value <0.001 and a fold change of 1.3. All quantitative results are available at the NIH GEO database (GEO #GSE42955). DAVID programme was used to classify genes functionally associated with the aim to explore alterations in these functional categories following the published protocol for DAVID .
Real-time quantitative PCR analysis
We performed a quantitative real-time polymerase chain reaction (RT-qPCR) on frozen heart specimens from pathological and control subjects. Reverse transcription was carried out using 1 µg total RNA and Superscript III (Invitrogen Ltd, UK) according to the manufacturer’s protocol. The resulting cDNA was used as the template for RT-qPCR in a high-throughput thermocycler (ViiATM 7 Real-Time PCR System, Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The following TaqMan® probes were used: SCN2B (Hs00394952_m1), KCNJ5 (Hs00942581_m1), KCNJ8 (Hs00958961_m1), CLIC2 (Hs01574555_m1), CLCN3 (Hs00923161_m1), CACNB2 (Hs00167861_m1), and CACNA1C (Hs00167681_m1). Quantification of gene expression was normalized to GAPDH (Hs99999905_m1), PGK1 (Hs99999906_m1), and TFRC (Hs00951083_m1) as endogenous controls. And as a positive control of the RT-qPCR experiment, we analyzed the gene expression level of the genes KCND3 (Hs00542597_m1) and ATPA2A (Hs00544877_m1) which have shown a downregulation in human HF [16-20] (Figure S1). Relative gene expression levels were calculated using the 2-ΔΔCT method .
Homogenization of samples and protein determination
Thirty milligrams of frozen left ventricles were transferred into Lysing Matrix D tubes designed for use with the FastPrep-24 homogenizer (MP Biomedicals, USA) in total protein extraction buffer (2% SDS, 10 mM EDTA, 6 mM Tris-HCl, pH 7.4) with protease inhibitors (25 µg/mL aprotinin and 10 µg/mL leupeptin). The homogenates were centrifuged and the supernatants aliquoted. The protein content of aliquots was determined using Peterson’s modification of the micro Lowry method, using bovine serum albumin (BSA) as a standard .
Gel electrophoresis and Western blot analysis
Protein samples for detection of SCN2B and KCNJ5 were separated by Bis-Tris electrophoresis on 4–12% polyacrylamide gels under reducing conditions. After electrophoresis, the proteins were transferred from the gel to a PVDF membrane using the iBlot Dry Blotting System (Invitrogen Ltd, UK) for Western blot analyses. The membrane was blocked all night at 4°C with 1% BSA in Tris-buffer solution containing 0.05% Tween 20 and then for 2 h with a primary antibody in the same buffer. The primary detection antibodies used were anti-SCN2B rabbit polyclonal antibody (1:200), and anti-KCNJ5 rabbit polyclonal antibody (1:500). Anti-GAPDH mouse monoclonal antibody (1:1000) was used as a loading control. All antibodies used were from Abcam (Cambridge, UK).
Bands were visualized using an acid phosphatase-conjugated secondary antibody and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP, Sigma) substrate system. Finally, the bands were digitalized using an image analyzer (DNR Bio-Imagining Systems, Israel) and quantified by the GelQuant Pro (v12.2) program.
Ingenuity Pathway Analysis (IPA) software (Ingenuity® Systems, www.ingenuity.com) was used to detect the biological pathways of the differentially expressed ion channel genes using the human Refseq IDs as input. Biological groups that were significantly associated with the genes of interest (p < 0.05) were identified.
Data are presented as the mean ± standard deviation (SD). The Kolmogorov–Smirnov test was used to analyze the normal distribution of the variables. Comparisons between 2 groups were performed using Student’s t-test, and Pearson’s correlation coefficient was calculated to analyze the association between variables. Analyses were considered significant when p < 0.05. All statistical analyses were performed using SPSS software v. 20 for Windows (IBM SPSS Inc., Chicago, IL, USA).
Clinical characteristics of patients
Samples from 12 explanted human hearts from patients diagnosed with DCM undergoing cardiac transplantation and 5 non-diseased donor hearts as CNT samples were used in the microarray profiling analysis. All the patients were men with a mean age of 48 ± 9 years, a mean NYHA functional classification of III-IV, and previously diagnosed significant comorbidities, including hypertension and hypercholesterolemia. Table 1 shows the clinical characteristics of patients according to the etiology of DCM.
|DCM (n = 12)||DCM (n = 21)|
|Age (years)||48 ± 9||49 ± 14|
|Gender male (%)||100||96|
|NYHA class||3.5 ± 0.4||3.2 ± 0.4|
|BMI (kg/m2)||26 ± 4||27 ± 7|
|Hemoglobin (mg/mL)||13 ± 2||13 ± 2|
|Hematocrit (%)||40 ± 6||39 ± 6|
|Total cholesterol (mg/dL)||158 ± 45||136 ± 41|
|EF (%)||20 ± 6||22 ± 7|
|FS (%)||10 ± 3||12 ± 4|
|LVESD (mm)||71 ± 10||62 ± 10|
|LVEDD (mm)||79 ± 9||71 ± 10|
|LV mass index (g/cm2)||206 ± 52||191 ± 53|
We increased the sample size to improve the analysis up to 21 DCM and 8 CNT hearts for the RT-PCR validation assay, the clinical characteristics of these DCM patients are also shown in Table 1.
