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Underexpression of CACNA1C Caused by Overexpression of microRNA-29a Underlies the Pathogenesis of Atrial Fibrillation

Background The objective of this study was to investigate the molecular mechanism of atrial fibrillation (AF), as well as the negative regulatory relationship between miR-29a-3p and CACNA1C. Material/Methods We searched the online miRNA database (www.mirdb.org) and identified the miR-29a-3p binding sequence within the 3′-UTR of the target gene, and then conducted luciferase assay to verify it. The cells were transfected with miR-29a-3p and ICa,L was determined in those cells. Results We validated CACNA1C to be the direct target gene of miR-29a-3p. We also established the negative regulatory relationship between miR-29a-3p and CACNA1C via studying the relative luciferase activity. We also conducted real-time PCR and Western blot analysis to study the mRNA and protein expression level of CACNA1C among different groups of cells treated with scramble control, 30nM miR-29a-3p mimics, and 60nM miR-29a-3p mimics, indicating a negative regulatory relationship between miR-29a-3p and CACNA1C. We next analyzed whether miR-29a-3p transfection in cardiomyocytes produced the effects on the ICa,L induced by electrical remodeling, and found a tonic inhibition of IBa by endogenous miR-29a-3p in atrial myocytes. Conclusions We validated the negative regulation between miR-29a-3p and CACNA1C, and found that miR-29a-3p might a potential therapeutic target in the treatment of AF.


Background
Atrial fibrillation (AF) is characterized by high incidence, prevalence, and mortality [1]. Increasing evidence has shown that uneven distribution of the vagus and sympathetic nerves, together with cardiac autonomic nerve remodeling (ANR), which is characterized by regeneration of nerve, are involved in the pathogenesis of both chronic and acute AF [2,3]. Structural remodeling, electricity, and ANR interact and promote each other, resulting in a vicious cycle that leads to AF [4,5]. The main risk factor related to myocardial-associated cerebrovascular events [6,7], especially fast atrial rates, results in progressive atrial structural and electrical changes (remodeling) that favor arrhythmia recurrence and maintenance. Fibrosis ranks among the most important structural changes associated with atrial remodeling [6,7]. Characterized by a marked shortening of the atrial action potential duration (APD) and refractoriness, electrical remodeling results from changes in Ca2+ and K+ channel densities [6][7][8][9][10]. Reduction of the density of the L-type Ca2+ current (I Ca,L), which is generated by channels consisting of a2d (CACNA2D), a1c, b2 (CACNB2) or Cav1.2 (encoded by CACNA1C) subunits, is a hallmark of the electrical remodeling [7,8]. Importantly, the molecular mechanisms underlying the upregulation of I Ca,L has not been completely elucidated yet. Some researchers consider that it is because of a reduction of expression of mRNA CACNB2 and CACNA1C,9,11 whereas others suggested post-transcriptional mechanisms, such as protein dephosphorylation [12].
MicroRNAs (miRNAs) are a class of small non-coding RNAs (ncRNAs) consisting of 21-24 nucleotides, which function in posttranscriptional regulation of gene expression and RNA silencing [13]. MRNA changes, driven by miRNAs, might be observed in AF patients. Some researchers carried out microarray experiments using samples from AF patients [14,15], while others have validated key roles for miRNAs, such as miR-1, miR-328, and miR-26, in AF pathogenesis [16][17][18]. These studies have added to our understanding of the factors involved in early AF development. MicroRNAs are important in a wide range of cardiac remodeling processes [19], and there is increasing evidence for participation in AF [20]; its upregulation contributes to AF stabilization because of electrical remodeling [21,22].
It was reported that dysregulation of CACNA1C is responsible for the development of AF [23]. In addition, miR-29a-3p and miR-3135b were shown to be differentially expressed in AF tissue samples [24], and we found the both miR-29a-3p and miR-3135b target CACNA1C. In this study, we aimed to validate the miRNA and mRNA regulatory relationship, and to explore the role of the miR-29a-3p and CACNA1C in the pathogenesis of AF.

Human atrial samples
The tip of the right atrial appendage was removed as part of the general surgical procedure in patients undergoing valve replacement, including 25 patients with AF and 25 without AF, open-heart surgery for bypass grafting, or a combination of both after written informed consent was obtained. The diagnosis of AF was reached by evaluating medical records and 12-lead electrocardiogram findings. Those who had other cardiovascular diseases, such as heart failure, mitral valve stenosis/regurgitation, or cardiomyopathy, were excluded from this study. All biopsies were performed at the same site, which is at the tip of the right atrial appendage. The samples were flash frozen in liquid nitrogen after collection in accordance with protocols approved by the ethics committee of the No. 7 People's Hospital in Zhengzhou City.

Quantitative real-time PCR
iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA) was applied to perform reverse transcription in order to measure levels of miR-29a-3p and mRNA of CACNA1C. To measure levels of miR-NAs, reverse transcription and quantitative real-time (qRT)-PCR were carried out in accordance with the instructions provided by the manufacturer. Mastercycler ep realplex (Eppendorf, Hamburg, Germany), PerfeCTa ® SYBR ® Green FastMix ® (Quanta Biosciences, Gaithersburg, MD) were employed to perform qRT-PCR reactions in 96-well plates in triplicate. The 2 -DDCT method was applied to calculate the relative expression levels after normalization to U6.

Western blot
The culture cells and tissue samples were homogenized and lysed by lysis buffer, and the lysates were loaded onto the 10% PAGE gel. The separated proteins were transferred onto a PVDF membrane (Millipore), and the blots were incubated with the primary antibodies. Primary antibodies against b-actin (Cell Signaling Technology, MA, USA) at a final concentration of 1: 1000 and against CACNA1C (Abcam, Cambridge, UK) at a final concentration of 1:250 were used to carry out Western blot analysis. Blots were washed 3 times and incubated with secondary antibody (Cell Signaling Technology, MA, USA). We then utilized Quantity One software (Bio-Rad, Hercules, CA, USA) to measure the band intensity (area × OD) of each group in order to quantify the Western blot bands. Data were normalized to the control values.

Cell transfection and luciferase assays
We purchased pGL3 with luciferase and inserted amplified CACNA1C full-length 3'UTR with a miR-29a-3p binding site deletion (pGL3-CACNA1C-UTR) or mutant CACNA1C full-length 3'UTR (pGL3-CACNA1C-UTR-mutant) from Shanghai Biobuy Biotech Co., Ltd. (Shanghai, China). Sequencing was performed to confirm the plasmids. The constructed vectors transfected the cells with the help of lipofectamine 2000 (Invitrogen, CA, USA). We added 50 ng of pRL-TK (Promega, Madison, WI, USA) to correct the efficiency of transfection in each group. Dual-Luciferase Reporter Assay System (Promega) was used to measure luciferase activity. We expressed the activity of promoter as the ratio of Firefly luciferase activity to Renilla luciferase activity.

MiRNA target prediction
Computational prediction of putative targets for miR-29a-3p was performed by searching www.mirdb.org, using the target prediction algorithms.

Transfection studies
We transfected the HL-1 cells with miR-29a-3p mimics (Genepharma, Shanghai, China) in accordance with the manufactures' protocol and took fluorescein-conjugated scrambled siRNA as control. We transfected them in accordance with the manufacturer's protocol. MiR-29a-3p mimics were transfected into HL-1 with the help of lipofectamine 2000 (Invitrogen).

Calcium current recordings
With the whole-cell patch-clamp technique (micropipette resistance <3.5 MΩ), we recorded the currents in human HL-1 cells and atrial myocytes at room temperature. We compensated for series resistance when ³80% compensation was achieved. No significant voltage errors (<5 mV) were caused by rising series resistance with the micropipettes used under our experimental conditions.

Statistical analysis
Fisher's exact test and 2-tailed Student's t-test were used where appropriate. Data are presented as mean ±SEM and p<0.05 was considered statistically significant, except for the miRNA microarray data analyzed by LC Sciences (Houston, TX) using a validated method.