The control group used for the microarray profiling was comprised of 80 % men with 55 ± 3 years. And in the increased sample size used for the validation process, 60% were men with 45 ± 14 years.
Gene expression profiling
A gene expression microarray was performed to determine gene expression differences between the DCM and CNT groups. The results of the microarray experiment are shown in Table S1. A quality control for hybridization was carried out before the statistical analysis obtained using Partek Genomics Suite. Multivariate analysis, in the form of principal component analysis (PCA) was used to compare the expression profile of the sample groups based on their comprehensive expression profiles. The score plot obtained showed that 21.2 % of the differences among the sample groups could be explained by PCA component 1, 10.4 % by PCA component 2, while PCA component 3 explained 9.95 % of the differences (Figure 1A). A hierarchical clustering in both dimensions (samples and genes) showed clear differentiation between the pathological and control groups without any degree of overlap (Figure 1B).
The comparison of DCM patients with the CNT group showed 503 genes differentially expressed (p-value <0.001 and fold change >1.3), of which 201 were upregulated and 302 were downregulated (Table S1).
Among these differentially expressed genes, 13 belonged to the cardiac voltage-gated ion channel activity functional category according to the DAVID programme (Table S2). These genes are responsible for ion trafficking involved in cardiac contraction, an important process compromised in DCM. As this functional category has yet to be characterized in DCM, we focused on 7 of these ion channels (SCN2B, KCNJ5, KCNJ8, CLIC2, CLCN3, CACNB2, and CACNA1C) in this study, based on the described relationship of these channels with the contraction process (Table 2).
|SCN2B||Sodium channel subunit beta-2||2.03||5.20 x 10-6|
|KCNJ5||G protein-activated inward rectifier potassium channel 4||-1.95||6.44 x 10-4|
|KCNJ8||ATP-sensitive inward rectifier potassium channel 8||-1.44||6.72 x 10-3|
|CLIC2||Chloride intracellular channel protein 2||-1.77||4.87 x 10-4|
|CLCN3||H(+)/Cl(-) exchange transporter 3||-1.39||7.82 x 10-3|
|CACNB2||Voltage-dependent L-type calcium channel subunit beta-2||-1.51||1.92 x 10-3|
|CACNA 1C||Voltage-dependent L-type calcium channel subunit alpha-1C||-1.37||9.95 x 10-3|
Real-time quantitative PCR analysis
RT-qPCR was performed to validate the results obtained in the microarray profiling experiment using both the same samples used in the microarray and new samples for a total of 21 DCM and 8 CNT subjects. It was shown that SCN2B was upregulated, while KCNJ5, KCNJ8, CLIC2, and CACNB2 were downregulated in DCM compared to CNT (Figure 2), confirming the microarray results with regard to fold change and significance. However, the expression of CACNA1C and CLCN3 was not significantly altered in the RT-qPCR analysis.
The graph depicts the values obtained in microarrays and relative mRNA levels obtained using RT-qPCR normalized to the mRNA expression of 3 housekeeping genes (GAPDH, PGK1, and TFRC), respectively. The error bar represents the standard error of the mean (SEM) for DCM (n =12) and CNT (n = 5) samples in microarray data, and for DCM (n =21) and CNT (n = 8) samples in RT-qPCR data. * p < 0.05 vs. CNT.
Protein expression analysis
To analyze if the changes observed in gene expression were translated into changes at protein level, we performed a Western blot experiment of the two most differentially expressed genes SCN2B and KCNJ5. We did not found statistically significant differences between the DCM group and the CNT group in the SCN2B protein levels (78 ± 19 vs. 100 ± 31, respectively), and the same results were obtained comparing these two groups in the KCNJ5 protein levels (128 ± 34 vs. 100 ± 27, respectively) (data not shown).
To test whether the differentially expressed genes clustered into groups based on the biological process, or were related to one another, IPA was used. By applying the recommended parameters, we obtained a network that included the SCN2B, KCNJ5, KCNJ8, and CACNB2 genes, and an additional network with the CLIC2 gene.
In the first network (Figure 3A), CACNB2 showed direct interactions with gene families related to Ca2+ ion channels, such as the CACN and CACNB gene families. RIM1, a gene that regulates the voltage-gated calcium channels, is also associated with CACNB2. Finally, the sodium channel SCN2B is also closely related to CACNB2 in this network through the Ca2+ channel CACNA1B. The potassium ion channel genes KCNJ5 and KCNJ8 interact with some of the 17 members of the inward rectifier K+ KCNJ family.
A. First network including SCN2B, KCNJ5, KCNJ8 and CACNB2 genes (network score = 11). B. Second network with CLIC2 gene (network score = 3). Color intensity is correlated with fold change, green means downregulation and red overexpression. Straight lines indicate direct gene-to-gene interactions and dashed lines indirect interactions.
In the CLIC2 network (Figure 3B), the inhibition of RYR1 and RYR2 (ryanodine receptor 1 and 2, respectively) was shown. In addition, CLIC2 was related to TRAPPC2 (trafficking protein particle complex 2). Finally, the ubiquitin system is related to CLIC2 through NEDD4 (E3 ubiquitin-protein ligase NEDD4), NEDD4L (E3 ubiquitin-protein ligase NEDD4-like), and UBC (ubiquitin C).
In the present study we carried out a microarray profiling of LV tissue from patients with DCM to investigate differential gene expression of ion channel genes in DCM compared to CNT group. Ordog et al. reported the gene expression of several ion channel subunits in healthy human cardiomyocytes, particularly comparing the ion channel gene expression between atrium and ventricle . However, there have been no studies that have analyzed the expression level of genes related to these ion channels in human DCM and with a suitable sample size. Consequently, we focused on examining the expression levels of cardiac ion channels relevant to the contraction process.
Microarray experiments are a suitable method for analyzing the global expression of genes involved in human diseases, such as HF, showing alterations in gene expression profiles [23-26]. Moreover, there have been many studies that have used this method to examine expression levels of genes related to the EC process that occurs in muscle cells [27-29]. Besides, there are studies analyzing failing and non-failing human hearts establishing gender differences in electrophysiological gene expression, and using these data to predict the electrophysiological remodeling [30,31]. The DAVID gene functional classification tool allows sorting large gene lists into functionally related gene groups with an enrichment score, and summarizes the major biological importance of these gene groups.
An alteration at the gene level in cardiac ion channels could produce an imbalance in the currents of the different ions involved in the contraction process of the cardiomyocyte. Since DCM is a disease resulting in the impairment of the cardiac contraction process [32,33], we aimed to study the alterations in the gene expression profile of 7 ion channels in DCM by comparing with control subjects and using a large sample size.
The results showed an upregulation of the SCN2B sodium channel and a downregulation of the potassium channels KCNJ5 and KCNJ8, chloride channels CLIC2 and CLCN3, and calcium channels CACNA1C and CACNB2. The differential mRNA expression levels of the genes SCN2B, KCNJ5, KCNJ8, CLIC2, and CACNB2 were validated by RT-qPCR, while the calcium channel CACNA1C and the chloride channel CLCN3 were not. The fact that these two genes could not be validated might be due to the high variability in DCM disease [34,35].
SCN2B was the only gene upregulated in DCM. However, when we measured the protein levels, we observed no significant changes in SCN2B protein. This sodium channel forms the β2-subunit of the voltage-gated, cardiac-specific sodium channel (SCN5A), which is responsible for the initiation of action potentials in the myocyte . The α-subunit encoded by SCN5A forms the pore, and 2 auxiliary β-subunits (β1 and β2) modulate channel gating and cell surface expression levels and interact with the extracellular matrix and cell adhesion molecules. Moreover these auxiliary subunits play a key role in the regulation of the cardiac AP [36-37]. Therefore, differential expression of the α and β subunits can contribute to the ion flux alterations in HF. Our results show an upregulation of SCN2B gene, but a decreasing trend not significant compared to CNT in the level of SCN2B protein. This decreasing tendency has also been observed in other studies . Possibly, this could be explained by control mechanisms such as post-translational modifications or degradation systems that occur in DCM patients. Furthermore, the absence of higher levels of SCN2B protein are consistent with the studies that show a reduction in the sodium current produced by the pore forming subunit SCN5A in HF .
Potassium channels KCNJ5 and KCNJ8 were downregulated in DCM, while the protein level of KCNJ5 remained unchanged. This observation could be explained by possible regulation mechanisms for the KCNJ5 protein. These mechanisms may be required to fine-tune levels of KCNJ5 activity and control its effect on cells, including potential post-transcriptional modifications. The KCNJ5-encoded protein Kir 3.4 can form both a homodimer and/or a heterodimer and is activated through various receptors coupled to G proteins modulating the channel complex opening . Several studies confirm the importance of Kir3.4 to form a functional potassium channel . The Kir 6.1 protein, encoded by KCNJ8, forms a heterodimer with the subunit Kir 6.2, establishing an entire pore complex . In a KCNJ8 knockout mouse, a progressive impairment in cardiac output was seen . In analyzing the functions of these potassium channels, it seems that downregulating one or both of these in DCM patients could impair the current of K+ ions through the plasma membrane of the cardiomyocyte, provoking an alteration in the EC process, and consequently diminishing the ability of the heart to contract accordingly.
The CLIC2 gene encodes a protein belonging to the ubiquitous glutathione transferase structural family. These proteins are capable of transitioning from the aqueous phase into a phospholipid membrane, where they can function as ion channels . There are studies that propose a regulatory role for CLIC2 in the ryanodine receptor channel RYR2 [45,46]. RYR2 encodes a Ca2+ channel protein anchored to the sarcoplasmic reticulum (SR) in cardiomyocytes that triggers Ca2+ release to the cytoplasm during the contraction process. CLIC2 acts as an inhibitor of RYR2 by binding directly and depressing Ca2+ release under resting conditions, thus favoring low cytoplasmic Ca2+ concentrations during diastole [45,47]. Takano et al. identified a mutation in CLIC2 that resulted in abnormal cardiac function dependent on RYR channel activity . This failure to inhibit RYR2, and thus increased cytoplasmic Ca2+ levels due to the downregulation of CLIC2, may alter the relaxation process in the myocyte and consequently explain the impaired EC process in DCM.