CACNA1C was a target of miR-29a-3p
Voltage-operated L-type Ca 2+ channels (LCCs) play a critical role in the electrical and mechanical functions of the heart, and disorders of LCCs have been linked to atrial fibrillation (AF).
A characteristic of AF is atrial electric remodeling associated with profound reduction of L-type Ca 2+ current and shortening of the action potential duration. Atrial LCC is a multi-subunit complex consisting of the performing a1 subunit (encoded by CACNA1C) and auxiliary subunits, such as Cavb2 and a2 d, in which the function of CACNA1C would affect the atrial fibrillation. As shown in Figure 1, we therefore used online miRNA target prediction tools to search the regulatory miR-NA of CACNA1C, and consequently identified CACNA1C as the candidate target gene of miR-3135b and miR-29a-3p in HL-1 cells with the 'seed sequence' in the 3'UTR of CACNA1C. To confirm expression of miR-3135b and miR-29a-3p in AF tissues, we evaluated the expression of miR-3135b and miR-29a-3p in 80 AF and normal frozen heart tissues using quantitative reverse transcriptase PCR (qRT-PCR). As shown in Figure 2, the expression level of miR-3135b was comparable in the 2 groups, while the expression level of miR-29a-3p in AF tissues was significantly higher than in controls.
To validate the regulatory relationship between miR-29a-3p and miR-3135b and CACNA1C, we also conducted luciferase activity reporter assay in HEK293 cells. We found that only the luciferase activity from the cells co-transfected with miR-29a-3p and CACNA1C 3'UTR decreased significantly ( Figure 3A), while cells co-transfected with miR-3135band CACNA1C 3'UTR were comparable ( Figure 3B). The results confirmed that CACNA1C was a validated target of miR-29a-3p.
To determine whether the expression of CACNA1C is controlled by miR-29a-3p, we collected tissue samples from the patients with or without AF, we found that miR-29a-3p levels were dramatically increased ( Figure 2B), and also found a significant decrease in CACNA1C mRNA ( Figure 4A) and protein ( Figure 4B) in atrial tissues with AF compared to those without AF. These findings indicate the negative regulatory relationship between miR-29a-3p and CACNA1C.
To further validate the hypothesis of the negative regulatory relationship between miR-29a-3p and CACNA1C, we investigated       Figure 5A) and mRNA expression level ( Figure 5B) of atrial myocytes treated with 30nM miR-29a-3p mimics were clearly lower than the scramble control, while those of the cells treated with 60nM miR-29a-3p mimics were even lower than in the 30nM treatment group, validating the negative regulatory relationship between the miRNA and mRNA.
MiR-29a-3p reduces the density of L-type calcium currents qPCR experiments demonstrated that AF myocytes displayed greater miR-29a3p expression than non-AF myocytes and that there was a correlation between I Ca,L density and miR-29a-3p expression. We next analyzed whether miR-29a-3p transfection in atrial myocytes cells produced the effects on the I Ca,L induced by electrical remodeling.
Currents through L-type Ca 2+ channels were recorded in I Ca,Lpredominant cells using Ba 2+ as the charge carrier (I Ba ). I Ba traces were recorded by applying the above protocol in cells transfected or not with miR-29a-3p mimic (30 nmol/L). As can be observed in Figure 6A, current density reached its maximum value at +20 mV (n=30) and transfection of miR-29a-3p mimic significantly reduced I Ba density (n=33; P=0.02) and slowed the time course of inactivation (P<0.01), without modifying activation kinetics ( Figure 6A, 6B). I Ba density was not modified by the negative control miRNA (30 nmol/L; n=15), but was significantly increased by antimiR-29a-3p (30 nmol/L, n=15; Figure 6C), demonstrating a tonic inhibition of I Ba by endogenous miR-29a-3p in atrial myocytes.  Figure 5. We investigated the mRNA/protein expression level of CACNA1C of atrial myocytes treated with scramble control, 30nM miR-29a-3p mimics, and 60nM miR-29a-3p mimics. The CACNA1C protein (A) and mRNA expression level (B) of atrial myocytes treated with 30nM miR-29a-3p mimics were clearly lower than in the scramble control, while those of the sample group treated with 60nM miR-29a-3p mimics were even lower than in the 30nM treatment group, validating the negative regulatory relationship between miR-499a and PFGFRB.