Our results showed a downregulation of the calcium channel CACNB2, while the expression of CACNA1C was not altered. The calcium channel encoded by the CACNB2 gene is a membrane-associated guanylate kinase (MAGUK) protein that constitutes the β2 subunit of the L-type cardiac calcium channel CACNA1C. L-type calcium channels allow the influx of Ca2+ to the cytoplasm and are critical for controlling both cardiac excitability and EC coupling . The pore forming subunit α contains the voltage sensor and is encoded by the CACNA1C gene, but its expression and functional properties are influenced by auxiliary subunits such as β2 [49,50]. Indeed, Yamaguchi et al. demonstrated that the β2 subunit increases the channel density and facilitates channel opening. This important role for the β2 subunit in the Ca2+ channel has been evidenced by its implication in several cardiovascular diseases such as short QT syndrome or Brugada syndrome [51,52]. Many groups have shown that mutations in genes encoding different β2 subunits and the pore forming α subunit are related to the pathology. The downregulation observed in the CACNB2 gene may not properly inactivate the Ca2+ current through the CACNA1C α subunit, thereby altering the suitable cytoplasmic concentration of Ca2+ ions for the EC of cardiac muscle.
The gene expression profile of cardiac channels analyzed in this work has shown a general downregulation of all types of channels studied, with the exception of the sodium channel SCN2B, which is upregulated. This observation could explain the pathological process that occurs in DCM patients, where a general impairment of the contraction process may exist. As mentioned above, although no significant changes in its protein level have been found in our studies, the upregulation SCN2B gene could modify the sodim current. Therefore, it would be very interesting to address further protein expression studies to know if exists a regulation mechanism in these ion channel proteins related to clinical implications in DCM patients. The downregulation of KCNJ5 and KCNJ8 impairs the K+ current; and the downregulation of CACNB2 and CLIC2 leads to an increase in cytoplasmic Ca2+ ions, suggesting an altered time course for myocyte shortening and relaxation in DCM and a compromise in cardiac contractibility.
In addition, the sodium channel SCN2B is functionally coupled to CACNB2. It has been shown that a mutation in CACNB2b, another β subunit of the calcium channel, together with a mutation in SCN5A, underlies cardiac conduction disease . Interestingly, there are other studies linking ion channels to each other through the observation of a complex interaction between the cardiac subunits of the sodium and transient potassium channels by coimmunoprecipitation experiments .
In the IPA analysis, we found two networks connecting different families of ion channels. In the first network, CACNB2 showed interactions with the CACN gene family that comprises CACNA1A, CACNA1C, CACNA1F, CACNA1S, and CACNB4, which provides instructions for forming functional calcium channels [55,56]. CACNB2 also interacts with the CACNB gene family (CACNB genes 1–4) that encodes MAGUK proteins and that function as auxiliary β subunits in the assembly and gating of voltage-gated Ca2+ channels . Finally, CACNB2 is associated with the regulator of the voltage-gated calcium channels RIM1 , and with the sodium channel SCN2B, through its interaction with CACNA1B, since it has been described that a mutation in CACNB2b, and a mutation in SCN5A underlie cardiac conduction disease, as mentioned above .. The potassium ion channel genes KCNJ5 and KCNJ8 interact with some of the 17 members of the inward rectifier K+ KCNJ family, including KCNJ3, KCNJ6, KCNJ9, and KCNJ11.
Another network revealed in the IPA analysis showed that CLIC2 is an inhibitor of RYR1 and RYR2, which function as calcium release channels in the SR by this chloride channel . In addition, CLIC2 is related to TRAPPC2, which is involved in the endoplasmic reticulum-to-Golgi transport vesicles . Finally, the ubiquitin system is related to CLIC2 through NEDD4, NEDD4L, and UBC (ubiquitin C), which regulate the interaction between the motor neurons and the muscle  or the current of ion channels [62-64].
All these data reveal a communication between cardiac ion channels, where a minimum alteration could produce a general injury in the normal function of the heart, affecting the EC coupling.
A common limitation of the studies that use cardiac tissues from end-stage failing human hearts is the fact that there is a high variability in disease etiology and treatment. To make our study population etiologically homogeneous, we chose DCM patients that did not report any family history of the disease. In addition, the patients used in this study were on conventional therapy and certain treatments may influence ion channel mRNA levels. Moreover, our tissue samples are confined to transmural left ventricle apex, so our findings could not be generalized to all layers and regions of the left ventricle, as well as to only cardiomyocytes. However our group has extensively used samples from human LV tissue and in techniques as electron microscopy it has been shown the presence of a high number of cardiomyocytes in these samples [65-68].
In conclusion, in our study we analyzed the gene expression of ion channels involved in cardiac muscle contraction in DCM patients compared with CNT group. The changed expression levels shown in the ion channel genes might partly underlie the altered shortening and relaxation process observed in this pathology. Our results may constitute the basis to modulate the contractibility impairment observed in DCM, associated with differential mRNA levels in ion channel genes.
Gene expression of KCND3 and ATPA2A as positive controls of RT-qPCR experiments. The graph shows the relative mRNA levels of RT-qPCR experiment normalized to the mRNA expression of 3 housekeeping genes (GAPDH, PGK1, and TFRC). The error bar represents the standard error of the mean (SEM) for DCM (n =21) and CNT (n = 8) samples in RT-qPCR data. * p < 0.05; ** p < 0.01.
Gene expression differences between DCM and CNT groups. p < 0.01 and FC ≥ 1.3.
Conceived and designed the experiments: MMMN ERL AO ET. Performed the experiments: MMMN ERL AO ET. Analyzed the data: MMMN AO MO MP. Contributed reagents/materials/analysis tools: PGP JAM FE LMD FL JRGJ. Wrote the manuscript: MMMN AO MR.
- 1. Taylor MR, Carniel E, Mestroni L (2006) Cardiomyopathy, familial dilated. Orphanet J Rare Dis 1: 27. doi:https://doi.org/10.1186/1750-1172-1-27. PubMed: 16839424.
- 2. Jefferies JL, Towbin JA (2010) Dilated cardiomyopathy. Lancet 375: 752-762. doi:https://doi.org/10.1016/S0140-6736(09)62023-7. PubMed: 20189027.
- 3. Elliott P, Andersson B, Arbustini E, Bilinska Z, Cecchi F et al. (2008) Classification of the cardiomyopathies: a position statement from the European Society Of Cardiology Working Group on Myocardial and Pericardial Diseases. Eur Heart J 29: 270-276. PubMed: 17916581.
- 4. Ordög B, Brutyó E, Puskás LG, Papp JG, Varró A et al. (2006) Gene expression profiling of human cardiac potassium and sodium channels. Int J Cardiol 111: 386-393. doi:https://doi.org/10.1016/j.ijcard.2005.07.063. PubMed: 16257073.
- 5. Balse E, Steele DF, Abriel H, Coulombe A, Fedida D et al. (2012) Dynamic of ion channel expression at the plasma membrane of cardiomyocytes. Physiol Rev 92: 1317-1358. doi:https://doi.org/10.1152/physrev.00041.2011. PubMed: 22811429.
- 6. Bers DM (2002) Cardiac excitation-contraction coupling. Nature 415: 198-205. doi:https://doi.org/10.1038/415198a. PubMed: 11805843.
- 7. Cortés R, Rivera M, Roselló-Lletí E, Martínez-Dolz L, Almenar L et al. (2012) Differences in MEF2 and NFAT transcriptional pathways according to human heart failure aetiology. PLOS ONE 7: e30915. doi:https://doi.org/10.1371/journal.pone.0030915. PubMed: 22363514.
- 8. Börjesson SI, Elinder F (2008) Structure, function, and modification of the voltage sensor in voltage-gated ion channels. Cell Biochem Biophys 52: 149-174. doi:https://doi.org/10.1007/s12013-008-9032-5. PubMed: 18989792.
- 9. Bers DM, Pogwizd SM, Schlotthauer K (2002) Upregulated Na/Ca exchange is involved in both contractile dysfunction and arrhythmogenesis in heart failure. Basic Res Cardiol 97 Suppl 1: I36-I42. PubMed: 12479232.
- 10. Catterall WA (1988) Structure and function of voltage-sensitive ion channels. Science 242: 50-61. doi:https://doi.org/10.1126/science.2459775. PubMed: 2459775.
- 11. Catterall WA (2000) Structure and regulation of voltage-gated Ca2+ channels. Annu Rev Cell Dev Biol 16: 521-555. doi:https://doi.org/10.1146/annurev.cellbio.16.1.521. PubMed: 11031246.
- 12. Catterall WA (2000) From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 26: 13-25. doi:https://doi.org/10.1016/S0896-6273(00)81133-2. PubMed: 10798388.
- 13. Macrae DJ (2007) The Council for International Organizations and Medical Sciences (CIOMS) guidelines on ethics of clinical trials. Proc Am Thorac Soc 4: 176-179. doi:https://doi.org/10.1513/pats.200701-011GC. PubMed: 17494727.
- 14. Swedberg K, Cleland J, Dargie H, Drexler H, Follath F et al. (2005) Guidelines for the diagnosis and treatment of chronic heart failure: executive summary (update 2005): The Task Force for the Diagnosis and Treatment of Chronic Heart Failure of the European Society of Cardiology. Eur Heart J 26: 1115-1140. doi:https://doi.org/10.1093/eurheartj/ehi204. PubMed: 15901669.
- 15. Huang da W, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4: 44-57. PubMed: 19131956.
- 16. Kääb S, Dixon J, Duc J, Ashen D, Näbauer M et al. (1998) Molecular basis of transient outward potassium current downregulation in human heart failure: a decrease in Kv 4.3 mRNA correlates with a reduction in current density. Circulation 98: 1383-1393. doi:https://doi.org/10.1161/01.CIR.98.14.1383. PubMed: 9760292.
- 17. Partemi S, Batlle M, Berne P, Berruezo A, Campos B et al. (2013) Analysis of the arrhythmogenic substrate in human heart failure. Cardiovasc Pathol 22: 133-140. doi:https://doi.org/10.1016/j.carpath.2012.07.003. PubMed: 23036686.
- 18. Mercadier JJ, Lompré AM, Duc P, Boheler KR, Fraysse JB et al. (1990) Altered Sarcoplasmic Reticulum Ca2+-ATPase Gene Expression in the Human Ventricle during End-Stage. Heart Failure - J Clin Invest 85: 305-309.
- 19. Dally S, Bredoux R, Corvazier E, Andersen JP, Clausen JD et al. (2006) Ca2+-ATPases in non-failing and failing heart: evidence for a novel cardiac sarco/endoplasmic reticulum Ca2+-ATPase 2 isoform (SERCA2c). Biochem J 395: 249–258. doi:https://doi.org/10.1042/BJ20051427. PubMed: 16402920.