Discussion
In this study we validated CACNA1C as a direct gene of miR-29a-3p, and established the negative regulatory relationship between miR-29a-3p and CACNA1C by using luciferase activity. We confirmed the negative regulatory relationship between miR-29a-3p and CACNA1C by treating the cells with miR-29a-3p mimics. We also found a tonic inhibition of I Ba by endogenous miR-29a-3p in atrial myocytes.
Recent evidence indicated that miRNAs are involved in I Ca,L downregulation. In this line, miR-1 and miR-328 decrease I Ca,L expression by binding to 3'UTR of CACNA1C [17,25]. In contrast to patients in sinus rhythm (SR), expression of miR-29a-3p increased in the samples from chronic AF (CAF) patients [26]. Adam et al. [27] found a 2.5-fold upregulation of miR-29a-3p expression in left atrial appendages from patients with AF compared with samples from patients with SR. They believed that miR-29a-3p took part in atrial fibrosis formation and contributed to the structural remodeling induced by AF [27,28]. Indeed, cardiac fibroblasts have a higher expression level of miR-29a-3p, where it is implicated in atrial profibrillatory remodeling and activation of profibrotic pathways [29] in experimental models of heart failure [30]. However, miR-29a-3p expression in cardiac myocytes has not been consistently observed, and how it affects the electrical remodeling is currently unknown. We hypothesized that miR-29a-3p is upregulated in myocytes and is expressed in human atrial myocytes from patients with AF compared with those in SR, playing a role in the AF-related I Ca,L decrease. In this study we used online miRNA target prediction tools to search for the regulatory miRNA of CACNA1C, and consequently identified CACNA1C as the candidate target gene of miR-3135b and miR-29a-3p in HEK293 cells with the 'seed sequence' in the 3'UTR of CACNA1C. To confirm expression of miR-3135b and miR-29a-3p in AF tissues, we evaluated the expression of miR-3135b and miR-29a-3p in 80 AF and normal frozen heart tissues using quantitative reverse transcriptase PCR (qRT-PCR). We showed that the expression level of miR-3135b was comparable in the 2 groups, while the expression level of miR-29a-3p in AF tissues was significantly higher than in controls. Furthermore, we performed luciferase assay to confirm that CACNA1C was a validated target of miR-29a-3p.
Encoded by CACNA1C gene, protein CaV1.2 is a1-subunits of cardiac L-type calcium channel (LTCC) and comprises the ion permeating subunit, which determines the main pharmacological and biophysical properties of the channel [31]. A consistent reduction of I Ca,L density has been observed in in AF experimental models as well as in CAF patients [32], and it is believed that the process was triggered by Ca 2+ overload secondary to the rapid atrial rates [33]. The molecular mechanisms underlying I Ca,L downregulation are complex and may include impaired Cav1.2 protein trafficking induced by impaired Src kinase activity, ankyrin-B dysfunction, ankyrin-B dysfunction, enhanced Cav1.2 a-subunit S-nitrosylation, Ca 2+ channel dephosphorylation because of serine/threonine protein phosphatase activation, Ca 2+ channel dephosphorylation because of serine/threonine protein phosphatase activation, activation of the Ca 2+ / calmodulin/calcineurin/nuclear factor of activated T cells system, causing transcriptional downregulation of the Cav1.2 asubunit or a zinc-binding protein [7,[34][35][36]. In addition to all these mechanisms, miRNAs have been recently proposed to be involved in AF-related I Ca,L downregulation [17,37,38]. Only miR-328 has been shown to regulate Ca 2+ channel a1c and b subunits expression among all the miRNAs that are upregulated in CAF patients [17]. MiR-328 is increased in right atrial appendages from CAF patients but not in those with SR.
Overexpression of miR-328 through transgenic manipulation in mice and adenoviral infection in canine atria enhanced AF susceptibility, decreased I Ca,L density, and shortened atrial APD as a result of the repression of CACNA1C expression. Other miRNAs, such as miR-26 and miR-1, have been reported to play important roles in AF-related electrical remodeling, mainly by targeting Kir2.1 channels [16,18,39]. Both miRNAs take part in the shortening of the APD as well as the AF-related increase of the inward rectifier. Consequently, we consider that all these miRNAs (miR-1, miR-29a-3p, miR-26, and miR-328) may contribute to AF-related electrical remodeling and eventually to the perpetuation of the arrhythmia. In this study, we investigated the mRNA/protein expression level of CACNA1C of atrial myocytes treated with scramble control, 30nM miR-29a-3p mimics, and 60nM miR-29a-3p mimics. The CACNA1C protein ( Figure 5A) and mRNA expression level ( Figure 5B) of atrial myocytes treated with 30nM miR-29a-3p mimics were clearly lower than in the scramble control, while those of the sample group treated with 60nM miR-29a-3p mimics were even lower than in the 30nM treatment group, validating the negative regulatory relationship between the miRNA and mRNA. In addition, we observed that current density reached its maximum value at +20 mV (n=30) and transfection of miR-29a-3p mimic significantly reduced I Ba density (n=33; P=0.02) and slowed the time course of inactivation (P=0.005), without modifying activation kinetics ( Figure 6A, 6B). I Ba density was not modified by the negative control miRNA (30 nmol/L; n=15), but was significantly increased by antimiR-29a-3p (30 nmol/L, n=15; Figure 6C), demonstrating a tonic inhibition of I Ba by endogenous miR-29a-3p in atrial myocytes.
The present study has certain limitations. Firstly, the sample size was relative small and further studies with larger populations and different ethnic backgrounds are warranted. Secondly, no animal model was used in this study, which makes the study less persuasive. Further animal experiments, especially in transgenic animal models, is need to confirm the conclusions drawn in this study.