- 20. Arai M, Alpert NR, MacLennan DH, Barton P, Periasamy M (1993) Alterations in sarcoplasmic reticulum gene expression in human heart failure. A possible mechanism for alterations in systolic and diastolic properties of the failing myocardium. Circ Res 72: 463-469. doi:https://doi.org/10.1161/01.RES.72.2.463. PubMed: 8418995.
- 21. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402-408. doi:https://doi.org/10.1006/meth.2001.1262. PubMed: 11846609.
- 22. Winters AL, Minchin FR (2005) Modification of the Lowry assay to measure proteins and phenols in covalently bound complexes. Anal Biochem 346: 43-48. doi:https://doi.org/10.1016/j.ab.2005.07.041. PubMed: 16197913.
- 23. Szmit S, Jank M, Maciejewski H, Grabowski M, Glowczynska R et al. (2010) Gene expression profiling in peripheral blood nuclear cells in patients with refractory ischaemic end-stage heart failure. J Appl Genet 51: 353-368. doi:https://doi.org/10.1007/BF03208866. PubMed: 20720311.
- 24. Barth AS, Kumordzie A, Frangakis C, Margulies KB, Cappola TP et al. (2011) Reciprocal transcriptional regulation of metabolic and signaling pathways correlates with disease severity in heart failure. Circ Cardiovasc Genet 4: 475-483. doi:https://doi.org/10.1161/CIRCGENETICS.110.957571. PubMed: 21828333.
- 25. Ivandic BT, Mastitsky SE, Schönsiegel F, Bekeredjian R, Eils R et al. (2012) Whole-genome analysis of gene expression associates the ubiquitin-proteasome system with the cardiomyopathy phenotype in disease-sensitized congenic mouse strains. Cardiovasc Res 94: 87-95. doi:https://doi.org/10.1093/cvr/cvs080. PubMed: 22308238.
- 26. Prat-Vidal C, Gálvez-Montón C, Nonell L, Puigdecanet E, Astier L et al. (2013) Identification of temporal and region-specific myocardial gene expression patterns in response to infarction in Swine. PLOS ONE 8: e54785. doi:https://doi.org/10.1371/journal.pone.0054785. PubMed: 23372767.
- 27. Kim JI, Kim IK (2012) Probing regulatory proteins for vascular contraction by deoxyribonucleic Acid microarray. Korean Circ J 42: 479-486. doi:https://doi.org/10.4070/kcj.2012.42.7.479. PubMed: 22870082.
- 28. Zhou Y, Gong B, Kaminski HJ (2012) Genomic profiling reveals Pitx2 controls expression of mature extraocular muscle contraction-related genes. Invest Ophthalmol Vis Sci 53: 1821-1829. doi:https://doi.org/10.1167/iovs.12-9481. PubMed: 22408009.
- 29. Dasgupta T, Stillwagon SJ, Ladd AN (2013) Gene expression analyses implicate an alternative splicing program in regulating contractile gene expression and serum response factor activity in mice. PLOS ONE 8: e56590. doi:https://doi.org/10.1371/journal.pone.0056590. PubMed: 23437181.
- 30. Ambrosi CM, Yamada KA, Nerbonne JM, Efimov IR (2013) Gender differences in electrophysiological gene expression in failing and non-failing human hearts. PLOS ONE 8: e54635. doi:https://doi.org/10.1371/journal.pone.0054635. PubMed: 23355885.
- 31. Walmsley J, Rodriguez JF, Mirams GR, Burrage K, Efimov IR et al. (2013) mRNA expression levels in failing human hearts predict cellular electrophysiological remodeling: A population- based simulation study. PLOS ONE 8: e56359. doi:https://doi.org/10.1371/journal.pone.0056359. PubMed: 23437117.
- 32. Morgan JP, Erny RE, Allen PD, Grossman W, Gwathmey JK (1990) Abnormal intracellular calcium handling, a major cause of systolic and diastolic dysfunction in ventricular myocardium from patients with heart failure. Circulation 81: III21-32.
- 33. Zhang HB, Li RC, Xu M, Xu SM, Lai YS et al. (2013) Ultrastructural uncoupling between T-tubules and sarcoplasmic reticulum in human heart failure. Cardiovasc Res 98: 269-270. doi:https://doi.org/10.1093/cvr/cvt030. PubMed: 23405000.
- 34. Maron BJ, Towbin JA, Thiene G, Antzelevitch C, Corrado D et al. (2006) Contemporary definitions and classification of the cardiomyopathies: an American Heart Association Scientific Statement from the Council on Clinical Cardiology, Heart Failure and Transplantation Committee; Quality of Care and Outcomes Research and Functional Genomics and Translational Biology Interdisciplinary Working Groups; and Council on Epidemiology and Prevention. Circulation 113: 1807-1816. doi:https://doi.org/10.1161/CIRCULATIONAHA.106.174287. PubMed: 16567565.
- 35. Fatkin D (2011) Guidelines for the diagnosis and management of familial dilated cardiomyopathy. Heart Lung Circ 20: 691-693. doi:https://doi.org/10.1016/j.hlc.2011.07.008. PubMed: 21885340.
- 36. Isom LL (2001) Sodium channel beta subunits: anything but auxiliary. Neuroscientist 7: 42-54. doi:https://doi.org/10.1177/107385840100700108. PubMed: 11486343.
- 37. Dhar Malhotra J, Chen C, Rivolta I, Abriel H, Malhotra R et al. (2001) Characterization of sodium channel alpha- and beta-subunits in rat and mouse cardiac myocytes. Circulation 103: 1303-1310. doi:https://doi.org/10.1161/01.CIR.103.9.1303. PubMed: 11238277.
- 38. Zicha S, Maltsev VA, Nattel S, Sabbah HN, Undrovinas AI (2004) Post-transcriptional alterations in the expression of cardiac sodium channel subunits in chronic heart failure. J Mol Cell Cardiol 37: 91-100. doi:https://doi.org/10.1016/j.yjmcc.2004.04.003. PubMed: 15242739.
- 39. Ufret-Vincenty CA, Baro DJ, Lederer WJ, Rockman HA, Quinones LE et al. (2001) Role of sodium channel deglycosylation in the genesis of cardiac arrhythmias in heart failure. J Biol Chem 276: 28197-28203. doi:https://doi.org/10.1074/jbc.M102548200. PubMed: 11369778.
- 40. Nobles M, Sebastian S, Tinker A (2010) HL-1 cells express an inwardly rectifying K+ current activated via muscarinic receptors comparable to that in mouse atrial myocytes. Pflugers Arch 460: 99-108. doi:https://doi.org/10.1007/s00424-010-0799-z. PubMed: 20186548.
- 41. Hedin KE, Lim NF, Clapham DE (1996) Cloning of a Xenopus laevis inwardly rectifying K+ channel subunit that permits GIRK1 expression of IKACh currents in oocytes. Neuron 16: 423-429. doi:https://doi.org/10.1016/S0896-6273(00)80060-4. PubMed: 8789957.
- 42. Cui Y, Giblin JP, Clapp LH, Tinker A (2001) A mechanism for ATP-sensitive potassium channel diversity: Functional coassembly of two pore-forming subunits. Proc Natl Acad Sci U S A 98: 729-734. doi:https://doi.org/10.1073/pnas.98.2.729. PubMed: 11136227.
- 43. Kane GC, Lam CF, O'Cochlain F, Hodgson DM, Reyes S et al. (2006) Gene knockout of the KCNJ8-encoded Kir6.1 K(ATP) channel imparts fatal susceptibility to endotoxemia. FASEB J 20: 2271-2280. doi:https://doi.org/10.1096/fj.06-6349com. PubMed: 17077304.
- 44. Cromer BA, Gorman MA, Hansen G, Adams JJ, Coggan M et al. (2007) Structure of the Janus protein human CLIC2. J Mol Biol 374: 719-731. doi:https://doi.org/10.1016/j.jmb.2007.09.041. PubMed: 17945253.
- 45. Dulhunty AF, Pouliquin P, Coggan M, Gage PW, Board PG (2005) 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 390: 333-343. doi:https://doi.org/10.1042/BJ20042113. PubMed: 15916532.
- 46. Dulhunty AF, Hewawasam R, Liu D, Casarotto MG, Board PG (2011) Regulation of the cardiac muscle ryanodine receptor by glutathione transferases. Drug Metab Rev 43: 236-252. doi:https://doi.org/10.3109/03602532.2010.549134. PubMed: 21323602.
- 47. Jalilian C, Gallant EM, Board PG, Dulhunty AF (2008) Redox potential and the response of cardiac ryanodine receptors to CLIC-2, a member of the glutathione S-transferase structural family. Antioxid Redox Signal 10: 1675-1686. doi:https://doi.org/10.1089/ars.2007.1994. PubMed: 18522493.
- 48. Takano K, Liu D, Tarpey P, Gallant E, Lam A et al. (2012) An X-linked channelopathy with cardiomegaly due to a CLIC2 mutation enhancing ryanodine receptor channel activity. Hum Mol Genet 21: 4497-4507. doi:https://doi.org/10.1093/hmg/dds292. PubMed: 22814392.
- 49. Chien AJ, Carr KM, Shirokov RE, Rios E, Hosey MM (1996) Identification of palmitoylation sites within the L-type calcium channel beta2a subunit and effects on channel function. J Biol Chem 271: 26465-26468. doi:https://doi.org/10.1074/jbc.271.43.26465. PubMed: 8900112.
- 50. Yamaguchi H, Okuda M, Mikala G, Fukasawa K, Varadi G (2000) Cloning of the beta(2a) subunit of the voltage-dependent calcium channel from human heart: cooperative effect of alpha(2)/delta and beta(2a) on the membrane expression of the alpha(1C) subunit. Biochem Biophys Res Commun 267: 156-163. doi:https://doi.org/10.1006/bbrc.1999.1926. PubMed: 10623591.
- 51. Antzelevitch C, Pollevick GD, Cordeiro JM, Casis O, Sanguinetti MC et al. (2007) Loss-of-function mutations in the cardiac calcium channel underlie a new clinical entity characterized by ST-segment elevation, short QT intervals, and sudden cardiac death. Circulation 115: 442-449. doi:https://doi.org/10.1161/CIRCULATIONAHA.106.668392. PubMed: 17224476.
- 52. Cordeiro JM, Marieb M, Pfeiffer R, Calloe K, Burashnikov E et al. (2009) Accelerated inactivation of the L-type calcium current due to a mutation in CACNB2b underlies Brugada syndrome. J Mol Cell Cardiol 46: 695-703. doi:https://doi.org/10.1016/j.yjmcc.2009.01.014. PubMed: 19358333.
- 53. Hu D, Barajas-Martinez H, Nesterenko VV, Pfeiffer R, Guerchicoff A et al. (2010) Dual variation in SCN5A and CACNB2b underlies the development of cardiac conduction disease without Brugada syndrome. Pacing Clin Electrophysiol 33: 274-285. doi:https://doi.org/10.1111/j.1540-8159.2009.02642.x. PubMed: 20025708.
- 54. Deschênes I, Armoundas AA, Jones SP, Tomaselli GF (2008) Post-transcriptional gene silencing of KChIP2 and Navbeta1 in neonatal rat cardiac myocytes reveals a functional association between Na and Ito currents. J Mol Cell Cardiol 45: 336-346. doi:https://doi.org/10.1016/j.yjmcc.2008.05.001. PubMed: 18565539.
- 55. Bidaud I, Mezghrani A, Swayne LA, Monteil A, Lory P (2006) Voltage-gated calcium channels in genetic diseases. Biochim Biophys Acta 1763: 1169-1174. doi:https://doi.org/10.1016/j.bbamcr.2006.08.049. PubMed: 17034879.
- 56. Arikkath J, Campbell KP (2003) Auxiliary subunits: essential components of the voltage-gated calcium channel complex. Curr Opin Neurobiol 13: 298-307. doi:https://doi.org/10.1016/S0959-4388(03)00066-7. PubMed: 12850214.
- 57. Ebert AM, McAnelly CA, Handschy AV, Mueller RL, Horne WA et al. (2008) Genomic organization, expression, and phylogenetic analysis of Ca2+ channel beta4 genes in 13 vertebrate species. Physiol Genomics 35: 133-144. doi:https://doi.org/10.1152/physiolgenomics.90264.2008. PubMed: 18682574.
- 58. Weiss N, Sandoval A, Kyonaka S, Felix R, Mori Y et al. (2011) Rim1 modulates direct G-protein regulation of Ca(v)2.2 channels. Pflugers Arch 461: 447-459. doi:https://doi.org/10.1007/s00424-011-0926-5. PubMed: 21331761.
- 59. Board PG, Coggan M, Watson S, Gage PW, Dulhunty AF (2004) CLIC-2 modulates cardiac ryanodine receptor Ca2+ release channels. Int J Biochem Cell Biol 36: 1599-1612. doi:https://doi.org/10.1016/j.biocel.2004.01.026. PubMed: 15147738.
- 60. Sacher M, Kim YG, Lavie A, Oh BH, Segev N (2008) The TRAPP complex: insights into its architecture and function. Traffic 9: 2032-2042. doi:https://doi.org/10.1111/j.1600-0854.2008.00833.x. PubMed: 18801063.
- 61. Yang B, Kumar S (2010) Nedd4 and Nedd4-2: closely related ubiquitin-protein ligases with distinct physiological functions. Cell Death Differ 17: 68-77. doi:https://doi.org/10.1038/cdd.2009.84. PubMed: 19557014.
- 62. Rougier JS, van Bemmelen MX, Bruce MC, Jespersen T, Gavillet B et al. (2005) Molecular determinants of voltage-gated sodium channel regulation by the Nedd4/Nedd4-like proteins. Am J Physiol Cell Physiol 288: C692-C701. PubMed: 15548568.
- 63. Van Bemmelen MX, Rougier JS, Gavillet B, Apothéloz F, Daidié D et al. (2004) Cardiac voltage-gated sodium channel Nav1.5 is regulated by Nedd4-2 mediated ubiquitination. Circ Res 95: 284-291. doi:https://doi.org/10.1161/01.RES.0000136816.05109.89. PubMed: 15217910.
- 64. Mia S, Munoz C, Pakladok T, Siraskar G, Voelkl J et al. (2012) Downregulation of Kv1.5 K channels by the AMP-activated protein kinase. Cell Physiol Biochem 30: 1039-1050. doi:https://doi.org/10.1159/000341480. PubMed: 23221389.
- 65. Cortés R, Roselló-Lletí E, Rivera M, Martínez-Dolz L, Salvador A et al. (2010) Influence of heart failure on nucleocytoplasmic transport in human cardiomyocytes. Cardiovasc Res 85: 464-472. doi:https://doi.org/10.1093/cvr/cvp336. PubMed: 19819881.
- 66. Cortés R, Rivera M, Roselló-Lletí E, Martínez-Dolz L, Almenar L et al. (2012) Differences in MEF2 and NFAT transcriptional pathways according to human heart failure aetiology. PLOS ONE 7: e30915. doi:https://doi.org/10.1371/journal.pone.0030915. PubMed: 22363514.
- 67. Roselló-Lletí E, Rivera M, Cortés R, Azorín I, Sirera R et al. (2012) Influence of heart failure on nucleolar organization and protein expression in human hearts. Biochem Biophys Res Commun 418: 222-228. doi:https://doi.org/10.1016/j.bbrc.2011.12.151. PubMed: 22244875.
- 68. Tarazón E, Rivera M, Roselló-Lletí E, Molina-Navarro MM, Sánchez-Lázaro IJ et al. (2012) Heart failure induces significant changes in human pore complex of human cardiomyocytes. PLOS ONE 7: e48957. doi:https://doi.org/10.1371/journal.pone.0048957. PubMed: 23152829.